Citation
Infectious Bursal Disease Virus in Wild Turkeys and Sandhill Cranes of Florida

Material Information

Title:
Infectious Bursal Disease Virus in Wild Turkeys and Sandhill Cranes of Florida
Creator:
Candelora, Kristen L
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (66 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Wildlife Ecology and Conservation
Committee Chair:
Percival, Henry F.
Committee Members:
Spalding, Marilyn G.
Giuliano, William M.
Graduation Date:
8/11/2007

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Birds ( jstor )
Blood ( jstor )
Diseases ( jstor )
Feces ( jstor )
Infectious bursal disease virus ( jstor )
Poultry ( jstor )
Quails ( jstor )
Ranches ( jstor )
Wild birds ( jstor )
Wildlife Ecology and Conservation -- Dissertations, Academic -- UF
americana, bursal, canadensis, crane, disease, florida, gallopavo, georgia, grus, infectious, meleagris, sandhill, turkey, virus, whooping, wild
Lake County ( local )
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Wildlife Ecology and Conservation thesis, M.S.

Notes

Abstract:
Captive-reared whooping cranes (Grus americana) released into Florida for the resident reintroduction project experienced unusually high mortality and morbidity during the 1997/98 and 2001/02 release seasons. Infectious bursal disease virus (IBDV) serotype 2 is currently under investigation as the factor that precipitated the mortality events. A small percentage of whooping cranes have been exposed to IBDV in the captive setting. However, many more are being exposed post-release, and prevalence of exposure increases with age or length of time the birds are in the wild in Florida. No studies have been published on the prevalence of IBDV in wild birds of North America. The goal of this study is to provide baseline data that can be used to create effective protocols and take measures to ensure that this virus does not impact the recovery of the endangered whooping crane. To determine if wild exposure to IBDV serotype 2 is possible, captive sentinel chickens were monitored for exposure to the virus on whooping crane release sites in central Florida during the 2003/04 and 2004/05 release seasons. Wild exposure is possible as chickens on both sites became exposed to IBDV serotype 2. To examine the potential for exposure of whooping cranes from other wildlife reservoirs, blood samples were collected from wild turkeys (Meleagris gallopavo) and sandhill cranes (Grus canadensis) in 21 counties throughout Florida and 2 counties in southern Georgia. There is potential for whooping cranes, both resident and migratory, to become exposed to this virus through contact with wild birds as wild turkeys and sandhill cranes in 8 counties in Florida and 1 county in southern Georgia have been exposed to the virus. In addition, there is a significant age effect on seroprevalence in sandhill cranes. These findings are consistent with chicks having a shorter exposure time and immature immune system. The presence of higher seroprevalence and higher titers in older birds suggests that there is constant re-exposure or that birds remain carriers of the virus. To investigate the original source and history of the virus in wild birds of Florida, archived serum samples collected from sandhill cranes were tested for antibodies to IBDV serotype 2. Although I was unable to demonstrate that sandhill cranes in Florida were exposed to IBDV prior to the introduction of captive-reared cranes, the high prevalence and wide distribution of the virus in both sandhill cranes and wild turkeys suggest that the virus has been in Florida for quite some time. Many of the sites where blood was collected from wild turkeys and sandhill cranes overlap with the current distribution of whooping cranes in Florida. The presence of this virus in wild birds in these areas is especially concerning for the resident flock of whooping cranes because they nest and raise their chicks in Florida. The effect of exposure on whooping crane chicks is unknown at this time. However, the impact to young chickens suggests that if whooping crane chicks hatched in the wild are exposed to the virus at an early age, chick survival could be greatly reduced. Conversely, the relatively high titers maintained by adults may be passed on to the chicks and protect them at this otherwise vulnerable period in their lives. ( en )
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 (M.S.)--University of Florida, 2007.
Local:
Adviser: Percival, Henry F.
Statement of Responsibility:
by Kristen L Candelora.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Candelora, Kristen L. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Classification:
LD1780 2007 ( lcc )

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Surnter County L 9~Y j ake County Release Site



Polk County Release Site ~ Three Lakes WMA



Palmdale







Figure 4-1. Captive-reared sandhill crane and whooping crane release sites (depicted with
triangles) and dispersal areas (depicted with dots). Polk and Lake County release
sites are locations where sentinel chickens were placed (Chapter 2).


Table 4-1. Statistical results for exposure prevalence by age of archived sandhill crane samples
collected in Alachua and Osceola counties from May 1992 to March 1998.
Age n Mean SE Median Range
Adult 41 1:104 27 1:64 1:4 to 1:1024
Subadult 36 1:28 4 1:32 1:2 to 1:128
Juvenile 31 1:12 3 1:04 1:0 to 1:64


Gilchrist County


Payn es Prairie


Marion County


Levy County c "











County n
Highlands 11
Sumter 13
Hernando, Pasco, Sumter, Lake 44
Lake 16
Madison, Wakulla, Leon, Gadsen, 11
Jefferson
Osceola 74
Orange 8
Osceola 23
Polk, Pasco 8


Table 3-2. Number of blood samples collected from wild turkeys using rocket nets from
December 2003 through January 2007.
Location County n
Caravelle Ranch WMA Putnam 71
Ordway/Swisher Preserve Putnam 21
Lykes Brothers Ranch Glades 18
Sharp's Ranch DeSoto 13
2-Rivers Ranch Hillsborough 13
Three Lakes WMA Osceola 7
Private Plantations, South Georgia Thomas, Grady 19


Table 3-1. Continued
Location
Hickory Hammock WMA and private property
Lake Panasoffkee WMA
Richloam WMA
Seminole Forest WMA
Tall Timbers Research Station and private
property
Three Lakes WMA
Tosohatchee WMA
Triple N Ranch WMA
Upper Hillsborough WMA





































Figure 2-4. Collecting blood from a chicken via the medial metatarsal vein.









BIOGRAPHICAL SKETCH

Kristen Lee Candelora was born in Gainesville, FL in 1974. She grew up in Tampa,

graduating from Chamberlain High School in 1992. Kristen earned a B.A. in psychology from

the University of North Carolina at Wilmington in 1997. Following graduation, she returned to

Tampa to work as a Crisis Counselor on a Baker Act Unit.

Kristen earned a B.S. in wildlife ecology and conservation from the University of Florida

in 2002. Upon graduation, she began work as a Whooping Crane Biologist for the Florida Fish

and Wildlife Conservation Commission (FWC). After working for FWC for two years, Kristen

entered the Wildlife Ecology and Conservation M. S. program at the University of Florida. She

continued working for FWC while completing her master' s proj ect. She received the Best

Student Paper award at the 10th North American Crane Workshop in Zacatecas, Mexico and the

Florida Chapter of The Wildlife Society spring meeting in Cocoa Beach, Florida.

Upon completion of her M.S. program, Kristen began work as the Private Lands

Coordinator for the Upland Ecosystem Restoration Proj ect.









CHAPTER 1
INTTRODUCTION

The range of the whooping crane (Grus amnericana) once extended from central Canada

south to Mexico, and from Utah to the Atlantic coast. In 1865, the estimated population size was

700-1,400 birds (U.S. Fish and Wildlife Service 1994). Habitat loss, unregulated hunting, and

specimen collection had severe negative impacts, and by 1937 the population was reduced to a

single non-migratory flock in southwestern Louisiana and a single migratory flock that wintered

on the Gulf coast of Texas and nested in an unknown location (later discovered to be Wood

Buffalo National Park in Canada). By 1950, the non-migratory flock had been extirpated and

just 34 birds remained in the migratory flock (U.S. Fish and Wildlife Service 1994).

Congress passed the Endangered Species Preservation Act in 1966, and the whooping

crane was listed as threatened with extinction in 1967. In that same year the Canadian Wildlife

Service and the U.S. Fish and Wildlife Service began collecting eggs from birds in the Wood

Buffalo flock, to generate a captive breeding flock of whooping cranes. The goal was to

propagate whooping cranes, and reintroduce their offspring into the areas from which they were

extirpated. Reintroduction of captive-reared whooping cranes began in 1993 when the Florida

Fish and Wildlife Conservation Commission, in cooperation with several government and private

agencies, released birds on the Kissimmee Prairie in central Florida with the goal of establishing

a resident, non-migratory flock (U.S. Fish and Wildlife Service 1994).

Captive-reared whooping cranes released into Florida for the resident reintroduction

proj ect experienced unusually high mortality and morbidity during the 1997/98 and 2001/02

release seasons. Exposure to infectious bursal disease virus (IBDV) was documented, and may

have been the precipitating factor for these mortality events (Spalding et al. 2006). The purpose

of this study is to provide baseline data on the potential for exposure of whooping cranes to


















Surnter Count
Hernando Cou ntI M Orange County

Polk Countb Osoeola County














Figure 3-2. Blood collection sites for Florida sandhill crane samples collected during 2004, 2005,
and 2006 (n=53).









CHAPTER 2
SENTIENEL CHICKENS

Introduction

Infectious bursal disease virus (IBDV) is a common poultry virus. It is well documented

that birds in domestic operations are being exposed worldwide, and transmission mechanisms are

fairly well understood in this setting (Lukert and Saif 2003). Although there are no published

studies confirming that wild exposure to IBDV has occurred in North America, or demonstrating

that transmission mechanisms exist, Spalding et al. (2006) found that captive-reared whooping

cranes (Grus amnericana) had been exposed to IBDV following their release into Florida. To test

the hypothesis that wild exposure in Florida is possible without direct contact with potentially

infected whooping cranes, sentinel chickens confirmed to be free of disease were monitored for

exposure to IBDV.

Materials and Methods

Six-week-old specific pathogen free (SPF) leghorn chickens were purchased from

Charles River Laboratories, Inc. (251 Ballardvale Street Wilmington, MA 01887-1000). Blood

samples were collected upon arrival to confirm the sentinel chickens had not previously been

exposed to IBDV. Chickens were housed in 32x10xl2 Tomahawk Raccoon/Feral Cat Live

Traps (PO Box 323, Tomahawk, WI, 54487) and provided with fresh food and water daily.

Lake County Release Site

The first cohort of captive-reared whooping cranes for the 2003/04 release season arrived

at the release site on December 8, 2003. The birds were brailed and placed in a portable pre-

release pen. Brails were removed on December 21, 2003 and the birds were free to leave the pen

(Figure 2-1). Three of these birds were positive for exposure to IBDV serotype 2 upon arrival at

the release site. No birds seroconverted while in the pen. The second cohort arrived on












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ........._..... ...............7..____ ......


LIST OF FIGURES .............. ...............8.....


AB S TRAC T ......_ ................. ............_........9


CHAPTER


1 INTRODUCTION ................. ...............11.......... ......


Mortality Events .............. ... ......... ............1
Epidemiology of Infectious Bursal Disease .............. .....................13
Impact of Disease on Population Recovery ....._ .....___ .........__ ...........1
Obj ectives ................. ...............17...___ ......

2 SENTIENEL CHICKENS ............. ...... .__ ...............18..


Introducti on ............. ...... ._ ...............18...
M materials and M ethods .............. ...............18....
Lake County Release Site............... ...............18..
Polk County Release Site .............. ...............20....
Blood Collection and Analysis............... ...............20
Bursal Fluid Aspiration .............. ...............21....
R e sults............... ... ....__ ....... ... .... .... ..............2
Lake County: December 2003 through April 2004 ......____ ........._ ...............22
Polk County: December 2004 through May 2005 ......____ ...... .. ............. ..22
Discussion ............. ...... ._ ...............23...


3 INFECTIOUS BURSAL DISEASE INT WILD BIRDS OF FLORIDA ................. ...............30


Introducti on ................. ...............30.................
Use of Poultry Farms ................... .. .. ........ .. ......... ............3
Human Activity and Disposal of Poultry Products ................. ................. ..........3 1
Contact with W ild Bird s ................. ...............3.. 3.............
Materials and Methods .............. ...............35....
Wild Turkey................. ...............35
Florida Bobwhite Quail .............. ...............37....
Florida Sandhill Crane............... ...............38.
Re sults ................ ...............39.................
Wild Turkey................. ...............39
Florida Bobwhite Quail .............. ...............41....
Florida Sandhill Crane............... ...............41.










whooping crane chicks is unknown at this time. However, the impact to young chickens

suggests that if whooping crane chicks hatched in the wild are exposed to the virus at an early

age, this could greatly reduce chick survival potential. Conversely, the relatively high titers

maintained by adults may be passed on to the chicks and protect them at this otherwise

vulnerable period in their lives.

Age Effect on Seroprevalence

Significantly higher exposure prevalence and average titer levels were found in adult

sandhill cranes captured for this study and in archived samples, when compared to juvenile

cranes. These findings are consistent with the findings of Spalding et al. (2006) that exposure

prevalence in whooping cranes increased with age. The lower seroprevalence and titer levels in

juveniles could be explained by juvenile cranes having a shorter exposure time and immature

immune system. The higher seroprevalence and titer levels in older birds suggest that there is

constant re-exposure or that birds remain carriers of the virus. However, there is also the

possibility that this age effect on seroprevalence reflects decreased survival of sandhill cranes

infected at a young age (Schettler et al. 2001, Garvin et al. 2004). The effect of exposure on

sandhill crane chicks is unknown at this time. However, the impact to young chickens suggests

that if sandhill crane chicks hatched in the wild are exposed to the virus at an early age, chick

survival could be greatly reduced. If exposed chicks are less likely to survive then they are

consequently less likely to be sampled, biasing the chick samples toward birds that have not been

exposed to the virus. Therefore, investigation into the pathogenicity of IBDV in sandhill cranes

is warranted.

Variation in Exposure Prevalence among Sites

Prevalence of exposure in wild turkeys and archived sandhill crane samples varied

among sites. Future research should investigate exposure prevalence in relation to potential



































Figure 2-5. Aspirating bursal fluid from the bursa of fabricius.










(U.S. Fish and Wildlife Service 1994). It is estimated that these remaining birds were highly

interrelated, having an effective population size of only 1.2 birds (Jones et al. 2002). Because

this was the founder population, all whooping cranes that exist today suffer reduced genetic

diversity (Glenn et al. 1999).

In the conservation of small, already compromised populations such as the whooping

crane, immunocompromising diseases such as IBDV could be especially problematic (Thorne

and Williams 1988, Lafferty and Gerber 2002). Once infected with such a disease, host

organisms become susceptible to secondary infection by common but normally benign disease

organisms (Hollmen et al. 2000).

The potential for transmission of IBDV between domestic poultry and wild birds is an

additional concern for the resident flock of whooping cranes in Florida as disease can be an issue

in the recovery of species when the potential for transmission between domestic animals and

endangered species exists. The African wild dog (Lycaon pictus) population has declined

considerably in recent decades, and in 2002 was estimated at less than 5,500 individuals.

Domestic canine diseases such as rabies and canine distemper, possibly transmitted by dogs from

local villages, were some of the suggested causes of this decline (van de Bildt et al. 2002).

According to Thorne and Williams (1988) canine distemper was also common in domestic dogs

in Wyoming. In 1985, a drastic decline was noted in the last known wild population of black-

footed ferrets (M~ustela nigripes). To investigate the cause of the decline, ferrets were captured

from widely separated locations. The fact that all died in captivity of canine distemper, indicated

that the disease was widespread. The 18 known survivors of the disease outbreak were captured

and placed in a captive breeding program. The free-ranging colony was essentially extirpated.













90% -1 OYear 1
SYear 2
80%

70%

60%

50%

40%

30%

20%

10%
%o 0%o 0%o 0%
0%
Caravelle Half Moon Richloam Three Lakes Triple N Ranch
Ranch

Figure 3-4. Wild turkey exposure prevalence (% birds with titer level >1:32) at five sample sites
where 10 or more samples were collected in multiple years.


Table 3-4. P-values for difference in yearly exposure prevalence and average titer level in wild
turkeys at sites where more than 10 samples were collected.
Location nl n2 Average Titer Exposure Prevalence
Level
Caravelle Ranch WMA 36 in 2004 35 in 2005 P<0.0001 P=0.0364
Half Moon WMA 15 in 2004 19 in 2005 P=0.0002 P=0.0011
Richloam WMA 16 in 2004 28 in 2006 NSD P=0.0401
Three Lakes WMA 21 in 2005 53 in 2006 P=0.0362 P=0.0012
Triple N Ranch WMA 11 in 2005 12 in 2006 P=0.0080 P=0.0002


100%









Kruskal-Wallis nonparametric ANOVA was used. Post-hoc analysis on each pair of treatments

was done using the Wilcoxon rank sum test.

Exposure prevalence and average titer level of wild turkeys sampled in this study

(reported in Chapter 3) differed significantly among locations. Analysis of archived samples

allowed for further investigation of this result. To determine if mean titer level differed between

sandhill cranes captured in Alachua and Osceola counties, a Wilcoxon rank sum test was used.

To determine if likelihood of exposure (# samples with titer level > 1:32) was independent of

location, a likelihood ratio (G) test for independence was used. All tests were 2-tailed and

considered significant at P I 0.05. Analysis was performed using the statistical software JMP 7

(SAS 2007).

Results

Of the three samples collected from wild sandhill cranes in Lake County in 1993, all birds

were exposed to IBDV serotype 2. Titer levels ranged from 1:32 to 1:128. None of the samples

collected from captive-reared sandhill cranes released on Three Lakes WMA in 1991 had titer

levels high enough to indicate exposure. Titer levels ranged from 1:0 to 1:4.

Forty-six percent of samples collected from wild sandhill cranes in Alachua and Osceola

counties had titer levels high enough to indicate exposure to IBDV serotype 2 (n = 108, median

= 1:16, range = 1:0 to 1:1024, mean = 1:52, SE = 11i). Sixty-three percent of adults, 56% of

subadults, and 13% of juveniles had been exposed to the virus (Table 4-1, Figure 4-2). Juveniles

had significantly lower exposure prevalence than both adults (P<0.0001) and subadults

(P<0.0002), but there was not a significant difference between adults and subadults. Average

titer level differed among all age groups (P<0.0001) with adults having significantly higher

average titer levels than subadults (P=0.0043) and juveniles (P<0.0001), and subadults having

significantly higher average titer levels than juveniles (p=0.0001).









Obj ectives

I could find no published study confirming that wild birds in the United States have

become exposed to IBDV. However, results from the epidemiological study by Spalding et al.

(2006) indicated that some captive-reared whooping cranes had been exposed to IBDV following

their release into Florida. Therefore, the first obj ective was to test the hypothesis that wild

exposure to IBDV is possible without direct contact with potentially infected captive-reared

whooping cranes.

To investigate how wild exposure may occur I tested the hypothesis that contact with

wild birds is a potential exposure mechanism. For contact with wild birds to be a valid exposure

mechanism, wild birds that whooping cranes share habitat with post-release must be positive for

IBDV exposure. Therefore, the second objective was to determine if such species had been

exposed to the virus and to assess the prevalence of exposure among those species.

The original source of IBDV in wild bird of Florida is unknown. There are many

possibilities related to domestic poultry operations such as: transmission by people carrying the

virus on contaminated footwear, inappropriate disposal of poultry products, and use of poultry

litter as fertilizer in the agricultural industry. Another option is that captive-reared cranes were

exposed to the virus in captivity and they introduced the virus to wild birds of Florida post-

release. However, this possibility might be eliminated if it could be shown that wild birds in

Florida had been exposed to the virus prior to the release of captive-reared cranes. Therefore, the

third obj ective was to test the hypothesis that wild sandhill cranes (Grus Cana~densis) in Florida

were exposed to the virus prior to the release of captive-reared cranes, or in areas where contact

with captive-reared cranes was highly unlikely.









exposed as well serotypee not identified). The authors did not investigate the source of exposure

in these wild birds, but suggested that it could have been the domestic poultry (Nawathe et al.

1978).

When blood samples were collected from 11 species of wild water birds in Western

Australia, evidence of exposure to IBDV serotypee not identified) was found in 7 species

(Wilcox et al. 1983). Antibodies to the virus were most commonly detected in black ducks

(Ana~s superciliosa) from the Perth area. The authors reported that farm ponds used to collect

drainage from poultry sheds are common on commercial poultry farms in Perth, and that black

ducks had been observed using these fresh water ponds.

Sera from king penguins (Aptenodytes patagonicus) on Possession Island of the Crozet

Archipelago in the South Indian Ocean were examined for antibodies to IBDV serotypes 1 and 2.

Chicks and adults had been exposed to both serotypes of the virus. For many years there was a

poultry yard with domestic chickens and ducks in the scientific station on Possession Island.

Although the authors did not know whether any of the domestic poultry had been exposed to

IBDV, these domestic birds did have daily contact with wild birds (Gauthier-Cleric et al. 2002).

Human Activity and Disposal of Poultry Products

It has also been suggested that human activity and disposal of poultry products could be

sources of exposure for wild birds. Gauthier-Cleric et al. (2002) reported that since the 1960s the

beach of the king penguin colony where birds were found to have been exposed to IBDV, was

the main landing point for people, equipment, and food destined for the research station.

Although the authors did not investigate potential exposure mechanisms, the virus may have

been introduced by human activity on the island or by sewage from the poultry yard that was

discharged untreated into a field.
















15 --


10 -cnlc













0 2 4 8 16 32 64 128 256 1024
Titer Level

Figure 4-2. Frequency of titer levels for juvenile, subadult, and adult archived sandhill crane
samples collected in Alachua and Osceola counties from May 1992 to March 1998
(n=108).


Table 4-2. Statistical results for exposure prevalence by county of archived sandhill crane
samples collected in Alachua and Osceola counties from May 1992 to March 1998.
County n Mean SD Median Range
Alachua 55 1:70 149 1:32 1:2 to 1:1024
Osceola 53 1:33 53 1:16 1:0 to 1:256










LIST OF FIGURES


Figure page

2-1 Timeline for Lake County release site. .............. ...............26....

2-2 Chicken trap locations on Lake County release site. ............. ...............27.....

2-4 Collecting blood from a chicken via the medial metatarsal vein. .............. ................28

2-5 Aspirating bursal fluid from the bursa of fabricius. ....._.__._ .... ... .__. ................. .29

3-1 Blood collection sites for wild turkey samples collected from December 2003
through January 2007. ............ ...............43.....

3-2 Blood collection sites for Florida sandhill crane samples collected during 2004,
2005, and 2006............... ...............45..

3-3 Frequency of titer levels for all wild turkey samples collected from December 2003
through January 2007............... ...............46..

