Biology and colonization of the sand fly Lutzomyia diabolica (Hall) (Diptera: Psychodidae) with notes on its potential r...

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Title:
Biology and colonization of the sand fly Lutzomyia diabolica (Hall) (Diptera: Psychodidae) with notes on its potential relationship to human cutaneous leishmaniasis in Texas, USA
Uncontrolled:
Lutzomyia diabolica
Physical Description:
xv, 244 leaves : ill. ; 28 cm.
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English
Creator:
Lawyer, Phillip G., 1945-
Publication Date:

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Subjects / Keywords:
Psychodidae   ( lcsh )
Leishmaniasis, Cutaneous   ( lcsh )
Insects as carriers of disease   ( lcsh )
Insects -- Texas   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1984.
Bibliography:
Includes bibliographical references (leaves 229-242).
Statement of Responsibility:
by Phillip G. Lawyer.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000481004
notis - ACP8566
oclc - 11908120
System ID:
AA00003417:00001

Full Text










BIOLOGY AND COLONIZATION OF THE SAND FLY
Lutzomyia diabolica (HALL) (DIPTERA: PSYCHODIDAE)
WITH NOTES ON ITS POTENTIAL RELATIONSHIP TO HUMAN
CUTANEOUS LEISHMANIASIS IN TEXAS, USA





By

PHILLIP G. LAWYER


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












UNIVERSITY OF FLORIDA


1984































TO MY "FOREVER" FAMILY












ACKNOWLEDGEMENTS


Producing this dissertation involved the skills of many people

and, although I accept full responsibility for its contents, I do not

claim all the credit.

I am indebted to the United States Army for providing the

opportunity to achieve this long time goal. I gratefully acknowledge

the selfless contributions of members of my graduate committee. Dr.

Jerry F. Butler, chairman, provided continuous instructional guidance,

moral and material support throughout the course of this study. Dr.

David G. Young was a true mentor, providing many unanswered questions

and the principal direction for my research; his enthusiasm for

natural science in general and medical entomology in particular was

very contagious. Dr. Stephen G. Zam gave freely of his time and

expertise in parasitology and protozoology and offered valuable

suggestions. I genuinely appreciate such a rare individual as Dr. A.

G. B. Fairchild for his wisdom, historical perspective, and his

ability as an editor to make kid gloves from a sow's ear. LTC(P) John

Reinert served on the committee for the first year and, in concert

with the other committee members, helped outline a demanding schedule

of courses.

Sincere thanks go to Ms. Diana Simon, Mrs. Debra Boyd, and other

members of the laboratory staff for the many administrative and

logistical details I was able to take for granted. I extend special






thanks to Ms. Edna Mitchell for long hours of tedious work devoted to

maintenance of sand fly and rodent colonies, and to conducting life

cycle experiments. Mr. Ray Hale and Ms. Gabrielle Hodson assisted

with the photography and graphics, and Mrs. Margo Duncan offered

valuable suggestions in designing tables and figures. Ms. Debra Akin

generously shared her time and technical expertise in electron

microscopy. Mrs. Adele Koehler helped type the final manuscript.

I am grateful for the friendship of fellow graduate students and

cohorts, Dr. Richard Endris, Dr. Peter. Perkins, Dr. Richard Johnson,

CPT Terry Klein, Mr. Eric Milstrey, Mr. Bruce Alexander, and Mr.

Charles "Ben" Beard. Their suggestions and encouragement were

appreciated and their humor was therapeutic.

Finally, I wish to express my love and deepest gratitude to my

wife, Joyce, for her unwaivering love, support and sacrifice, and for

periodically reminding me of my true priorities. I thank my wonderful

children, Natalie, Juliet, Nathan, Joshua, Aaron, and Andrew, for the

three year's worth of afternoons, evenings, and weekends that could

have been theirs.












TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS.... ......................................... iii

LIST OF TABLES.............................................. vii

LIST OF FIGURES................ .......... ..... .... .......... ix

ABSTRACT.................................................... xiv

CHAPTER

1 SAND FLIES AND LEISHMANIASIS............................ 1

Introduction .......................................... 1
Historical Review of the Incrimination of Major
Vectors of Leishmaniasis.............................. 2
Old World Leishmaniasis............................ 7
New World Leishmaniasis........ .................. 13
Leishmaniasis in the United States of America....... 19
Statement of Objectives................................ 24

2 SAND FLIES ASSOCIATED WITH HUMAN CUTANEOUS LEISHMANIASIS
IN TEXAS: OBSERVATIONS ON THEIR BIOLOGY WITH SPECIAL
REFERENCE TO Lutzomyia diabolica (Hall).................. 26

Introduction ........................................ ... 26
General........................................... 26
Human Case Histories............................... 27
Description of Study Sites.......................... 33
Materials and Methods................................... 43
Determination of Sand Fly Fauna..................... 43
Processing and Maintenance of Wild-Caught Sand
Flies and Recovery of Eggs........................ 47
Results................................................ .. 49
Species of Sand Flies Present....................... 49
Processing and Maintenance of Wild-Caught Sand
Flies and Recovery of Eggs........................ 56
Accessory Glands and Parity......................... 58
Natural Parasite Infections......................... 61
Discussion and Conclusions. ............................. 66
Determination of Sand Fly Fauna..................... 66
Potential Vectors ................................. 81
Processing and Maintenance of Wild-Caught Sand
Flies and Recovery of Eggs........................ 81







Page

Accessory Glands and Parity........................ 83
Natural Parasite Infections......................... 86

3 LABORATORY COLONIZATION OF Lutzomyia diabolica
WITH NOTES ON ITS BIOLOGY IN THE LABORATORY
(DIPTERA: PSYCHODIDAE) ................................. 91

Introduction........................... .. .......... 91
Materials and Methods................................... 92
General....................................... 92
Immature Stages..................................... 93
Adults ................... .......... ...... ........ 97
Results .................................................. 100
General Observations .............................. 100
Immature Stages..................................... 103
Adults.......................................... 124
Age-Specific Life Table........................... 134
Discussion and Conclusions.............................. 136
General............... ...... ...... ..... .............. 136
Immature Stages.................................... 137
Adults....................... .................... 151
Age-Specific Life Table........................... 157

4 EXPERIMENTAL TRANSMISSION OF Leishmania mexicana BY
BITES OF Lutzomyia diabolica (HALL) AND Lutzomyia
shannoni (DYAR) WITH NOTES ON THE EXTRINSIC
DEVELOPMENT OF THE PARASITE............................. 162

Introduction.................................... ... 162
Materials and Methods.................................... 163
Infection of Sand Flies and Parasite Development.... 163
Ultrastructure Studies............................. 166
Transmission Trials................................. 167
Results ................................................ 168
Infection of Sand Flies and Parasite Development.... 168
Ultrastructure Studies.............................. 188
Transmission Trials ................................. 190
Discussion and Conclusions ............................... 206
Infection of Sand Flies and Parasite Development.... 206
Ultrastructure Studies.............................. 215
Transmission of Leishmaniasis....................... 218

5 SUMMARY AND RECOMMENDATIONS.............................. 226

REFERENCES........................................................ 229

BIOGRAPHICAL SKETCH......................................... 243












LIST OF TABLES


Table Page

1-1. Proven or suspected sand fly vectors of leishmaniasis
in the New World.................................... 3

2-1. Summary of Lutzomyia sand fly surveys conducted
in vicinities of human case sites of cutaneous
leishmaniasis in south central Texas................... 50

2-2. Number of male and female Lutzomyia diabolica in
selected collections from three sites using six
trapping methods............................. ........ 51

2-3. Fecundity summary of wild-caught Lutzomyia diabolica
females from south central Texas....................... 59

2-4. Summary of preoviposition intervals, postcapture and
postoviposition longevities of wild-caught Lutzomyia
diabolica females from south central Texas............. 60

2-5. Incidence of natural parasite infections in 341 female
Lutzomyia diabolica collected in south central
Texas ................................................ 64

3-1. Summary of fecundity, incubation time, and hatching
for generations 1 through 9 and 13 in a laboratory
colony of Lutzomyia diabolica.......................... 105

3-2. Comparison of life-cycle parameters of immature
Lutzomyia diabolica reared at 240C and 270C............ 107

3-3. Duration of larval and pupal stages in generations 1
through 9 and 13 of a laboratory colony of Lutzomyia
diabolica.............................................. 112

3-4. Comparison of immature development times for male and
female Lutzomyia diabolica reared individually to
adult stage at 24oC and 27C ........................... 114

3-5. Comparison of life-cycle parameters of sand flies
(Lutzomyia diabolica) reared on 5 larval diets......... 117

3-6. Summary of oviposition and longevity data for
generations 2 through 9 and 13 in laboratory colony
of Lutzomyia diabolica................................ 130






Table Page

3-7. Age-specific life table for laboratory-reared
Lutzomyia diabolica based on observations of
several generations.................................. 135

4-1. Infection rates in laboratory-reared Lutzomyia
diabolica and Lu. shannoni sand flies fed on
hamsters with hTstiocytomas due to Leishmania
mexicana (strain WR-411).............................. 169

4-2. Distribution of Leishmania mexicana (strain WR-411)
in the alimentary tract of Lutzomyia diabolica
dissected post mortem after feeding on infected
hamsters......... .... ........................... ....... 172

