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Identification of Potential Mosquito Vectors of West Nile Virus to Horses in North Central Florida

Permanent Link: http://ufdc.ufl.edu/UFE0022088/00001

Material Information

Title: Identification of Potential Mosquito Vectors of West Nile Virus to Horses in North Central Florida
Physical Description: 1 online resource (136 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: West Nile virus (family Flaviviridae, genus flavivirus WNV) is of concern in the US and Florida because the virus causes disease in humans and horses. Since 1999, there have been 23,925 clinical human cases of WNV in the United States (1999-2007). Prevention and reduction of cases requires a clear understanding of the WNV transmission cycle, but much of the needed information is lacking. It is still unknown which mosquito species transmit WNV to horses. This study integrated field investigations with laboratory studies to identify possible mosquito vectors of WNV to horses in north central Florida. The primary objectives of this research were to compare the abundance and seasonality of mosquito species collected near horses, and to characterize host preference of potential vectors. An additional aim was to evaluate extrinsic risk factors of WNV to Florida horses. The extrinsic factors of interest included farm management, farm ecology, and the entomological conditions associated with each farm. A questionnaire that focused on potential risk factors was mailed to the owners of all horses tested for arbovirus from 2001 to 2003. Vaccination was the factor most strongly associated with a protective effect for WNV disease outcome in horses. The factors that were associated with an increased risk of WNV in horses were fan use in the stable, mosquito activity, and dead birds on the property. Blood meal identification and virus screening were done in order to determine which mosquito species, if any, were involved in WNV transmission to horses. Mosquitoes were collected for a period of 26 months from a horse research area in north central Florida. DNA was extracted from the abdomen of the blood fed mosquitoes to test for the presence of avian, mammalian, and reptilian blood using PCR with different primer sets. The blood meals were confirmed with sequencing. The non-blood-fed mosquitoes were sorted into pools of up to 50 mosquitoes and screened for WNV, SLEV, and EEEV by Real-Time quantitative RT-PCR. A total of 45,851 mosquitoes (twenty three species) were collected, 252 of which had visible blood meals. Twelve mosquito species (fifty eight individuals) were positive for horse DNA. St. Louis encephalitis virus was detected in one pool of Mansonia titillans collected on September 26, 2006. This study was able to identify several mosquito species feeding on horses and risk factors associated with WNV disease. The vaccine can protect horses against WNV disease if administered two weeks prior to exposure and if a booster is administered yearly.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Day, Jonathan F.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022088:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022088/00001

Material Information

Title: Identification of Potential Mosquito Vectors of West Nile Virus to Horses in North Central Florida
Physical Description: 1 online resource (136 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: West Nile virus (family Flaviviridae, genus flavivirus WNV) is of concern in the US and Florida because the virus causes disease in humans and horses. Since 1999, there have been 23,925 clinical human cases of WNV in the United States (1999-2007). Prevention and reduction of cases requires a clear understanding of the WNV transmission cycle, but much of the needed information is lacking. It is still unknown which mosquito species transmit WNV to horses. This study integrated field investigations with laboratory studies to identify possible mosquito vectors of WNV to horses in north central Florida. The primary objectives of this research were to compare the abundance and seasonality of mosquito species collected near horses, and to characterize host preference of potential vectors. An additional aim was to evaluate extrinsic risk factors of WNV to Florida horses. The extrinsic factors of interest included farm management, farm ecology, and the entomological conditions associated with each farm. A questionnaire that focused on potential risk factors was mailed to the owners of all horses tested for arbovirus from 2001 to 2003. Vaccination was the factor most strongly associated with a protective effect for WNV disease outcome in horses. The factors that were associated with an increased risk of WNV in horses were fan use in the stable, mosquito activity, and dead birds on the property. Blood meal identification and virus screening were done in order to determine which mosquito species, if any, were involved in WNV transmission to horses. Mosquitoes were collected for a period of 26 months from a horse research area in north central Florida. DNA was extracted from the abdomen of the blood fed mosquitoes to test for the presence of avian, mammalian, and reptilian blood using PCR with different primer sets. The blood meals were confirmed with sequencing. The non-blood-fed mosquitoes were sorted into pools of up to 50 mosquitoes and screened for WNV, SLEV, and EEEV by Real-Time quantitative RT-PCR. A total of 45,851 mosquitoes (twenty three species) were collected, 252 of which had visible blood meals. Twelve mosquito species (fifty eight individuals) were positive for horse DNA. St. Louis encephalitis virus was detected in one pool of Mansonia titillans collected on September 26, 2006. This study was able to identify several mosquito species feeding on horses and risk factors associated with WNV disease. The vaccine can protect horses against WNV disease if administered two weeks prior to exposure and if a booster is administered yearly.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Day, Jonathan F.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022088:00001


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86a173483d9948ccc54ee2a24e5590d7
f8c51512a3b0197021ff2b52311a0e48565b7c87







IDENTIFICATION OF POTENTIAL MOSQUITO VECTORS OF WEST NILE VIRUS TO
HORSES IN NORTH CENTRAL FLORIDA




















By

LESLIE MICHELLE VIGUERS RIOS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008






























2008 Leslie Michelle Viguers Rios

































To my husband, Salvador Rios Madrigal









ACKNOWLEDGMENTS

I thank my committee, colleagues, friends and family for the necessary support and

encouragement to finish this dissertation. Dr. James Maruniak provided laboratory space,

reagents, and daily interaction. Dr. Maureen Long provided the stable trap, laboratory space, and

laboratory help. She provided reagents, and allowed me access to a multi-year database to

conduct the extrinsic risk analysis. Dr. Shue spent many hours with me on statistical analyses

and for that I'm very grateful. I thank Dr. Alejandra Maruniak for her insight and guidance. She

read manuscripts, helped with trouble shooting when assays were not working, and kept a

positive attitude gently leading me and other students in the lab toward success. I thank Dr.

Roxanne Connelly for superior editing comments and helpful writing tips. I am grateful for the

mentorship of Dr. Jonathan Day who is an excellent field ecologist and was always supporting

and encouraging throughout the entire dissertation process.

My friends and colleagues were of enormous assistance providing both emotional support

and intellectual stimulus. Erin Vrzal has been a great friend and she provided me with

mosquitoes for blood feeding analysis and for fluorescent release trials. John Herbert helped

with making graphs and statistical analysis. Dr. Karla Addesso read my dissertation and

provided editing comments.

My mom has been my greatest advocate. Her belief in me helped me believe in myself.

My husband helped me keep perspective of what is truly important in life. Lastly, my daughter's

sweet smile reminded me to stay in the present and enjoy each day as it comes.










TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ..............................................................................................................4

L IS T O F T A B L E S .................................................................................8

LIST OF FIGURES .................................. .. .... ...... ................. 10

A B S T R A C T ........................................... ................................................................. 1 1

CHAPTER

1 INTRODUCTION AND REVIEW OF THE LITERATURE .............. ............... 13

In tro du cto ry Statem en t ............................................... ................................ .................... 13
In tro du ctio n ................... ...................1...................4..........
T ran sm mission C y cle ................................................................15
C lin ical D disease ................................................................18
Human ...........................................18
E q u in e ................... ...................1...................8..........
A v ia n ............... .... ..............................................................1 9
O th er V ertab rates ................................................................................ 19
E pidem iology and E cology ............................................................................................... 20
Invertebrate Hosts (vector) .................................. .......................... ............. ........ 21
Vertebrate Hosts (Reservoir/Amplification Host) .......................................................... 24
H u m an ............................... ............................................2 5
Surveillance and Detection of West Nile Virus ....... ............. ...... ........... 26
D iag n o stic s .........................................................................2 7
R isk F a cto rs .............. ...... ....................... ...............................................................2 9
Prediction of Human Cases .............. .......... ........ ........29
B lood M eal A analysis ................................................................... 30

2 EXTRINSIC RISK FACTORS ASSOCIATED WITH WEST NILE VIRUS
INFECTION IN FLORIDA HORSES ..................................................34

M materials an d M eth o d s ...........................................................................................................3 6
Arbovirus Case Information .................. .................. ........ .. ........ ................ 36
R etrospectiv e Surv ey ........................................................................ .............................36
C ase D efin ition ................................................................................ 3 7
Statistical A n aly sis ...............................................................................................3 8
R results ............... ........ ......... ........ ................... ................ 39
Farm and Sample Submission Information .................................................39
Arbovirus Infection Prevention ......................................................... .............. 40
Stable Characteristics and Farm Ecology .........................................................42
D iscussion..................... .....................................................................43









3 MOSQUITOES COLLECTED IN LIGHT TRAPS, RESTING BOXES, AND HORSE-
BAITED TRAPS IN N ORTH FLORIDA ............................................................................. 56

Introduction ................... .......................................................... ................. 56
M materials and M methods ........................ ...... .......... .............. .... .... ......59
Study Site and M osquito Collection Protocol .................................................................59
Fluorescent M osquito Release and Recapture...................................... ............... 61
R esu lts ................... ................................................................. ................62
D isc u ssio n ................... .............................................................. ................6 4

4 ARBOVIRUS SURVEILLANCE: MOSQUITO POOLS, SENTINEL CHICKENS,
A N D H O R SE S. ..............................................................................76

M materials and M methods ...................................... .. ......... ......... .....79
S entin el A n im als ....................................................... ................ 7 9
M osquito C ollections........... .......................................................... .... .. .. ........ 80
M meteorological Data ....................................... .......... ...... ............ 81
R e su lts ............................ ............... .............. .................................. 82
S entin el A n im als ....................................................... ................ 82
M osquito C ollections........... ... ............................................................ ...... .............82
M meteorological D ata .................................................. ..... ...............84
D iscu ssion ......................................................... .............................. 84

5 BLOOD MEAL IDENTIFICATION OF MOSQUITOES COLLECTED FROM
LIGHT TRAPS IN NORTH CENTRAL FLORIDA (2004-2006) ......................................98

M materials M methods .............. ........................................................... ........... 100
B lood Fed M osquito C ollections....................................................................... ..... 100
B lood M eal Identification............................................ ....................................... 10 1
R e su lts ................... ...............................................................................10 3
D isc u ssio n ................... ............................................................................1 0 4

6 HOST FEEDING, VIRUS SURVEILLANCE AND FUTURE EXPERIMENTS ..............109

S u m m a ry ................... .............................................................................1 0 9
H ost F feeding .............................................................................................. 109
Su rv eillan ce ......................................................... .................. ................. 1 1 1
M icroenvironm ent and W eather......... ................. ........................................... ...............112
Extrinsic Risk Factors of WNV to Horses............... ........... .. ....................112
C onclu sions.......... .............................. ...............................................113

APPENDIX

A ARBOVIRUS CASE INFORMATION FORM ..............................................................114

B EN CEPH A LITIS SU R V EY .................................................................. .......................116

C SU R V EY R EQ U E ST LETTER ........................................ ............................................120









L IST O F R E F E R E N C E S .............................................................................. ..........................12 1

B IO G R A PH IC A L SK E T C H ............................................................................... ............... ..... 136









LIST OF TABLES


Table page

2-1 Outline of information submitted by Arboviral Case Form (ACF) and by
retrospective mail survey (RMS). Veterinarians submitted data on all horses tested
for arboviruses in the state via the A CF..................................... .......................... 50

2-2 Total number of horses exhibiting signs of encephalitis and test results for WNV
from 2001 to 2005 .................................................................... .......... 51

2-4 Gender of horses tested for WNV from 2001-2004 .....................................................51

2-5 Results of logistic regression analysis factors associated with WNV among horses
with clinical signs in the state of Florida between 2001 and 2003...................................52

2-6 Stable characteristics and farm ecology for horses classified as West Nile virus
diagnosed (WNVD) or negative (WNVN) by the Florida Department of Agriculture
and Consum er Services 2001 to 2003 ...................................................... .............. 53

3-1 Blood meals of mosquitoes collected in the horse-baited stable trap. A subsample (n
= 50) of the total stable trap catch (n = 525) in 2005 and 2006 was analyzed.
(Results of the blood meal analysis from mosquitoes collected outside the stable trap
are presented in C chapter 5.) ....................................................................... ..................72

3-2 Mosquitoes collected in the mark-release-recapture study in the horse-baited stable
trap. Red marked mosquitoes were released inside the stall in groups of 100 each
date. Green marked mosquitoes were released 5 m outside the stall in groups of 100
each d ate ............... .............. .............. ................................................7 3

3-3 Comparison of mosquito catch in the horse-baited stable trap and an adjacent light
trap. Culex sp. include Cx. quinquefasciatus (16) Cx. salinarius (3) and Cx. erraticus
(10). Anopheles sp. include An. quadrimaculatus (9) and An. crucians (7)....................74

3-4 Total number of five mosquito species caught each study year. .......................................75

4-1 Number of arbovirus positive sentinel chickens and horses in Alachua County in
2005 and 2006..................................... .................. ............... ........... 91

4-2 Mosquito collections by trap location from October 2004 though October 2005 (13
months). Mosquitoes were collected from light trap collections.................................92

4-3 Mosquito collections by trap location from November 2005 though November 2006
(13 months) Mosquitoes were collected from light trap collections.............................. 92

5-1 Primer sets in PCR used to amplify DNA from vertebrate hosts. .................................107









5-2 Identification of blood meals from mosquitoes collected in Gainesville, FL, October
2004 to N ovem ber 2006......... ......... ......... .......... .......................... ............... 108









LIST OF FIGURES


Figure pe

2-1 Total WNVD horse cases reported in Florida between 2001-2003..............................55

3-1 Location of the four paired light traps and resting boxes marked as T1, T2, T3, and
T 4 ............................................................................................. . 6 9

3-2 Measurements of the stall that held the horse in 2005. A single stall was modified
for the trap in a six-stall barn. ................................................ ................................ 70

3-3 Stable trap design 2006. 30 cm lengths of PVC pipe with a 15.2cm diameter cut in
half with openings of 1.9cm were placed along two sides of the stall for mosquito
entry ................... ...................7...................1......... .

3-4 The species that represented at least 1% or more of the total trap catch between
October 2004 and November 2006. ...... ......................................................................73

3-5 The most abundant mosquito species collected at the study site are represented over
the two trapping seasons from October 2004 to November 2006. ...................................74

4-1 Location of the four paired light traps and resting boxes marked as T1, T2, T3, and
T4...... ............................................. ........90

4-2 The species that represented at least 1% or more of the total trap catch between
October 2004 and N ovem ber 2006 ....................................................... ............... 91

4-3 M monthly deviations from normal for rainfall .......................................... ............... 93

4-4 Comparison by month of the four light trap locations average mosquito trap catch.
Bars followed by a different letter are significant at P < 0.05......................... ...............94

4-5 Temporal distribution of the seven most abundant mosquito species collected at the
University of Florida Veterinary School (March through December 2005)....................94

4-6 Temporal distribution of three Culex mosquito species collected at the University of
Florida Veterinary School (March through December 2005).........................................95

4-7 Temporal distribution of seven most abundant mosquito species collected at the
University of Florida Veterinary School (May through November 2006). .....................96

4-7 Temporal distribution of three Culex mosquito species collected at the University of
Florida Veterinary School (May through November 2006). ...........................................97









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

IDENTIFICATION OF POTENTIAL MOSQUITO VECTORS OF WEST NILE VIRUS TO
HORSES IN NORTH CENTRAL FLORIDA


By

Leslie Michelle Viguers Rios

May 2008

Chair: Jonathan Day
Major: Entomology and Nematology

West Nile virus (family Flaviviridae, genus flavivirus WNV) is of concern in the US and Florida

because the virus causes disease in humans and horses. Since 1999, there have been 23,925

clinical human cases of WNV in the United States (1999-2007). Prevention and reduction of

cases requires a clear understanding of the WNV transmission cycle, but much of the needed

information is lacking. It is still unknown which mosquito species transmit WNV to horses.

This study integrated field investigations with laboratory studies to identify possible mosquito

vectors of WNV to horses in north central Florida. The primary objectives of this research were

to compare the abundance and seasonality of mosquito species collected near horses, and to

characterize host preference of potential vectors. An additional aim was to evaluate extrinsic

risk factors of WNV to Florida horses. The extrinsic factors of interest included farm

management, farm ecology, and the entomological conditions associated with each farm. A

questionnaire that focused on potential risk factors was mailed to the owners of all horses tested

for arbovirus from 2001 to 2003. Vaccination was the factor most strongly associated with a

protective effect for WNV disease outcome in horses. The factors that were associated with an

increased risk of WNV in horses were fan use in the stable, mosquito activity, and dead birds on









the property. Blood meal identification and virus screening were done in order to determine

which mosquito species, if any, were involved in WNV transmission to horses. Mosquitoes were

collected for a period of 26 months from a horse research area in north central Florida. DNA

was extracted from the abdomen of the blood fed mosquitoes to test for the presence of avian,

mammalian, and reptilian blood using PCR with different primer sets. The blood meals were

confirmed with sequencing. The non-blood-fed mosquitoes were sorted into pools of up to 50

mosquitoes and screened for WNV, SLEV, and EEEV by Real-Time quantitative RT-PCR. A

total of 45,851 mosquitoes (twenty three species) were collected, 252 of which had visible blood

meals. Twelve mosquito species (fifty eight individuals) were positive for horse DNA. St. Louis

encephalitis virus was detected in one pool ofMansonia titillans collected on September 26,

2006. This study was able to identify several mosquito species feeding on horses and risk factors

associated with WNV disease. The vaccine can protect horses against WNV disease if

administered two weeks prior to exposure and if a booster is administered yearly.









CHAPTER 1
INTRODUCTION AND REVIEW OF THE LITERATURE

Introductory Statement

In Florida, West Nile virus (family Flaviviridae, genus Flavivirus, WNV) continues to

threaten the health of humans and domestic animals. Between 1999 and 2007, 23,925 human

cases of WNV disease have occurred in the United States {2007 1905 /id}. West Nile virus is

considered an emerging infectious disease (EID) in the United States (NIAID 2008). An EID is

a disease that is newly recognized or a previously known pathogen that has spread in incidence

or geographic range (Lederberg et al. 1992). As WNV becomes locally established, recurrent

epidemics occurring seasonally in the summer through fall will likely continue to occur (Hayes

et al. 2005). Florida may be especially susceptible to WNV epidemics due to the subtropical

climate and the endemic status of another closely related arbovirus, St. Louis encephalitis virus

(family Flaviviridae, genus Flavivirus, SLEV). Additionally, Florida plays an important role in

the equine industry (FDACS 2008); many breeding horses are located within the state and are at

risk of infection. The focus of this dissertation was on the ecology of WNV in Florida with

respect to mosquito vectors and animal disease. Unlike other states where human infection has

and now predominates, animal infection has been the most prominent feature in Florida WNV

encroachment. Thus laboratory and field studies investigating the epidemiology of WNV in

horses and their potential vectors in a region of high WNV activity during encroachment, north

central Florida, can contribute to our overall understanding of WNV in Florida. The purpose of

the following literature review is to provide a background of relevant WNV literature and present

a summary of WNV surveillance and detection in Florida. The goal of this dissertation is to

strengthen understanding of the WNV vector-host interactions in an area where susceptible hosts

(horses) are present. The primary objectives of this research were to document the mosquito









species collected near horses, identify the seasonal distribution of these mosquito species, and

characterize their host preferences. The central testable hypothesis of this work is that

microhabitats and ii e u/her conditions dictate the local mosquito species present, and some of

these species may be capable of transmitting arboviruses to clinically susceptible hosts.


Introduction

West Nile virus is an enveloped, single stranded, positive sense RNA virus in the family

Flaviviridae {ICTVdB Management, 2006 19 /id}. The first isolation of WNV was from a

febrile woman in the West Nile District of Uganda in 1937 (Smithburn et al. 1940). Cases of

West Nile fever, (WNV infection resulting in fever, headache, and/or rash) have been regularly

reported in Africa, West Asia, and the Middle East. West Nile virus was recognized as a cause

of central nervous system (CNS) infections such as meningitis and encephalitis when a number

of people became sick in Israel in 1951 (Work et al. 1955). In horses, summer neurological

syndromes were observed since the early 1900s and equine cases caused by WNV were first

identified in the early 1960s in France (Murgue et al. 2001). Since its introduction to the United

States in 1999, WNV has been a growing public health concern in the western hemisphere {2008

2281 /id}. The strain of WNV introduced into New York in 1999 (NY99) was fist isolated from

an American Crow (Corvus ossifragus Brehm). This strain was sequenced and found to have

over 98% homology with a WNV strain isolated from a goose in Israel in 1998 (Brinton 2002).

Since its introduction in the United States, WNV has spread throughout the US, Canada, Mexico,

Central and South America and the Caribbean (Reisen and Brault 2007)

The phylogenetic relationship of WNV strains is broken into two lineages based on amino

acid substitutions or deletions in the envelope protein (Brinton 2002). Lineage 1 is associated

with all cases of severe disease and it is the most widespread of the lineages including the









introduced strain to the United States (NY99). Lineage 2 is restricted to Africa, and has never

been the cause of severe disease or death, but is associated with WN fever (Brinton 2002).

Serologically, WNV is most closely related to flaviviruses in the Japanese encephalitis complex,

which includes Japanese encephalitis virus, Murray Valley encephalitis, Alfuy virus, and St.

Louis encephalitis virus (SLEV) (Brinton 2002). A subtype of WNV, Kunjin virus, is found in

Australia and Southeast Asia (Hayes et al. 2005).

Transmission Cycle

West Nile virus is an arthropod-borne virus (arbovirus) with a natural transmission cycle

between mosquito vectors and wild birds that serve as amplification hosts. Mosquitoes in the

genus Culex have been widely implicated as primary vectors of WNV (Andreadis et al. 2001,

Hayes 1988, Nasci et al. 2001b, Trock et al. 2001, Turell et al. 2001). Culex univittatus

Theobald is considered the primary vector in Africa and Culexpipiens L in Europe (Hubalek and

Halouzka 1999). In Asia Cx. vishnui is the primary vector. The primary North American

vectors of WNV are Culex spp. with great regional variation. In the northeast, Cx. pipiens is the

primary vector; in the southeast Cx. quinquefasciatus is considered an important vector, and in

the west, Cx. tarsalis appears to be the primary vector (Sardelis et al. 2001, Goddard et al. 2002,

Kilpatrick et al. 2005). Other species may be important depending on geographic location and

environmental conditions (Kilpatrick et al. 2005). In Florida, Cx. nigripalpus is a primary vector

for WNV (Rutledge et al. 2003).

West Nile virus is zoonotic and is maintained in complex life cycles involving birds as

the primary vertebrate amplification host and mosquitoes as the principle arthropod vector. The

transmission cycle of WNV does not affect humans or domestic animals until the virus escapes

its transmission/amplification focus via either the amplifying vector or other mosquito with

epidemic potential (Campbell et al. 2002). Humans and domestic animals can develop clinical









illness but are considered dead end hosts because they do not frequently produce sufficient

viremia to infect mosquitoes, and therefore, do not contribute to the transmission cycle (Hayes et

al. 2005). A transient viremia was documented in horses experimentally infected in Egypt

(Schmidt et al. 1963), and a similar study by Bunning et al. (2002) found low level titers (a

maximum viremia of 10 3.0 PFU/mL) which were insufficient to infect Aedes albopictus. High

titers were found in the brain and spinal cord but not in the blood (Bunning et al. 2002). Even if

this low viremia results in transmission to a mosquito, a lower infective titer is correlated with a

reduced transmission rate overall (Bunning et al. 2002).

Humans and horses are susceptible to infection when the virus has become amplified

throughout the resident avifauna. Amplification involves a cascade of virus transmission

between infected birds and competent mosquito vectors. If the proper environmental conditions

persis, this bird to mosquito to bird amplification cycle can result in a large number of infective

mosquitoes. After a period of efficient virus amplification, often late in the summer, there is

transmission to the human and horse population (Petersen et al. 2003). Direct human-to-human

transmission does not occur, although direct transmission has been documented for birds (Austin

et al. 2004, Banet-Noach et al. 2003), and farmed alligators (Jacobson et al. 2005). Horizontal

transmission (non-vector) in humans is possible through breast milk, blood donation, trans-

placental transmission, and organ transplant (Hayes and O'Leary 2004).

An understanding of arboviral transmission cycles begins with the correct identification of

biologically significant vectors. West Nile virus has been isolated from 62 mosquito species

collected in North America {2007 1905 /id}. It is unlikely that many of these mosquito species

play a significant role in the transmission of WNV (Hayes et al. 2005). In order to be implicated

as a vector, a mosquito must fit the following four criteria. 1) The mosquito must be









physiologically able to replicate the virus and to infect a naive host (vector competence). 2) The

mosquito must survive the extrinsic incubation period, which is the time necessary for the virus

to replicate in the mosquito (10 to 20 days for WNV). 3) The mosquito must feed on a

susceptible host. And 4) in order to be a bridge vector (one taking the virus from the mosquito-

bird cycle and transmitting to secondary hosts) it must be an indiscriminate feeder. There has

been laboratory confirmation of vector competence of several species (Turell et al. 2005). In

Florida, there are 80 mosquito species (Darsie and Morris 2003), and very few meet the criteria

of a vector outlined above.

West Nile virus undergoes four phases in the yearly cycle of transmission: maintenance,

amplification, early transmission, and late transmission (Shaman et al. 2003). Several abiotic

factors or non-living components of the environment are considered determinants for viral levels

seen in host and vector populations each year. In peninsular Florida, the maintenance phase is

from January to March, the amplification phase is from April to June, early transmission is from

July to September, and late transmission is from October to December (Shaman et al. 2003).

During the maintenance phase the virus survives the peninsular Florida dry season. It is not clear

where the virus is during this phase, but low level transmission between mosquito vectors and

susceptible wild birds is probable. Virus may be maintained throughout the winter in

overwintering mosquitoes, chronic infection in birds, and by continued enzootic transmission

(Reisen and Brault 2007). Drought conditions limit the available water and bring mosquito

populations into contact with susceptible wild birds, thus facilitating WNV amplification. Once

rainfall increases, infected mosquitoes are able to disperse into new habitats. Infected female

mosquitoes then transmit WNV when feeding on a susceptible host. During years when drought









brings mosquito and bird populations into close proximity the amplification phase can be quite

intense and early transmission is more often seen (Shaman et al. 2005).

Clinical Disease

Human

The most clinically susceptible hosts of WNV appear to be humans, horses and corvids.

The symptoms of disease in humans range from sub-clinical, to encephalitis, coma, or even death

(Petersen and Marfin 2002). In 80% of the cases WNV infection is sub-clinical (Mostashari et

al. 2001). The remaining 20% of infected people will develop symptoms ranging from mild

(headache, body ache, and flu-like symptoms) to severe (severe headache, stiff neck,

convulsions, coma, and death) (Bernard and Kramer 2001, Petersen and Roehrig 2001). The

incubation period (time from infection to onset of systems) lasts about 3 to 14 days with

symptoms lasting between a few days and a few weeks (Jeha et al. 2003, Mackenzie et al. 2004)

West Nile fever refers to less severe cases that are self-limited and often resolve within a

week. West Nile encephalitis and West Nile meningitis are more severe forms of the disease that

affect the nervous system and may persist for over a month (Hayes et al. 2005). Encephalitis

refers to an inflammation of the brain and meningitis is an inflammation of the membrane around

the brain and the spinal cord (Mostashari et al. 2001). The people at highest risk of severe

disease outcome are those over 50 years of age, or that are immune compromised. Less than 1%

of all infected persons will develop severe disease (Hayes et al. 2005).

Equine

Clinical symptoms in horses and other equids (ponies, donkeys, mules) range from

asymptomatic to fatal (Ostlund et al. 2001, Porter et al. 2003). Approximately 10% of infected

horses and other equids develop clinical symptoms. The clinical signs in horses are most

commonly ataxia, weakness, and changes in mental state (Cantile et al. 2000). Early reports









during the U.S. WNV outbreak predicted an incubation period from 3 to 14 days and symptoms

lasting between a few days to a few weeks (Ostlund et al. 2001). In experimental inoculations,

horses become viremic between 2 and 5 days after infection and develop clinical disease between

9 and 14 days post inoculation, which is consistent in all methods of infection including needle,

mosquito and intrathecal routes. The outcome is fatal in 35 to 45% of clinically affected horses

(Bunning et al. 2002, Long et al. 2007). In New York a seropositive rate of 29% was

documented when asymptomatic stable mates of confirmed horses were tested (Trock et al.

2001). The increased rate of seroprevalence was likely an indication of the increased arboviral

activity in the area.

Avian

Symptoms in birds infected with WNV may range from asymptomatic to fatal (Komar,

2003). Avian mortality in the Old World was relatively uncommon prior to the introduction of

WNV to North America in 1999. The neurological invasion of WNV in domestic geese (1997),

and in a flock of storks (1998) in Israel, are among the few reports of WNV causing death in

birds in the Old World (Malkinson et al. 2002, McLean et al. 2002). In North America there

have been 198 species of birds reported to be susceptible to a fatal outcome when infected with

WNV (Komar et al. 2003). Corvids (Passiformes) are especially susceptible to infection. Signs

of infection include lethargy, recumbency, and hemorrhage (Komar 2003). During epizootics,

(outbreaks in the bird population) there is a high rate of natural infection in birds (Work et al.

1955, McIntosh and Jupp 1982, Malkinson and Banet 2002, Komar 2003). Multiple tissues are

damaged with infection and the cause of death is likely multiple organ failure (Komar 2003).

Other Vertabrates

Experimental infections of WNV in other domestic animals have shown that development

of viral titer and clinical signs are relatively rare (Blackburn et al. 1989, McLean et al. 2002.









Sheep that were fed on by WNV infected mosquitoes did not mount a viremia (McLean et al.

2002). In a seroprevelence study, in eastern Slovakia, WNV antibodies were detected in 1% of

608 sheep screened (Hubalek and Halouzka 1999, McLean et al. 2002). Calves that were

experimentally infected did not produce viremia. In a seroprevalence study in Romania, 4.9% of

sheep, 4.1% of cattle, and 12% of goats had HI antibody for WNV (Hubalek and Halouzka 1999,

McLean et al. 2002, Murgue et al. 2002). Dogs that were inoculated subcutaneously with WNV

developed antibody titers and one dog developed a low titer viremia (Blackburn et al. 1989). A

survey of dogs in South Africa found 46% of 377 dogs screened had HI antibodies against WNV

(Blackburn, 1989, McLean et al. 2002). A water buffalo fed on by infective WNV mosquitoes

did not produce detectable viremia, and in a seroprevalence study 72% of water buffalo sampled

had neutralizing WNV antibody (McLean et al. 2002). Farmed alligators are susceptible to fatal

infection in North America (Jacobson et al. 2005) and develop extremely high viral loads in the

blood. Lake frogs in Russia are apparently competent reservoirs for WNV (Hubalek and

Halouzka 1999, McLean et al. 2002).

Epidemiology and Ecology

The numbers of human cases, horse cases, and positive surveillance reports were highest in

Florida between 2001 and 2003, and since that time have decreased. The pattern of WNV

dispersal in the United States has usually displayed a three-year cycle (Reisen and Brault 2007).

The entry year is often followed by transmission at epidemic levels (in Florida the highest levels

occurred two years after WNV was first reported in 2003 with 82 human cases). A decrease in

WNV activity is observed after a large epidemic occurs in a new geographic setting (Reisen and

Brault 2007). Interestingly, as WNV becomes established in a new geographic focus, reports of

SLEV activity in the area decline (Reisen and Brault 2007), although this was not the case in

Florida where sentinel chicken seroconversions to SLEV > WNV in 2006. The decline of SLEV









in many areas is most likely due to the partial cross protection against WNV provided by

previous exposure to SLEV in both birds and mammals (Tesh et al. 2002, Fang and Reisen

2006). West Nile virus epidemics (just as SLEV epidemics) require a number of complex

ecological factors to be in place. West Nile virus may also be subject to epidemiological

conditions such as local bird population susceptibility, rainfall patterns, and mosquito vector

dynamics for transmission to occur.

Invertebrate Hosts (vector)

The enzootic (within animal) WNV transmission cycle includes an avian reservoir

(amplification host) and a mosquito vector. After feeding on an infectious blood meal, WN

virions enter the mosquito midgut and infection of the midgut epithelium may follow (Brinton

2002). In a competent vector (an arthropod capable of becoming infective), the virus replicates

in the cells of the midgut epithelium and subsequently is released into the body of the mosquito

resulting in a disseminated infection (Scholle et al. 2004). Virus then enters other organs,

including the salivary glands, via the hemolymph. After replication in the salivary glands

transmission to a host by probing or taking a subsequent blood meal can occur (Scholle et al.

2004). Differences in both midgut and salivary gland infection and escape barriers may explain

variations in mosquito vector competence.

Additional biological routes of infection include transovarial (entry of virus into mosquito

eggs during oviposition) and venereal (female to male) transmission. Mid-winter isolations of

WNV from overwintering Culex mosquitoes demonstrates the potential of the virus to persist

until spring and emerge with mosquitoes to reestablish an enzootic transmission cycle in the area

(Nasci et al. 2001a). Vertical transmission may contribute to maintenance of WNV (Miller et al.

2000). Mid-winter isolations of WNV are most likely from a mosquito undergoing diapause

(hibernation physiology and behavior) and because normally the female does not first blood feed,









it can be reasonably assumed a mid-winter infection of WNV was acquired transovarially

(Komar 2003). Alternatively, Culex infected by feeding on a viremic vertebrate host may have

survived the winter. Transovarial transmission of WNV and preservation of the virus in

hibernating mosquitoes are not thought to play an important role in the maintenance of the virus

in nature, but the potential of alternative routes of transmission such as vertical transmission do

exist.

The most important mosquito genus in terms of WNV transmission is Culex. The majority

of WNV field isolations have been from Culex mosquitoes, and in field studies, Culex spp. have

in repeated investigations, the highest minimum infection rates (MIR) relative to other mosquito

species (Nasci et al. 2002, Kilpatrick et al. 2005). Because of the midgut barrier, Culex

mosquitoes do not have the highest vectoral capacity (physiological ability to transmit the virus)

as compared to container breeding Aedes spp. and Ochleratatus spp. Despite a lower vectoral

capacity, other factors such as mosquito density, biting preference and seasonal activity makes

Culex species the most important mosquito genus in WNV transmission (Nasci et al. 2002,

Kilpatrick et al. 2005). Finally, each mosquito species may demonstrate a range in vectoral

capacity because ambient temperature, infective dose (from blood), and length of extrinsic

incubation period influence the efficiency of a vector under field conditions.

Several Culex species are involved in the transmission cycle of WNV and preferentially

feed on birds thereby amplifying the virus in avian populations. Ornithiphilic (avian-feeding)

species such as Culex nigripalpus, Culexpipiens, Culex quinquefasciatus, and Culex tarsalis are

considered maintenance and amplification vectors of WNV (Turell et al. 2005). Host-shifting

behavior (a host preference switch from birds in the spring to mammals in the fall) seen in Cx.

nigripalpus, Cx. tarsalis, and Cx. quinquefasciatus may drive WNV transmission to human and









horse populations late in summer and early fall by brining the virus from its point of focal

transmission out to exposed hosts (Kilpatrick et al. 2005). This occurs when mosquitoes first

feed on an avian host and become infected. After completing the extrinsic incubation period the

mosquito may transfer the virus at a subsequent blood meal by probing a susceptible mammalian

host.

An infective mosquito can deliver approximately 104.3 plaque forming units (PFU)/mL of

WN virus to a host, with a range of viral titer (amount of virus in the salivary glands) among

individual mosquitoes and species (Vanlandingham et al. 2004). Aedes albopictus is a

competent vector of WNV. Aedes albopictus experimentally infected with WNV developed

titers between 106.6 to 107.9 PFU per mosquito. When subsequently fed on horses, this titer was

sufficient to infect the majority of horses, which developed a low-level viremia ranging from

101.0- 102.7 PFU/mL (Bunning et al. 2002). None of the horses (n = 12) were able to re-infect

mosquitoes. The minimum host viremia capable of infecting a mosquito vector varies by

mosquito species, but the relationship between susceptibility to WNV infection is dose

dependent and approaches 0 below 104.0 PFU/mL (Reisen et al. 2005). Serum titers below 104.3

PFU/mL are not capable of infecting most mosquito species when feeding on rabbits with low-

level viremia (Tiawsirisup et. al 2005). Culex tarsalis is considered one of the most efficient

vectors and when fed on a blood meal containing 104.9 PFU/mL the number of mosquitoes that

became infected ranged between 0%-36% (Hayes et al. 2005). Concentrations of 1071 PFU/mL

are required before 74%-100% of Cx. tarsalis become infected when fed on an infectious blood

meal (Hayes et al. 2005). The threshold necessary to infect Cx. pipienes and Cx.

quinquefasciatus is 1050 PFU/mL (Allison et al. 2004). It is possible that hosts that maintain a

low viremia (below the threshold) for an extended amount of time may encounter many









mosquitoes and successfully infect a small number of them (Lord et al. 2006). These instances

could be considered as secondary routes of transmission in the WNV cycle.

Vertebrate Hosts (Reservoir/Amplification Host)

The most important amplification hosts of WNV are avian. Laboratory studies have

shown that member of the orders Passeriformes (song birds), Charadriiformes (shorebirds),

Strigiformes (owls), and Flaconiformes (hawks) develop blood virus levels sufficient to infect

most feeding mosquitoes (Komar 2003, Komar et al. 2003). Passerines, such as common

grackles (Quiscalus quiscula), house finches (Carpodacus mexicanus), and house sparrows

(Passer domesticus) are capable of infecting many mosquitoes (Komar 2003). Serosurveys have

demonstrated that house sparrows are frequently infected with WNV (up to 60%), may develop a

high viral titer of sufficient duration and magnitude to infect vector mosquitoes, and are

abundant (Komar et al. 2001, Komar et al. 2003, Godsey et al. 2005). These attributes allow

house sparrows to potentially serve as important amplifying hosts. Crows may experience up to

100% mortality in some outbreaks (Komar et al. 2001) and their rapid fatality likely limits their

reservoir potential. Some resident birds were found to have seroprevelence rates of 20 to 50% in

the epicenter of WNV outbreaks (Komar et al. 2001) which in migratory birds the

seroprevelence was 0.8% (McLean et al. 2002). The importance of migratory birds in dispersing

WNV remains uncertain, but it has been suggested that movement of resident birds,

nonmigratory birds, and migratory birds may contribute to the spread of WNV (Reed et al.

2003, Petersen et al. 2003).

Field observations of direct bird-to-bird transmission have not been made, but laboratory

tests confirm this probability. Infected birds caged with uninfected birds are able to spread

WNV (McLean et al. 2002); the mode of transmission is likely low-level viral shedding per os

(oral) or per cloaca (cloacal). Oral transmission in crows and geese has been documented by









ingestion of infected water, mosquitoes, or carrion (Langevin et al. 2001, McLean et al. 2002,

Banet-Noach et al. 2003).

Although WNV has been isolated from some mammals, and there have been occasional

reports of mammals spiking sufficient viremia to infect mosquitoes, in general mammals are

commonly considered dead-end hosts because they do not usually spike a sufficient viremia to

infect a feeding mosquito and thereby do not contribute to the continuation of the virus cycle

(Hayes 1988, Bunning et al. 2002). Rabbits were found to be capable of infecting various

mosquito species and developing a short-lived viremia of up to 105.8 PFU/mL (Tiawsirisup et al.

2005). Other mammals experimentally infected such as horses, cats, dogs, and mice rarely

exhibit titers above 104.0 PFU/mL whereas birds such as passerines can exceed 106.0 PFU/mL for

a few days (Tiawsirisup et al. 2005). Corvids are the most susceptible to infection producing

high viremia of over 10100 PFU/mL (Reisen et al. 2005). The corvids usually are moribund

(approaching death) after 5-6 days postinoculation. Blood virus levels in naturally infected rock

pigeons ranged from 102.3 PFU/mL 107.2 PFU/mL (Allison et al. 2004). Chickens remain

valuable in sentinel programs because, even though chicks can develop a substantial viremia, the

average adult viremia is <104.0 PFU/mL, which is insufficient to infect most mosquitoes

(Langevin et al. 2001).

Human

In humans, patients develop an average viremia of 0.1 PFU/mL (ranging from 0.06-0.50

PFU/mL) (Montgomery et al. 2006). Blood screened from donors in the US in 2002 had a

maximum titer of 103.2 PFU/mL (Hayes et al. 2005). This level is safely below the viremia

required to infect most efficient vectors. Therefore, humans do not likely contribute to the WNV

transmission cycle and can be considered dead-end hosts. Despite findings that some children in









Israel spiked viremias sufficient to infect mosquitoes, humans are still considered dead-end hosts

(Hayes and O'Leary 2004).

Seroprevelence of WNV in a 1999 New York study (Mostashari et al. 2001) was 1 in 150

infections resulted in meningitis or encephalitis. A 2000 study in New York again found a

similar result (CDC 2001). The results of the two serosurveys were consistent with a previous

study in Romania (1996) indicating that 1 in 140 to 320 infections led to these clinical outcomes

(Tsai et al. 1998). The case fatality rate in the US in 2002 for human cases of WNV with

meningitis was 2%, and the case fatality rate for those with encephalitis was 12% (O'Leary et al.

2004).

Surveillance and Detection of West Nile Virus

Florida has had an arthropod borne virus (arbovirus) surveillance program in place since

1977 to track the amplification and transmission of mosquito-borne viruses including eastern

equine encephalitis virus (EEEV) and St Louis encephalitis virus (SLEV) (Day and Stark 1996).

The Florida state department of health (DOH), division of environmental health, coordinates the

surveillance program. The Interagency Arbovirus Surveillance Network reports to the DOH and

is composed of several local, state and federal agencies, which are involved with the surveillance

and control of arboviral diseases.

Upon its arrival in the United States, WNV was easily added to the existing surveillance

program with the addition of WNV-specific laboratory diagnostics. Because SLEV and WNV

are antigenically related, cross-reactions are observed with some serologic tests and so plaque

reduction neutralization testing (PRNT) is done to distinguish the two viruses. Due to the

correlation of WNV-positive dead bird reporting and local WNV transmission, dead bird

reporting has become a valuable surveillance tool in the United States (Eidson et al. 2001a,

Eidson et al. 2001b, Nasci et al. 2002).









Horses have been found positive for IgM antibody 8 to 10 days after infection with WNV.

The IgM antibodies may persist for 2 to 3 months, most horses only have antibody for 3-4 weeks

which makes this test ideal for detection of recent infection. West Nile virus neutralizing

antibodies can persist for years after infection (Durand et al. 2002). Horses are not currently

used as part of an active WNV surveillance program in the United States, but data is collected

passively on all confirmed horse cases in the U.S. by the CDC. In New York State horse

positives were unreliable in the prediction of human cases of WNV (Trock et al. 2001). It is not

yet known whether WNV surveillance in horses can predict human cases in Florida, but horse

cases that are reported to local health departments are used as part of arbovirus surveillance.

Blackmore et al. (2003) reported that the epicenter of the 2001 WNV outbreak in Florida horses

was in Jefferson County. From Jefferson County, the outbreak spread east, west, and south to a

total of 40 Florida counties with confirmed horse cases. In the counties reporting both horse and

human cases, the horse cases preceded the human cases by one to four weeks (Blackmore et al.

2003). Because horse cases generally precede human arboviral infections, local identification of

horse cases is an important part of the passive surveillance network in Florida and the U.S.

Diagnostics

Routine arbovirus surveillance methods include screening of mosquito pools by viral

isolation, or by antigen detection. Viral isolation is typically carried out in the cell line C6/36

(Aedes albopictus) or in Vero cells. Cell culture procedures detect live virus in the sample.

Enzyme Linked Immunosorbent Assay (ELISA) can detect viral antigen in mosquito pools,

avian tissues, and human tissues. Frequently, viral nucleic acid detection methods such as real

time quantitative RT-PCR are used by local health departments for screening of mosquito pools

{Stark, 2006 20 /id}. The VecTestTM (Medical Analysis Systems, Camarillo, CA) is a rapid

immunochromatographic test developed for the detection of viral antigen. The VecTest can be









used directly in a mosquito pool homogenate, eliminating the need for lengthy laboratory

preparation.

Vero cell culture and RT-PCR were used to confirm the index case (initial case identified

in an outbreak) in the United States (Huang et al. 2002). When cell culture is used for WNV

isolation, it is usually in conjunction with RT-PCR because the latter is more sensitive. Using

RT-PCR, one infected mosquito can be detected in a pool of 50 mosquitoes (a standard

procedure for surveillance of mosquito populations) because the limit of detection in RT-PCR is

40 RNA copies (Shi et al. 2001). This sensitivity is more than adequate for WNV screening

since a mosquito capable of transmitting virus contains more than 105 PFU of virus (Hadfield et

al. 2001). Lanciotti and Kerst (2001) found that nucleic acid amplification assays were far more

sensitive for screening mosquito pools than relying on cell culture alone. The use of RT-PCR

increased the detection of virus and significantly decreased the amount diagnostic laboratory

time (Lanciotti et al. 2000, Lanciotti and Kerst 2001).

Diagnostic methods for WNV detection in horses changed after the introduction of a

vaccine. In 1999, the primary diagnostic tool was the plaque-reduction neutralization test

(PRNT) of equine serum for confirmation of WNV infection and virus isolation from equine

brain or spinal cord tissue (Ostlund et al. 2001). To update the diagnostic tools available the

(Ig)M-capture enzyme-linked immunosorbent assay (MAC-ELISA) was developed (Ostlund et

al. 2001). The assay was modeled after the EEEV MAC-ELISA. Upon experimental challenge

in horses, Immunoglobulin (Ig) M isotype anti-WNV antibodies become detectable 8-10 days

after infection and persist up to two months (Ostlund et al. 2001). This test has a sensitivity and

specificity of 91.2% and 99.7%, respectively, for confirming recent infection in equids with









encephalitis (Long et al. 2006). West Nile virus neutralizing antibodies may be detectable in

horse sera for years after infection (Durand et al. 2002).