3-4 Wild turkey exposure prevalence (% birds with titer level >1:32) at five sample sites
where 10 or more samples were collected in multiple years. ............. .....................4

3-5 Frequency of titer levels for Florida sandhill cranes captured in 2004, 2005, and
2006............... ...............48..

3-6 Titer level results for serum/filter paper comparisons. ............. ...............49.....









Mealworms could be transferred from the poultry house to the wild if litter infested with

mealworms is spread on the fields, and presence of the lesser mealworm has been confirmed in

numerous counties throughout Florida (Dunford and Kaufman 2006).









CHAPTER 5
SYNTHESIS AND SIGNIFICANCE

Wild turkeys and sandhill cranes throughout Florida have been exposed to IBDV

serotype 2. The virus has been present in Florida for at least 15 years and is available to infect

susceptible hosts. Because we know so little about the distribution of this virus in the

environment and its mode of transmission, it is imperative that we conduct further research in

order to learn what, if any, steps can be taken to minimize the effects of this virus on the survival

of endangered whooping cranes. The presence of the virus in Florida could not be linked with

certainty to the reintroduction proj ect, but the evidence is consistent with the virus being present

in the environment for a long time.

Implications for the Whooping Crane Reintroduction Project

Many of the wild turkey and sandhill crane blood collection sites overlap with areas

where whooping cranes are currently found or have been found in the past. Therefore whooping

cranes, both resident and migratory, could come in contact with wild turkeys and sandhill cranes

that have been exposed to IBDV serotype 2. In addition, although these findings do not rule out

other potential exposure mechanisms, they do suggest that post-release interaction with wild

birds of Florida is one potential exposure mechanism for whooping cranes involved in the

1997/98 and 2001/02 mortality events.

The presence of this virus in wild turkeys and sandhill cranes of Florida is especially

concerning for the resident flock of whooping cranes because they nest and raise their chicks in

Florida. When chickens are exposed to IBDV between 3 and 6 weeks of age, symptoms rapidly

appear and mortality rates can approach 30%. When chickens are exposed before 3 weeks of age

a severe, prolonged immunosuppression results, leaving the birds vulnerable to normally benign

disease agents (Lukert and Saif 2003). The effect of exposure to IBDV on prefiedgling










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Howie, R. I., and J. Thorsen. 1981. Identification of a strain of infectious bursal disease virus
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Jackwood, D. J., Y. M. Saif, P. D. Moorhead, and R. N. Dearth. 1981. Infectious bursal disease
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Jackwood, D. J., Y. M. Saif, and J. H. Hughes. 1982. Characteristics and serologic studies of
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Jackwood, D. J., R. E. Gough, and S. E. Sommer. 2005. Neucleotide and amino acid sequence
analysis of a birnavirus isolated from penguins. Veterinary Record 156: 550-552.

Jones, K. L., T. C. Glenn, R. C. Lacy, J. R. Pierce, N. Unruh, C. M. Mirande, and F. Chavez-
Ramirez. 2002. Refining the whooping crane studbook by incorporating microsatellite
DNA and leg-banding analyses. Conservation Biology 16: 789-799.

Lafferty, K. D., and L. R. Gerber. 2002. Good medicine for conservation biology: The
intersection of epidemiology and conservation theory. Conservation Biology 16: 593-604.

Lukert, P. D., and Y. M. Saif. 2003. Infectious bursal disease. Pages 161-179 in Y. M. Saif,
editor. Diseases of Poultry. Eleventh edition. Iowa State University Press, Ames, IA,
USA.









counties in southern Georgia. There is potential for whooping cranes, both resident and

migratory, to become exposed to this virus through contact with wild birds as wild turkeys and

sandhill cranes in 8 counties in Florida and 1 county in southern Georgia have been exposed to

the virus. In addition, there is a significant age effect on seroprevalence in sandhill cranes.

These findings are consistent with chicks having a shorter exposure time and immature immune

system. The presence of higher seroprevalence and higher titers in older birds suggests that there

is constant re-exposure or that birds remain carriers of the virus.

To investigate the original source and history of the virus in wild birds of Florida, archived

serum samples collected from sandhill cranes were tested for antibodies to IBDV serotype 2.

Although I was unable to demonstrate that sandhill cranes in Florida were exposed to IBDV

prior to the introduction of captive-reared cranes, the high prevalence and wide distribution of

the virus in both sandhill cranes and wild turkeys suggest that the virus has been in Florida for

quite some time.

Many of the sites where blood was collected from wild turkeys and sandhill cranes overlap

with the current distribution of whooping cranes in Florida. The presence of this virus in wild

birds in these areas is especially concerning for the resident flock of whooping cranes because

they nest and raise their chicks in Florida. The effect of exposure on whooping crane chicks is

unknown at this time. However, the impact to young chickens suggests that if whooping crane

chicks hatched in the wild are exposed to the virus at an early age, chick survival could be

greatly reduced. Conversely, the relatively high titers maintained by adults may be passed on to

the chicks and protect them at this otherwise vulnerable period in their lives.









seven 9 to 10-month-old captive-reared sandhill cranes were released. Survivors dispersed to

Gilchrist, Levy, Marion, Putnam, and Sumter counties (Nesbitt and Carpenter 1993) (Figure 4-

1). In 1991, 15 captive-reared sandhill cranes ranging from 1 to 2 years-of-age were placed in a

holding pen on Kanapaha Prairie in Alachua County (Figure 4-1). The 11 surviving birds were

then moved to a release pen on the Prairie Unit of Three Lakes WMA in Osceola County (Figure

4-1). Some experimental birds interacted with wild sandhill cranes, and two formed pairs with

wild sandhill cranes (Nesbitt and Folk 1992). In 1993, the first captive-reared whooping cranes

were released at Three Lakes WMA.

Materials and Methods

I was unable to locate any blood samples collected prior to 1971. As a result I could not

test the hypothesis that wild birds in Florida were exposed to IBDV serotype 2 prior to the

release of captive-reared cranes. Instead, I analyzed samples collected from wild birds in an area

where contact with captive-reared cranes was highly unlikely, and hypothesized that these birds

had been exposed to the virus.

From 1991 2000, Dr. Marilyn Spalding archived 477 wild sandhill crane serum samples

collected in 7 counties (Alachua, Citrus, Lake, Levy, Marion, Osceola, Sumter) in Florida.

Samples were eliminated from consideration if they were collected in a county after captive-

reared cranes had been released there or were known to have dispersed there. Unfortunately, this

eliminated from consideration all but 3 samples collected in Lake County in 1993 (Figure 4-1).

These samples were tested for antibodies to IBDV serotype 2. The archived samples also

included serum collected from captive-reared sandhill cranes prior to their release on Three

Lakes WMA in 1991. Samples for 10 of the 11 birds released were tested for antibodies to

IBDV serotype 2.









CHAPTER 4
ARCHIVED SAMPLES

Introduction

The original source of IBDV serotype 2 in wild bird of Florida is unknown. There are

many possibilities related to domestic poultry operations such as transmission of the virus by

poultry workers on contaminated footwear, inappropriate disposal of poultry products, and the

use of litter containing feces as fertilizer in agricultural operations. But once it was determined

that some cranes in captive-rearing facilities had been exposed to IBDV, there was concern that

captive-reared cranes may be responsible for introducing the virus to wild birds of Florida

(Hartup and Sellers 2006). There have been instances when captive-bred animals exposed to a

pathogen in the captive facility, exposed wild animals in and around the release site to those

pathogens (Spalding et al.1996, Snyder et al. 1996, Woodford and Rossiter 1994). This

possibility could be eliminated if it was determined that wild birds in Florida had been exposed

to the virus prior to the release of captive-reared cranes, or in areas where contact with captive-

reared cranes was highly unlikely.

Sandhill cranes reared at Patuxent Wildlife Research Center in Laurel, Maryland were

sporadically released into Florida from 1971 1991 as preliminary trials to develop release

techniques for captive-reared whooping cranes. Fourteen 5-month-old captive-reared sandhill

cranes were released in 1971 near Palmdale in Glades County (Figure 4-1). None of these birds

were observed associating with wild sandhill cranes and all died within 3 months without leaving

the immediate area (Nesbitt 1978). From 1974 1976, 4 captive-reared sandhill cranes ranging

from 6 months to 4 years-of-age were released on Paynes Prairie in Alachua County (Figure 4-

1). Three died and 1 paired with a wild sandhill crane and set up a territory on Paynes Prairie

(Nesbitt 1978). Additional releases took place on Paynes Prairie from 1986 1987. Twenty-









CHAPTER 3
INFECTIOUS BURSAL DISEASE IN WILD BIRDS OF FLORIDA

Introduction

Infectious bursal disease virus has been well studied in commercial poultry operations,

but very little is known about the prevalence or exposure mechanisms in wild birds (Lukert and

Saif 2003). Although I could find no published literature on the incidence of IBDV in wild birds

of North America, studies done in Antarctica, Australia, Crozet Archipelago in the Indian Ocean,

Finland, Ireland, Japan, Nigeria, and Spain indicate that wild birds worldwide are being exposed

to the virus (Nawathe et al. 1978, Wilcox et al. 1983, Gardner et al. 1997, Ogawa et al. 1998,

Hollmen et al. 2000, Campbell 2001, Hoffe et al. 2001, Gauthier-Clerc et al. 2002). Anecdotal

evidence from these studies suggests that exposure to IBDV in wild birds of North America may

be the result of spill-over, the transmission of contagious agents from reservoir animal

populations (often domesticated species) to wildlife occupying the same area (Daszak and

Cunningham 2000). Potential transmission mechanisms are the use of poultry farms, human

activity, disposal of poultry products, the use of poultry litter as fertilizer, and contact with

infected wild birds.

Use of Poultry Farms

The farm environment provides valuable habitat for wildlife, and wild birds in North

America may become exposed to IBDV through their use of poultry farms. This includes both

large commercial operations where wild birds may use drainage ponds and small farms with

free-range poultry where direct contact with wild birds is possible. At the poultry farm of the

National Veterinary Research Institute in Nigeria, evidence of exposure to IBDV serotypee not

identified) was found in six of 50 wild birds captured on the farm. Chickens on the poultry farm

housed in the commercial type setting and those kept as free ranging "back-yard" birds had been









Disposal of poultry products is suspected as the source of exposure in common elders

(Somateria mollissima) and herring gulls (Larus argentatus) in two mixed species breeding

colonies along the Finnish coast. The colony that was close to human development had

significantly greater prevalence of exposure to IBDV serotype 1. Herring gulls from this colony

were observed foraging at a nearby landfill. The authors proposed that gulls foraging at the

landfill may have come in contact with the virus through waste from poultry farms, and then

transmitted the virus when they returned to feed their young (Hollmen et al. 2000).

Human activity is suspected in exposure of two species of Antarctic penguins

(Aptenodytes forsteri and Pygoscelis adeliae) to IBDV serotypee not identified) as evidence of

exposure was found in colonies near centers of human activity, but none of the samples collected

from penguins in a remote and rarely visited site had antibodies to the virus. Authors suggested

that the virus may be spread by people on their footwear, clothing, equipment, or vehicles as they

move around Antarctica. They also proposed that inappropriate disposal of imported poultry

products may have been involved as wild birds could have become exposed when scavenging on

waste and then transmitted the virus to other birds in the area (Gardner et al. 1997).

Use of Poultry Litter as Fertilizer

Poultry litter containing feces is used as fertilizer in the agricultural industry, and this

practice may be involved in transmission of IBDV from domestic operations to the wild. The

virus can be transmitted in chickens through contact with infected feces, but IBDV does not

survive the Maryland Method of dead bird composting, otherwise known as two stage

composting (Murphy 1990). Therefore, the material itself if properly composted is probably not

the source of infection. But lesser mealworms (Alphitobius diaperinus Panzer)~PPP~~~~PPP~~~PPP commonly

inhabit poultry houses where they live in poultry droppings and litter, and the IBDV serotypee



































To my parents, Richard and Betty Candelora; without their support, none of this would have
been possible.










back, and a Kendall Monoj ect 16G x 1-1/2 aluminum hub blunt needle (tyco/Healthcare, Two

Ludlow Park Dr., Chicopee, MA, 01022) was inserted into the vaginal opening of the vent, at a

slightly downward angle. The needle was inserted until it dropped down into the bursa of

fabricius (Figure 2-5). Fluid was aspirated and then inj ected into a sterile solution of phosphate

buffer saline. The solution was frozen and sent to the lab for evaluation.


Results

Lake County: December 2003 through April 2004

Seroprevalence did not differ significantly between groups exposed and not exposed to

crane feces (P = 0.148). Of the 4 chickens in the non-exposure group, three seroconverted and

one was possibly exposed. Of the three chickens with titer levels high enough to indicate

exposure, two had a titer level of 1:64 and one had a titer level of 1:256 indicating recent

exposure. Two of these chickens seroconverted after being on the release site for 101 days. One

chicken seroconverted after being on the release site for 133 days (Figure2-1).

Of the four chickens in the fecal exposure group, one became exposed to IBDV serotype

2, two were possibly exposed, and one was not exposed. The chicken with a titer level high

enough to indicate viral exposure seroconverted after being on the release site for 101 days

(Figure 2-1). The chicken that did not seroconvert is the bird that spent the least amount of time

on the release site. It was found dead in its cage after having been on the release site for 79 days

(Figure 2-1). Necropsy did not reveal cause of death, and no virus was isolated from its tissues.

Polk County: December 2004 through May 2005

Eight sentinel chickens were placed on the release site, but a raccoon killed one within

the first 2 weeks. Data were analyzed on the 7 remaining chickens. Two chickens became

exposed to the virus, one was possibly exposed, and four were not exposed. Of the two chickens









pathogenic to these species (Lukert and Saif 2003). It is Type 2 that appears to have been

involved in the whooping crane mortality event of 2001/02 (Spalding et al. 2006).

Anecdotal evidence suggests IBDV may be pathogenic to other avian species, but no

study has directly linked exposure to clinical illness in any species other than chickens.

Evidence of exposure to IBDV serotypee not identified) was found in Adelie penguin (Pgyoscelis

adeliae) chicks following a mortality event where an infectious agent was suspected to be the

cause. Clinical disease was not apparent at that time, but authors suggested that further

investigation was warranted (Gardner et al. 1997). Populations of common eider (Sontateria

nzollissinta) in the Baltic Sea have declined significantly in some areas and juvenile mortality

was reported to be the primary cause for this decline (Hollmen et al. 2000). Although authors

reported no evidence of illness, exposure to IBDV serotype I was confirmed and exposure

prevalence was highest for elders nesting in areas where duckling survival was low (Hollmen et

al. 2000). In addition, following a mortality event of African black-footed penguins (Spheniscus

dentersus) and macaroni penguins (Eudyptes chrysolophus) in a zoo in the United Kingdom,

Jackwood et al. (2005) were able to isolate IBDV serotype 2 from birds involved. The role of

this virus in the mortality event was unknown, and authors suggested that further study should be

conducted to determine pathogenicity of IBDV in these species.

Impact of Disease on Population Recovery

Interactions between disease and population density play an important role in regulating

animal populations, generally without threat of eliminating the population completely (Lafferty

and Gerber 2002). This is especially true for large populations that occupy large geographic

areas (Woodroffe 1999). As a population expands, animals are forced to live in closer proximity

to one another and share resources with an increasing number of conspecifics. In some situations

these circumstances lead to an increase in stress and a decrease in general condition, leaving









was small (n = 8). Second, it is possible that I did not collect any infected feces. Previous

research indicates that chickens shed the virus in feces for 14 days (Ahad 2002). It is unknown if

or how long whooping cranes may shed the virus in feces, but it is possible that exposed cranes

were not shedding the virus at time of collection.

Regardless of whether or not chickens in the fecal exposure group were exposed to

infected crane feces, some chickens in the non-exposure group did seroconvert. This suggests

that the virus is present in the environment and available to infect susceptible hosts. Therefore,

further research into wild exposure mechanisms with insects acting as the vector is warranted. A

strain of IBDV serotypee not identified) was isolated from mosquitoes (Aedes vexans) trapped in

southwestern Ontario in 1976 (Howie and Thorsen 1981). This species is found throughout the

United States and utilizes a wide range of habitat types (O'Malley 1990). Whooping cranes are

known to have contracted other mosquito born viruses such as Eastern Equine Encephalitis and

West Nile virus (M. G. Spalding, University of Florida, unpublished data). Dung beetles may be

another insect of interest as whooping cranes in Florida have become infected with a nematode

that utilizes dung beetles as an intermediate host (Varela et al. 2001).

Another potential exposure mechanism is the use of poultry litter containing feces as

fertilizer, a practice used regularly on the property adj oining the Lake County release site.

Although the virus can be transmitted in chickens through contact with infected feces, research

indicates that IBDV does not survive the Maryland Method of dead bird composting, otherwise

known as two stage composting (Murphy 1990). Therefore the material itself if properly

composted is probably not the source of infection. But the virus has been isolated from adult

lesser mealworms (Alphitobius diaperinus Panzer) which commonly inhabit poultry houses,

living in poultry droppings and litter (McAllister et al. 1995, Dunford and Kaufman 2006).









Florida Bobwhite Quail

None of the 11 fi1ter paper samples collected on Babcock-Webb WMA were completely

saturated with blood. All were analyzed, and each had a titer level of 1:0. However, of the 16

serum and fi1ter paper samples compared, none resulted in matching titer levels. For 6 birds the

filter paper method overestimated antibody titer level, and for 10 birds it underestimated

antibody titer level (Figure 3-7).

Florida Sandhill Crane

Overall, 7.5% of sandhill cranes were exposed to IBDV serotype 2 (n = 53, mean = 1:10,

SE = 3, median = 1:4, range = 1:0 to 1:128,). Exposure prevalence (p=0.0025) and average titer

level (p=0.0155) were significantly higher in adults than juveniles (Figure 3-6). Forty-three

percent of adults were exposed to the virus (mean = 1:37, SE = 17, median = 1:16, range = 1:0 to

1:128). Two percent of juveniles were exposed to the virus (mean = 1:6, SE = 1, median = 1:3,

range = 1:0 to 1:32).

Discussion

The hypothesis that wild turkeys and sandhill cranes in Florida have been exposed to

IBDV serotype 2 was supported. Overall prevalence of exposure in wild turkeys remained

constant during the 2003/04 and 2004/05 sampling years and then decreased thereafter,

suggesting that viral occurrence may be cyclic in nature. This was supported by exposure

prevalence decreasing significantly between years on all sites tested except Caravelle Ranch

WMA where exposure prevalence increased. But this is the only site where both years used for

comparison were during the sampling years (2003/04 and 2004/05) when overall exposure

prevalence remained constant.









Florida Sandhill Crane

To determine if antibodies to IBDV serotype 2 were present in Florida sandhill cranes, 53

blood samples were collected in seven counties in central Florida: Hernando, Highlands, Lake,

Orange, Osceola, Polk, and Sumter (Figure 3-2). Seven samples were opportunistically collected

from adult sandhill cranes. Four samples were obtained from dead or injured birds (3 from

Osceola County and 1 from Orange County), 2 were collected from nuisance birds (1 from

Highlands County and 1 from Lake County), and 1 was acquired from an extremely tame bird

that we were able to hand grab while capturing chicks at Moss Park in Orange County.

Forty-six samples were collected from pre-fledgling sandhill crane chicks captured

during the 2004, 2005, and 2006 breeding seasons (3 from Hernando County, 7 from Polk

County, 2 from Sumter County, 30 from Osceola County, and 4 from Orange County). Chicks

ranged in age from approximately 25 to 65 days old.

Capture teams of 3 to 5 wildlife biologists drove through known crane-breeding areas

looking for families. Chicks selected for capture were at least 2 weeks of age, and no older than

approximately 70 days so they could not fly. If the chicks could be captured safely, the team

drove as close as possible to the family and ran out and captured the chicks by hand.

Between 1 and 2 mL of blood were collected from the medial metatarsal vein using a 25-

gauge needle. After collection blood was transferred to a lithium heparinized vacutainer.

Pictures were taken of the chicks' head, wings, and body so that age estimate could be

confirmed. Chicks judged to be at least 50 days old were banded with color bands and/or

aluminum FWS bands. Average handling time was 15 minutes, with a range from 6 to 29

minutes. Handling time for each chick varied based on the number of chicks (single or twins),

the ease with which I was able to get a blood sample, whether the chick(s) was old enough to be

banded, and the capture team's level of experience. All blood samples collected from sandhill









Florida Bobwhite Quail

I hoped to obtain blood samples from harvested Florida bobwhite quail in a manner

similar to that of harvested wild turkeys. However, due to the smaller size of bobwhite quail I

was unable to get a sufficient serum sample by collecting blood in a heparinized vacutainer post

mortem, spinning it down, and separating the serum. Therefore, I conducted a pilot study to test

the feasibility of collecting blood with fi1ter paper strips. This method allows for the

determination of antibody titer level with a much smaller amount of blood. First, I investigated

whether enough blood could be collected from dead quail to perform the analysis. Then I

examined whether this method provided an accurate measure of antibody titer level for IBDV

serotype 2.

To examine whether enough blood could be collected from harvested Florida bobwhite

quail to perform the analysis, 11 samples were collected from birds harvested in November of

2004 on Babcock-Webb WMA in Charlotte County. Collectors were instructed to cut the

jugular or brachial vein and saturate 100 diameter Nebuto blood fi1ter strips (Advantec MFS,

Inc., 6691 Owens Dr., Pleasanton, CA, 94588) with as much blood as possible. The goal was to

completely saturate the fi1ter paper strip with blood. Then the filter paper strip was placed in a

small plastic bottle with a desiccant pack (Schleicher & Schuell BioScience, 10 Optical Ave.,

Keene, NH, 03431). Bottles with filter paper strips and desiccant were then sent to the lab for

analysis.

To test the accuracy of the antibody titer level resulting from samples collected with filter

paper strips, serum and filter paper samples were compared for 4 harvested turkeys and 12 live

chickens. Blood was collected in a heparinized vacutainer. Then a filter paper strip was placed

in the vial and saturated with blood. The remaining blood in the vial was spun down, and the

serum separated. Each sample was analyzed for antibodies to IBDV serotype 2.









not identified) has been isolated from adult lesser mealworms up to 14 days after exposure

(McAllister et al. 1995, Dunford and Kaufman 2006). Mealworms could be transferred from the

poultry house to the wild if litter and feces infested with mealworms is spread on fields as

fertilizer (Dunford and Kaufman 2006). Presence of the lesser mealworm has been confirmed in

Alachua, Broward, Charlotte, Clay, Dade, Hillsborough, Indian River, Manatee, Marion, Orange,

Pasco, Pinellas, Polk, Putnam, and Volusia counties and probably occurs throughout the state of

Florida (Dunford and Kaufman 2006).