4-3. Distribution of Leishmania mexicana (strain WR-411)
in the alimentary tract of Lutzomyia shannoni
dissected post mortem after feeding on infected
hamsters..................... .................... 173

4-4. Description and location of Leishmania mexicana
(strain WR-411) in the alimentary tract of laboratory-
reared Lutzomyia diabolica 12 hrs to 8+ days after
an infecting blood meal................................ 174

4-5. Distribution of Leishmania mexicana amazonensis
(strain untyped) in the alimentary tract of Lutzomyia
diabolica dissected post mortem after feeding
on histiocytomas of infected hamsters................ 185

4-6. Distribution of Leishmania braziliensis guyanensis
(strain MHOM/SR/80/CUMC 1) in the alimentary tract of
Lutzomyia diabolica dissected post mortem after
feeding on a histiocytoma on the tail of a white
mouse.................................................. 186

4-7. Results of transmission trials of Leishmania
mexicana (strain WR-411) by bites of laboratory-
reared Lutzomyia diabolica........................... 196

4-8. Results of postfeeding dissections of laboratory-
reared Lutzomyia diabolica sand flies involved in
experimental transmissions of Leishmania mexicana
(strain WR-411) to Syrian hamsters.................... 200

4-9. Results of transmission trials of Leishmania
mexicana (strain WR-411) by bites of laboratory-
reared Lutzomyia shannoni............................. 201

4-10. Results of postfeeding dissections of laboratory-
reared Lutzomyia shannoni sand flies involved
in experimental transmissions of Leishmania mexicana
(strain WR-411) to Syrian hamsters.................... 203


viii













LIST OF FIGURES


Figure Page

2-1. Distribution of autochthonous human cases of
cutaneous leishmaniasis in Texas and locations
of study sites......................................... 28

2-2. Cutaneous lesion due to Leishmania mexicana on
ear lobe of patient B............................................ 31

2-3. Cutaneous lesion due to Leishmania mexicana on
thigh of patient C..................................... 31

2-4. Cutaneous lesion due to Leishmania mexicana on
cheek of patient D .................................... 32

2-5. Habitat typical of that found at Garner State Park,
Concan, Uvalde County, Texas.......................... 38

2-6. Habitat at Garner State Park, Concan, Uvalde County,
Texas, showing public latrine resting station.......... 38

2-7. Habitat typical of that found at Seminole Canyon
State Park, Val Verde County, Texas.................... 39

2-8. Habitat at Fawcett Boy Scout Camp, Barksdale,
Edwards County, Texas, showing open latrine resting
station........................................ ....... 39

2-9. Farmland surrounding the home of patient D in the
rural community of D'Hanis, Medina County, Texas....... 40

2-10. Hunting dogs in a kennel behind the home of
patient D in D'Hanis, Medina County, Texas............. 40

2-11. Wood pile located behind the home of patient D in
D'Hanis, Medina County, Texas.......................... 41

2-12. Hog pen located about 80 m west of the home of
patient D in D'Hanis, Medina County, Texas............. 41

2-13. Romer ranch, near Devine, Medina County, Texas,
home of the paternal grandparents of patient B......... 42

2-14. Neighborhood of patient B in northwest San
Antonio, Bexar County, Texas........................... 42






Figure Page

2-15. Neighborhood of patient A in southeast San
Antonio, Bexar County, Texas.......................... 44

2-16. Shannon trap erected behind the home of patient D,
D'Hanis, Medina County, Texas.......................... 46

2-17. Lutzomyia diabolica female............................ 53

2-18. Dismantled woodrat (Neotoma) nest near the home of
patient D in D'Hanis, Medina County, Texas, where
specimens of Lutzomyia anthophora were found........... 57

2-19. Paired accessory glands of a gravid female Lutzomyia
diabolica showing dense granular material.............. 62

2-20. Condition of accessory glands in relation to number
of eggs deposited.................................... 63

2-21. Unidentified flagellates in the midgut of a
Lutzomyia diabolica female collected at Garner
State Park, Uvalde County, Texas, June 1982............ 67

2-22. Gamont of aseptate gregarine in the abdominal
cavity of a female Lutzomyia diabolica collected
at Garner State Park, Uvalde County, Texas, June 1982.. 67

2-23. Gametocyst of an aseptate gregarine attached to
the accessory gland of a female Lutzomyia
diabolica collected at Garner State Park,
Uvalde County, Texas, June 1982....................... 68

2-24. Gregarine oocysts spilling from the accessory glands
of a female Lutzomyia diabolica collected
at Garner State Park, Uvalde County, Texas,
June 1982............................................. 68

2-25. Microsporidian spores in the hemocoel of a female
Lutzomyia diabolica collected at Garner
State Park, Uvalde County, Texas, June 1982........... 69

2-26. Microsporidian spore germinated in vitro by
addition of 0.2 M KC1 (pH 9) to T ~secting medium....... 69

2-27. Scanning electronmicrograph of mite (Eustigmaeus
sp.) found on the abdomen of a female Lutzomyia
diabolica collected at Garner State Park,
Uvalde County, Texas, June 1982........................ 70

2-28. Scanning electronmicrograph of mite (Eustigmaeus
sp.) found on the abdomen of a female Lutzomyia
diabolica collected at Garner State Park,
Uvalde County, Texas, June 1982........................ 70







Figure Page


2-29. Known geographical distribution of the sand fly
Lutzomyia diabolica in Texas........................... 73

2-30. Known geographical distribution of the sand fly
Lutzomyia anthophora in Texas......................... 80

2-31. Known geographical distribution of the sand fly
Lutzomyia texana in Texas.............................. 80

3-1. Bloodfeeding female sand flies, Lutzomyia
diabolica............................................ 98

3-2. Number of adult progeny per parent female of
Lutzomyia diabolica reared during the first nine
generations of a laboratory colony..................... 101

3-3. Total number of Lutzomyia diabolica adults per
generation in a laboratory colony...................... 102

3-4. Generation times for generations 1 through 9 and 13
in a laboratory colony of Lutzomyia diabolica.......... 104

3-5. Life cycle of the Phlebotomine sand fly
Lutzomyia diabolica (Hall)............................. 110

3-6. Survivorship curves for immature Lutzomyia
diabolica reared at 240C and 270C...................... 113

3-7. Graphic comparison of immature development times
for male and female Lutzomyia diabolica reared
individually to the adult stage at 24C and 270C....... 115

3-8. Graphic comparison of immature development times for
Lutzomyia diabolica reared on five diet regimens....... 118

3-9. Proportion of diapausing eggs in batches laid
outdoors by Lutzomyia diabolica females between
June and December at Gainesville, Florida............. 120

3-10. Duration of the egg stage in outdoor-reared
Lutzomyia diabolica according to month of
oviposition at Gainesville, Florida.................... 121

3-11. Winter diapause development and spring emergence
in 33 Lutzomyia diabolica egg batches
deposited outdoors at Gainesville, Florida, between
August and December in 1982 and in 1983................ 123

3-12. Adult emergence patterns of Lutzomyia diabolica
in a laboratory colony (1st and 13th colony
generations)................... .................... 128







Figure Page

3-13. Adult emergence patterns of Lutzomyia diabolica
reared individually at 24C and 27............... ... 127

3-14. Emergence patterns of 12th generation Lutzomyia
diabolica adults reared at 270C and 87% RH
on five larval diets.................................. 128

4-1. Histiocytoma on the ear of a hamster infected with
Leishmania mexicana................................... 165

4-2. Infecting sand flies in a three-sleeved isolation
chamber by feeding them on a leishmanial
histiocytoma on a hamster's ear........................ 165

4-3. Diagram of a sand fly alimentary tract................ 171

4-4. Development of Leishmania mexicana in
Lutzomyia diabolica.................................. 180

4-5. Long-slender promastigotes of Leishmania mexicana
swimming freely in the hind triangle of an
infected sand fly, Lutzomyia diabolica................ 181

4-6. Short-broad promastigotes ("haptomonads") of
Leishmania mexicana spewing into the dissecting
medium at the site of a rupture in the gut wall of
a sand fly, Lutzomyia diabolica....................... 181

4-7. Short-broad, dividing promastigotes ("haptomonads")
massed at the stomodeal valve in the gut of a
female sand fly, Lutzomyia diabolica, infected
with Leishmania mexicana............................... 182

4-8. Severed anterior aspect of the cardia in a female
sand fly, Lutzomyia diabolica, showing massive
numbers of Leishmania mexicana promastigotes
("haptomonads") attached to the stomodeal valve........ 182

4-9. Long-slender, nondividing, binucleated prornastigote
seen in a 5-day infection of Leishmania
mexicana in Lutzomyia diabolica........................ 183

4-10. Extremely long mononucleated form seen in a 5-day
infection of Leishmania mexicana in
Lutzomyia diabolica.................................. .... 183

4-11. Large peanut-shaped flagellate observed in infected
and uninfected laboratory-reared Lutzomyia
diabolica females............................ ... .... 187







Figure Page

4-12. Electronmicrograph of Leishmania mexicana
anastigotes in large vehicles within a macrophage
cell of a hamster infected by the bite of a
female Lutzomyia diabolica ............................ 189

4-13. Electronmicrograph of saggital and transverse
sections of Leishmania mexicana amastigotes in
macrophage cells of an infected hamster................ 192

4-14. Electronmicrograph of Leishmania mexicana
promastigotes in the abdominal midgut of an
infected Lutzomyia diabolica female.................... 193

4-15. Electronmicrograph of Leishmania mexicana
pronastigotes in the cardia (thoracic midgut) of
an infected Lutzomyia diabolica female................. 195

4-16. Lesion on the ear of a hamster approximately two
weeks after it was bitten by a female Lutzomyia
diabolica that was experimentally infected with
Leishmania mexicana................................. 199


xiii












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


BIOLOGY AND COLONIZATION OF THE SAND FLY
Lutzomyia diabolica (HALL) (DIPTERA: PSYCHODIDAE)
WITH NOTES ON ITS POTENTIAL RELATIONSHIP TO HUMAN
CUTANEOUS LEISHMANIASIS IN TEXAS, USA

By .