Neutralizing antibodies to WNV may persist for more than two years following infection.

Neutralizing antibodies can also be passed from mare to foal via the colostrum (milk). Due to

the properties of neutralizing antibodies, the MAC-ELISA is an important diagnostic tool to

identify recently infected horses in areas where previous infection has occurred because the IgM

antibody response wanes more rapidly than neutralizing antibodies to WNV. Additionally, the

MAC-ELISA is capable of producing reliable results even in vaccinated horses (Porter et al.

2003). The ELISA detects antibodies to WNV and can indicate if the horse had been exposed

even without clinical symptoms.

Risk Factors

Because arboviruses are maintained in complex cycles of avian hosts (reservoirs) and

mosquito vectors, a number of factors must be in place for epidemic transmission to occur.

Abiotic factors greatly affect the year-to-year transmission patterns observed by facilitating the

interactions of infective mosquitoes and susceptible hosts. Weather influences WNV

transmission by affecting the distribution and abundance of mosquito vectors and the time of

extrinsic incubation period (Reiter 1988). There is abundant research on the predictive factors to

human arboviral outbreaks and the most reliable indicators are rainfall patterns, sentinel chicken

conversions, and a large juvenile bird population (Ruiz et al. 2004, Day and Lewis 1991). The

single most important risk factor to human transmission is an abundant infective mosquito

population (Komar 2003).

Prediction of Human Cases

The principle reason for the active surveillance of arboviruses is to protect the public.

Various surveillance tools including mosquito collection, dead bird testing, sentinel chickens,









and horse cases (Blackmore et al. 2003) are used to monitor arboviral amplification and

transmission. Together these surveillance techniques are used to make public health decisions,

by comparing arboviral activity to baseline historical data, in order to protect the public against

an outbreak of a particular arbovirus (FDOH 2007). A public health advisory (by radio,

television and print) can be released that advises people to stay indoors during hours of heavy

mosquito activity, reduce exposure to mosquitoes, and take preventative measures. Preventative

measures include wearing protective clothing, and using chemical repellants. When necessary,

public health measures can be implemented as was done in Florida during the 1990 outbreak

caused by SLEV (Day 2001). All factors including human cases are part of the assessment risk

resulting in a health advisory. Reliance only on human disease results in dissemination of

information after an epidemic is often well underway. Reliable prediction assists policy and

regulatory officials focus control efforts that reduce the possibility of human cases before any

disease occurs. In Florida, animal, mosquito, and chicken seroconversion data is compiled

weekly and released by the Florida Department of Health. In addition representatives from the

Arbovirus Interagency Task Force discusses additional options for media release and public

health advisories relating to the weekly data.

Blood Meal Analysis

Knowledge of mosquito host feeding patterns provides insight to viral transmission cycles

through investigations of the role of a vector mosquito in enzootic transmission among avian

hosts or epidemic transmission outside of this cycle to mammalian hosts. Techniques in blood

meal analysis have been changing since the early 1920s and have included direct observation of

feeding mosquitoes, host-baited trap catches, and serological and genetic based techniques (Ngo

and Kramer 2003). The most common serological and genetic based techniques have been the

precipitin test, the Enzyme Linked ImmunoSorbent Assay (ELISA) and Polymerase Chain









Reaction (PCR) assays (Ngo and Kramer 2003). Polymerase Chain Reaction amplification of

host DNA followed by sequencing is becoming a common method for blood meal detection and

has several advantages over precipitin tests and ELISA. Due to the sensitivity of the PCR, a very

small amount of DNA can be used as template so even partially engorged mosquitoes can yield a

blood meal confirmation. Additionally, with serological tests such as precipitin and ELISA, anti-

sera must be prepared for each potential host species allowing blood meal confirmation to only a

limited number of species. With the advent of web-based databases such as GenBank, it is now

possible to compare nucleotide sequences and determine the exact identity arthropod blood

meals.

In precipitin tests, the blood meal suspension is mixed with the antiserum of various

vertebrates and if a reaction is observed then a precipitate will form and the blood meal is

considered positive for that host type (Tempelis 1989). The ELISA uses a species-specific

antibody that will react with the blood meal and result in a color change signally that binding of

the antibody has occurred. These techniques can identify blood meals to general groups of

animals such as avian vs. mammal and within mammalian hosts, human, cow, horse, etc.

Genetic methods allow for species-specific identification for birds to the species level. Using

restriction fragment length polymorphism (RFLP) analysis, Kirstein and Gray (1996) were able

to identify genera level mammalian blood meals from Ixodidus ticks. Heteroduplex analysis

(HDA) helped classify mosquito-feeding patterns in the Tennessee valley area to the species

level (Lee et al. 2002a). Sequencing blood meal polymerase chain reaction (PCR) product is

another way of determining to the source of the blood meal to the species level. The most

common technique today relies on the genetic characterization of the blood meal to determine

the host. Typically primers in the cytochrome b genome are used to amplify a vertebrate-specific









region from the blood meal DNA. The product of the PCR is sequenced and then matched with

known published sequences in the BLAST database of GenBank (NCBI 2008). Host DNA can

be detected for up to 72 hours after the mosquito takes the blood meal (Ngo and Kramer 2003).

Many blood meal analysis studies focus on avian hosts for purposes of understanding the

commonly fed upon reservoirs in the WNV transmission cycle (Lee et al. 2002a, Ngo and

Kramer 2003). Others have done work on avian and mammalian hosts (Apperson et al. 2002,

Molaei et al. 2006). Cupp et al. (2004) studied the potential role of reptiles in the WNV

transmission cycle by analyzing blood fed mosquitoes that fed on reptilian hosts.

The collection of blood fed mosquitoes is often done by the method of vacuum aspiration

and the use of resting boxes (Edman 1971, Edman 1979). Baited CDC light traps generally

attract host seeking mosquitoes, but may collect females that are partially engorged, fully

engorged, and gravid. Aspiration collections are made in vegetation, natural or man-made

structures, and in likely resting habitats such as around tree roots or from resting boxes. The

vacuum sucks the mosquitoes into a collection container with a screen to hold them in until they

are transferred to another container (Holck and Meek 1991). In Florida, vacuum aspiration has

been used to collect mosquitoes in the genera Culex, Aedes, Anopheles, Coquillettidia,

Mansonia, and Psorophora (Niebylski et al. 1994) Day and Curtis 1993, (Edman 1971, Edman

1979). The CDC light traps generally use white light, although alternate colors may be used to

increase catch and the traps are frequently baited with CO2 to attract host seeking mosquitoes

(Service 1976). As mosquitoes approach the trap, a fan-generated air current pulls them into a

collection bag (Sudia and Chamberlain 1988). Incandescent lights have been shown to be

attractive to Uranotaenia sapphirina (Osten Saken), Anopheles crucians (Wiedemann), Aedes

vexans (Meigen), Anopheles quadrimaculatus Say, Cx. nigripalpus, and Culex in the subgenus









Melanoconion (Love and Smith 1957, Burkett et al. 1998). To maximize catch, the optimal time

to run the trap is coincident with maximum flight activity, the crepuscular period (dusk and

dawn) (Bidlingmayer 1967).

Resting boxes are designed to mimic a natural resting habitat (Edman et al. 1968). They

often attract blood-engorged females seeking a dark resting place to digest the blood meal.

Mosquitoes most often enter in the morning and may leave during the day as the temperatures

rise (Edman et al. 1968).

Many critical questions regarding risk factors of WNV transmission to horses exist.

Although an efficacious vaccine is available, horse cases continue to occur annually throughout

the nation. The study of extrinsic risk factors to horses will help horse owners better understand

the environmental risk factors and farm management practices associated with an increased risk

of WNV transmission. This knowledge can help broaden our understanding of the epidemiology

and ecology of WNV in Florida. Additionally, a full understanding of vector host interactions is

still incomplete. The work presented here helps identify the mosquito species feeding on horses.

Knowledge of blood feeding habits can be used along with vector competence studies and

mosquito life history studies, to incriminate potential mosquito vectors of WNV to horses in

Florida.









CHAPTER 2
EXTRINSIC RISK FACTORS ASSOCIATED WITH WEST NILE VIRUS INFECTION IN
FLORIDA HORSES

Since its introduction to the United States in 1999, West Nile virus (family Flaviviridae,

genus Flavivirus, WNV) has been a growing public health concern. West Nile virus is a

zoonotic (naturally transmitted between vertebrate animals and humans) arthropod-borne virus

(arbovirus). West Nile virus is maintained in a complex life cycle involving a primary vertebrate

host (passerine birds) and a primary arthropod vector (Culex mosquitoes). Susceptible wild birds

and vector mosquitoes amplify WNV in foci where mosquito and bird populations are sympatric.

Culex mosquitoes have been widely implicated as the primary vector of WNV (Andreadis et al.

2001, Hayes 1988, Nasci et al. 2001b, Trock et al. 2001, Turell et al. 2001). The natural cycle of

WNV does not affect humans or domestic animals unless the virus escapes its amplification

focus. This occurs when infective mosquitoes disperse from amplification foci and bite a

susceptible secondary (horses or humans) host (Campbell et al. 2002, Petersen et al. 2003.

Humans and horses can develop clinical illness, but are considered dead end hosts because they

do not produce sufficient viremia to infect mosquitoes, and therefore, do not contribute to the

amplification cycle by infecting additional vector mosquitoes (Bunning et al. 2002, Hayes et al.

2005). Direct (non-vector) transmission has been documented for birds (Austin et al. 2004,

Banet-Noach et al. 2003) and farmed alligators (Jacobson et al. 2005). Direct human-to-human

transmission is limited to infection through blood transfusion, breast milk, and organ transplant

(Hayes et al. 2005).

In Florida, WNV continues to threaten the health of humans and horses. Between 2001

and 2006, there were a total of 1,082 WNV horse cases reported in the state {2007 1906 /id}.

Florida plays an important role in the equine industry; many breeding horses are located within

the state and are at risk of infection even with the availability of three commercially licensed









vaccines. Despite the availability of a vaccine protecting horses against infection with eastern

equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, EEEV), horse cases are

reported regularly. As WNV becomes established, recurrent epidemics and epizootics will most

likely occur (Komar 2003). Although Florida has not yet experienced a major human epidemic

of WNV, the endemic and epidemic presence of St. Louis encephalitis (family Flaviviridae,

genus Flavivirus, SLEV), which shares an epidemiology similar to that of WNV, suggests that

the necessary ecological variables are present in Florida to support future epidemics caused by

WNV.

The annual occurrence of WNV infection in horses corresponds with the cycling of the

virus in amplification hosts and mosquito vectors in enzootic habitats {2003 645 /id}. Human

and equine cases of WNV peak in Florida in late summer and decline after November. Several

extrinsic factors such as rainfall (Shaman et al. 2005), avian population dynamics (Ward et al.

2006), and temperature (Dohm et al. 2002) have been correlated with WNV outbreaks in humans

and horses. However, there are few data regarding the extrinsic factors related to the ecology of

horse farms and the risks associated with farm management practices and disease manifestation

in horses. Retrospective studies have been performed that examine clinical disease associated

with infection, treatments, and outcomes {Salazar, 2004 342 /id { Schuler, 2004 270 /id} Epp et

al. 2007).

This study is an analysis of extrinsic risk factors that were collected for horses tested for

arboviral infection in Florida between 2001 and 2003. This particular study focuses not only on

clinical signs but also on the ecology of horse farms where WNV cases were reported. Given

that year round management of horses reflects the subtropical Florida climate, there are likely

unique factors that intersect with horse husbandry that create risk for horses. Since WNV is a









reportable disease in humans and horses in Florida, all veterinarians are required to submit an

arbovirus case information form (ACF) to the Florida Department of Agriculture and Consumer

Services (FDACS). This highly detailed form allowed for the development of a database and the

opportunity for a follow-up survey of horse owners.

The primary objective of this research was to identify factors contributing to the total

WNV equine cases from 2001 to 2003 in Florida. The factors of interest in this study were farm

management, farm ecology, and the entomological conditions associated with each farm. The

central hypothesis, that risk factors for WNV transmission in horses are related to the availability

of mosquito larval habitat, animal housing conditions, and animal management practices was

investigated.

Materials and Methods

Arbovirus Case Information

The arbovirus case information form (ACF) provided specific information about each

horse tested including stable location, signalment (clinical signs and symptoms), individual

history (age, sex, breed), date of onset of clinical signs, and date of testing. Space was provided

on the form to note any other clinical signs and a brief history of clinical presentation of the

horse (Appendix A). The FDACS provided copies of the ACF information to the Emerging

Diseases and Arbovirus Research and Test Program (EDART) at the University Of Florida

College Of Veterinary Medicine in Gainesville, Florida for data entry and analysis for all horses

tested in Florida from 2001 to 2005. All data were entered into a database (Microsoft Excel and

Access, Microsoft Corporation, Redmond, Washington) and coded for statistical analysis.

Retrospective Survey

Information was taken from the ACF to create a follow-up survey (Appendix B), which

was mailed to the owners of all horses that were tested for arboviruses from 2001to 2003. A









50% return of the questionnaire was the targeted response rate. The questionnaire focused on

horse husbandry, farm management, and farm ecology (Table 2-1). Respondents were asked to

check the most appropriate category in response to each question. A cover letter describing the

objectives of the study and a promise of anonymity to participants accompanied the

questionnaire (Appendix C). Reminder postcards were sent two and four weeks following the

initial mailing. A second mailing was sent to the owners who did not respond to the initial

survey within two months of the first mailing. Two reminder postcards were mailed out two and

four weeks following the second mailing.

Case Definition

Positive Horses. A confirmed horse case was defined as manifestation of WNV clinical

signs (Appendix A) and one or more of the following: isolation of WNV from tissue, blood, or

CSF; detection of a positive IgM antibody to WNV by MAC-ELISA in a single serum test, and

in the first year (2001) of WNV encroachment, a four-fold rise intheWNV plaque reduction

neutralization test (PRNT). After 2001, it was presumed that vaccinated horses would have

neutralizing antibody precluding the usefulness of the PRNT. All confirmed positive horse cases

in the state of Florida from 2001-2003 (n = 534) were analyzed as the positive group in the study

of WNV risk factors associated with the farm environment.

Negative Horses. A WNV-negative horse failed to meet the above criteria for WNV

infection based on serological testing and/or post-mortem analysis. All horses that tested

negative for WNV in the state of Florida from 2001-2003 (n = 402) were analyzed as the

comparison control group in the study of WNV risk factors associated with the farm

environment.









Statistical Analysis

Responses to survey questions were categorical and statistical analysis for independence

was performed with bivariate analysis (SPSS v 15, Chicago, IL). Fisher's exact test was used on

variables containing fewer than five responses in a contingency table cell. A Chi-square (x2) test

was used for analysis of independence between nominal variables that consisted of two or more

categories and contained variables with greater than five responses per contingency table cell.

Odds ratios were calculated for the dichotomous variables that were significant with x2 statistics

or logistic regression (P < 0.05) in the survey. A cross-table was used to stratify variables and

create a contingency table to compare the relationships between variables. Stratified analysis

was used to compare WNV test outcomes with the time since last vaccination and the frequency

of vaccination separately. A logistic regression analysis was performed using WNV disease

status as the outcome (dependent) variable. The data that were included in the regression

analysis were from the combined results of the survey and the ACF. Observational

(independent) variables tested included sex, vaccination status (those positively indicated on the

ACF, all unknowns were treated as missing data points), and each environmental variable.

The logistic regression analysis presented here was adjusted by controlling for vaccination

status to clearly identify the risk/protective effects of the environmental variables associated with

the individual farm (Table 2-5). These data were not as powerful when separated by year since

the separation reduced the sample size and subsequently increased the standard deviation. For

all the variables examined (arbovirus prevention, stable characteristics, and farm ecology) the

years were combined to keep sample size robust.

All statistical analyses were performed with commercially available software (Minitab v

14, State College, Pennsylvania; EPI-Calc v 1.02, Brixton Books, Brixton, UK; SPSS v 15,

Chicago, IL). Multivariate analyses were performed for extrinsic factors using cross-tables and









logistic regression models. The presence of clinical WNV symptoms was the dependent variable

for each statistical test. Cross-tables were used to compare two contingency tables and stratify

the results to compare two variables such as vaccination status and disease outcome.

Results

The Florida Department of Agriculture and Consumer Services (FDACS) compiles

information provided by veterinarians in the state of Florida on every horse tested for viral

encephalitis. The veterinarians use an arboviral case information form (ACF) at the time of

testing a symptomatic horse (Table 2-1). Between 2001 and 2005 there were 2,824 horses that

were classified as either WNV diagnosed (WNVD, n = 1,386 (49%)) or WNV negative (WNVN,

n = 1,438) based on clinical symptoms and serological testing (Table 2-2). The retrospective

mail survey was sent to all owners of horses tested from 2001 to 2003 (n = 2,501) of which 936

(37%) were completed and returned for 534 positive horses and 402 negative horses.

Farm and Sample Submission Information

West Nile virus positive horses were reported in 55 of 67 counties in Florida from 2001 to

2003. Cases began in the summer of each year (2001 to 2003) and peaked in the fall

(September) followed by a sharp decline in the winter (Figure 2-1). Each year the cases were

seasonal except in 2002, when cases were reported throughout the year.

There was a significant (P = 0.045) difference of age structure of positive horses between

2001 and the other study years (2002 to 2004). In 2001, young and old horses were affected,

whereas in 2002-2004, horses between 1 and 5 years of age were most commonly reported as

WNV-positive. In 2001, seven (1.2%) WNV-positive horses were < 1 year old, 324 (56%) were

between 1 and 5 years old, 99 (17.8%) were between 5-10 years old, and 145 (25%) were greater

than 10 years of age. From 2002 to 2004, 36 horses (9.6%) were < 1 years old, 135 (35%) were









between 1 and 5 years old, 181 (47.3%) were between 5-10 years old, and 31 (8.1%) were

greater than 10 years old (Table 2-3).

Quarter horses were the most common breed tested in both the WNVD and the WNVN

groups, and there were no significant differences in breed representation in either group. The

frequency of WNV testing in most other breeds was similar between groups. There were 626

(45%) females, 680 (48%) geldings, and 93 (7%) stallions that were WNVD. This sex

distribution was significantly different (P < 0.001) from the WNVN horses where 510 (47%)

females, 388 (35%) geldings, and 200 (18%) stallions were tested (Table 2-4). The male to

female ratio was about equal in both groups, but significantly more geldings were diagnosed

positive for WNV.

Arbovirus Infection Prevention

The prevention of WNV infection in Florida horses included vaccination, use of insect

repellents, and barrier protection with fly sheets (a protective covering secured to the horse)

(Table 2-1). Insecticides containing permethrin were the most commonly used products for the

protection of horses from biting arthropods. There was no statistical difference between the

WMVD and WNVN groups regarding the use of permethrin products. Frequency of use of all

insect repellents (spray or lotion) was similar between the two analyzed groups. Fly sheets were

not frequently used in either group.

Eighty percent of the tested horses had a known vaccination history. Between 2001 and

2003, 1,368 horses were WNVD and 1,184 were WNVN. Forty two percent of all tested horses

received a vaccine for WNV prior to the onset of illness. To obtain more detailed information

from the owners they were asked if the horse was vaccinated and how many times the horse had

been vaccinated each year.









In 2001 there was an increased association of WNVD horses with vaccination (Table 2-5).

In 2002 and 2003 the vaccine showed a protective effect. The data were stratified by the number

of times the horse was vaccinated and by the time since last vaccination (within 2 weeks, under 6

months, between 6-12 months, or >12 months) in order to more closely examine the relationship

between vaccination and disease. Eighty-five (9.6%) of the WNVD horses were vaccinated two

weeks prior to the onset of illness, 297 (34%) were vaccinated between two weeks and 6 months,

47 (5.3%) were vaccinated between 6-12 mo, and 16 (1.8%) were vaccinated >12 months prior

to onset of illness. Horses were not protected from infection if the vaccine was received within

two weeks of WN disease onset or if the vaccine was received over one year prior to WN disease

onset. For the group of horses that were vaccinated in the time frame of more than two weeks to

under one year prior to the onset of WN disease, the vaccine did decrease the incidence of WNV

(Table 2-5).

Owners were asked to indicate the number of times each horse received a WNV vaccine

and were given a choice of one to four times. The average response for the combined years of

2001 through 2003 was once (25%), twice (55%), three times (15%), and 4 times (4%). Horses

that received only one dose of vaccine did not have protection to WNV. A protective association

was seen for horses that were given two or more doses of vaccine (Table 2-5).

Because the timing of vaccine administration was closely associated with protection

against WNV infection, vaccination data were sorted into two groups: effective and non-

effective vaccine doses received. The effective group was considered to be the group of horses

that received the vaccine more than two weeks and less than six months before the onset of

illness. When the group of horses that received the vaccine in this time frame was compared to

their vaccination status, a strong protective effect of the vaccine was seen (Table 2-5).









Stable Characteristics and Farm Ecology

The construction material of a stable was significantly (P =0.045) associated with WNV

infection. For the WNVD group, stables were made of solid wood and cement for 158 (29%)

horses, boards with openings for 146 (27%), and an open shed for 73 (13%). The WNVN group

had 131 (33%) stables made of solid wood and cement, 79 (20%) made of boards with openings,

and 60 (15%) in an open shed (Table 2-6). The logistic model did not indicate a significant

correlation with type of stable material and incidence of WNV infection. There was no

significant difference if the horse was kept in a pasture versus a paddock. Additionally no

significant difference of WN disease incidence was seen for the frequency of stall cleaning.

The presence of fans in a stall (P = 0.04) and the frequency of fan use (P = 0.05) were

significantly correlated with WN disease incidence in horses. In the WNVD group 184 (34%)

stables had fans, 70 (13%) used the fans all day, 103 (19%) used fans only when necessary, and

358 (66%) did not have fans. In the WNVN group 111 (28%) stables had fans, 54 (13%) used

the fans all day, 49 (12%) used fans only when necessary, and 290 (72%) did not have fans.

Fans were significantly correlated with WNV in the logistic regression (Table 2-5). The

presence of fans increased the risk of WNV by 80%. The duration of use (only when necessary

or all the time) was not significant in the logistic regression.

Mosquito larval habitats are associated with standing water, thus the retrospective survey

attempted assessed the types of water that were present on the property to determine their

significance in relation to WNV status in horses (Table 2-6). No association was detected for the

type of water source or the presence of temporary or permanent water bodies on the farm in the

X2 analysis. In the logistic regression model, natural water on the property has a protective

effect of reducing WNV by half (Table 2-5). The type of water associated with risk could not be

determined because respondents were allowed to mark multiple answers on the questionnaire.









Debris and tree canopy can also provide adult mosquito resting habitat; however, there was no

association with the presence of debris piles or tree canopy in the pasture or near the barn.

The presence of dead birds on the property was significantly (P = 0.003) associated with

WNV activity. These birds were reported by the owner and may or may not have been positive

with WNV. Additionally, other horses becoming ill with WNV on the property were also

significantly (P = 0.048) associated with WNV-positive horses.

The respondents were asked to mark their perceived level of mosquito activity (none,

minimal, moderate, and severe). The level of mosquito activity on the farm was significantly (P

= 0.016) correlated with an increased risk of WNV in the logistic regression model. The

minimum level of mosquito activity increased risk of WNV by 128% when compared to no

activity (Table 2-5). The other levels of mosquito activity also showed an increased risk, as

indicated from the odds ratio, but were not associated with an increased risk of WNV strongly

enough to be significant in the regression model.

Discussion

The purpose of this study was to describe the extrinsic risk factors associated with WNV

infection and to develop recommendations for prevention of WNV infection in Florida horses.

This study is distinctive because it is an extensive analysis of risk factors performed on WNV

positive and negative horses. West Nile virus was first reported in Florida during June of 2001

and the WNV vaccine was conditionally released for use in Florida and throughout North

America in August of 2001. The implication of the timing of virus introduction in Florida and

vaccine release is that the Florida equine population represented a completely naive population

relative to WNV exposure in 2001. In the northeastern US, horses had been exposed to WNV

since 1999, and in the western US, the vaccine was released prior to the first reported horse cases

of WNV. In the face of the WNV outbreak many Florida horses were vaccinated. The results of









this study suggest that horses vaccinated during the summer of 2001 may not have had adequate

time for optimal immunity to develop following vaccination and prior to the onset of clinical

symptoms. In fact in a naive population, there may even be a negative impact of vaccination at

the time of exposure.

Since the release of the WNV vaccine in 2001, there have been high numbers of horse

cases in western regions of the United States that did not experience WNV activity until after the

vaccine was available. Just as cases of eastern equine encephalitis virus (EEEV; family

Togaviridae, genus Alphavirus) occur annually in Florida despite the availability of a vaccine,

there is likely to remain a group of unvaccinated horses that are susceptible to WNV infection

each year. Furthermore, the continued annual transmission of WNV in the U.S. despite the

availability of three vaccines against WNV supports the argument that the virtual disappearance

of WNV transmission to horses in Florida since 2004 is related to WNV transmission patterns in

Florida rather than to a completely protected equine population.

The arboviral case information form (ACF) allowed tracking of disease and gathering of

signalment (clinical signs and symptoms) and demographic data for the 2,824 horses tested from

2001 to 2005 in the state of Florida. The IgM capture ELISA was the most commonly used test

to classify WNV disease status in horses. The gold standard for arbovirus diagnosis is regarded

as neutralizing antibody testing (PRNT) (Farfan-Ale et al. 2006). Neutralizing antibody testing

cannot be used reliably for horses that have been vaccinated because the horse vaccine consists

of formalin-inactivated whole-virion virus eliciting an IgG and neutralizing antibody response

(Porter et al. 2004).

A significantly higher number of geldings than mares or stallions were WNVD in this

study. Epp et al. (2005) recorded the gender of horses affected by WNV in Saskatchewan, but









did not report a significant relationship between WN disease incidence and gender. Other

studies have reported a higher incidence of WNV in male horses than females {Tber A.A., 1996

3 /id} Ostlund et al. 2001). In a serosurvey in France there were no significant differences based

on gender in subclinical WN disease in horses (Durand et al. 2002). In a study of risk factors of

death in clinically affected horses, males were more often diagnosed with WNV; however,

females were 2.9 times more likely to die from WNV than males {Salazar, 2004 342 /id}.

Testosterone levels in males may attract more mosquitoes and increase the likelihood of WNV

transmission to males. Alternatively, stable conditions may vary by gender, for example

stallions may be stabled indoors more often then geldings, thereby increasing mosquito exposure

to animals pastured outdoors more frequently.

A thorough analysis of the vaccination history of horses tested in this study was made.

The vaccine had a protective effect against WNV infection in those horses that were vaccinated

two times in a period of two weeks to one year prior to infection. In contrast Salazar et al.

(2004) reported that one vaccine dose of the same vaccine provided protection. It is possible that

the horses in my analysis did not experience the same amount of time frame after the single

vaccine and before WNV exposure, as did the horses in the Salazar et al. study. The results from

both of these studies are only applicable to the formalin inactivated whole virion vaccine, which

was the only available vaccine on the market for the duration of the owner survey portion of this

study from 2001 to 2003. In 2008 there are three licensed vaccines on the market with different

modes of action, which result in varying duration of immunity that may interact with other

environmental factors differently from the outcome reported in the present study.

In addition to vaccination, arbovirus prevention measures include barrier protection and

repellent use. Barrier methods, such as flysheets, were not often used and were not significantly









associated with WNV infection. Repellents were often used and included pyrethins, natural oils,

and Skin-so-Soft lotion. DEET is not and active ingredient in insect repellents sold for use on

horses due to documented adverse skin reactions (Palmer 1969). The most common type of

repellent applied to horses was a pyrethrum-based insecticide. There was not a significant

association with repellent use and WNV infection; however the linear regression was close to

significant for a protective effect against WNV infection, and the association may merit further

study.

Fans in the stable greatly increased the risk of WNV infection. The important factor in the

increased risk of WNV was only whether fans were used or not. It is likely that there is an

association of duration of fan use and disease outcome. Duration of fan use may have been

confounded by other variables because this factor was significant in multivariable analyses.

Most fans used in stables are small, non-industrial indoor fans. The strength of these small fans

may not be enough to create a breeze that will prevent mosquito flight and blood feeding on the

horses in the stable. However, the small fans do aid in the dispersal of CO2 and other chemical

odors that act as cues for host seeking mosquitoes (Bowen, 1991). When located inside a stall,

the fans may increase the range at which mosquitoes can detect the horse and attract them in

from a longer distance to blood feed. Fan use likely corresponds with human activity in the barn.

Because of fire risks, workers are likely to only use the fans in the day when they are present in

the barn. Fan use may not correlate with highest mosquito activity periods (between sunset and

sunrise) but probably serves to effectively disseminate strong odors from the barn into the

surrounding environment. If the mosquitoes can detect host odor from a greater distance, a

higher number of mosquitoes may be able to successfully locate and blood feed on the stabled

horses.









Natural water on the property was significantly associated with a reduced risk of WNV

infection. The most common type of water on the property was a stream, river, or moving body

of water, which was not suitable for mosquito larval development. This resulted in the apparent

protective association of natural water on the property. The presence of a stream on the property

was associated with a reduced risk of WNV infection in horses, but no other types of water were

significantly associated with WNV infection. No other association was detected for the type of

water source or the presence of temporary or permanent water on the property. This was

unexpected due to the close association of mosquito larval habitats and adult mosquito

abundance with rates of arboviral transmission. Debris and tree canopy can also provide suitable

adult mosquito resting habitats; however, there was no association with the presence of debris

piles or tree canopy over the pasture or barn with WNV infection.

Abundant mosquito populations are a necessary prerequisite for transmission of WNV

(Zyzak et al. 2002). The minimum perceived level of mosquito activity was significant for

increased risk of WNV infection. Other levels of perceived mosquito activity had an odds ratio

demonstrating an increased risk for WNV infection but were not significant in the linear

regression model. Important factors associated with epidemics are mosquito population size and

age (Lord and Day 2001), mosquito infection rates, and mosquito transmission rates (Reeves et

al. 1961, Rutledge et al. 2003). When compared to no mosquito activity, a minimum level of

mosquito activity was associated with a 128% increased risk of WNV infection (Table 2-5). The

correlation of mosquito activity and WNV transmission is associated with the minimum infection

rates (MIRs), blood feeding activity, and transmission rates, which are all key factors in viral

transmission to vertebrate hosts.









Stalls constructed from solid wood or cement were associated with a higher risk of WNV

infection in horses than were stalls constructed from boards with openings or open sheds. In a

study performed by the USDA in 1999 and 2000, pasture management was associated with

higher rates of WNV disease than indoor stabling. The previous study examined horses exposed

in the northeastern U.S. Stalling in a hot, humid environment may actually provide an

environment for at least equal feeding of mosquitoes compared to the pasture. A solid stable

construction, combined with lack of environmental temperature control may be putting horses at

an increased risk of mosquito exposure. Additionally, the solid stall construction may provide

resting sites for adult mosquitoes.

Guptill et al. (2003) showed avian deaths were associated with an increased level of WN

viral activity and could serve as a warning of human infection. In my study, dead birds on the

property were a powerful indicator of WNV transmission risk (Table 2-5). If a dead bird

(regardless of WNV disease status) was seen on the property there was a strong indication of

WNV activity in the immediate area (assuming that the bird did not disperse far from its original

infection site) and was associated with a 97% increased risk of WNV infection to horses. Other

ill equids (due to WNV) on the property were similarly a good risk indicator (Table 2-5).

This study could have been improved by including a group of clinically normal horses as

controls in the analysis. All horses in this study showed some type of clinical manifestation

consistent with a neurological infection. Some of the horses in the WNVN group were positive

for EEEV and this may have somewhat reduced the power of the study to evaluate risk of WNV.

In conclusion, WNV disease in Florida horses appears to be primarily related to

vaccination status. As of 2008, there are three efficacious vaccines available and if horses

receive two doses according to manufacturers instructions, the incidence of equine WNV could









be effectively controlled (Ng et al. 2003, Seino et al. 2007). The use of fans in the stable should

be examined more closely with a focus on the type of fan, time of day used, and other factors

that may provide a more conclusive correlation with use and WNV infection. Some other

variables that warrant further study are the use of insecticide or repellent and the canopy cover

which both had close to significant associations in the regression model with WNV infection. In

areas of high vector activity, reduction of vector larval habitat and limiting horse exposure to

mosquitoes remain important prevention methods for Florida equines. Dead birds on the

property and other ill equids should be noted and considered an indicator of viral activity in the

area. Precautions against mosquito exposure should be taken to protect horses on the property

when dead birds have been reported nearby, especially if the horses are unvaccinated.









Table 2-1. Outline of information submitted by Arboviral Case Form (ACF) and by retrospective
mail survey (RMS). Veterinarians submitted data on all horses tested for arboviruses
in the state via the ACF.
Farm/Sample Submission Source
County of origin ACS
Horse origin ACS
Sample(s) submitted ACS
Date of onset of clinical signs ACS
Date of testing ACS

Signalment and History
Age ACS
Sex ACS
Breed ACS
Use RMS

Arbovirus Prevention
Vaccination ACS/RMS
Frequency of vaccination RMS
Fly spray frequency RMS
Fly spray type RMS
Barrier protection with flysheet RMS

Stable Characteristics and Farm Ecology
Type of stable structure RMS
Duration of outside activity RMS
Frequency of stall cleaning RMS
Manure handling RMS
Presence of debris RMS
Tree canopy characteristics RMS
Vector activity (mosquito abundance) RMS
Presence of dead birds RMS
These data were submitted to the Florida Department of Agriculture and Consumer Services as
part of reporting requirements in the state of Florida for all horses exhibiting symptoms of
encephalitis. Another source of data was the RMS filled out by the owner of the horses tested;
the surveys were returned to the College of Veterinary Medicine.









Table 2-2. Total number of horses exhibiting signs of encephalitis and test results for WNV from
2001 to 2005
ACF ACF RMS RMS
WNVD WNVN WNVD WNVN
2001 651 382 234 138
2002 643 323 265 95
2003 73 429 35 169
2004 7 173
2005 12 131
Total 1386 1438 534 402
(Source: ACF) and number of mail surveys (RMS) returned from owners of horses tested
between 2001 and 2003.


Table 2-3. Age of WNVD horses in Florida 2001-2004
Ages 2001 2002 2003 2004 Total
<1 7 34 2 0 43
1-2 133 24 9 1 167
2-3 122 28 4 0 154
3-4 46 34 1 0 81
4-5 23 29 5 1 58
5-6 16 21 4 0 41
6-7 22 28 4 1 55
7-10 61 101 20 2 184
>10 145 9 20 2 176
Total 575 308 69 7 959
Source: ACF

Table 2-4. Gender of horses tested for WNV from 2001-2004.
Female Male Stallion
2001
WNVD 279 273 59
WNVN 156 134 49
2002
WNVD 310 273 32
WNVN 22 22 20
2003
WNVD 37 34 2
WNVN 222 144 95
2004
WNVD 0 100 0
WNVN 110 88 36
2001-2004
WNVD 626 680 93
WNVN 510 388 200
Source: ACF









Table 2-5. Results of logistic regression analysis factors associated with WNV among horses
with clinical signs in the state of Florida between 2001 and 2003.
Variable Category OR 95%CI P value
Vaccination <0.001


Vaccinated 2wks-
6mo


Received two doses


Protective vaccine


Fans in stable


Natural water


Dead birds


Other animals ill


Mosquito activity


2001
2002
2003


Yes
No

Yes
No

Yes
No

Yes
No

Yes
No


Yes
No


None
Mild
Moderate
Severe


1.68
0.34
0.42


0.06
1

0.48
1

0.18
1

1.79
1

0.48
1

1.97
1

2.42
1

1
2.28
1.67
1.18


1.12-1.83
0.67-0.93
0.31-0.00


0.04-0.82


0.01-0.69


0.09-0.98


1.22-2.53


0.31-0.75


1.27-3.47


1.01-5.79


0.002


<0.001


<0.001


0.013


0.001


0.003


0.048


0.016


1.35-3.52
0.57-1.74
0.82-1.20









Table 2-6. Stable characteristics and farm ecology for horses classified as West Nile virus
diagnosed (WNVD) or negative (WNVN) by the Florida Department of Agriculture
and Consumer Services 2001 to 2003.
Variable WNVD WNVN
Where was horse primarily turned out? (# responses [%])
Pasture 359 (66) 250 (62)
Grass paddock 66 (12) 48 (12)
Sand paddock 64(12) 46(11)
Unanswered 54 (10) 58 (14)

How often are the stalls cleaned? (# responses [%])
Monthly 12 (2) 14 (3)
Twice a month 2 (0) 7 (2)
Weekly 33 (6) 20 (5)
Daily 240(44) 157(39)
Not applicable/unanswered 256(47) 204(50)

Are there fans in the stable? (# responses [%]) *
Yes 184(34) 111(28)
No 358(66) 290(72)
Unanswered 1(0) 1 (0)

How often are the fans run? (# responses [%])
All the time 70(13) 54(13)
Only when necessary 103 (19) 49 (12)
Never 0(0) 1 (0)
Not applicable/unanswered 370 (68) 298 (74)


What is the stable made of? (# responses [%])
Boards with openings 158 (29) 131 (33)
Solid wood or cement 146(27) 79(20)
Open shed 73 (13) 60 (15)
Unanswered 166(31) 132(32)

After rain, is there temporary standing water on the property?
(# responses [%])
Yes 257(47) 192(48)
No 285(52) 209(52)
Unanswered 1(0) 1 (0)

Tree canopy cover (# responses [%])
None 65 (12) 52 (13)









Table 2-6. Continued
Over barn
Over pasture
Over both
Unanswered


38 (7) 29 (7)
227 (42) 160 (40)
163 (30)110(27)
50(9) 51(13)


How many debris piles exist near the stable, or area of horse
activity? (# responses [%])
0
1
2


More than 3
Unanswered


Severity of mosquito/fly activity (# responses [%])
None
Mild
Moderate
Severe
Unanswered

Were there any dead birds on the property? (# responses [%])
Yes
No
Unanswered


291 (54) 189 (47)
128 (24) 82 (20)
34 (6) 26 (6)
11(2) 11(3)
27 (5) 33 (8)
52 (9) 61(15)

*
72(13) 40(10)
208 (38) 148 (37)
145 (27) 107 (27)
71 (13) 37(9)
47 (9) 70(17)

*
98(18) 69(17)
444(82)332(83)
1 (0) 1 (0)


* Significant P<0.05








































4,

N


2 3 4 5 6 7
Month of report


Total


2001


8 9 10 11 12


2002


2003


Figure 2-1. Total WNVD horse cases reported in Florida between 2001-2003.









CHAPTER 3
MOSQUITOES COLLECTED IN LIGHT TRAPS, RESTING BOXES, AND HORSE-BAITED
TRAPS IN NORTH FLORIDA

Introduction

West Nile virus (WNV; family Flaviviridae: genus Flavivirus) is a pathogen that is

primarily maintained between birds and mosquitoes in enzootic transmission cycles, but is also

sometimes transmitted to mammals, including horses and humans (Petersen and Roehrig 2002).

The virus and disease have been present in the United States since 1999 and have continued to

spread throughout North and Central America and throughout the Caribbean Basin causing

annual outbreaks (Reisen and Brault 2007). An efficacious horse vaccine has been available

since 2001, but horse cases continue to occur annually throughout the transmission zone. In

Florida alone, 1,082 horses were diagnosed with WN infection between 2001 and 2007 (USDA-

APHIS 2007). Florida has a large equine industry with over 299,000 horses in the state

potentially at risk for WNV infection (FDACS 2007). Estimates of asymptomatic (subclinical)

WNV infection in horses have ranged from 1.2% (Lorono-Pino et al. 2003) to 58% (Durand et

al. 2002). Of the clinically infected horses, 35-40% of the cases result in death. West Nile virus

is a reportable disease in Florida but because a large proportion of infected horses do not show

symptoms of infection it is likely that horse cases are underreported.

West Nile virus is maintained in enzootic foci where mosquito and bird populations are in

close proximity (Campbell et al. 2002). The primary (enzootic) cycle involves avian hosts and

ornithophilic mosquitoes and the secondary cycle involves non-avian hosts and epizootic vector

mosquitoes which are sometimes linked between both cycles. Vertebrate hosts that facilitate

WNV epidemics are termed amplification or reservoir hosts (Kilpatrick et al. 2006).

Amplification hosts spike a high viremia for a short duration of time; reservoir hosts may sustain

a low level viremia for a long duration and aid in maintaining the virus through periods of low









mosquito activity. Avian amplification hosts remain infective for WNV for one to three days

(Komar et al. 2003). The transmission of virus between infectious amplification birds and vector

mosquitoes (primarily Culex spp) results in amplification of the virus. After sufficient

amplification in the bird population, the virus escapes its focus when infective mosquitoes

disperse into habitats where they may come into contact with a susceptible non-avian host.

Epizootic vectors such as Culex nigripalpus (Theobald), Culex salinarius Coquillett, Aedes

vexans (Meigen) and Coquillettidiaperturbans (Walker), which are opportunistic feeders,

transmit WNV to horses and humans (Campbell et al. 2002, Samui et al. 2003). Isolations of

WNV from natural mosquito populations in Florida have been reported in Cx. nigripalpus,

Mansonia titillans (Walker), Ochlerotatus taeniorhynchus (Wiedemann), and Deinocerites

cancer Theobald (FDOH 2007, CDC 2007). Culex nigripalpus is considered an epidemic and

epizootic vector of St. Louis encephalitis virus (SLEV; family Flaviviridae, genus Flavivirus) in

Florida, which shares a similar epidemiology to WNV (Shaman et al. 2005, Zyzak et al. 2002).

A thorough knowledge of the biology, ecology, and behavior of mosquito vectors is

essential for understanding WNV transmission, amplification, epizootics, and epidemics. The

blood feeding behavior of many mosquito species has been studied (Apperson et al. 2002, Lee et

al. 2002, Ngo and Kramer 2003) and this information combined with mosquito susceptibility to

viral infection and mosquito trapping data can help identify possible mosquito vectors of WNV

to horses.

Livestock-baited traps have been widely used to identify the presence, seasonal abundance,

and host preference of mosquitoes. One of the first portable stable traps used for the collection

of mosquitoes was the Magoon trap, designed in 1935, and with various modifications, the trap

remains widely used today (Service, 1976). Samui et al. (2003) classifies a mosquito species as









an important horse feeder if it frequently enters a horse-baited stable trap and if a large

percentage of the individuals entering the horse-baited stable trap are blood fed when they are

collected. In areas of eastern equine encephalitis virus (EEEV; family Togaviridae, genus

Alphavirus) transmission, a mosquito that is a competent EEEV vector, feeds on a horse may

serve as an epizootic vector from the amplification host to the horse (Samui et al. 2003). Data

from horse-baited traps can help identify which mosquito species are present in a locality and

which mosquito species feed on horses. An understanding of these mosquito/host interactions

can help augment existing knowledge of potential vector species for arboviruses to infect horses.

Of particular interest in this are those species that are physiologically competent for EEEV or

WNV, have had field isolations of EEEV or WNV, and have been temporally associated with

EEEV or WNV transmission foci.

The purpose of this study was to determine the identity and seasonal abundance of

mosquito species attracted to horses in a study area in north Florida. To accomplish this, a two-

year mosquito surveillance project was designed to provide information about the seasonal

abundance and spatial distribution of mosquito species near horses maintained at the study area.

These data were collected to evaluate mosquito seasonal variability and host seeking behavior

compared to WNV transmission (n = 1) and EEEV transmission (n = 10) to horses in north

Florida in 2005 and 2006. Three clinical WNV horse cases were reported at the study site in

2001 and, based in part on this observation, the site was chosen for the present study. The

relative abundance and species composition of mosquito fauna collected from and around horses

at the north Florida study site are reported here and compared with light trap and resting box

collections made at the same site during the same time period.









Materials and Methods

Study Site and Mosquito Collection Protocol

The study site for this project was located at the University of Florida Veterinary School,

in Alachua County in north central Florida (29038' N, 82o20' W). Four CDC light traps (John

W. Hock Company, Gainesville, FL) (Sudia and Chamberlain 1988), four resting boxes (Moussa

1966), and a horse-baited stable trap (Bates 1944) were used to collect mosquitoes. The site was

chosen based on a history of WNV transmission to horses (three clinical cases in 2001),

abundant mosquito habitats, a reliable population of horses (n = 50) permanently pastured at the

site, and a ranch-style operation surrounded by urban development.

The study area was mostly open grass bordered by a sylvan habitat supporting nighttime

mosquito flight activity and providing many mosquito-resting habitats. Bivens Arm Nature Park

bordered the site to the south. The nature park is a 57-acre urban wetland (43 acres of aquatic

habitat) bordered by upland mixed forest (14 acres) (Fig. 3-1). The main feature of the park is a

lake surrounded by a live oak hammock. A 1500 sq-ft retention pond was located on the north

side of the study site. After a rainfall event, standing water would accumulate in the pastureland

at the site. The water would drain slowly into the retention pond over a period of several days

unless rainfall continued.

The research horses (n = 50) at the site were kept in outdoor pastures and the property had

an 80-stall equine hospital on the west side. The hospital's outside lights were left on at night

and veterinary students and doctors had access to the hospital and were sometimes present

throughout the night. Both of these factors may have influenced the abundance and species

composition of mosquitoes around the hospital at night.

In 2005, the stable trap was located in a single barn containing six stalls with a large

hallway in the middle. Each stall had two 30.5cm by 30.5cm windows, one opening to the









outside of the stable and the other opening into the hallway. One stall (36.5m3) was modified

into a horse-baited stable trap following the design of Bates (1944) (Figure 3-2). Mosquito

netting was secured to all sides of the stall and across the ceiling. Two horizontal 30.5cm baffles

were placed along the windows on the outside and inside of the stall 1.21 m from the ground

(Figure 3-2). The baffles consisted of a cut foam mattress pad glued to the side of the stall to

make a V-shaped opening of 20-cm to the outside and converging to a 2.5-cm opening into the

trap. The floor of the stall was covered with sawdust that was cleaned after each use.