Contact with Wild Birds

Wild birds for whom the virus is not pathogenic may act as reservoirs of the virus

(Nawathe et al. 1978, Wilcox et al. 1983). To address this question, van den Berg et al. (2001)

performed experimental infections of 3-6 week old commercial pheasants (Pha~sianus

colchicus), grey partridges (Perdix perdix), Japanese quail (Coturnix coturnix jponica), and

guinea fowl (Numida meleagris) using the very virulent strain of IBDV serotypee 1). These

species were chosen because they are closely related evolutionarily to domestic fowl, and

because they are commonly released for hunting or ornamental purposes.

None of these species exhibited clinical signs of illness. Guinea fowl were found to be

fully refractory to infection. Some pheasants and partridges seroconverted, but none excreted the

virus. Quail were susceptible to infection and shed the virus in feces for up to 7 days. Therefore,

Japanese quail have the potential to act as a reservoir of the very virulent strain serotype 1 virus.

However, results from this study do not support the findings of Weisman and Hitchner (1978)

who found Coturnix quail (species not specified) to be refractory to IBDV, although they used

the Classical Strain serotype 1 virus. Therefore Coturnix quail appear capable of transmitting

and being a reservoir of at least one strain of IBDV.





































O 2007 Kristen L. Candelora









INFECTIOUS BURSAL DISEASE VIRUS INT WILD TURKEYS AND SANDHILL CRANES
OF FLORIDA



















By

KRISTEN L. CANDELORA


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007









cranes were handled in the same manner, and evaluated using the same criteria as was used for

wild turkeys.

To determine if likelihood of exposure (# birds with titer level > 1:32) was independent

of age, a likelihood ratio (G) test for independence was used. To determine if average titer level

differed by age, a Wilcoxon rank sum test was used. All tests were 2-tailed and considered

significant at P I 0.05. Analysis was performed using the statistical software JMP 7 (SAS

Institute 2007).

Results

Wild Turkey

Overall, 6% of wild turkeys were exposed to IBDV serotype 2 (n = 596, mean = 1:7, SE =

1, median = 1:2, range = 1:0 to 1:256, Figure 3-3). Evidence of exposure was found in 8

counties in Florida (Hernando, Highlands, Lake, Osceola, Pasco, Polk, Putnam, Sumter) and 1

county in southern Georgia (Grady).

Exposure prevalence (% samples with titer level >1:32) was 13% in 2003/04, 13% in

2004/05, 1% in 2005/06, and 0% in 2006/07 (Table 3-3). To investigate whether exposure

prevalence and average titer level differed significantly between years, I analyzed the Hyve sites

where 10 or more samples were collected in multiple years: Caravelle Ranch WMA-2004 vs.

2005, Half Moon WMA-2004 vs. 2006, Richloam WMA-2004 vs. 2006, Three Lakes WMA-

2005 vs. 2006, and Triple N Ranch WMA-2005 vs. 2006. Exposure prevalence differed

significantly between years at all sites, and average titer level differed significantly between

years at all sites except Richloam WMA (Table 3-4). Exposure prevalence and titer level

decreased over time at all sites except Caravelle Ranch WMA where there was an increase

(Figure 3-4).









statistical software JMP 7 (SAS Institute 2007). The test was 2-tailed and considered significant

at P <0.05.

Polk County Release Site

Five captive-reared whooping cranes arrived for release on December 8, 2004 and were

debrailed on December 22, 2004 (Figure 2-3). None of the captive-reared cranes were positive

for IBDV exposure upon arrival. One bird seroconverted while in the pen (M. G. Spalding,

University of Florida, unpublished data).


Eight SPF chickens were placed in cages on December 11, 2004 and remained on the

release site through May 2005 (Figure 2-3). All 8 chickens were placed in the same area and

none were exposed to crane feces. The cages were placed in a small oak hammock surrounded

by improved pasture.

Blood Collection and Analysis

Blood collection and analysis methods were the same for SPF chickens on both release

sites. A blood sample was collected from each chicken approximately every 2 weeks. One to 2

mL of blood was collected from the medial metatarsal vein (Figure 2-4). A 27-gauge needle was

used on young chickens, and a 25-gauge needle was used when they were full grown. After

collection, blood was transferred into a lithium heparinized vacutainer. All samples were kept

cold until they were spun down and the serum collected. The serum was frozen until it was sent

to the Poultry Diagnostic & Research Center (College of Veterinary Medicine, University of

Georgia, 953 College Station Road, Athens, GA 30602) for evaluation.

All serum samples were tested for IBDV serotype 2 antibodies. Infectious bursal disease

serotype 2 virus neutralizations, using the beta procedure (constant virus/diluted serum), were

performed in primary chicken embryo fibroblast (CEF) cultures prepared from 9-11 day old SPF





Table 3-3.
Year


2003/04 95 1:14 4 1:02 1:0 to 1:256
2004/05 128 1:12 2 1:04 1:0 to 1:128
2005/06 353 1:05 0.3 1:02 1:0 to 1:32
2006/07 20 1:00 0.1 1:00 1:0 to 1:2


200 m


0 +


0 2 4 8 16 32 64 128 256
Titer Level

Figure 3-3. Frequency of titer levels for all wild turkey samples collected from December 2003
through January 2007 (n=596).


Statistical results for yearly exposure prevalence in wild turkeys.
n Mean SE Median Range









sources of infection such as domestic poultry facilities and small family farms with free-ranging

back yard poultry. However, it is important that the research proj ect control for effect of year as

viral occurrence may be cyclic in nature.

Transmission Mechanisms

There are 2 subspecies of the sandhill crane found in Florida. The Florida sandhill crane

(Grus canadensis pratensis) is resident, and the greater sandhill crane (Grus canadensis tabida)

is migratory. These 2 subspecies interact when greater sandhill cranes are in Florida during the

winter. The role that greater sandhill cranes play in the epidemiology of IBDV in wild birds of

Florida is unknown, and certainly warrants further investigation. Out of the 108 archived

samples analyzed, 6 came from greater sandhill cranes. One of these birds had been exposed to

the virus. Therefore, greater sandhill cranes may become exposed to the virus on their wintering

grounds in Florida and carry the virus north with them. Or it is possible that greater sandhill

cranes were exposed first, and are the original source of the virus in wild birds of Florida. Either

way, this is a potential transmission mechanism for IBDV throughout the flyway.

The possibility that wild birds were exposed to the virus as a result of domestic poultry

operations or disposal of poultry products has not been addressed in this study but certainly

remains a potential source of exposure. The use of poultry litter containing feces as fertilizer in

Florida' s agricultural industry remains one potential mechanism for transmission of the virus

from domestic operations to the wild. Farmers should be educated on the importance of properly

composting chicken litter to eliminate the possibility of disease transfer. In addition,

investigation into methods that minimize the transfer of mealworms within the litter could

greatly reduce the potential for transmission from domestic operations to the wild. Research into

potential insect vectors is needed as well, and should focus on mealworms, dung beetles, and

mosquitoes.










exposed to the virus post-release. Very little is known about the incidence of IBDV outside of

poultry operations, especially in North America. Therefore prevalence of the virus in wild birds,

the etiology of the virus, and its transmission mechanism are unknown. The goals of this study

are to investigate whether wild exposure is possible, how wild exposure may occur, and the

origin of the virus in wild birds of Florida.

Epidemiology of Infectious Bursal Disease

Infectious bursal disease is an acute, highly contagious, viral disease that is common on

poultry farms. The pathogenicity, resistance to disinfectants, and persistence in the environment

make it one of the most important viruses of domestic poultry throughout the world (Lukert and

Saif 2003). Infected chickens shed the virus in their feces, and can transmit the virus for at least

14 days (Ahad 2002). Water, feed, and droppings taken from infected pens in poultry houses

were found to be infectious for 52 days (Benton et al. 1967).

There are two forms of this disease, clinical and subclinical. For chickens, the period of

greatest susceptibility to clinical disease is between 3 and 6 weeks of age. The incubation period

is very short, and clinical signs are seen within 2-3 days of exposure. Symptoms such as loss of

appetite, dehydration, and diarrhea rapidly appear and the mortality rate can be as high as 30%

(Lukert and Saif 2003). In chickens infected before 3 weeks of age, a severe, prolonged

immunosuppression occurs (Lukert and Saif 2003). The suppression of their immune system is

similar to the effect of HIV on humans, leaving the birds vulnerable to a variety of ailments that

normally would not be life threatening (Allan et al. 1972). In this weakened state, the birds are

also more susceptible to predation.

There are two serotypes of IBDV. Type 1 occurs in all major poultry producing areas

worldwide, and can cause acute mortality/immunosuppression in chickens. Type 2 is widespread

in poultry flocks in the United States. It is found in both chickens and turkeys, but is non-
















-Ordway-Swisher Presenre

AndewsWMACaravelle Ranch WM~A 4%~
Half M~oon WMALtcyc 24%--- Seminole Foret WVMA
Lake Panasoffee WMA 8%-
,Tosohatchee WnMA
Brooksvl lie -
Richloam WMA 5"/rP Bull Creek WMdA 6%~
Green Swamp WMA# IIp~ I~ ~; Triple N Ranch WMA 30%6
?-Rivers Rand
'Three Lakes WMA 5%g
.1pper '-lmlrorough WMA 139
Hickory Hammock WMrA
AvonR Park Bo mbing Ran ge4?-
Dupuis WMA
Sharp's Ranch
Co rbett WM A
Fisheating Creek W
Lykes Brothers Ran








Figure 3-1. Blood collection sites for wild turkey samples collected from December 2003
through January 2007 (n=596). Exposure prevalence is listed beside each location.
Locations with no percent listed had 0% exposure prevalence.


Table 3 -1. Number of blood samples collected from harvested wild turkeys during the 2004,
2005, and 2006 spring turkey seasons.
Location County n


Choctawhatchee River WMA


tTall Tim~bers Re~search Station
-South Geor la Plantations 5%u


Andrews WMA
Avon Park Bombing Range
Private properties near Brooksville
Bull Creek WMA
Choctawhatchee River WMA and private
property
Corbett WMA
Dupuis WMA
Fisheating Creek WMA
Green Swamp WMA
Half Moon WMA


Levy
Polk, Highlands
Hernando
Osceola
Holmes


Palm Beach
Martin, Palm Beach
Glades
Polk, Sumter, Lake, Pasco
Sumter










McAllister, J. C., C. D. Steelman, L. A. Newberry, and J. K. Skeeles. 1995. Isolation of
infectious bursal disease virus from the lesser mealworm, Alphitobius diaperinus (Panzer).
Poultry Science 74(1): 45-49.

Murphy, D. W. 1990. Disease transfer studies in a dead bird composer. Pages 25-29 in
Proceedings of the 1990 National Poultry Waste Management Symposium. 3 October-4
October 1990, North Carolina, USA.

Nawathe, D. R., O. Onunkwo, and I. M. Smith. 1978. Serological evidence of infection withthe
virus of infectious bursal disease in wild and domestic birds in Nigeria. Veterinary Record
102: 444.

Nesbitt, S. A. 1978. Notes on suitability of captive-reared sandhill cranes for release into the
wild. Proceedings of North American Crane Workshop: 85-88.

Nesbitt, S. A., and M. J. Folk. 1992. Whooping crane reintroduction in Florida final
performance report. Florida Game and Freshwater Fish Commission, Research
Laboratory, Gainesville, FL, USA.

Nesbitt, S. A., and J. W. Carpenter. 1993. Survival and movements of greater sandhill cranes
experimentally released in Florida. Journal of Wildlife Management 57(4): 673-679.

O'Brien, S. J., M. E. Roelke, L. Marker, A. Newman, C. A. Winkler, D. Meltzer, L. Cooley, J. F.
Evermann, M. Bush, and D. E. Wildt. 1985. Genetic basis for species vulnerability in the
cheetah. Science 277: 1428-1434.

Ogawa, M., T. Wakuda, T. Yamaguchi, K. Murata, and A. Setiyono. 1998. Seroprevalence of
infectious bursal disease virus in free-living wild birds in Japan. Journal of Veterinary
Medical Science 60(11): 1277-1297.

O'Malley, C. M. 1990. Aedes vexans (Meigen): an old foe. Proceedings of New Jersey Mosquito
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SAS Institute 2007. JMP 7. SAS Institute, Cary, North Carolina, USA.

Schettler, E., T. Langgemach, P. Sommer, J. Streich, and K. Frolich. 2001. Seroepizootiology
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Smith, B. L. 2001. Winter feeding of elk in western North America. Journal of Wildlife
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Snyder, N. F. R., S. R. Derrickson, S. R. Beissinger, J. W. Wiley, T. B. Smith, W. D. Toone, and
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Conservation Biology 10(2): 338-348.










LIST OF TABLES


Table page

3-1 Number of blood samples collected from harvested wild turkeys during the 2004,
2005, and 2006 spring turkey seasons. ............. ...............43.....

3-2 Number of blood samples collected from wild turkeys using rocket nets from
December 2003 through January 2007. ............. ...............44.....

3-3 Statistical results for yearly exposure prevalence in wild turkeys .................. ...............46

3-4 P-values for difference in yearly exposure prevalence and average titer level in wild
turkeys. ................. ...............47._ _._......

4-1 Statistical results for exposure prevalence by age of archived sandhill crane samples
collected in Alachua and Osceola counties from May 1992 to March 1998. ................... .56

4-2 Statistical results for exposure prevalence by county of archived sandhill crane
samples collected in Alachua and Osceola counties from May 1992 to March 1998.......57









Van den Berg et al. (2001) concluded that persistence of IBDV in wild bird populations is

unlikely to occur and that the source of infection has to be found in poultry farms or the

environment. Captive-reared whooping cranes released in Florida spend the majority of their

time on farms and ranches. These range from large ranches that specialize in cattle and crops, to

smaller farms where owners keep "back-yard" chickens. In addition, the use of litter from

poultry operations as fertilizer is common on some farms. Therefore it is possible that whooping

crane exposure may be a result of their use of the farm environment, or exposure to wild birds

that act as reservoirs of the virus.

For contact with wild birds to be a potential exposure mechanism for whooping cranes, it

first needs to be shown that wild birds utilizing the same habitat have been exposed to the virus.

I tested the hypothesis that wild turkeys (M~eleagris gallopavo), Florida sandhill cranes (Grus

canadensis pratensis), and Florida bobwhite quail (Colinus virginianus florid ana)~~~ddd~~~ have been

exposed to IBDV. These 3 species were chosen based on likelihood of interaction (based on

personal observations made during the 4 years I monitored whooping cranes), and previous

research regarding susceptibility to IBDV in closely related domestic species

I found no previous research on susceptibility of sandhill cranes to IBDV. They were

included because they are closely related to whooping cranes (both in genus Grus), and are the

species I observed captive-reared whooping cranes interacting with most regularly after release.

Wild turkeys were chosen because previous research indicates that domestic turkeys can be

carriers of IBDV. When domestic turkeys are exposed to IBDV they respond serologically and

are capable of transmitting the virus, but do not develop clinical disease (Giambrone et al. 1978,

Weisman and Hitchner 1978, Jackwood et al. 1981, Barnes et al. 1982). If wild turkeys are also

carriers, then they are likely candidates to be natural reservoirs of the virus. In addition, IBDV









February 5, 2004 and brails were removed on February 18, 2004 (Figure 2-1). None of these

cranes were positive for exposure to IBDV serotype 2 upon arrival. One bird seroconverted

while in the pen (M. G. Spalding, University of Florida, unpublished data).

Eight SPF chickens were placed in cages when the first cohort arrived for release

(December 8, 2003) and remained on the site through April 2004 (Figure 2-1). To increase the

chance for viral exposure for another investigation, the chickens were separated into two groups

of four and placed 1.07 km apart. One group of chickens was placed 1.47 km from the release

site, along the edge of a small pine forest (Figure 2-2). This group of chickens was exposed to

crane feces collected from the vicinity of the release pen. The second group of chickens was

placed 0.51 km from the release site, along the edge of an oak hammock (Figure 2-2). This

group was not exposed to crane feces. To minimize cross contamination when checking the

sentinel chickens the protocol was to always visit the non-exposure group first.

Feces were collected from areas surrounding the feeders used by cranes after leaving the

release pen. All were used by the current year' s release birds, past years release birds, and a few

wild sandhill cranes. For each collection attempt, I monitored the feeder being used by the most

current year' s captive-reared whooping cranes. I watched the birds, noting when and where one

defecated. Feces were collected when cranes left the feeder area. When there were multiple

feces in the vicinity, all were collected in an attempt to ensure collection of the target feces.

Feces were placed in the cages of the exposure group when the chickens had been on the site for

43 days, 50 days, and 56 days (Figure 2-1). No feces were collected after the arrival of the

second cohort of captive-reared whooping cranes.

To determine whether the likelihood of becoming exposed to IBDV was independent of

exposure to crane feces, a likelihood ratio (G) test for independence was employed using the









by the feet. An incision, 2.5-5.1 cm in length, was made in the right side of the neck. This

would cut the jugular vein, releasing blood. A lithium heparinized vacutainer was held beneath

the incision and as much blood as possible was collected. From December 2003 through January

2007, 162 blood samples were collected from live birds captured with rocket nets (Table 3-2).

Between 1 and 2 mL of blood was collected from the medial metatarsal vein using a 25-gauge

needle. After collection, blood was transferred into a lithium heparinized vacutainer.

All blood samples were kept cold until they were spun down and the serum collected. The

serum was frozen until it was sent to the Poultry Diagnostic & Research Center (College of

Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA 30602) for

evaluation. All serum samples were tested for IBDV serotype 2 antibodies using the beta

procedure (constant virus/diluted serum) described in Chapter 2.

It is unknown what titer level indicates true exposure to IBDV. Previous studies of wild

birds have considered titer levels ranging from 1:16 to 1:80 as evidence of exposure (Wilcox et

al. 1983, Gardner et al. 1997, Ogawa et al. 1998, Hollmen et al. 2000, van den Berg 2001). For

the purposes of this study I assumed a titer level of 1:16 or less was not indicative of exposure to

the virus. Birds with a titer level of 1:32 or greater were considered exposed to the virus.

The Kruskal-Wallis nonparametric ANOVA and Wilcoxon rank sum tests were used to

determine if average titer level differed by location and year respectively. Post-hoc comparisons

were performed using Wilcoxon rank sum tests. To determine if likelihood of exposure (# birds

with titer level > 1:32) was independent of location and year, a likelihood ratio (G) test for

independence was used. Post-hoc comparisons were performed using the likelihood ratio test as

well. All tests were 2-tailed and considered significant at P I 0.05. Analysis was performed

using the statistical software JMP 7 (SAS Institute 2007).









chicken embryos. Sera were heat inactivated at 56oC for 45 minutes, followed by centrifugation

at 1200 x g for 10 minutes. Fifty microliters of serum was added to the first well of each row

(rows A, B, C, D, E, F, G, and H) in a tissue culture-treated 96 well plate. Serotype 2 IBDV

antigen was diluted to contain 100-500 Tissue Culture Infectious Doseso/50pl~.

Subsequently, 50 CLL of diluted antigen was added to all wells (columns 1-1 1), except

wells in column 12 (this served as a cell control). Serial dilutions were prepared by mixing the

serum and antigen in column 1 and transferring 50 CLL to column 2. Pipette tips were changed

and contents in column 2 were mixed and 50 CLL was transferred to column 3. This was repeated

through column 10 where contents were mixed and 50 CLL were discarded. Column 11 contained

antigen only and was the virus control. Positive serotype 2 IBDV sera and negative sera were

included in assay as controls. One hundred and ninety CLL of CEFs were added to all wells.

Cells were incubated at 37oC for 5 days, cell culture media decanted, cells fixed with methanol

for 1 minute and stained with crystal violet for 1 minute. Virus neutralizing titer was recorded as

the reciprocal dilution of the last well exhibiting no cytopathic effect.

It is unknown what titer level indicates true exposure to IBDV. Previous studies of wild

birds have considered titer levels ranging from 1:16 to 1:80 as evidence of exposure (Wilcox et

al. 1983, Gardner et al. 1997, Ogawa et al. 1998, Hollmen et al. 2000). For the purposes of this

study I assumed a titer level of 1:8 or less indicated no exposure to the virus. Birds with a titer

level of 1:16 were considered possibly exposed. A titer level of 1:32 or greater was assumed to

be indicative of exposure.

Bursal Fluid Aspiration

Seventeen bursal fluid samples were collected from SPF chickens on the Polk County

release site in an attempt to isolate the IBDV serotype 2. The bursa of fabricius is a sac-like

extension of the hindgut, located on the dorsal side of the cloaca. The chicken was placed on its










Spalding, M. G., J. M. Kinsella, S. A. Nesbitt, M. J. Folk, and G. W. Foster. 1996. Helminth
and arthropod parasites of experimentally introduced whooping cranes in Florida. Journal
of Wildlife Diseases 32(1): 44-50.

Spalding, M. G., H. S. Sellers, B. K. Hartup, and G. H. Olsen. 2006. A wasting syndrome in
released whooping cranes in Florida associated with infectious bursal disease titers.
Proceedings of North American Crane Workshop 10 In Press: 00-00.

Thome, T. E., and E. S. Williams. 1988. Disease and endangered species: the black-footed
ferret as a recent example. Conservation Biology 2(1): 66-74.

U. S. Fish and Wildlife Service. 1994. Whooping Crane Recovery Plan. Albuquerque, New
Mexico, USA.

van de Bildt, M. W. G., T. Kuiken, A. M. Visee, S. Lema, T. R. Fitzjohn, and A. Osterhaus.
2002. Distemper outbreak and its effect on African wild dog conservation. Emerging
Infectious Diseases 8: 211-213.

Van den Berg, T. P., A. Ons, M. Dolores, M., and J. F. Rodriguez. 2001. Experimental
inoculation of game/ornamental birds with a very virulent strain of IBDV. Pages 236-246
in Proceedings of the International Symposium on Infectious Bursal Disease and Chicken
Infectious Anaemia. 2001, Rauischholzhausen, Germany.