Phillip G. Lawyer

December 1984

Chairman: Dr. Jerry F. Butler
Cochairman: Dr. David G. Young
Major Department: Entomology and Nematology

A survey for potential sand fly vectors of human cutaneous

leishmaniasis was conducted within an endemic focus of the disease in

south central Texas, USA. Five species of Lutzomyia, including one

new species, were collected and eight new county records established.

Lutzomyia diabolica (Hall), the only anthropophilic sand fly

encountered, was the most commonly collected, accounting for 99% of

the total catch. This species was taken in light trap collections

throughout the frost-free season at a case site in D'Hanis, Texas. No

natural leishmanial infections were observed in more than 600

dissections of wild-caught female Lu. diabolica. Several natural

infections of nonleishmanial parasites are reported for the first

time.

The first productive laboratory colony of Lutzomyia diabolica was

established and detailed studies of the fly's biology were conducted


xiv







through 16 generations. A new larval diet, developed from a modified

horn fly [Haematobia irritans (Linn.)] diet, reduced the average

immature development time by 50%, to about 33 days. Quiescence and

diapause occurred in both the egg and larval stages, and lasted as

long as 270 days in the egg stage of outside-reared sand flies.

Infection rates of 88% and 95% in Lu. diabolica and Lu. shannoni,

respectively, were obtained by feeding the flies on leishmanial

histiocytomas of laboratory-infected hamsters. The development of

Leishmania mexicana (strain WR-411) in Lu. diabolica is described in

detail.

Ultrastructure studies of L. mexicana amastigotes and

promastigotes revealed that the subpellicular microtubule number

varies widely and is not a good criterion for distinguishing the

Leishmania species.

For the first time, transmission experiments demonstrated that Lu.

diabolica and Lu. shannoni are able to transmit L. mexicana to hamsters

by individual bites. Transmission occurred after a period of parasite

multiplication and development, during which time massive infections

were established initially in the midgut, then in the cardia and at

the stomodeal valve. These gave rise to short-slender, highly active

promasitgotes that spread throughout the alimentary tract and invaded

the pharynx and mouthparts.

Based on the findings of this study, Lutzomyia diabolica is the

suspected vector of human cutaneous leishmaniasis in Texas.













CHAPTER 1
SAND FLIES AND LEISHMANIASIS



Introduction


The common name "sand fly" has been used confusingly in the

literature for members of two families of Diptera, the

Ceratopogonidae and the Psychodidae. In this work "sand fly"

refers only to the members of Psychodidae belonging to the subfamily

Phlebotominae.

Sand flies are important as vectors of several human pathogens

including phleboviruses (e.g., sand fly fever), bartonellosis

(Carrion's disease) and, most notably, leishmaniasis, a complex of

diseases caused by various species and subspecies of unicellular

hemoflagellates in the genus Leishmania Ross (Adler and Theodor,

1957). Leishmaniasis is widely distributed in most tropical and

subtropical countries, extending through Central and South America,

Central and Southeast Asia, India, China, the Mediterranean Basin, and

Africa (Lainson, 1982). Until recently the disease was believed to be

absent from North America north of Mexico, but the confirmation of

several autochthonous human cases in Texas since 1968 has dispelled

that belief (Simpson et al., 1968; Shaw et al., 1976; Gustafson et al.,

1984). In 1981 the World Health Organization (WHO) estimated that 400

thousand new cases of leishmaniasis occur annually throughout the

world, but this may be an underestimation. Leishmaniasis is probably

second in importance only to malaria among the protozoan diseases in







terms of human suffering and economics (Lainson, 1982). Selection of

leishmaniasis by WHO as one of six diseases of man warranting a

special program of study has led to increasing interest in sand flies,

the only known natural vectors of the disease (Killick-Kendrick, 1978;

WHO, 1981). It should be noted that workers in Oklahoma fed nymphal

ticks [Rhipicephalus sanguineus (Latrielle)] on dogs infected with L.

infantum and found that the ticks remained culture positive for the

parasite for at least one month postmolting (Fox, pers. comm., 1984).

Culture-positive adult ticks that subsequently fed on uninfected

puppies, transferred Leishmania to the puppies. Although this

demonstrates that ticks can experimentally transmit leishmaniasis, it

may not occur under natural circumstances.

Previous work on sand flies has been mostly taxonomic. More

recently, however, interest has turned to the biology of sand flies,

the most important practical aspect being their relationship with

leishmanial parasites. The role as vectors of most of the 53 species

or subspecies of sand flies thought to transmit leishmaniasis to man

requires confirmation (Killick-Kendrick, 1978). Of the 21 species of

New World sand flies reported or suspected as being natural hosts of

Leishmania spp. infecting man, only six have been definitely

incriminated as vectors (Table 1-1). Prior to this study no

anthropophilic species in the USA had been incriminated in the

transmission of leishmaniasis.



Historical Review of the Incrimination of
Major Vectors of Leishmaniasis


Cutaneous and visceral leishmaniases are apparently ancient

afflictions of man. As early as the first century AD, in Central








Table 1-1.


Proven or suspected sand fly vectors of leishmaniasis in
the New World, by country (Killick-Kendrick, 1978; WHO,
1984).


Leishmania Lutzomyia sand fly
Country parasites vectors


Belize

Bolivia

Brazil

Brazil

Brazil

Brazil

Brazil

Brazil

Brazil

Brazil

Brazil

Brazil

Brazil

Colombia

Colombia

Costa Rica

Costa Rica

Dominican Republic

Ecuador

Ecuador

El Salvador

French Guiana

Guatemala

Guatemala


m. m.

d. c.

d. c.

b. b.

b. b.

b. b.

b. b.

b. b.

b. b.

b. b.

b. g.

b. s.

m.

d. c.

b.

b.

b.



b.

b.



b. 1.

d. c.

m.


olmeca2

longipalpis3

longipalpis2

amazonensis3

intermedia2

migonei3

parensis3
.3
pessoai3

wellcomei3

whitmani3

anduzei3

umbratilis2

flaviscutellata3

longipalpis3

trapidoi3

shannoni3

ylephiletor3

christophei3

hartmanni3

trapidoi3

longipalpis3

umbratilis3

longipalpis3

olmeca3








Table 1-1. Continued.


Leishmania Lutzomyia sand fly
Country parasites 1 vectors


Honduras

Mexico

Mexico

Nicaragua

Panama

Panama

Panama

Panama

Paraguay

Peru

Peru

Surinam

USA

Venezuela

Venezuela

Venezuela


L. d. c.

L. d. c.

L. m.

L. d. c.

L. b. p.

L. b. p.

L. b. p.

L. b. p.

L. d. c.

L. p.

L. .

L. b. g.

L. m.

L. d. c.

L. m. a.

L. m. g.


Leishmania species: L. m. m. = L. mexicana mexicana, L. d. c. =
L. donovani chagasi, L. b. b. = L. braziliensis braziliensis, L. b. g. =
L. braziliensis guyanensis, L. m. = L. mexicana, L. b. p. = L. brazil-
Tensis panamensis, L. e. = L. peruviana, L. m. a. = L. mexicana
amazonensis, L. m. 1. = L. mexicana garnhami.

Proven vector.

Suspected vector.


longipalpis3

longipalpis3

olmeca2

longipalpis3

gomezi

panamensis3

trapidoi2

ylephiletor3

longipalpis3

peruensis3

verrucarum3

umbratilis3

diabolica3

longipalpis3

flaviscutellata3

townsendi3







Asia, the cutaneous disease was referred to as "Balkh Sore" (after a

town in north Afghanistan, near the Russian border), and by early

travelers as "Aleppo Boil" in Syria and "Baghdad Boil" in Iraq

(Lainson, 1982). In the Americas, Peruvian and Ecuadorean pottery

from the era 400 to 900 AD depicts human faces with mutilations very

similar to those caused by cutaneous and mucocutaneous leishmaniasis.

Spanish historians at the time of the conquest described severely

mutilating sores on the faces of Peruvian Indians (Lainson, 1982).

Because the major signs and symptoms of visceral leishmaniasis

resemble those of several other tropical diseases, it is difficult to

trace clear-cut references to this disease in ancient writings in

either the Old or the New World. The disease first attracted public

attention in 1882 when Clark, of the Sanitary Commission of India,

gave an account of 100 cases of a severe form of malarial cachexia,

depopulating areas of the Garo Hills, Assam. Natives of the area

called the disease "kala-azar" (black fever) and it appears that it

was known to them as early as 1869 (Strong, 1945). Epidemics of what

must have been the same disease, under the name of "Burdwan fever,"

occurred in lower Bengal from 1854 to 1875, causing a quarter of a

million deaths (Strong, 1945). During this same period in the

Mediterranean region it was referred to as "infectious splenic anemia"

or "infantile splenic anemia" (Lainson, 1982).