The barn used in 2005 was not available for the study in 2006, so a change in stall

location occurred. The design of the stall was almost the same, only small modifications were

made and the stall was located 14 m south of the stable used in 2005 (Figure 3-1). The stall was

a standard portable stall design measuring 3.65m3 x 3.65m x of 3.65m on the low side angled up

to 4.26m on the high angle side. The portable stable trap was large enough to house an adult

horse. The bottom half of the trap was constructed of wooden boards and the upper half of

vertical steel bars with a slanted tin roof. To modify the stall following the design of Bates

(1944), the entire stall was sealed with mosquito netting. To construct the entrances into the

trap, 30cm lengths of PVC pipe with a 15.2cm outer diameter were cut in half longitudinally and

secured along two sides of the stall at a height of 1.21 m (Figure 3-3). The diameter of the

openings were 15.2 cm to the outside with a 1.9 cm opening cut along the inside of the pipe

facing into the stall to act as a baffle allowing mosquitoes entrance into the stall and limiting

escape. The floor of the stable trap was covered with sawdust and was cleaned after each use.

The study commenced on October 6, 2004 and ended on December 5, 2006. The stable

trap collections were conducted from May 2005 through November 2005 for the first stable trap

and from May 2006 through November 2006 for the second. Because the horse had freedom to









move about in the stall and was provided with food and water, the stable trap could be run

overnight (IACUC approval #E248). Four CDC light traps (John W. Hock, Gainesville, Fl)

baited with approximately 1 kg of dry ice were placed in different habitats at the site. In relation

to the stable trap, the light traps were placed as follow: 1) in a highly wooded area (300 m south),

2) by a 1,500 sq-ft retention pond (50 m north), 3) in an oak stand (50 m west) and 4) by the

horse stable trap (10 m east). A resting box was paired with each of the four light traps (Figure

3-1). Each resting box was constructed of plywood in the form of a cube 30.5 cm on each side

and one open side. The open side was covered with a square of mosquito netting that could be

secured to the sides by Velcro. The cover was left open over night and in the morning secured to

trap mosquitoes inside prior to aspiration. The outside of each box was painted black with

acrylic paint, and the inside was a deep red color to provide a dark space for resting mosquitoes.

All traps were set twice a week at about 1600 hr. Traps were retrieved the following morning at

approximately 0800 hr.

The stable trap and the resting boxes were aspirated with a backpack aspirator (Dvacc,

John Hock, Gainesville, FL) followed by a small hand held aspirator (Bioquip, Rancho

Dominguez, CA) to reach into the smaller spaces. Collection bags from each trap were placed

individually in a -700C freezer and then the contents were transferred to Petri dishes, labeled, and

stored at 700C until the mosquitoes were identified to species on a chill table and sorted into

pools by species, date, and trap type. All blood fed mosquitoes were stored separately, and

empty females were stored in pools of up to 50 and tested for virus (results of the viral analysis

are reported in Chapter 4).

Fluorescent Mosquito Release and Recapture

To evaluate mosquito escape from and entrance into the two stable traps used in this study

eight marked mosquito release trials were performed. In 2005 and 2006, colony reared Culex









quinquefasciatus females (USDA-Gaineseville, Fl) were colored red and green with Dayglo

fluorescent dye. The mosquitoes were knocked down by cooling to 40C and gently shaken in a

container lightly coated with fluorescent powder to dust the mosquitoes and cover them in the

dye. During these studies in 2005 (n = 4) and 2006 (n = 4), 100 green mosquitoes were released

outside of the stall and 100 red mosquitoes were released inside the stall at 1700 hr. In 2005, red

mosquitoes were released inside the stall once in the presence of a horse and three times in the

absence of a horse. In 2006, mosquito releases were made three times in the presence of a horse

and once in the absence of a horse. In 2005, an un-baited light trap was set in an adjacent stall

next to the stable trap. No other horses were present in the stalls next to the stable trap during

these tests.

Colonized Culex quinquefasciatus were chosen for the mosquito release trials because this

species naturally occurred at the study site and a laboratory colony was easily assessable. Culex

quinquefasciatus is an opportunistic feeder (Elizondo-Quiroga et al. 2006). Females from the

laboratory colony were fed bovine blood, demonstrating their willingness to feed on mammalian

blood and colony males and females were provided continual access to a 10% sugar solution for

flight and maintenance energy. Prior to experimental release, the mosquitoes were provided only

water for 24h to ensure maximum host seeking behavior upon release.

Results

During the study 45,851 mosquitoes were captured in the three trap types: light traps,

45,271; horse-baited stable traps, 526; and resting boxes, 55. Twenty-three mosquito species

were captured in light traps, seven in the horse-baited stable traps, and three in the resting boxes.

Totals for the nine most abundant species are illustrated in Figure 3-4. All seven species

collected inside the horse-baited trap blood fed on the horse. These were all confirmed as horse

blood meals by PCR analysis (Table 3-1).









Fluorescently dyed Culex quinquefasciatus females were released four times each year to

determine the recapture rate inside the stable trap and to validate mosquito entry and exit from

the stable trap. The number of mosquitoes recaptured inside the stall when a horse was present,

varied from 16% (16/100) to 41% (41/100). When no horse was present the number recaptured

the following morning varied from 1% (1/100) to 5% (5/100) (Table 3-2). For mosquitoes

released outside the stall, there was an entry and recapture rate of varying from 3% (3/100) to 7%

(7/100) when a horse was present. None of the marked mosquitoes entered the stall when a

horse was not present nor were any of the marked mosquitoes recaptured in the un-baited light

trap located in the adjacent stall.

During the first 13 months of the study (Oct. 2004 to Oct. 2005) Cx. nigripalpus was the

most abundant mosquito species collected. The high Cx. nigripalpus numbers were recorded

during the months of October and November 2004 (Figure 3-5). The next most abundant

mosquito species collected during the first half of the study were Anopheles crucians

(Wiedemann), followed by Ma. titillans, Oc. infirmatus, Cx. erraticus, and Cx. salinarius (Figure

3-4). After November 2004, the number of mosquitoes captured decreased dramatically. In

March of 2005 An. crucians populations began to increase followed by an increase in April 2005

of Oc. infirmatus. The abundance of both of these species decreased after July 2005. The

number ofMa. titillans increased in the early fall and remained high until the end of December

2005 (Figure 3-5). When mosquito numbers and species composition were compared between

the light trap and the horse- baited trap (mosquitoes collected inside the horse stall), a preference

ofMa. titillans for the horse was observed over the light trap (Table 3-3).

Mosquito abundance and species diversity patterns observed at the study site during the

second 13 months of the study (November 2005 to November 2006) (Figure 3-5). The most









abundant mosquito species collected during this time period was Ma. titillans, followed by An.

crucians, and Cx. erraticus. During 2006, females of three mosquito species entered the horse-

baited stable trap: Ma. titillans, Cq. perturbans, and Cx. erraticus. Of the three species that

entered the trap, only Cq. perturbans was collected in higher numbers in the horse-baited stall

than in the light trap located near it (Table 3-3).

A comparison of the total number and species composition of the mosquito collections

made during 2005 and 2006 appears in Table 3-4. Culex nigripalpus numbers in all trap

collections declined dramatically from 10,530 in 2005 to 135 in 2006. Culex salinarius also

declined from 1,494 in 2005 to 498 in 2006. Culex erraticus was much more abundant the

second year and increased from 397 in 2005 to 3,108 in 2006. Culex erraticus was collected

throughout the entire summer of 2006. Mansonia titillans was abundant during the autumn of

both years. Mansonia titillans was less abundant in 2005 (n =2,153) than in 2006 (n = 10,134).

Anopheles crucians was abundant during the winter and spring of 2005 (n = 2,979) and 2006 (n

= 4,633). Its numbers declined in early summer and it virtually disappeared by August of both

years.

Discussion

Seven of the 23 mosquito species collected during this study entered the horse-baited

stable trap. The four light traps used during this study were responsible for 98% of the total

mosquito catch. The light trap located next to the stable trap collected approximately the same

number of mosquitoes as the stable trap did (light trap, n = 515; stable trap n = 526). Olson et al.

(1968) found nine of thel6 species that they studied entered a livestock-baited trap in a small

farming community in Utah. In their study, light trap collections accounted for over 90% of the

total trap catch with the exception of Anophelesfreeborni. In contrast, Carpenter and Peyton

(1952) found a total of 3,391 mosquitoes collected in light traps over a one-year period compared









with 65,323 mosquitoes collected in a horse-baited stable trap at the same site. Perhaps the

discrepancies observed between this study and the other two studies discussed above are due to

trap design and/or trap location. Because the total trap collections were about equal in the stable

trap and the light trap, lends credence to a well-placed stable trap in this study.

All seven mosquito species collected in the horse-baited stable trap in my study have had

associated field isolations ofWNV (CDC 2007). This does not mean that all seven are 1)

competent vectors and/or 2) important epizootic vectors, but field isolation of an arbovirus is one

criterion used to identify a mosquito species that has had contact with a WNV positive host and

is a potential arboviral vector. Polymerase Chain Reaction analysis of blood meals from the

mosquitoes captured in the horse-baited stable traps used in my study confirmed that all of the

mosquitoes that entered the stable trap blood fed on the horse contained in the trap (Table 3-1).

All seven mosquito species (Cx. salinarius, Cx. quinquefasciatus, Cx. erraticus, An. crucians,

An. quadrimaculatus, Ma. titillans, and Cq. perturbans) that entered the trap blood fed on the

horse and can therefore be considered horse feeders at this study site (Samui et al. 2003). A goal

of this study was to identify mosquito species that were likely to blood feed on horses at the site;

further studies should be conducted to evaluate the potential of these mosquito species to

transmit WNV and EEEV to horses in nature.

Mansonia titillans and Cq. perturbans showed a preference for the horse-baited stable trap

compared with the adjacent light trap (Table 3-3). Olson et al. (1968) found the most abundant

mosquito species present at their study site were the ones that entered the livestock-baited trap.

In my study in 2006 Cq. perturbans composed only 6% of the total collection but was collected

in about equal numbers in the stable trap (n =47) and the adjacent light trap (n = 43). Mansonia

titillans was the most common mosquito captured in the stable trap during both years of my









study. The next most abundant species collected in the stable trap were Cx. quinquefasciatus in

2005 and Cq. perturbans in 2006. Mansonia titillans has been positive for WNV isolations in

the field (CDC 2007); however, the detection of WNV in a given mosquito species does not

mean that the species is a vector of WNV. Population density, host preference, feeding behavior,

longevity, seasonal activity, viral susceptibility, and vector competence must also be considered

when attempting to determine the status of a mosquito species as an important vector (Sardelis et

al. 2001). Mansonia titillans will blood feed on avian hosts, but has a strong preference for

mammals (Edman 1971). Members of the genus Mansonia have a long flight range of up to 2.5

km (Macdonald et al. 1990). A long flight range enhances the vector capacity of a bridge vector,

if the species is an otherwise efficient and competent arboviral vector, by bringing the virus out

of its amplification focus into different habitats where transmission can occur (Moncayo and

Edman 1999).

Coquillettidiaperturbans was collected frequently at the study site (n = 2,747). This

species is considered an important bridge vector for EEEV (Chamberlain et al. 1954, Boromisa

et al. 1987, Vaidyanathan et al. 1997). Eastern equine encephalitis virus has been isolated from

pools of field collected Cq. perturbans (Nasci et al. 1993, Andreadis et al. 1998). This species is

considered mammophilic, but also feeds on birds (Edman 1971). Because of blood feeding

preference, collection in the horse-baited stable trap, and virus isolations field-collected females,

Cq. perturbans may play a role in EEEV transmission in north central Florida. Coquillettidia

perturbans has been demonstrated to be an inefficient laboratory vector of WNV and could

occasionally play a secondary role in WNV transmission in the field (Sardelis et al. 2001). The

Culex species that entered the horse-baited stable trap, did so in much lower numbers than their

overall abundance as indicated by light trap collections at my study site. All of the Culex species









that entered the trap (n = 29 in 2005; n = 6 in 2006) blood fed on the horse indicating that these

mosquitoes feed on horses in nature.

When a horse was present in the stable trap, 16% (16/100) to 41% (41/100) of

fluorescently marked released mosquitoes were recovered the following day. Failure to recover

100% of the marked mosquitoes may have resulted from mosquito escape, or from death due to

horse defensive measures including tail swipes, biting, and pawing with the feet. Very few (1%,

1/100 to 7%, 7/100) marked mosquitoes were recovered when no horse was present in the stable

trap. It is likely the released mosquitoes actively and successfully searched for an exit from the

unoccupied stable trap. Additionally, the fact that no mosquitoes (0/400) entered the stable trap

when a horse was not present demonstrates that the horse itself acted as an attractant and that

mosquitoes were not attracted to lingering odors left in the stall.

Very few marked mosquitoes entered the stable trap (16/400) when a horse was present

during the mark-release-recapture trials. The collections inside the stall during both trapping

seasons were about equal to the adjacent light trap for the same time period (Table 3-3). In

mark-release-recapture trials, mosquito numbers recaptured are often low (Conway et al. 1974,

Kramer et al. 1995, Reisen et al. 2003). The two reasons that affect recapture rates the most are

wind speed and mosquito source (laboratory reared versus locally collected) (Reisen et al. 2003).

The average wind speed on the nights of the marked mosquito release trials were between 4.52

mph and 15.07 mph.

Caution must be used when interpreting results from collections made by light traps, horse-

baited stable traps, and resting box because the collections may not reflect true mosquito

abundance (Huffaker et al. 1943). The most accurate mosquito population estimate is made by

combining the results of multiple trap collection types (Huffaker et al. 1943, Bidlingmayer 1967)









to account for variation due to species biases of traps, biases due to trap location or the influence

of meteorological conditions. Despite overall low mosquito numbers in the stable trap

collections, the collection information can be useful when combined with other trap types used

during this study. Simultaneously using various trapping methods can provide a reliable

measurement of mosquito abundance and diversity (Huffaker, 1943, Bidlingmayer 1967).

Seven mosquito species fed on the horse maintained in a stable trap and the largest

mosquito collections were made during the fall of both study years. Mansonia titillans was the

most frequently collected mosquito in the horse-baited stable trap. Mansonia titillans and Cq.

perturbans were the only two species at the site that were collected in higher number in the

stable trap than in the adjacent light trap. Both of these species should be considered in future

studies as potential vectors of WNV and EEEV, respectively, in north Florida. To my

knowledge, laboratory studies have not been completed to evaluate the vector competency of

Ma. titillans for WNV. Such studies would provide valuable information to supplement the

results of my study. The possible role ofMa. titillans as a potential vector of WNV to horses

should be investigated. Coquillettidiaperturbans may play an important role in the transmission

of EEEV to horses (Morris 1988) and may play a secondary role in WNV transmission (Sardelis

et al. 2001). Coquillettidiaperturbans is considered a bridge vector of EEEV (Crans and

Schulze 1986, (Morris 1988) and it is likely that this species may play a role in EEEV

transmission as an epizootic vector to horses in north central Florida.

The differences in mosquito numbers collected at the site each year are most likely due to

variation in the weather conditions. In 2005 the weather was wetter than average (reported in

chapter 4), and in 2006 Florida experienced a prolonged drought. The number of Cx. nigripalpus

declined dramatically after the first few months of the study. The collections began in October









2004 and the hurricane season had been particularly active for Florida earlier in the year. Only a

few weeks after the final hurricane of the season did trapping begin and it is possible that the

large numbers of Cx. nigripalpus were correlated with the rainfall associated with this time

period.


Figure 3-1. Location of the four paired light traps and resting boxes marked as T1, T2, T3, and
T4. The Chen barn was modified for the stable trap in 2005, and a portable stall was
erected 14m south of the Chen barn during the 2006 season. The sentinel chicken
flock was located adjacent to the stable trap in 2006.































36.5 m


Figure 3-2. Measurements of the stall that held the horse in 2005. A single stall was modified for
the trap in a six-stall barn.










i ..

-Ni...Z

* "SfM^R 4 ljf'Jy I I(f)a~ Y ^^^^^^^ ^ ____
^^^*<---1()~im... j


a a M.. a


r .


Figure 3-3. Stable trap design 2006. 30 cm lengths of PVC pipe with a 15.2cm diameter cut in
half with openings of 1.9cm were placed along two sides of the stall for mosquito
entry. The rest of the trap was sealed with mosquito netting.
























Table 3-1. Blood meals of mosquitoes collected in the horse-baited stable trap. A subsample (n
= 50) of the total stable trap catch (n = 525) in 2005 and 2006 was analyzed.
(Results of the blood meal analysis from mosquitoes collected outside the stable trap
are presented in Chapter 5.)
Species # tested # confirmed (%) result
Cx. salinarius 2 1 (50) horse
Cx. quinquefasciatus 3 2 (66) horse
Cx. erraticus 7 5 (71) horse
An. crucians 1 1 (100) horse
An. quadrimaculatus 4 1 (25) horse
Ma. titillans 20 11 (55) horse
Cq. perturbans 13 7 (54) horse

























< "
"o"~


Figure 3-4. The species that represented at least 1% or more of the total trap catch between
October 2004 and November 2006.




Table 3-2. Mosquitoes collected in the mark-release-recapture study in the horse-baited stable
trap. Red marked mosquitoes were released inside the stall in groups of 100 each
date. Green marked mosquitoes were released 5 m outside the stall in groups of 100
each date.
Collection Horse Mosquito # recaptured # recaptured
Date present color inside stall outside stall
6/11/2005 Yes Red 26 1


Green
9/16/2005 No Red
Green
9/23/2005 No Red
Green
9/30/2005 No Red
Green
9/16/2006 Yes Red
Green
9/21/2006 Yes Red
Green
9/28/2006 Yes Red
Green
9/30/2006 No Red
Green


7
3
0
1
0
4
0
41
3
16
5
25
1
5
0


1













2500





0


S1000

500
E
z 0




Cx nigripalpus O Cx erratiucs E Ma titillans U Cq pertubans E An crucians


Figure 3-5. The most abundant mosquito species collected at the study site are represented over
the two trapping seasons from October 2004 to November 2006.


Table 3-3. Comparison of mosquito catch in the horse-baited stable trap and an adjacent light
trap. Culex sp. include Cx. quinquefasciatus (16) Cx. salinarius (3) and Cx. erraticus
(10). Anopheles sp. include An. quadrimaculatus (9) and An. crucians (7).
May to Oct 2005 Horse Stable May to Nov 2006 Horse Stable
Stall Light Trap Stall Light Trap
Culex sp. 29 43 Culex erraticus 6 213
Anopheles sp. 18 22 Mansonia titillans 96 171
Ps. ciliata 1 0 Cq. perturbans 47 43
Mansonia titillans 330 13
Cq. perturbans 1 9










Table 3-4. Total number of five mosquito species caught each study year.
Mosquito species 2005 2006 Total
Ma. titillans 4,430 7,529 11,959
Cx. nigripalpus 10,530 135 10,665
Cx. erraticus 1,572 3,102 4,674
Cx. salinarius 1,498 500 1,998
An. crucians 2,991 5,158 8,149
Total 21,021 16,424 37,445









CHAPTER 4
ARBOVIRUS SURVEILLANCE: MOSQUITO POOLS, SENTINEL CHICKENS, AND
HORSES.

Florida has had an arthropod borne virus (arbovirus) surveillance program in place since

1977 to track the amplification and transmission of mosquito-borne viruses including eastern

equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) Highlands J (family

Togaviridae, genus Alphavirus, HJ) and St Louis encephalitis virus (SLEV; Flaviviridae, genus

Flavivirus) (Day and Stark 1996). The Florida Department of Health (FDOH), Division of

Environmental Health, coordinates the surveillance program. The Interagency Arbovirus

Surveillance Network reports to the FDOH and is composed of several local, state and federal

agencies, which are involved with the surveillance and control of arboviral diseases.

Upon its arrival in the United States, West Nile virus (WNV; family Flaviviridae, genus

Flavivirus) was easily added to the existing surveillance program with the addition of WNV-

specific laboratory diagnostics. Because SLEV and WNV are antigenically related, cross-

reactions are observed with some serologic tests and so plaque reduction neutralization testing

(PRNT) is done to distinguish the two viruses. During Florida's first reported WNV

transmission season (2001), virus was recorded in 65 of 67 counties (Blackmore et al. 2003). In

both the northeastern U.S. and in Florida, wild bird mortality was the most sensitive viral activity

indicator (Blackmore et al. 2003). In 2001, wild bird mortality was the first indication of viral

presence in 54 of the 65 counties in Florida where WNV was detected (Blackmore et al. 2003).

Due to the correlation of WNV-positive dead bird reporting and local WNV transmission, dead

bird reporting has become a valuable surveillance tool in the United States (Eidson et al. 2001a,

(Nasci et al. 2002).

Like SLEV, the natural cycle of WNV involves Culex mosquitoes and wild birds.

However, unlike SLEV, WNV causes high rates of mortality in certain families of birds.









Members of the family Corvidae (crows, magpies, ravens, and jays) are particularly susceptible

to fatal infection (Nasci et al. 2002). Chickens in the northeastern United States are not

considered reliable indicators of human disease because seroconversions occurred after human

cases had already appeared (Crans and Schulze, 1986 Cherry et al. 2001). For this reason, New

York does not have a sentinel chicken program in place. In California and Florida, however,

sentinel chickens are an indispensable component of arboviral surveillance because viral

positives in chickens are closely associated with and predictive of human cases (Day and Lewis

1991, Reisen et al. 1994).

Horses are not currently bled as part of an active WNV surveillance program in the United

States. In New York State horse positives were unreliable in the prediction of human cases of

WNV (Trock et al. 2001). It is not yet known whether WNV surveillance in horses can predict

human cases in Florida, but horse cases that are reported to local health departments are used as

part of arbovirus surveillance. Blackmore et al. (2003) reported that the epicenter of the 2001

WNV outbreak in Florida horses was in Jefferson County. From Jefferson County, the outbreak

spread east, west, and south to a total of 40 Florida counties with confirmed horse cases. In the

counties reporting both horse and human cases, the horse cases preceded the human cases by one

to four weeks (Blackmore et al. 2003).

Weather conditions greatly affect mosquito populations and consequently arboviral activity

(Wegbreit et al. 2000, DeGaetano 2005, Pecoraro et al. 2007). Drought in the spring followed by

summer rain is closely associated with transmission of SLEV and WNV in Florida (Day and

Stark 1996, Day 2001, Shaman et al. 2005). Drought brings mosquito and bird populations into

close proximity by limiting the available water. Epizootic amplification may occur under these

circumstances if an abundant mosquito population is available to feed on susceptible wild birds.









Under conditions of prolonged drought, however, virus transmission is greatly reduced (Day and

Lewis 1991). Several critical factors have been outlined as criteria that create a high epidemic

risk for arbovirus transmission in south Florida. They include a large population of susceptible

wild birds, severe drought in the spring followed by a wet summer, and the continuation of dry

and wet patterns throughout the summer that focus virus transmission between the mosquito

vectors and vertebrate hosts (Day and Lewis 1991). The predictive factors outlined here are

based on aspects affecting Culex nigripalpus populations and dynamics. The weather pattern of

rain followed by drought synchronizes the oviposition and blood feeding of Cx. nigripalpus and

subsequently virus transmission (Day and Curtis 1993). Culex nigripalpus plays a major role in

arbovirus transmission in the southern part of the state. It is necessary to make similar

evaluations of the relationship between weather patterns and mosquito populations with virus

transmission patterns in the northern part of the state where Cx. nigripalpus populations are

typically much lower (Zyzak et al. 2002). Because Cx. nigripalpus is not as common in north

Florida as it is in south Florida, weather conditions such as a mild spring may increase the

population of Cx. nigripalpus in north Florida (Zyzak et al. 2002); or it is possible that other

Culex species are playing a larger role in virus transmission.

There were three aims of this study. The first aim was to compare arboviral activity in

Alachua County with the seasonal dynamics and abundance of mosquito species present at a

north Florida site. The second aim of the study was to correlate the temporal patterns of viral

activity and mosquito abundance with abiotic environmental factors including rainfall,

temperature, and wind speed. The third aim was to examine mosquito abundance at four

microenvironments within a site. To accomplish this goal a two-year surveillance project was

designed to provide information about the seasonal patterns of arbovirus activity in relation to









the abundance of mosquito species at a study site in north central Florida. The data were

collected to compare WNV, SLEV, and EEEV activity in north Florida with the mosquitoes

collected at the study site. Arbovirus activity included sentinel chicken seroconversion and

mosquito pool positives during the study period.

Materials and Methods

Sentinel Animals

The study site was located at the University of Florida Veterinary School, in Alachua

County in north central Florida (2938' N, 82o20' W) (see chapter 3 Materials and Methods).

Three equines were used as arboviral sentinels at the site between May and November of 2005

and 2006. The sentinel horses had blood samples taken weekly to screen for the presence of

antibody for WNV, EEEV, and HJ (IACUC approval # E312). Ten ml of blood was taken from

the jugular vein with a vacutainer needle and drawn directly into a 10 mL blue top (3.8% Na

citrate) vacutainer tube then centrifuged for 10 min at 600 g. The serum was tested at the

University of Florida Emerging Disease Arboviral Research and Testing (EDART) laboratory

with the Immunoglobulin M antibody-capture Enzyme-Linked Immunosorbent Assay (MAC-

ELISA) for detection of viral antibody.

Two of the sentinel horses were blood donors (research horses kept on the veterinary

school property for blood donation as needed, IACUC#A712) and were maintained permanently

in an open field near a sylvan habitat (Figure 4-1). The third animal was a pony that was used

twice a week as a bait animal in a stable trap. The pony was maintained in a pasture during

periods when it was not housed in the stable trap. The pony was located on the opposite side of

the study site, 600 m west of the blood donors (Figure 4-1).

Sentinel chickens were added to the protocol (IACUC approval # E248) from May through

November of 2006. Two white Leghorn chickens were housed in a 1.8m X 0.9m cage. The cage









was located in a field 100 m west of a horse pasture (Figure 4-1). It was placed next to a horse

stall that was used as a stable trap (Chapter 3). The chickens were bled weekly from the brachial

vein. Collections were taken from alternate wings each time the chicken was bled in order to

allow healing. One ml of blood was collected in a gel separator vial with a 25-gauge needle and

was centrifuged for 10 min at 600 g. The resulting sera were delivered to the Alachua County

Health Department the same day they were collected. The samples were shipped with other

Alachua County sentinel chicken blood samples to the Florida Department of Health Bureau of

Laboratories in Tampa, where they were tested for Flavivirus and Alphavirus hemagglutination

inhibition (HI) antibodies. The Department of Health routinely tests any resulting positive serum

samples to identify WNV, SLEV, EEEV, or HJ antibody by IgM enzyme immunoassays and

plaque reduction neutralization tests. The Alachua County health department received a weekly

report of the results of the chicken serum tests, from the FDOH for all chickens tested in the

County.

Mosquito Collections

Mosquito collections at the study site began on October 6, 2004 and continued to November

30, 2006. The traps were operated to two times a week (Figure 4-1) at 1600 hr and picked up the

following morning at 0800 hr. Centers for Disease Control light traps (John W. Hock,

Gainesville, Fl) baited with approximately 1 kg of dry ice were placed in four different habitats

at the site: at the edge of Bivens Arm Park, next to the retention pond, under a small stand of oak

trees, and by the horse stable trap (Figure 4-1). To analyze trap catch differences in four

microhabitats, a Kruskal-Wallis one-way analysis of variance was used (Minitab version 15,

State College, PA). PROC GLIMMIX analysis was done to compare the trap location

microhabitats by month and to evaluate mosquito species collection between the two years of the

study (SAS version 9.1, Cary, NC).









Mosquito collections were sorted by species and date of collection and stored in pools of

up to 50 at -700C. Pools were homogenized in 1 ml diluent of Phosphate Buffered Saline (PBS)

with 4% Fetal Bovine Serum (FBS) by placing two copper BBs in the vial and vortexing. After

a 10 min centrifugation at 11,356g to separate the mosquito solids, 200 tl of supernatant was

transferred to a new tube for RNA extraction with Trizol following the manufacture's protocol

(Molecular Research Center Inc., Cincinnati, OH). The remaining sample, not used in the RNA

extraction, was stored at -70 C for possible cell culture if a positive result in RT-PCR was

found. The RNA isolated with an RNeasy mini kit (QIAGEN, Valencia, CA) from the mosquito

pools, and was tested for WNV, SLEV, and EEEV using quantitative Real Time RT-PCR

(Lanciotti and Kerst 2001, Stark and Kanzanis 2007).

Four hundred microliters of the RNA extraction homogenate, and 600 tl L 15 media (5%

FBS, 15 [tg/mL gentomyacin, 200 units/mL penicillin, streptomycin, fungizone) was added to a

T25 cm2 flask of Vero cells (2.0 x 106 cells). The cells were rocked and incubated at 370 C for

Ih. Four mL L15 media was added and the cells were observed every 24h for 7d.

Meteorological Data

In order to address the relationship between weather and mosquito population dynamics,

daily weather conditions including rainfall, wind speed, and average temperatures were accessed

from the Florida Automated Weather Network (FAWN) recording station in Gainesville, FL

(29 39'N, 82 30'W). A 50-year mean of precipitation and temperature was calculated from

1951-2000 by compiling data from the National Oceanic and Atmospheric Association (NOAA)

archives. Monthly deviations from normal for precipitation and temperature were calculated by

subtracting recorded values from the 50-year mean monthly values. Paired t-tests were used to

evaluate differences between the two trapping season's temperature, precipitation, and vector









abundance. Linear regression was used to examine independent relationships between average

mosquito catch per trap night and weekly average precipitation, temperature, and wind speed.

Results

Sentinel Animals

None of the five sentinel animals (two horses, one pony, and two sentinel chickens)

maintained at the study site seroconverted to an arboviral agent during the study period. During

the same time period, the sentinel chickens maintained by Alachua County Public Health Unit

showed the following seroconversion activity: 2005, 16 WNV, 47 EEEV, and 3 HJ; 2006, 0

WNV, 15 EEEV, and 1 HJ (Table 4-1). A confirmed arboviral infection in a Florida horse is

classified as a reportable disease in which case the attending veterinarian must report the case to

the Florida Department of Agriculture and Consumer Services in Tallahassee. Between May 1

and August 29 2005, nine horses in Alachua County were confirmed as EEEV positive. One

horse was positive for WNV; this report came on October 20, 2005. In 2006, no WNV horse

cases were reported in Alachua County. Only a single EEEV horse case was reported on July 31

in Alachua County in 2006 (Table 4-1).

Mosquito Collections

Three hundred fifty nine mosquito pools (n = 13,809 total mosquitoes; 7 species) were

tested for WNV, EEEV, and SLEV. One pool of 50 Ma. tttillans, collected in September 26,

2006 was positive for SLEV (minimum infection rate of 0.254). The attempt to grow the SLEV

in Vero cell culture was unsuccessful. After 7d of observation no cytopathology was observed in

the cells. The mosquito pools were collected over a period of 26 months (October 2004 through

November 2006). During the same time period, Alachua County did not submit mosquito pools

to the FDOH for testing. In the state of Florida in 2005 there were 1,603 mosquito pools tested

from 11 counties. Five mosquito pools from three Florida counties (Monroe, Pinellas, and









Sarasota) tested positive for WNV and ten mosquito pools from four Florida counties (Escambia,

Sarasota, St. Johns, and Volusia) tested positive for EEEV. In 2006, there were no positive

mosquito pools (n = 1,253) in the state of Florida.

The most frequently collected mosquito species at the study site was Mansonia titillans

(29%) followed by Culex nigripalpus (25%) (Figure 4-2). The seasonal distribution of the seven

most abundant species was compared with horse and chicken seroconversions for WNV and

EEEV in Alachua County (Figures 4-5 to 4-8). In 2005, the abundance of Cx. erraticus

increased in February and March and the first EEEV chicken seroconversion was reported in

April. In 2006, Cx. erraticus abundance increased in May and the first EEEV chicken

serocnversion was at the end of May (Figures 4-6 and 4-8). The number ofMa. titillans

increased in the fall, which was when the last of the chicken seroconversions and horse cases in

both 2005 and 2006 were seen.

When trap location was examined by Kruskal-Wallis and Proc Glimmix analysis, the

highest numbers of mosquitoes were trapped in the Bivens Arm Forest and the lowest numbers

were obtained by the horse stable (Table 4-2, Figure 4-4). The collection of Anopheles crucians

(P = 0.001), Cx. erraticus (P = 0.045), and Ps. columbiae (P = 0.04) varied significantly by

location. There was no significant difference in the number ofMa. titillans, Cx. nigripalpus, Cx.

quinquefasciatus, and Oc. infirmatus caught at each trap location. During each month of the

study, the trap located in the Bivens Arm Forest collected significantly more mosquitoes that

traps number 2 and 3. No significant difference in trap catch were seen between trap 1 (Bivens

Arm) and trap 4 (the retention pond) in November and December, but all other months trap 1

caught significantly more mosquitoes than trap 4 (Figure 4-4). Trap year (year 1, October 2004

to October 2005 and year 2, November 2005 to November 2006) was significantly different for









collections of An. quadrimaculatus, Cq. perturbans, Cx. nigripalpus, Cx. salinarius, and Oc.

infirmatus.

Meteorological Data

December 2005 was abnormally wet, with 48.6 cm of rainfall above the expected 50 yr

(1951-2000) mean for Alachua County (Figure 4-3A). During the winter months of January

through March 2005, the observed monthly rainfall amounts were 3 to 5 cm below average

(Figure 4-3A). In 2006, the winter months of January through March were unusually wet,

accumulating over 20 cm of rainfall above normal. In March 2006, a drought began and

continued through September. During 2006, North Florida experienced a prolonged drought that

resulted in a total of 15.4 cm less rainfall than average (Figure 4-3A). The mean daily rainfall

patterns were significantly different between 2005 and 2006 (2005 mean 5.00 mm, 2006 mean

2.77 mm, t(364) = 1.66, P = 0.048).

The temperatures ranged from 1 to 3 OC cooler than average for most of the study period

(Figure 4-3B). Each month was colder than expected when compared to the long term means

except for January of each year and April of 2006. The mean monthly temperatures were not

significantly different between 2005 and 2006 (2005 mean 66.280 C, 2006 mean 66.230 C, t(ll)=

0.064, P = 0.47). There were no significant correlations of temperature, wind speed, or rainfall

with mosquito abundance.

Discussion

Although arbovirus (WNV, SLEV, and EEEV) activity was minimal in Florida during the

years of this study, information gathered during inter-epidemic years is valuable to the complete

understanding of mosquito-borne disease epidemiology (Hay et al. 2000). Virus transmission is

dependent on the presence of an abundant and old mosquito population (Zyzak et al. 2002) and

mosquito reproduction and mortality are directly influenced by meteorological conditions.









Weather conditions including temperature and rainfall directly affect vector population

distribution and abundance (Hay et al. 2000). During the spring of 2006 there was a prolonged

drought in north Florida that reduced the abundance of mosquitoes and minimized arbovirus

transmission (FDOH 2007).

The year-to-year differences in mosquito abundance and diversity at the study site are

likely related to local weather patterns. The weather was wetter than average in north Florida in

March through June and in October through December of 2005. Conversely, in 2006 there was a

prolonged drought in Florida and very little arboviral activity was reported throughout the state.

At the study site, some mosquito species (Cx. nigripalpus P = 0.0015, Cx. salinarius P = 0.0003,

An. quadrimaculatus P = 0.0002) were collected in significantly fewer numbers in 2006 when

compared to 2005. Transmission of SLEV and WNV are closely associated with rainfall

patterns (Day 2001, Shaman et al 2005). The data presented here support the conclusion of drier

years reducing the number of potentially infective mosquitoes (Day and Lewis 1991). The total

range of mosquito habitat is dependent upon the presence of available bodies of water and humid

daytime resting habitats. Therefore, a reduction in the number of infective mosquitoes decreases

the likelihood of arbovirus transmission.

The trap located in Bivens Arm Forest (trap 1) collected significantly more An. crucians,

Cx. erraticus and Ps. columbiae than the other three trap locations. Sylvan microhabitats may

support a greater population of mosquitoes because they provide a daytime resting habitat and

retain a high humidity. Additionally, the aquatic environment of Bivens Arm Lake provided

larval habitat. Animals located near such a habitat may experience a greater number of mosquito

bites by these species. The more open habitats of the oak stand, the stable, and the retention

pond had significantly fewer mosquitoes collected and this is likely because these areas did not









retain high daytime humidity levels to support daytime resting. Therefore, the mosquitoes would

need to fly much further from the daytime resting habitat of the forest to encounter a host in

these open habitats.

There were no significant differences in the number of numbers of Cx. nigripalpus, Ma.

titillans, and Cx. quinquefasciatus collected at each trap location. Two of the most abundant

species collected at the study site, Ma. titillans and Cx. nigripalpus, were collected equally at all

four trap locations. Day et al. (1991) found parous Cx. nigripalpus females collected in

abundance in open habitats. They reported that abundance was especially high during wet

summers when normally dry habitat became moist and humid allowing mosquitoes access to

hosts. When surveying the flight capacity of blood-engorged mosquitoes, Edman and

Bidlingmayer (1969), found Cx. nigripalpus in higher numbers in wooded habitats compared to

open habitats. In my study, the fact that no significant difference of Cx. nigripalpus abundance

occurred in the four trap microhabitats is consistent with the literature that this species can be

collected in high numbers in either open or wooded habitats. The ubiquitous nature of Cx.

nigripalpus at the study site may increase the likelihood of a single mosquito encountering both a

reservoir host and a susceptible host. Mansonia titillans was abundant at this site in the late fall

and was not significantly correlated with trap location. The lake at Bivens Arm contains

abundant aquatic flora (waterhyacinth, Eichhornia crassipes (Mart.) Solms., and waterlettuce,

Pistia stratiotes L.) necessary for the larval development ofMa. titillans populations. In a

dispersal study of several Mansonia species, individuals were re-captured from 0.5 to 2.4 km

from the release point (Macdonald et al. 1990). This flight distance is adequate to explain why

approximately equal collections ofMa. titillans were obtained in all the habitats surveyed.









Location of sentinel chicken sites is an important factor when initiating an arbovirus

surveillance program (Day et al. 1991). The historical enzootic activity of WNV at this site was

a key factor for choosing to place sentinel chickens there. None of the sentinel animals (three

equines and two chickens) at the site was positive for WNV, SLEV, HJ, or EEEV during this

study. Although the sample size was small with a total of five animals being screened, there

were no WNV seroconversions of sentinel chickens in Alachua County in 2006 (Table 4-1).

Moreover there were no horse or human WNV cases in Alachua County in 2006. Because

sentinel animals in Florida provide the most accurate and timely indication of field transmission,

the lack of seroconversions at our site may be an indication of low-level virus circulation in the

area in 2005 and 2006.

Although collected in large numbers, Ma. titillans did not show a temporal correlation in

abundance with the EEEV or WNV transmission season in Alachua County in 2005 and 2006

(Figure 4-4). West Nile virus has been isolated from field caught Ma. titillans (CDC 2007), but

no work has been published regarding their vector competence (Turell et al. 2005). Vector

capacity is determined not only from natural infection and demonstrated laboratory transmission,

but also biological factors such as biting preference, length of life, and timing of adult activity

are all fundamentally tied to vector capacity (DeFoliart et al 1987). Because Ma. titillans did not

appear at the study site until EEEV and WNV transmission had already begun in Alachua

County in 2005 it may not play a major vector role in arbovirus epidemics in north Florida.

Despite the lack of temporal correlation with EEEV and WNV transmission at the site, more

research is likely warranted as Ma. titillans is considered a vector of Venezuelan Equine

Encephalitis virus (VEEV; Family Togaviridae, genus alphavirus) in Central and South America

(Mendez et al. 2001, Turell et al. 2000). Mansonia dyari may be a maintenance vector of SLEV









in Panama (Gorgas Memorial Laboratory 1979, as cited by Lounibos et al. 1990). Members of

the genus Mansonia in Africa have had several WNV isolations reported (Traore-Lamizana et al.

2001). Furthermore, several species of Mansonia are likely involved in the transmission of

Japanese Encephalitis virus (JEV; family Flaviviridae, genus Flavivirus,) in Asia (Arunachalam

et al. 2004). This is the first positive identification of SLEV from Ma. titillans in Florida. The

failure to isolate virus in cell culture may have been because no cryogenic protection (i.e.

DMSO) was added to the homogenate. Freezing and thawing the sample reduces viable virions

because ice crystals break the envelope. Another potential reason virus was not isolated is that

the mosquito pools may have been stored at -200 C for a period of time and research indicates

that -70 C is the optimal storage temperature for virus detection of mosquito pools (Turell et al.

2002).

Culex erraticus populations increased at the study site a few weeks to a month prior to

EEEV transmission in Alachua County. In Alabama EEEV isolations from field caught Cx.

erraticus have been reported (Cupp et al. 2003). The natural isolations in the southeastern

United States and temporal correlation may indicate that this species is involved in EEEV

transmission in north Florida. Culex nigripalpus was active at low levels throughout the duration

of the study. In 2005 in Florida, there were three WNV isolations from Cx. nigripalpus (2 in

Pinellas and 1 in Sarasota County) and transmission has been documented in Jefferson County,

Florida (Rutledge et al. 2003). However, at our study site the population of Cx. nigripalpus after

November 2004 remained low (n < 50/trap night) for the duration of the study. Perhaps the low

numbers were related to weather conditions unfavorable for mosquito development.

Having only four light traps in different locations at the site was a limitation of the study.

A better assessment of microhabitat could have been made if the traps were moved within a









single microhabitat randomly to exclude the possibility of a trapping-out effect. A second

limitation of the study was the fact that only five sentinel animals were screened for arbovirus

activity. If more animals were used, there would have been a better chance of observing virus

activity had there been any. A comparison of arboviral activity in Alachua County with the

mosquitoes present at the study site was possible because the county maintains sentinel flocks

and horse cases are recorded. However, in this study, mosquitoes were collected in one place

and transmission occurred in another, making interpretation more difficult. The seasonal

abundance of Cx. erraticus and Cx. nigripalpus at this site increased prior to EEEV and WNV

transmission in Alachua County. These two species should be further considered as potential

vectors in north Florida of EEEV and WNV respectively.












































Figure 4-1. Location of the four paired light traps and resting boxes marked as T1, T2, T3, and
T4. The Chen barn was modified for the stable trap in 2005, and a portable stall was
erected 14m south of the Chen barn during the 2006 season.















90










An quadrimaculatus
2%

2%
Oc infirmatus
5%


.Cq perturbans
6%


Mansonia
29%


;x erraticus
10%


Cx nigripalpus
24%


Figure 4-2. The species that represented at least 1% or more of the total trap catch between
October 2004 and November 2006


Table 4-1. Number of arbovirus positive sentinel chickens and horses in Alachua County in 2005
and 2006.
Chicken Horse Chicken Horse
Arbovirus 2005 2005 2006 2006 Total
EEE 47 9 15 1 72
WNV 16 1 0 0 17
SLE 0 0 0 0 0
HJ 3 0 1 0 4
Total 66 10 16 1 93















Table 4-2. Mosquito collections by trap location from October 2004 though October 2005 (13
months). Mosquitoes were collected from light trap collections..
Light traps (Oct 2004-
Oct 2005) Location
Oak
Species Woods Pond trees Stable Total
Cx. salinarius 928 219 296 55 1498
Cx. nigripalpus 7060 1441 1952 77 10530
Cx. erraticus 1291 136 101 44 1572
Oc. infirmatus 1753 281 50 10 2094
An. crucians 2377 461 117 36 2991
An. quadrimaculatus 444 120 36 19 619
Mansonia titillans 1851 1819 527 233 4430
Total 15704 4477 3079 474 23734

Table 4-3. Mosquito collections by trap location from November 2005 though November 2006
(13 months). Mosquitoes were collected from light trap collections.
Light traps (Nov 2005-
Nov 2006) Location
Oak
Species Woods Pond trees Stable Total
Cx. salinarius 339 109 31 21 500
Cx. nigripalpus 112 14 6 3 135
Cx. erraticus 2455 206 224 217 3102
Oc. infirmatus 273 48 3 1 325
An. crucians 4939 141 55 23 5158
An. quadrimaculatus 236 14 32 4 286
Mansonia titillans 2845 2262 2194 228 7529
Total 11199 2794 2545 497 17035


































ON D J F M A M J J A S ON D J F M A M J J A S O N


1.5
1
0.5
0
-0.5
-1

-1.5
-2
-2.5
-3
-3.5


ONDJ FMAMJ JASONDJ FMAMJ JASON

Months (Oct. 04 to Nov. 06)
Figure 4-3. Monthly deviations from normal for rainfall A) and temperature B) for October 2004
through November 2006 in Alachua County Florida. Observed monthly values were
subtracted from a 50-year mean (1951-2000) to determine the deviations from
normal.











a
----. I-------------


a a
c a a a a a


0 0. -
a
S0.3
Sbb b
c. L b bI b bb b

0I1 bbb bbb b bbb bb -L
S.0.1. .


dPA


*Lighttrap 1


* Lighttrap 2 Lightlrap 3 Lighttrap 4


Figure 4-4. Comparison by month of the four light trap locations average mosquito trap catch.
Bars followed by a different letter are significant at P < 0.05.