Varela, A., JM. Kinsella, and M. G. Spalding. 2001. Presence of encysted immature nematodes
in a released whooping crane (Grus americana). Journal of Zoo and Wildlife Medicine 32
(4): 523-525.

Weisman, J., and S. B. Hitchner. 1978. Infectious bursal disease virus infection attempts in
turkeys and cotumix quail. Avian Diseases 22: 604-608.

Wilcox, G. E., R. L. P. Flower, W. Baxendale, and J. S. Mackenzie. 1983. Serological survey of
wild birds in Australia for the prevalence of antibodies to egg drop syndrome 1976 (EDS-
76) and infectious bursal disease viruses. Avian Pathology 12:135-139.

Wiley, N. 2005. Bobwhite management and hunting in Florida' s ranchlands: an overview of
rules and regulations. Proceedings of quail management short course 1: 76-80.

Woodford, M. H., and P. B. Rossiter. 1994. Disease risks associated with wildlife translocation
projects. Pages 178-198 in Olney, P. J. S., Mace, G. M. and A. T. C. Feistner, editors.
Creative Conservation. Chapman & Hall, London, UK.

Woodroffe, R. 1999. Managing disease threats to wild mammals. Animal Conservation 2: 185-
193.










Samples collected in Alachua County had significantly higher average titer level

(P=0.0291) and exposure prevalence (P=0.0307) than those collected in Osceola County. In

Alachua County, 54% of samples had titer levels high enough to indicate exposure, and earliest

evidence of exposure came from samples collected on May 7, 1992. (Table 4-2, Figure 4-3). In

Osceola County, 3 8% of birds had titer levels high enough to indicate exposure, and earliest

evidence of exposure came from samples collected on October 1, 1992 (Table 4-2, Figure 4-3).

Discussion

All 3 sandhill crane samples collected in Lake County prior to the release/dispersal of

captive-reared cranes to that area, had titer levels high enough to indicate exposure to IBDV

serotype 2. Although this may be evidence that the release of captive-reared cranes was not the

original source of the virus, I cannot discount the possibility that wild birds having contact with

captive-reared cranes dispersed to the area. Therefore, I am unable to rule out the possibility that

captive-reared cranes were the original source of the virus in wild birds of Florida. However,

because 10 of the 11 captive-reared sandhill cranes released on Three Lakes WMA in 1991 did

not have titer levels high enough to indicate exposure (1 crane was not tested), we can be fairly

confident that this particular release was not the source of the virus in wild birds of Florida.


Significantly higher exposure prevalence and average titer levels were found in adult

sandhill cranes when compared to juvenile cranes. Subadults were intermediate between adults

and juveniles as there was no statistical difference in prevalence of exposure between adults and

subadults, but there was a significant difference in mean titer level. This is because the maj ority

of subadult samples indicating exposure (75%) had a titer level of 1:32, the lowest possible titer

level to indicate exposure. There were very few samples at the higher end of the spectrum, with









Site specific exposure prevalence ranged from 0% to 30%, with 14 sites having no

detectable evidence of exposure (Figure 3-1). Exposure prevalence (P=0.0003) and average titer

level (P<0.0001) differed significantly between the sample sites. When performing individual

comparisons I eliminated all sites where less than 10 samples were collected (Andrews WMA,

Choctawhatchee River WMA, Tosohatchee WMA, and Upper Hillsborough WMA). The two

sites with the highest exposure prevalence, Triple N Ranch WMA (3 0%) and Half Moon WMA

(24%), did not differ significantly from each other but had significantly higher exposure

prevalence than most other sites. Exposure prevalence on Triple N Ranch WMA was

significantly greater than exposure prevalence on all sites except Brooksville (8%) and Lake

Panasoffkee WMA (8%). Exposure prevalence on Half Moon WMA was significantly greater

than exposure prevalence on all sites except South Georgia (5%), Brooksville, and Lake

Panasoffkee WMA.


When locations were combined into regions (area with a 10.5 mile radius) there was still

a significant difference in exposure prevalence (P=0.0020) and average titer level (P<0.0001).

The two regions with the highest exposure prevalence were the one that included Half Moon

WMA and the one that included Triple N Ranch WMA. Exposure prevalence decreased to 19%

on Half Moon WMA and 9% on Triple N Ranch WMA. This caused the exposure prevalence of

Half Moon WMA to become significantly higher than that of Triple N Ranch WMA. When

locations were combined the number of sites that Half Moon WMA was significantly greater

than was reduced from 18 to 14, and the number of sites that Triple N Ranch WMA was

significantly greater than was reduced from 19 to 10. The only site that Triple N Ranch WMA

did not differ from alone but differed from when combined by region was the site that was

combined with Half Moon WMA.










LIST OF REFERENCES


Ahad, A. 2002. Isolation and pathogenic characterizations of IBDV isolate from an outbreak of
IBD in a rural poultry unit in Bangladesh. Thesis, The Royal Veterinary and Agricultural
University, Copenhagen, Denmark and Bangladesh Agricultural University, Mymensingh,
Bangladesh.

Allan, W. H., J. T. Faragher, and G. A. Cullen. 1972. Immunosuppression by the infectious
bursal agent in chickens immunized against Newcastle disease. Veterinary Records 90:
511-512.

Barnes, H. J., J. Wheeler, and D. Reed. 1982. Serologic evidence of infectious bursal disease
virus infection in lowa turkeys. Avian Diseases 26: 560-565.

Benton, W. J., M. S. Cover, J. K. Rosenberger, and R. S. Lake. 1967. Physicochemical
properties of the infectious bursal agent (IBA). Avian Disease 1 1: 438-445.

Berger, J. 1990. Persistence of different-sized populations: an empirical assessment of rapid
extinctions in bighorn sheep. Conservation Biology 4: 91-98.

Campbell, G. 2001. Investigation into evidence of exposure to infectious bursal disease virus
(IBDV) and chick infectious anaemia virus (CIAV) in wild birds in Ireland. Pages 230-
23 5 in Proceedings of the International Symposium on Infectious Bursal Disease and
Chicken Infectious Anaemia, 2001, Rauischholzhausen, Germany.

Chin, R. P., R. Yamamoto, L. Weiqing, K. M. Lam, and T. B. Farver. 1984. Serological survey
of infectious bursal disease virus serotypes 1 and 2 in California turkeys. Avian Diseases
28(4): 1026-1036.

Daszak, P., and A. A. Cunningham. 2000. Emerging infectious diseases of wildlife--threats to
biodiversity and human health. Science 287(5452): 443-449.

Dunford, J. C., and P. E. Kaufman. 2006. Lesser mealworm, Alphitobius diaperinus.
University of Florida Institute of Food and Agricultural Sciences Publication Number
EENY-367.

Folk, M. J., S. A. Nesbitt, J. M. Parker, M. G. Spalding, S. B. Baynes, and K. L. Candelora.
2006. Current status of nonmigratory whooping cranes (Grus amnericana) in Florida.
Proceedings of North American Crane Workshop 10 In Press: 00-00.

Gardner, H., K. Kerry, M. Riddle, S. Brouwer, and L. Gleeson. 1997. Poultry virus infection in
Antarctic penguins. Nature 387: 245.

Garvin, M. C., K. A. Tarvin, L. M. Stark, G. E. Woolfenden, J. W. Fitzpatrick, and J. F. Day.
2004. Arboviral infection in two species of wild jays (Aves: Corvidae): Evidence for
population impacts. J. Med. Entomol. 41(2): 215-225.













Discussion ................. ...............41.................


4 ARCHIVED SAMPLES ................. ...............50........... ....


Introducti on ................. ...............50.................
Materials and Methods .............. ...............51....
Re sults ................ ...............53.................
Discussion ................. ...............54.................


5 SYNTHESIS AND SIGNIFICANCE .............. ...............59....


Implications for the Whooping Crane Reintroduction Proj ect ....._____ ....... ....__..........59
Age Effect on Seroprevalence .............. ...... ...............60
Variation in Exposure Prevalence among Sites ................. ...............60...............
Transmission Mechanisms............... ...............6


LIST OF REFERENCES ................. ...............62................


BIOGRAPHICAL SKETCH .............. ...............66....









individuals more susceptible to disease while bringing them closer to animals from which they

may contract it (Smith 2001). A decline in population density is often the result. At lower

densities transmission of disease is usually reduced and diseases are spread more slowly, if at all

(Lafferty and Gerber 2002). The disease's role in population regulation then becomes negligible.

The impact of disease on small populations can be very different, posing a serious threat

to the persistence of such populations. If hosts are killed more quickly than they can breed,

population growth rates decline (Woodruff 1999). This perpetuates the small population's

vulnerability to extinction by stochastic events. One example comes from the long-term

monitoring of bighorn sheep (Ovis canadensis). Berger (1990) found that small populations of

50 animals or less were more prone to extinction than populations numbering 100 animals or

more. This is a concern for the Florida resident population of whooping cranes which now

number approximately 60 individuals (Folk et al. 2006).

The threat of extinction caused by disease is also greater in small populations because

they are more likely to have experienced inbreeding, and limited genetic diversity can lead to a

decrease in disease resistance (Glenn et al. 1999). These populations could therefore experience

greater mortality than a genetically diverse population, and disease has proven to be an issue in

some species involved in reintroduction programs when there is a decline or reduction of genetic

diversity in captive-bred animals (Glenn et al. 1999). In 1982 and 1983, 18 cheetahs (Acinonyx

jubatus) in a captive breeding facility died from feline infectious peritonitis. It is unusual for this

disease to cause such high mortality. O'Brien et al. (1985) suggested that mortality was so

significant because the cheetahs lacked an effective immune response as a result of inbreeding.

This concern also applies to whooping cranes because the population experienced a severe

genetic bottleneck in the early 1940's when the migratory flock declined to just 15 or 16 birds













68
64 -
O Filter Paper Strip
60-
56 -1 I 5 Serum
52-
48-
44-
40 -- -
S36-
~32-
S28-
24-
20-
16-












Figure 3-6. Titer level results for serum/filter paper comparisons.










To gain further insight into prevalence of exposure and length of time the virus has been

present in wild sandhill cranes of Florida, 108 archived serum samples collected in Alachua

County (northern Florida) and Osceola County (central Florida) from May 1992 March 1998

were tested for antibodies to IBDV serotype 2. These samples came from 98 individuals.

All serum samples were sent to the Poultry Diagnostic & Research Center (College of

Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA 30602) for

evaluation. They were tested for IBDV serotype 2 antibodies using the beta procedure (constant

virus/diluted serum) described in Chapter 2.

It is unknown what titer level indicates true exposure to IBDV. Previous studies of wild

birds have considered titer levels ranging from 1:16 to 1:80 as evidence of exposure (van den

Berg 2001, Hollmen et al. 2000, Ogawa et al. 1998, Gardner et al., 1997, Wilcox et al. 1983).

For the purposes of this study I assumed that a titer level of 1:16 or less was not indicative of

exposure to the virus. Birds with a titer level of 1:32 or greater were considered exposed to the

V1TUS.

I detected an age effect on seroprevalence in the sandhill crane samples collected for this

study (reported in Chapter 3); however, the adult sample size was small (n = 7). Analysis of the

archived samples separated by age allowed for further investigation of this finding. Samples

were separated into age categories. Juveniles ranged in age from 55 days to 10 months.

Subadults ranged from 12 months to 2.7 years. Adults ranged from 3 to 10+ years. To

determine if likelihood of exposure (# samples with titer level > 1:32) was independent of age,

the likelihood ratio (G) test for independence was used. Post-hoc analysis on each pair of

treatments was done using the same test. To determine if average titer level differed by age, a









IBDV from other wildlife reservoirs, so that effective protocols can be created and measures

taken to ensure that this virus does not impact the recovery of the endangered whooping crane.

Mortality Events

In 1997/98, 14 of 22 captive-reared whooping cranes died within 6 months of their

release. Another mortality event occurred during the 2001/02 release season. Seventy percent of

birds released were affected. Of the 27 birds released 14 died, 5 others became sick and two of

these had to be taken into permanent captivity. Birds from 4 different captive-rearing facilities in

the United States and Canada were involved (Spalding et al 2006). A number of potential causes

were considered (food, water, environmental toxins, herbicides, West Nile virus, coccidiosis, St.

Louis and eastern equine encephalitis) and were ruled out (M. G. Spalding, University of Florida,

unpublished data). Finally, because there was evidence of immunosuppression and the pattern of

illness seemed to be restricted to young of the year, blood samples were tested for exposure to

IBDV.

The pattern of birds testing positive matched well with those that were sick or died,

suggesting that IBDV may have been the factor precipitating the mortality events (Spalding et al.

2006). As a result, an epidemiological study was initiated. This included testing archived blood

samples from whooping cranes for exposure to IBDV, the initiation of a monitoring program for

all captive-reared whooping cranes released into Florida and their offspring, and testing of

whooping cranes in the captive flock. Results showed that some birds had been exposed to

IBDV in the captive setting (Hartup and Sellers 2006). However, many more were exposed after

their release into Florida and prevalence of exposure increased with age or length of time a bird

was in the wild (Spalding et al. 2006).

Infectious bursal disease poses a significant threat to the successful recovery of the

endangered whooping crane, and captive-reared whooping cranes released in Florida are being










that became exposed, one seroconverted after being on the release site for 29 days, the other

seroconverted after 66 days (Figure 2-1). One had a titer level of 1:256, indicating recent

exposure.

The IBDV serotype 2 was not isolated from any bursal fluid samples. Comparison of

titer level from blood collected on the same day as bursal fluid revealed that only one of the

seventeen samples was collected from a bird having a titer level high enough to indicate

exposure.

Discussion

Some sentinel chickens on both release sites seroconverted to IBDV serotype 2,

supporting the hypothesis that wild transmission of IBDV in Florida is possible. The hypothesis

is strengthened by the failure of exposure to potentially infected feces from whooping cranes to

increase the seroconversion rate of the chickens. It is possible, but unlikely, that the chickens

became infected directly from the captive-reared whooping cranes. Cranes may have visited the

chicken cages, although this was never observed. The virus may have been carried by people

when feeding or handling the chickens, although protocol dictated that chickens were visited

before entering the release area to minimize this possibility.

The hypothesis is further supported by the fact that chickens on the Polk County release

site became exposed to IBDV, yet none of the captive-reared whooping cranes released were

positive for IBDV serotype 2 antibodies upon arrival at the release site. However, one of the

captive-reared whooping cranes seroconverted while in the pen. Seroconversion could be the

result of wild exposure or it is possible that the crane was exposed in captivity, but did not

seroconvert until after it was put in the pen.

The finding that likelihood of exposure was not significantly affected by contact with

potentially infected feces must be interpreted with caution for 2 reasons. First, the sample size












Dec 8, 2003: Whooping Crane Cohort
1 placed in pen on the release site. Dec 8, 2003: Sentinel chickens placed
Three cranes were positive for IBDV in cages on the release site
exposure upon arrival.

Dec 21, 2003: Whooping Crane
Cohort 1 released from pen. No cranes
became exposed to IBDV while in the
pen.
Jan 20, 2004: Sentinel chickens in
Exposure Group exposed to crane feces

Jan 27, 2004: Sentinel chickens in
Exposure Group exposed to crane feces

Feb 2, 2004: Sentinel chickens in
Exposure Group exposed to crane feces

Feb 5, 2004: Whooping Crane Cohort
2 placed in pen on the release site.
None of these cranes were positive for
IBDV exposure upon arrival.

Feb 18, 2004: Whooping Crane
Cohort 2 released from pen. One crane-
seroconverted while in the pen.

Feb 27, 2004: Mortality of sentinel
chicken in Fecal Exposure Group

March 19, 2004: Seroconversion of 1
sentinel chicken in Fecal Exposure
Group and 2 chickens in Non-exposure
Group
April 20, 2004: Seroconversion of 1
-sentinel chicken in Non-exposure
Group
April 23, 2004: Sentinel chickens
removed from release site

Figure 2-1. Timeline for Lake County release site.









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

INFECTIOUS BURSAL DISEASE VIRUS INT WILD TURKEYS AND SANDHILL CRANES
OF FLORIDA

By

Kristen L. Candelora

August 2007

Chair: H. Franklin Percival
Major: Wildlife Ecology and Conservation

Captive-reared whooping cranes (Grus amnericana) released into Florida for the resident

reintroduction project experienced unusually high mortality and morbidity during the 1997/98

and 2001/02 release seasons. Infectious bursal disease virus (IBDV) serotype 2 is currently

under investigation as the factor that precipitated the mortality events. A small percentage of

whooping cranes have been exposed to IBDV in the captive setting. However, many more are

being exposed post-release, and prevalence of exposure increases with age or length of time the

birds are in the wild in Florida. No studies have been published on the prevalence of IBDV in

wild birds of North America. The goal of this study is to provide baseline data that can be used

to create effective protocols and take measures to ensure that this virus does not impact the

recovery of the endangered whooping crane.

To determine if wild exposure to IBDV serotype 2 is possible, captive sentinel chickens

were monitored for exposure to the virus on whooping crane release sites in central Florida

during the 2003/04 and 2004/05 release seasons. Wild exposure is possible as chickens on both

sites became exposed to IBDV serotype 2. To examine the potential for exposure of whooping

cranes from other wildlife reservoirs, blood samples were collected from wild turkeys (M~eleagris

gallopavo) and sandhill cranes (Grus canadensis) in 21 counties throughout Florida and 2



























0 2 4 8 16 32 64 128 256
Titer Level
Figure 3-5. Frequency of titer levels for Florida sandhill cranes captured in 2004, 2005, and
2006 (n=53).


O Juveniles


I I


SAdults


I n


































Figure 2-2. Chicken trap locations on Lake County release site.


Dec 8, 2004: Whooping cranes
placed in pen on release site. No
cranes were positive for IBDV
exposure upon arrival.

Dec 11, 2004: Sentinel chickens
placed in cages on the release site

Dec 22, 2004: Whooping cranes
released from pen. One crane-
seroconverted while in the pen.

Jan 19. 2005 Ser.coni\ version of 1
sentinel c~hick~en

Feb 15. 200(5 Ser~oconi version of 1
sentinel c~hick~en


May 3 2005: Sentinel chickens
removed from release site

Figure 2-3. Timeline for Polk County release site.









I was unable to assess site specific variation in exposure prevalence of sandhill cranes

due to small samples sizes, but prevalence of exposure in wild turkeys did vary among sites.

However, this result must be interpreted with caution as 12 of the 14 sites with no detectable

evidence of exposure were sites sampled only in 2006, a year with extremely low exposure

prevalence statewide. If viral occurrence is cyclic in nature then differences in site specific

exposure may be the result of populations being in different phases of this cycle.

Adult sandhill cranes captured for this study had significantly higher seroprevalence and

average titer level than juveniles. Although the adult sample size was small, this is consistent

with findings of Spalding et al. (2006) that exposure prevalence in whooping cranes increases

with age.

I was unable to assess the prevalence of exposure to IBDV serotype 2 in Florida

bobwhite quail. Collecting a sufficient amount of blood for analysis from harvested birds proved

difficult, and results from the fi1ter paper comparative study demonstrated that the resulting titer

levels were not accurate. Therefore the use of filter paper strips was not a reliable method to

determine the antibody titer level for IBDV serotype 2 in Florida bobwhite quail.

Hunters involved in the pilot study on Babcock-Webb WMA were cooperative and

exhibited interest in being involved in the investigation of disease prevalence in Florida

bobwhite quail. Large numbers of quail are harvested each year and present an opportunity to

investigate disease issues in this species. Alternative methods for collecting blood from

harvested quail that could be used for disease studies should be explored. But it appears that

future research investigating prevalence of IBDV would have to involve sacrificing quail

specifically for the disease study or live trapping to collect blood samples.









no subadult birds having a titer level higher than 1:128. In contrast, 31% of adults had titer

levels greater than 1:128 and only 19% had a titer level of 1:32.

The significant trend toward higher titer levels as the birds age suggests that there is

constant re-exposure or that birds remain carriers of the virus. If sandhill cranes are re-exposed

to the virus throughout their lifetime they could mount a more effective immune response with

each subsequent exposure thus leading to higher titer levels as the birds age. Although unknown

for IBDV in cranes, birds that are carriers of a virus could have a latent infection that results in

intermittent shedding of the virus throughout their life. This could result in higher titer levels as

the birds age as well.









serotype 2 has been detected in domestic turkeys in the United States (Jackwood et al. 1982,

Chin et al. 1984). Although I observed minimal interaction between wild turkeys and whooping

cranes, wild turkeys were observed foraging in the same pastures used by whooping cranes.

Florida bobwhite quail also share habitat with whooping cranes. Although I never

observed direct interaction between these two species, both were observed using edge habitat in

agricultural areas. No studies have been published on prevalence of IBDV in this species of

quail. However, previous research indicates that Coturnix quail are capable of transmitting at

least 1 strain of the virus, serotype 1 (van den Berg 2001). Although Coturnix quail are in a

different family (Old World quail of the family Phasianidae) than bobwhite quail (New World

Quail of the Family Odontophoridae), it is possible that they are capable of transmitting the virus

as well. In addition, there is another mode of transmission of the virus from a domestic source

into the wild in Florida as pen-raised bobwhite quail are often released for dog training and

hunting (Wiley 2005).

Materials and Methods

Wild Turkey

To determine if populations of wild turkey had been exposed to IBDV, 596 blood

samples were collected from wild turkeys at 24 locations in 21 counties throughout Florida

(DeSoto, Gadsden, Glades, Hernando, Highlands, Holmes, Jefferson, Lake, Leon, Levy,

Wakulla, Madison, Martin, Orange, Osceola, Palm Beach, Pasco, Polk, Putnam, Sumter,

Wakulla) and 3 adj oining plantations in Thomas and Grady counties in southern Georgia (Figure

3-1).

Two methods were used to collect these samples. During the 2004, 2005, and 2006

spring turkey-hunting seasons, 434 blood samples were collected from hunter-harvested wild

turkeys (Table 3-1). When hunters brought a bird to the check station, it was hung upside down















12-
SOsceola County





,8-


0 6-











0 2 4 8 16 32 64 128 256 1024

Titer Level

Figure 4-3. Frequency of titer levels for archived sandhill crane samples collected in Alachua
and Osceola counties from May 1992 to March 1998 (n=108).