Visceral leishmaniasis apparently was unrecognized as a distinct

disease in Latin America until the time of the first parasitologically

proven case, in Paraguay in 1913 (Migone, 1913). This fact, coupled

with clinical, epidemiological, and biochemical similarities between

Mediterranean and American visceral leishmaniasis, suggests that it was







introduced since the European discovery of the New World (Lainson,

1982).

Possibly the first written account implicating sand flies in the

transmission of human pathogens, including leishmaniasis, appeared in

1764 in a sort of almanac published in Lima, Peru,under the direction

of Cosme Bueno, distinguished physician, mathematician and geographer

(Herrer and Christensen, 1975). In El Conocimiento de los Tiempos he

discussed the folklore regarding the natural transmission of verruga

(bartonellosis) and corrosive facial ulcers (uta, a form of cutaneous

leishmaniasis), reporting that both diseases originate from the bite

of a small insect called "Uta" (sand fly). More than a century later,

Mitford reported on cutaneous leishmaniasis in the Middle East and

considered the possible participation of some insect in the transmis-

sion of Aleppo boil, although its exact role was not clearly indicated

(Lewis, 1978). In 1904 Rogers discovered that the causative agent of

Indian kala-azar (visceral leishmaniasis) developed into a leptomonad

flagellate in culture. He also noted that similar organisms (e.g.,

Leptomonas) had been found in insects (mosquitoes), tangentially sug-

gesting an insect vector of kala-azar (Rogers, 1904). The ensuing

search for vectors of kala-azar (Leishmania donovani) and oriental

sore (Leishmania tropica) included a wide range of suspects including

bed bugs, fleas, mosquitoes, house flies, sand flies, hippoboscids,

and even leeches. In 1905, the Sergents and Pressat, attracted by the

coincidental distribution of sand flies and leishmaniasis, indepen-

dently suggested that these insects were probable vectors of oriental

sore (Sergent et al., 1914; Kirk and Lewis, 1955). Wenyon (1912)

reviewed the advances made in the knowledge of leishmaniasis in





-7-


the Old and New Worlds and supported the suggestion that some sort

of insect was involved in its transmission. He and others

unsuccessfully attempted to demonstrate leishmaniasis transmission

using fleas (Ctenocephalides canis and Pulex irritans), mosquitoes

(Stegomyia fasciata = Aedes aegypti), bed-bugs (Cimex sp.), and sand

flies (Phlebotomus sp.). On another occasion, Wenyon (1911)

dissected a number of wild-caught sand flies from Aleppo and observed

"Herpetomonas" flagellates in about 6% of the specimens; he

acknowledged the possibility that what-appeared to be harmless

parasites of sand flies might, in fact, be developmental forms of

L. tropica. Wenyon's discovery marked the beginning of intensified

efforts by numerous researchers to study all aspects of the parasitic

relationship between Leishmania and the sand fly host. For the next

30 years investigations progressed mainly on two fronts, in North

Africa and Palestine with oriental sore, and in India with kala-azar.


Old World Leishmaniasis


Leishmania tropica cutaneouss leishmaniasis, "oriental sore").

In a note on the etiology of oriental sore in Mesopotamia, Patton

(1919) believed that P. papatasi Scopoli and probably P. minutus

Rondani were carriers of the parasite. Acton (1919) showed that the

distribution on the body of oriental sores corresponded to the

distribution of bites by Phlebotomus. In 1921, Sergent et al.,

working in Algeria, first described the transmission of oriental sore

to a human. They divided 559 sand flies into 23 batches, crushed them

in saline and inoculated the resulting suspensions into the arms of 23

volunteers. The flies had been collected in Biskra, an endemic center







of the disease 600 km south of Algiers where the disease does not

occur. Only one of the inoculations produced positive results (Adler

and Theodor, 1925).

Adler and Theodor (1925), studying Phlebotomus in Jericho

(Jordan) found heavy "Herpetomonas" infections in four female sand

flies out of nearly 400 dissected. Material from one of these

infected flies was inoculated into the forearm of a volunteer and a

small papule containing Leishman-Donovan bodies (amastigotes) was

subsequently produced. Following this successful transmission of

oriental sore, they conducted feeding experiments to determine if

"Herpetomonas tropica" was capable of developing in P. papatasi after

a feed on an oriental sore, and whether or not the parasite remained

infective to man after passing through the sand fly. Dissections were

made two to seven days after the infective feed; 16 sand flies were

found to be infected. Material from these artificially infected flies

was inoculated into volunteers, none of whom subsequently showed signs

of infection. Although, Adler and Theodor felt that their experiments

had provided sufficient proof of the role of sand flies as vectors of

oriental sore, this view was criticized by Wenyon (Lainson, 1982), who

emphasized the need to transmit oriental sore by natural bite of the

sand fly. Adler and Theodor replied by ingeniously demonstrating the

capability of the sand fly to transmit Leishmania by bite, although it

was not from man to man (Adler, 1928). Phlebotomus papatasi were fed

through a rabbit-skin membrane on a culture of L. tropica, and eight

days later were allowed to feed through another membrane on sterile,

inactivated rabbit serum. Some of this serum was sown into blood-agar

medium used for culturing Leishmania. Flagellates were




-9-


present six days later. The same authors may have actually

transmitted Leishmania to man by bite of the sand fly one year later.

They fed experimentally infected P. sergenti Parrot on a number of

volunteers and obtained a positive lesion on the arm of one man. It

should be noted that the incubation period was so long that the

individual had visited endemic areas of oriental sore before

appearance of the lesion and therefore could possibly have acquired

the infection there (Adler and Theodor, 1929). Finally, 20 years

after Sergent and colleagues artificially transmitted oriental sore by

scarification, Adler and Ber (1941) proved without question that

artificially infected P. papatasi can transmit oriental sore to a

human by bite. The key to this success appears to have been in

feeding the sand flies on flagellates suspended in three parts 2.7

percent saline and one part defibrinated blood.

Leishmania donovani (visceral leishmaniasis, "kala-azar"). In

1915 Mackie, convinced that a relationship existed between kala-azar

and some biting insect, attempted to make a hut to hut insect census

in kala-azar-infected villages. His team collected body lice, head

lice, bed bugs, mosquitoes, sand flies and even leeches in the bedding

or on the persons of patients who were proven to have active kala-

azar; these specimens were carefully examined for Leishmania

parasites. All specimens were negative except the sand flies which

contained "Herpetomonas parasites" (probably Leishmania), "bodo-like

parasites," and "sporozoan-like parasites." Not realizing how close

he must have been to linking sand flies with kala-azar, Mackie stated:

"This long series of negative results rather tends to check enthusiasm

for the insect-borne hypothesis of kala-azar. The only insect




-10-


which has given any return for work spent on it is the sand fly and I

am of the opinion that the relation of this insect to disease would

repay further investigation" (1915, p. 949).

Perhaps stimulated by Mackie's suggestion and following the

example of Sergent et al. (1921) in their work on oriental sore

in Algeria, Sinton (1922) studied the distribution of Indian sand

flies and found that the distribution of kala-azar showed striking

congruence with that of P. argentipes Annandale and Brunetti; he

considered this sand fly to be the most likely vector. In 1924, a

"Kala-azar Commission" was set up under the directorship of

Christophers, and its members were soon successful in rearing this

most likely suspect in the laboratory. Once a laboratory colony was

established, Knowles et al. (1924) fed specimens of these sand flies

on patients with kala-azar and found that a heavy flagellate infection

developed in P. argentipes; they noted that the same degree of

infection did not occur in other Phlebotomus species. The following

year Shortt et al. (1926), working with L. donovani, demonstrated that

in P. argentipes the infection passes forward in the fly to the buccal

cavity and proboscis. For the next five years, Shortt and co-workers

of the Kala-azar Commission conducted three series of transmission

experiments using human volunteers and Chinese hamsters as hosts.

They fed an astounding 79,939 artificially infected P. argentipes on

either human volunteers or hamsters but reported negative results as

follows:

The failure of any of the subjects of experiment to show
infection with kala-azar is very difficult to explain if
the theory of Phlebotomus transmission is to be maintained. We
can only suppose that some essential factor in the process of
infection has been omitted in our experiments which is present
under natural conditions, or that the vast amount of labour
expended by us during a period of 5 years has been expended




-11-


on an insect which is not an essential link in the chain of
infection. (Shortt et al., 1930, p. 929)

It appears that this final report was written before all the results

were in, for on February 19, 1931, the following telegram from New

Delhi was received by Nature magazine: "Lieut-Col. Shortt reports

successful transmission of Leishmania donovani to Chinese hamsters by

bites of artificially infected Phlebotomus argentipes. Hamster bitten

repeatedly during twelve months; generalized infection found seventeen

months after experiment began" (Shortt, 1931, p. 308). Their

persistence had paid off.

The kala-azar commission continued its efforts to transmit kala-

azar to man by bite of P. argentipes but met with uniformly negative

results (Swaminath et al., 1942). It was only after Smith et al.