1800
1600
1400
1200
1000
800
400
200
0


Collection dalte 2005

* Cxnigripalpus aCxsalinarius Cxerraliucs Malillans
OCqperlubans nAn ucians Ocinfirmatus


Figure 4-5. Temporal distribution of the seven most abundant mosquito species collected at the
University of Florida Veterinary School (March through December 2005). E, Eastern
Equine Encephalitis in Alachua County sentinel chickens (n = 47); Eh, Eastern
Equine Encephalitis in Alachua County horses (n = 9); W, West Nile virus in Alachua
County sentinel chickens (n = 16); Wh, West Nile virus in Alachua County horse (n =
1).


a a


a




Dab


'0


0


Month


00
) 61


f E EI E
~ ^ ,
,11 y


I













100
.E E E EEEEE EE E EEE E E
80 ,fi-: --------E -,e- -E c --E E--------------
0 JAf k E EkIk ~EE E E W
C) 70 w wwww
0 w www
s w
0 50
S40
30



Collection date 2005
..10





Collection date 2005

SCx nigripalpus Cx salinarius Cxerratiucs


Figure 4-6. Temporal distribution of three Culex mosquito species collected at the University of
Florida Veterinary School (March through December 2005). E, Eastern Equine
Encephalitis in Alachua County sentinel chickens (n = 47); Eh, Eastern Equine
Encephalitis in Alachua County horses (n = 9); W, West Nile virus in Alachua
County sentinel chickens (n = 16); Wh, West Nile virus in Alachua County horse (n
1).













.2 2000
S 1800 E E E E E E
S1600
o 1400
8 200
1 000
80
a600
c 40
C- 200
I-




Collection date 2006

*Cxnigripalpus Cxsalinarius Cxerratiucs o Matitillans
QCqpertubans mAn crucians =Ocinfirmatus


Figure 4-7. Temporal distribution of seven most abundant mosquito species collected at the
University of Florida Veterinary School (May through November 2006). E, Eastern
Equine Encephalitis in Alachua County sentinel chickens (n = 15); Eh, Eastern
Equine Encephalitis in an Alachua County horse (n = 1).














300


250
200
150
100
50
0


Collection date 20 06



Collection date 2006


I Cxnigripalpus BCxsalinarius BCxerratiucs


Figure 4-7. Temporal distribution of three Culex mosquito species collected at the University of
Florida Veterinary School (May through November 2006). E, Eastern Equine
Encephalitis in Alachua County sentinel chickens (n = 15); Eh, Eastern Equine
Encephalitis in an Alachua County horse (n = 1).


E E EI E E
EE


E









CHAPTER 5
BLOOD MEAL IDENTIFICATION OF MOSQUITOES COLLECTED FROM LIGHT TRAPS
IN NORTH CENTRAL FLORIDA (2004-2006).

The dynamics of vector and host interactions are an integral part of understanding disease

transmission. Knowledge of vector host feeding patterns provides insight to viral transmission

cycles by identifying possible host preferences. Techniques in blood meal analysis have been

changing since the early 1920s and have included direct observation of feeding mosquitoes,

quantification by capture in host-baited traps, and serological and genetic based techniques (Ngo

and Kramer 2003). The most common serological and genetic based techniques have been the

precipitin test, the Enzyme Linked ImmunoSorbent Assay (ELISA) and Polymerase Chain

Reaction (PCR) assays (Ngo and Kramer 2003). Polymerase Chain Reaction amplification of

host DNA followed by sequencing is becoming a common method for blood meal detection and

has several advantages over precipitin tests and ELISA. Due to the sensitivity of the PCR, a very

small amount of DNA can be used as template so even partially engorged mosquitoes can yield a

blood meal confirmation. Additionally, with serological tests such as precipitin and ELISA, anti-

sera must be prepared for each potential host species allowing blood meal confirmation to only a

limited number of species. With the advent of web-based databases such as GenBank, it is now

possible to compare nucleotide sequences and identify sources of arthropod blood meals.

Many studies of mosquito blood meals have emphasized the avian host identifications (Lee

et al. 2002; Ngo and Kramer 2004). Other studies have focused on mosquito feeding patterns by

distinguishing between avian and mammalian derived blood meals (Apperson et al. 2002).

Mosquitoes that are primarily ornithophilic, such as Culiseta melenura (Coquillett) for eastern

equine encephalitis virus (family Togaviridae, genus Alphavirus, EEEV) (Scott et al. 1984), play

a major role in the amplification of arboviruses. Some mosquito species in the genus Culex

prefer avian hosts and are important enzootic vectors of St. Louis encephalitis virus (family









Flaviviridae, genus Flavivirus, SLEV) (Tsai and Mitchell 1989). Culex nigripalpus Theobald is

an opportunistic feeder and may serve as an important vector of SLEV and West Nile virus

(family Flaviviridae, genus Flavivirus, WNV) in Florida (Day 2001). Additionally, Culex

salinarius Theobald may play a secondary role in the transmission of both SLEV and WNV

during times of the year Cx. nigripalpus is less abundant (Day 2001). Another important factor

for epidemic arboviral transmission is the well-documented host switching behavior of Cx.

nigripalpus (Edman 1974). Culex nigripalpus feeds preferentially on birds in the winter and

spring and shows an increased preference for mammalian hosts in the later part of the summer

and into the fall.

Entomological measures of arboviral transmission risk can be estimated by considering

mosquito abundance and age, biting preference, field isolations of virus, and vector competence

(Molaei et al. 2006). The primary Culex mosquito vector of WNV differs by geographic location

(Hayes and Gubler 2006). Research in Florida suggests that Culex nigripalpus is an important

enzootic and epidemic vector of WNV to humans (Rutledge et al. 2003, Shaman et al. 2005).

However, research is lacking on which mosquito species may potentially transmit WNV to

horses in Florida.

Horses are susceptible to WNV infection and each year fatal WNV horse cases are

reported in the US. The purpose of this study was to determine the mosquito blood feeding

patterns at a site where horses were stabled outdoors year round. To accomplish this goal a two-

year project was designed to collect blood fed mosquitoes located near horses. The results of

this study may provide valuable insight to potential mosquito species vectoring WNV to horses

in north Florida.









Materials Methods


Blood Fed Mosquito Collections

All blood fed mosquito collections were made at a site (29 38' N, 82 20' W) in Alachua

County in North Florida. Horses (n = 50) were maintained in outdoor pastures and in the spring,

when mares delivered, the total number of horses on the property increased to approximately 70.

The site was an open pastureland surrounded by urban development (Chapter 3 Materials and

Methods). Mosquitoes were collected twice a week using four CDC light traps and a backpack

aspirator (John Hock Co., Gainesville, FL) (Chapter 3 Materials and Methods). Mosquitoes

were aspirated from four resting boxes that were paired with a light trap and from the outside of

the horse-baited stable trap. Additional resting collections were taken from 10-minute ground

aspirations in the surrounding vegetation next to light trap number 1 (Figure 3-1). Light trap

number 1 was located within the sheltered habitat of trees, along the edge of dense vegetation

and open pasture where horses were always present. Light trap number 2 was located under a

small oak stand consisting of seven mature trees. Vegetation and under story at this trap site was

sparse and standing water was present after a rainfall event. Light trap number 3 was placed

adjacent to a horse stable. The grass surrounding the stable was mowed short and no trees were

located in the vicinity. Light trap number 4 was set next to a 1500 sq ft retention pond with

aquatic vegetation including cattails (Typha latifolia L), rushes (Family Juncaceae), and

waterlettuce (Pistia stratiotes L.) (Figure 3-1). All mosquitoes taken from aspirator and light

trap collections were immediately transported to the laboratory, where they were stored at -80C.

They were then counted on a chill table and identified and sorted by species according to Darsie

and Morris (2003).

All blood engorged females were separated from the collection and stored individually at

-800C. Each mosquito was identified to species and the size and stage of the bloodmeal were









recorded. The size of the bloodmeal was categorized according to Edman et al. (1975), the sizes

were: trace (no distention of the abdomen), 1/4, 1/2, 3/4, and 1 (fully fed). The stage (estimation

of days after a blood meal based on appearance of the abdomen) of the bloodmeal was

categorized according to Sella (Detinova 1962) on a one to seven scale, one (unfed) to seven

(fully gravid) scale.

Blood Meal Identification.

The extraction and PCR procedures were validated and optimized with positive controls.

The mosquitoes used as positive controls were Cx. quinquefasciatus and Cx. nigripalpus that

were obtained from the USDA (United States Department of Agriculture CMAVE), Gainesville

colonies. These mosquitoes were starved for 24 h prior to blood feeding. Culex

quinquefasciatus was fed cow blood from a sausage lining and Cx. nigripalpus was allowed to

feed on a live chicken (feeding that is a normal part of colony maintenance at the USDA). The

engorged female mosquitoes were frozen at 200 C. After it was validated that the primer sets

were successfully amplifying the target DNA, the remaining aliquot from the known host DNA

extaction was used as the positive control in subsequent PCRs. An unengorged female mosquito

was used as the negative control to ensure that no invertebrate DNA was amplified in the PCR.

Blood fed mosquitoes were thawed and placed on a Kimwipe tissue. The abdomen was

separated from the thorax by using sterile pipette tips to sever the tegument and isolate the

bloodmeal from all extraneous material. The DNA from the mosquito abdomen was isolated

using a phenol chloroform extraction (Levy et al. 2002). The DNA pellet was resuspended in 10

[tl of ImM Tris, and stored at -70 OC until later analysis.

Two separate PCR reactions were used to amplify the DNA template. Each PCR

contained a distinct primer set from the cytochrome b region of the mitochondrial genome.









Primers were chosen based on previously published blood meal analyses, one primer set was

designed to amplify avian blood (Cicero and Johnson 2001) and the other primer set was

designed to amplify mammalian blood (Ngo and Kramer 2003) (Table 5-1). A third set of

vertabrate specific primers was used to amplify the sequences if results could not be obtained

from the first PCR attempt (Cupp et al. 2004) (Table 5-1).

Mammalian and avian PCR assays were run in a final volume of 25 tl. Each reaction

contained 0.5 mM dNTPs, 3mM MgC12 and 1.2 units of Taq polymerase (Invitrogen, Carlsbad,

CA). The avian assay contained a final primer concentration of 15 pmole per reaction and the

mammalian assay contianed 5 pmole of each primer per reaction. Amplification conditions for

the avian PCR were 5 min at 93 C with 45 cycles of 94C for 30 s, 50C for 30 s, and 72C for 1

min 30 s with a final extension of 3 min at 72C.

The amplification cycle of the mammalian PCR was equalivant to the avian PCR

conditions, with the exception of the melting temperature, which was lowered to 48C. The

vertebrate specific assay conditions were 2 min at 94C with 55 cycles of 94C for 45 s, 50C for

50 s, and 72C for 1 min with a final extension of 7 min at 72C. The products were visualized

on a 1% agarose gel stained with ethidium bromide under UV light.

The bands of expected size (508 bp for avian and 772 bp for mammalian) were cut from

the gel and the DNA was extracted using the QIAquick PCR Purification Kit (Qiagen, Valencia,

CA). The purified DNA was sequenced using BigDye Terminator Kit version 1.1 (ABI prism,

Foster City, CA). The Interdisciplinary Center for Biotechnology Research (ICBR) Sequencing

Core at the University of Florida loaded the sequenced DNA and ran the product on a gel. The

electropherograms (base pair sequence information) were edited using Sequencher version 4.1.2









software (Macintosh). The edited sequences were compared to the nucleotide database with

BLAST analysis software available through NCBI.

Results

From a total of 45,326 mosquitoes collected in CDC light traps and resting collections, 242

(0.53%) were engorged. Of the 23 mosquito species identified at the study site, 14 species were

blood fed. A confirmed host blood meal match was obtained for 143 samples of the 242 (59%)

blood fed mosquitoes collected. The blood meals were identified to species or in some cases

order and represented mammalian (95%, 136/143), reptilian (2%, 3/143), and avian (3%, 4/143)

hosts (Table 5-2).

The results of the blood meal analysis are summarized in Table 5-2. Of the blood meals

obtained from reptiles, the turtle blood meals were isolated from Culex erraticus and

Ochleratatus infirmatus and the anole blood meal was isolated from Culex nigripalpus. A mixed

blood meal was isolated from one Aedes vexans that had fed on both a human and a horse. With

the avian primer set there was non-specific amplification of cow and reptilian hosts. The blood

meals (n = 9) isolated from Cx. salinarius were from horse and human. The most common blood

meal isolated from Cx. nigripalpus was horse (58%); the other blood meals were from human,

raccoon, cow and anole. Mansonia titillans fed on horse, human, mouse, cow, and there was a

single isolation from a chicken (Table 5-2).

Non-specific amplification with the avian primer set occurred. A PCR amplified DNA

band of the correct size was obtained in both assays with the mammalian primers and the avian

primers for two samples. Prior to sequencing this was thought to be a mixed blood meal, where

the mosquito had partially engorged on two separate hosts. Upon sequencing, both matched up

100% with Bos taurus, the domestic cow. A control sample of cow blood was run with both

primer sets and amplified in each assay. Additionally, the avian primer set amplified the two









reptilian derived blood meals. This primer set was designed to be avian specific, but non-target

amplification did occur. However, the unfed mosquito used as a negative control never showed

any amplification, and all the avian controls (chicken, dove, and vulture) amplified consistently.

Based on the consistency of amplification in the positive controls of avian blood, and the absence

of amplification of the mosquito negative controls, it appears that there was no interaction with

these primers and mosquito DNA, and when avian blood was present, it amplified.

Discussion

Blood fed mosquitoes were collected in light traps, resting traps, and aspirator collections.

Host identification studies usually focus on collection of mosquitoes from resting sites because

these areas yield the highest number of blood fed individuals (Nasci 1984, Apperson et al. 2002).

Therefore, the relatively large number of blood fed mosquitoes collected from light traps (n =

199) in this study was unexpected. Light traps specifically attract host-seeking females, and for

this reason the majority of mosquitoes collected are normally unfed. However, Ngo and Kramer

(2003) successfully used light traps in their study of blood meal identification. The light traps in

this study likely collected most of the blood fed individuals because the available vegetation was

inadequate to serve as a resting site. Most of the field site vegetation was located at the trap

number 1, which was along the edge of the pasture. The vegetation was not as dense and

probably not able to retain the high relative humidity characteristic of a suitable resting habitat

(Day 2001).

To increase sample size all specimens containing any trace of blood were processed,

regardless of Sella stage. A number of samples did not result in DNA amplification; the old age

of the blood meal may have contributed to this. Studies of the time limit of blood meal detection

have varied with reports of a maximum of 72 hours (Ngo and Kramer 2003) to a maximum of 7

days (Lee et al. 2002) before the blood meal was too far digested to amplify in PCR. The range









in detection limit is likely due to variation in individual mosquito species rate of blood digestion.

Trace blood meals frequently amplified and the age of the blood meal, based on Sella stage,

appeared to be a more important factor than size of the blood meal in this study. Blood meals at

stage 6 only amplified 4 times and 16 blood meals did not. Because PCR is extremely sensitive,

DNA extracted from a small blood meal, but not from an old blood meal, was adequate for

amplification.

In this study there were only three avian blood meals amplified and three of these were

chicken blood meals almost certainly obtained from the sentinel chickens being held adjacent to

the stable trap. It is probable the height of the traps affected the diversity of mosquitoes caught.

Traps placed in the tree canopy will capture higher numbers of mosquitoes that are likely feeding

on avian hosts (Anderson et al. 2004). The trap height of 1 meter was chosen to target those

mosquitoes active at the ground level and those most likely to feed on horses.

Horses were pastured in outdoor paddocks and were available to mosquito species active

throughout the day and night. Horses were the most common host blood meal isolated in this

study. It has been documented that Cx. nigripalpus is a widely opportunistic feeder and has a

host range of birds, mammals, and reptiles (Day and Curtis 1994). Edman (1974) found a shift

in the feeding pattern of Cx. nigripalpus from avian hosts in the spring to mammalian hosts in

the fall. However, in this study Cx. nigripalpus only fed only on mammalian and reptilian blood.

The sample size of 20 Cx. nigripalpus amplified blood meals was not sufficient to detect host

switching at this site. The frequent (9/20) horse feeding by Cx. nigripalpus, found at this site

along with previous research supporting the role of Cx. nigripalpus in WNV transmission,

warrants further research into the importance of this species in WNV transmission cycles in

north Florida (Rutledge et al. 2003; Shaman et al. 2005).









Culex salinarius has been implicated in transmission of SLEV and EEEV (Slaff 1990;

Cupp et al. 2004). This species is documented as readily feeding on horses {Samui, 2003 2335

/id}, and has the ability to travel up to 2.0 km in 1.5 hr (LaSalle and Dakin 1982), allowing it to

easily disperse out into the open pasture areas to feed. The blood meals (n = 9) from Cx.

salinarius at this site were exclusively on horses and humans. Culex salinarius is considered a

general feeder attacking indiscriminately both birds and mammals including humans (Andreadis

et al. 2001). However, in open agricultural habitats Cx. salinarius has been documented feeding

exclusively on mammals (Edman 1974). Because Cx. salinarius has had field isolations of

WNV, is a competent laboratory vector of WNV (Turell et al. 2005) and feeds on avian and

mammalian hosts it should be further studied in north Florida as a possible epidemic vector.

Furthermore, Cx. salinarius is seasonally abundant in the early spring correlating with the

amplification phase of WNV and is continually collected through June (Figures 4-6 and 4-8).

Mansonia titillans was the most frequently collected of the all the species represented at

this site. From the 41 amplified blood meals from Ma. titillans, 26 were from horse. The

remaining blood meals were from raccoon, mouse, cow, human, and chicken. The role ofMa.

titillans in WNV transmission has not been well studied. But the fact that there was an avian

isolation and it readily feeds on horses may be worth investigating further. The avian isolation,

however, was from a chicken located at ground level and not an amplification host. Finally, the

seasonal abundance ofMa. titillans does not correlate with amplification, or early transmission

phases of WNV (Figures 4-5 and 4-7).

There were 53 human blood meals derived from this sample set isolated from eight

mosquito species (Table 5-2). With students and workers outside or in open barns throughout










the day and evening, a host-seeking mosquito at this site would have frequent opportunity to

encounter a human and obtain a blood meal.

As we examine the many epidemiological factors of disease transmission, the clues

regarding host preference in wild caught mosquitoes will help broaden our understanding of

vector-host interactions. By better understanding which mosquitoes play a key role in the

transmission cycle, new control measures can be developed that are more efficient and target

specific species. With tools such as DNA-based amplification, the host species are more readily

and accurately able to be identified. The host preference information, when combined with the

existing knowledge of mosquito biology, aids the effort to improve arbovirus surveillance and

prevention strategies through control programs and public health advisories.

Table 5-1. Primer sets in PCR used to amplify DNA from vertebrate hosts.
Product
Name Sequence size (bp) Reference
Avian 5'-GACTGTGACAAAATCCCNTTCCA-3' 508 Cicero and
5'-GGTCTTCATCTYHGGYTTACAAGAC-3' Johnson (2001)

Mammalian 5'-CGAAGCTTGATATGAAAAACCATCGTTG-3' 772 Ngo and
5'-TGTAGTTRTCWGGGTCHCCTA-3' Kramer (2003)

Invertabrate 5'-CCCCTCAGAATGATATTTGTCCTCA-3' 228 Cupp et al.
5'-GCHGAYACHWVHHYHGCHTTYTCHTC-3' (2004)
H =A, C, orT; Y = CorT; V = A, C, orG;N =A, T, C, orG ; R = GorA; W= AorT.















Table 5-2. Identification of blood meals from mosquitoes collected in Gainesville, FL, October
2004 to November 2006.


Mosquito Species
Ae. vexans
An. crucians


An. quadrimaculatus
Cx. salinarius
Cx. erraticus

Cx. quinquefasciatus
Cx. nigripalpus
Ma. titillans

Cq. perturbans
Oc. infirmatus
Oc. mitchellae
Ps. columbiae
Ps. ciliata
Totals


# # confirmed
tested (%)
4 4(100)
4 4(100)
1 1(100)
14 9(55)
30 27 (90)


3
36
88

15
9
2
28
3
237


1 (33)
20 (55)
41 (56)

8(53)
7 (78)
1 (50)
19(68)
1 (33)
143


Results
3 Human; 1 Mixed Horse and Human
3 Human; 1 Horse
1 Horse
4 Horse; 5 Human
1 Night Heron; 1 Box Turtle; 6 Horse;
3 Raccoon; 16 Human
1 Chicken
1 Anole; 2 Cow; 9 Horse; 3 Raccoon; 5 Human
1 Chicken; 2 Mouse;3 Cow; 4 Raccoon;
5 Human; 26 Horse
1 Armadillo; 1 Deer; 6 horse
1 Box Turtle; 2 Horse; 2 Human; 2 Raccoon
1 Horse
1 Chicken; 1 Deer; 3 Horse; 14 Human
1 Horse


Horse (Equus caballus), Human (Homo sapiens), Raccoon (Procyon lotor), Cow (Bos taurus),
Chicken (Gallus gallus), Deer (Odocoileus virginianus), Mouse (Mus musculus), Night heron
(Nycticorax nycticorax), Armadillo (Dasypuis novemcintus), Turtle (Terrapene carolina), and
Anole (Anolis trinitatis)









CHAPTER 6
HOST FEEDING, VIRUS SURVEILLANCE AND FUTURE EXPERIMENTS

Summary

West Nile virus (Family Flaviviridae, genus Flavivirus, WNV) has become endemic in the

US and the western hemisphere (Komar and Clark 2006). The introduction of WNV into the

New World has provided a unique opportunity to study the spread and epidemiology of an

arbovirus in a new geographic setting. The impact of WNV on horses and humans has facilitated

collaborations between the veterinary and human medical fields. It has also sparked the

development of new diagnostic and surveillance techniques that may help the United States

prepare for future disease introductions. Many critical questions about potential mosquito

vectors of WNV, the effects of microhabitat and weather on WNV amplification and

transmission, and the blood feeding patterns of potential WNV vectors remain unanswered.

Host Feeding

Of the 23 mosquito species collected in CDC light traps, resting boxes, and a horse-baited

stable trap, during my study, at least some individual females from 14 species had blood fed.

Twelve species (fifty eight individuals) were positive for horse DNA: Aedes vexans, Anopheles

crucians, Anopheles quadrimaculatus, Culex nigripalpus, Culex salinarius, Culex erraticus,

Mansonia titillans, Coquillettidia perturbans, Ochleratatus infirmatus, Ochleratatus mitchellae,

Psorophora columbia, and Psorophor ciliata. Individual females from eight mosquito species

(Ma. titillans, Cx. erraticus, Cx. salinarius, Cx.nigripalpus Ae. vexans, An. crucians, Oc.

infirmatus, and Ps. columbiae) blood fed on human (n = 48). Mansonia titillans blood fed

primarily on mammals at my Alachua County, Florida study site. There was, however, a single

Ma. titillans blood meal identification from a chicken. IfMa. titillans is a competent vector of

WNV, then it could, under certain circumstances, serve as an epizootic or epidemic vector to









horses and humans in north Florida. Further work, such as vector competence studies and WNV

screening of mosquito pools, can help clarify the potential role of this species in WNV

transmission. There have been WNV isolations from Ma. titillans, but to my knowledge no work

has been done to test vector competence. A maj or factor that may preclude Ma. titillans from

vectoring WNV to humans and horses is that the seasonal abundance peaks late in the fall.

Mansonia titillans is not active early in the spring during WNV amplification in the bird

population. Instead, this species becomes active later in the season and is not likely to encounter

a WNV positive bird prior to blood feeding on horses and humans.

Culex erraticus is another species that may be worth studying further because this species

is an opportunistic feeder, was found at the site frequently, and is considered a competent vector

of EEEV (Cupp et al. 2004). While competence for one type of arbovirus does not necessarily

correlate with competence for another (Hardy et al. 1983), this species has had several isolations

of WNV in the US. Vector competency studies of Cx. erraticus for WNV should be done.

Vector competency studies indicate that other members of the genus Culex (Cx. nigripalpus, Cx.

quinquefasciatus, and Cx. tarsalis) are moderate to excellent vectors of WNV (Turell et al.

2005). Laboratory studies show that Cx. erraticus is a long-lived species, which could contribute

to its potential as a WNV vector (Kline et al. 1987).

The horse-baited stable trap used in this study attempted to collect a representation of

mosquitoes entering the trap throughout the entire night. Many studies collect for short periods

of time directly off the horse or bait animal. These collections record the species of biting flies

that are attracted to the bait animal, but may not truly represent the species that are feeding on

the animal. In this study the majority of the mosquitoes (518/528, 98%) that were collected from

the horse-baited stable trap were blood fed. When the blood was analyzed by PCR it was









confirmed that, all the blood meals were derived from the horse (Chapter 5). Overall, the trap

collected a small number of mosquitoes and future experiments could include modifications of

the construction of the openings to allow for easier entrance of more mosquitoes and to make it

more difficult for them to exit. In an experimental release of mosquitoes inside the stall, many

were able to escape from the trap. Perhaps by adding an upward sloping baffle on the interior of

the trap, fewer mosquitoes would have been able to exit the trap.

Surveillance

None of the sentinel animals (three horses, two chickens) tested positive for any of the

arboviruses being monitored (SLEV, EEE, HJ, and WNV) in 2005 and 2006. Sentinel chickens

are routinely utilized in arbovirus surveillance programs in the state of Florida and are

considered reliable predictors of arboviral activity in an area (Day and Lewis 1991). The

location for monitoring sentinel animals was chosen because horse cases occurred at the site in

2001. This site would probably be appropriate to continue monitoring for virus in future studies,

perhaps the addition of more sentinel chickens to the flock would increase the possibility of

arbovirus detection.

From the mosquito pools (n = 359) tested for arbovirus (WNV, SLEV and EEEV), there

was one positive SLEV identification. To my knowledge this is the first time SLEV has been

isolated from a pool ofMa. titillans. Mansonia titillans is involved with transmission of an

alphavirus Venezuelan equine encephalomyelitis virus (family Togaviridae, genus Alphavirus,

VEEV) (Mendez et al. 2001, Turell et al. 2000). Future studies determining vector competence

of Mansonia mosquitoes for WNV and SLEV would be valuable to determine if this genus may

play a secondary role in transmission of arboviruses in Florida.









Microenvironment and Weather

As part of this study, microenvironment and weather were correlated with mosquito

abundance and diversity. Several differences were found for trap location at the study site, with

the highest mosquito trap location being in Bivens Forest (Chapter 4). Weather conditions

studied (rainfall, temperature and wind speed) were not significantly correlated with mosquito

species abundance. This may have been due to the yearly meteorological differences at the site

and may take a longer study period to establish how the climate affects the mosquito populations

at this site. Previous field studies conducted in warm tropical climates have found an association

between meteorological and landscape conditions and the incidence of mosquito borne disease

(Reisen et al. 1993, Dhileepan 1996, Hu et al. 2006). In the United States, Mirimontes et al.

(2006) found that high temperature and agricultural land use was associated with an increased

incidence of WNV. In Georgia, urbanization was found to increase risk of human WNV

infection (Gibbs et al. 2006). In Texas, mosquito vector populations were correlated with

temperature, precipitation, and canopy cover (Bolling et al. 2005). In Rhode Island, precipitation

was the factor most closely associated with arbovirus activity (Takeda et al. 2003), and in

Florida, spring drought followed by rain was specifically associated with incidence of WNV

(Shaman et al. 2005). Given the multiple correlations of WNV activity with climatological (long

term weather) conditions, perhaps the collection of weather data at this site for a longer period of

time would be informative.

Extrinsic Risk Factors of WNV to Horses

In the study of the extrinsic risk factors of WNV to horses, several factors were close to

significant and warrant further study. The presence of water on the property should be

investigated to determine the association of type of water present and risk of WNV infection in

horses. Additionally, it would be interesting to study the relationship of fan use and mosquito









activity. Time of day the fans are used and the type of fan used could be compared

experimentally with the abundance of mosquitoes entering a stable. Investigating these factors

could not only help in evaluating WNV risk to horses, but could also be useful in further

describing mosquito population dynamics in an agricultural setting. The strongest protective

factor to horses for WNV was vaccination status. The negative comparison group in this study

showed signs of arboviral infection and may not have represented a true negative control. A case

control study would be a useful future study.

Conclusions

Mosquitoes commonly fed on horses, and several Culex spp were abundant and temporally

correlated with arboviral activity in north central Florida. Future experiments that focus on

arbovirus transmission to horses should consider Cx. erraticus, Cx. nigripalpus, and Cx.

salinarius as possible epizootic vectors of EEEV and WNV to horses. The seasonal abundance

of these three mosquitoes, and their vector competence in a laboratory setting combined with the

blood meals from horse make them strong candidates as potential arboviral vectors to horses in

Florida. The single most important preventative measure a horse owner can take is to vaccinate

the horse against WNV twice a year. This is especially important in Florida where mosquito

activity can occur year round.












APPENDIX A
ARBOVIRUS CASE INFORMATION FORM


Florida Department of Agriculture & Consumer Services
Division of Animal Industry
Bureau of Animal Disease Control

Arboviral Encephalitis
Case Information Form


Contact:

Dr. Michael A. Short
Equine Programs
Rm 329, 407 S. Calhoun St.
Tallahassee, FL 32399-0800
850/410-0901; Fax: 410-0919


585.145, Florida Statutes www.doacs.state.fl.us/ai
Note: All documents and attachments submitted with this request are subject to public review pursuant to Chapter 119, F.S.


Submitter: Please send this completed form along with collected samples to the Kissimmee
Diagnostic Laboratory at: 2700 N John Young Pkwy, Kissimmee, FL 34741_Phone (321) 697-1400


FOR LAB USE ONLY


If submitting split samples, send copies of completed form (both pages) to each laboratory used. If samples are not being submitted,
please send the completed form to Dr. Michael A. Short, Division of Animal Industry, Fax 850-410-0919. Hard copies can be mailed to
the address shown above.


Premises GPS (5 decimal digits)
Premises GPS (5 decimal digits)


Latitude


Longitude


Date Reported


Premises ID Number


FDACS/USDA Veterinanan(s) or Inspector(s) Assigned
Name Title/Occupation

Business/Affiliation

Mailing Address Physical Address (if different)

rC
0

Phone # Fax #

Mobile # Pager#

Email

Name Title/Occupation

S Premises/Farm Name
0

E Mailing Address Physical Address (if different) (Where Horse Resides)
0

I Phone # Fax #

E
S Mobile # Pager#
U.
Email


DACS-09125 Rev. 12/05
Page 1 of 2


CHARLES H. BRONSON
COMMISSIONER















Arboviral Encephalitis
Case Information Form (continued)


Name/Animal Identification Date of onset of clinical symptoms

0
c
Breed Age

Sex (Male/Female/Gelding) Vaccination Status (History)

0 Status of Horse: Alive Dead Critical Date of Death: Buried? Yes No
L Recovering as of (Date):
0
I Showing clinical symptoms? Yes No Method of Death: Natural causes
Euthanasia Other:
Number of samples taken. Date samples taken:

Samples submitted to FDACS Kissimmee Diagnostic Laboratory

S Sample type: Blood Brain Other: Date Sent:
Samples submitted to USDA National Veterinary Services Laboratory (NVSL)

S Sample type: Blood Brain Other: Date Sent:
Samples submitted to Florida DOH Laboratory

Sample type: Blood Brain Other: Date Sent:

History:

0
>,

.S-
r Clinical Presentation:
0 -Apprehension Other:
S Depression
Elevated Temperature
SHead Shaking
4 _Muscle Twitching
CL. Incoordination
S Weakness of Hind Limbs
-- Inability to Stand
.- Aimless Wandering
0 _Head Pressing
Listlessness

Comments/Additional Information:
Attach additional pages as needed.


DACS-09125 Rev. 12/05
Page 2 of2


Florida Department of Agriculture and Consumer Services


Division of Animal Industry/Office of the State Veterinarian









APPENDIX B
ENCEPHALITIS SURVEY


1. What best describes the activity of the horse that became ill:

2. How many years has the horse that became ill been at this address:

3. How many horses were on the property when it became ill?

4. How many other horses become ill/displayed neurological symptoms in:
2000 2001 2002 2003

5. How many other horses have been diagnosed with WNV or EEE:
2000 2001 2002 2003

6. Did the horse survive its clinical symptoms?
If its answer is no to question 6, skip to question 13.

7. Did the horse recover to complete activity after its clinical signs?
If yes, how long did it take? (months)

8. A. There are changes in your horses personality?
B. Does the horse act depressed?
C. Are there gait abnormalities?
D. Does the horse shake spontaneously?
E. Does the horse shake after being ridden?
F. Does the horse act weak or is incapable of maintaining its own weight?
G. Is there a loss of muscle mass?
H. If there is muscle loss is it in a specific area or general loss?

9. If the horse survived, do you still posses it?

10. If the horse was sold, did you receive the expected value?

11. If you did not receive the expected value, estimate your loss.

12. If the horse was sold, how much did the illness increase your investment?

13. If the horse did not survive, estimate its replacement value.

14. When the horse was ill, what were your veterinarian costs?

15. If you had to miss work, estimate your loss?

16. A. When the horse became ill was vaccinated against WNV?
B. If the answer is yes, report when month/year.









C. Were the other horses on the property vaccinated against WNV?


D. How many times were the horses vaccinated in the 2001?
Once Twice Three times Four times
E. How many times were the horses vaccinated in the 2002?
Once Twice Three times Four times
F. How many times were the horses vaccinated in the 2003?
Once Twice Three times Four times
G. What months were they vaccinated?
H. Was the horse that became ill vaccinated against EEE?
I. If the answer is if, report when month/year.
J. Were the other horses on the property vaccinated against EEE?
K. How many times were the horses vaccinated in the 2001?
Once Twice Three times Four times
L. How many times were the horses vaccinated in the 2002?
Once Twice Three times Four times
M. How many times were the horses vaccinated in the 2003?
Once Twice Three times Four times
N. What months were they vaccinated?

17. Who applied the vaccine for WNV?
Myself Veterinarian Other
Who applied the vaccine for EEE?
Myself Veterinarian Other

18. What was the cost to vaccinate on a per horse basis against WNV and EEE in
2000? 2001? 2002? 2003?

19. What best describes the way in which the horse was treated when ill?
SStabled all day and night. __ Stabled and let out 2-4 hours during the day.
SStabled in the afternoon/outside in the morning and night.
SStabled all day/outside all night. Stabled at night/outside all day.
Stabled and let out 2-4 hours in the evening Outside 24 hours

20. When let out, the horse was in: Pasture (0.5 acres) __ Grass Paddock
Sand Paddock

21. If stabled, how often were the stalls cleaned? __ Every month 2 times a
month
Every week Daily

22. Are there fans in the stable?
If yes, how often are they run? All day Only when necessary
Never









23. What best describes the cover of the stable? boards with openings
Solid wood or cement __ Open shed

24. After rain, is there temporary standing water on the property?

25. Mark the different water sources for the horse. Bucket automatic
natural
water tank


26. Describe the type of water that exists on the property. __ River Stream
Lagoon or pool Marsh or wetland
27. What best describes the canopy cover? Over barn Over grass
Over both

28. How many piles of debris exist near the stable, or the area of horse activity?
29. When in the stable, do you notice great, medium, or minimum activity of flies and/or
mosquitoes? Severe Medium Minimum None

30. What best describes the contents of the repellant used during the time in which the
horse was ill? Permethrin Citronella Skin so soft
Organic
How frequently was the repellant used? As needed __ once daily __ 2-3
times a week
SWeekly Monthly __ None

31. How often did you use a flysheet? Never __ Occasionally Continuously

32. During the time of your horse's illness, did you notice any dead birds on your
property?
Sno yes
During that same time, were there other ill animals? If yes, please list:



Reproductive effects. Please it responds if there was breeding activity on the
premises where its horse resided during the time of its disease.


33. Was there any breeding activity on the property since 2000? No Yes
2001? No Yes 2002? No Yes 2003? No
Yes

34. In 2000, how many mares checked pregnant at 30 days?
In 2001, how many of these mares foaled?
In 2001, how many mares checked pregnant at 30 days?









In 2002, how many of these mares foaled?
In 2002, how many mares checked pregnant at 30 days?
In 2003, how many of these mares foaled?
In 2003, how many mares checked pregnant at 30 days?
In 2004 how many of these mares foaled?

35. Did you vaccinate your mare against the WNV during pregnancy? no
yes

36. Were there any abortions after the vaccine against WNV was administered during
the gestation or at the end of the pregnancy? __ no __ yes
If the answer is yes, were other vaccines that were administered at the same time or
shortly
before the abortion? __ no __ yes

37. Have there been abortions during the months of autumn independently of vaccines
administered on the property or farm? __ no __ yes







APPENDIX C
SURVEY REQUEST LETTER




UNIVERSITY OF

SFLORIDA


February 18, 2004

To whom it may concern,

In an effort to gain critical information on the West Nile Virus and Eastern Equine Encephalitis
outbreaks since 2001 in Florida, the University of Florida, in collaboration with the Florida
Department of Agriculture and Consumer Services and the United States Department of
Agriculture, are asking for your assistance in filling out the enclosed survey. This information
will help us understand the natural course of mosquito transmitted encephalitis that threatens
Florida horses yearly.
Your horse was reported to have exhibited clinical signs consistent with either West Nile virus or
Eastern Equine Encephalitis virus. Although these signs may or may not have been confirmed
by testing, the enclosed survey verifies vaccination information submitted at the time of testing,
as well as, gathering further information regarding herd health and management. This will allow
us to identify risk and management factors that we can then make recommendations about to the
horse owning public.
Your participation in this study is of great importance, and your response is much appreciated.
As a token of our appreciation, please use the enclosed gift certificate.
Enclosed is a postage-paid envelope so that you can return the survey to the University of Florida
as soon as possible. You may also fax this information to Ashley Cunningham at 352-392-8289.


We hope that you will assist us in this important endeavor.
Respectfully,



The Veterinary Class of 2008
Maureen Long, DVM, PhD, DACVIM-LA
Assistant Professor
Large Animal Clinical Sciences
Enclosures (2)









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Kulasekera, L. D. Kramer, and N. Komar. 2001. West Nile outbreak among horses in
New York state, 1999 and 2000. Emerg. Inf. Dis. 7: 745-747.

Tsai, T. F., F. Popovici, C. Cernescu, G. L. Campbell, and N. I. Nedelcu, 1998. West Nile
encephalitis epidemic in southeastern Romania. Lancet. 352: 767-771


Turell, M. J., J. W. Jones, M. R. Sardelis, D. J. Dohm, R. E. Coleman, D. M. Watts, R.
Fernandes, C. Calampa, and T. A. Klein. 2000. Vector competence of Peruvian
mosquitoes (Diptera: Culicidae) for epizootic and enzootic strains of Venezuelan equine
encephalomyelitis virus. J Med Entomol. 37: 835-839.

Turell, M. J., D. J. Dohm, M. R. Sardelis, M. L. Oguinn, T. G. Andreadis, and J. A. Blow.
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transmit West Nile Virus. J. Med. Entomol. 42: 57-62.

Turell, M. J., M. L. O'Guinn, D. J. Dohm, and J. W. Jones. 2001. Vector competence of
North American Mosquitoes (Diptera: Culicidae) for West Nile virus. J. Med. Entomol.
38:130-134.

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Polymerase Chain Reaction from Mosquito (Diptera: Culicidae) Pools. J. Med. Ent. 39:
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mosquitoes (Diptera:Culicidae) from Massachusetts for a sympatric isolate of eastern
equine encephalomyelitis virus. J. Med. Ent. 34: 346-352.

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the roosting behavior of birds affect transmission dynamics of West Nile virus?
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BIOGRAPHICAL SKETCH

Leslie Rios was born in Seattle Washington. She received her bachelor's degree from

Western Washington University in 1998. She continued her education at Oregon State

University studying entomology. She received her master's degree in 2000. She then worked

for the University of Alabama at Birmingham. There she studied West Nile virus in mosquito

and bird populations. It was there that she fell in love with the field of medical entomology and

went on to receive her PhD at the University of Florida.