ACKNOWLEDGMENTS

I thank Larry Perrin, Roger Shields, and Jeremy Olson for allowing me to participate in

the research proj ect investigating the use of camera traps as a potential method for wild turkey

population estimation. This collaboration not only contributed samples to the project, but also

resulted in alternate collection methods that ultimately were responsible for the maj ority of

samples collected. I also thank my committee members, Marilyn Spalding, Franklin Percival,

Bill Guiliano, and Steve Nesbitt for their guidance and patience. I am especially grateful to

Marilyn Spalding who spent countless hours guiding the direction of the proj ect and reviewing

my thesis, and contributed archived sandhill crane samples for analysis. My sincere thanks go to

the Florida Fish and Wildlife Conservation Commission (FWC) biologists who were integral in

acquiring data from wild turkey populations on Wildlife Management Areas in Florida (David

Blood, Pam Boody, Mike Brooks, Victor Chavez, Matthew Chopp, Jean-Marie Conners, Nancy

Dwyer, Tim Farley, Steve Glass, Lewis Gonzales, Linda King, Tom O'Neil, Jayde Roof,

Nicholas Snavely, Valerie Sparling, Rick Spratt, Grant Steelman, Justin Nolte, and Jason

Williams) and the check station operators working on those areas. I acknowledge the FWC

Whooping Crane Reintroduction Program biologists Jeannette Parker, Marty Folk, Steve Baynes,

and Kathy Chappell who went above and beyond the call of duty collecting data from wild

Florida sandhill cranes and sentinel chickens. I thank Holly Sellers for analyzing all samples

collected for this study, and Jamie Miller for her technical and administrative assistance. I thank

Charlie Luthin and the Natural Resources Foundation of Wisconsin for their generous support.

Most of all my tremendous gratitude goes to my parents, Richard and Betty Candelora, for

making my schooling a priority and for always giving me their constant support in every way.




Full Text

PAGE 1

1 INFECTIOUS BURSAL DISEASE VIRUS IN WILD TURKEYS AND SANDHILL CRANES OF FLORIDA By KRISTEN L. CANDELORA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Kristen L. Candelora

PAGE 3

3 To my parents, Richard and Betty Candelora; wi thout their support, none of this would have been possible.

PAGE 4

4 ACKNOWLEDGMENTS I thank Larry Perrin, Roger Shie lds, and Jeremy Olson for al lowing me to participate in the research project inve stigating the use of camera traps as a potential method for wild turkey population estimation. This collaboration not only contributed samples to the project, but also resulted in alternate collection methods that ul timately were responsible for the majority of samples collected. I also thank my committee me mbers, Marilyn Spalding, Franklin Percival, Bill Guiliano, and Steve Nesbitt for their guidance and patience. I am especially grateful to Marilyn Spalding who spent countless hours guiding the direction of the project and reviewing my thesis, and contributed archived sandhill crane samples for analysis. My sincere thanks go to the Florida Fish and Wildlife Conservation Comm ission (FWC) biologists w ho were integral in acquiring data from wild turkey populations on W ildlife Management Areas in Florida (David Blood, Pam Boody, Mike Brooks, Victor Chavez, Matthew Chopp, Jean-Marie Conners, Nancy Dwyer, Tim Farley, Steve Glass, Lewis Gon zales, Linda King, Tom ONeil, Jayde Roof, Nicholas Snavely, Valerie Sparling, Rick Spra tt, Grant Steelman, Justin Nolte, and Jason Williams) and the check station operators work ing on those areas. I acknowledge the FWC Whooping Crane Reintroduction Program biologists J eannette Parker, Marty Folk, Steve Baynes, and Kathy Chappell who went above and beyond th e call of duty collectin g data from wild Florida sandhill cranes and sentinel chickens. I thank Holly Sellers for analyzing all samples collected for this study, and Jamie Miller for her t echnical and administrative assistance. I thank Charlie Luthin and the Natural Resources Foundation of Wisconsin for their generous support. Most of all my tremendous gratitude goes to my parents, Richard and Betty Candelora, for making my schooling a priority and for always giving me their constant support in every way.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................11 Mortality Events............................................................................................................... ......12 Epidemiology of Infectious Bursal Disease...........................................................................13 Impact of Disease on Population Recovery............................................................................14 Objectives..................................................................................................................... ..........17 2 SENTIENEL CHICKENS......................................................................................................18 Introduction................................................................................................................... ..........18 Materials and Methods.......................................................................................................... .18 Lake County Release Site................................................................................................18 Polk County Release Site................................................................................................20 Blood Collection and Analysis........................................................................................20 Bursal Fluid Aspiration...................................................................................................21 Results........................................................................................................................ .............22 Lake County: December 2003 through April 2004.........................................................22 Polk County: December 2004 through May 2005...........................................................22 Discussion..................................................................................................................... ..........23 3 INFECTIOUS BURSAL DISEASE IN WILD BIRDS OF FLORIDA.................................30 Introduction................................................................................................................... ..........30 Use of Poultry Farms.......................................................................................................30 Human Activity and Disposal of Poultry Products.........................................................31 Contact with Wild Birds..................................................................................................33 Materials and Methods.......................................................................................................... .35 Wild Turkey.................................................................................................................... .35 Florida Bobwhite Quail...................................................................................................37 Florida Sandhill Crane.....................................................................................................38 Results........................................................................................................................ .............39 Wild Turkey.................................................................................................................... .39 Florida Bobwhite Quail...................................................................................................41 Florida Sandhill Crane.....................................................................................................41

PAGE 6

6 Discussion..................................................................................................................... ..........41 4 ARCHIVED SAMPLES.........................................................................................................50 Introduction................................................................................................................... ..........50 Materials and Methods.......................................................................................................... .51 Results........................................................................................................................ .............53 Discussion..................................................................................................................... ..........54 5 SYNTHESIS AND SIGNIFICANCE....................................................................................59 Implications for the Whooping Crane Reintroduction Project...............................................59 Age Effect on Seroprevalence................................................................................................60 Variation in Exposure Prevalence among Sites......................................................................60 Transmission Mechanisms......................................................................................................61 LIST OF REFERENCES............................................................................................................. ..62 BIOGRAPHICAL SKETCH.........................................................................................................66

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7 LIST OF TABLES Table page 3-1 Number of blood samples collected fr om harvested wild turkeys during the 2004, 2005, and 2006 spring turkey seasons...............................................................................43 3-2 Number of blood samples collected from wild turkeys using rocket nets from December 2003 through January 2007..............................................................................44 3-3 Statistical results for yearly e xposure prevalence in wild turkeys.....................................46 3-4 P-values for difference in yearly exposure prevalence and average titer level in wild turkeys........................................................................................................................ ........47 4-1 Statistical results for ex posure prevalence by age of ar chived sandhill crane samples collected in Alachua and Osceola co unties from May 1992 to March 1998.....................56 4-2 Statistical results for exposure prev alence by county of archived sandhill crane samples collected in Alachua and Osceola counties from May 1992 to March 1998.......57

PAGE 8

8 LIST OF FIGURES Figure page 2-1 Timeline for Lake County release site...............................................................................26 2-2 Chicken trap locations on Lake County release site..........................................................27 2-4 Collecting blood from a chicken vi a the medial metatarsal vein.......................................28 2-5 Aspirating bursal fluid fr om the bursa of fabricius............................................................29 3-1 Blood collection sites for wild turkey samples collected from December 2003 through January 2007. ......................................................................................................43 3-2 Blood collection sites for Florida sa ndhill crane samples collected during 2004, 2005, and 2006................................................................................................................. ..45 3-3 Frequency of titer levels for all wild turkey samples collected from December 2003 through January 2007.........................................................................................................46 3-4 Wild turkey exposure prevalen ce (% birds with titer level 1:32) at five sample sites where 10 or more samples were collected in multiple years.............................................47 3-5 Frequency of titer levels for Florid a sandhill cranes captured in 2004, 2005, and 2006........................................................................................................................... .........48 3-6 Titer level results for se rum/filter paper comparisons.......................................................49

PAGE 9

9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INFECTIOUS BURSAL DISEASE VIRUS IN WILD TURKEYS AND SANDHILL CRANES OF FLORIDA By Kristen L. Candelora August 2007 Chair: H. Franklin Percival Major: Wildlife Ecology and Conservation Captive-reared whooping cranes ( Grus americana) released into Florida for the resident reintroduction project e xperienced unusually high mortalit y and morbidity during the 1997/98 and 2001/02 release seasons. Inf ectious bursal disease virus (IBDV) serotype 2 is currently under investigation as the factor that precipitated the mortality events. A small percentage of whooping cranes have been exposed to IBDV in the captive setting. However, many more are being exposed post-release, and pr evalence of exposure increases w ith age or length of time the birds are in the wild in Florida. No studies have been published on the prevalence of IBDV in wild birds of North America. The goal of this st udy is to provide baseline data that can be used to create effective protocols and take measures to ensure that this virus does not impact the recovery of the endangered whooping crane. To determine if wild exposure to IBDV serot ype 2 is possible, captive sentinel chickens were monitored for exposure to the virus on wh ooping crane release sites in central Florida during the 2003/04 and 2004/05 release seasons. W ild exposure is possible as chickens on both sites became exposed to IBDV serotype 2. To examine the potential for exposure of whooping cranes from other wildlife reservoirs, blood sa mples were collected from wild turkeys ( Meleagris gallopavo ) and sandhill cranes ( Grus canadensis ) in 21 counties throughout Florida and 2

PAGE 10

10 counties in southern Georgia. There is pot ential for whooping cranes both resident and migratory, to become exposed to this virus throug h contact with wild bird s as wild turkeys and sandhill cranes in 8 counties in Florida and 1 coun ty in southern Georgia have been exposed to the virus. In addition, there is a significant age effect on sero prevalence in sandhill cranes. These findings are consistent with chicks having a shorter exposure time and immature immune system. The presence of higher seroprevalence an d higher titers in older bi rds suggests that there is constant re-exposure or that bird s remain carriers of the virus. To investigate the original source and history of the virus in wild bird s of Florida, archived serum samples collected from sandhill cranes we re tested for antibodies to IBDV serotype 2. Although I was unable to demonstrate that sandh ill cranes in Florida were exposed to IBDV prior to the introduction of captiv e-reared cranes, the high preval ence and wide distribution of the virus in both sandhill cranes and wild turkeys suggest that th e virus has been in Florida for quite some time. Many of the sites where blood was collected from wild turkeys and sandhill cranes overlap with the current distribution of w hooping cranes in Florida. The presence of this virus in wild birds in these areas is especia lly concerning for the resident flock of whooping cranes because they nest and raise their chicks in Florida. The effect of e xposure on whooping crane chicks is unknown at this time. However, the impact to young chickens suggests that if whooping crane chicks hatched in the wild are exposed to the vi rus at an early age, chick survival could be greatly reduced. Conversely, the relatively high titers maintained by adults may be passed on to the chicks and protect them at this othe rwise vulnerable period in their lives.

PAGE 11

11 CHAPTER 1 INTRODUCTION The range of the whooping crane ( Grus americana ) once extended from central Canada south to Mexico, and from Utah to the Atlantic coast. In 1865, the es timated population size was 700-1,400 birds (U.S. Fish and Wildlife Service 1994). Habitat loss, unregulated hunting, and specimen collection had severe negative imp acts, and by 1937 the population was reduced to a single non-migratory flock in southwestern Louisi ana and a single migrator y flock that wintered on the Gulf coast of Texas and nested in an unknown location (later discovered to be Wood Buffalo National Park in Canada). By 1950, th e non-migratory flock had been extirpated and just 34 birds remained in the migratory flock (U.S. Fish and Wildlife Service 1994). Congress passed the Endangered Species Preservation Act in 1966, and the whooping crane was listed as threatened with extinction in 1967. In that same year the Canadian Wildlife Service and the U.S. Fish and Wildlife Servi ce began collecting eggs from birds in the Wood Buffalo flock, to generate a captive breedi ng flock of whooping cranes. The goal was to propagate whooping cranes, and reintroduce their offs pring into the areas from which they were extirpated. Reintroduction of captive-reared whooping cranes be gan in 1993 when the Florida Fish and Wildlife Conservation Commission, in c ooperation with several government and private agencies, released birds on the Ki ssimmee Prairie in central Florid a with the goal of establishing a resident, non-migratory flock (U.S. Fish and Wildlife Service 1994). Captive-reared whooping cranes released in to Florida for the resident reintroduction project experienced unusually high mortality and morbidity during the 1997/98 and 2001/02 release seasons. Exposure to in fectious bursal disease virus (IBDV) was documented, and may have been the precipitating factor for these mo rtality events (Spalding et al. 2006). The purpose of this study is to provide baseline data on the potential for exposur e of whooping cranes to

PAGE 12

12 IBDV from other wildlife reservoirs, so that e ffective protocols can be created and measures taken to ensure that this virus doe s not impact the recovery of the e ndangered whooping crane. Mortality Events In 1997/98, 14 of 22 captive-reared whooping cran es died within 6 months of their release. Another mortality ev ent occurred during the 2001/02 rele ase season. Seventy percent of birds released were affected. Of the 27 birds re leased 14 died, 5 others became sick and two of these had to be taken into permanent captivity. Bi rds from 4 different cap tive-rearing facilities in the United States and Canada were involved (Spaldi ng et al 2006). A number of potential causes were considered (food, water, envi ronmental toxins, herbicides, West Nile virus, coccidiosis, St. Louis and eastern equine encephali tis) and were ruled out (M. G. Spalding, University of Florida, unpublished data). Finally, because there was evidence of immunosuppression and the pattern of illness seemed to be restricted to young of the year, blood samples were tested for exposure to IBDV. The pattern of birds testing positive matched well with those that were sick or died, suggesting that IBDV may have been the factor precipitating the mort ality events (Spalding et al. 2006). As a result, an epidemiological study was initiated. This included testing archived blood samples from whooping cranes for exposure to IB DV, the initiation of a monitoring program for all captive-reared whooping cran es released into Florida and their offspring, and testing of whooping cranes in the captive flock. Results showed that some birds had been exposed to IBDV in the captive setting (Hart up and Sellers 2006). However, many more were exposed after their release into Florid a and prevalence of exposure increased w ith age or length of time a bird was in the wild (Spalding et al. 2006). Infectious bursal disease poses a significan t threat to the successf ul recovery of the endangered whooping crane, and cap tive-reared whooping cranes rel eased in Florida are being

PAGE 13

13 exposed to the virus post-release. Very little is known about the incidence of IBDV outside of poultry operations, especially in No rth America. Therefore prevalence of the virus in wild birds, the etiology of the virus, and its transmission mechanism are unknown. The goals of this study are to investigate whether wild exposure is pos sible, how wild exposure may occur, and the origin of the virus in wild birds of Florida. Epidemiology of Infectious Bursal Disease Infectious bursal disease is an acute, highl y contagious, viral dis ease that is common on poultry farms. The pathogenicity, resistance to di sinfectants, and persiste nce in the environment make it one of the most important viruses of domestic poultry throughout the world (Lukert and Saif 2003). Infected chickens shed the virus in their feces, and can transmit the virus for at least 14 days (Ahad 2002). Water, feed, and droppings taken from infected pens in poultry houses were found to be infectious for 52 days (Benton et al. 1967). There are two forms of this disease, clinical and subclinical. For ch ickens, the period of greatest susceptibility to clinical disease is betw een 3 and 6 weeks of age. The incubation period is very short, and clinical signs are seen within 2 days of exposure. Symptoms such as loss of appetite, dehydration, and diarrhea rapidly appear and the mort ality rate can be as high as 30% (Lukert and Saif 2003). In chickens infected before 3 weeks of age, a severe, prolonged immunosuppression occurs (Lukert and Saif 2003). The suppression of their immune system is similar to the effect of HIV on humans, leaving the birds vulnerable to a variety of ailments that normally would not be life threatening (Allan et al 1972). In this weakened state, the birds are also more susceptible to predation. There are two serotypes of IBDV. Type 1 occurs in all major poultry producing areas worldwide, and can cause acute mortality/immunos uppression in chickens. Type 2 is widespread in poultry flocks in the United States. It is found in both chickens and turkeys, but is non-

PAGE 14

14 pathogenic to these species (Lukert and Saif 2003). It is Type 2 that appears to have been involved in the whooping crane mortality event of 2001/02 (Spalding et al. 2006). Anecdotal evidence suggests IBDV may be pa thogenic to other avian species, but no study has directly linked exposure to clinical illness in any sp ecies other than chickens. Evidence of exposure to IBDV (serotype not identified) was found in Adelie penguin ( Pgyoscelis adeliae ) chicks following a mortality event where an infectious agent was suspected to be the cause. Clinical disease was not apparent at that time, but authors suggested that further investigation was warranted (Gardner et al. 1997). P opulations of common eider ( Somateria mollissima ) in the Baltic Sea have declined significan tly in some areas and juvenile mortality was reported to be the primary cause for this de cline (Hollmen et al. 2000). Although authors reported no evidence of illness, exposure to IBDV serotype 1 was confirmed and exposure prevalence was highest for eiders nesting in areas where duckling survival was low (Hollmen et al. 2000). In addition, followi ng a mortality event of Afri can black-footed penguins ( Spheniscus demersus ) and macaroni penguins ( Eudyptes chrysolophus ) in a zoo in the United Kingdom, Jackwood et al. (2005) were able to isolate IBDV serotype 2 from birds involved. The role of this virus in the mortality ev ent was unknown, and authors suggested that further study should be conducted to determine pathogenicity of IBDV in these species. Impact of Disease on Population Recovery Interactions between disease and population density play an important role in regulating animal populations, generally wit hout threat of elimin ating the population completely (Lafferty and Gerber 2002). This is espe cially true for large populations that occupy large geographic areas (Woodroffe 1999). As a popula tion expands, animals are forced to live in closer proximity to one another and share resources with an increas ing number of conspecifics. In some situations these circumstances lead to an increase in st ress and a decrease in general condition, leaving

PAGE 15

15 individuals more susceptible to disease while br inging them closer to animals from which they may contract it (Smith 2001). A decline in popula tion density is often the result. At lower densities transmission of disease is usually reduced and diseases are spread more slowly, if at all (Lafferty and Gerber 2002). The diseases role in population regula tion then becomes negligible. The impact of disease on small populations can be very different, pos ing a serious threat to the persistence of such populat ions. If hosts are killed more quickly than they can breed, population growth rates decline (Woodruff 1999) This perpetuates the small populations vulnerability to extinction by stochastic ev ents. One example comes from the long-term monitoring of bighorn sheep ( Ovis canadensis ). Berger (1990) found that small populations of 50 animals or less were more prone to extin ction than populations numbering 100 animals or more. This is a concern for the Florida re sident population of w hooping cranes which now number approximately 60 indi viduals (Folk et al. 2006). The threat of extinction caused by disease is also greater in small populations because they are more likely to have experienced inbreed ing, and limited genetic diversity can lead to a decrease in disease resistance (G lenn et al. 1999). These populat ions could therefore experience greater mortality than a genetical ly diverse population, and disease has proven to be an issue in some species involved in reintroduc tion programs when there is a d ecline or reduc tion of genetic diversity in captive-br ed animals (Glenn et al. 1999) In 1982 and 1983, 18 cheetahs ( Acinonyx jubatus ) in a captive breeding faci lity died from feline infectious pe ritonitis. It is unusual for this disease to cause such high mort ality. OBrien et al (1985) suggested that mortality was so significant because the cheetahs lacked an effectiv e immune response as a result of inbreeding. This concern also applies to whooping cranes because the population experienced a severe genetic bottleneck in the early 1940 s when the migratory flock d eclined to just 15 or 16 birds

PAGE 16

16 (U.S. Fish and Wildlife Service 199 4). It is estimated that these remaining birds were highly interrelated, having an effective population size of only 1.2 birds (Jones et al. 2002). Because this was the founder population, al l whooping cranes that exist today suffer reduced genetic diversity (Glenn et al. 1999). In the conservation of small, already compromised populations such as the whooping crane, immunocompromising diseases such as IBDV could be especially problematic (Thorne and Williams 1988, Lafferty and Gerber 2002). On ce infected with such a disease, host organisms become susceptible to secondary infection by common but normally benign disease organisms (Hollmen et al. 2000). The potential for transmission of IBDV betw een domestic poultry and wild birds is an additional concern for the resident flock of whooping cranes in Florida as di sease can be an issue in the recovery of species when the potentia l for transmission between domestic animals and endangered species exists. The African wild dog ( Lycaon pictus ) population has declined considerably in recent decades, and in 2002 wa s estimated at less than 5,500 individuals. Domestic canine diseases such as rabies and canine distemper, possibly transmitted by dogs from local villages, were some of the suggested causes of this decline (van de Bildt et al. 2002). According to Thorne and Williams (1988) canine distemper was also common in domestic dogs in Wyoming. In 1985, a drastic decline was not ed in the last known wild population of blackfooted ferrets ( Mustela nigripes ). To investigate the cause of th e decline, ferrets were captured from widely separated locations. The fact that al l died in captivity of canine distemper, indicated that the disease was widespread. The 18 known su rvivors of the disease outbreak were captured and placed in a captive breeding program. The free-ranging colony was essentially extirpated.

PAGE 17

17 Objectives I could find no published study confirming that wild birds in the United States have become exposed to IBDV. However, results fr om the epidemiological study by Spalding et al. (2006) indicated that some captiv e-reared whooping cranes had b een exposed to IBDV following their release into Florid a. Therefore, the first objective wa s to test the hypothesis that wild exposure to IBDV is possible without direct cont act with potentially in fected captive-reared whooping cranes. To investigate how wild exposure may occur I tested the hypothesis that contact with wild birds is a potential exposure mechanism. Fo r contact with wild birds to be a valid exposure mechanism, wild birds that whooping cranes shar e habitat with post-release must be positive for IBDV exposure. Therefore, the second objective was to determine if such species had been exposed to the virus and to assess the pr evalence of exposure among those species. The original source of IBDV in wild bi rd of Florida is unknown. There are many possibilities related to domestic poultry operations such as : transmission by people carrying the virus on contaminated footwear, inappropriate disposal of pou ltry products, and use of poultry litter as fertilizer in the agricu ltural industry. Another option is that captive-reared cranes were exposed to the virus in captivity and they introduced the virus to wild birds of Florida postrelease. However, this possibili ty might be eliminated if it coul d be shown that wild birds in Florida had been exposed to the virus prior to the release of captive-reared cranes. Therefore, the third objective was to test the hypo thesis that wild sandhill cranes ( Grus Canadensis ) in Florida were exposed to the virus prior to the release of captive-reared cr anes, or in areas where contact with captive-reared cranes was highly unlikely.