(1940) devised the technique of keeping the flies alive after

oviposition by feeding on the juice of boiled raisins that Swaminath

et al. (1942), using this technique, were able to forge the final link

of evidence incriminating P. argentipes as the insect vector of kala-

azar in India. They suggested that feeding the flies on fruit juices

acted either by increasing the virulence of the parasites or

increasing the parasitemia, thus enabling them to reach the anterior

part of the midgut (cardia) more rapidly.

Leishmania donovani infantum (infantile visceral leishmaniasis).

The coincidental distributions of visceral leishmaniasis and the sand

fly P. chinensis Newstead in China north of the Yangtze River, pointed

to this insect as the vector of the disease. Young and Hertig (1926),

in North China, dissected hundreds of field-caught P. chinensis, P.

sergenti, and P. perturbans de Meijere and examined them for the presence

of flagellates; all specimens were negative. They also attempted to





-12-


incriminate these three species by experimentally infecting laboratory-

bred sand flies, feeding them on kala-azar patients or infected

hamsters, and allowing them to refeed on uninfected hamsters. The

rates of infection were 85.3%, <2%, and 0% for P. chinensis,

P. sergenti, and P. perturbans, respectively. A small percentage of

these infected flies took a second blood meal, but transmission was

unsuccessful. Similar studies were conducted by Sun et al. (1936) and

Sun and Wu (1937) in which 7 of 21 P. chinensis, collected in houses

with cases of visceral leishmaniasis, were infected with

promasitgotes. Successful transmission to hamsters by the bite of

P. chinensis was finally reported by Feng and Chung (1941).

In the Cevennes, in southern France, Rioux and colleagues

accumulated overwhelming epidemiological evidence regarding the

suspected role of P. ariasi Tonnoir as the vector of infantile

visceral leishmaniasis. Not until 1979, however, did they confirm

their suspicions by transmitting the disease to a dog by the bite of

an experimentally-infected sand fly (Rioux, et al., 1979).

Leishmania major cutaneouss leishmaniasis, wet sore). It is

unclear when L. major was first transmitted experimentally by bite of

a sand fly because early workers recognized only one leishmanial

species that was divided into two subspecies, "minor" and "major"

causing "dry" and "wet" oriental sore, respectively. Perfil'ev (1968)

claimed that the first successful transmission of cutaneous

leishmaniasis by bite of a sand fly was accomplished in 1941 by

Kryukova in experiments with gerbils. Laboratory-bred P. papatasi

were infected with cutaneous leishmaniasis by feeding on a histocytoma

on a gerbil's ear and were subsequently allowed to take a second blood





-13-


meal from an uninfected gerbil. Lesions developed on the second

gerbil 15 days post infective feed. In 1927 Koshevnikova and

coworkers showed that the principal reservoir host of L. tropica in

Central Asia is man and that the host of L. major is the gerbil

(Rhombomys opimus) (Perfil'ev, 1968). Therefore, it seems safe to

assume that Kryukova's results represent the first experimental

transmission of L. major by the bite of a sand fly, because the

original pathogen was obtained from gerbils caught in Turkmenia.


New World Leishmaniasis


In the New World, the task of vector incrimination has been

complicated by three factors: the extremely wide variety of sand fly

species (about 327 Lutzomyia compared to about 101 Phlebotomus in the

Old World), the failure to appreciate the multiplicity of leishmanial

parasites on the part of some workers, and the difficulty of working

in a dense tropical rain forest (Lainson, 1982).

Leishmania donovani chagasi (American visceral leishmaniasis).

The peridomestic nature of American visceral leishmaniasis due to

L. donovani chagasi facilitated the incrimination of Lu.1 longipalpis

(Lutz and Neiva) as the main vector of this disease. Its geographic

distribution coincides with visceral leishmaniasis throughout Latin

America.

In 1936, Evandro Chagas found Lu. longipalpis in the house of the

first case of visceral leishmaniasis to be studied in South America,

in Sergipe, Brazil (Lainson, 1982). This prompted Chagas and others


1The abbreviation Lu. for Lutzomyia is used to avoid confusion with L.
for Leishmania.




-14-


to feed Lu. longipalpis on infected dogs. Promastigotes developed in

the guts of these sand flies and were infective upon subsequent

inoculation into uninfected hamsters (Lainson, 1982). With the

accidental death of Evandro Chagas in 1940, interest in visceral

leishmaniasis also seemed to die, not to be resurrected until 1954

when Deane and Deane, investigating serious outbreaks of the disease

in the state of Ceara, Brazil, found wild-caught Lu. longipalpis

heavily infected with promastigotes believed to be L. donovani. They

also noted highly active flagellates in the biting mouthparts of other

Lu. longipalpis they had fed on a naturally-infected fox eight days

previously (Lainson, 1982). Although the promastigotes seen by the

Deanes were not proven to be L. d. chagasi, the epidemiological

evidence was so strong that there remained little doubt as to the

importance of this sand fly (Lainson, 1982).

Further evidence to incriminate this peridomestic vector came in

1977 when Lainson and colleagues achieved five separate transmissions

of L. d. chagasi in hamsters, by bite of laboratory-bred insects which

had previously ingested amastigotes in artificially infected rabbit

blood (Lainson et al., 1977).

Leishmania mexicana (American cutaneous leishmaniasis, bay sore,

chiclero's ulcer). Experimental infection of sylvatic sand flies with

parasites causing American cutaneous leishmaniasis has been

accomplished in a wide range of species, but experimental transmission

has been difficult to achieve because of the wide choice of potential

vectors. Strangways-Dixon and Lainson (1962) infected wild-caught

females of nine species of sand flies in Belize by feeding them on

hamsters exhibiting skin lesions due to L. m. mexicana. The flies then




-15-


were fed on human volunteers, one of whom developed a small lesion 17

days after one insect made a 30 second bloodless probe. The vector

was initially identified as "P. paraensis" Costa Lima, but was later

reexamined by Williams (1983) and determined to be most similar to

Lu. panamensis (Shannon) rather than paraensis. To demonstrate a natural

infection of leishmaniasis in sand flies and to strengthen their case

for incriminating the vector (Lu. panamensis), Strangways-Dixon and

Lainson (1962), collected approximately 270 wild female sand flies,

triturated them in sterile Locke's solution and inoculated half of

the suspension into the back of an uninfected hamster. Two months

later a small dermal swelling was noted at the site of the

inoculation. Stained smears from the lesion revealed leishmaniae

(Strangways-Dixon and Lainson, 1962). This experiment showed that

some wild-caught sand flies were naturally infected, but since all

specimens collected were pooled, it was not determined which species

was (were) infected. Subsequent dissections of 334 newly caught, man-

biting Lutzomyia revealed epimastigote-like infections in two specimens,

one each of Lu. ovallesi (Ortiz) and Lu. cruciata (Coq.). In view of

the scanty infections, the authors felt that these more likely

represented Leptomonas or Herpetomonas rather than Leishmania

promastigotes.

In Brazil, wild-caught Lu. longipalpis and Lu. renei (Martins,

Falcao, and da Silva), allowed to feed on a strain of Leishmania

isolated from a human patient in Belize, also successfully transmitted

the parasite (Coelho and Falcao, 1962).

Williams (1966a) collected wild sand flies in Belize and fed them

on a leishmanial lesion on a hamster. Two to three days later, the




-16-


flies fed on the forearm of a human volunteer. Two lesions were

produced about four weeks later, and in both cases the vector fly was

Lu. cruciata.

In the same country, Disney (1966) designed a trap to catch sand

flies attracted to rodents and found that Lu. olmeca olmeca (Vargas

and Najera) was highly attracted to them. Later, using this

technique, he found Lu. o. olmeca naturally infected with L. m.

mexicana (Disney, 1968). In neighboring Yucatan Peninsula, Biagi et

al. (1965) confirmed the importance of Lu. o. olmeca as a vector of

leishmaniasis, and transmitted the parasite to a volunteer by bite of

a naturally infected fly (Lainson, 1982).

Lainson and Shaw (1968) demonstrated that the vector of

L. mexicana amazonensis in the Amazon forests of Brazil is

Lu. flaviscutellata (Mang.), a species highly attracted to the rodent

Proechimys guyanensis, the principal reservoir host. Of 7,322 flies

dissected between 1968 and 1973, 45 or 0.6% were infected with

promastigotes. Parasites from 18 of these infected flies were

inoculated into hamsters and 15 of the inoculations produced typical

L. mexicana amazonensis infections (Ward et al., 1973). These workers

also transmitted the disease from hamster to hamster on four occasions

using laboratory-bred Lu. flaviscutellata (Ward et al., 1977). From

these findings it was concluded that Lu. flaviscutellata is the

principal, and probably only, vector of L. mexicana amazonensis in the

Amazon region (Lainson and Shaw, 1979).

Leishmania brazilienensis panamensis (Panamanian cutaneous

leishmaniasis). Natural flagellate infections have been recorded in

numerous neotropical sand fly species, but it was not until extensive




-17-


studies were carried out at the Gorgas Memorial Laboratory in Panama

that they were proven to be Leishmania (Hertig and McConnell, 1963;

Johnson et al., 1963). After many years of patient work, these

researchers found natural promastigote infections in more than 400

man-biting Panamanian sand flies and incriminated three species,

Lu. trapidoi (Fairchild and Hertig), Lu. ylephiletor (Fairchild and

Hertig) and Lu. gomezi (Nitz.) as vectors of Panamanian cutaneous

leishmaniasis, L. braziliensis panamensis (Johnson et al., 1963).