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1 IDENTIFICATION OF POTENTIAL MOSQUITO VECTORS OF WEST NILE VIRUS TO HORSES IN NORTH CENTRAL FLORIDA By LESLIE MICHELLE VIGUERS RIOS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Leslie Michelle Viguers Rios

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3 To my husband, Salvador Rios Madrigal

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4 ACKNOWLEDGMENTS I thank my comm ittee, colleagues, friends and family for the necessary support and encouragement to finish this dissertation. Dr. James Maruniak provided laboratory space, reagents, and daily inte raction. Dr. Maureen Long provided the stable trap, laboratory space, and laboratory help. She provided reagents, and a llowed me access to a multi-year database to conduct the extrinsic risk analysis. Dr. Shue spent many hour s with me on statistical analyses and for that Im very grateful. I thank Dr. Alej andra Maruniak for her insight and guidance. She read manuscripts, helped with trouble shooti ng when assays were not working, and kept a positive attitude gently leading me and other stud ents in the lab toward success. I thank Dr. Roxanne Connelly for superior editing comments a nd helpful writing tips. I am grateful for the mentorship of Dr. Jonathan Day who is an excellent field ecologist and was always supporting and encouraging throughout the en tire dissertation process. My friends and colleagues were of enorm ous assistance providing both emotional support and intellectual stimulus. Er in Vrzal has been a great fri end and she provided me with mosquitoes for blood feeding analysis and for fl uorescent release trials. John Herbert helped with making graphs and statistical analysis. Dr. Karla Addesso read my dissertation and provided editing comments. My mom has been my greatest advocate. Her belief in me helped me believe in myself. My husband helped me keep perspective of what is truly important in life Lastly, my daughters sweet smile reminded me to stay in the pr esent and enjoy each day as it comes.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.......................................................................................................................10 ABSTRACT...................................................................................................................................11 CHAPTER 1 INTRODUCTION AND REVIEW OF THE LITERATURE............................................... 13 Introductory Statem ent......................................................................................................... ..13 Introduction................................................................................................................... ..........14 Transmission Cycle............................................................................................................. ...15 Clinical Disease......................................................................................................................18 Human.............................................................................................................................18 Equine..............................................................................................................................18 Avian...............................................................................................................................19 Other Vertabrates.............................................................................................................19 Epidemiology and Ecology..................................................................................................... 20 Invertebrate Hosts (vector).............................................................................................. 21 Vertebrate Hosts (Reservoi r/Amplification Host)........................................................... 24 Human.............................................................................................................................25 Surveillance and Detecti on of West Nile Virus ...................................................................... 26 Diagnostics.............................................................................................................................27 Risk Factors.............................................................................................................................29 Prediction of Human Cases............................................................................................. 29 Blood Meal Analysis............................................................................................................ ..30 2 EXTRINSIC RISK FACTORS ASSOCI ATED W ITH WEST NILE VIRUS INFECTION IN FLORIDA HORSES...................................................................................34 Materials and Methods...........................................................................................................36 Arbovirus Case Information............................................................................................ 36 Retrospective Survey....................................................................................................... 36 Case Definition................................................................................................................37 Statistical Analysis.......................................................................................................... 38 Results.....................................................................................................................................39 Farm and Sample Submission Information..................................................................... 39 Arbovirus Infection Prevention....................................................................................... 40 Stable Characteristics and Farm Ecology........................................................................ 42 Discussion...............................................................................................................................43

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6 3 MOSQUITOES COLLECTED IN LIGHT TRAPS, RE STING BOXES, AND HORSEBAITED TRAPS IN NORTH FLORIDA.............................................................................. 56 Introduction................................................................................................................... ..........56 Materials and Methods...........................................................................................................59 Study Site and Mosquito Collection Protocol................................................................. 59 Fluorescent Mosquito Release and Recapture................................................................. 61 Results.....................................................................................................................................62 Discussion...............................................................................................................................64 4 ARBOVIRUS SURVEILLENCE: MOSQUITO POOLS, SENTINEL CHICKENS, AND HORSES..................................................................................................................... ..76 Materials and Methods...........................................................................................................79 Sentinel Animals.............................................................................................................79 Mosquito Collections....................................................................................................... 80 Meteorological Data........................................................................................................ 81 Results.....................................................................................................................................82 Sentinel Animals.............................................................................................................82 Mosquito Collections....................................................................................................... 82 Meteorological Data........................................................................................................ 84 Discussion...............................................................................................................................84 5 BLOOD MEAL IDENTIFICATION OF MOSQUITOES COLLECTED FROM LIGHT TRAPS IN NORTH CE NTRAL FLORIDA (2004-2006)........................................ 98 Materials Methods................................................................................................................100 Blood Fed Mosquito Collections...................................................................................100 Blood Meal Identification.............................................................................................. 101 Results...................................................................................................................................103 Discussion.............................................................................................................................104 6 HOST FEEDING, VIRUS SURVEILLANCE AND FUTURE EXPERIMENTS.............. 109 Summary...............................................................................................................................109 Host Feeding.........................................................................................................................109 Surveillance................................................................................................................... .......111 Microenvironment and Weather........................................................................................... 112 Extrinsic Risk Factors of WNV to Horses............................................................................ 112 Conclusions...........................................................................................................................113 APPENDIX A ARBOVIRUS CASE INFORMATION FORM................................................................... 114 B ENCEPHALITIS SURVEY.................................................................................................116 C SURVEY REQUEST LETTER...........................................................................................120

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7 LIST OF REFERENCES.............................................................................................................121 BIOGRAPHICAL SKETCH.......................................................................................................136

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8 LIST OF TABLES Table page 2-1 Outline of information submitted by Arboviral Case Form (ACF) and by retrospec tive mail survey (RMS). Veterinarians submitted data on all horses tested for arboviruses in the state via the ACF............................................................................. 50 2-2 Total number of horses exhibiting signs of encephalitis and test results for WNV from 2001 to 2005 .............................................................................................................. 51 2-4 Gender of horses test ed for WN V from 2001-2004...........................................................51 2-5 Results of logistic regression analysis factors associated with WN V among horses with clinical signs in the state of Florida between 2001 and 2003.................................... 52 2-6 Stable characteristics and farm ecology for horses classified as We st Nile virus diagnosed (WNVD) or negative (WNVN) by th e Florida Department of Agriculture and Consumer Services 2001 to 2003................................................................................ 53 3-1 Blood meals of mosquitoes collected in th e horse-baited stable trap. A subsample ( n = 50) of the total stable trap catch ( n = 525) in 2005 and 2006 was analyzed. (Results of the blood meal analysis from mos quitoes collected outside the stable trap are presented in Chapter 5.)...............................................................................................72 3-2 Mosquitoes collected in the mark-releas e-recaptu re study in the horse-baited stable trap. Red marked mosquitoes were releas ed inside the stall in groups of 100 each date. Green marked mosquitoes were released 5 m outside the st all in groups of 100 each date.............................................................................................................................73 3-3 Comparison of mosquito catch in the horse -baited stable trap a nd an adjacent light trap. Culex sp. include Cx quinquefasciatus (16) Cx. salinarius (3) and Cx. erraticus (10). Anopheles sp. include An. quadrimaculatus (9) and An. crucians (7)...................... 74 3-4 Total number of five mosquito species caught each study year........................................ 75 4-1 Number of arbovirus positive sentinel chicke ns and horses in Alachua County in 2005 and 2006....................................................................................................................91 4-2 Mosquito collections by trap locatio n from October 2004 though October 2005 (13 months). Mosquitoes w ere collect ed from light trap collections...................................... 92 4-3 Mosquito collections by trap location from Nove mber 2005 though November 2006 (13 months) Mosquitoes were collect ed from light trap collections.................................. 92 5-1 Primer sets in PCR used to amplify DNA fro m vertebrate hosts................................... 107

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9 5-2 Identification of blood meals from mosqu itoes collected in Gainesville, FL, October 2004 to November 2006................................................................................................... 108

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10 LIST OF FIGURES Figure page 2-1 Total WNVD horse cases reported in Florida between 2001-2003................................... 55 3-1 Location of the four pair ed light traps and resting boxes ma rked as T1, T2, T3, and T4.......................................................................................................................................69 3-2 Measurements of the stall that held th e horse in 2005. A single stall was modified for the trap in a six-stall barn. ............................................................................................ 70 3-3 Stable trap design 2006. 30 cm lengths of PVC pipe with a 15.2cm diameter cut in half with openings of 1.9cm were placed along two sides of the stall for mosquito entry...................................................................................................................................71 3-4 The species that represented at least 1% or more of the total trap catch between October 2004 and November 2006. ...................................................................................73 3-5 The most abundant mosquito species coll ected at the study site are represented over the two trapping seasons from October 2004 to N ovember 2006..................................... 74 4-1 Location of the four pair ed light traps and resting boxes ma rked as T1, T2, T3, and T4.......................................................................................................................................90 4-2 The species that represented at least 1% or more of the total trap catch between October 2004 and November 2006 ....................................................................................91 4-3 Monthly deviations from normal for rainfall..................................................................... 93 4-4 Comparison by month of the four light trap locations av erage mosquito trap catch. Bars followed by a different lett er are significant at P < 0.05. ..........................................94 4-5 Temporal distribution of the seven most abundant mosquito speci es collected at the University o f Florida Veterinary School (March through December 2005)...................... 94 4-6 Temporal distribution of three Culex mosquito species collected at th e University of Florida Veterinary School (March through December 2005)............................................95 4-7 Temporal distribution of seven most a bundant mosquito species collected at the University of Florida Veterinary School (May through November 2006)........................ 96 4-7 Temporal distribution of three Culex mosquito species collected at th e University of Florida Veterinary School (May through November 2006).............................................. 97

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IDENTIFICATION OF POTENTIAL MOSQUITO VECTORS OF WEST NILE VIRUS TO HORSES IN NORTH CENTRAL FLORIDA By Leslie Michelle Viguers Rios May 2008 Chair: Jonathan Day Major: Entomology and Nematology West Nile virus (family Flaviviridae, genus flavivirus WNV) is of concern in the US and Florida because the virus causes disease in humans and horses. Since 1999, there have been 23,925 clinical human cases of WNV in the United St ates (1999-2007). Preven tion and reduction of cases requires a clear understand ing of the WNV transmission cy cle, but much of the needed information is lacking. It is still unknown wh ich mosquito species transmit WNV to horses. This study integrated field inves tigations with laboratory studies to identify possible mosquito vectors of WNV to horses in north central Florida. The primary objectives of this research were to compare the abundance and seasonality of mos quito species collected near horses, and to characterize host preference of potential vectors. An additional aim was to evaluate extrinsic risk factors of WNV to Florid a horses. The extrinsic factor s of interest included farm management, farm ecology, and the entomological conditions associated with each farm. A questionnaire that focused on potential risk factors was mailed to the owners of all horses tested for arbovirus from 2001 to 2003. Vaccination was the factor most strongly associated with a protective effect for WNV disease outcome in horses. The factors that were associated with an increased risk of WNV in horses were fan use in the stable, mosquito activity, and dead birds on

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12 the property. Blood meal identif ication and virus screening were done in order to determine which mosquito species, if any, were involved in WNV transmission to horses. Mosquitoes were collected for a period of 26 months from a horse research area in north central Florida. DNA was extracted from the abdomen of the blood fed mo squitoes to test for the presence of avian, mammalian, and reptilian blood usin g PCR with different primer sets. The blood meals were confirmed with sequencing. The non-blood-fed mos quitoes were sorted into pools of up to 50 mosquitoes and screened for WNV, SLEV, a nd EEEV by Real-Time quantitative RT-PCR. A total of 45,851 mosquitoes (twenty three species) were collected, 252 of which had visible blood meals. Twelve mosquito species (fifty eight in dividuals) were positive for horse DNA. St. Louis encephalitis virus was detected in one pool of Mansonia titillans collected on September 26, 2006. This study was able to identify several mos quito species feeding on horses and risk factors associated with WNV disease. The vaccine can protect horses against WNV disease if administered two weeks prior to exposure a nd if a booster is administered yearly.

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13 CHAPTER 1 INTRODUCTION AND REVIEW OF THE LITERATURE Introductory Statement In Florida, W est Nile virus (family Flaviviridae genus Flavivirus WNV) continues to threaten the health of humans and dome stic animals. Between 1999 and 2007, 23,925 human cases of WNV disease have occurred in the Unit ed States {2007 1905 /id}. West Nile virus is considered an emerging infectious disease (EID) in the United States (NIAID 2008). An EID is a disease that is newly recognized or a previously known pathogen that has spread in incidence or geographic range (Lederberg et al. 1992). As WNV becomes locally established, recurrent epidemics occurring seasonally in the summer th rough fall will likely continue to occur (Hayes et al. 2005). Florida may be especially suscep tible to WNV epidemics due to the subtropical climate and the endemic status of another closely related arbovirus, St. Louis encephalitis virus (family Flaviviridae genus Flavivirus SLEV). Additionally, Florida plays an important role in the equine industry (FDACS 2008); many breeding hor ses are located within the state and are at risk of infection. The focus of this disser tation was on the ecology of WNV in Florida with respect to mosquito vectors and animal disease. Unlike other states where human infection has and now predominates, animal infection has been the most prominent feature in Florida WNV encroachment. Thus laboratory and field stud ies investigating the ep idemiology of WNV in horses and their potential vector s in a region of high WNV activity during encroachment, north central Florida, can contribute to our overall und erstanding of WNV in Florida. The purpose of the following literature review is to provide a background of relevant WNV literature and present a summary of WNV surveillance and detection in Fl orida. The goal of this dissertation is to strengthen understanding of the WNV vector-host interactions in an area where susceptible hosts (horses) are present. The primary objectives of this research were to document the mosquito

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14 species collected near horses, identify the seas onal distribution of these mosquito species, and characterize their host preferences. The central testable hypothesi s of this work is that microhabitats and weather conditions dictate the lo cal mosquito species present, and some of these species may be capable of transmitting ar boviruses to clinically susceptible hosts. Introduction We st Nile virus is an enveloped, single stra nded, positive sense RNA virus in the family Flaviviridae {ICTVdB Management, 2006 19 /id}. The first isolation of WNV was from a febrile woman in the West Nile District of Uganda in 1937 (Smithburn et al. 1940). Cases of West Nile fever, (WNV infection resulting in fever, headache, and/or rash) have been regularly reported in Africa, West Asia, and the Middle East West Nile virus was recognized as a cause of central nervous system (CNS) infections such as meningitis and encephalitis when a number of people became sick in Israel in 1951 (Work et al. 1955). In horses, summer neurological syndromes were observed since the early 1900s and equine cases caused by WNV were first identified in the early 1960s in France (Murgue et al. 2001). Since its introduction to the United States in 1999, WNV has been a growing public health concern in the western hemisphere {2008 2281 /id}. The strain of WNV introduced into New York in 1999 ( NY99) was fist isolated from an American Crow (Corvus ossifragus Brehm). This strain was sequenced and found to have over 98% homology with a WNV strain isolated from a goose in Israel in 1998 (Brinton 2002). Since its introduction in the United States, WNV has spread throughout the US, Canada, Mexico, Central and South America and the Caribbean (Reisen and Brault 2007) The phylogenetic relationship of WNV strains is broken into two lineages based on amino acid substitutions or deletions in the envelope protein (Brinton 2002). Lineage 1 is associated with all cases of severe disease and it is th e most widespread of the lineages including the

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15 introduced strain to the United St ates (NY99). Lineage 2 is restricted to Africa, and has never been the cause of severe disease or death, but is associated with WN fever (Brinton 2002). Serologically, WNV is most closel y related to flaviviruses in the Japanese encephalitis complex, which includes Japanese encephalitis virus, Murr ay Valley encephalitis, Alfuy virus, and St. Louis encephalitis virus (SLEV) (B rinton 2002). A subtype of WNV, Kunjin virus, is found in Australia and Southeast Asia (Hayes et al. 2005). Transmission Cycle West Nile virus is an arthropod-borne virus (arbovirus) with a natural transmission cycle between m osquito vectors and wild birds that serve as amplification hosts. Mosquitoes in the genus Culex have been widely implicated as prim ary vectors of WNV (Andreadis et al. 2001, Hayes 1988, Nasci et al. 2001b, Trock et al. 2001, Turell et al. 2001). Culex univittatus Theobald is considered the primary vector in Africa and Culex pipiens L in Europe (Hubalek and Halouzka 1999). In Asia Cx. vishnui is the primary vector. The primary North American vectors of WNV are Culex spp. with great regional variation. In the northeast, Cx. pipiens is the primary vector; in the southeast Cx. quinquefasciatus is considered an important vector, and in the west, Cx. tarsalis appears to be the primary vector (Sardelis et al. 2001, Goddard et al. 2002, Kilpatrick et al. 2005). Othe r species may be important de pending on geographic location and environmental conditions (Kilpatrick et al. 2005). In Florida, Cx. nigripalpus is a primary vector for WNV (Rutledge et al. 2003). West Nile virus is zoonotic and is maintain ed in complex life cycl es involving birds as the primary vertebrate amplification host and mos quitoes as the principl e arthropod vector. The transmission cycle of WNV does not affect huma ns or domestic animals until the virus escapes its transmission/amplification focus via either the amplifying vector or other mosquito with epidemic potential (Campbell et al. 2002). Humans and domestic animals can develop clinical

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16 illness but are considered dead end hosts becau se they do not frequently produce sufficient viremia to infect mosquitoes, and therefore, do not contribute to the transm ission cycle (Hayes et al. 2005). A transient viremia was documented in horses experimentally infected in Egypt (Schmidt et al. 1963), and a similar study by B unning et al. (2002) found low level titers (a maximum viremia of 10 3.0 PFU/mL) which were insufficient to infect Aedes albopictus High titers were found in the brain and spinal cord but not in the blood (Bunning et al. 2002). Even if this low viremia results in transmission to a mos quito, a lower infective t iter is correlated with a reduced transmission rate overall (Bunning et al. 2002). Humans and horses are susceptible to infec tion when the virus has become amplified throughout the resident avifauna. Amplifica tion involves a cascade of virus transmission between infected birds and competent mosquito v ectors. If the proper environmental conditions persis, this bird to mosquito to bird amplifica tion cycle can result in a large number of infective mosquitoes. After a period of efficient virus am plification, often late in the summer, there is transmission to the human and horse population (Petersen et al. 2003). Direct human-to-human transmission does not occur, although direct tr ansmission has been documented for birds (Austin et al. 2004, Banet-Noach et al. 2003), and farmed alligators (Jacobson et al. 2005). Horizontal transmission (non-vector) in humans is po ssible through breast m ilk, blood donation, transplacental transmission, and organ transp lant (Hayes and OLeary 2004). An understanding of arboviral transmission cycles begins with the corre ct identification of biologically significant vect ors. West Nile virus has been isolated from 62 mosquito species collected in North America {2007 1 905 /id}. It is unlikely that ma ny of these mosquito species play a significant role in the tran smission of WNV (Hayes et al. 2005). In order to be implicated as a vector, a mosquito must fit the following four criteria. 1) The mosquito must be

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17 physiologically able to replicate th e virus and to infect a nave host (vector competence). 2) The mosquito must survive the extrinsic incubation period, which is the time necessary for the virus to replicate in the mosquito (10 to 20 days for WNV). 3) The mosquito must feed on a susceptible host. And 4) in order to be a bridge vector (one taking the virus from the mosquitobird cycle and transmitting to secondary hosts) it must be an indiscriminate feeder. There has been laboratory confirmation of vector competen ce of several species (Tur ell et al. 2005). In Florida, there are 80 mosquito sp ecies (Darsie and Morri s 2003), and very few meet the criteria of a vector outlined above. West Nile virus undergoes four phases in the yearly cycle of transmission: maintenance, amplification, early transmission, and late transmission (Shaman et al. 2003). Several abiotic factors or non-living com ponents of the environment are considered determinants for viral levels seen in host and vector populations each year. In peninsular Fl orida, the maintenance phase is from January to March, the amplification phase is from April to June, early transmission is from July to September, and late transmission is from October to December (Shaman et al. 2003). During the maintenance phase the virus survives the peninsular Florida dry se ason. It is not clear where the virus is during this phase, but low level transmission between mosquito vectors and susceptible wild birds is probable. Virus may be maintained throughout the winter in overwintering mosquitoes, chroni c infection in birds, and by c ontinued enzootic transmission (Reisen and Brault 2007). Drought conditions li mit the available water and bring mosquito populations into contact with sus ceptible wild birds, thus fac ilitating WNV amplification. Once rainfall increases, infected mosquitoes are able to disperse into new habitats. Infected female mosquitoes then transmit WNV when feeding on a susceptible host. During years when drought

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18 brings mosquito and bird populations into close proximity the amplification phase can be quite intense and early transmission is more often seen (Shaman et al. 2005). Clinical Disease Human The mo st clinically susceptible hosts of WNV appear to be humans, horses and corvids. The symptoms of disease in humans range from subclinical, to encephalitis, coma, or even death (Petersen and Marfin 2002). In 80% of the cases WNV infection is sub-clinical (Mostashari et al. 2001). The remaining 20% of infected people will develop symptoms ranging from mild (headache, body ache, and flu-like symptoms) to severe (s evere headache, stiff neck, convulsions, coma, and death) (B ernard and Kramer 2001, Petersen and Roehrig 2001). The incubation period (time from infection to onset of systems) lasts about 3 to 14 days with symptoms lasting between a few days and a few weeks (Jeha et al. 2003, Mackenzie et al. 2004) West Nile fever refers to less severe cases that are self-limited and often resolve within a week. West Nile encephalitis and West Nile meni ngitis are more severe forms of the disease that affect the nervous system and may persist for over a month (Hayes et al. 2005). Encephalitis refers to an inflammation of the brain and meni ngitis is an inflammation of the membrane around the brain and the spinal cord (M ostashari et al. 2001). The peopl e at highest risk of severe disease outcome are those over 50 years of age, or that are immune compro mised. Less than 1% of all infected persons will develop se vere disease (Hayes et al. 2005). Equine Clinical symptoms in horses and other e quids (ponies, donkeys, m ules) range from asymptomatic to fatal (Ostlund et al. 2001, Porter et al. 2003). Approxima tely 10% of infected horses and other equids develop clinical symptoms. The clini cal signs in horses are most commonly ataxia, weakness, and changes in mental state (Cantile et al. 2000). Early reports

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19 during the U.S. WNV outbreak predicted an incu bation period from 3 to 14 days and symptoms lasting between a few days to a few weeks (Ostlund et al. 2001). In experimental inoculations, horses become viremic between 2 and 5 days after infection and develop c linical disease between 9 and 14 days post inoculation, which is consistent in all methods of inf ection including needle, mosquito and intrathecal routes. The outcome is fatal in 35 to 45% of c linically affected horses (Bunning et al. 2002, Long et al. 2007). In New York a seropositive rate of 29% was documented when asymptomatic stable mates of confirmed horses were tested (Trock et al. 2001). The increased rate of seroprevalence was likely an indication of the increased arboviral activity in the area. Avian Symp toms in birds infected with WNV may range from asymptomatic to fatal (Komar, 2003). Avian mortality in the Old World was re latively uncommon prior to the introduction of WNV to North America in 1999. The neurologic al invasion of WNV in domestic geese (1997), and in a flock of storks (1998) in Israel, are among the few reports of WNV causing death in birds in the Old World (Malki nson et al. 2002, McLean et al. 2002). In North America there have been 198 species of birds reported to be su sceptible to a fatal outcome when infected with WNV (Komar et al. 2003). Corvid s (Passiformes) are especially su sceptible to infection. Signs of infection include lethargy, recumbency, and hemorrhage (Komar 2003). During epizootics, (outbreaks in the bird population) there is a high rate of natural infection in birds (Work et al. 1955, McIntosh and Jupp 1982, Malkinson and Banet 2002, Komar 2003). Multiple tissues are damaged with infection and the cause of deat h is likely multiple organ failure (Komar 2003). Other Vertabrates Experime ntal infections of WNV in other domestic animals have shown that development of viral titer and clin ical signs are relatively rare (Blackburn et al. 1989, McLean et al. 2002.

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20 Sheep that were fed on by WNV infected mosqu itoes did not mount a viremia (McLean et al. 2002). In a seroprevelence study, in eastern Slovakia, WNV antibodie s were detected in 1% of 608 sheep screened (Hubalek and Halouzka 1999, McLean et al. 2002). Calves that were experimentally infected did not produce viremia. In a seroprevalence study in Romania, 4.9% of sheep, 4.1% of cattle, and 12% of goats had HI antibody for WNV (Hubal ek and Halouzka 1999, McLean et al. 2002, Murgue et al. 2002). Dogs that were inocul ated subcutaneously with WNV developed antibody titers and one dog developed a low titer viremia (Blackburn et al. 1989). A survey of dogs in South Africa found 46% of 377 dogs screened had HI antibodies against WNV (Blackburn, 1989, McLean et al. 2002). A water buffalo fed on by infective WNV mosquitoes did not produce detectable viremia, and in a seroprevalence study 72% of water buffalo sampled had neutralizing WNV antibody (McLean et al. 2002). Farmed alligators are susceptible to fatal infection in North America (Jacobson et al. 2005 ) and develop extremely high viral loads in the blood. Lake frogs in Russia are apparently competent reservoirs for WNV (Hubalek and Halouzka 1999, McLean et al. 2002). Epidemiology and Ecology The numbers of human cases, horse cases, and positive surveillance reports were highest in Florida between 2001 and 2003, and since that ti me have decreased. The pattern of WNV dispersal in the United States has usually displa yed a three-year cycle (Reisen and Brault 2007). The entry year is often followed by transmission at epidemic levels (in Florida the highest levels occurred two years after WNV wa s first reported in 2003 with 82 human cases). A decrease in WNV activity is observed after a large epidemic occurs in a new geographic setting (Reisen and Brault 2007). Interestingly, as WNV becomes esta blished in a new geographic focus, reports of SLEV activity in the area decline (Reisen and Brault 2007), although this was not the case in Florida where sentinel chicken seroconversions to SLEV > WNV in 2006. The decline of SLEV

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21 in many areas is most likely due to the pa rtial cross protection against WNV provided by previous exposure to SLEV in both birds and mammals (Tesh et al. 2002, Fang and Reisen 2006). West Nile virus epidemics (just as S LEV epidemics) require a number of complex ecological factors to be in place. West Nile virus may also be subject to epidemiological conditions such as local bird population suscepti bility, rainfall patterns, and mosquito vector dynamics for transmission to occur. Invertebrate Hosts (vector) The enzootic (within anim al) WNV transmi ssion cycle includes an avian reservoir (amplification host) and a mosquito vector. After feeding on an infectious blood meal, WN virions enter the mosquito midgut and infecti on of the midgut epithelium may follow (Brinton 2002). In a competent vector (an arthropod capable of becoming infective), the virus replicates in the cells of the midgut epithelium and subseque ntly is released into the body of the mosquito resulting in a disseminated infection (Scholle et al. 2004). Virus then enters other organs, including the salivary glands, via the hemolymph. After rep lication in the salivary glands transmission to a host by probing or taking a subs equent blood meal can occur (Scholle et al. 2004). Differences in both midgut and salivary gland infection and escape barriers may explain variations in mosquito vector competence. Additional biological routes of in fection include transovarial (e ntry of virus into mosquito eggs during oviposition) and venere al (female to male) transmissi on. Mid-winter isolations of WNV from overwintering Culex mosquitoes demonstrates the pot ential of the virus to persist until spring and emerge with mosquitoes to reesta blish an enzootic transmission cycle in the area (Nasci et al. 2001a). Vertical transmission may contribute to maintenance of WNV (Miller et al. 2000). Mid-winter isolations of WNV are most likely from a mosquito undergoing diapause (hibernation physiology and behavi or) and because normally the fe male does not first blood feed,

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22 it can be reasonably assumed a mid-winter infection of WNV was ac quired transovarially (Komar 2003). Alternatively, Culex infected by feeding on a vire mic vertebrate host may have survived the winter. Transova rial transmission of WNV and preservation of the virus in hibernating mosquitoes are not thou ght to play an important role in the maintenance of the virus in nature, but the potential of alternative routes of transmission such as vertical transmission do exist. The most important mosquito genus in terms of WNV transmission is Culex. The majority of WNV field isolati ons have been from Culex mosquitoes, and in field studies, Culex spp. have in repeated investigations, the highest minimum infec tion rates (MIR) relative to other mosquito species (Nasci et al. 2002, Kilpatrick et al. 2005). Because of the midgut barrier, Culex mosquitoes do not have the highest vectoral capacity ( physiological ability to transmit the virus) as compared to container breeding Aedes spp. and Ochleratatus spp. Despite a lower vectoral capacity, other factors such as mosquito dens ity, biting preference and seasonal activity makes Culex species the most important mosquito ge nus in WNV transmission (Nasci et al. 2002, Kilpatrick et al. 2005). Finally, each mosquito species may demonstrate a range in vectoral capacity because ambient temperature, infec tive dose (from blood), a nd length of extrinsic incubation period influence the efficiency of a vector under fi eld conditions. Several Culex species are involved in the transmi ssion cycle of WNV an d preferentially feed on birds thereby amplifying the virus in avian populations. Ornithiphilic (avian-feeding) species such as Culex nigripalpus, Culex pipiens, Culex quinquefasciatus, and Culex tarsalis are considered maintenance and amplification vector s of WNV (Turell et al. 2005). Host-shifting behavior (a host preference switch from birds in the spring to mammals in the fall) seen in Cx. nigripalpus, Cx. tarsalis, and Cx. quinquefasciatus may drive WNV transmission to human and

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23 horse populations late in summer and early fall by brining the virus from its point of focal transmission out to exposed hosts (Kilpatrick et al. 2005). This occurs when mosquitoes first feed on an avian host and become infected. Af ter completing the extrinsic incubation period the mosquito may transfer the virus at a subse quent blood meal by probing a susceptible mammalian host. An infective mosquito can deliver approximately 104.3 plaque forming units (PFU)/mL of WN virus to a host, with a range of viral titer (amount of vi rus in the salivary glands) among individual mosquitoes and species (Vanlandingham et al. 2004). Aedes albopictus is a competent vector of WNV. Aedes albopictus experimentally infected with WNV developed titers between 106.6 to 107.9 PFU per mosquito. When subsequently fed on horses, this titer was sufficient to infect the majority of horses, wh ich developed a low-level viremia ranging from 101.0 102.7 PFU/mL (Bunning et al. 2002) None of the horses ( n = 12) were able to re-infect mosquitoes. The minimum host viremia capable of infecting a mosquito vector varies by mosquito species, but the relationship between susceptibility to WNV infection is dose dependent and approaches 0 below 104.0 PFU/mL (Reisen et al. 2005). Serum titers below 104.3 PFU/mL are not capable of infec ting most mosquito species when feeding on rabbits with lowlevel viremia (Tiawsirisup et. al 2005). Culex tarsalis is considered one of the most efficient vectors and when fed on a blood meal containing 104.9 PFU/mL the number of mosquitoes that became infected ranged between 0%-36% (Hayes et al. 2005). Concentrations of 107.1 PFU/mL are required before 74%-100% of Cx. tarsalis become infected when fed on an infectious blood meal (Hayes et al. 2005). The threshold necessary to infect Cx. pipienes and Cx. quinquefasciatus is 105.0 PFU/mL (Allison et al. 2004). It is possible that hosts that maintain a low viremia (below the threshold) for an extended amount of time may encounter many

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24 mosquitoes and successfully infect a small number of them (Lord et al. 2006). These instances could be considered as secondary routes of transmission in the WNV cycle. Vertebrate Hosts (Reservoir/Amplification Host) The most i mportant amplification hosts of WNV are avian. Laboratory studies have shown that member of the orders Passeriform es (song birds), Charadriiformes (shorebirds), Strigiformes (owls), and Flaconiformes (hawks) develop blood virus levels sufficient to infect most feeding mosquitoes (Komar 2003, Komar et al. 2003). Passerines, such as common grackles (Quiscalus quiscula ), house finches (Carpodacus mexicanus ), and house sparrows ( Passer domesticus) are capable of infecting many mosquitoes (Komar 2003). Serosurveys have demonstrated that house sparrows are frequently infected with WNV (up to 60%), may develop a high viral titer of sufficient dur ation and magnitude to infect vector mosquitoes, and are abundant (Komar et al. 2001, Komar et al. 2003, G odsey et al. 2005). These attributes allow house sparrows to potentially serve as important amplifying hosts. Crows may experience up to 100% mortality in some outbreaks (Komar et al. 2001) and their rapid fatality likely limits their reservoir potential. Some resident birds were found to have seroprevelen ce rates of 20 to 50% in the epicenter of WNV outbr eaks (Komar et al. 2001) wh ich in migratory birds the seroprevelence was 0.8% (McLean et al. 2002). The importance of migratory birds in dispersing WNV remains uncertain, but it has been suggest ed that movement of resident birds, nonmigratory birds, and migratory birds may contribute to the spread of WNV (Reed et al. 2003, Petersen et al. 2003). Field observations of direct bi rd-to-bird transmission have not been made, but laboratory tests confirm this probability. Infected birds caged with uninfected birds are able to spread WNV (McLean et al. 2002); the mode of transmi ssion is likely low-level viral shedding per os (oral) or per cloaca (cloacal). Oral transmission in crows and geese has been documented by

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25 ingestion of infected water, mosquitoes, or carrion (Langevin et al. 2001, McLean et al. 2002, Banet-Noach et al. 2003). Although WNV has been isolated from some mammals, and there have been occasional reports of mammals spiking sufficient viremia to infect mosquitoes, in general mammals are commonly considered dead-end hosts because they do not usually spike a sufficient viremia to infect a feeding mosquito and thereby do not cont ribute to the continua tion of the virus cycle (Hayes 1988, Bunning et al. 2002). Rabbits were found to be capable of infecting various mosquito species and developing a short-lived viremia of up to 105.8 PFU/mL (Tiawsirisup et al. 2005). Other mammals experimentally infected su ch as horses, cats, dogs, and mice rarely exhibit titers above 104.0 PFU/mL whereas birds such as passerines can exceed 106.0 PFU/mL for a few days (Tiawsirisup et al. 2005). Corvids ar e the most susceptible to infection producing high viremia of over 1010.0 PFU/mL (Reisen et al. 2005). The corvids usually are moribund (approaching death) after 5-6 days postinoculation. Blood virus levels in naturally infected rock pigeons ranged from 102.3 PFU/mL 107.2 PFU/mL (Allison et al. 2004). Chickens remain valuable in sentinel programs because, even though chicks can develop a substantial viremia, the average adult viremia is <104.0 PFU/mL, which is insufficient to infect most mosquitoes (Langevin et al. 2001). Human In humans, patients develop an average virem ia of 0.1 PFU/mL (ranging from 0.06-0.50 PFU/mL) (Montgomery et al. 2006). Blood screened from donors in the US in 2002 had a maximum titer of 103.2 PFU/mL (Hayes et al. 2005). This level is safely below the viremia required to infect most efficient vectors. Th erefore, humans do not likely contribute to the WNV transmission cycle and can be considered dead-e nd hosts. Despite findings that some children in

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26 Israel spiked viremias sufficient to infect mosquitoes, humans are still considered dead-end hosts (Hayes and OLeary 2004). Seroprevelence of WNV in a 1999 New York st udy (Mostashari et al. 2001) was 1 in 150 infections resulted in meningitis or encepha litis. A 2000 study in New York again found a similar result (CDC 2001). The re sults of the two sero surveys were consistent with a previous study in Romania (1996) indicating that 1 in 140 to 320 infections led to these clinical outcomes (Tsai et al. 1998). The case fatality rate in the US in 2002 for human cases of WNV with meningitis was 2%, and the case fatality rate for those with encephalitis was 12% (O'Leary et al. 2004). Surveillance and Detection of West Nile Virus Florida ha s had an arthropod borne virus (ar bovirus) surveillance program in place since 1977 to track the amplification and transmission of mosquitoborne viruses including eastern equine encephalitis virus (EEEV) and St Louis encephalitis virus (SLEV) (Day and Stark 1996). The Florida state department of health (DOH), di vision of environmental health, coordinates the surveillance program. The Interagency Arboviru s Surveillance Network reports to the DOH and is composed of several local, state and federal agencies, which are involved with the surveillance and control of arboviral diseases. Upon its arrival in the United States, WNV wa s easily added to the existing surveillance program with the addition of WNV-specific laboratory diagnos tics. Because SLEV and WNV are antigenically related, crossreactions are observed with some serologic tests and so plaque reduction neutralization testing (P RNT) is done to distinguish th e two viruses. Due to the correlation of WNV-positive dead bird reporti ng and local WNV transmission, dead bird reporting has become a valuable surveillance tool in the United States (Eidson et al. 2001a, Eidson et al. 2001b, Na sci et al. 2002).

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27 Horses have been found positive for IgM antibody 8 to 10 days after infection with WNV. The IgM antibodies may persist for 2 to 3 mont hs, most horses only have antibody for 3-4 weeks which makes this test ideal for detection of recent infection. West Nile virus neutralizing antibodies can persist for years after infection (Durand et al. 2002). Horses are not currently used as part of an active WNV surveillance program in the United States, but data is collected passively on all confirmed horse cases in the U.S. by the CDC. In New York State horse positives were unreliable in the prediction of human cases of WNV (Trock et al. 2001). It is not yet known whether WNV surveillance in horses can predict human cases in Florida, but horse cases that are reported to local h ealth departments are used as pa rt of arbovirus surveillance. Blackmore et al. (2003) reported that the epicenter of the 2001 WNV outbreak in Florida horses was in Jefferson County. From Jefferson County, th e outbreak spread east, west, and south to a total of 40 Florida counties with confirmed horse cases. In the counties reporting both horse and human cases, the horse cases preceded the human cases by one to four weeks (Blackmore et al. 2003). Because horse cases generally precede huma n arboviral infections, lo cal identification of horse cases is an important part of the passive surveillance network in Florida and the U.S. Diagnostics Routine arbovirus surv eillance me thods includ e screening of mosquito pools by viral isolation, or by antigen detection. Viral isolation is typically carried out in the cell line C6/36 ( Aedes albopictus) or in Vero cells. Cell culture proce dures detect live virus in the sample. Enzyme Linked Immunosorbent Assay (ELISA) can detect viral antigen in mosquito pools, avian tissues, and human tissues. Frequently, vira l nucleic acid detection methods such as real time quantitative RT-PCR are used by local hea lth departments for screening of mosquito pools {Stark, 2006 20 /id}. The VecTest (Medical Analysis Systems, Camarillo, CA) is a rapid immunochromatographic test developed for the det ection of viral antigen. The VecTest can be

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28 used directly in a mosquito pool homogenate eliminating the need for lengthy laboratory preparation. Vero cell culture and RT-PCR were used to c onfirm the index case (initial case identified in an outbreak) in the United States (Huang et al. 2002). When cell culture is used for WNV isolation, it is usually in conjunc tion with RT-PCR because the latter is more sensitive. Using RT-PCR, one infected mosquito can be detect ed in a pool of 50 mosquitoes (a standard procedure for surveillance of mosquito populatio ns) because the limit of detection in RT-PCR is 40 RNA copies (Shi et al. 2001). This sensitiv ity is more than adequate for WNV screening since a mosquito capable of transmitting virus contains more than 105 PFU of virus (Hadfield et al. 2001). Lanciotti and Kerst (2001) found that nucleic acid amplification assays were far more sensitive for screening mosquito pools than re lying on cell culture alone. The use of RT-PCR increased the detection of virus and significan tly decreased the amount diagnostic laboratory time (Lanciotti et al. 2000, Lanciotti and Kerst 2001). Diagnostic methods for WNV detection in hor ses changed after the introduction of a vaccine. In 1999, the primary diagnostic tool was the plaque-reduction neutralization test (PRNT) of equine serum for confirmation of WN V infection and virus is olation from equine brain or spinal cord tis sue (Ostlund et al. 2001). To update the diagnostic tools available the (Ig)M-capture enzyme-linked im munosorbent assay (MAC-ELISA) was developed (Ostlund et al. 2001). The assay was modeled after the EEEV MAC-ELISA. Upon experimental challenge in horses, Immunoglobulin (Ig) M isotype anti-WNV antibodies become detectable 8-10 days after infection and persist up to two months (Ostlund et al. 2001). This test has a sensitivity and specificity of 91.2% and 99.7%, respectively, for confirming recent infection in equids with

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29 encephalitis (Long et al. 2006). We st Nile virus neutralizing anti bodies may be detectable in horse sera for years after inf ection (Durand et al. 2002). Neutralizing antibodies to WN V may persist for more than two years following infection. Neutralizing antibodies can also be passed from mare to foal via the colostrum (milk). Due to the properties of neutralizing an tibodies, the MAC-ELISA is an important diagnostic tool to identify recently infected horses in areas wher e previous infection has occurred because the IgM antibody response wanes more rapidly than neutra lizing antibodies to WNV. Additionally, the MAC-ELISA is capable of producing reliable resu lts even in vaccinated horses (Porter et al. 2003). The ELISA detects antibodies to WNV and can indicate if the horse had been exposed even without clinical symptoms. Risk Factors Because arboviruses are maintained in com p lex cycles of avian hosts (reservoirs) and mosquito vectors, a number of factors must be in place for epidemic transmission to occur. Abiotic factors greatly affect the year-to-year transmission patterns observed by facilitating the interactions of infective mosquitoes and susceptible hosts. Weather influences WNV transmission by affecting the distribution and ab undance of mosquito ve ctors and the time of extrinsic incubation period (Reiter 1988). There is abundant re search on the predictive factors to human arboviral outbreaks and the most reliable indi cators are rainfall patterns, sentinel chicken conversions, and a large juveni le bird population (Ruiz et al. 2004, Day and Lewis 1991). The single most important risk factor to human transmission is an abundant infective mosquito population (Komar 2003). Prediction of Human Cases The principle reason for the ac tive surveillance of arboviruses is to pr otect the public. Various surveillance tools includi ng mosquito collection, dead bi rd testing, sentinel chickens,

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30 and horse cases (Blackmore et al. 2003) are used to monitor arboviral amplification and transmission. Together these surveillance techniqu es are used to make public health decisions, by comparing arboviral activity to baseline historical data, in order to pr otect the public against an outbreak of a particular arbovirus (FDOH 2007). A public health advisory (by radio, television and print) can be re leased that advises people to stay indoors during hours of heavy mosquito activity, reduce exposure to mosquitoes and take preventative measures. Preventative measures include wearing protective clothing, and using chemical repellants. When necessary, public health measures can be implemented as was done in Florida during the 1990 outbreak caused by SLEV (Day 2001). All f actors including human cases are part of the assessment risk resulting in a health advisor y. Reliance only on human diseas e results in dissemination of information after an epidemic is often well unde rway. Reliable prediction assists policy and regulatory officials focus control efforts that reduce the possibil ity of human cases before any disease occurs. In Florida, animal, mosquito, and chicken seroconversion data is compiled weekly and released by the Florida Department of Health. In addition re presentatives from the Arbovirus Interagency Task Force discusses ad ditional options for media release and public health advisories relating to the weekly data. Blood Meal Analysis Knowledge of mo squito host feed ing patterns provides insight to viral transmission cycles through investigations of the role of a vector mosquito in enzootic transmission among avian hosts or epidemic transmission outside of this cycle to mammalia n hosts. Techniques in blood meal analysis have been changing since the ea rly 1920s and have included direct observation of feeding mosquitoes, host-baited trap catches, an d serological and genetic based techniques (Ngo and Kramer 2003). The most common serological and genetic based techniques have been the precipitin test, the Enzyme Linked ImmunoSor bent Assay (ELISA) and Polymerase Chain

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31 Reaction (PCR) assays (Ngo and Kramer 2003). Polymerase Chain Reaction amplification of host DNA followed by sequencing is becoming a common method for blood meal detection and has several advantages over precip itin tests and ELISA. Due to the sensitivity of the PCR, a very small amount of DNA can be used as template so even partially engorged mosquitoes can yield a blood meal confirmation. Additionally, with serol ogical tests such as pr ecipitin and ELISA, antisera must be prepared for each potential host species allowing blood meal confirmation to only a limited number of species. With the advent of web-based databases such as GenBank, it is now possible to compare nucleotide sequences and determine the exact identity arthropod blood meals. In precipitin tests, the bl ood meal suspension is mixed w ith the antiserum of various vertebrates and if a reaction is observed then a precipitate will form and the blood meal is considered positive for that host type (Tempe lis 1989). The ELISA uses a species-specific antibody that will react with the blood meal and result in a color change signally that binding of the antibody has occurred. These techniques can identify blood meals to general groups of animals such as avian vs. mammal and within mammalian hosts, human, cow, horse, etc. Genetic methods allow for species-specific identi fication for birds to the species level. Using restriction fragment length polymorphism (RFLP) an alysis, Kirstein and Gray (1996) were able to identify genera level mammalian blood meals from Ixodidus ticks. Heteroduplex analysis (HDA) helped classify mosquito-feeding patterns in the Tennessee valley area to the species level (Lee et al. 2002a). Sequencing blood meal polymerase chain reaction (PCR) product is another way of determining to the source of th e blood meal to the species level. The most common technique today relies on the genetic char acterization of the blo od meal to determine the host. Typically primers in the cytochrome b genome are used to amplify a vertebrate-specific

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32 region from the blood meal DNA. The product of the PCR is sequenced and then matched with known published sequences in the BLAST databa se of GenBank (NCBI 2008). Host DNA can be detected for up to 72 hours af ter the mosquito takes the blo od meal (Ngo and Kramer 2003). Many blood meal analysis studies focus on av ian hosts for purposes of understanding the commonly fed upon reservoirs in the WNV tr ansmission cycle (Lee et al. 2002a, Ngo and Kramer 2003). Others have done work on avian and mammalian hosts (Apperson et al. 2002, Molaei et al. 2006). Cupp et al (2004) studied the potential role of reptiles in the WNV transmission cycle by analyzing blood fed mo squitoes that fed on reptilian hosts. The collection of blood fed mosquitoes is of ten done by the method of vacuum aspiration and the use of resting boxes (Edman 1971, Edman 1979). Baited CDC light traps generally attract host seeking mosquitoes but may collect females that are partially engorged, fully engorged, and gravid. Aspirati on collections are made in vegetation, natural or man-made structures, and in likely resting habitats such as around tree roots or from resting boxes. The vacuum sucks the mosquitoes into a collection cont ainer with a screen to hold them in until they are transferred to another container (Holck and Meek 1991). In Florida, vacuum aspiration has been used to collect mo squitoes in the genera Culex, Aedes, Anopheles, Coquillettidia, Mansonia, and Psorophora (Niebylski et al. 1994) Day and Curtis 1993, (Edman 1971, Edman 1979). The CDC light traps generally use white li ght, although alternate colors may be used to increase catch and the traps are frequently baited with CO2 to attract host seeking mosquitoes (Service 1976). As mosquitoes approach the trap, a fan-generate d air current pulls them into a collection bag (Sudia and Chamberlain 1988). In candescent lights have been shown to be attractive to Uranotaenia sapphirina (Osten Saken), Anopheles crucians (Wiedemann), Aedes vexans (Meigen), Anopheles quadrimaculatus Say, Cx. nigripalpus, and Culex in the subgenus

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33 Melanoconion (Love and Smith 1957, Burkett et al 1998). To maximize catch, the optimal time to run the trap is coincident with maximum flight activity, the cre puscular period (dusk and dawn) (Bidlingmayer 1967). Resting boxes are designed to mimic a natura l resting habitat (Edman et al. 1968). They often attract blood-engorged females seeking a dark resting place to digest the blood meal. Mosquitoes most often enter in the morning and may leave during the day as the temperatures rise (Edman et al. 1968). Many critical questions regard ing risk factors of WNV transmission to horses exist. Although an efficacious vaccine is available, horse cases continue to occur annually throughout the nation. The study of extrinsic risk factors to horses will help horse owners better understand the environmental risk factors and farm management practices associated with an increased risk of WNV transmission. This know ledge can help broaden our unde rstanding of the epidemiology and ecology of WNV in Florida. Additionally, a full understanding of vector host interactions is still incomplete. The work presented here helps identify the mosquito speci es feeding on horses. Knowledge of blood feeding habits can be used along with vector competence studies and mosquito life history studies, to incriminate potential mosquito vectors of WNV to horses in Florida.

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34 CHAPTER 2 EXTRINSIC RISK FACTORS ASSOCIATED WI TH WEST NILE VIRUS INFECTION IN FLORIDA HORSES Since its introduction to the United Stat es in 1999, West Nile virus (family Flaviviridae genus Flavivirus, WNV) has been a growing public hea lth concern. West Nile virus is a zoonotic (naturally transmitted between vertebra te animals and humans) arthropod-borne virus (arbovirus). West Nile virus is maintained in a complex life cycle involv ing a primary vertebrate host (passerine birds) and a primary arthropod vector ( Culex mosquitoes). Susceptible wild birds and vector mosquitoes amplify WN V in foci where mosquito and bird populations are sympatric. Culex mosquitoes have been widely implicated as the primary vector of WNV (Andreadis et al. 2001, Hayes 1988, Nasci et al. 2001b, Trock et al. 2001, Turell et al. 2001). The natural cycle of WNV does not affect humans or domestic animal s unless the virus escapes its amplification focus. This occurs when infective mosquitoes disperse from amplification foci and bite a susceptible secondary (horses or humans) host (Campbell et al. 2002, Petersen et al. 2003. Humans and horses can develop clin ical illness, but are considered dead end hosts because they do not produce sufficient viremia to infect mosqu itoes, and therefore, do not contribute to the amplification cycle by infecting additional vector mosquitoes (Bunning et al. 2002, Hayes et al. 2005). Direct (non-vector) transmission has be en documented for birds (Austin et al. 2004, Banet-Noach et al. 2003) and farmed alligators (Jacobson et al. 2005). Direct human-to-human transmission is limited to inf ection through blood transfusion, breast milk, and organ transplant (Hayes et al. 2005). In Florida, WNV continues to threaten the health of humans and horses. Between 2001 and 2006, there were a total of 1,082 WNV horse cases reported in the state {2007 1906 /id}. Florida plays an important role in the equine industry; many breeding hors es are located within the state and are at risk of infection even with the availability of three commercially licensed

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35 vaccines. Despite the availability of a vaccine protecting horses against infection with eastern equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, EEEV), horse cases are reported regularly. As WNV becomes established, recurrent epidemics and epizootics will most likely occur (Komar 2003). Alt hough Florida has not yet experi enced a major human epidemic of WNV, the endemic and epidemic pres ence of St. Louis encephalitis (family Flaviviridae genus Flavivirus, SLEV), which shares an epidemiology si milar to that of W NV, suggests that the necessary ecological variables are present in Florida to support future epidemics caused by WNV. The annual occurrence of WNV in fection in horses corresponds with the cycling of the virus in amplification hosts and mosquito vect ors in enzootic habita ts {2003 645 /id}. Human and equine cases of WNV peak in Florida in la te summer and decline af ter November. Several extrinsic factors such as rainfall (Shaman et al. 2005), avian population dynamics (Ward et al. 2006), and temperature (Dohm et al. 2002) have been correlated with WNV outbreaks in humans and horses. However, there are few data regarding the extrinsic factors related to the ecology of horse farms and the risks associated with farm management practices and disease manifestation in horses. Retrospective studies have been perf ormed that examine clinical disease associated with infection, treatments, and outcomes {S alazar, 2004 342 /id} {Schuler, 2004 270 /id} Epp et al. 2007). This study is an analysis of extrinsic risk f actors that were collected for horses tested for arboviral infection in Florida between 2001 and 2003. This particular study focuses not only on clinical signs but also on the ecology of horse farms where WNV cases were reported. Given that year round management of horses reflects the subtropical Florida climate, there are likely unique factors that intersect with horse husbandry that create risk for horses. Since WNV is a

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36 reportable disease in humans and horses in Flor ida, all veterinarians are required to submit an arbovirus case information form (ACF) to the Fl orida Department of Agriculture and Consumer Services (FDACS). This highly de tailed form allowed for the development of a database and the opportunity for a follow-up survey of horse owners. The primary objective of this research was to identify factors contributing to the total WNV equine cases from 2001 to 2003 in Florida. The factors of interest in this study were farm management, farm ecology, and the entomological conditions associated with each farm. The central hypothesis, that risk factors for WNV transmission in horses are related to the availability of mosquito larval habitat, animal housing c onditions, and animal management practices was investigated. Materials and Methods Arbovirus Case Information The arbovirus case information for m (ACF) provided specific information about each horse tested including stable location, signalment (clinical si gns and symptoms), individual history (age, sex, breed), date of onset of clinical signs, and date of testing. Space was provided on the form to note any other clinical signs and a brief history of clinical presentation of the horse (Appendix A). The FDACS provided copies of the ACF information to the Emerging Diseases and Arbovirus Research and Test Progr am (EDART) at the University Of Florida College Of Veterinary Medicine in Gainesville, Florida for data entry and analysis for all horses tested in Florida from 2001 to 2005. All data were entered into a database (Microsoft Excel and Access, Microsoft Corporation, Redmond, Washington) and coded fo r statistical analysis. Retrospective Survey Information was taken from the ACF to create a follow-up survey (Appendix B), which was mailed to the owners of a ll horses that were tested for arboviruses from 2001to 2003. A

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37 50% return of the questionnaire was the targeted response rate The questionnaire focused on horse husbandry, farm management, and farm ecol ogy (Table 2-1). Respondents were asked to check the most appropriate category in response to each question. A cover letter describing the objectives of the study and a promise of anonymity to participants accompanied the questionnaire (Appendix C). Reminder postcards were sent two and four weeks following the initial mailing. A second mailing was sent to th e owners who did not respond to the initial survey within two months of the first mailing. Two reminder postcards were mailed out two and four weeks following the second mailing. Case Definition Positive Horses. A confirmed horse case was defined as manifestation of WNV clinical signs (Appendix A) and one or more of the follo wing: isolation of WNV from tissue, blood, or CSF; detection of a positive IgM antibody to WNV by MAC-ELISA in a single serum test, and in the first year (2001) of W NV encroachment, a four-fold rise intheWNV plaque reduction neutralization test (PRNT). After 2001, it was presumed that vaccinated horses would have neutralizing antibody precluding the usefulness of the PRNT. All confirmed positive horse cases in the state of Florida from 2001-2003 ( n = 534) were analyzed as the positive group in the study of WNV risk factors associated with the farm environment. Negative Horses. A WNV-negative horse failed to meet the above criteria for WNV infection based on serological te sting and/or post-mortem analysis. All horses that tested negative for WNV in the state of Florida from 2001-2003 ( n = 402) were analyzed as the comparison control group in th e study of WNV risk factors associated with the farm environment.