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18 CHAPTER 2 SENTIENEL CHICKENS Introduction Infectious bursal disease virus (IBDV) is a common poultry virus. It is well documented that birds in domestic operati ons are being exposed worldwide, and transmission mechanisms are fairly well understood in this setting (Lukert and Saif 2003). Although there are no published studies confirming that wild expos ure to IBDV has occurred in No rth America, or demonstrating that transmission mechanisms exist, Spalding et al. (2006) found that captive-reared whooping cranes ( Grus americana ) had been exposed to IBDV following their release into Florida. To test the hypothesis that wild exposure in Florida is possible without direct contact with potentially infected whooping cranes, sentinel chickens confirmed to be free of disease were monitored for exposure to IBDV. Materials and Methods Six-week-old specific pathogen free (SPF) leghorn chickens were purchased from Charles River Laboratories, Inc. (251 Balla rdvale Street Wilmington, MA 01887-1000). Blood samples were collected upon arrival to confirm th e sentinel chickens had not previously been exposed to IBDV. Chickens were housed in 32x10x12 Tomahawk Raccoon/Feral Cat Live Traps (PO Box 323, Tomahawk, WI, 54487) and provi ded with fresh food and water daily. Lake County Release Site The first cohort of captive-reared whooping cranes for the 2003/04 release season arrived at the release site on December 8, 2003. The bird s were brailed and placed in a portable prerelease pen. Brails were removed on December 2 1, 2003 and the birds were free to leave the pen (Figure 2-1). Three of these birds were positiv e for exposure to IBDV serotype 2 upon arrival at the release site. No birds seroconverted while in the pen. The second cohort arrived on

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19 February 5, 2004 and brails were removed on Fe bruary 18, 2004 (Figure 2-1). None of these cranes were positive for exposure to IBDV serot ype 2 upon arrival. One bird seroconverted while in the pen (M. G. Spalding, Univers ity of Florida, unpublished data). Eight SPF chickens were placed in cages wh en the first cohort arrived for release (December 8, 2003) and remained on the site thr ough April 2004 (Figure 2-1). To increase the chance for viral exposure for another investigatio n, the chickens were separated into two groups of four and placed 1.07 km apart. One group of chickens was placed 1.47 km from the release site, along the edge of a small pine forest (Figur e 2-2). This group of chickens was exposed to crane feces collected from the vicinity of the release pen. The second group of chickens was placed 0.51 km from the release s ite, along the edge of an oak hammock (Figure 2-2). This group was not exposed to crane feces. To mi nimize cross contamination when checking the sentinel chickens the protocol was to always visit the non-exposure group first. Feces were collected from areas surrounding th e feeders used by cranes after leaving the release pen. All were used by the current years release birds, past years release birds, and a few wild sandhill cranes. For each collection attempt, I monitored the feeder being used by the most current years captive-reared whooping cranes. I watched the birds, noting when and where one defecated. Feces were collected when cranes le ft the feeder area. When there were multiple feces in the vicinity, all were co llected in an attempt to ensure collection of the target feces. Feces were placed in the cages of the exposure gr oup when the chickens had been on the site for 43 days, 50 days, and 56 days (Figure 2-1). No feces were collected af ter the arrival of the second cohort of captive-rear ed whooping cranes. To determine whether the likelihood of becoming exposed to IBDV was independent of exposure to crane feces, a like lihood ratio (G) test for independence was employed using the

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20 statistical software JMP 7 (SAS Institute 2007). The test was 2tailed and considered significant at P 0.05. Polk County Release Site Five captive-reared whooping cranes arrive d for release on December 8, 2004 and were debrailed on December 22, 2004 (Figure 2-3). None of the captive-reared cranes were positive for IBDV exposure upon arrival. One bird sero converted while in the pen (M. G. Spalding, University of Florida, unpublished data). Eight SPF chickens were placed in cages on December 11, 2004 and remained on the release site through May 2005 (Figur e 2-3). All 8 chickens were placed in the same area and none were exposed to crane feces. The cages were placed in a small oak hammock surrounded by improved pasture. Blood Collection and Analysis Blood collection and analysis methods were th e same for SPF chickens on both release sites. A blood sample was collected from each chicken approximately every 2 weeks. One to 2 mL of blood was collected from the medial metatarsal vein (Fi gure 2-4). A 27-gauge needle was used on young chickens, and a 25-gauge needle was used when they were full grown. After collection, blood was transferred in to a lithium heparinized vacuta iner. All samples were kept cold until they were spun down and the serum co llected. The serum was frozen until it was sent to the Poultry Diagnostic & Research Center (Col lege of Veterinary Medicine, University of Georgia, 953 College Station Roa d, Athens, GA 30602) for evaluation. All serum samples were tested for IBDV serot ype 2 antibodies. Infectious bursal disease serotype 2 virus neutralizations, using the beta procedure (constant virus/diluted serum), were performed in primary chicken embryo fibroblast (CEF) cultures prepared from 9-11 day old SPF

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21 chicken embryos. Sera were heat inactivated at 56 C for 45 minutes, followed by centrifugation at 1200 x g for 10 minutes. Fifty microliters of serum was added to the first well of each row (rows A, B, C, D, E, F, G, and H) in a tissu e culture-treated 96 well plate. Serotype 2 IBDV antigen was diluted to contain 100-5 00 Tissue Culture Infectious Dose50/50 l. Subsequently, 50 L of diluted antigen was added to all wells (columns 1-11), except wells in column 12 (this served as a cell control) Serial dilutions were prepared by mixing the serum and antigen in column 1 and transferring 50 L to column 2. Pipette tips were changed and contents in column 2 were mixed and 50 L was transferred to column 3. This was repeated through column 10 where contents were mixed and 50 L were discarded. Column 11 contained antigen only and was the virus control. Positi ve serotype 2 IBDV sera and negative sera were included in assay as controls One hundred and ninety L of CEFs were added to all wells. Cells were incubated at 37 C for 5 days, cell culture media d ecanted, cells fixed with methanol for 1 minute and stained with crysta l violet for 1 minute. Virus ne utralizing titer was recorded as the reciprocal dilution of the last well exhibiting no cytopathic effect. It is unknown what titer level indicates true e xposure to IBDV. Previous studies of wild birds have considered titer leve ls ranging from 1:16 to 1:80 as evidence of exposure (Wilcox et al. 1983, Gardner et al. 1997, Ogawa et al. 1998, Hollm en et al. 2000). For the purposes of this study I assumed a titer level of 1:8 or less indicated no exposure to the virus. Birds with a titer level of 1:16 were considered possibly exposed. A titer level of 1:32 or greater was assumed to be indicative of exposure. Bursal Fluid Aspiration Seventeen bursal fluid samples were colle cted from SPF chickens on the Polk County release site in an attempt to isolate the IBDV se rotype 2. The bursa of fabricius is a sac-like extension of the hindgut, located on the dorsal side of the cloaca. The chicken was placed on its

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22 back, and a Kendall Monoject 16G x 1-1/2 aluminum hub blunt needle (tyco/Healthcare, Two Ludlow Park Dr., Chicopee, MA, 01022) was inserted into the vaginal open ing of the vent, at a slightly downward angle. The needle was in serted until it dropped down into the bursa of fabricius (Figure 2-5). Fluid was aspirated and th en injected into a ster ile solution of phosphate buffer saline. The solution was frozen and sent to the lab for evaluation. Results Lake County: December 2003 through April 2004 Seroprevalence did not differ significantly between groups exposed and not exposed to crane feces (P = 0.148). Of the 4 chickens in the non-exposure group, three seroconverted and one was possibly exposed. Of the three chicke ns with titer levels high enough to indicate exposure, two had a titer level of 1:64 and one had a titer level of 1:256 indicating recent exposure. Two of these chickens seroconverted af ter being on the release site for 101 days. One chicken seroconverted after being on the release site fo r 133 days (Figure2-1). Of the four chickens in the fecal exposu re group, one became exposed to IBDV serotype 2, two were possibly exposed, and one was not ex posed. The chicken with a titer level high enough to indicate viral exposure seroconverted after being on the release site for 101 days (Figure 2-1). The chicken that did not seroconver t is the bird that spent the least amount of time on the release site. It was found dead in its cage after having been on the release site for 79 days (Figure 2-1). Necropsy did not reveal cause of de ath, and no virus was isolated from its tissues. Polk County: December 2004 through May 2005 Eight sentinel chickens were placed on the release site, but a raccoon killed one within the first 2 weeks. Data were analyzed on th e 7 remaining chickens. Two chickens became exposed to the virus, one was possibly exposed, a nd four were not exposed. Of the two chickens

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23 that became exposed, one seroconv erted after being on the releas e site for 29 days, the other seroconverted after 66 days (Figure 2-1). On e had a titer level of 1:256, indicating recent exposure. The IBDV serotype 2 was not isolated from any bursal fluid samples. Comparison of titer level from blood collected on the same day as bursal fluid revealed that only one of the seventeen samples was collected from a bird having a titer level high enough to indicate exposure. Discussion Some sentinel chickens on both release sites seroconverted to IBDV serotype 2, supporting the hypothesis that wild transmission of IBDV in Florida is po ssible. The hypothesis is strengthened by the failure of exposure to pote ntially infected feces fr om whooping cranes to increase the seroconversion rate of the chickens. It is possible, but unlikely, that the chickens became infected directly from the captive-reared whooping cranes. Cranes may have visited the chicken cages, although this was never observed. The virus may have been carried by people when feeding or handling the chickens, although pr otocol dictated that chickens were visited before entering the release area to minimize this possibility. The hypothesis is further supported by the fact that chickens on the Polk County release site became exposed to IBDV, yet none of the captive-reared whooping cr anes released were positive for IBDV serotype 2 antibodies upon arrival at the release site. However, one of the captive-reared whooping cranes se roconverted while in the pen. Seroconversion could be the result of wild exposure or it is possible that the crane was ex posed in captivity, but did not seroconvert until after it was put in the pen. The finding that likelihood of exposure was not significantly affect ed by contact with potentially infected feces must be interpreted wi th caution for 2 reasons. First, the sample size

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24 was small (n = 8). Second, it is possible that I did not collect any infected feces. Previous research indicates that chickens shed the virus in feces for 14 days (Ahad 2002). It is unknown if or how long whooping cranes may shed the virus in feces, but it is possible that exposed cranes were not shedding the virus at time of collection. Regardless of whether or not chickens in the fecal exposure group were exposed to infected crane feces, some chickens in the non -exposure group did seroconvert. This suggests that the virus is present in the environment and available to infect susceptible hosts. Therefore, further research into wild exposure mechanisms w ith insects acting as the vector is warranted. A strain of IBDV (serotype not identified) was isolated from mosquitoes ( Aedes vexans ) trapped in southwestern Ontario in 1976 (Howie and Thor sen 1981). This species is found throughout the United States and utilizes a wide range of ha bitat types (OMalley 1990 ). Whooping cranes are known to have contracted other mosquito born vi ruses such as Eastern Equine Encephalitis and West Nile virus (M. G. Spalding, University of Florida, unpublished data). Dung beetles may be another insect of interest as whooping cranes in Florida have b ecome infected with a nematode that utilizes dung beetles as an intermed iate host (Varela et al. 2001). Another potential exposure mechanism is the use of poultry litter containing feces as fertilizer, a practice used regularly on the prope rty adjoining the Lake County release site. Although the virus can be transmitted in chickens through contact with infected feces, research indicates that IBDV does not surv ive the Maryland Method of d ead bird composting, otherwise known as two stage composting (Murphy 1990). Th erefore the material itself if properly composted is probably not the source of infection. But the virus has been isolated from adult lesser mealworms ( Alphitobius diaperinus Panzer ) which commonly inhabit poultry houses, living in poultry droppings and litter (McAllister et al. 1995, Dunford and Kaufman 2006).

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25 Mealworms could be transferred from the poultry house to the wild if litter infested with mealworms is spread on the fields, and presence of the lesser mealworm has been confirmed in numerous counties throughout Florida (Dunford and Kaufman 2006).

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26 Dec 8, 2003 : Whooping Crane Cohort 1 placed in pen on the release site. Three cranes were positive for IBDV exposure upon arrival. Dec 8, 2003 : Sentinel chickens placed in cages on the release site Dec 21, 2003 : Whooping Crane Cohort 1 released from pen. No cranes became exposed to IBDV while in the pen. Jan 20, 2004 : Sentinel chickens in Exposure Group exposed to crane feces Jan 27, 2004 : Sentinel chickens in Exposure Group exposed to crane feces Feb 2, 2004 : Sentinel chickens in Exposure Group exposed to crane feces Feb 5, 2004 : Whooping Crane Cohort 2 placed in pen on the release site. None of these cranes were positive for IBDV exposure upon arrival. Feb 18, 2004 : Whooping Crane Cohort 2 released from pen. One crane seroconverted while in the pen. Feb 27, 2004 : Mortality of sentinel chicken in Fecal Exposure Group March 19, 2004 : Seroconversion of 1 sentinel chicken in Fecal Exposure Group and 2 chickens in Non-exposure Group April 20, 2004 : Seroconversion of 1 sentinel chicken in Non-exposure Group April 23, 2004 : Sentinel chickens removed from release site Figure 2-1. Timeline for Lake County release site.

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27 Figure 2-2. Chicken trap locations on Lake County release site. Dec 8, 2004 : Whooping cranes placed in pen on release site. No cranes were positive for IBDV exposure upon arrival. Dec 11, 2004 : Sentinel chickens placed in cages on the release site Dec 22, 2004 : Whooping cranes released from pen. One crane seroconverted while in the pen. Jan 9, 2005 : Seroconversion of 1 sentinel chicken Feb 15, 2005 : Seroconversion of 1 sentinal chicken May 3, 2005 : Sentinel chickens removed from release site Figure 2-3. Timeline for Polk County release site.

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28 Figure 2-4. Collecting blood from a chic ken via the medial metatarsal vein.

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29 Figure 2-5. Aspirating bu rsal fluid from the bursa of fabricius.

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30 CHAPTER 3 INFECTIOUS BURSAL DISEASE IN WILD BIRDS OF FLORIDA Introduction Infectious bursal disease virus has been well studied in commercial poultry operations, but very little is known about the prevalence or exposure mechanisms in wild birds (Lukert and Saif 2003). Although I could find no published litera ture on the incidence of IBDV in wild birds of North America, studies done in Antarctica, Australia, Crozet Archipelago in the Indian Ocean, Finland, Ireland, Japan, Nigeria, an d Spain indicate that wild bi rds worldwide are being exposed to the virus (Nawathe et al. 1978, Wilcox et al. 1983, Gardner et al. 1997, Ogawa et al. 1998, Hollmen et al. 2000, Campbell 2001, Hofle et al. 2001, Gauthier-Clerc et al. 2002). Anecdotal evidence from these studies suggests that exposure to IBDV in wild birds of North America may be the result of spill-over, the transmission of contagious agents from reservoir animal populations (often domesticated species) to wildli fe occupying the same area (Daszak and Cunningham 2000). Potential transmission mechan isms are the use of poultry farms, human activity, disposal of poultry produc ts, the use of poultry litter as fertilizer, and contact with infected wild birds. Use of Poultry Farms The farm environment provides valuable hab itat for wildlife, and wild birds in North America may become exposed to IBDV through their use of poultry farms. This includes both large commercial operations where wild birds may use drainage ponds and small farms with free-range poultry where direct contact with wild birds is possible. At the poultry farm of the National Veterinary Research In stitute in Nigeria, evidence of exposure to IBDV (serotype not identified) was found in six of 50 wild birds capt ured on the farm. Chic kens on the poultry farm housed in the commercial type setting and those kept as free ranging back-yard birds had been

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31 exposed as well (serotype not identified). The authors did not investigate the source of exposure in these wild birds, but suggested that it could have been th e domestic poultry (Nawathe et al. 1978). When blood samples were collected from 11 species of wild water birds in Western Australia, evidence of exposure to IBDV (ser otype not identified) was found in 7 species (Wilcox et al. 1983). Antibodies to the virus we re most commonly dete cted in black ducks ( Anas superciliosa ) from the Perth area. The authors re ported that farm ponds used to collect drainage from poultry sheds ar e common on commercial poultry fa rms in Perth, and that black ducks had been observed using these fresh water ponds. Sera from king penguins ( Aptenodytes patagonicus ) on Possession Island of the Crozet Archipelago in the South Indian Ocean were exam ined for antibodies to IBDV serotypes 1 and 2. Chicks and adults had been exposed to both sero types of the virus. For many years there was a poultry yard with domestic chickens and ducks in the scientific st ation on Possession Island. Although the authors did not know whether any of the domestic poultry had been exposed to IBDV, these domestic birds did have daily contact with wild birds (Gauthie r-Cleric et al. 2002). Human Activity and Disposal of Poultry Products It has also been suggested that human activ ity and disposal of poultry products could be sources of exposure for wild bird s. Gauthier-Cleric et al. (2002) reported that since the 1960s the beach of the king penguin colony where birds were found to have been exposed to IBDV, was the main landing point for people, equipment, and food destined for the research station. Although the authors did not investigate potenti al exposure mechanisms, the virus may have been introduced by human activity on the island or by sewage fr om the poultry yard that was discharged untreated into a field.

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32 Disposal of poultry products is suspected as the source of exposure in common eiders ( Somateria mollissima ) and herring gulls ( Larus argentatus ) in two mixed species breeding colonies along the Finnish coast. The col ony that was close to human development had significantly greater prev alence of exposure to IBDV serotype 1. Herring gulls from this colony were observed foraging at a nearby landfill. The authors proposed that gulls foraging at the landfill may have come in contact with the vi rus through waste from poultry farms, and then transmitted the virus when they returned to feed their young (Hollmen et al. 2000). Human activity is suspected in exposure of two species of Antarctic penguins ( Aptenodytes forsteri and Pygoscelis adeliae ) to IBDV (serotype not id entified) as evidence of exposure was found in colonies n ear centers of human activity, but none of the samples collected from penguins in a remote and rarely visited site had antibodies to the vi rus. Authors suggested that the virus may be spread by people on their footwear, clothing, e quipment, or vehicles as they move around Antarctica. They also proposed th at inappropriate disposal of imported poultry products may have been involved as wild birds could have become exposed when scavenging on waste and then transmitted the virus to othe r birds in the area (G ardner et al. 1997). Use of Poultry Litter as Fertilizer Poultry litter containing feces is used as fert ilizer in the agricultural industry, and this practice may be involved in transmission of IBDV from domestic operations to the wild. The virus can be transmitted in chickens through c ontact with infected feces, but IBDV does not survive the Maryland Method of dead bird composting, otherwise known as two stage composting (Murphy 1990). Therefore, the material itself if properly co mposted is probably not the source of infection. But lesser mealworms ( Alphitobius diaperinus Panzer ) commonly inhabit poultry houses where they live in poultr y droppings and litter, and the IBDV (serotype

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33 not identified) has been isolat ed from adult lesser mealworm s up to 14 days after exposure (McAllister et al. 1995, Dunford a nd Kaufman 2006). Mealworms could be transferred from the poultry house to the wild if litte r and feces infested with mealworms is spread on fields as fertilizer (Dunford and Kaufman 2006). Presence of the lesser mealworm has been confirmed in Alachua, Broward, Charlotte, Clay, Dade, Hillsbo rough, Indian River, Manatee, Marion, Orange, Pasco, Pinellas, Polk, Putnam, and Volusia countie s and probably occurs throughout the state of Florida (Dunford and Kaufman 2006). Contact with Wild Birds Wild birds for whom the virus is not pat hogenic may act as reservoirs of the virus (Nawathe et al. 1978, Wilcox et al. 1983). To address this question, van den Berg et al. (2001) performed experimental infections of 3 week old commercial pheasants ( Phasianus colchicus ), grey partridges ( Perdix perdix ), Japanese quail ( Coturnix coturnix japonica ), and guinea fowl ( Numida meleagris ) using the very virulent strain of IBDV (serotype 1). These species were chosen because they are closely related evolutionarily to domestic fowl, and because they are commonly released fo r hunting or ornamental purposes. None of these species exhibited clinical signs of illness. Guinea fowl were found to be fully refractory to infection. Some pheasants and partridges seroconverte d, but none excreted the virus. Quail were susceptible to infection and shed the virus in feces for up to 7 days. Therefore, Japanese quail have the potential to act as a reservoir of the very vi rulent strain serotype 1 virus. However, results from this study do not suppor t the findings of Weis man and Hitchner (1978) who found Coturnix quail (species not specif ied) to be refractory to IBDV, although they used the Classical Strain serot ype 1 virus. Therefore Coturnix quail appear capable of transmitting and being a reservoir of at leas t one strain of IBDV.