Christensen et al. (1969) also found Lu. panamensis infected with

promastigotes, but there is some question as to the identity of those

promastigotes. Lainson (1982) believed that some of these infections

were due either to Endotrypanum (a blood parasite of sloths which also

develops as promastigotes in sand flies) or to nonhuman Leishmania,

such as Lu. hertigi of porcupines.

McConnell (1963) cultured flagellates from wild-caught sand flies

and determined, without question, that Lu. trapidoi harbored

promastigotes of L. b. panamensis. Furthermore, the observation that

Lu. trapidoi is largely arboreal led to the incrimination of sloths as

the major reservoir of L. braziliensis panamensis (Lainson, 1982).

Leishmania braziliensis guyanensis ("pian-bios"). In Surinam,

Wijers and Linger (1966) caught large numbers of anthropophilic sand

flies off human bait in areas where "pian-bois," due to L. b.

guyanensis, is endemic. The most common species recorded was

Lu. squamiventris (Lutz and Neiva), but all dissections of this species

proved negative for leishmaniae. Numerous promastigote infections

were found in dissections of another species, "Lu. anduzei," found

resting on tree trunks. Their "anduzei" probably represented




-18-


Lu. umbratilis (Ward and Fraiha) since it was found resting on tree trunks

(Ward and Fraiha, 1977). However, the role of this sand fly as a

vector remained speculative, since attempts to infect a hamster with

the parasite failed.

Lainson et al. (1976), observed a 7% infection rate in "Lu.

anduzei" (now Lu. umbratilis Ward and Fraiha) taken mostly from large

tree trunks during their studies of the epidemiology of "pian-bois" in

the Monte Dourado region, Para State, Brazil. Intradermal inoculations

of the flagellates into hamsters produced infections in all cases.

The parasite was shown to be identical biologically and biochemically

with that causing the disease in man. Lu. umbratilis was subsequently

incriminated as the vector of L. b. guyanensis in the neighboring

state of Amazonas, Brazil, by Arias and Freitas in 1977. Lainson et

al. (1976) reported heavy promastigote infections in seven specimens

of Lu. whitmani (Antunes and Coutinho), another tree trunk-inhabiting

sand fly. Unlike Lu. umbratilis, this sand fly was not particularly

anthropophilic, however, and they suggested that its importance is

probably limited to secondary transmission among wild animal

reservoirs.

Leishmania braziliensis braziliensis (mucocutaneous

leishmaniasis, "espundia"). Incrimination of sand fly vectors of L.

b. braziliensis has been more difficult than with the other American

forms because of the poor growth of the parasite in laboratory animals

and in culture media, and because of difficulties in establishing

productive colonies of suspected vectors for transmission experiments.

Forattini et al. (1972), however, successfully infected hamsters with

promastigotes (believed to be L. b. braziliensis) found in two




-19-


naturally infected sand flies, Lu. intermedia (Lutz and Neiva) and Lu.

pessoai (Coutinho and Barretto) in Brazil. The former species is found

in low, secondary forests and is known also to invade houses, while

the latter is essentially sylvatic, but has been collected in houses

up to 300 m from the forest edge. With this in mind, it was suggested

that cutaneous leishmaniasis in southern Brazil may have a

peridomestic transmission, the original source of the infection being

in nearby wooded areas (Lainson, 1982).

In Serra dos Carajas, Pard State,-North Brazil, where both

cutaneous and mucocutaneous leishmaniasis are serious public health

problems, Lainson et al. (1973) concluded that Lu. welcome

(Fraiha, Shaw, and Lainson) was a major vector to man. Attempts to

infect hamsters by inoculation with parasites isolated from naturally

infected Lu. wellcomei were largely unsuccessful, but it was shown

that the parasite was the same as that infecting man in the same area.

Lu. wellcomei was considered to be of particular importance because of

the avidity with which it attacks man both during the night and the

day.



Leishmaniasis in the United States of America


Few studies have been conducted on the epidemiology of

leishmaniasis in the USA. Prior to 1976 leishmaniasis was not

generally thought to occur autochthonously in the USA, and

potential sand fly vectors were reported so rarely as to be considered

of little medical consequence.

McEwen (1914) reported the first case of leishmaniasis in the

USA, referring to it as "oriental sore." There is little doubt that




-20-


he was dealing with American cutaneous leishmaniasis since the patient

acquired the lesion while traveling in South America (Stewart and

Pilcher, 1945). At that early date, differentiation between oriental

sore and American cutaneous leishmaniasis had not been made, nor had

any vectors been incriminated.

In 1919, Parman drew attention to a "Phlebotomus" species in

Uvalde, Texas,and noted that it attacked man. He did not attempt to

implicate it in disease transmission. This fly, described and named

Phlebotomus (Brumptomyia) diabolicus by Hall in 1936, represented the

first confirmed anthropophilic species of the genus Phlebotomus (now

Lutzomyia) from the USA. Lindquist (1936) stated that this species

was not a major pest in Uvalde, but that it frequently caused some

annoyance to people in southwestern Texas. He described a

peridomestic fly that often entered dwellings and other buildings to

feed on man and domestic animals. At that time, species of

Phlebotomus had been incriminated in the transmission of Phlebotomus

fever but were only suspected to play a role in the transmission of

leishmaniasis. Consequently, the fly was considered to be of little

importance other than being a nuisance.

Prior to 1943, approximately 30 human cases of cutaneous

leishmaniasis (oriental sore) had been reported from the USA and

Canada (Dwork, 1942), none of which (with one possible exception), were

autochthonous. The possible exception was a case reported by Gelber

(1942) in a 53-year-old woman from southern California who had a

typical leishmanial lesion on her left cheek. This case was thought

to be autochthonous because the woman had not left the country for 13

years. But since she had made an earlier tour of Europe and the





-21-


Mediterranean, the possibility cannot be ruled out that she might have

contracted the disease outside the USA.

Only three cases of mucocutaneous leishmaniasis had been reported

in the USA prior to 1943, one of which was presumed to be

autochthonous (Stewart and Pilcher, 1943). Benedek (1940) reported a

case of mucocutaneous leishmaniasis in a man from Chicago, which he

considered the first autochthonous case of leishmaniasis in the USA.

However, there is some question as to the extent of the patient's

travel, and thus there is room for doubt.

Stewart and Pilcher (1943) reported on a case of cutaneous

leishmaniasis in a 6-year-old Mexican-American boy who lived on a

ranch near Alice, Texas and had never traveled more than 60 miles from

his home. The authors, obviously aware of the recent incrimination of

sand flies as the vectors of both visceral and cutaneous leishmaniasis

in the Old World, mentioned that three'species of "Phlebotomus"

(Lutzomyia) had been identified in the USA, all of which were found

within or near Texas. They astutely concluded that the apparent

rarity of the disease in the United States was probably not real and

that the return of military and civilian personnel from endemic

centers was further reason for keeping American leishmaniasis in mind.

This was probably the first truly autochthonous case of leishmaniasis

reported in this country, although Wenyon disputed the identity of the

parasite stating: "The microphotograph illustrating the paper is a

good one, but though suggestive of Leishmania, it is not absolutely

convincing" (1945, p. 712).

Addis (1945a) described a new species of sand fly from Texas,

"Phlebotomus anthophorus" (Lu. anthophora), from specimens collected





-22-


at Uvalde, Texas,in the same locality where Lu. diabolica was found.

The female flies were collected in the mornings while feeding on

domestic rabbits. Attempts to feed this fly on man were unsuccessful.

Packchanian (1946) reviewed distributions and habits of the six

species of sand flies then known to occur in the USA. He drew

attention to the fact that sand flies were known vectors of

leishmaniasis in the Old World and were known to have been infected

naturally or experimentally with leishmaniasis in numerous localities

in Latin America. He stated that in all probability the species found

in the USA represented potential vectors of leishmaniasis and that

this important problem remained to be investigated under rigid

experimental conditions.

In 1968, Simpson et al. reported the first well documented, and

undisputed, autochthonous case of leishmaniasis in the USA. The

patient was a 64-year-old Mexican-American woman from San Benito,

Texas, who had a 57 year history of chronically active, disseminated

anergic cutaneous leishmaniasis.

Shaw et al. (1976) described two more autochthonous human cases

of cutaneous leishmaniasis in Texas. The first occurred in 1972 in a

74-year-old woman from Dilworth, Gonzales County,and the second in a

56-year-old man from Kenedy, Karnes County. The first patient owned a

ranch a few miles from her home and visited it daily to feed the

cattle and some stray dogs. She related that she frequently saw

swarms of gnats and small black flies, but did not remember any

specific insect bites, especially in relation to her skin lesions.

Aside from short visits to northern Mexico she had had no foreign

travel (Shaw et al., 1976). The second patient lived in a primitive




-23-


shack in rather unsanitary conditions. He reported no travel outside

the USA except for two half-day visits to Nuevo Laredo, Mexico. The

authors conducted epidemiologic studies in the neighborhoods and

communities surrounding the two case sites but collected no sand

flies. They concluded that conditions may be proper in south central

Texas for arthropod-borne transmission of cutaneous leishmaniasis, and

that the suspected endemicity of the disease should be confirmed by

further studies.