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38 Statistical Analysis Responses to survey questions were categoric al and statistical analysis for independence was performed with bivariate analysis (SPSS v 15, Ch icago, IL). Fishers exact test was used on variables containing fewer than five responses in a contingenc y table cell. A Chi-square ( 2) test was used for analysis of independence between no minal variables that consisted of two or more categories and contained variables with greater than five responses per contingency table cell. Odds ratios were calculated for the dichotom ous variables that were significant with 2 statistics or logistic regression (P < 0.05) in the survey. A cross-table was used to stratify variables and create a contingency table to compare the relations hips between variables. Stratified analysis was used to compare WNV test outcomes with th e time since last vaccin ation and the frequency of vaccination separately. A logistic regre ssion analysis was perfor med using WNV disease status as the outcome (dependent) variable. Th e data that were included in the regression analysis were from the combined results of the survey and the ACF. Observational (independent) variables tested in cluded sex, vaccination status (those positively indicated on the ACF, all unknowns were treated as missing data points), and each environmental variable. The logistic regression analysis presented he re was adjusted by controlling for vaccination status to clearly identify the risk/protective eff ects of the environmental va riables associated with the individual farm (Table 2-5). These data we re not as powerful when separated by year since the separation reduced the sample size and subs equently increased the st andard deviation. For all the variables examined (arbovirus prevention stable characteristics, and farm ecology) the years were combined to k eep sample size robust. All statistical analyses were performed with commercially available software (Minitab v 14, State College, Pennsylvania; EPI-Calc v 1.02, Brixton Books, Brixton, UK; SPSS v 15, Chicago, IL). Multivariate analyses were perfor med for extrinsic factors using cross-tables and

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39 logistic regression models. The presence of clin ical WNV symptoms was the dependent variable for each statistical test. Cross-tables were used to compare two contingency tables and stratify the results to compare two variables such as vaccination status and disease outcome. Results The Florida Departme nt of Agriculture and Consumer Services (FDACS) compiles information provided by veterinarians in the stat e of Florida on every horse tested for viral encephalitis. The veterinarians use an arboviral case information form (ACF) at the time of testing a symptomatic horse (Table 2-1). Between 2001 and 2005 there were 2,824 horses that were classified as either WNV diagnosed (WNVD, n = 1,386 (49%)) or WN V negative (WNVN, n = 1,438) based on clinical symptoms and serologi cal testing (Table 2-2) The retrospective mail survey was sent to all owners of horses tested from 2001 to 2003 ( n = 2,501) of which 936 (37%) were completed and returned for 534 positive horses and 402 negative horses. Farm and Sample Submission Information West Nile virus positive horses were reported in 55 of 67 counties in Florida from 2001 to 2003. Cases began in th e summer of each year (2001 to 2003) and peaked in the fall (September) followed by a sharp decline in the winter (Figure 2-1). Each year the cases were seasonal except in 2002, when cases were reported throughout the year. There was a significant (P = 0.045) difference of age structure of positive horses between 2001 and the other study years (2002 to 2004). In 2001, young and old horses were affected, whereas in 2002-2004, horses between 1 and 5 year s of age were most commonly reported as WNV-positive. In 2001, seven (1.2%) WNV-positiv e horses were < 1 year old, 324 (56%) were between 1 and 5 years old, 99 (17.8%) were betw een 5-10 years old, and 145 (25%) were greater than 10 years of age. From 2002 to 2004, 36 horses (9.6%) were < 1 years old, 135 (35%) were

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40 between 1 and 5 years old, 181 (47.3%) were between 5-10 years old, and 31 (8.1%) were greater than 10 years old (Table 2-3). Quarter horses were the most common breed tested in both the WNVD and the WNVN groups, and there were no signifi cant differences in breed repres entation in either group. The frequency of WNV testing in most other breeds was similar between groups. There were 626 (45%) females, 680 (48%) geldings, and 93 (7%) stallions that were WNVD. This sex distribution was significantly different (P < 0.001) from th e WNVN horses where 510 (47%) females, 388 (35%) geldings, and 200 (18%) stallions were tested (Table 2-4). The male to female ratio was about equal in both groups, but significantly more geld ings were diagnosed positive for WNV. Arbovirus Infection Prevention The prevention of WNV infecti on in Florida horses include d vaccination, use of insect repellents, and barrier protection with fly sheet s (a protective covering secured to the horse) (Table 2-1). Insecticides containing permethrin were the most commonly used products for the protection of horses from biting arthropods. Th ere was no statistical difference between the WMVD and WNVN groups regarding the use of perm ethrin products. Frequency of use of all insect repellents (spray or lotion) was similar between the two analyzed groups. Fly sheets were not frequently used in either group. Eighty percent of the tested horses had a known vaccination history. Between 2001 and 2003, 1,368 horses were WNVD and 1,184 we re WNVN. Forty two percent of all tested horses received a vaccine for WNV prior to the onset of illness. To obtain more detailed information from the owners they were asked if the horse was vaccinated and how many times the horse had been vaccinated each year.

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41 In 2001 there was an increased association of WNVD horses with vaccin ation (Table 2-5). In 2002 and 2003 the vaccine showed a protective e ffect. The data were stratified by the number of times the horse was vaccinated and by the time since last vaccination (within 2 weeks, under 6 months, between 6-12 months, or >12 months) in order to more closely examine the relationship between vaccination and disease. Eighty-five (9.6%) of the WNVD horses were vaccinated two weeks prior to the onset of illness, 297 (34%) we re vaccinated between two weeks and 6 months, 47 (5.3%) were vaccinated between 6-12 mo, and 16 (1.8%) were vaccinated >12 months prior to onset of illness. Horses were not protected from infection if the vaccine was received within two weeks of WN disease onset or if the vaccine was received over one year prior to WN disease onset. For the group of horses that were vaccinated in the time fr ame of more than two weeks to under one year prior to the onset of WN disease, the vaccine did decreas e the incidence of WNV (Table 2-5). Owners were asked to indicate the number of times each horse received a WNV vaccine and were given a choice of one to four times. The average response for the combined years of 2001 through 2003 was once (25%), twice (55%), thre e times (15%), and 4 times (4%). Horses that received only one dose of vaccine did not ha ve protection to WNV. A protective association was seen for horses that were given two or more doses of vaccine (Table 2-5). Because the timing of vaccine administrati on was closely associated with protection against WNV infection, vaccination data were sorted into two groups: effective and noneffective vaccine doses received. The effective group was consider ed to be the group of horses that received the vaccine more than two weeks and less than si x months before the onset of illness. When the group of horses that received the vaccine in this time frame was compared to their vaccination status, a strong protective eff ect of the vaccine was seen (Table 2-5).

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42 Stable Characteristics and Farm Ecology The construction ma terial of a stable was si gnificantly (P =0.045) associated with WNV infection. For the WNVD group, stables were made of solid wood and cement for 158 (29%) horses, boards with openings for 146 (27%), and an open shed for 73 (13%). The WNVN group had 131 (33%) stables made of solid wood and cement, 79 (20%) made of boards with openings, and 60 (15%) in an open shed (Table 2-6). The logistic model did not indicate a significant correlation with type of stable material a nd incidence of WNV infection. There was no significant difference if the horse was kept in a pasture versus a paddock. Additionally no significant difference of WN disease incidence wa s seen for the frequency of stall cleaning. The presence of fans in a stall (P = 0.04) and the frequency of fan use (P = 0.05) were significantly correlated with WN disease incidence in horses. In the WNVD group 184 (34%) stables had fans, 70 (13%) used the fans all da y, 103 (19%) used fans only when necessary, and 358 (66%) did not have fans. In the WNVN gr oup 111 (28%) stables had fans, 54 (13%) used the fans all day, 49 (12%) used fans only when necessary, and 290 (72%) did not have fans. Fans were significantly correlated with WNV in the logistic regression (Table 2-5). The presence of fans increased the risk of WNV by 80%. The duration of use (only when necessary or all the time) was not significant in the logistic regression. Mosquito larval habitats are associated with standing water, thus th e retrospective survey attempted assesed the types of water that we re present on the propert y to determine their significance in relation to WNV stat us in horses (Table 2-6). No association was detected for the type of water source or the pres ence of temporary or permanent wa ter bodies on the farm in the X2 analysis. In the logistic re gression model, natural water on the property has a protective effect of reducing WNV by half (Tab le 2-5). The type of water asso ciated with risk could not be determined because respondents were allowed to mark multiple answers on the questionnaire.

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43 Debris and tree canopy can also provide adult mo squito resting habitat; however, there was no association with the presence of debris piles or tree canopy in the pasture or near the barn. The presence of dead birds on the property was significantly (P = 0.003) associated with WNV activity. These birds were reported by the owner and may or may not have been postitive with WNV. Additionally, other horses becoming ill with WNV on the property were also significantly (P = 0.048) associated with WNV-positive horses. The respondents were asked to mark their perceived level of mosquito activity (none, minimal, moderate, and severe). The level of mosquito activity on the farm was significantly (P = 0.016) correlated with an increased risk of WNV in the logistic regression model. The minimum level of mosquito activity increased risk of WNV by 128% when compared to no activity (Table 2-5). The other levels of mosquito activity also showed an increased risk, as indicated from the odds ratio, but were not associ ated with an increased risk of WNV strongly enough to be significant in the regression model. Discussion The purpose of this study was to describe the extrinsic risk factors associated with WNV infection and to develop recomm endations for pr evention of WNV infection in Florida horses. This study is distinctive because it is an extensive analysis of risk factors performed on WNV positive and negative horses. West Nile virus wa s first reported in Florida during June of 2001 and the WNV vaccine was conditionally releas ed for use in Florida and throughout North America in August of 2001. The im plication of the timing of viru s introduction in Florida and vaccine release is that the Florida equine popul ation represented a completely nave population relative to WNV exposure in 2001. In the north eastern US, horses had been exposed to WNV since 1999, and in the western US, the vaccine was re leased prior to the firs t reported horse cases of WNV. In the face of the WNV outbreak many Florida horses were vaccinated. The results of

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44 this study suggest that horses vaccinated duri ng the summer of 2001 may not have had adequate time for optimal immunity to develop following v accination and prior to the onset of clinical symptoms. In fact in a nave population, there ma y even be a negative impact of vaccination at the time of exposure. Since the release of the WNV vaccine in 2001, there have been high numbers of horse cases in western regions of the United States th at did not experience WNV activity until after the vaccine was available. Just as cases of eas tern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) occur annually in Florida despite the availability of a vaccine, there is likely to remain a group of unvaccinate d horses that are susceptible to WNV infection each year. Furthermore, the continued annual transmission of WNV in the U.S. despite the availability of three vaccines against WNV supports th e argument that the virtual disappearance of WNV transmission to horses in Florida since 2004 is related to WNV transmission patterns in Florida rather than to a complete ly protected equine population. The arboviral case information form (ACF) allo wed tracking of diseas e and gathering of signalment (clinical signs and symptoms) and dem ographic data for the 2,824 horses tested from 2001 to 2005 in the state of Florida. The IgM ca pture ELISA was the most commonly used test to classify WNV disease status in horses. The gold standard for arbovirus diagnosis is regarded as neutralizing antibody testing (P RNT) (Farfan-Ale et al. 2006). Neutralizing antibody testing cannot be used reliably for horses that have b een vaccinated because the horse vaccine consists of formalin-inactivated whole-virion virus e liciting an IgG and neut ralizing antibody response (Porter et al. 2004). A significantly higher number of geldings th an mares or stallions were WNVD in this study. Epp et al. (2005) recorded the gender of horses affected by WNV in Saskatchewan, but

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45 did not report a significant re lationship between WN disease incidence and gender. Other studies have reported a higher incidence of W NV in male horses than females {Tber A.A., 1996 3 /id} Ostlund et al. 2001). In a serosurvey in France there were no significant differences based on gender in subclinical WN disease in horses (Durand et al. 2002). In a study of risk factors of death in clinically affected horses, males we re more often diagnosed with WNV; however, females were 2.9 times more likely to die from WNV than males {Salazar, 2004 342 /id}. Testosterone levels in males may attract more mosquitoes and increas e the likelihood of WNV transmission to males. Alternatively, stable conditions may vary by gender, for example stallions may be stabled indoors more often then geldings, thereby increasing mosquito exposure to animals pastured outdoors more frequently. A thorough analysis of the vaccination history of horses tested in this study was made. The vaccine had a protective effect against WNV infection in those horses that were vaccinated two times in a period of two weeks to one year pr ior to infection. In contrast Salazar et al. (2004) reported that one vaccine dose of the same vaccine provided protecti on. It is possible that the horses in my analysis did not experience the same amount of time frame after the single vaccine and before WNV exposure, as did the horses in the Salazar et al. study. The results from both of these studies are only appl icable to the formalin inactivated whole virion vaccine, which was the only available vaccine on the market for th e duration of the owner survey portion of this study from 2001 to 2003. In 2008 there are three li censed vaccines on the ma rket with different modes of action, which result in varying duration of immunity th at may interact with other environmental factors differently from th e outcome reported in the present study. In addition to vaccination, arbovirus preventi on measures include ba rrier protection and repellent use. Barrier methods, su ch as flysheets, were not often used and were not significantly

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46 associated with WNV infection. Repellents were often used and included pyrethins, natural oils, and Skin-so-Soft lotion. DEET is not and active ingredie nt in insect repellents sold for use on horses due to documented adverse skin reactions (Palmer 1969). The most common type of repellent applied to horses was a pyrethrum-based insecticide. There was not a significant association with repellent use and WNV infection; however the linear regression was close to significant for a protective effect against WNV in fection, and the association may merit further study. Fans in the stable greatly increased the risk of WNV infection. The im portant factor in the increased risk of WNV was only whet her fans were used or not. It is likely that there is an association of duration of fan use and disease outcome. Duration of fan use may have been confounded by other variables because this factor was significant in multivariable analyses. Most fans used in stables are small, non-industria l indoor fans. The strength of these small fans may not be enough to create a breeze that will pr event mosquito flight and blood feeding on the horses in the stable. However, the sma ll fans do aid in the dispersal of CO2 and other chemical odors that act as cues for host seeking mosquitoes (Bowen, 1991). When located inside a stall, the fans may increase the range at which mosqu itoes can detect the horse and attract them in from a longer distance to blood fee d. Fan use likely corresponds with human activity in the barn. Because of fire risks, workers are likely to only use the fans in the day when they are present in the barn. Fan use may not correlate with highest mosquito activity period s (between sunset and sunrise) but probably serves to effectively disseminate strong odors from the barn into the surrounding environment. If the mosquitoes can detect host odor from a greater distance, a higher number of mosquitoes may be able to succ essfully locate and blood feed on the stabled horses.

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47 Natural water on the property was significantly associated with a reduced risk of WNV infection. The most common type of water on the property was a stream, river, or moving body of water, which was not suitable for mosquito larval development. This resulted in the apparent protective association of natural water on the property. The presence of a stream on the property was associated with a reduced risk of WNV infec tion in horses, but no othe r types of water were significantly associated with WNV infection. No other association was detected for the type of water source or the presence of temporary or permanent water on the property. This was unexpected due to the close association of mo squito larval habitats and adult mosquito abundance with rates of arboviral transmission. Debris and tree ca nopy can also provide suitable adult mosquito resting habitats; however, there wa s no association with th e presence of debris piles or tree canopy over the pasture or barn with WNV infection. Abundant mosquito populations are a necessa ry prerequisite for transmission of WNV (Zyzak et al. 2002). The minimum perceived le vel of mosquito activity was significant for increased risk of WNV infection. Other levels of perceived mosquito activity had an odds ratio demonstrating an increased risk for WNV infection but were not significant in the linear regression model. Important f actors associated with epidemic s are mosquito population size and age (Lord and Day 2001), mosquito infection rates, and mosquito transmission rates (Reeves et al. 1961, Rutledge et al. 2003). When compared to no mosquito activity, a minimum level of mosquito activity was associated with a 128% incr eased risk of WNV infection (Table 2-5). The correlation of mosquito activity and WNV transmi ssion is associated with the minimum infection rates (MIRs), blood feedi ng activity, and transmission rates, wh ich are all key f actors in viral transmission to vertebrate hosts.

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48 Stalls constructed from solid wood or cement were associated with a higher risk of WNV infection in horses than were stalls constructe d from boards with openings or open sheds. In a study performed by the USDA in 1999 and 2000, pa sture management was associated with higher rates of WNV dis ease than indoor st abling. The previous study examined horses exposed in the northeastern U.S. Stalling in a hot humid environment may actually provide an environment for at least equal f eeding of mosquitoes compared to the pasture. A solid stable construction, combined with lack of environmen tal temperature control may be putting horses at an increased risk of mosquito exposure. Addi tionally, the solid stall construction may provide resting sites for adult mosquitoes. Guptill et al. (2003) showed avian deaths were associated with an increased level of WN viral activity and could serve as a warning of hum an infection. In my study, dead birds on the property were a powerful indicator of WNV transmission risk (Tab le 2-5). If a dead bird (regardless of WNV diseas e status) was seen on the property there was a strong indication of WNV activity in the immediate area (assuming that th e bird did not disperse far from its original infection site) and was associated with a 97% incr eased risk of WNV infec tion to horses. Other ill equids (due to WNV) on the property were similarly a good risk indicator (Table 2-5). This study could have been improved by includ ing a group of clinical ly normal horses as controls in the analysis. All horses in this study showed some type of clinical manifestation consistent with a neurological infection. Some of the horses in the WNVN group were positive for EEEV and this may have somewhat reduced the power of the study to ev aluate risk of WNV. In conclusion, WNV disease in Florida hor ses appears to be primarily related to vaccination status. As of 2008, there are three efficacious vaccines available and if horses receive two doses according to manufacturers inst ructions, the incidence of equine WNV could

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49 be effectively controlled (Ng et al. 2003, Seino et al. 2007). The us e of fans in the stable should be examined more closely with a focus on the ty pe of fan, time of day used, and other factors that may provide a more conclusive correlation with use and WNV infection. Some other variables that warrant further study are the use of insecticide or repell ent and the canopy cover which both had close to significant associations in the regression model with WNV infection. In areas of high vector activity, re duction of vector larval habitat and limiting horse exposure to mosquitoes remain important prevention methods for Florida equines. Dead birds on the property and other ill equids should be noted and c onsidered an indicator of viral activity in the area. Precautions against mosquito exposure sh ould be taken to protect horses on the property when dead birds have been reported nearby, especially if the horses are unvaccinated.

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50 Table 2-1. Outline of information submitted by Arboviral Case Form (ACF) and by retrospective mail survey (RMS). Veterinarians submitted data on all horses tested for arboviruses in the state via the ACF. Farm/Sample Submission Source County of origin ACS Horse origin ACS Sample(s) submitted ACS Date of onset of clinical signs ACS Date of testing ACS Signalment and History Age ACS Sex ACS Breed ACS Use RMS Arbovirus Prevention Vaccination ACS/RMS Frequency of vaccination RMS Fly spray frequency RMS Fly spray type RMS Barrier prot ection with flysheet RMS Stable Characteristics and Farm Ecology Type of stable structure RMS Duration of outside activity RMS Frequency of stall cleaning RMS Manure handling RMS Presence of debris RMS Tree canopy characteristics RMS Vector activity (mosquito abundance) RMS Presence of dead birds RMS These data were submitted to the Florida Departme nt of Agriculture and Consumer Services as part of reporting requirements in the state of Florida for all horses exhibiting symptoms of encephalitis. Another source of data was the RMS filled out by the owner of the horses tested; the surveys were returned to the College of Veterinary Medicine.

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51 Table 2-2. Total number of horses exhibiting signs of encephalitis and test results for WNV from 2001 to 2005 ACF ACF RMS RMS WNVD WNVN WNVDWNVN 2001 651 382 234 138 2002 643 323 265 95 2003 73 429 35 169 2004 7 173 2005 12 131 Total 1386 1438 534 402 (Source: ACF) and number of mail surveys (RMS ) returned from owners of horses tested between 2001 and 2003. Table 2-3. Age of WNVD horses in Florida 2001-2004 Ages 2001 2002 20032004Total <1 7 34 2043 1-2 133 24 91167 2-3 122 28 40154 3-4 46 34 1081 4-5 23 29 5158 5-6 16 21 4041 6-7 22 28 4155 7-10 61 101 202184 >10 145 9 202176 Total 575 308 697959 Source: ACF Table 2-4. Gender of horses tested for WNV from 2001-2004. Female Male Stallion 2001 WNVD 279 273 59 WNVN 156 134 49 2002 WNVD 310 273 32 WNVN 22 22 20 2003 WNVD 37 34 2 WNVN 222 144 95 2004 WNVD 0 100 0 WNVN 110 88 36 2001-2004 WNVD 626 680 93 WNVN 510 388 200 Source: ACF

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52 Table 2-5. Results of logistic regression anal ysis factors associated with WNV among horses with clinical signs in the st ate of Florida between 2001 and 2003. Variable Category OR 95%CI P value Vaccination <0.001 2001 1.68 1.12-1.83 2002 0.34 0.67-0.93 2003 0.42 0.31-0.00 Vaccinated 2wks6mo 0.002 Yes 0.06 0.04-0.82 No 1 Received two doses <0.001 Yes 0.48 0.01-0.69 No 1 Protective vaccine <0.001 Yes 0.18 0.09-0.98 No 1 Fans in stable 0.013 Yes 1.79 1.22-2.53 No 1 Natural water 0.001 Yes 0.48 0.31-0.75 No 1 Dead birds 0.003 Yes 1.97 1.27-3.47 No 1 Other animals ill 0.048 Yes 2.42 1.01-5.79 No 1 Mosquito activity 0.016 None 1 Mild 2.28 1.35-3.52 Moderate 1.67 0.57-1.74 Severe 1.18 0.82-1.20

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53 Table 2-6. Stable characteristics and farm ecol ogy for horses classified as West Nile virus diagnosed (WNVD) or negative (WNVN) by th e Florida Department of Agriculture and Consumer Services 2001 to 2003. Variable WNVDWNVN Where was horse primarily turned out? (# responses [%]) Pasture 359 (66) 250 (62) Grass paddock 66 (12) 48 (12) Sand paddock 64 (12) 46 (11) Unanswered 54 (10) 58 (14) How often are the stalls clean ed? (# responses [%]) Monthly 12 (2) 14 (3) Twice a month 2 (0) 7 (2) Weekly 33 (6) 20 (5) Daily 240 (44) 157 (39) Not applicable/unanswered 256 (47) 204 (50) Are there fans in the stable? (# responses [%]) Yes 184 (34) 111 (28) No 358 (66) 290 (72) Unanswered 1 (0) 1 (0) How often are the fans run? (# responses [%]) All the time 70 (13) 54 (13) Only when necessary 103 (19) 49 (12) Never 0 (0) 1 (0) Not applicable/unanswered 370 (68) 298 (74) What is the stable made of? (# responses [%]) Boards with openings 158 (29) 131 (33) Solid wood or cement 146 (27) 79 (20) Open shed 73 (13) 60 (15) Unanswered 166 (31) 132 (32) After rain, is there temporary st anding water on the property? (# responses [%]) Yes 257 (47) 192 (48) No 285 (52) 209 (52) Unanswered 1 (0) 1 (0) Tree canopy cover (# responses [%]) None 65 (12) 52 (13)

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54 Table 2-6. Continued Over barn 38 (7) 29 (7) Over pasture 227 (42)160 (40) Over both 163 (30)110 (27) Unanswered 50 (9) 51 (13) How many debris piles exist near the stable, or area of horse activity? (# responses [%]) 0 291 (54)189 (47) 1 128 (24)82 (20) 2 34 (6) 26 (6) 3 11 (2) 11 (3) More than 3 27 (5) 33 (8) Unanswered 52 (9) 61 (15) Severity of mosquito/fly ac tivity (# responses [%]) None 72 (13) 40 (10) Mild 208 (38)148 (37) Moderate 145 (27)107 (27) Severe 71 (13) 37 (9) Unanswered 47 (9) 70 (17) Were there any dead birds on the property? (# responses [%]) Yes 98 (18) 69 (17) No 444 (82)332 (83) Unanswered 1 (0) 1 (0) Significant P<0.05

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55 0 50 100 150 200 250 300 350 400 123456789101112 Month of reportNumber of WNV horse case S er ie s2 S er ies 1 S er ies 3 Ser ies 4 Figure 2-1. Total WNVD horse cases reported in Florida between 2001-2003. Total 2001 2002 2003

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56 CHAPTER 3 MOSQUITOES COLLECTED IN LIGHT TRA PS, RESTING B OXES, AND HORSE-BAITED TRAPS IN NORTH FLORIDA Introduction West Nile virus (W NV; family Flaviviridae : genus Flavivirus ) is a pathogen that is primarily maintained between birds and mosquitoes in enzootic transmission cycles, but is also sometimes transmitted to mammals, including ho rses and humans (Petersen and Roehrig 2002). The virus and disease have been present in the United States since 1999 and have continued to spread throughout North and Central America and throughout the Caribbean Basin causing annual outbreaks (Reisen and Brault 2007). An e fficacious horse vaccine has been available since 2001, but horse cases continue to occur an nually throughout the transmission zone. In Florida alone, 1,082 horses were diagnosed w ith WN infection between 2001 and 2007 (USDAAPHIS 2007). Florida has a large equine industry with over 299,000 horses in the state potentially at risk for WNV infection (FDACS 2007). Estimates of asymptomatic (subclinical) WNV infection in horses have ranged from 1.2% (Lorono-Pino et al. 2003 ) to 58% (Durand et al. 2002). Of the clinically infected horses, 35-40 % of the cases result in death. West Nile virus is a reportable disease in Florida but because a large proportion of inf ected horses do not show symptoms of infection it is likely that horse cases are underreported. West Nile virus is maintained in enzootic fo ci where mosquito and bird populations are in close proximity (Campbell et al. 2002). The pr imary (enzootic) cycle involves avian hosts and ornithophilic mosquitoes and the secondary cycl e involves non-avian host s and epizootic vector mosquitoes which are sometimes linked between both cycles. Vertebrate hosts that facilitate WNV epidemics are termed amplification or reservoir hosts (Kilpat rick et al. 2006). Amplification hosts spike a high vi remia for a short duration of tim e; reservoir hosts may sustain a low level viremia for a long duration and aid in maintaining the virus through periods of low

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57 mosquito activity. Avian amplification hosts re main infective for WNV for one to three days (Komar et al. 2003). The transmission of virus be tween infectious amplification birds and vector mosquitoes (primarily Culex spp) results in amplification of the virus. After sufficient amplification in the bird population, the virus escapes its focus when infective mosquitoes disperse into habitats where th ey may come into contact with a susceptible non-avian host. Epizootic vectors such as Culex nigripalpus (Theobald), Culex salinarius Coquillett, Aedes vexans (Meigen) and Coquillettidia perturbans (Walker), which are opportunistic feeders, transmit WNV to horses and humans (Campbell et al. 2002, Samui et al. 2003). Isolations of WNV from natural mosquito populations in Florida have been reported in Cx. nigripalpus, Mansonia titillans (Walker), Ochlerotatus taeniorhynchus (Wiedemann), and Deinocerites cancer Theobald (FDOH 2007, CDC 2007). Culex nigripalpus is considered an epidemic and epizootic vector of St. Louis encephalitis virus (SLEV; family Flaviviridae genus Flavivirus) in Florida, which shares a similar epidemiology to WNV (Shaman et al. 2005, Zyzak et al. 2002). A thorough knowledge of the biology, ecology, a nd behavior of mosquito vectors is essential for understanding WNV tr ansmission, amplification, epizootics, and epidemics. The blood feeding behavior of many mo squito species has been studied (Apperson et al. 2002, Lee et al. 2002, Ngo and Kramer 2003) and this information combined with mosquito susceptibility to viral infection and mosquito trapping data can help identify possible mo squito vectors of WNV to horses. Livestock-baited traps have been widely used to identify the presence, seasonal abundance, and host preference of mosquitoes. One of the fi rst portable stable traps used for the collection of mosquitoes was the Magoon trap, designed in 1935, and with various modifications, the trap remains widely used today (Service, 1976). Samui et al. (2003) classifies a mosquito species as

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58 an important horse feeder if it frequently enters a horse-baite d stable trap and if a large percentage of the individuals entering the horse-b aited stable trap are bl ood fed when they are collected. In areas of eastern equi ne encephalitis virus (EEEV; family Togaviridae, genus Alphavirus ) transmission, a mosquito that is a co mpetent EEEV vector, feeds on a horse may serve as an epizootic vector from the amplifica tion host to the horse (Samui et al. 2003). Data from horse-baited traps can help identify which mosquito species are present in a locality and which mosquito species feed on horses. An understanding of these mosquito/host interactions can help augment existing knowledge of potential vect or species for arboviruses to infect horses. Of particular interest in this are those species that are physio logically competent for EEEV or WNV, have had field isolations of EEEV or WNV, and have been temporally associated with EEEV or WNV transmission foci. The purpose of this study was to determin e the identity and s easonal abundance of mosquito species attracted to hor ses in a study area in north Florid a. To accomplish this, a twoyear mosquito surveillance project was designe d to provide information about the seasonal abundance and spatial distribution of mosquito species near horses maintained at the study area. These data were collected to evaluate mosquito seasonal variability and host seeking behavior compared to WNV transmission (n = 1) and EEEV transmission ( n = 10) to horses in north Florida in 2005 and 2006. Three clinical WNV horse cases were reported at the study site in 2001 and, based in part on this observation, the site was chosen for the present study. The relative abundance and species composition of mosquito fauna collected from and around horses at the north Florida study site are reported here and compared with light trap and resting box collections made at the same site during the same time period.

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59 Materials and Methods Study Site and Mosquito Collection Protocol The study site for this project was located at the University of Florida Veterinary School, in Alachua County in no rth central Florida (29o38 N, 82o20 W). Four CDC light traps (John W. Hock Company, Gainesville, FL) (Sudia and Chamberlain 1988), four resting boxes (Moussa 1966), and a horse-baited stable trap (Bates 1944) we re used to collect mosquitoes. The site was chosen based on a history of WNV transmission to horses (three clinical cases in 2001), abundant mosquito habitats, a reliable population of horses ( n = 50) permanently pastured at the site, and a ranch-style operation su rrounded by urban development. The study area was mostly open grass bordered by a sylvan habitat supporting nighttime mosquito flight activity and provi ding many mosquito-resting habita ts. Bivens Arm Nature Park bordered the site to the south. The nature park is a 57-acre urban wetla nd (43 acres of aquatic habitat) bordered by upland mixed fo rest (14 acres) (Fig. 3-1). The main feature of the park is a lake surrounded by a live oak hammock. A 1500 sq-ft retention pond was located on the north side of the study site. After a rainfall event, standing water would accumulate in the pastureland at the site. The water would drain slowly into the retention pond over a period of several days unless rainfall continued. The research horses (n = 50) at the site were kept in outdoor pastures and the property had an 80-stall equine hospital on the west side. Th e hospitals outside lights were left on at night and veterinary students and doctors had access to the hospital and were sometimes present throughout the night. Both of these factors ma y have influenced the abundance and species composition of mosquitoes around the hospital at night. In 2005, the stable trap was located in a singl e barn containing six stalls with a large hallway in the middle. Each stall had two 30.5cm by 30.5cm windows, one opening to the

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60 outside of the stable and the other opening into the hallway. One stall (36.5m3) was modified into a horse-baited stab le trap following the design of Ba tes (1944) (Figure 3-2). Mosquito netting was secured to all sides of the stall and across the ceili ng. Two horizontal 30.5cm baffles were placed along the windows on the outside an d inside of the stall 1.21 m from the ground (Figure 3-2). The baffles consisted of a cut foam mattress pad glued to the side of the stall to make a V-shaped opening of 20-cm to the outside and converging to a 2.5-cm opening into the trap. The floor of the stall was covered with sawdust that was cleaned after each use. The barn used in 2005 was not available fo r the study in 2006, so a change in stall location occurred. The design of the stall was almost the same, only small modifications were made and the stall was located 14 m south of the stable used in 2005 (Figur e 3-1). The stall was a standard portable stall design measuring 3.65m3 x 3.65m x of 3.65m on th e low side angled up to 4.26m on the high angle side. The portable st able trap was large enough to house an adult horse. The bottom half of the trap was cons tructed of wooden boards and the upper half of vertical steel bars with a slan ted tin roof. To modify the stall following the design of Bates (1944), the entire stall was sealed with mosquito netting. To construct the entrances into the trap, 30cm lengths of PVC pipe with a 15.2cm outer diameter were cut in half longitudinally and secured along two sides of the st all at a height of 1.21 m (Figur e 3-3). The diameter of the openings were 15.2 cm to the outside with a 1.9 cm opening cut along the inside of the pipe facing into the stall to act as a baffle allowing mosquitoes entrance into the stall and limiting escape. The floor of the stable trap was covered with sawdust and was clean ed after each use. The study commenced on October 6, 2004 and ended on December 5, 2006. The stable trap collections were conducted from May 2005 th rough November 2005 for the first stable trap and from May 2006 through November 2006 for the second. Because the horse had freedom to

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61 move about in the stall and was provided with food and water, the stable trap could be run overnight (IACUC approval #E248). Four CDC light traps (John W. Hock, Gainesville, Fl) baited with approximately 1 kg of dry ice were placed in different habitats at the site. In relation to the stable trap, the light traps were placed as follow: 1) in a highly wooded area (300 m south), 2) by a 1,500 sq-ft retention pond (50 m north), 3) in an oak stand (50 m west) and 4) by the horse stable trap (10 m east). A resting box was pa ired with each of the four light traps (Figure 3-1). Each resting box was constructed of plyw ood in the form of a cube 30.5 cm on each side and one open side. The open side was covered with a square of mosquito netting that could be secured to the sides by Velcro. The cover was le ft open over night and in the morning secured to trap mosquitoes inside prior to aspiration. The outside of each box was painted black with acrylic paint, and the inside was a deep red color to provide a dark space for resting mosquitoes. All traps were set twice a week at about 1600 hr. Traps were retrieved the following morning at approximately 0800 hr. The stable trap and the resting boxes were aspirated with a backpack aspirator (Dvacc, John Hock, Gainesville, FL) followed by a sm all hand held aspirator (Bioquip, Rancho Dominguez, CA) to reach into the smaller spaces. Collection bags from each trap were placed individually in a -70C freezer and then the conten ts were transferred to Petri dishes, labeled, and stored at 70C until the mosquitoes were identi fied to species on a chill table and sorted into pools by species, date, and trap type. All blood fed mosquitoes were stored separately, and empty females were stored in pools of up to 50 and tested for virus (results of the viral analysis are reported in Chapter 4). Fluorescent Mosquito Release and Recapture To evaluate mosquito escape from a nd entrance into the two stable tr aps used in this study eight marked mosquito release trials were performed. In 2005 and 2006, colony reared Culex

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62 quinquefasciatus females (USDA-Gaineseville, Fl) were colored red and green with Dayglo fluorescent dye. The mosquitoes were knocked dow n by cooling to 4C and gently shaken in a container lightly coated with fluorescent powder to dust the mos quitoes and cover them in the dye. During these studies in 2005 ( n = 4) and 2006 ( n = 4), 100 green mosquitoes were released outside of the stall and 100 red mosquitoes were released inside the stall at 1700 hr. In 2005, red mosquitoes were released inside the stall once in the presence of a horse and three times in the absence of a horse. In 2006, mosquito releases we re made three times in the presence of a horse and once in the absence of a horse. In 2005, an un-ba ited light trap was set in an adjacent stall next to the stable trap. No othe r horses were present in the stalls next to the stable trap during these tests. Colonized Culex quinquefasciatus were chosen for the mosquito release trials because this species naturally occurred at the study site and a laboratory colony was easily assessable. Culex quinquefasciatus is an opportunistic feeder (Elizondo-Qu iroga et al. 2006). Females from the laboratory colony were fed bovine blood, demonstrating their willingness to feed on mammalian blood and colony males and females were provide d continual access to a 10% sugar solution for flight and maintenance energy. Prior to experiment al release, the mosquitoes were provided only water for 24h to ensure maximum host seeking behavior upon release. Results During the study 45,851 mosquitoes were captured in the three trap types: light traps, 45,271; horse-baited stable traps, 526; and restin g boxes, 55. Twenty-three mosquito species were captured in light traps, seven in the horse-ba ited stab le traps, and thr ee in the resting boxes. Totals for the nine most abundant species ar e illustrated in Figure 3-4. All seven species collected inside the horse-baited trap blood fed on the horse. Thes e were all confirmed as horse blood meals by PCR analysis (Table 3-1).

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63 Fluorescently dyed Culex quinquefasciatus females were released four times each year to determine the recapture rate inside the stable tr ap and to validate mosquito entry and exit from the stable trap. The number of mosquitoes recaptured inside the stall when a horse was present, varied from 16% (16/100) to 41% (41/100). When no horse was present the number recaptured the following morning varied from 1% (1/100) to 5% (5/100) (Table 3-2). For mosquitoes released outside the stall, there was an entry and recapture rate of varying from 3% (3/100) to 7% (7/100) when a horse was present. None of th e marked mosquitoes en tered the stall when a horse was not present nor were any of the marked mosquitoes recaptured in the un-baited light trap located in the adjacent stall. During the first 13 months of the study (Oct. 2004 to Oct. 2005) Cx. nigripalpus was the most abundant mosquito species collected. The high Cx. nigripalpus numbers were recorded during the months of October and November 2004 (Figure 3-5). The next most abundant mosquito species collected during the first half of the study were Anopheles crucians (Wiedemann), followed by Ma. titillans, Oc. infirmatus, Cx. erraticus, and Cx. salinarius (Figure 3-4). After November 2004, the number of mos quitoes captured decreased dramatically. In March of 2005 An. crucians populations began to increase foll owed by an increase in April 2005 of Oc. infirmatus The abundance of both of these speci es decreased after July 2005. The number of Ma. titillans increased in the early fall and remained high until the end of December 2005 (Figure 3-5). When mosquito numbers a nd species composition were compared between the light trap and the horsebaited trap (mosquitoes collected inside the hor se stall), a preference of Ma. titillans for the horse was observed over the light trap (Table 3-3). Mosquito abundance and species diversity patt erns observed at the study site during the second 13 months of the study (November 2005 to November 2006) (Figure 3-5). The most

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64 abundant mosquito species collect ed during this time period was Ma. titillans, followed by An. crucians, and Cx. erraticus. During 2006, females of three mosquito species en tered the horsebaited stable trap: Ma. titillans, Cq. perturbans, and Cx. erraticus Of the three species that entered the trap, only Cq. perturbans was collected in higher number s in the horse-baited stall than in the light trap loca ted near it (Table 3-3). A comparison of the total number and species composition of the mo squito collections made during 2005 and 2006 appears in Table 3-4. Culex nigripalpus numbers in all trap collections declined dramatically from 10,530 in 2005 to 135 in 2006. Culex salinarius also declined from 1,494 in 2005 to 498 in 2006. Culex erraticus was much more abundant the second year and increased from 397 in 2005 to 3,108 in 2006. Culex erraticus was collected throughout the entire summer of 2006. Mansonia titillans was abundant during the autumn of both years. Mansonia titillans was less abundant in 2005 ( n =2,153) than in 2006 ( n = 10,134). Anopheles crucians was abundant during the wi nter and spring of 2005 ( n = 2,979) and 2006 ( n = 4,633). Its numbers declined in early summer and it virtually disappeared by August of both years. Discussion Seven of the 23 mosquito species collected during this study entered the horse-baited stable trap. The four light tr aps used during this study were re sponsible for 98% of the total mosquito catch. The light trap located next to th e stable trap collected approximately the same number of mosquitoes as the stable trap did (light trap, n = 515; stable trap n = 526). Olson et al. (1968) found nine of the16 species that they studied entered a liv estock-baited trap in a small farming community in Utah. In their study, light trap collections accounted for over 90% of the total trap catch with the exception of Anopheles freeborni. In contrast, Carpenter and Peyton (1952) found a total of 3,391 mosquito es collected in light traps over a one-year period compared

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65 with 65,323 mosquitoes collected in a horse-baited stable trap at the same site. Perhaps the discrepancies observed between th is study and the other two studies discussed above are due to trap design and/or trap location. Because the total trap collections were ab out equal in the stable trap and the light trap, lends credence to a well-placed stable trap in this study. All seven mosquito species collected in the ho rse-baited stable trap in my study have had associated field isolations of WNV (CDC 2007). This does not mean that all seven are 1) competent vectors and/or 2) importa nt epizootic vectors, but field isolation of an arbovirus is one criterion used to identify a mosquito species that has had contact with a WNV positive host and is a potential arboviral vector. Polymerase Ch ain Reaction analysis of blood meals from the mosquitoes captured in the horse-baited stable tr aps used in my study confirmed that all of the mosquitoes that entered the stable trap blood fed on the horse contained in the trap (Table 3-1). All seven mosquito species ( Cx. salinarius, Cx. quinquefasciatus, Cx. erraticus, An. crucians, An. quadrimaculatus, Ma. titillans, and Cq. perturbans ) that entered the trap blood fed on the horse and can therefore be considered horse feeders at this study site (Sam ui et al. 2003). A goal of this study was to identify mo squito species that were likely to blood feed on horses at the site; further studies should be conducted to evaluate the potential of these mosquito species to transmit WNV and EEEV to horses in nature. Mansonia titillans and Cq. perturbans showed a preference for the horse-baited stable trap compared with the adjacent light trap (Table 3-3). Olson et al. (1968) found the most abundant mosquito species present at their study site were the ones that entered the livestock-baited trap. In my study in 2006 Cq. perturbans composed only 6% of the tota l collection but was collected in about equal numbers in the stable trap ( n =47) and the adjacent light trap ( n = 43). Mansonia titillans was the most common mosquito captured in the stable trap during both years of my

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66 study. The next most abundant species collected in the stable trap were Cx. quinquefasciatus in 2005 and Cq. perturbans in 2006. Mansonia titillans has been positive for WNV isolations in the field (CDC 2007); however, th e detection of WNV in a given mosquito species does not mean that the species is a vector of WNV. Po pulation density, host preference, feeding behavior, longevity, seasonal activity, viral susceptibility, and vect or competence must also be considered when attempting to determine the status of a mosqu ito species as an important vector (Sardelis et al. 2001). Mansonia titillans will blood feed on avian hosts, but has a strong preference for mammals (Edman 1971). Members of the genus Mansonia have a long flight range of up to 2.5 km (Macdonald et al. 1990). A long flight range e nhances the vector capac ity of a bridge vector, if the species is an otherwise efficient and co mpetent arboviral vector, by bringing the virus out of its amplification focus into different habitats where transmission can occur (Moncayo and Edman 1999). Coquillettidia perturbans was collected frequently at the study site ( n = 2,747). This species is considered an impor tant bridge vector for EEEV (C hamberlain et al. 1954, Boromisa et al. 1987, Vaidyanathan et al. 1997 ). Eastern equine encephalitis virus has been isolated from pools of field collected Cq. perturbans (Nasci et al. 1993, Andreadis et al. 1998). This species is considered mammophilic, but also feeds on bird s (Edman 1971). Because of blood feeding preference, collection in the horse-baited stable tr ap, and virus isolations field-collected females, Cq. perturbans may play a role in EEEV transm ission in north central Florida. Coquillettidia perturbans has been demonstrated to be an ineffi cient laboratory vect or of WNV and could occasionally play a secondary role in WNV transm ission in the field (Sardelis et al. 2001). The Culex species that entered the horse-baited stable tr ap, did so in much lower numbers than their overall abundance as indicated by light trap collections at my study site. All of the Culex species

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67 that entered the trap ( n = 29 in 2005; n = 6 in 2006) blood fed on the horse indicating that these mosquitoes feed on horses in nature. When a horse was present in the stable trap, 16% (16/100) to 41% (41/100) of fluorescently marked released mosquitoes were recovered the following da y. Failure to recover 100% of the marked mosquitoes may have resulted from mosquito escape, or from death due to horse defensive measures including tail swipes, b iting, and pawing with the feet. Very few (1%, 1/100 to 7%, 7/100) marked mosquitoes were recovered when no horse was present in the stable trap. It is likely the released mosquitoes activ ely and successfully searched for an exit from the unoccupied stable trap. Additiona lly, the fact that no mosquitoes (0/400) entered the stable trap when a horse was not present demons trates that the horse itself act ed as an attractant and that mosquitoes were not attracted to lin gering odors left in the stall. Very few marked mosquitoes entered the stab le trap (16/400) when a horse was present during the mark-release-recapture trials. The co llections inside the stall during both trapping seasons were about equal to the adjacent light tr ap for the same time period (Table 3-3). In mark-release-recapture trials, mosquito numbers recaptured are often low (Conway et al. 1974, Kramer et al. 1995, Reisen et al. 2003). The two r easons that affect recapture rates the most are wind speed and mosquito source (la boratory reared versus locally co llected) (Reisen et al. 2003). The average wind speed on the nights of the mark ed mosquito release trials were between 4.52 mph and 15.07 mph. Caution must be used when interpreting result s from collections made by light traps, horsebaited stable traps, and resting box because th e collections may not reflect true mosquito abundance (Huffaker et al. 1943). The most accurate mosquito population estimate is made by combining the results of multiple trap collecti on types (Huffaker et al. 1943, Bidlingmayer 1967)

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68 to account for variation due to spec ies biases of traps, biases due to trap location or the influence of meteorological conditions. Despite overall low mosquito numbers in the stable trap collections, the collection information can be usef ul when combined with other trap types used during this study. Simultaneously using various trapping methods can provide a reliable measurement of mosquito abundance and di versity (Huffaker, 1943, Bidlingmayer 1967). Seven mosquito species fed on the horse maintained in a stable trap and the largest mosquito collections were made during the fall of both study years. Mansonia titillans was the most frequently collected mosquito in the horse-baited stable trap. Mansonia titillans and Cq. perturbans were the only two species at the site th at were collected in higher number in the stable trap than in the adjacent light trap. Both of these species should be considered in future studies as potential vectors of WNV and EEEV, respectively, in north Florida. To my knowledge, laboratory studies have not been comple ted to evaluate the vector competency of Ma. titillans for WNV Such studies would provide valu able information to supplement the results of my study. The possible role of Ma. titillans as a potential vector of WNV to horses should be investigated. Coquillettidia perturbans may play an important role in the transmission of EEEV to horses (Morris 1988) and may play a secondary role in WNV transmission (Sardelis et al. 2001). Coquillettidia perturbans is considered a bridge vector of EEEV (Crans and Schulze 1986, (Morris 1988) and it is likely th at this species may play a role in EEEV transmission as an epizootic vector to horses in north central Florida. The differences in mosquito numbers collected at the site each year are most likely due to variation in the weather conditions. In 2005 the weather was wetter than average (reported in chapter 4), and in 2006 Florida experien ced a prolonged drought. The number of Cx. nigripalpus declined dramatically after the first few months of the study. The collections began in October

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69 2004 and the hurricane season had been particularly active for Florida earlier in the year. Only a few weeks after the final hurricane of the season did trapping begin and it is possible that the large numbers of Cx. nigripalpus were correlated with the rain fall associated with this time period. Figure 3-1. Location of the four paired light traps and resting boxes marked as T1, T2, T3, and T4. The Chen barn was modified for the stable trap in 2005, and a portable stall was erected 14m south of the Chen barn duri ng the 2006 season. The sentinel chicken flock was located adjacent to the stable trap in 2006. 2006 stable trap and sentinelchickenflock 2005 stable

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70 Figure 3-2. Measurements of the st all that held the horse in 2005. A single stall was modified for the trap in a six-stall barn. 30.5 cm 30.5 cm 36.5 m 36.5 m

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71 Figure 3-3. Stable trap design 2006. 30 cm lengths of PVC pipe w ith a 15.2cm diameter cut in half with openings of 1.9cm were placed along two sides of the stall for mosquito entry. The rest of the trap was sealed with mosquito netting.