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34 Van den Berg et al. (2001) concluded that pe rsistence of IBDV in wild bird populations is unlikely to occur and that the source of inf ection has to be found in poultry farms or the environment. Captive-reared whooping cranes rel eased in Florida spend the majority of their time on farms and ranches. These range from large ranches that specialize in cattle and crops, to smaller farms where owners keep back-yard chickens. In addition, the use of litter from poultry operations as fertilizer is common on some farms. Theref ore it is possible that whooping crane exposure may be a result of their use of the farm environment, or exposure to wild birds that act as reservoirs of the virus. For contact with wild birds to be a potential exposure mechanism for whooping cranes, it first needs to be shown that wild birds utilizing the same habitat ha ve been exposed to the virus. I tested the hypothesis that wild turkeys ( Meleagris gallopavo ), Florida sandhill cranes ( Grus canadensis pratensis ), and Florida bobwhite quail ( Colinus virginianus floridana ) have been exposed to IBDV. These 3 species were chos en based on likelihood of interaction (based on personal observations made during the 4 year s I monitored whooping cranes), and previous research regarding susceptibility to IBDV in closely related domestic species I found no previous research on susceptibility of sandhill cr anes to IBDV. They were included because they are closely relate d to whooping cranes (both in genus Grus ), and are the species I observed captive-re ared whooping cranes inte racting with most regul arly after release. Wild turkeys were chosen because previous research indicates that domestic turkeys can be carriers of IBDV. When domestic turkeys are exposed to IBDV they respond serologically and are capable of transmitting the virus, but do not develop clinical disease (Giambrone et al. 1978, Weisman and Hitchner 1978, Jackwood et al. 1981, Barn es et al. 1982). If wild turkeys are also carriers, then they are likely candidates to be na tural reservoirs of the virus. In addition, IBDV

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35 serotype 2 has been detected in domestic turk eys in the United States (Jackwood et al. 1982, Chin et al. 1984). Although I observed minima l interaction between w ild turkeys and whooping cranes, wild turkeys were observed foraging in the same pastures used by whooping cranes. Florida bobwhite quail also share habita t with whooping cranes Although I never observed direct interaction between these two species, both were observed using edge habitat in agricultural areas. No studies have been published on prevalence of IBDV in this species of quail. However, previous research indicates that Coturnix quail are capable of transmitting at least 1 strain of the virus, serotype 1 (van den Berg 2001). Although Coturnix quail are in a different family (Old World quail of the family Phasianidae) than bobw hite quail (New World Quail of the Family Odontophoridae), it is possible that they are capable of transmitting the virus as well. In addition, there is another mode of transmission of the virus from a domestic source into the wild in Florida as pen-raised bobwhite quail are of ten released for dog training and hunting (Wiley 2005). Materials and Methods Wild Turkey To determine if populations of wild tu rkey had been exposed to IBDV, 596 blood samples were collected from wild turkeys at 24 locations in 21 c ounties throughout Florida (DeSoto, Gadsden, Glades, Hernando, Highl ands, Holmes, Jefferson, Lake, Leon, Levy, Wakulla, Madison, Martin, Orange, Osceola, Palm Beach, Pasco, Polk, Putnam, Sumter, Wakulla) and 3 adjoining plantations in Thomas and Grady counties in southern Georgia (Figure 3-1). Two methods were used to collect these samples. During the 2004, 2005, and 2006 spring turkey-hunting seasons, 434 blood samples we re collected from hunter-harvested wild turkeys (Table 3-1). When hunters brought a bi rd to the check station, it was hung upside down

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36 by the feet. An incision, 2.5.1 cm in length, was made in the right side of the neck. This would cut the jugular vein, releasing blood. A lit hium heparinized vacutainer was held beneath the incision and as much blood as possible was collected. From December 2003 through January 2007, 162 blood samples were collected from live birds captured with rocket nets (Table 3-2). Between 1 and 2 mL of blood was collected from the medial metatarsal vein using a 25-gauge needle. After collection, blood was transferre d into a lithium hepari nized vacutainer. All blood samples were kept cold until they were spun down and the serum collected. The serum was frozen until it was sent to the Poultr y Diagnostic & Research Center (College of Veterinary Medicine, University of Georgia, 953 College St ation Road, Athens, GA 30602) for evaluation. All serum samples were tested fo r IBDV serotype 2 antibodies using the beta procedure (constant virus/diluted se rum) described in Chapter 2. It is unknown what titer level indicates true e xposure to IBDV. Previous studies of wild birds have considered titer leve ls ranging from 1:16 to 1:80 as evidence of exposure (Wilcox et al. 1983, Gardner et al. 1997, Ogawa et al. 1998, Hollmen et al. 2000, van den Berg 2001). For the purposes of this study I assume d a titer level of 1:16 or less was not indicative of exposure to the virus. Birds with a titer leve l of 1:32 or greater were considered exposed to the virus. The Kruskal-Wallis nonparametric ANOVA and Wilcoxon rank sum tests were used to determine if average titer level differed by locatio n and year respectively. Post-hoc comparisons were performed using Wilcoxon rank sum tests. To determine if likelihood of exposure (# birds with titer level 1:32) was independent of location and year, a likelihood ratio (G) test for independence was used. Post-hoc comparisons were performed us ing the likelihood ratio test as well. All tests were 2-tailed and considered significant at P 0.05. Analysis was performed using the statistical software JMP 7 (SAS Institute 2007).

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37 Florida Bobwhite Quail I hoped to obtain blood samples from harv ested Florida bobwhite quail in a manner similar to that of harvested wild turkeys. However, due to th e smaller size of bobwhite quail I was unable to get a sufficient serum sample by co llecting blood in a hepa rinized vacutainer post mortem, spinning it down, and separating the serum. Therefore, I conducted a pilot study to test the feasibility of collecting blood with filter paper strips. This method allows for the determination of antibody titer le vel with a much smaller amount of blood. First, I investigated whether enough blood could be collected from d ead quail to perform the analysis. Then I examined whether this method provided an accu rate measure of antibody titer level for IBDV serotype 2. To examine whether enough blood could be co llected from harvested Florida bobwhite quail to perform the analysis, 11 samples were co llected from birds harv ested in November of 2004 on Babcock-Webb WMA in Char lotte County. Collectors we re instructed to cut the jugular or brachial vein and saturate 100 diam eter Nebuto blood filter strips (Advantec MFS, Inc., 6691 Owens Dr., Pleasanton, CA, 94588) with as much blood as possible. The goal was to completely saturate the filter paper strip with bl ood. Then the filter paper strip was placed in a small plastic bottle with a desi ccant pack (Schleicher & Schuell BioScience, 10 Optical Ave., Keene, NH, 03431). Bottles with filt er paper strips and desiccant we re then sent to the lab for analysis. To test the accuracy of the antibody titer level resulting from samples collected with filter paper strips, serum and filter paper samples we re compared for 4 harvested turkeys and 12 live chickens. Blood was collected in a heparinized v acutainer. Then a filter paper strip was placed in the vial and saturated with blood. The re maining blood in the vial was spun down, and the serum separated. Each sample was analyzed for antibodies to IBDV serotype 2.

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38 Florida Sandhill Crane To determine if antibodies to IBDV serotype 2 were present in Florida sandhill cranes, 53 blood samples were collected in seven counties in central Florida: Hernando, Highlands, Lake, Orange, Osceola, Polk, and Sumter (Figure 3-2). Seven samples we re opportunistically collected from adult sandhill cranes. Four samples were obtained from dead or injured birds (3 from Osceola County and 1 from Orange County), 2 we re collected from nuisance birds (1 from Highlands County and 1 from Lake County), and 1 was acquired from an extremely tame bird that we were able to hand grab while capturi ng chicks at Moss Park in Orange County. Forty-six samples were collected from pre-fledgling sandhill cr ane chicks captured during the 2004, 2005, and 2006 breeding seasons (3 from Hernando County, 7 from Polk County, 2 from Sumter County, 30 from Osceola C ounty, and 4 from Orange County). Chicks ranged in age from approximately 25 to 65 days old. Capture teams of 3 to 5 wildlife biologi sts drove through known crane-breeding areas looking for families. Chicks selected for capture were at least 2 weeks of age, and no older than approximately 70 days so they could not fly. If the chicks could be cap tured safely, the team drove as close as possible to the family and ran out and captured the chicks by hand. Between 1 and 2 mL of blood we re collected from the medial metatarsal vein using a 25gauge needle. After collection blood was transf erred to a lithium hepa rinized vacutainer. Pictures were taken of the chicks head, wi ngs, and body so that age estimate could be confirmed. Chicks judged to be at least 50 days old were banded with color bands and/or aluminum FWS bands. Average handling time was 15 minutes, with a range from 6 to 29 minutes. Handling time for each chick varied base d on the number of chicks (single or twins), the ease with which I was able to get a blood sa mple, whether the chick(s) was old enough to be banded, and the capture teams le vel of experience. All blood samples collected from sandhill

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39 cranes were handled in the same manner, and eval uated using the same criteria as was used for wild turkeys. To determine if likelihood of e xposure (# birds wi th titer level 1:32) was independent of age, a likelihood ratio (G) test for independence was used. To determine if average titer level differed by age, a Wilcoxon rank sum test was use d. All tests were 2-tailed and considered significant at P 0.05. Analysis was performed using th e statistical software JMP 7 (SAS Institute 2007). Results Wild Turkey Overall, 6% of wild turkeys were exposed to IBDV serotype 2 (n = 596, mean = 1:7, SE = 1, median = 1:2, range = 1:0 to 1:256, Figure 3-3). Evidence of exposure was found in 8 counties in Florida (Hernando, Highlands, Lake, Osceola, Pasco, Polk, Putnam, Sumter) and 1 county in southern Ge orgia (Grady). Exposure prevalence (% samples with titer level 1:32) was 13% in 2003/04, 13% in 2004/05, 1% in 2005/06, and 0% in 2006/07 (Table 3-3). To investigate whether exposure prevalence and average tite r level differed significantly between years, I analyzed the five sites where 10 or more samples were collected in multiple years: Caravelle Ranch WMA-2004 vs. 2005, Half Moon WMA-2004 vs. 2006, Richloam WMA-2004 vs. 2006, Three Lakes WMA2005 vs. 2006, and Triple N Ranch WMA-2005 vs. 2006. Exposure prevalence differed significantly between years at al l sites, and average titer level differed significantly between years at all sites except Rich loam WMA (Table 3-4). Expos ure prevalence and titer level decreased over time at all sites except Ca ravelle Ranch WMA where there was an increase (Figure 3-4).

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40 Site specific exposure prevalence ranged from 0% to 30%, with 14 sites having no detectable evidence of exposure (Figure 3-1). Exposure prevalence (P=0. 0003) and average titer level (P<0.0001) differed significan tly between the sample sites. When performing individual comparisons I eliminated all sites where less than 10 samples were collected (Andrews WMA, Choctawhatchee River WMA, Tosohatchee WM A, and Upper Hillsborough WMA). The two sites with the highest exposure prevalence, Triple N Ranch WMA (30%) and Half Moon WMA (24%), did not differ significantly from each other but had significantly higher exposure prevalence than most other sites. Expos ure prevalence on Triple N Ranch WMA was significantly greater than exposure prevalence on all sites except Brooksville (8%) and Lake Panasoffkee WMA (8%). Exposure prevalence on Half Moon WMA was si gnificantly greater than exposure prevalence on all sites except South Georgia (5%), Brooksville, and Lake Panasoffkee WMA. When locations were combined into regions (a rea with a 10.5 mile radius) there was still a significant difference in exposure prevalen ce (P=0.0020) and average titer level (P<0.0001). The two regions with the highest exposure pr evalence were the one th at included Half Moon WMA and the one that included Triple N Ranch WMA. Exposure prevalence decreased to 19% on Half Moon WMA and 9% on Trip le N Ranch WMA. This caused the exposure prevalence of Half Moon WMA to become significantly higher th an that of Triple N Ranch WMA. When locations were combined the number of sites that Half Moon WMA wa s significantly greater than was reduced from 18 to 14, and the num ber of sites that Tr iple N Ranch WMA was significantly greater than was reduced from 19 to 10. The only site that Triple N Ranch WMA did not differ from alone but differed from when combined by region was the site that was combined with Half Moon WMA.

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41 Florida Bobwhite Quail None of the 11 filter paper samples collected on Babcock-Webb WMA were completely saturated with blood. All were analyzed, and each had a titer level of 1:0. However, of the 16 serum and filter paper samples compared, none result ed in matching titer le vels. For 6 birds the filter paper method overestimated antibody titer level, and for 10 birds it underestimated antibody titer level (Figure 3-7). Florida Sandhill Crane Overall, 7.5% of sandhill cranes were exposed to IBDV serotype 2 (n = 53, mean = 1:10, SE = 3, median = 1:4, range = 1:0 to 1:128,). Exposure prevalence (p=0. 0025) and average titer level (p=0.0155) were significantl y higher in adults than juven iles (Figure 3-6). Forty-three percent of adults were exposed to the virus (mean = 1:37, SE = 17, median = 1:16, range = 1:0 to 1:128). Two percent of juveniles were exposed to the virus (mean = 1:6, SE = 1, median = 1:3, range = 1:0 to 1:32). Discussion The hypothesis that wild turkeys and sandhill cranes in Florida have been exposed to IBDV serotype 2 was supported. Overall preval ence of exposure in wild turkeys remained constant during the 2003/04 and 2004/05 sampli ng years and then decreased thereafter, suggesting that viral occurrence may be cyclic in nature. This was supported by exposure prevalence decreasing significantly between years on all sites tested except Caravelle Ranch WMA where exposure prevalence increased. But this is the only site where both years used for comparison were during the sampling years ( 2003/04 and 2004/05) when overall exposure prevalence remained constant.

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42 I was unable to assess site specific varia tion in exposure prevalence of sandhill cranes due to small samples sizes, but prevalence of exposure in wild turkeys did vary among sites. However, this result must be interpreted with caution as 12 of the 14 si tes with no detectable evidence of exposure were sites sampled only in 2006, a year with extremely low exposure prevalence statewide. If viral occurrence is cyclic in nature then differences in site specific exposure may be the result of populations being in different phases of this cycle. Adult sandhill cranes captured for this st udy had significantly higher seroprevalence and average titer level than juvenile s. Although the adult sample size was small, this is consistent with findings of Spalding et al (2006) that exposure preval ence in whooping cranes increases with age. I was unable to assess the prevalence of exposure to IBDV serotype 2 in Florida bobwhite quail. Collecting a sufficient amount of blood for analysis from harvested birds proved difficult, and results from the filter paper comparat ive study demonstrated that the resulting titer levels were not accurate. Therefore the use of f ilter paper strips was not a reliable method to determine the antibody titer le vel for IBDV serotype 2 in Florida bobwhite quail. Hunters involved in the pilot study on Babcock-Webb WMA were cooperative and exhibited interest in being involved in the investigation of disease prevalence in Florida bobwhite quail. Large numbers of quail are harvested each year and presen t an opportunity to investigate disease issues in this species. Alternative me thods for collecting blood from harvested quail that could be used for disease studies should be explored. But it appears that future research investigati ng prevalence of IBDV would have to involve sacrificing quail specifically for the disease study or live trapping to collect blood samples.

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43 Figure 3-1. Blood collection sites for wild tu rkey samples collected from December 2003 through January 2007 (n=596). Exposure preval ence is listed beside each location. Locations with no percent listed had 0% exposure prevalence. Table 3-1. Number of blood samples collected from harvested wild turkeys during the 2004, 2005, and 2006 spring turkey seasons. Location County n Andrews WMA Levy 5 Avon Park Bombing Range Polk, Highlands 25 Private properties near Brooksville Hernando 13 Bull Creek WMA Osceola 34 Choctawhatchee River WMA and private property Holmes 8 Corbett WMA Palm Beach 17 Dupuis WMA Martin, Palm Beach 14 Fisheating Creek WMA Glades 16 Green Swamp WMA Polk, Sumter, Lake, Pasco 53 Half Moon WMA Sumter 41

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44 Table 3-1. Continued Location County n Hickory Hammock WMA and pr ivate property Highlands 11 Lake Panasoffkee WMA Sumter 13 Richloam WMA Hernando, Pasco, Sumter, Lake 44 Seminole Forest WMA Lake 16 Tall Timbers Research Station and private property Madison, Wakulla, Leon, Gadsen, Jefferson 11 Three Lakes WMA Osceola 74 Tosohatchee WMA Orange 8 Triple N Ranch WMA Osceola 23 Upper Hillsborough WMA Polk, Pasco 8 Table 3-2. Number of blood samples collected from wild turkeys using rocket nets from December 2003 through January 2007. Location County n Caravelle Ranch WMA Putnam 71 Ordway/Swisher Preserve Putnam 21 Lykes Brothers Ranch Glades 18 Sharps Ranch DeSoto 13 2-Rivers Ranch Hillsborough 13 Three Lakes WMA Osceola 7 Private Plantations, South Georgia Thomas, Grady 19

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45 Figure 3-2. Blood collection site s for Florida sandhill crane samples collected during 2004, 2005, and 2006 (n=53).

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46 0 25 50 75 100 125 150 175 200 0248163264128256Titer Level# of Samples Figure 3-3. Frequency of titer levels for all wild turkey samples collected from December 2003 through January 2007 (n=596). Table 3-3. Statistical results for yearly exposure prevalence in wild turkeys. Year n Mean SE MedianRange 2003/04 95 1:14 4 1:021:0 to 1:256 2004/05 128 1:12 2 1:041:0 to 1:128 2005/06 353 1:05 0.3 1:021:0 to 1:32 2006/07 20 1:00 0.1 1:001:0 to 1:2

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47 0%0%0% 0%0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Caravelle Ranch Half MoonRichloamThree LakesTriple N Ranch Year 1 Year 2 Figure 3-4. Wild turkey exposure prev alence (% birds with titer level 1:32) at five sample sites where 10 or more samples were co llected in multiple years. Table 3-4. P-values for differen ce in yearly exposure prevalence and average titer level in wild turkeys at sites where more than 10 samples were collected. Location n1 n2 Average Titer Level Exposure Prevalence Caravelle Ranch WMA 36 in 200435 in 2005P<0.0001P=0.0364 Half Moon WMA 15 in 200419 in 2005P=0.0002P=0.0011 Richloam WMA 16 in 200428 in 2006NSDP=0.0401 Three Lakes WMA 21 in 200553 in 2006P=0.0362P=0.0012 Triple N Ranch WMA 11 in 200512 in 2006P=0.0080P=0.0002

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48 0 2 4 6 8 10 12 14 0248163264128256Titer LevelNumber of Samples Juveniles Adults Figure 3-5. Frequency of titer levels for Fl orida sandhill cranes captured in 2004, 2005, and 2006 (n=53).

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49 0000000000 00 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68t u rk e y turkey t u rkey tu rk ey chi c ken ch ick en chicken chi ck en c h ick en chi ck en ch ick en chi c ken ch ick en chi c ken ch ick en chickenTiter Level Filter Paper Strip Serum Figure 3-6. Titer level results for serum/filter paper comparisons.

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50 CHAPTER 4 ARCHIVED SAMPLES Introduction The original source of IBDV serotype 2 in wild bird of Florida is unknown. There are many possibilities related to domes tic poultry operations such as transmission of the virus by poultry workers on contaminated footwear, inappr opriate disposal of poultry products, and the use of litter containing feces as fertilizer in ag ricultural operations. But once it was determined that some cranes in captive-rearing facilities had been exposed to IBDV, there was concern that captive-reared cranes may be re sponsible for introducing the viru s to wild birds of Florida (Hartup and Sellers 2006). There have been inst ances when captive-bred animals exposed to a pathogen in the captive facility, exposed wild animals in and ar ound the release site to those pathogens (Spalding et al.1996, Snyder et al.1996, Woodford and Rossiter 1994). This possibility could be elim inated if it was determined that wild birds in Florida had been exposed to the virus prior to the release of captive-reared cranes, or in areas where contact with captivereared cranes was highly unlikely. Sandhill cranes reared at Patuxent Wildlife Research Center in Laurel, Maryland were sporadically released into Fl orida from 1971 1991 as preliminar y trials to develop release techniques for captive-reared whooping cranes. Fourteen 5-month-old captive-reared sandhill cranes were released in 1971 near Palmdale in Gl ades County (Figure 4-1). None of these birds were observed associating with wild sandhill cranes and all died within 3 months without leaving the immediate area (Nesbitt 1978). From 1974 1976, 4 captive-reared sandhill cranes ranging from 6 months to 4 years-of-age were releas ed on Paynes Prairie in Alachua County (Figure 41). Three died and 1 paired with a wild sandh ill crane and set up a te rritory on Paynes Prairie (Nesbitt 1978). Additional releases took plac e on Paynes Prairie from 1986 1987. Twenty-

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51 seven 9 to 10-month-old captive-reared sandhill cran es were released. Survivors dispersed to Gilchrist, Levy, Marion, Putnam, and Sumter co unties (Nesbitt and Carpenter 1993) (Figure 41). In 1991, 15 captive-reared sandhill cranes rangi ng from 1 to 2 years-of-age were placed in a holding pen on Kanapaha Prairie in Alachua County (Figure 4-1). The 11 surviving birds were then moved to a release pen on the Prairie Unit of Three Lakes WMA in Osceola County (Figure 4-1). Some experimental birds interacted with wild sandhill cranes, and two formed pairs with wild sandhill cranes (Nesbitt and Folk 1992). In 1993, the first captive -reared whooping cranes were released at Three Lakes WMA. Materials and Methods I was unable to locate any blood samples collect ed prior to 1971. As a result I could not test the hypothesis that wild bi rds in Florida were exposed to IBDV serotype 2 prior to the release of captive-reared cranes. Instead, I analyzed samples co llected from wild birds in an area where contact with captive-reared cranes was hi ghly unlikely, and hypothe sized that these birds had been exposed to the virus. From 1991 2000, Dr. Marilyn Spalding archived 477 wild sandhill crane serum samples collected in 7 counties (Alachua, Citrus, Lake Levy, Marion, Osceola, Sumter) in Florida. Samples were eliminated from consideration if they were collected in a county after captivereared cranes had been released there or were kn own to have dispersed ther e. Unfortunately, this eliminated from consideration all but 3 samples collected in Lake County in 1993 (Figure 4-1). These samples were tested for antibodies to IBDV serotype 2. The archived samples also included serum collected from captive-reared sa ndhill cranes prior to their release on Three Lakes WMA in 1991. Samples for 10 of the 11 birds released were tested for antibodies to IBDV serotype 2.