Interest in the epidemiology of leishmaniasis in Texas mounted

slowly until, in 1980, cutaneous leishmaniasis (L. mexicana mexicana)

was diagnosed in a 11-year-old boy from Uvalde, Texas, the same

locality in which the first confirmed anthropophilic species of sand

fly in the USA (Lu. diabolica) was found (Gustafson et al., 1984).

The boy was presumed to have contracted the disease near his home or

while on camping trips in south central Texas, since his travel had

been limited.

In that same year, Anderson et al. (1980) reported endemic canine

leishmaniasis in dogs near Oklahoma City, Oklahoma. The parasite most

closely resembles L. donovani infantum (Kocan et al., 1984). These

instances further emphasize the need to study the epidemiology of

leishmaniasis in the USA.

In 1981, Perkins (1982) demonstrated for the first time that an

anthropophilic USA sand fly, Lu. shannoni, could be experimentally

infected with an indigenous strain of L. mexicana (strain WR-411,

Uvalde, Texas) by feeding them on histocytomas on infected hamsters.

Although transmission was not accomplished at that time, the ground

work was laid for further studies. Later that same year Endris et al.





-24-


(Endris, pers. comm., 1984) demonstrated the ability of the non-

anthropophilic, rodent-feeding sand fly, Lu. anthophora, to transmit

L. mexicana (strain WR-411, Uvalde, Texas) from infected to uninfected

hamsters by bite. This was the first report of a native USA species

of sand fly transmitting leishmaniasis by bite. The authors suggested

that L. mexicana could be maintained in a wild rodent population by

Lu. anthophora from which it could then be transmitted to man by other

sympatric anthropophilic sand flies such as Lu. diabolica.

Finally, in 1983, Gustafson reported three confirmed and one

suspected case of cutaneous leishmaniasis from south central Texas

(Gustafson et al., 1984).

The vectors of this disease in Texas are unknown and until the

present study, no attempts had been made to incriminate any

anthropophilic sand fly from areas of Leishmania endemicity in the

USA.



Statement of Objectives


This study was undertaken to investigate the life history and

biology of the sand fly Lutzomyia diabolica (Hall) and its possible

role in the transmission of human cutaneous leishmaniasis in Texas.

For comparison, the vector capacity of Lu. shannoni (Dyar), another

anthropophilic sand fly, was investigated in conjunction with that of

Lu. diabolica. The specific objectives of the study were to

1. conduct a field survey of potential vector sand flies in

vicinities of recent human case sites of leishmaniasis in Texas;

2. study the field biology of Lu. diabolica and collect wild

stock for a laboratory colony;




-25-

3. establish a productive colony of Lu. diabolica in the

laboratory;

4. study the biology and life history of Lu. diabolica under

laboratory conditions;

5. investigate, under controlled laboratory conditions, the

vector capacity of Lu. diabolica, as compared with Lu. shannoni, for

leishmaniasis; and

6. examine by means of the electron microscope the morphology of

L. mexicana promastigotes found in the.sand fly vector.











CHAPTER 2
SAND FLIES ASSOCIATED WITH HUMAN CUTANEOUS LEISHMANIASIS
IN TEXAS: OBSERVATIONS ON THEIR BIOLOGY WITH SPECIAL
REFERENCE TO Lutzomyia diabolica (HALL)



Introduction


General


Recent reports of human cutaneous leishmaniasis acquired in south

central Texas strongly suggest that the disease is endemic there (Shaw

et al., 1976; Gustafson et al., 1984). Because sand flies are the

only known natural vectors of leishmaniasis, a knowledge of their

field biology and host associations is of paramount importance in

epidemiologic studies of this disease. Six species of sand flies are

known to occur in Texas; these are Lutzomyia anthophora (Addis), Lu.

californica (Fairchild and Hertig), Lu. diabolica (Hall), Lu. oppidana

(Dampf), Lu. texana (Dampf), and Lu. vexator (Coq.) (Young and

Perkins, 1984). Only one, Lu. diabolica, is known to be

anthropophilic. This field study is the first to investigate sand

flies in Texas associated with specific human case sites of cutaneous

leishmaniasis.

The investigation was initiated in 1982 with a survey trip in

late spring and early summer (4-28 June) to south central Texas. The

principal study site was Garner State Park near Concan in Uvalde

County. Secondary sites included Rio Frio in Real County, Seminole

Canyon State Park in Val Verde County, and Fawcett Boy Scout Camp near


-26-





-27-


Barksdale in Edwards County. The field work was conducted in

cooperation with Drs. D. G. Young, G. B. Fairchild, and R. G. Endris,

all from the University of Florida, Gainesville, FL. A second survey

trip was taken in early fall of 1983 (19-30 September), the principal

field study site being in and around the rural community of D'Hanis in

Medina County. Secondary sites included Garner State Park, the Romer

Ranch south of Devine in Medina County, and two sites within the city

limits of San Antonio in Bexar County. During the second trip the

able assistance of Mr. T. Long, zoonoti.c technician, Region 9, Texas

State Health Department, was greatly appreciated. The objectives

were to

1. conduct a survey of potential vector sand flies in the

vicinities of recent human case sites of leishmaniasis in Texas (A

case site, as used herein, is defined as the home environs of a

confirmed leishmaniasis patient, or localities where that individual

camped or otherwise visited within three months prior to the onset of

disease symptoms.); and

2. study the field biology of Lu. diabolica and collect wild

stock for a laboratory colony.



Human Case Histories


Eight autochthonous human cases of cutaneous leishmaniasis have

been reported in south central Texas, four of which have occurred since

1980 (Gustafson et al., 1984) (Fig. 2-1). These latter four were the

only cases investigated during this study and are described below. Por-

tions of the following unpublished case histories were graciously provided

by Dr. T. Gustafson, the investigating Texas State epidemiologist. The





-28-


S= autochthonous human case of
cutaneous leishmaniasis

= case site and study site

** study site

El cases reported since 1980


Figure 2-1.


Distribution of autochthonous human cases of cutaneous
leishmaniasis in Texas and locations of study sites.




-29-


remaining information was obtained through personal interviews with

patients or their family members. A complete discussion of the case

histories and results of serologic tests are provided by Gustafson et

al. (1984).

Patient A. The patient was an 11-year-old white male from Uvalde,

Texas, who noticed an ulcerating lesion on his left cheek beginning

February, 1980. In May, 1980, a biopsy was performed which showed

amastigotes in dermal macrophages. Leishmania mexicana was cultured

from biopsy material that was sent to Walter Reed Army Institute of

Research. Patient A had never traveled outside the USA except for

occasional one-day trips to the border city of Ciudad Acuna, Mexico.

In the month prior to onset, he had participated in a camping trip at

Fawcett Boy Scout Camp in Edwards County. The family had no pets.

Patient B. The patient was a 56-year-old white female from a

suburban neighborhood in southeast San Antonio, Texas, who first

noticed a small lesion on her left ear in November 1982 (Fig. 2-2).

She distinctly remembered waking one morning with an itching ear and

noticed a drop of blood on her ear lobe and on her pillow. She

suspected that she had been bitten by an insect. Her bed was located

next to an open, screened window. A biopsy performed in February,

1983, showed amastigotes in dermal macrophages by light and electron

microscopy. Patient B had traveled out of the USA as a military

dependent more than ten years previously and had visited Chihuahua in

northern Mexico in June, 1982. She had one pet dog.

Patient C. The patient was a 5-year-old white male from a

suburban neighborhood in northeast San Antonio, Texas, who first

noticed an enlarging papule on his left thigh beginning in November





-30-


1982 (Fig. 2-3). When the lesion persisted, an excisional biopsy was

performed which showed amastigotes in large vesicles within

macrophages by both light and electron microscopy. The patient had

never traveled outside the USA. He frequently spent the week ends at

the ranch of his paternal grandparents in Devine, Texas. The family

had one pet dog, and the boy's grandparents had three hunting dogs.

Patient D. The patient was a 10-year-old white male from the

rural farming community of D'Hanis, Texas, who developed a lesion near

his right eye brow and another on his right cheek in December, 1982

(Fig. 2-4). When the lesions persisted, an excisional biopsy was

performed which showed amastigotes in large vesicles within

macrophages by light microscopy. Presence of kinetoplasts was

confirmed by electron microscopy. Promastigotes were recovered from

an aspirate of the eyebrow lesion, but these died before further

identification could be performed. The patient had never traveled

outside of Texas and had lived all of his life in Medina County with

only occasional trips to Uvalde, San Antonio, and Seguin, Texas. He

regularly accompanied his father on coyote hunting trips near his

home. The family had ten hunting dogs. The patient's mother recalled

seeing tiny hopping flies in the house, around the light fixture, and

members of the family reported being bitten by tiny gnats. When shown

a live female Lu. diabolica the mother said that it was the same as

those she had seen in the house.

A suspected case, with history very similar to the others, was

reported from Hondo, Medina County, Texas, within 12 miles of the home

of Patient D. The patient was a 4-year-old white male who first

noticed a lesion on the bridge of his nose beginning in January, 1983.




-31-


Figure 2-2.


Figure 2-3.


Cutaneous lesion due to Leishmania mexicana on ear lobe
of patient B. Photo courtesy Dr. T. Gustafson.


Cutaneous lesion due to Leishmania mexicana on thigh of
patient C. Photo courtesy of Dr. T. Gustafson.




-32-


Figure 2-4. Cutaneous lesion due to Leishmania mexicana on cheek of
patient D. Photo courtesy of Dr. T. Gustafson.