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72 Table 3-1. Blood meals of mosquitoes collected in the horse-baited stable trap. A subsample (n = 50) of the total stable trap catch ( n = 525) in 2005 and 2006 was analyzed. (Results of the blood meal analysis from mos quitoes collected outside the stable trap are presented in Chapter 5.) Species # tested # confirmed (%) result Cx. salinarius 2 1 (50) horse Cx. quinquefasciatus 3 2 (66) horse Cx. erraticus 7 5 (71) horse An. crucians 1 1 (100) horse An. quadrimaculatus 4 1 (25) horse Ma. titillans 20 11 (55) horse Cq. perturbans 13 7 (54) horse

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73 12433 10665 8156 4690 2747 2419 2001 915 904 0 2000 4000 6000 8000 10000 12000 14000 M a. titillans Cx nigri pa lpus An. c r ucian s Cx er rati c us Cq. per t ur ba ns Oc. infirmatus Cx. salinarius A n. qu adr i m ac ul at us P s c o lum bi ae Figure 3-4. The species that repres ented at least 1% or more of the total trap catch between October 2004 and November 2006. Table 3-2. Mosquitoes collected in the mark-rel ease-recapture study in the horse-baited stable trap. Red marked mosquitoes were releas ed inside the stall in groups of 100 each date. Green marked mosquitoes were released 5 m outside the st all in groups of 100 each date. Collection Date Horse present Mosquito color # recaptured inside stall # recaptured outside stall 6/11/2005 Yes Red 26 1 Green 7 9/16/2005 No Red 3 Green 0 9/23/2005 No Red 1 Green 0 9/30/2005 No Red 4 Green 0 9/16/2006 Yes Red 41 Green 3 9/21/2006 Yes Red 16 Green 5 9/28/2006 Yes Red 25 1 Green 1 9/30/2006 No Red 5 Green 0

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74 0 500 1000 1500 2000 2500O-04 N-0 4 D-0 4 J-05 F-05 M -05 A-05 M0 5 J -05 J -05 A-05 S -0 5 O-0 5 N-0 5 D-05 J -06 F 06 M -0 6 A -0 6 M -06 J -0 6 J -06 A -0 6 S-06 O0 6 N -06DateNumber Mosquitoes/trap night Cx nigripalpus Cx erratiucs Ma titillans Cq pertubans An crucians Figure 3-5. The most abundant mosquito species collected at the study si te are represented over the two trapping seasons from October 2004 to November 2006. Table 3-3. Comparison of mosquito catch in the horse-baited stable trap and an adjacent light trap. Culex sp. include Cx. quinquefasciatus (16) Cx. salinarius (3) and Cx. erraticus (10). Anopheles sp. include An. quadrimaculatus (9) and An. crucians (7). May to Oct 2005 Horse Stable May to Nov 2006 Horse Stable Stall Light Trap Stall Light Trap Culex sp. 29 43 Culex erraticus 6 213 Anopheles sp. 18 22 Mansonia titillans 96 171 Ps. ciliata 1 0 Cq. perturbans 47 43 Mansonia titillans 330 13 Cq. perturbans 1 9

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75 Table 3-4. Total number of five mosqu ito species caught each study year. Mosquito species 20052006 Total Ma. titillans 4,4307,529 11,959 Cx. nigripalpus 10,530135 10,665 Cx. erraticus 1,5723,102 4,674 Cx. salinarius 1,498500 1,998 An. crucians 2,9915,158 8,149 Total 21,02116,424 37,445

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76 CHAPTER 4 ARBOVIRUS SURVEILLENCE: MOSQUITO POOLS, SENTINEL CHICKENS, AND HORSES. Florida has had an arthropod borne virus (arbovirus) surveillance program in place since 1977 to track the amplification and transmission of mosquitoborne viruses including eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus ) Highlands J (family Togaviridae, genus Alphavirus, HJ) and St Louis encephalitis virus (SLEV; Flaviviridae, genus Flavivirus ) (Day and Stark 1996). The Florida Depa rtment of Health (FDOH), Division of Environmental Health, coordinates the surv eillance program. The Interagency Arbovirus Surveillance Network reports to the FDOH and is composed of several local, state and federal agencies, which are involved with the surv eillance and control of arboviral diseases. Upon its arrival in the United States West Nile virus (WNV; family Flaviviridae, genus Flavivirus ) was easily added to the existing surveillance program with the addition of WNVspecific laboratory diagnostics. Because SLEV and WNV are antigenically related, crossreactions are observed with some serologic test s and so plaque reducti on neutralization testing (PRNT) is done to distinguish the two viruses. During Fl oridas first reported WNV transmission season (2001), virus was recorded in 65 of 67 countie s (Blackmore et al. 2003). In both the northeastern U.S. and in Florida, wild bi rd mortality was the most sensitive viral activity indicator (Blackmore et al. 2003). In 2001, wild bird mortality wa s the first indication of viral presence in 54 of the 65 counties in Florida wher e WNV was detected (Blackmore et al. 2003). Due to the correlation of WNV-positive dead bi rd reporting and local WNV transmission, dead bird reporting has become a valu able surveillance tool in the United States (Eidson et al. 2001a, (Nasci et al. 2002). Like SLEV, the natural cycle of WNV involves Culex mosquitoes and wild birds. However, unlike SLEV, WNV causes high rates of mortality in certain families of birds.

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77 Members of the family Corvidae (crows, magpies, ravens, and jays ) are particularly susceptible to fatal infection (Nasci et al. 2002). Chicke ns in the northeastern United States are not considered reliable indicators of human disease because seroconversions occurred after human cases had already appeared (Crans and Schulze, 1986 Cherry et al. 2001). For this reason, New York does not have a sentinel chicken program in place. In Californi a and Florida, however, sentinel chickens are an indispensable component of arboviral surveillance because viral positives in chickens are closely associated with and predictive of human cases (Day and Lewis 1991, Reisen et al. 1994). Horses are not currently bled as part of an active WNV surveillance program in the United States. In New York State horse positives were unreliable in the prediction of human cases of WNV (Trock et al. 2001). It is not yet known whether WNV surveillance in horses can predict human cases in Florida, but horse cases that are reported to local health departments are used as part of arbovirus surveillance. Blackmore et al. (2003) reported that the epicenter of the 2001 WNV outbreak in Florida horses was in Jeffers on County. From Jefferson County, the outbreak spread east, west, and south to a total of 40 Florida counties with confirmed horse cases. In the counties reporting both horse and human cases, th e horse cases preceded the human cases by one to four weeks (Blackmore et al. 2003). Weather conditions greatly affect mosquito popu lations and consequently arboviral activity (Wegbreit et al. 2000, DeGaetano 2005, Pecoraro et al. 2007). Drought in the spring followed by summer rain is closely associated with transm ission of SLEV and WNV in Florida (Day and Stark 1996, Day 2001, Shaman et al. 2005). Drought br ings mosquito and bird populations into close proximity by limiting the available water. Epizootic amplification may occur under these circumstances if an abundant mosquito population is available to feed on susceptible wild birds.

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78 Under conditions of prolonged drought, however, vi rus transmission is greatly reduced (Day and Lewis 1991). Several critical fact ors have been outlined as crite ria that create a high epidemic risk for arbovirus transmission in south Florida. They include a large population of susceptible wild birds, severe drought in the spring follo wed by a wet summer, and the continuation of dry and wet patterns throughout the summer that fo cus virus transmission between the mosquito vectors and vertebrate hosts (D ay and Lewis 1991). The predictive factors outlined here are based on aspects affecting Culex nigripalpus populations and dynamics. The weather pattern of rain followed by drought synchronizes th e oviposition and blood feeding of Cx. nigripalpus and subsequently virus transmission (Day and Curtis 1993). Culex nigripalpus plays a major role in arbovirus transmission in the southern part of th e state. It is necessary to make similar evaluations of the rela tionship between weather patterns and mosquito populations with virus transmission patterns in the nor thern part of the state where Cx. nigripalpus populations are typically much lower (Zyzak et al. 2002). Because Cx. nigripalpus is not as common in north Florida as it is in south Flor ida, weather conditions such as a mild spring may increase the population of Cx. nigripalpus in north Florida (Zyzak et al. 2002); or it is possible that other Culex species are playing a larger role in virus transmission. There were three aims of this study. The first aim was to compare arboviral activity in Alachua County with the seasonal dynamics and abundance of mosquito species present at a north Florida site. The second ai m of the study was to correlate the temporal patterns of viral activity and mosquito abundance with abiotic environmental factors including rainfall, temperature, and wind speed. The third aim was to examine mosquito abundance at four microenvironments within a site. To accomplish this goal a two-year surveillance project was designed to provide information a bout the seasonal patterns of arbovirus activity in relation to

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79 the abundance of mosquito species at a study site in north central Florida. The data were collected to compare WNV, SL EV, and EEEV activity in north Florida with the mosquitoes collected at the study site. Arbovirus activity included sentinel chicken seroconversion and mosquito pool positives during the study period. Materials and Methods Sentinel Animals The study site was located at the University of Florida Veterinary School, in Alachua County in north central Florida (29o38 N, 82o20 W) (see chapter 3 Materials and Methods). Three equines were used as arboviral sentinels at the site between Ma y and November of 2005 and 2006. The sentinel horses had blood samples taken weekly to screen for the presence of antibody for WNV, EEEV, and HJ (IACUC approval # E312). Ten ml of blood was taken from the jugular vein with a vacutainer needle and drawn directly into a 10 mL blue top (3.8% Na citrate) vacutainer tube then centrifuged for 10 min at 600 g. The serum was tested at the University of Florida Emerging Disease Arbovi ral Research and Testing (EDART) laboratory with the Immunoglobulin M antibody-capture Enzyme-Linke d Immunos orbent Assay (MACELISA) for detection of viral antibody. Two of the sentinel horses were blood donors (research horses kept on the veterinary school property for blood donation as needed, IACUC#A712) and were maintained permanently in an open field near a sylvan habitat (Figure 4-1). The third animal was a pony that was used twice a week as a bait animal in a stable tr ap. The pony was maintained in a pasture during periods when it was not housed in the stable tr ap. The pony was located on the opposite side of the study site, 600 m west of the blood donors (Figure 4-1). Sentinel chickens were added to the protoc ol (IACUC approval # E248) from May through November of 2006. Two white Leghorn chickens were housed in a 1.8m X 0.9m cage. The cage

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80 was located in a field 100 m west of a horse pasture (Figure 4-1). It was placed next to a horse stall that was used as a stable tr ap (Chapter 3). The chickens we re bled weekly from the brachial vein. Collections were taken from alternate wi ngs each time the chicken was bled in order to allow healing. One ml of blood wa s collected in a gel separator vi al with a 25-gauge needle and was centrifuged for 10 min at 600 g. The resulting sera were delivered to the Alachua County Health Department the same day they were co llected. The samples were shipped with other Alachua County sentinel chicken blood samples to the Florida Department of Health Bureau of Laboratories in Tampa, where they were tested for Flavivirus and Alphavirus hemagglutination inhibition (HI) antibodies. The Department of H ealth routinely tests any resulting positive serum samples to identify WNV, SLEV, EEEV, or HJ antibody by IgM enzyme immunoassays and plaque reduction neutralization tests. The Alach ua County health department received a weekly report of the results of the chic ken serum tests, from the FDOH for all chickens tested in the County. Mosquito Collections Mosquito collections at the study site began on October 6, 2004 and continued to November 30, 2006. The traps were operated to two tim es a w eek (Figure 4-1) at 1 600 hr and picked up the following morning at 0800 hr. Centers for Disease Control light tr aps (John W. Hock, Gainesville, Fl) baited with approximately 1 kg of dry ice were placed in four different habitats at the site: at the edge of Bi vens Arm Park, next to the retention pond, under a small stand of oak trees, and by the horse stable tr ap (Figure 4-1). To analyze trap catch differences in four microhabitats, a Kruskal-Wallis one-way analysis of variance was used (Minitab version 15, State College, PA). PROC GLIMMIX analys is was done to compare the trap location microhabitats by month and to evaluate mosquito species collection between the two years of the study (SAS version 9.1, Cary, NC).

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81 Mosquito collections were sort ed by species and date of collection and stored in pools of up to 50 at -70C. Pools were homogenized in 1 ml diluent of Phosphate Buffered Saline (PBS) with 4% Fetal Bovine Serum (F BS) by placing two copper BBs in th e vial and vortexing. After a 10 min centrifugation at 11,356g to se parate the mosquito solids, 200 l of supernatant was transferred to a new tube fo r RNA extraction with Trizol following the manu factures protocol (Molecular Research Center Inc ., Cincinnati, OH). The remaining sample, not used in the RNA extraction, was stored at C for possible cell culture if a positive result in RT-PCR was found. The RNA isolated with an RNeasy mini k it (QIAGEN, Valencia, CA) from the mosquito pools, and was tested for WNV, SLEV, and EEEV using quantitative Real Time RT-PCR (Lanciotti and Kerst 2001, Stark and Kanzanis 2007). Four hundred microliters of the RNA extraction homogenate, and 600 l L15 media (5% FBS, 15 g/mL gentomyacin, 200 units/mL penicillin, streptomycin, fungizone) was added to a T25 cm2 flask of Vero cells (2.0 x 106 cells). The cells were rocked and incubated at 37 C for 1h. Four mL L15 media was added and the cells were observed every 24h for 7d. Meteorological Data In order to address the relationship betw een weather and m osquito population dynamics, daily weather conditions includi ng rainfall, wind speed, and averag e temperatures were accessed from the Florida Automated Weather Network (F AWN) recording station in Gainesville, FL (29 39N, 82 30W). A 50-year mean of precip itation and temperature was calculated from 1951-2000 by compiling data from the National O ceanic and Atmospheric Association (NOAA) archives. Monthly deviations from normal for precipitation and temperature were calculated by subtracting recorded values from the 50-year mean monthly values. Paired t-tests were used to evaluate differences between the two trapping s easons temperature, prec ipitation, and vector

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82 abundance. Linear regression was used to exam ine independent relationships between average mosquito catch per trap night and weekly averag e precipitation, temperature, and wind speed. Results Sentinel Animals None of the five sentinel animals (two horses, one pony, and two sentinel chickens) maintained at the study site seroconverted to an arboviral agent during the study period. During the same time period, the sentinel chickens ma intained by Alachua County Public Health Unit showed the following seroconversion activ ity: 2005, 16 WNV, 47 EEEV, and 3 HJ; 2006, 0 WNV, 15 EEEV, and 1 HJ (Table 4-1). A confir med arboviral infection in a Florida horse is classified as a reportable disease in which case the attending veterinarian must report the case to the Florida Department of Agricu lture and Consumer Services in Tallahassee. Between May 1 and August 29 2005, nine horses in Alachua Count y were confirmed as EEEV positive. One horse was positive for WNV; this report came on October 20, 2005. In 2006, no WNV horse cases were reported in Alachua County. Only a single EEEV horse case was reported on July 31 in Alachua County in 2006 (Table 4-1). Mosquito Collections Three hundred fifty nine mosquito pools ( n = 13,809 total mosquito es; 7 species) were tested for WNV, EEEV, a nd SLEV. One pool of 50 Ma. tttillans, collected in September 26, 2006 was positive for SLEV (minimum infection rate of 0.254). The attempt to grow the SLEV in Vero cell culture was unsucce ssful. After 7d of observation no cytopathology was observed in the cells. The mosquito pools were collected over a period of 26 mont hs (October 2004 through November 2006). During the same time period, Alachua County did not submit mosquito pools to the FDOH for testing. In the state of Flor ida in 2005 there were 1,603 mosquito pools tested from 11 counties. Five mosquito pools from three Florida counties (M onroe, Pinellas, and

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83 Sarasota) tested positive for WNV and ten mosqu ito pools from four Florida counties (Escambia, Sarasota, St. Johns, and Volusia) tested pos itive for EEEV. In 2006, there were no positive mosquito pools (n = 1,253) in the stat e of Florida. The most frequently collected mosquito species at the study site was Mansonia titillans (29%) followed by Culex nigripalpus (25%) (Figure 4-2). The seas onal distribution of the seven most abundant species was compared with hor se and chicken seroconversions for WNV and EEEV in Alachua County (Figures 4-5 to 4-8). In 2005, the abundance of Cx. erraticus increased in February and March and the firs t EEEV chicken seroconvers ion was reported in April. In 2006, Cx. erraticus abundance increased in May and the first EEEV chicken serocnversion was at the end of May (F igures 4-6 and 4-8). The number of Ma. titillans increased in the fall, which was when the last of the chicken seroconversions and horse cases in both 2005 and 2006 were seen. When trap location was examined by Kruskal-Wallis and Proc Glimmix analysis, the highest numbers of mosquitoes were trapped in the Bivens Arm Forest and the lowest numbers were obtained by the horse stable (Table 4-2, Figure 4-4). The collection of Anopheles crucians (P = 0.001), Cx. erraticus (P = 0.045), and Ps. columbiae (P = 0.04) varied significantly by location. There was no significan t difference in the number of Ma. titillans, Cx. nigripalpus, Cx. quinquefasciatus, and Oc. infirmatus caught at each trap locati on. During each month of the study, the trap located in the Bive ns Arm Forest collected signif icantly more mosquitoes that traps number 2 and 3. No significant difference in trap catch were seen between trap 1 (Bivens Arm) and trap 4 (the retention pond) in Novemb er and December, but all other months trap 1 caught significantly more mosquitoes than trap 4 (Figure 4-4). Trap year (year 1, October 2004 to October 2005 and year 2, November 2005 to N ovember 2006) was significantly different for

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84 collections of An. quadrimaculatus, Cq. perturbans, Cx. nigripalpus, Cx. salinarius, and Oc. infirmatus Meteorological Data December 2005 was abnorm ally wet, with 48.6 cm of rainfall above the expected 50 yr (1951-2000) mean for Alachua County (Figure 4-3A ). During the winter months of January through March 2005, the observed monthly rainfa ll amounts were 3 to 5 cm below average (Figure 4-3A). In 2006, the winter months of January through March were unusually wet, accumulating over 20 cm of rainfall above normal. In March 2006, a drought began and continued through September. During 2006, North Florida experienced a prolonged drought that resulted in a total of 15.4 cm less rainfall than av erage (Figure 4-3A). The mean daily rainfall patterns were significantly different between 2005 and 2006 (2005 mean 5.00 mm, 2006 mean 2.77 mm, t(364) = 1.66, P = 0.048). The temperatures ranged from 1 to 3 C cool er than average for most of the study period (Figure 4-3B). Each month was colder than ex pected when compared to the long term means except for January of each year and April of 2006. The mean m onthly temperatures were not significantly different between 2005 and 2006 (2005 mean 66.28 C, 2006 mean 66.23 C, t(11) = 0.064, P = 0.47). There were no sign ificant correlations of temper ature, wind speed, or rainfall with mosquito abundance. Discussion Although arbovirus (WNV, SLEV, and EEEV) activ ity was m inimal in Florida during the years of this study, information gathered during inte r-epidemic years is valuable to the complete understanding of mosquito-borne disease epidemiology (H ay et al. 2000). Virus transmission is dependent on the presence of an abundant and old mosquito population (Z yzak et al. 2002) and mosquito reproduction and mortality are directly influenced by meteorological conditions.

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85 Weather conditions including temperature and rainfall directly aff ect vector population distribution and abundance (Hay et al. 2000). During the spring of 2006 there was a prolonged drought in north Florida that reduced the abundance of mosquitoes and minimized arbovirus transmission (FDOH 2007). The year-to-year differences in mosquito abundance and diversity at the study site are likely related to local weather patt erns. The weather was wetter th an average in north Florida in March through June and in October through D ecember of 2005. Conversely, in 2006 there was a prolonged drought in Florida and very little arbo viral activity was reported throughout the state. At the study site, some mosquito species ( Cx. nigripalpus P = 0.0015, Cx. salinarius P = 0.0003, An. quadrimaculatus P = 0.0002) were collected in signifi cantly fewer numbers in 2006 when compared to 2005. Transmission of SLEV and WNV are closely associated with rainfall patterns (Day 2001, Shaman et al 2005). The data presented here support th e conclusion of drier years reducing the number of potentially infectiv e mosquitoes (Day and Lewis 1991). The total range of mosquito habitat is dependent upon the presence of available bodies of water and humid daytime resting habitats. Therefore, a reduction in the number of infective mosquitoes decreases the likelihood of arbovirus transmission. The trap located in Bivens Arm Forest (trap 1) collected significantly more An. crucians, Cx. erraticus and Ps. columbiae than the other three trap loca tions. Sylvan microhabitats may support a greater population of mosquitoes because they provide a daytime resting habitat and retain a high humidity. Add itionally, the aquatic environment of Bivens Arm Lake provided larval habitat. Animals located near such a ha bitat may experience a greater number of mosquito bites by these species. The more open habitats of the oak stand, the stable, and the retention pond had significantly fewer mosquitoes collected and this is likely because these areas did not

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86 retain high daytime humidity levels to support da ytime resting. Therefore, the mosquitoes would need to fly much further from the daytime resti ng habitat of the forest to encounter a host in these open habitats. There were no significant differenc es in the number of numbers of Cx. nigripalpus, Ma. titillans, and Cx. quinquefasciatus collected at each trap locati on. Two of the most abundant species collected at the study site, Ma. titillans and Cx. nigripalpus, were collected equally at all four trap locations. Day et al. (1991) found parous Cx. nigripalpus females collected in abundance in open habitats. They reported th at abundance was especially high during wet summers when normally dry habitat became moist and humid allowing mosquitoes access to hosts. When surveying the flight capacity of blood-engorged mo squitoes, Edman and Bidlingmayer (1969), found Cx. nigripalpus in higher numbers in wooded habitats compared to open habitats. In my study, the fact that no significant difference of Cx. nigripalpus abundance occurred in the four trap microhabitats is consiste nt with the literature th at this species can be collected in high numbers in either open or wooded habitats. The ubiquitous nature of Cx. nigripalpus at the study site may increas e the likelihood of a single mo squito encountering both a reservoir host and a susceptible host. Mansonia titillans was abundant at this site in the late fall and was not significantly correlate d with trap location. The lake at Bivens Arm contains abundant aquatic flora (waterhyacinth, Eichhornia crassipes (Mart.) Solms., and waterlettuce, Pistia stratiotes L.) necessary for the larval development of Ma. titillans populations. In a dispersal study of several Mansonia species, individuals were re -captured from 0.5 to 2.4 km from the release point (Macdonald et al. 1990). This flight dist ance is adequate to explain why approximately equal collections of Ma. titillans were obtained in all the habitats surveyed.

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87 Location of sentinel ch icken sites is an important fact or when initiating an arbovirus surveillance program (Day et al. 199 1). The historical enzootic activity of WNV at this site was a key factor for choosing to place sentinel chickens there. None of the sentinel animals (three equines and two chickens) at the site was pos itive for WNV, SLEV, HJ, or EEEV during this study. Although the sample size was small with a total of five animals being screened, there were no WNV seroconversions of sentinel chic kens in Alachua County in 2006 (Table 4-1). Moreover there were no horse or human WNV cases in Alachua County in 2006. Because sentinel animals in Florida provide the most accu rate and timely indication of field transmission, the lack of seroconversions at our site may be an indication of low-level virus circulation in the area in 2005 and 2006. Although collected in large numbers, Ma. titillans did not show a temporal correlation in abundance with the EEEV or WNV transmissi on season in Alachua County in 2005 and 2006 (Figure 4-4). West Nile virus ha s been isolated from field caught Ma. titillans (CDC 2007), but no work has been published regarding their vect or competence (Turell et al. 2005). Vector capacity is determined not only from natural in fection and demonstrated laboratory transmission, but also biological factors such as biting preference, length of life, and timing of adult activity are all fundamentally tied to vector cap acity (DeFoliart et al 1987). Because Ma. titillans did not appear at the study site until EEEV and WNV transmission had already begun in Alachua County in 2005 it may not play a major vector ro le in arbovirus epidemics in north Florida. Despite the lack of temporal correlation with EEEV and WNV transmissi on at the site, more research is likely warranted as Ma. titillans is considered a vector of Venezuelan Equine Encephalitis virus (VEEV; Family Togaviridae, genus alphavirus) in Central and South America (Mendez et al. 2001, Ture ll et al. 2000). Mansonia dyari may be a maintenance vector of SLEV

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88 in Panama (Gorgas Memorial Laboratory 1979, as cited by Lounibos et al. 1990). Members of the genus Mansonia in Africa have had seve ral WNV isolations reporte d (Traore-Lamizana et al. 2001). Furthermore, several species of Mansonia are likely involved in the transmission of Japanese Encephalitis virus (JEV; family Flaviviridae, genus Flavivirus, ) in Asia (Arunachalam et al. 2004). This is the first po sitive identification of SLEV from Ma. titillans in Florida. The failure to isolate virus in cell culture may ha ve been because no cryogenic protection (i.e. DMSO) was added to the homogenate. Freezing a nd thawing the sample reduces viable virions because ice crystals break the e nvelope. Another potential reason virus was not isolated is that the mosquito pools may have been stored at -20 C for a period of time and research indicates that -70 C is the optimal storage temperature fo r virus detection of mosquito pools (Turell et al. 2002). Culex erraticus populations increased at the study s ite a few weeks to a month prior to EEEV transmission in Alachua County. In Al abama EEEV isolations from field caught Cx. erraticus have been reported (Cupp et al. 2003). Th e natural isolations in the southeastern United States and temporal correlation may indi cate that this species is involved in EEEV transmission in north Florida. Culex nigripalpus was active at low leve ls throughout the duration of the study. In 2005 in Florida, th ere were three WNV isolations from Cx. nigripalpus (2 in Pinellas and 1 in Sarasota County) and tran smission has been documented in Jefferson County, Florida (Rutledge et al. 2003). Howeve r, at our study site the population of Cx. nigripalpus after November 2004 remained low (n < 50/trap night) for the duration of the study. Perhaps the low numbers were related to weather conditions unfavorable for mosquito development. Having only four light traps in di fferent locations at the site was a limitation of the study. A better assessment of microhabitat could have b een made if the traps were moved within a

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89 single microhabitat randomly to exclude the pos sibility of a trapping-out effect. A second limitation of the study was the fact that only five sentinel animals were screened for arbovirus activity. If more animals were used, there would have been a better chance of observing virus activity had there been any. A comparison of arboviral activity in Alachua County with the mosquitoes present at the study site was possibl e because the county maintains sentinel flocks and horse cases are recorded. However, in th is study, mosquitoes were collected in one place and transmission occurred in another, making interpretation more difficult. The seasonal abundance of Cx. erraticus and Cx. nigripalpus at this site increased prior to EEEV and WNV transmission in Alachua County. These two species should be further considered as potential vectors in north Florida of EEEV and WNV respectively.

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90 Figure 4-1. Location of the four paired light traps and resting boxes marked as T1, T2, T3, and T4. The Chen barn was modified for the stable trap in 2005, and a portable stall was erected 14m south of the Chen barn during the 2006 season. 2006 stable trap and sentinelchickenflock 2005 stable Blood Donors

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91 An crucians 18% Cq perturbans 6% Cx erraticus 10% Cx nigripalpus 24% Cx salinarius 4% Mansonia 29% Ps columbia 2% Oc infirmatus 5% An quadrimaculatus 2% Figure 4-2. The species that repres ented at least 1% or more of the total trap catch between October 2004 and November 2006 Table 4-1. Number of arbovirus positive sentinel chickens and horses in Alachua County in 2005 and 2006. Chicken Horse Chicken Horse Arbovirus 2005 2005 2006 2006 Total EEE 47 9 151 72 WNV 16 1 00 17 SLE 0 0 00 0 HJ 3 0 10 4 Total 66 10 161 93

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92 Table 4-2. Mosquito collections by trap lo cation from October 2004 though October 2005 (13 months). Mosquitoes were collected from light trap collections.. Light traps (Oct 2004Oct 2005) Location Species Woods Pond Oak trees Stable Total Cx. salinarius 928 219 296 55 1498 Cx. nigripalpus 7060 1441 1952 77 10530 Cx. erraticus 1291 136 101 44 1572 Oc. infirmatus 1753 281 50 10 2094 An. crucians 2377 461 117 36 2991 An. quadrimaculatus 444 120 36 19 619 Mansonia titillans 1851 1819 527 233 4430 Total 15704 4477 3079 474 23734 Table 4-3. Mosquito collections by trap lo cation from November 2005 though November 2006 (13 months). Mosquitoes were collected from light trap collections. Light traps (Nov 2005Nov 2006) Location Species Woods Pond Oak trees Stable Total Cx. salinarius 339 109 31 21 500 Cx. nigripalpus 112 14 6 3 135 Cx. erraticus 2455 206 224 217 3102 Oc. infirmatus 273 48 3 1 325 An. crucians 4939 141 55 23 5158 An. quadrimaculatus 236 14 32 4 286 Mansonia titillans 2845 2262 2194 228 7529 Total 11199 2794 2545 497 17035

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93 A -20 -10 0 10 20 30 40 50 60 ONDJFMAMJJASONDJFMAMJJASONRainfall Dev. from normal (cm) B Figure 4-3. Monthly deviations fr om normal for rainfall A) and te mperature B) for October 2004 through November 2006 in Alachua County Flor ida. Observed monthly values were subtracted from a 50-year mean (1951-2000) to determine the deviations from normal.

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94 Figure 4-4. Comparison by month of the four light trap locations average mosquito trap catch. Bars followed by a different letter are significant at P < 0.05. Figure 4-5. Temporal distribution of the seven mo st abundant mosquito sp ecies collected at the University of Florida Veterinary School (March through December 2005). E, Eastern Equine Encephalitis in Alachua County sentinel chickens ( n = 47); Eh Eastern Equine Encephalitis in Alachua County horses (n = 9); W, West Nile virus in Alachua County sentinel chickens ( n = 16); Wh West Nile virus in Alachua County horse ( n = 1).

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95 Figure 4-6. Temporal distribution of three Culex mosquito species collected at the University of Florida Veterinary School (March th rough December 2005). E, Eastern Equine Encephalitis in Alachua C ounty sentinel chickens (n = 47); Eh Eastern Equine Encephalitis in Alachua County horses (n = 9); W, West Nile virus in Alachua County sentinel chickens ( n = 16); Wh West Nile virus in Alachua County horse ( n = 1).

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96 Figure 4-7. Temporal distribution of seven most abundant mosquito species collected at the University of Florida Veterinary School (May through November 2006). E, Eastern Equine Encephalitis in Alachua County sentinel chickens ( n = 15); Eh Eastern Equine Encephalitis in an Alachua County horse ( n = 1).

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97 Figure 4-7. Temporal distribution of three Culex mosquito species collected at the University of Florida Veterinary School (May thro ugh November 2006). E, Eastern Equine Encephalitis in Alachua C ounty sentinel chickens (n = 15); Eh Eastern Equine Encephalitis in an Alachua County horse ( n = 1).

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98 CHAPTER 5 BLOOD MEAL IDENTIFICATION OF MOS QUITOES COLLECTE D FROM LIGHT TRAPS IN NORTH CENTRAL FL ORIDA (2004-2006). The dynamics of vector and host interactions are an integral part of understanding disease transmission. Knowledge of vector host feedi ng patterns provides insight to viral transmission cycles by identifying possi ble host preferences. Techniques in blood meal analysis have been changing since the early 1920s and have include d direct observation of feeding mosquitoes, quantification by capture in host-baited traps, an d serological and genetic based techniques (Ngo and Kramer 2003). The most common serological and genetic based techniques have been the precipitin test, the Enzyme Linked ImmunoSor bent Assay (ELISA) and Polymerase Chain Reaction (PCR) assays (Ngo and Kramer 2003). Polymerase Chain Reaction amplification of host DNA followed by sequencing is becoming a common method for blood meal detection and has several advantages over precip itin tests and ELISA. Due to the sensitivity of the PCR, a very small amount of DNA can be used as template so even partially engorged mosquitoes can yield a blood meal confirmation. Additionally, with serol ogical tests such as pr ecipitin and ELISA, antisera must be prepared for each potential host species allowing blood meal confirmation to only a limited number of species. With the advent of web-based databases such as GenBank, it is now possible to compare nucleotide sequences and id entify sources of arthropod blood meals. Many studies of mosquito blood meals have em phasized the avian host identifications (Lee et al. 2002; Ngo and Kramer 2004). Other studie s have focused on mosquito feeding patterns by distinguishing between avian and mammalian de rived blood meals (Apperson et al. 2002). Mosquitoes that are primaril y ornithophilic, such as Culiseta melenura (Coquillett) for eastern equine encephalitis virus (family Togaviridae, genus Alphavirus, EEEV) (Scott et al. 1984), play a major role in the amplificat ion of arboviruses. Some mo squito species in the genus Culex prefer avian hosts and are impor tant enzootic vectors of St. Louis encephalitis virus (family

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99 Flaviviridae, genus Flavivirus, SLEV) (Tsai and Mitchell 1989). Culex nigripalpus Theobald is an opportunistic feeder and may serve as an im portant vector of SLEV and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in Florida (Day 2001). Additionally, Culex salinarius Theobald may play a secondary role in the transmission of both SLEV and WNV during times of the year Cx. nigripalpus is less abundant (Day 2001). Another important factor for epidemic arboviral transmission is the well-documented host switching behavior of Cx. nigripalpus (Edman 1974). Culex nigripalpus feeds preferentially on bi rds in the winter and spring and shows an increased preference for mamma lian hosts in the later part of the summer and into the fall. Entomological measures of arboviral transmi ssion risk can be estimated by considering mosquito abundance and age, biting preference, field isolations of virus, and vector competence (Molaei et al. 2006). The primary Culex mosquito vector of WNV differs by geographic location (Hayes and Gubler 2006). Res earch in Florid a suggests that Culex nigripalpus is an important enzootic and epidemic vector of WNV to huma ns (Rutledge et al. 2003, Shaman et al. 2005). However, research is lacking on which mosqu ito species may potentially transmit WNV to horses in Florida. Horses are susceptible to WNV infection and each year fatal WNV horse cases are reported in the US. The purpose of this study was to determine the mosquito blood feeding patterns at a site where horses were stabled ou tdoors year round. To accomplish this goal a twoyear project was designed to coll ect blood fed mosquitoes located near horses. The results of this study may provide valuable insight to poten tial mosquito species vectoring WNV to horses in north Florida.

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100 Materials Methods Blood Fed Mosquito Collections All blood fed mosquito collections were m ade at a site (29 38 N, 82 20 W) in Alachua County in North Florida. Horses ( n = 50) were maintained in out door pastures and in the spring, when mares delivered, the total number of horses on the property in creased to approximately 70. The site was an open pastureland surrounded by urban development (Cha pter 3 Materials and Methods). Mosquitoes were collected twice a w eek using four CDC light traps and a backpack aspirator (John Hock Co., Gain esville, FL) (Chapter 3 Materials and Methods). Mosquitoes were aspirated from four resting boxes that were paired with a light trap and from the outside of the horse-baited stable trap. Additional res ting collections were taken from 10-minute ground aspirations in the surrounding vegetation next to light trap number 1 (Fi gure 3-1). Light trap number 1 was located within the sheltered habi tat of trees, along the e dge of dense vegetation and open pasture where horses were always present. Light tr ap number 2 was located under a small oak stand consisting of seven mature trees. Vegetation and under story at this trap site was sparse and standing water was pres ent after a rainfall event. Li ght trap number 3 was placed adjacent to a horse stable. The grass surrounding the stable was mowed short and no trees were located in the vicinity. Light trap number 4 was set next to a 1500 sq ft retention pond with aquatic vegetation including cattails ( Typha latifolia L.) rushes (Family Juncaceae), and waterlettuce ( Pistia stratiotes L.) (Figure 3-1). All mosquitoes taken from aspirator and light trap collections were immediatel y transported to the la boratory, where they were stored at -80 C. They were then counted on a chill table and iden tified and sorted by species according to Darsie and Morris (2003). All blood engorged females were separated from th e collection and stored individually at -80C. Each mosquito was identified to species and the size and stage of the bloodmeal were

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101 recorded. The size of the bloodmeal was categori zed according to Edman et al. (1975), the sizes were: trace (no distention of the a bdomen), 1/4, 1/2, 3/4 and 1 (fully fed). The stage (estimation of days after a blood meal based on appearance of the abdomen) of the bloodmeal was categorized according to Sella (Detinova 1962) on a one to seven scale, one (unfed) to seven (fully gravid) scale. Blood Meal Identification. The extrac tion and PCR procedures were vali dated and optimized with positive controls. The mosquitoes used as positive controls were Cx. quinquefasciatus and Cx. nigripalpus that were obtained from the USDA (United States Department of Agriculture CMAVE), Gainesville colonies. These mosquitoes were starved for 24 h prior to blood feeding. Culex quinquefasciatus was fed cow blood from a sausage lining and Cx. nigripalpus was allowed to feed on a live chicken (feeding that is a normal part of colony ma intencance at the USDA). The engorged female mosquitoes were frozen at 20 C. After it was validated that the primer sets were successfully amplifying the target DNA, the remaining aliquot from the known host DNA extaction was used as the positive control in subsequent PCRs. An unengorged female mosquito was used as the negative control to ensure that no invertebrate DNA was amplified in the PCR. Blood fed mosquitoes were thawed and placed on a Kimwipe tissue. The abdomen was separated from the thorax by using sterile pipe tte tips to sever the te gument and isolate the bloodmeal from all extraneous material. The DNA from the mosquito abdomen was isolated using a phenol chloroform extraction (Levy et al. 2002). The DNA pellet was resuspended in 10 l of 1mM Tris, and stored at -70 C until later analysis. Two separate PCR reactions were used to amplify the DNA template. Each PCR contained a distinct primer set from the cyto chrome b region of the mitochondrial genome.