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52 To gain further insight into prevalence of exposure and length of time the virus has been present in wild sandhill cranes of Florida, 108 archived seru m samples collected in Alachua County (northern Florida) and Osceola County (central Florida) from May 1992 March 1998 were tested for antibodies to IBDV serotype 2. These samples came from 98 individuals. All serum samples were sent to the Poultry Diagnostic & Research Center (College of Veterinary Medicine, University of Georgia, 953 College St ation Road, Athens, GA 30602) for evaluation. They were tested for IBDV serotype 2 antibodies using the beta procedure (constant virus/diluted serum) described in Chapter 2. It is unknown what titer level indicates true e xposure to IBDV. Previous studies of wild birds have considered titer leve ls ranging from 1:16 to 1:80 as evidence of exposure (van den Berg 2001, Hollmen et al. 2000, Ogawa et al. 19 98, Gardner et al., 1997, Wilcox et al. 1983). For the purposes of this study I assumed that a ti ter level of 1:16 or less was not indicative of exposure to the virus. Birds with a titer level of 1:32 or greater were considered exposed to the virus. I detected an age effect on seroprevalence in the sandhill cr ane samples collected for this study (reported in Chapter 3); howev er, the adult sample size was small (n = 7). Analysis of the archived samples separated by age allowed for fu rther investigation of this finding. Samples were separated into age categories. Juveniles ranged in age from 55 days to 10 months. Subadults ranged from 12 months to 2.7 years. Adults ranged from 3 to 10+ years. To determine if likelihood of exposure (# samples with titer level 1:32) was independent of age, the likelihood ratio (G) test for independence wa s used. Post-hoc analysis on each pair of treatments was done using the same test. To de termine if average titer level differed by age, a

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53 Kruskal-Wallis nonparametric ANOVA was used. Post -hoc analysis on each pair of treatments was done using the Wilcoxon rank sum test. Exposure prevalence and average titer leve l of wild turkeys sampled in this study (reported in Chapter 3) differed significantly am ong locations. Analysis of archived samples allowed for further investigation of this result. To determine if mean titer level differed between sandhill cranes captured in Alachua and Osceola counties, a Wilcoxon rank sum test was used. To determine if likelihood of expos ure (# samples with titer level 1:32) was independent of location, a likelihood ratio (G) test for independ ence was used. All tests were 2-tailed and considered significant at P 0.05. Analysis was performed usi ng the statistical software JMP 7 (SAS 2007). Results Of the three samples collected from wild sa ndhill cranes in Lake County in 1993, all birds were exposed to IBDV serotype 2. Titer levels ranged from 1:32 to 1:128. None of the samples collected from captive-reared sandhill cranes rele ased on Three Lakes WMA in 1991 had titer levels high enough to indicate exposure. Ti ter levels ranged from 1:0 to 1:4. Forty-six percent of samples collected from wild sandhill cranes in Alachua and Osceola counties had titer levels high enough to indicate exposure to IBDV serotype 2 (n = 108, median = 1:16, range = 1:0 to 1:1024, mean = 1:52, SE = 11) Sixty-three percent of adults, 56% of subadults, and 13% of juveniles had been exposed to the virus (Table 4-1, Figure 4-2). Juveniles had significantly lower exposure prevalence than both adults (P<0.0001) and subadults (P<0.0002), but there was not a si gnificant difference between a dults and subadults. Average titer level differed among all age groups (P< 0.0001) with adults having significantly higher average titer levels than subadults (P=0.0043) and juveniles (P<0.0001), and subadults having significantly higher average ti ter levels than juveniles (p=0.0001).

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54 Samples collected in Alachua County had significantly higher average titer level (P=0.0291) and exposure prevalence (P=0.0307) than those collected in Osceola County. In Alachua County, 54% of samples had titer levels high enough to indicate exposure, and earliest evidence of exposure came from samples collecte d on May 7, 1992. (Table 4-2, Figure 4-3). In Osceola County, 38% of birds ha d titer levels high enough to i ndicate exposure, and earliest evidence of exposure came from samples collec ted on October 1, 1992 (Table 4-2, Figure 4-3). Discussion All 3 sandhill crane samples collected in Lake County prior to the release/dispersal of captive-reared cranes to that area, had titer levels high enoug h to indicate exposure to IBDV serotype 2. Although this may be evidence that th e release of captive-rear ed cranes was not the original source of the virus, I cannot discount the possibility that wild birds having contact with captive-reared cranes dispersed to th e area. Therefore, I am unable to rule out the possibility that captive-reared cranes were the original source of the virus in wild birds of Florida. However, because 10 of the 11 captive-re ared sandhill cranes released on Three Lakes WMA in 1991 did not have titer levels high enough to indicate exposure (1 crane was not tested), we can be fairly confident that this particular release was not the source of the virus in wild birds of Florida. Significantly higher exposure prevalence and average titer levels were found in adult sandhill cranes when compared to juvenile cranes Subadults were intermediate between adults and juveniles as there was no statistical differenc e in prevalence of exposure between adults and subadults, but there was a significant difference in mean titer level. This is because the majority of subadult samples indicating expo sure (75%) had a tite r level of 1:32, the lowest possible titer level to indicate exposure. There were very few samples at the higher end of the spectrum, with

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55 no subadult birds having a titer level higher than 1:128. In cont rast, 31% of adults had titer levels greater than 1:128 and only 19 % had a titer level of 1:32. The significant trend toward hi gher titer levels as the bird s age suggests that there is constant re-exposure or that birds remain carriers of the virus. If sandhill cranes are re-exposed to the virus throughout their lifetime they coul d mount a more effective immune response with each subsequent exposure thus leading to higher titer levels as the birds age. Although unknown for IBDV in cranes, birds that are carriers of a vi rus could have a latent infection that results in intermittent shedding of the virus throughout their life. This could result in higher ti ter levels as the birds age as well.

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56 Figure 4-1. Captive-reared sandhill crane and whooping crane release s ites (depicted with triangles) and dispersal areas (depicted with dots). Po lk and Lake County release sites are locations where sentinel chickens were placed (Chapter 2). Table 4-1. Statistical results fo r exposure prevalence by age of archived sandhill crane samples collected in Alachua and Osceola co unties from May 1992 to March 1998. Age n Mean SE Median Range Adult 41 1:104 27 1:641:4 to 1:1024 Subadult 36 1:28 4 1:321:2 to 1:128 Juvenile 31 1:12 3 1:041:0 to 1:64

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57 0 5 10 15 20 02481632641282561024Titer Level# of Samples Adults Subadults JuvenilesFigure 4-2. Frequency of titer levels for juveni le, subadult, and adult archived sandhill crane samples collected in Alachua and Osceo la counties from May 1992 to March 1998 (n=108). Table 4-2. Statistical results for exposure prevalence by county of archived sandhill crane samples collected in Alachua and Osceo la counties from May 1992 to March 1998. County n Mean SD Median Range Alachua 55 1:70 149 1:321:2 to 1:1024 Osceola 53 1:33 53 1:161:0 to 1:256

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58 0 2 4 6 8 10 12 14 02481632641282561024Titer Level# of Samples Alachua County Osceola County Figure 4-3. Frequency of titer levels for archived sandhill crane samples collected in Alachua and Osceola counties from May 1992 to March 1998 (n=108).

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59 CHAPTER 5 SYNTHESIS AND SIGNIFICANCE Wild turkeys and sandhill cranes throughout Florida have been exposed to IBDV serotype 2. The virus has been present in Florida for at least 15 years and is available to infect susceptible hosts. Because we know so little about the distribution of this virus in the environment and its mode of transmission, it is imperative that we conduct further research in order to learn what, if any, steps can be taken to minimize the effect s of this virus on the survival of endangered whooping cranes. The presence of th e virus in Florida coul d not be linked with certainty to the reintroduction project, but the ev idence is consistent with the virus being present in the environment for a long time. Implications for the Whooping Crane Reintroduction Project Many of the wild turkey a nd sandhill crane blood collection sites overlap with areas where whooping cranes are curren tly found or have been found in the past. Therefore whooping cranes, both resident and migratory, could come in contact with wild turkeys and sandhill cranes that have been exposed to IBDV serotype 2. In addition, although these findings do not rule out other potential exposure mechanisms, they do sugg est that post-release in teraction with wild birds of Florida is one potent ial exposure mechanism for w hooping cranes involved in the 1997/98 and 2001/02 mortality events. The presence of this virus in wild turkeys and sandhill cranes of Florida is especially concerning for the resident flock of whooping cranes because they nest and raise their chicks in Florida. When chickens are exposed to IBDV be tween 3 and 6 weeks of age, symptoms rapidly appear and mortality rates can approach 30%. Wh en chickens are exposed before 3 weeks of age a severe, prolonged immunosuppressi on results, leaving the birds vul nerable to normally benign disease agents (Lukert and Saif 2003). The effect of exposure to IBDV on prefledgling

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60 whooping crane chicks is unknown at this time. However, the impact to young chickens suggests that if whooping crane chic ks hatched in the wild are expos ed to the virus at an early age, this could greatly reduce chick survival pot ential. Conversely, the relatively high titers maintained by adults may be passed on to the chicks and protect them at this otherwise vulnerable period in their lives. Age Effect on Seroprevalence Significantly higher exposure prevalence and average titer levels were found in adult sandhill cranes captured for this study and in ar chived samples, when compared to juvenile cranes. These findings are consistent with the findings of Spalding et al. (2006) that exposure prevalence in whooping cranes incr eased with age. The lower seroprevalence and titer levels in juveniles could be explained by juvenile cran es having a shorter exposure time and immature immune system. The higher seroprevalence and tite r levels in older birds suggest that there is constant re-exposure or that bird s remain carriers of the virus. However, there is also the possibility that this age effect on seroprevalence reflects decreased survival of sandhill cranes infected at a young age (Schettler et al. 2001, Garvin et al. 2004) The effect of exposure on sandhill crane chicks is unknown at this time. However, the impact to young chickens suggests that if sandhill crane chicks hatche d in the wild are exposed to th e virus at an early age, chick survival could be greatly reduced. If exposed ch icks are less likely to survive then they are consequently less likely to be sampled, biasing th e chick samples toward birds that have not been exposed to the virus. Therefore, investigation into the pathogenicity of IBDV in sandhill cranes is warranted. Variation in Exposure Prevalence among Sites Prevalence of exposure in wild turkeys and archived sandhill crane samples varied among sites. Future research should investig ate exposure prevalence in relation to potential

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61 sources of infection such as domestic poultry faci lities and small family farms with free-ranging back yard poultry. However, it is important that the research project contro l for effect of year as viral occurrence may be cyclic in nature. Transmission Mechanisms There are 2 subspecies of the sandhill crane found in Florida. The Florida sandhill crane ( Grus canadensis pratensis ) is resident, and the greater sandhill crane ( Grus canadensis tabida ) is migratory. These 2 subspecies interact when greater sandhill cranes are in Florida during the winter. The role that greater sa ndhill cranes play in the epidem iology of IBDV in wild birds of Florida is unknown, and certainly warrants furt her investigation. Out of the 108 archived samples analyzed, 6 came from greater sandhill cran es. One of these birds had been exposed to the virus. Therefore, greater sandhill cranes ma y become exposed to the virus on their wintering grounds in Florida and carry the vi rus north with them. Or it is possible that greater sandhill cranes were exposed first, and are the original source of th e virus in wild birds of Florida. Either way, this is a potential transmission mech anism for IBDV throughout the flyway. The possibility that wild bird s were exposed to the virus as a result of domestic poultry operations or disposal of poultry products has not been addressed in this study but certainly remains a potential source of exposure. The use of poultry litter containing feces as fertilizer in Floridas agricultural industry remains one potential mechanism for transmission of the virus from domestic operations to the wild. Farmers should be educated on th e importance of properly composting chicken litter to e liminate the possibility of disease transfer. In addition, investigation into methods that minimize the tr ansfer of mealworms w ithin the litter could greatly reduce the potential for tr ansmission from domestic operations to the wild. Research into potential insect vectors is n eeded as well, and should focus on mealworms, dung beetles, and mosquitoes.

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62 LIST OF REFERENCES Ahad, A. 2002. Isolation and pathogenic characte rizations of IBDV isolate from an outbreak of IBD in a rural poultry unit in Bangladesh. Th esis, The Royal Veterinary and Agricultural University, Copenhagen, Denmark and Banglad esh Agricultural University, Mymensingh, Bangladesh. Allan, W. H., J. T. Faragher, and G. A. Cullen. 1972. Immunosuppression by the infectious bursal agent in chickens immunized against Ne wcastle disease. Veterinary Records 90: 511-512. Barnes, H. J., J. Wheeler, and D. Reed. 1982. Se rologic evidence of inf ectious bursal disease virus infection in Iowa turkey s. Avian Diseases 26: 560-565. Benton, W. J., M. S. Cover, J. K. Rosenberg er, and R. S. Lake. 1967. Physicochemical properties of the infectio us bursal agent (IBA). Av ian Disease 11: 438-445. Berger, J. 1990. Persistence of different-sized populations: an empirical assessment of rapid extinctions in bighorn sheep. Conservation Biology 4: 91-98. Campbell, G. 2001. Investigati on into evidence of e xposure to infectious bursal disease virus (IBDV) and chick infectious anaemia virus (C IAV) in wild birds in Ireland. Pages 230235 in Proceedings of the International Sym posium on Infectious Bursal Disease and Chicken Infectious Anaemia, 2001, Rauischholzhausen, Germany. Chin, R. P., R. Yamamoto, L. Weiqing, K. M. Lam, and T. B. Farver. 1984. Serological survey of infectious bursal disease vi rus serotypes 1 and 2 in Califor nia turkeys. Avian Diseases 28(4): 1026-1036. Daszak, P., and A. A. Cunningham. 2000. Emerging infectious diseases of wildlifethreats to biodiversity and human healt h. Science 287(5452): 443-449. Dunford, J. C., and P. E. Kaufman. 2006. Lesser mealworm, Alphitobius diaperinus University of Florida Institute of Food a nd Agricultural Sciences Publication Number EENY-367. Folk, M. J., S. A. Nesbitt, J. M. Parker, M. G. Spalding, S. B. Baynes, and K. L. Candelora. 2006. Current status of non migratory whooping cranes (G rus americana ) in Florida. Proceedings of North American Crane Workshop 10 In Press: 00-00. Gardner, H., K. Kerry, M. Riddle, S. Brouwer, a nd L. Gleeson. 1997. Poultry virus infection in Antarctic penguins. Nature 387: 245. Garvin, M. C., K. A. Tarvin, L. M. Stark, G. E. Woolfenden, J. W. Fitzpatrick, and J. F. Day. 2004. Arboviral infection in two species of w ild jays (Aves: Corvidae): Evidence for population impacts. J. Med. Entomol. 41(2): 215-225.

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63 Gauthier-Cleric, M., N. Eterra dossi, D. Toquin, M. Guittet, G. Kuntz, and Y. Le Maho. 2002. Serological survey of the king penguin, Aptenodytes patagonicus in Crozet Archipelago for antibodies to infectious bursal disease, influenza A and Newcastle disease viruses. Polar Biology 25: 316-319. Giambrone, J. J., O. J. Fletcher, P. D. Lukert, R. K. Page, and C. E. Eidson. 1978. Experimental infection of turkeys with in fectious bursal disease virus. Avian Diseases 22: 451-458. Glenn, T.C., W. Stephan, and M.J. Beaun. 1999. Effects of a population bottleneck on whooping crane mitochondrial DNA variation. Conservation Biology 13 (5): 1097-1107 Hartup, B. K., and H. S. Sellers. 2006. Serologi cal survey for infectious bursal disease virus exposure in captive cranes. Proceedings of North American Crane Workshop 10 In Press: 00-00. Hollmen, T., M. Kilpi, M. Hario, L. H. Creekmore, and M. R. Petersen. 2000. Infectious bursal disease virus antibodies in eider ducks and herring gulls. Condor 102: 688-691. Hofle, U., J. M. Blanco, and E. F. Kaleta. 2001. Neutralising antibod ies against infectious bursal disease virus in sera of free-living a nd captive birds of prey from central Spain (Preliminary Results). 2001. Pages 247-251 in Proceedings of the International Symposium on Infectious Bursal Disease and Chicken Infectious Anaemia. 2001, Rauischholzhausen, Germany. Howie, R. I., and J. Thorsen. 1981. Identification of a strain of infectious bursal disease virus isolated from mosquitoes. Canadian Jour nal of Comparative Me dicine 45: 315-320. Jackwood, D. J., Y. M. Saif, P. D. Moorhead, and R. N. Dearth. 1981. Infectious bursal disease virus and Alcaligenes faecalis infections in turkeys. Avian Diseases 26: 365-374. Jackwood, D. J., Y. M. Saif, and J. H. Hughes. 1982. Characteristics and serologic studies of two serotypes of infectious bursal disease viru s in turkeys. Avian Diseases 26 (4): 871882. Jackwood, D. J., R. E. Gough, and S. E. Somm er. 2005. Neucleotide and amino acid sequence analysis of a birnavirus isolated from penguins. Veterinary Record 156: 550-552. Jones, K. L., T. C. Glenn, R. C. Lacy, J. R. Pierce, N. Unruh, C. M. Mirande, and F. ChavezRamirez. 2002. Refining the whooping cr ane studbook by incorporating microsatellite DNA and leg-banding analyses. Conservation Biology 16: 789-799. Lafferty, K. D., and L. R. Gerber. 2002. Good medicine for cons ervation biology: The intersection of epidemiology and conserva tion theory. Conservation Biology 16: 593-604. Lukert, P. D., and Y. M. Saif. 2003. Infectious bursal disease. Pages 161-179 in Y. M. Saif, editor. Diseases of Poultry. Eleventh edit ion. Iowa State Univer sity Press, Ames, IA, USA.

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64 McAllister, J. C., C. D. Steelman, L. A. Newb erry, and J. K. Skeeles. 1995. Isolation of infectious bursal disease virus from the lesser mealworm, Alphitobius diaperinus (Panzer). Poultry Science 74(1): 45-49. Murphy, D. W. 1990. Disease transfer studies in a dead bird composter. Pages 25-29 in Proceedings of the 1990 National Poultry Wast e Management Symposium. 3 October-4 October 1990, North Carolina, USA. Nawathe, D. R., O. Onunkwo, and I. M. Smith. 1978. Serological eviden ce of infection withthe virus of infectious bursal diseas e in wild and domestic birds in Nigeria. Veterinary Record 102: 444. Nesbitt, S. A. 1978. Notes on suitability of capti ve-reared sandhill cranes for release into the wild. Proceedings of North American Crane Workshop: 85-88. Nesbitt, S. A., and M. J. Folk. 1992. W hooping crane reintroduction in Florida final performance report. Florida Game and Freshwater Fish Commission, Research Laboratory, Gainesville, FL, USA. Nesbitt, S. A., and J. W. Carpenter. 1993. Surv ival and movements of greater sandhill cranes experimentally released in Florida. Journal of Wildlife Management 57(4): 673-679. OBrien, S. J., M. E. Roelke, L. Marker, A. Newm an, C. A. Winkler, D. Me ltzer, L. Cooley, J. F. Evermann, M. Bush, and D. E. Wildt. 1985. Ge netic basis for species vulnerability in the cheetah. Science 277: 1428-1434. Ogawa, M., T. Wakuda, T. Yamaguchi, K. Mura ta, and A. Setiyono. 1998. Seroprevalence of infectious bursal disease virus in free-living wild birds in Japan. Journal of Veterinary Medical Science 60(11): 1277-1297. O'Malley, C. M. 1990. Aedes vexans (Meigen): an old foe. Proceedings of New Jersey Mosquito Control Association. Pages 90-95. SAS Institute 2007. JMP 7. SAS Institute, Cary, North Carolina, USA. Schettler, E., T. Langgemach, P. Sommer, J. Streich, and K. Frolich. 2001. Seroepizootiology of selected infectious disease agents in free -living birds of prey in Germany. Journal of Wildlife Diseases 37(1): 145 Smith, B. L. 2001. Winter feeding of elk in western North America. Journal of Wildlife Management 65(2): 173-190. Snyder, N. F. R., S. R. Derrickson, S. R. Beissi nger, J. W. Wiley, T. B. Smith, W. D. Toone, and B. Miller. 1996. Limitations of captive breeding in endangered species recovery. Conservation Biology 10(2): 338-348.

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65 Spalding, M. G., J. M. Kinsella, S. A. Nesbitt, M. J. Folk, and G. W. Foster. 1996. Helminth and arthropod parasites of expe rimentally introduced whooping cr anes in Florida. Journal of Wildlife Diseases 32(1): 44-50. Spalding, M. G., H. S. Sellers, B. K. Hartup, a nd G. H. Olsen. 2006. A wasting syndrome in released whooping cranes in Florida associated with infectious bur sal disease titers. Proceedings of North American Crane Workshop 10 In Press: 00-00. Thorne, T. E., and E. S. Williams. 1988. Disease and endangered species: the black-footed ferret as a recent example. Cons ervation Biology 2(1): 66-74. U. S. Fish and Wildlife Service. 1994. Whooping Crane Recovery Plan. Albuquerque, New Mexico, USA. van de Bildt, M. W. G., T. Kuiken, A. M. Visee, S. Lema, T. R. Fitzjohn, and A. Osterhaus. 2002. Distemper outbreak and its effect on African wild dog conservation. Emerging Infectious Diseases 8: 211-213. Van den Berg, T. P., A. Ons, M. Dolores, M ., and J. F. Rodriguez. 2001. Experimental inoculation of game/ornamental birds with a very virulent strain of IBDV. Pages 236-246 in Proceedings of the International Symposium on Infectious Bursal Disease and Chicken Infectious Anaemia. 2001, Rauischholzhausen, Germany. Varela, A., JM. Kinsella, and M. G. Spalding. 20 01. Presence of encysted immature nematodes in a released whooping crane (G rus americana). Journal of Zoo and Wildlife Medicine 32 (4): 523. Weisman, J., and S. B. Hitchner. 1978. Infecti ous bursal disease virus infection attempts in turkeys and coturnix quail. Avian Diseases 22: 604-608. Wilcox, G. E., R. L. P. Flower, W. Baxendale, and J. S. Mackenzie. 1983. Serological survey of wild birds in Australia for the prevalence of antibodies to egg drop syndrome 1976 (EDS76) and infectious bursal disease viruses. Avian Pathology 12:135-139. Wiley, N. 2005. Bobwhite management and hunti ng in Floridas ranchla nds: an overview of rules and regulations. Proceedings of quail management short course 1: 76-80. Woodford, M. H., and P. B. Rossiter. 1994. Diseas e risks associated with wildlife translocation projects. Pages 178-198 in Olney, P. J. S., Mace, G. M. and A. T. C. Feistner, editors. Creative Conservation. Chapman & Hall, London, UK. Woodroffe, R. 1999. Managing disease threats to wild mammals. Animal Conservation 2: 185193.

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66 BIOGRAPHICAL SKETCH Kristen Lee Candelora was born in Gainesv ille, FL in 1974. She grew up in Tampa, graduating from Chamberlain Hi gh School in 1992. Kristen earne d a B.A. in psychology from the University of North Carolina at Wilmington in 1997. Following graduation, she returned to Tampa to work as a Crisis Counselor on a Baker Act Unit. Kristen earned a B.S. in wildlife ecology and conservation from the University of Florida in 2002. Upon graduation, she began work as a Whooping Crane Biologist for the Florida Fish and Wildlife Conservation Commi ssion (FWC). After working fo r FWC for two years, Kristen entered the Wildlife Ecology and Conservation M.S. program at the University of Florida. She continued working for FWC while completing her masters project. She received the Best Student Paper award at the 10th North American Crane Workshop in Zacatecas, Mexico and the Florida Chapter of The Wildlife Society spri ng meeting in Cocoa Beach, Florida. Upon completion of her M.S. program, Kris ten began work as the Private Lands Coordinator for the Upland Ecosys tem Restoration Project.