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102 Primers were chosen based on previously publis hed blood meal analyses, one primer set was designed to amplify avian blood (Cicero and Johnson 2001) and the other primer set was designed to amplify mammalian blood (Ngo and Kr amer 2003) (Table 5-1). A third set of vertabrate specific primers was used to amplify the sequences if results could not be obtained from the first PCR attempt (Cupp et al. 2004) (Table 5-1). Mammalian and avian PCR assays were run in a final volume of 25 l. Each reaction contained 0.5 mM dNTPs, 3mM MgCl2 and 1.2 units of Taq polymerase (Invitrogen, Carlsbad, CA). The avian assay contained a final primer concentration of 15 pmole per reaction and the mammalian assay contianed 5 pmole of each primer per reaction. Amplification conditions for the avian PCR were 5 min at 93 C with 45 cycles of 94 C for 30 s, 50 C for 30 s, and 72 C for 1 min 30 s with a final extension of 3 min at 72 C. The amplification cycle of the mammalia n PCR was equalivant to the avian PCR conditions, with the exception of the melting temperature, which was lowered to 48 C. The vertebrate specific assay c onditions were 2 min at 94 C with 55 cycles of 94 C for 45 s, 50 C for 50 s, and 72 C for 1 min with a final extension of 7 min at 72 C. The products were visualized on a 1% agarose gel stained with ethidium bromide under UV light. The bands of expected size (508 bp for avia n and 772 bp for mammalian) were cut from the gel and the DNA was extracted using the QIA quick PCR Purification Kit (Qiagen, Valencia, CA). The purified DNA was sequenced using BigD ye Terminator Kit version 1.1 (ABI prism, Foster City, CA). The Interd isciplinary Center for Biotechno logy Research (ICBR) Sequencing Core at the University of Florida loaded th e sequenced DNA and ran the product on a gel. The electropherograms (base pair sequ ence information) were edited using Sequencher version 4.1.2

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103 software (Macintosh). The edited sequences we re compared to the nucleotide database with BLAST analysis software available through NCBI. Results From a total of 45,326 mosquitoes collected in CDC light traps and resting collections, 242 (0.53%) were engorged. Of the 23 mosquito species identified at the stud y site, 14 species were blood fed. A confirmed host blood meal match was obtained for 143 samples of the 242 (59%) blood fed mosquitoes collected. The blood meals we re identified to species or in some cases order and represented mammalian (95%, 136/143), reptilian (2%, 3/143), and avian (3%, 4/143) hosts (Table 5-2). The results of the blood meal analysis are su mmarized in Table 5-2. Of the blood meals obtained from reptiles, the turtle blood meals were isolated from Culex erraticus and Ochleratatus infirmatus and the anole blood meal was isolated from Culex nigripalpus A mixed blood meal was isolated from one Aedes vexans that had fed on both a human and a horse. With the avian primer set there was non-specific amplif ication of cow and reptilian hosts. The blood meals ( n = 9) isolated from Cx. salinarius were from horse and human. The most common blood meal isolated from Cx. nigripalpus was horse (58%); the other blood meals were from human, raccoon, cow and anole. Mansonia titillans fed on horse, human, mouse, cow, and there was a single isolation from a ch icken (Table 5-2). Non-specific amplification w ith the avian primer set occurred. A PCR amplified DNA band of the correct size was obtained in both as says with the mammalian primers and the avian primers for two samples. Prior to sequencing this was thought to be a mixed blood meal, where the mosquito had partially engorged on two se parate hosts. Upon sequencing, both matched up 100% with Bos taurus, the domestic cow. A control sample of cow blood was run with both primer sets and amplified in each assay. Additionally, the avian primer set amplified the two

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104 reptilian derived blood meals. This primer set was designed to be avian specific, but non-target amplification did occur. Howeve r, the unfed mosquito used as a negative contro l never showed any amplification, and all the avia n controls (chicken, dove, and vu lture) amplified consistently. Based on the consistency of amplification in th e positive controls of avian blood, and the absence of amplification of the mosquito negative controls, it appears that there was no interaction with these primers and mosquito DNA, and when avian blood was present, it amplified. Discussion Blood fed mosquitoes we re collected in light trap s, resting traps, and aspirator collections. Host identification studies usually focus on collect ion of mosquitoes from resting sites because these areas yield the highest number of blood fed individuals (Nasci 1984, Apperson et al. 2002). Therefore, the relatively large number of blood fed mosquitoes collected from light traps (n = 199) in this study was unexpected. Light traps specifically attract host-seeking females, and for this reason the majority of mos quitoes collected are normally unf ed. However, Ngo and Kramer (2003) successfully used light trap s in their study of blood meal id entification. The light traps in this study likely collected most of the blood fed individuals because the available vegetation was inadequate to serve as a resting site. Most of the field site ve getation was located at the trap number 1, which was along the edge of the pa sture. The vegetation was not as dense and probably not able to retain the hi gh relative humidity characteristic of a suitable resting habitat (Day 2001). To increase sample size all specimens c ontaining any trace of blood were processed, regardless of Sella stage. A number of samples did not result in DNA amp lification; the old age of the blood meal may have contributed to this. Studies of the time limit of blood meal detection have varied with reports of a maximum of 72 hours (Ngo and Kramer 200 3) to a maximum of 7 days (Lee et al. 2002) before the blood meal was t oo far digested to amplify in PCR. The range

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105 in detection limit is likely due to variation in i ndividual mosquito species ra te of blood digestion. Trace blood meals frequently amplified and the ag e of the blood meal, based on Sella stage, appeared to be a more important factor than size of the blood meal in this study. Blood meals at stage 6 only amplified 4 times and 16 blood meals did not. Because PCR is extremely sensitive, DNA extracted from a small blood meal, but not from an old blood meal, was adequate for amplification. In this study there were onl y three avian blood meals amplif ied and three of these were chicken blood meals almost certainly obtained from the sentinel chickens being held adjacent to the stable trap. It is probable the height of the traps affected th e diversity of mosquitoes caught. Traps placed in the tree canopy will capture higher numbers of mosquitoes that are likely feeding on avian hosts (Anderson et al. 2004). The trap height of 1 meter was chosen to target those mosquitoes active at the ground level and t hose most likely to feed on horses. Horses were pastured in outdoor paddocks and were available to mosquito species active throughout the day and night. Horses were the most common host blood meal isolated in this study. It has been documented that Cx. nigripalpus is a widely opportunistic feeder and has a host range of birds, mammals, and reptiles (Day and Curtis 1994). Edman (1974) found a shift in the feeding pattern of Cx. nigripalpus from avian hosts in the spring to mammalian hosts in the fall. However, in this study Cx. nigripalpus only fed only on mammalian and reptilian blood. The sample size of 20 Cx. nigripalpus amplified blood meals was not sufficient to detect host switching at this site. The fr equent (9/20) horse feeding by Cx. nigripalpus found at this site along with previous resear ch supporting the role of Cx. nigripalpus in WNV transmission, warrants further research into the importance of this species in WNV transmission cycles in north Florida (Rutle dge et al. 2003; Shaman et al. 2005).

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106 Culex salinarius has been implicated in transmission of SLEV and EEEV (Slaff 1990; Cupp et al. 2004). This species is documen ted as readily feeding on horses {Samui, 2003 2335 /id}, and has the ability to travel up to 2.0 km in 1.5 hr (LaSalle and Dakin 1982), allowing it to easily disperse out into the open past ure areas to feed. The blood meals ( n = 9) from Cx. salinarius at this site were exclus ively on horses and humans. Culex salinarius is considered a general feeder attacking indisc riminately both birds and mammals including humans (Andreadis et al. 2001). However, in open agricultural habitats Cx. salinarius has been documented feeding exclusively on mammals (Edman 1974). Because Cx. salinarius has had field isolations of WNV, is a competent laboratory vector of WNV (Turell et al. 2005) and feeds on avian and mammalian hosts it should be further studied in north Florida as a possible epidemic vector. Furthermore, Cx. salinarius is seasonally abundant in the early spring correl ating with the amplification phase of WNV and is continually collected through J une (Figures 4-6 and 4-8). Mansonia titillans was the most frequently collected of the all the species represented at this site. From the 41 am plified blood meals from Ma. titillans, 26 were from horse. The remaining blood meals were from raccoon, mouse, cow, human, and chicken. The role of Ma. titillans in WNV transmission has not b een well studied. But the fact that there was an avian isolation and it readily feeds on horses may be wort h investigating further. The avian isolation, however, was from a chicken located at ground level and not an amplification host. Finally, the seasonal abundance of Ma. titillans does not correlate with amplif ication, or early transmission phases of WNV (Figures 4-5 and 4-7). There were 53 human blood meal s derived from this sample set isolated from eight mosquito species (Table 5-2). With students and workers outside or in open barns throughout

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107 the day and evening, a host-seeking mosquito at this site would have frequent opportunity to encounter a human and obtain a blood meal. As we examine the many epidemiological factors of disease transmission, the clues regarding host preference in wild caught mosquitoes will help broaden our understanding of vector-host interactions. By better understanding which mosqu itoes play a key role in the transmission cycle, new control measures can be developed that are more efficient and target specific species. With tools such as DNA-based amplification, the host sp ecies are more readily and accurately able to be identified. The host preference information, when combined with the existing knowledge of mosquito biology, aids the effort to impr ove arbovirus surveillance and prevention strategies through control pr ograms and public health advisories. Table 5-1. Primer sets in PCR used to amplify DNA from vertebrate hosts. Product Name Sequence size (bp) Reference Avian 5'-GACTGTGACAAAATCCCNTTCCA-3' 508 Cicero and 5'-GGTCTTCATCTYHGGYTTACAAGAC-3' Johnson (2001) Mammalian 5'-CGAAGCTTGATATGAAAAACCATCGTTG-3' 772 Ngo and 5'-TGTAGTTRTCWGGGTCHCCTA-3' Kramer (2003) Invertabrate 5'-CCCCTCAGAATGATATTTGTCCTCA-3' 228 Cupp et al. 5'-GCHGAYACHWVHHYHGCHTTYTCHTC-3' (2004) H = A, C, or T; Y = C or T; V = A, C, or G; N =A, T, C, or G ; R = G or A ; W = A or T.

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108 Table 5-2. Identification of blood meals from mosquitoes collected in Gainesville, FL, October 2004 to November 2006. Mosquito Species # tested # confirmed (%) Results Ae. vexans 4 4 (100) 3 Human; 1 Mixed Horse and Human An. crucians 4 4 (100) 3 Human; 1 Horse An. quadrimaculatus 1 1 (100) 1 Horse Cx. salinarius 14 9 (55) 4 Horse; 5 Human Cx. erraticus 30 27 (90) 1 Night Heron; 1 Box Turtle; 6 Horse; 3 Raccoon; 16 Human Cx. quinquefasciatus 3 1 (33) 1 Chicken Cx. nigripalpus 36 20 (55) 1 Anole; 2 Cow; 9 Horse; 3 Raccoon; 5 Human Ma. titillans 88 41 (56) 1 Chicken; 2 M ouse;3 Cow; 4 Raccoon; 5 Human; 26 Horse Cq. perturbans 15 8 (53) 1 Armadillo; 1 Deer; 6 horse Oc. infirmatus 9 7 (78) 1 Box Turtle; 2 Ho rse; 2 Human; 2 Raccoon Oc. mitchellae 2 1 (50) 1 Horse Ps. columbiae 28 19 (68) 1 Chicken; 1 Deer; 3 Horse; 14 Human Ps. ciliata 3 1 (33) 1 Horse Totals 237 143 Horse ( Equus caballus ), Human ( Homo sapiens), Raccoon ( Procyon lotor ), Cow ( Bos taurus) Chicken ( Gallus gallus ), Deer ( Odocoileus virginianus ), Mouse ( Mus musculus ), Night heron ( Nycticorax nycticorax ), Armadillo ( Dasypuis novemcintus ), Turtle (Terrapene carolina), and Anole ( Anolis trinitatis)

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109 CHAPTER 6 HOST FEEDING, VIRUS SURVEILLANCE AND FUTURE EXPERIMENTS Summary West Nile virus (Fam ily Flaviviridae, genus Flavivirus, WNV) has become endemic in the US and the western hemisphere (Komar and Clark 2006). The introduction of WNV into the New World has provided a unique opportunity to study the spread and epidemiology of an arbovirus in a new geographic setting. The impact of WNV on horses and humans has facilitated collaborations between the veterinary and human medical fields. It has also sparked the development of new diagnostic and surveillance techniques that may help the United States prepare for future disease introductions. Many critical questions about potential mosquito vectors of WNV, the effects of microhabi tat and weather on WNV amplification and transmission, and the blood feeding patterns of potential WNV vectors remain unanswered. Host Feeding Of the 23 mosquito species collected in CDC light traps, resting boxes, and a horse-baited stable trap, during my study, at least some individual females from 14 species had blood fed. Twelve species (fifty eight indivi duals) were positive for horse DNA: Aedes vexans, Anopheles crucians, Anopheles quadrimaculatus, Culex nigr ipalpus, Culex salinarius, Culex erraticus, Mansonia titillans, Coquillettidia perturbans, Ochleratatus infirmatu s, Ochleratatus mitchellae, Psorophora columbia, and Psorophor ciliata. Individual females from eight mosquito species ( Ma. titillans, Cx. erraticus, Cx. salinarius, Cx.nigripalpus Ae. vexans, An. crucians, Oc. infirmatus, and Ps. columbiae ) blood fed on human (n = 48). Mansonia titillans blood fed primarily on mammals at my Alachua County, Fl orida study site. There was, however, a single Ma. titillans blood meal identification from a chicken. If Ma. titillans is a competent vector of WNV, then it could, under certain circumstances, se rve as an epizootic or epidemic vector to

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110 horses and humans in north Florida. Further wo rk, such as vector competence studies and WNV screening of mosquito pools, can help clarify the potential role of this species in WNV transmission. There have been WNV isolations from Ma. titillans, but to my knowledge no work has been done to test vector competen ce. A major factor that may preclude Ma. titillans from vectoring WNV to humans and horses is that th e seasonal abundance peaks late in the fall. Mansonia titillans is not active early in the spring dur ing WNV amplification in the bird population. Instead, this species b ecomes active later in the season and is not likely to encounter a WNV positive bird prior to blood feeding on horses and humans. Culex erraticus is another species that may be worth studying further because this species is an opportunistic feeder, was found at the site frequently, and is considered a competent vector of EEEV (Cupp et al. 2004). While competence fo r one type of arbovirus does not necessarily correlate with competence for another (Hardy et al 1983), this species has had several isolations of WNV in the US. Vector competency studies of Cx. erraticus for WNV should be done. Vector competency studies indicate that other members of the genus Culex (Cx. nigripalpus, Cx. quinquefasciatus, and Cx. tarsalis) are moderate to excellent ve ctors of WNV (Turell et al. 2005). Laboratory studies show that Cx. erraticus is a long-lived species which could contribute to its potential as a WNV vect or (Kline et al. 1987). The horse-baited stable trap used in this study attempted to collect a representation of mosquitoes entering the trap thr oughout the entire night. Many st udies collect for short periods of time directly off the horse or bait animal. Th ese collections record th e species of biting flies that are attracted to the bait animal, but may not truly represent the species that are feeding on the animal. In this study the majority of the mosquitoes (518/ 528, 98%) that were collected from the horse-baited stable trap were blood fed. When the blood was analyzed by PCR it was

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111 confirmed that, all the blood meals were derived from the horse (Chapter 5). Overall, the trap collected a small number of mosquitoes and futu re experiments could include modifications of the construction of the openings to allow for easier entrance of more mosquitoes and to make it more difficult for them to exit. In an experiment al release of mosquitoes inside the stall, many were able to escape from the trap. Perhaps by adding an upward sloping baffle on the interior of the trap, fewer mosquitoes would have been able to exit the trap. Surveillance None of the sentinel animals (three horses, two chickens) te sted positive for any of the arboviruses being monitored (SLEV, EEE, HJ, a nd WNV) in 2005 and 2006. Sentinel chickens are routinely utilized in ar bovirus surveillance programs in the state of Florida and are considered reliable predictors of arboviral activity in an area (Day and Lewis 1991). The location for monitoring sentinel animals was chosen because horse cases occurred at the site in 2001. This site would probably be appropriate to continue monitori ng for virus in future studies, perhaps the addition of more sentinel chickens to the flock would increa se the possibility of arbovirus detection. From the mosquito pools ( n = 359) tested for arbovirus (WNV, SLEV and EEEV), there was one positive SLEV identification. To my know ledge this is the first time SLEV has been isolated from a pool of Ma. titillans Mansonia titillans is involved with tr ansmission of an alphavirus Venezuelan equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, VEEV) (Mendez et al. 2001, Turell et al. 2000). Future studies determining vector competence of Mansonia mosquitoes for WNV and SLEV would be valuable to determine if this genus may play a secondary role in transmi ssion of arboviruses in Florida.

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112 Microenvironment and Weather As part of this study, mi croenvironment and weather were correlated with mosquito abundance and diversity. Several differences were found for trap location at the study site, with the highest mosquito trap location being in Bi vens Forest (Chapter 4). Weather conditions studied (rainfall, temperature and wind speed) were not significantly corr elated with mosquito species abundance. This may have been due to th e yearly meteorological differences at the site and may take a longer study period to establish ho w the climate affects the mosquito populations at this site. Previous field st udies conducted in warm tropical cl imates have found an association between meteorological and lands cape conditions and the inciden ce of mosquito borne disease (Reisen et al. 1993, Dhileepan 1996, Hu et al. 2006) In the United States, Mirimontes et al. (2006) found that high temperature and agricultural land use was associated with an increased incidence of WNV. In Georgia, urbaniza tion was found to increas e risk of human WNV infection (Gibbs et al. 2006). In Texas, mosquito vector populations were correlated with temperature, precipitatio n, and canopy cover (Bolling et al. 2005). In Rhode Island, precipitation was the factor most closely associated with arbovirus activity (Takeda et al. 2003), and in Florida, spring drought followed by rain was spec ifically associated w ith incidence of WNV (Shaman et al. 2005). Given the multiple correlat ions of WNV activity with climatological (long term weather) conditions, perhaps th e collection of weather data at this site for a longer period of time would be informative. Extrinsic Risk Factors of WNV to Horses In the study of the extrinsic risk factors of WN V to horses, several factors were close to significant and warrant further study. The presence of water on the property should be investigated to determine the asso ciation of type of water presen t and risk of WNV infection in horses. Additionally, it would be interesting to study the relationship of fan use and mosquito

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113 activity. Time of day the fans are used a nd the type of fan used could be compared experimentally with the abundance of mosquitoes entering a stable. Investigating these factors could not only help in evaluating WNV risk to horses, but could also be useful in further describing mosquito population dynamics in an agricultural setting. Th e strongest protective factor to horses for WNV was vaccination status The negative comparison group in this study showed signs of arboviral infection and may not have represente d a true negative control. A case control study would be a useful future study. Conclusions Mosquitoes commonly fed on horses, and several Culex spp were abundant and temporally correlated with arboviral activity in north central Florida. Future experiments that focus on arbovirus transmission to horses should consider Cx. erraticus, Cx. nigripalpus, and Cx. salinarius as possible epizootic vector s of EEEV and WNV to horses. The seasonal abundance of these three mosquitoes, and their vector competence in a laboratory setting combined with the blood meals from horse make them strong candidate s as potential arboviral vectors to horses in Florida. The single most important preventative measure a horse owner can take is to vaccinate the horse against WNV twice a year. This is es pecially important in Florida where mosquito activity can occur year round.

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114 APPENDIX A ARBOVIRUS CASE INFORMATION FORM

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115

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116 APPENDIX B ENCEPHALITIS SURVEY 1. What best describes the activity of the horse that became ill: 2. How many years has the horse t hat became ill been at this address: 3. How many horses were on the property when it became ill? 4. How many other horses become ill/di splayed neurological symptoms in: 2000 ____________ 2001 _________ 2002 _______ ___ 2003 ____________ 5. How many other horses have been diagnosed with WNV or EEE: 2000 ____________ 2001 _________ 2002 __________ 2003 ____________ 6. Did the horse survive its clinical symptoms? If its answer is no to question 6, skip to question 13. 7. Did the horse recover to complete activity after its clinical signs? If yes, how lo ng did it take? (months) 8. A. There are changes in your horses personality? B. Does the horse act depressed? C. Are there gait abnormalities? D. Does the horse shake spontaneously? E. Does the horse shake after being ridden? F. Does the horse act weak or is in capable of maintaining its own weight? G. Is there a loss of muscle mass? H. If there is muscle loss is it in a specific area or general loss? 9. If the horse survived, do you still posses it? 10. If the horse was sold, did you receive the expected value? 11. If you did not receive the expected value, estimate your loss. 12. If the horse was sold, how much di d the illness increase your investment? 13. If the horse did not survive, estimate its replacement value. 14. When the horse was ill, what were your veterinarian costs? 15. If you had to miss work, estimate your loss? 16. A. When the horse became ill was vaccinated against WNV? B. If the answer is yes, report when month/year.

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117 C. Were the other horses on the property vaccinated against WNV? D. How many times were the horses vaccinated in the 2001? _____Once ____ Twice _____Three times ____Four times E. How many times were the horses vaccinated in the 2002? _____Once ____ Twice _____Three times _____Four times F. How many times were the horses vaccinated in the 2003? _____Once ______Twice _____Three times ____Four times G. What months were they vaccinated? H. Was the horse that bec ame ill vaccinated against EEE? I. If the answer is if, report when month/year. J. Were the other horses on the property vaccinated against EEE? K. How many times were the horses vaccinated in the 2001? _____Once ____ Twice _____Three times ____Four times L. How many times were the horses vaccinated in the 2002? _____Once ____ Twice _____Three times _____Four times M. How many times were the horses vaccinated in the 2003? _____Once ______Twice _____Three times ____Four times N. What months were they vaccinated? 17. Who applied the vaccine for WNV? ______Myself _______Veterin arian ______Other Who applied the vaccine for EEE? ______Myself _______Veterin arian ______ Other 18. What was the cost to vaccinate on a per horse basis against WNV and EEE in 2000?___________ 2001?________ 2002?_________ 2003?_________ 19. What best describes the way in which the horse was treated when ill? _____ Stabled all day and night. _____ Stabled and let out 2-4 hours during the day. _____ Stabled in the afternoon/outside in the morning and night. _____ Stabled all day/outside all night.____ __ Stabled at night/outside all day. ______ Stabled and let out 2-4 hours in t he evening ______ Outside 24 hours 20. When let out, the horse was in: _____ Pasture (0.5 acres) _____ Grass Paddock _____ Sand Paddock 21. If stabled, how often were the stalls cleaned? _____ Every month _____ 2 times a month _____ every week _____Daily 22. Are there fans in the stable? If yes, how often are they run? _____All day ____Only when necessary _____ Never

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118 23. What best describes the cover of the stable? _____boards with openings _____Solid wood or cement _____Open shed 24. After rain, is there temporary standing water on the property? 25. Mark the different water sources for the horse. ____Bucket _____automatic ____natural ______water tank 26. Describe the type of wa ter that exists on the proper ty. _____ River ______ Stream _______ Lagoon or pool _______ Marsh or wetland 27. What best describes the canopy cover? _____Over barn ____Over grass _____Over both 28. How many piles of debris exist near t he stable, or the area of horse activity? 29. When in the stable, do y ou notice great, medium, or mini mum activity of flies and/or mosquitoes? ____Severe _____Medi um _______Minimum ____ None 30. What best describes the contents of the repe llant used during the time in which the horse was ill? ______Permethrin _____Citronella ______Skin so soft ______ Organic How frequently was the r epellant used? ___ As needed ____ once daily ____ 2-3 times a week ___ Weekly _____ Monthly _____ None 31. How often did you use a flysheet? ___ Never ____ Occasionally ___ Continuously 32. During the time of your horses il lness, did you notice any dead birds on your property? ____ no ____ yes During that same time, were there other ill animals? If yes, please list: ____________ ______________________ Reproductive effects. Please it responds if there was breeding activity on the premises where its horse resided dur ing the time of its disease. 33. Was there any breeding activity on the property since 2000? ____ No ____ Yes 2001? ____ No _____Yes 2002? ____ No _____Yes 2003? ____ No _____Yes 34. In 2000, how many mares checked pregnant at 30 days? ______ In 2001, how many of these mare s foaled? _______ In 2001, how many mare s checked pregnant at 30 days? ______

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119 In 2002, how many of these mare s foaled? _______ In 2002, how many mare s checked pregnant at 30 days? ______ In 2003, how many of these mare s foaled? _______ In 2003, how many mare s checked pregnant at 30 days? ______ In 2004 how many of these mares foaled? _______ 35. Did you vaccinate your mare agai nst the WNV during pregnancy? ____ no _____ yes 36. Were there any abortions after the vaccine against WNV was administered during the gestation or at the end of the pregnancy? _____ no ____ yes If the answer is yes, were other vaccines that were adm inistered at the same time or shortly before the abortion? _____ no _____ yes 37. Have there been abortions during the mont hs of autumn independently of vaccines administered on the property or farm? _____ no _____ yes

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120 APPENDIX C SURVEY REQUEST LETTER February 18, 2004 To whom it may concern, In an effort to gain critical information on th e West Nile Virus and Eastern Equine Encephalitis outbreaks since 2001 in Florida, the University of Florida, in collaboration with the Florida Department of Agriculture and Consumer Serv ices and the United States Department of Agriculture, are asking for your as sistance in filling out the enclos ed survey. This information will help us understand the natural course of mosquito transmitte d encephalitis that threatens Florida horses yearly. Your horse was reported to have ex hibited clinical signs consistent with either West Nile virus or Eastern Equine Encephalitis viru s. Although these signs may or may not have been confirmed by testing, the enclosed survey verifies vaccina tion information submitted at the time of testing, as well as, gathering further information regarding herd health and management. This will allow us to identify risk and management factors that we can then make recommendations about to the horse owning public. Your participation in this study is of great importance, and your response is much appreciated. As a token of our appreciation, please us e the enclosed gift certificate. Enclosed is a postage-paid envelope so that you can return the survey to the University of Florida as soon as possible. You may also fax this in formation to Ashley Cunningham at 352-392-8289. We hope that you will assist us in this important endeavor. Respectfully, The Veterinary Class of 2008 Maureen Long, DVM, PhD, DACVIM-LA Assistant Professor Large Animal Clinical Sciences Enclosures (2)

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121 LIST OF REFERENCES Allison, A.B., D.G. Mead, S. E. J. Gibbs, D.M. Hoffman, and D. E. Stallknecht. 2004. Wes t Nile virus viremia in wild rock pigeons. Emerg. Inf. Dis. 10: 2252-2255. Anderson, J. F., T. G. Andreadis, A. J. Main, and D. L. Kline. 2004. Prevalence of West Nile virus in tree canopy-inhabiting Culex pipiens and associated mosquitoes. Am. J. Trop. Med. Hyg. 71: 112-119. Andreadis, T.G., J. F. Anderson, S. J. Tirrell-Peck. 1998. Multiple is olations of eastern equine encephalitis and highla nds J viruses from mosquitoes (Diptera: Culicidae) during a 1996 epizootic in southeastern Connecticut. J Med Entomol. 1998. 35:296-302. Andreadis, T. G., J. F. Anders on, and C. R. Vossbrinck. 2001. Mosquito surveillance for W est N ile virus in Connecticut, 2000: Isolation from Culex pipiens, Cx. restuans, Cx. salinarius, and Culiseta melanura. Emerg. Inf. Dis. 7: 670-674. Apperson, C. S., B. A. Harrison, T. R. Unnasch, H. K. Hassan, W. S. Irby, H. M. Savage, S. E. Aspen, D. W. Watson, L. M. Rueda, B. R. Engber, and R. S. Nasci. 2002. Host-feeding habits of Culex and other mosquitoes (Diptera: Culicidae) in the Borough of Queens in New York City, with characters and techniques for identification of Culex mosquitoes. J. Med. Entomol. 39: 777-785. Austin, R.J., T. L. Whiting, R.A. Anderson, and M. A. Drebot. 2004. An outbreak of West Nile virus-associ ated disease in domestic geese ( Anser anser domesticus) upon initial introducti on to a geographic region, with evidence of bird to bird transm ission. Can. Vet. J. 45: 117-23. Banet-Noach C., L. Simanov, and M. Malkinson. 2003. Direct (non-vector) transmission of West Nile virus in geese. Avian Pathol. 32: 489-94. Bates, M. 1944. Notes on the construction and use of stable traps for mosquito studies. J. Nat. Malaria Soc. 3: 135-145. Bernard, K. A., and L. D. Kramer. 2001. West Nile virus activit y in the United States, 2001. Viral Immunol. 14: 319-338. Bidlingmayer, W. L. 1967. A comparison of trapping methods for adult mosquitoes: species response and environmental influence. J. Med. Entomol. 4: 200-220. Blackburn, N. K., F. Reyers, W. L. Berry, and A. J. Shepherd. 1989. Susceptibility of dogs to West Nile virus: a survey and pathogeni city trial. J. Comp. Pathol. 100: 59-66. Blackmore, C. G. M., L. M. Stark, W. C. Jeter, R. L. Oliveri, R. G. Brooks, L. A. Conti, and S. T. Wiersma. 2003. Surveillance results from the first

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122 West Nile virus transmission season in Florida, 2001. Am. J. Trop. Med. Hyg. 69:141--150. Bolling, B. G., J. H. Kennedy, and E. G. Zimmerman. 2005. Seasonal dynamics of four potential West Nile species in north-cen tral Texas. J. Vector Ecol. 30: 186-194. Boromisa, R. D., R. S. Copeland, and P. R. Grimstad. 1987. Oral transmission of eastern equine encephalomyelitis virus by a northern Indiana strain of Coquillettidia perturbans. J. Am. Mosq. Control Assoc. 3: 102-104. Bowen, M. F. 1991. The sensory physiology of host-se eking behavior in mosquitoes Ann. Rev. Entomol. 36: 139-158 Brinton, M.A. 2002. The molecular biology of West Nile vi rus: A new invader of the Western Hemisphere. Annu. Rev. Microbiol. 56: 371-402. Bunning M. L., R. A. Bowen, C. B. Cropp, K. G. Sullivan, B. T. Davis, N. Komar, M. S. Godsey, D. Baker, D. L. Hettler, D. A. Holmes, B. J. Biggerstaff, and C. J. Mitchell. 2002. Experimental infection of horses with West Nile virus. Emerg. Inf. Dis. 8: 380-383. Burkett, D. A., J. E. Butler, and D. L. Kline. 1998. Field evaluation of colored light emitting diodes as attractants for woodland mosquitoes an d other Diptera in north central Florida. J. Am. Mosq. Control. Assoc. 14: 186-195. Campbell, G. L., A. A Marfin, R. S. Lanciotti, and D. J. Gubler. 2002. West Nile virus. The Lancet 2: 519-529. Cantile, C., G. DiGuardo, C. Eleni, and M. Arispici. 2000. Clinical and neurological features of West Nile virus equine encephalo myelitis in Italy. Equine Vet J 32:31-35. Carpenter S. J., and E. L. Peyton. 1952. Mosquito studies in the Panama canal zone during 1949 and 1950 (Diptera, Culicidae). American Midland Naturalist 48: 673-682. [CDC] Centers for Disease Control and Prevention 2001. Serosurveys for West Nile virus infectionNew York and Connecticut C ounties, 2000. MMWR Morb Mortal Wkly Rep.50:37-39. [CDC] Centers for Disease Control and Prevention. 2003. West Nile virus. http:\\www.cdc.gov\ncdod\ dvbid\westnile\index.htm [CDC] Centers for Disease Control and Prevention. 2007. West Nile virus. http://www.cdc.gov/ncidod/dvbid/westni le/surv&controlCaseCount06_detailed.htm

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123 Chamberlain, R. W., R. K. Sikes, D. B. Nelson, and W. B. Sudia. 1954. Studies on the North American arthropod-borne encephalitis: VI. Quan titative determinations of virus-vector relationships. Am. J. Hyg. 60:278-285 Cherry, B., S. C. Trock, A. Glasser, L. Kramer, G. D. Ebel, C. Glaser, and J. R. Miller. 2001. Sentinel chikens as a surv eillance tool for West Nile virus in New York City, 2000. ANYAS 343-346. Conway G. R., M. Trpis and G. A. H. McClelland 1974 Population parameters of the mosquito Aedes aegypti estimated by mark-re lease-recapture in a suburban habitat in Tanzania. J. Anim. Ecol. 43: 289-304, Crans, W. J. and T. L. Schulze. 1986. Evidence incriminating Coquillettidia perturbans (Diptera: Culicidae) as an epizootic vector of eastern equine encepha litis. Isolation of EEE virus from C. perturbans during an epizootic among hors es in New Jersey. Bull. Soc. Vector Ecol. 11: 178. Cupp, E. W., K. Klingler, H. K. Hassan, L. M. Viguers, and T. R. Unnasch. 2003. Transmission of eastern equine encephalomye litis virus in central Al abama. Am. J. Trop. Med. Hyg. 68: 495-500. Cupp, E. W., D. Zhang, X. Yue, M. S. Cupp, C. Guyer, T. R. Sprenger, and T. R. Unnasch. 2004. Identification of reptilian and amphibian blood meals from mosquitoes in an eastern eauine encephalomyelitis virus focus in central Alabama. Am. J. Trop Med. Hyg. 71: 272-276. Darsie R. F., and C. D. Morris. 2003. Keys to the adult females and fourth instar larvae of the mosquitoes of Florida (Diptera, Culicidae). Technical Bulletin of the Florida Mosquito Control Association. E.O. Painter Printing Co. DeLeon Springs. Davis, B. S., G. J. Chang, B. Cropp, J. T. Roehrig, D. A. Martin, C. J. Mitchell, R. Bowen, and M. L. Bunning 2001. West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expre sses in vitro a noninfectious recombinant antigen that can be used in enzyme li nked immunosorbent assays. J. Virol. 75 : 4040 4047 Day, J. F. 2001. Predicting St. Louis encephalitis virus epid emics: lessons from recent, and not so recent, outbreaks. Annu. Rev. Entomol. 46: 111-138. Day, J. F. and G. A. Curtis. 1993. Annual emergence patterns of Culex nigripalpus females before, during, and after a widespread St. Loui s Encephalitis epidemic in South Florida. J. Am. Mosq. Control Assoc. 9: 249-255. Day, J. F. and G. A. Curtis. 1994. When it rains they soar and that makes Culex nigripalpus a dangerous mosquito. American Ent. 162-167.

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124 Day, J. F., and A. L. Lewis. 1991. An integrated approach to ar boviral surveillance in Indian River County, Florida. J. Florid a Mosq. Control Assoc. 62:46-52. Day, J. F., and L. M. Stark. 1996. Transmission patterns of St. L ouis encephalitis and eastern equine encephalitis viruses in Florid a: 1978-1993. J. Med. Entomol. 33: 132-139. DeGaetano A. T. 2005. Meteorological effects on adult mosquito ( Culex ) populations in metropolitan New Jersey. Int J Biometeorol. 49: 345-353. Detinova, T. S. 1962 Age-grading methods in Diptera of medical importance. WHO Monogr. 47: 216 pp. Dhileepan, K. 1996. Mosquito seasonality and arboviral di sease incidence in Murray Valley, southeast Australia. Med. Vet. Entomol. 10: 375-384. Dohm, D. J., M. L. OGuinn, and M. J. Turell. 2002. Effect of environmental temperature on the abilitiy of Culex pipiens (Diptera: Culicidae) to transm it West Nile virus. J. Med. Entomol. 39 : 221-225. Durand, B., V. Chevalier, R. Pouillot, J. Labie, I. Marendat, B. Murgue, H. Zeller, and S. Zientata. 2002. West Nile virus outbreak in horses, Southern France, 2000: results of a serosurvey. Emerg. Inf. Dis. 8:777-782. Edman, J. D. 1971. Host-feeding patterns of Florida mosquitoes I. Aedes, Anopheles, Coquillettidia, Mansonia and Psorophora. J. Med. Entomol. 8: 687-695. Edman, J. D. 1979. Host-feeding patterns of Florida mosquitoes (Diptera: Culiciadae) VI. Culex (Melanoconion). J. Med. Entomol. 15: 521-525. Edman, J. D., and W. L. Bidlingmayer, 1969. Flight capacity of blood-engorged mosquitoes. Mosquito News 29: 386-392 Edman, J. D., F. D. S. Evans, and J. A. Williams. 1968. Development of a diurnal resting box to collect Culiseta melanura (COQ.). Am. J. Trop. Med. Hyg. 17: 451-456. Eidson, M., N. Komar, F. Sorhage, R. Nelson, T. Talbot, F. Mostashari, R. McLean, and the West Nile Virus Avian Mortality Surveillance Group. 2001a. Crow deaths as a sentinel surveillance sy stem for West Nile virus in the Northeastern United States, 1999. Emerg. Inf. Dis. 7: 615-620. Eidson, M., L. Kramer, W. Stone, Y. Hagiwar a, K. Schmit, and the New York State West Nile Virus Avian Surveillance Team. 2001b. Dead bird surveillance as an early warning system for West Nile virus. Emerg. Inf. Dis. 7: 631-635. Elizondo-Quiroga, A., A. Flores-Suarez, D. El izondo-Quiroga, G. Ponce-Garcia, and B. J. Blitvich 2006. Host-feeding preference of Culex quinquefasciatus in Monterrey,

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125 Northeastern Mexico. J. Amer. Mosq. Conrtol Assoc. 22: 654-661. Epp, T. Y., C. Waldnre, K. West, F. A. Leighton, and H. G. Townsend. 2005. Efficacy of Vaccination for West Nile Virus in Saskatchewan Horses. In: 51 Annual Convention of the Am erican Association of Equine Pract itioners AAEP, 2005 Seattle, WA, USA, [Ed.]. Publisher: American Association of E quine Practitioners, Lexington KY. Internet Publisher: International Veterinary Information Service, Ithaca NY Epp, T., C. Waldner, K. West, and H. Townsend. 2007. Factors associated with West Nile virus disease fatalities in horses. Can Vet J. 48:1137-45. Fang, Y., and W. K. Reisen. 2006. Previous infection with West N ile or St. Louis encephalitis viruses provides cross protect ion during reinfection in house finches. Am J Trop Med Hyg. 75: 480-485. Farfn-Ale, J. A., B. J. Blitvich, N. L. Marle nnee, M. A. Loroo-Pino, F. Puerto-Manzano, J. E. Carca-Rejn, E. P. Rosado-Paredes, L. F. Flores-Flores, A. Ortega-Salazar, J. Chvez-Medina, J. C. Cremieux-Grimaldi, F. Correa-Morales, G. HernndesGaona, J. F. Mndez-Galvn, and B. J. Beaty. 2006. Antibodies to West Nile virus in asymptomatic mammals, birds, and reptiles in the Yucatan peninsula of Mexico. Am. J. Trop. Med. Hyg. 74: 908-914. [FDOH] Florida State Department of Health 2007 ArboVirus Interagency Task Force. http://www.doh.state.fl.us/Environment/comm unity/arboviral/pdf_files/UpdatedArboguid e.pdf Gibbs, S. E., M. C. Wimberly, M. Madden, J. Ma sour, M. J. Yabsley, and D. E. Stalknecht. 2006. Factors affecting the geographic distribution of West Nile virus in Georgia, USA: 2002-2004. Vector Borne Zoon. Dis. 6: 73-82. Goddard L. B., A. E. Roth, W. K. Reisen, and T. W. Scott. 2002. Vector competence of California mosquitoes for West Nile virus. Emerg. Inf. Dis. 8:1385-91. Godsey, M. S. Jr, R. Nasci, H. M. Savage, S. Aspen, R. King, A. M. Powers, K. Burkhalter, L. Colton, D. Charnetsky, S. Lasater, V. Taylor, and C. T. Palmisano. 2005. West Nile virus-infected mosquitoes, Loui siana, 2002. Emerg. Infect. Dis. 11: 1399-404. Gottfried, K. L., R. R. Gerhardt, R. S. Nasci, M. B. Crabtree, N. Karabatsos, K. L. Burkhalter, B. S. Davis, N. A. Panella, and D. J. Paulson. 2002. Temporal abundance, parity, survival rates, and arbovirus isolation of field-co llected container-inhabiting mosquitoes in eastern Tennessee. J. Am. Mosq. Control. Assoc. 18: 164-172. Guptill, S. C., K. G. Julian, G. L. Campbell, S. D. Price, and A. A. Marfin. 2003. Earlyseason avian deaths from West Nile virus as warnings of human infection. Emerg. Inf. Dis. 9: 483-484.

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126 Hadfield, T. L., M. Turell, M. P. Dempsey, J. David, and J. Park. 2001. Detection of West Nile virus in mosquitoes by RT-P CR. Molec. Cell. Probes. 15: 147-150. Hayes, C. G. 1988. West Nile fever. pp. 59-88 In T. P. Monath [ed.], The arboviruses: Epidemiology and ecology. CRC Press. Boca Raton. Hayes E. B., N. Komar, R. S. Nasci, S. P. Montgomery, D. R. OLeary, and G. L. Cambell. 2005. Epidemiology and transmission dynamics of West Nile virus disease. Emerg. Inf. Dis. 11: 1167-1173. Hayes, E. B. and D. R. OLeary. 2004. West Nile virus infectio n: a pediatric perspective Pediatrics. 113: 1375-1381. Hayes, E. B. and D. J. Gubler. 2006. West Nile virus: epidemiology and clinical features of an emerging epidemic in the United States. Annu. Rev. Med. 57: 181-94. Holck, A. R. and C. L. Meek. 1991. Comparison of sampling techniques for adult mosquitoes and other nematocera in open vegetation. J Entomol. Sci. 26: 231-236. Huang, C., B. Slatter, R. Rudd, N. Parchuri, R. Hull, M. Dupuis, and A. Hindburg. 2002. First isolation of West Nile virus from a patient with encephalitis in the United States. Emerg. Inf. Dis. 8:1367-1371. Hu, W. B., S. L. Tong, K. Mengersen, and B. Oldneburg. 2006. Rainfall, mosquito density and the transmission of Ross River virus: A time-series forecasting model. Ecol. Modeling 196: 505-514. Hubalek, Z., and J. Halouzka. 1999. West Nile fever-a reemerging mosquito-borne viral disease in Europe. Emerg. Inf. Dis. 5: 643-650. Jacobson, E. R., P. E. Ginn, J. M. Troutman, L. Farina, L. Sark, K. Klenk, K. L. Burkhalter, and N. Komar. 2005. West Nile virus infect ion in farmed American alligators ( Alligator mississippiensis) in Florida. J. Wildlife Dis. 41 : 96-106. Jeha, L. E., C. A. Sila, R. J. Lederman, R. A. Prayson, C. M. Isada, and S. M. Gordon. 2003. West Nile virus infection: a new acute paralytic illness. Neurology. 61: 55-59. Kilpatrick, A. M., P. Daszak, M. J. Jones, P. P. Marra, and L. D. Kramer. 2006. Host heterogeneity dominates West Nile viru s transmission. Proc Biol Sci. 273: 2327-2333. Kilpatrick, A. M., L. D Kramer, S. R. Campbell, E. O. Alleyne, A. P. Dobson, and P. Daszak. 2005 West Nile virus risk assessment a nd the bridge vector paradigm. Emerg. Inf. Dis. 11: 425-429.

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127 Kirstein, F., and J. S. Gray. 1996. A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus App. Env. Micro. 62: 4060-4065 Komar, N. 2003. West Nile virus: epidemiology and ecology in North America. Advances in Virus Research 61 : 185-234. Komar, N. and G. C. Clark. 2006. West Nile virus activity in Latin America and the Caribbean. Rev. Panam. Salud Publica. 19: 112-117. Komar, N., S. Langevin, S. Kinten, N. Nemeth, E. Edwards, D. Hettler, N. Davis, R. Bowen, and M. Bunning. 2003. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg. Inf. Dis. 9:311-322. Komar, N., N. A. Panella, J. E. Burns, S. W. Dusza, T. M. Mascarenhas, and T. O. Talbot. 2001. Serologic Evidence for West Nile Virus Infection in Birds in the New York City Vicinity During an Outbreak in 1999. Emerg. Inf. Dis. 7:621-625. Kramer, V. L., E. R. Carper, C. Beesley, and W. K. Reisen. 1995. Mark-Release-Recapture Studies with Aedes dorsalis (Diptera: Culicidae) in Coastal Northern California. J. Med. Entol. 32: 375-380. Lanciotti, R. S., and A. J. Kerst. 2001. Nucleic acid sequence-base d amplification assays for rapid detection of West Nile and St. Louis encephalitis viru ses. J. Clin. Microbiol. 39: 4506-4513. Lanciotti, R. S., A. J. Kerst, R. S. Nasci, M. S. Godsey, C. J. Mitchell, H. M. Savage, N. Komar, N. A. Panella, B. C. Allen, K. E. Volpe, B. S. Davis, and J. T. Roehrig. 2000. Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J. Clin. Microbiol. 38: 4066-4071. Langevin S. A., M. Bunning, B. Davis, and N. Komar. 2001. Experimental infection of chickens as candidate sentinel s for West Nile virus. Em erg. Inf. Dis. 7: 726-729. Lederberg J, R. E. Shope, and S. C. Oakes Jr [eds]. Institute of Medicine (U.S.). Committee on Emerging Microb ial Threats to Health. Emerging infections: microbial threats to health in the United States. Institute of Medicine, Washington DC: National Academy Press; 1992 PP 34-48. Lee, J. H., H. Hassan, G. Hill, E. W. Cupp, T. B. Higazi, C. J. Mitchell, M. S. Godsey, and T. R. Unnasch. 2002. Identification of mosquito avianderived blood meals by polymerase ch ain reaction-heter oduplex analysis. Am. J. Trop. Med. Hyg. 66: 599-604

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128 Lee, J. H., K. Tennessen, B. H. Lilly, and T. R. Unnasch. 2002. Simultaneous detection of three mosquito-borne encephalitis viruses (eastern equine, La Crosse and St. Louis) using a single tube multiplex reverse transcription-PCR assay. J. Am. Mosq. Control Assoc. 18: 26-31. Long, M. T., E. P. Gibbs, M. W. Mellencamp, R. A. Bowen, K. K. Seino, S. Zhang, S. E. Beachboard, and P. P. Humphrey. 2007. Efficacy, duration, and onset of immunogenicity of a West Nile virus vaccine, live Flavivirus chimera, in horses with a clinical disease challenge mode l. Equine Vet J. 39: 491-497. Long, M. T., W. Jeter, J. Hernande z, D. C. Sellon, D. Gosche, K. Gillis, E. Bille, and E. P. Gibbs. 2006. Diagnostic performance of the e quine IgM capture ELISA for serodiagnosis of West N ile virus infection. J Vet Intern Med. 20: 608-613. Lord, C. C., and J. F. Day. 2001. Simulation studies of St. Louis encephalitis and West Nile viruses: the impact of bird mortalit y. Vector Borne Zoonotic Dis. 1: 317-329. Lord, C. C., C. R. Rutledge, and W. J. Tabachnick. 2006. Relationships between host viremia and vector susceptibility for arbovi ruses. J. Med Ent. 43(3):623-630. Love, G. J., and W. W. Smith. 1957. Preliminary observations on the relation of light trap collections to mechanical sweep net collectio ns in sampling mosquito populations. Mosq. News. 17: 9-14. Macdonald, W. W., W. H. Cheong, K. P. Loong, and S. Mahadevan, 1990. A mark-releaserecapture experiment with Mansonia mosquitoes in Malaysia. Southeast Asian J Trop Med Public Health. 21: 424-429. Mackenzie, J. S., D. J. Gubler, and L. R. Petersen. 2004. Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat. Med. 10:98109. Malkinson, M. and C. Banet. 2002. The role of birds in the eco logy of West Nile virus in Europe and Africa. Curr. Top. Microbiol. Immunol. 267: 309-322. Malkinson, M., C. Banet, Y. Weisman, S. Pokamunski, R. King, M. T. Drouet, and V. Deubel. 2002. Introduction of West Nile virus in th e Middle East by migrating white storks. Emerg. Inf. Dis. 8: 392-397. Marfin, A. and D. J. Gubler. 2001. West Nile encephalitis: An emerging disease in the United States. Emerg. Inf. Dis. 33: 1713-1719. McIntosh, B. M., and P. G. Jupp. 1982. Ecological studies on West Nile virus in southern Africa. In: St. Geoge, T. D., B. H. Kay eds. Arbovirus Research in Australlia Proceedings from the 3rd Symposium 15-17 Fe bruary 1982. 82-84.

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136 BIOGRAPHICAL SKETCH Leslie Rios was born in Seat tle Wa shington. She received her bachelors degree from Western Washington University in 1998. Sh e continued her educat ion at Oregon State University studying entomology. She received he r masters degree in 2000. She then worked for the University of Alabama at Birmingham. There she studied West N ile virus in mosquito and bird populations. It was ther e that she fell in l ove with the field of medical entomology and went on to receive her PhD at the University of Florida.