-33-


When the lesion persisted the boy was seen by a physician who biopsied

it before a positive diagnosis could be rendered. Tissue samples and

saline aspirates taken from the biopsied lesion contained no

parasites. The boy had not traveled outside the USA, nor outside the

region during the previous year. The family had three pet dogs.

Serologic tests performed for each patient included indirect

fluorescent antibody test (IFA) and dot enzyme-linked immunosorbent

assays (ELISA) at Walter Reed Army Institute of Research, Wash., DC,

and fluorescent immunosorbent assays (FIAX) at Oklahoma State

University, Stillwater, Oklahoma (Gustafson et al., 1984). These tests,

performed between three and six months after onset, showed that all

four confirmed patients had detectable antibody titers to Leishmania.

The sister of Patient C and the mother of Patient D had detectable

titers by at least two serologic tests. One dog belonging to Patient

C and three dogs belonging to Patient D had detectable Leishmania

titers by at least two tests. However, all four dogs had antibody

titers to Trypanosoma cruzi, and the Leishmania titers may represent

cross-reactions (Gustafson et al., 1984).



Description of Study Sites


Eight study sites were selected based on previous collection

records of Lutzomyia from south central Texas (Eads et al., 1965; Easton,

et al., 1968; Young, 1972; Eads, 1978; Endris, 1982; Perkins, 1982) and

on their association with confirmed Leishmania patients (Fig. 2-1).

Three of the sites, A, B, and D, were close to the home environs of

patient A and he had camped at or near the sites three months prior to

onset of symptoms. Lutzomyia species were also known to occur at site




-34-

A (Young, 1972; Endris, 1982). Site B was selected on the basis of

1971 light trap collection records of 237 females from a single light

trap (Perkins, 1982). The remaining sites E, F, G, and H were the

actual home environs of confirmed leishmaniasis patients.

Surveys for potential sand fly vectors were conducted at all sites

during the course of the study. Emphasis was placed on qualitative

rather than quantitative sampling; no attempt was made to estimate

sand fly population densities.

Site A. Garner State Park, Concan, Uvalde County, Texas (4-28

June, 1982; 19-30 September, 1983; Fig. 2-5 and 2-6). This state park

is located 50 km north of Uvalde in the Frio River valley. It is

situated on the southern edge of the Edwards Plateau, on the Balcones

Escarpment, which separates the plateau from the Nueces plains to the

south. The habitat is characterized by grassy meadows dotted with

cedar (Juniperus sp.), live oak (Quercus virginiana), acacia (Acacia

sp.), pecan (Carya illinoensis), mesquite (Prosopis juliflora), and

elm (Ulmus crassifolia), with bald cypress (Cupressus arizonica)

bordering the Frio river. The park is surrounded by steep rocky

hills, the slopes of which are covered with cedar, wild cherry (Prunus

serotina ), persimmon (Diospyros texana), madrone (Arbutus texana),

and other Hill Country shrubs. Potential mammalian hosts for sand

flies include white tail deer (Odocoileus virginianus), coyote (Canis

latrans), fox (Vulpes sp.), bobcat (Lynx rufus), racoon (Procyon

lotor), porcupine (Erethizon dorsatum), skunk (Mephitis sp.), opossum

(Didelphus marsupialis), armadillo (Dasypus novemcinctus), jack rabbit

(Lepus californica), cottontail rabbit (Sylvilagus floridanus), and a

variety of rodents (Texas Parks and Wildlife Department, 1982a; Raisz,

1954; Kuchler, 1975).




-35-


The nearest weather station to Garner State Park is 50 km south at

Uvalde where the climate is fairly representative of the area

surrounding the park, characterized by hot, humid summers and

pleasantly mild winters (US Department of Commerce, 1965). Annual

precipitation averaged 63.1 cm between 1951 and 1960, with a range of

23.3 cm to 98.3 cm. The wettest periods of the year are May-June and

September-October. The hottest part of the year is July-August, with

extreme temperatures reaching a high of 430C, and a low around -150C,

reached in January and February. Relative humidity at noon, central

standard time, averages between 58% in January to 48% in July. Daily

fluctuations in RH occur especially during the warmer months, with a

gradual decrease in the afternoon until sunset, then a rapid increase

after dark (US Department of Commerce, 1965). Average daily maximum

and minimum temperatures for June 1982 were 350C and 220C,

respectively, and precipitation 14.4 cm (US Department of Commerce,

1982a). Average daily maximum and minimum temperatures for September

1983 were 340C and 210C, respectively, with 7.9 cm precipitation (US

Department of Commerce, 1983a).

Site B. Rio Frio, Real County, Texas (4-20 June, 1982). This

site is located approximately 20 km north of Site A, at the edge of a

small gorge carved by the Frio river. The habitat and climate are

very similar to that found at Garner State Park.

Site C. Seminole Canyon State Park, Val Verde County, Texas (10-

11 June, 1982; Fig. 2-7). This state historical park is located 75 km

west of Del Rio, Texas, a short distance downstream from the

confluence of the Rio Grande and Pecos Rivers. It is noted for its

rugged terrain, sparse vegetation and deep canyons. Situated in the




-36-


arid Pecos Shrub Savanna, its flora and fauna are derived from

elements of the Texas Hill Country, Tamaulipas Thorn Scrubland, and

the Chihuahua Desert (Texas Parks and Wildlife, 1982b). It is

considered a true desert habitat, receiving 25 cm or less

precipitation annually. The nearest weather station, Amistad Dam,

recorded average maximun and minimum temperatures for June 1982 of

360C and 230C, respectively, with daily temperatures frequently rising

above 38C during the summer months (US Department of Commerce,

1982a). Relative humidity was not measured, but probably averaged

below 30% during the summer. Mammalian inhabitants at the park are

limited to coyote, jack rabbit, armadillo, and small rodents.

Site D. Fawcett Boy Scout Camp, Barksdale, Edwards County, Texas

(11 and 22 June, 1982; Fig. 2-8). This site is located 7 km north of

Barksdale at a bend in the Nueces river. Also at the southern edge of

the Edwards plateau, the habitat resembles Garner State Park in most

features, but lacks steep, rocky hills. The nearest weather station

is about 20 km south at Camp Wood (US Department of Commerce, 1965).

Annual precipitation for the ten-year period of 1951-1960 averaged

59.8 cm with a range of 22.3 cm to 106.1 cm. Average daily maximum

and minimum temperatures during June 1982 were 330C and 210C,

respectively (US Department of Commerce, 1982a).

Site E. Myer's Farm, D'Hanis, Medina County, Texas (19-30

September, 1983). This farm, the home of patient D, is in a small

rural community 75 km west of San Antonio. The surrounding area is

characterized by low rolling farmland with live oak, pecan, and

mesquite trees bordering the fields and streams (Fig. 2-9).

Uncultivated areas are overgrown with cactus, mesquite and thorny

shrubs. One such area, about 50 m west of the farm house, contains a





-37-


number of woodrat (Neotoma) nests and armadillo burrows. Roughly 15 m

south of the house is a large equipment shed, behind which (to the

west) is the kennel in which are kept the family's ten hunting dogs

(Fig. 2-10). Between the house and the kennel are a large live oak

tree and a wood pile (Fig. 2-11). Approximately 80 m west of the

house are the hog pens (Fig. 2-12). Approximately 100 m behind (west

of) the house is a dry creek bed lined with mesquite trees, and beyond

that, a grove of live oak trees. The closest weather station is in

Hondo, 20 km to the east. The climate at this station is

representative of Medina County, with hot humid summers and pleasantly

mild, dry winters (US Department of Commerce, 1983b). Average annual

rainfall is 71.2 cm with heaviest rainfall received during April-June

and September-October. Mean length of the warm season is 263 days.

Mean dates of last occurrence of freezing temperatures in spring, and

first occurence in the fall are March 6 and Nov. 24, respectively.

Humidity at noon central standard time averages 58% in January, 56% in

April, 48% in July, and 55% in October. Average daily maximum and

minimum temperatures for September, 1983, were 320C and 260C,


respectively, with

ranged between 430C

minima of 270C and

Site F. Romer

September, 1983; Fi

grandparents of Pat

it is very similar

Site G. Romer


13.0 cm precipitation. Temperatures for the year

and -160C with ten-year average daily maxima and

130C (US Department of Commerce, 1983b).

Ranch, Devine, Medina County, Texas (19-30

g. 2-13). This is the home of the paternal

ient B. Approximately 10 km south east of Devine,

in general features to Site E in D'Hanis.

Residence, northwest San Antonio, Bexar County,


Texas (22 September, 1983; Fig. 2-14). This is the home of Patient B




-38-


Figure 2-5.


Figure 2-6.


Habitat typical of that found at Garner State Park, Concan,
Uvalde County, Texas.


Habitat at Garner Stake Park, Concan, Uvalde County, Texas,
showing public latrine resting station.




-39-


Figure 2-7.


Habitat typical of that found at
Val Verde County, Texas.


Seminole Canyon State Park,


Figure 2-8.


Habitat at Fawcett Boy Scout Camp, Barksdale, Edwards County,
Texas, showing open latrine resting station.




-40-


Figure 2-9.


Farmland surrounding the home of patient D in
community of D'Hanis, Medina County, Texas.


y1~

-~~=~P~ t~~b


Figure 2-10.


Hunting dogs in a kennel behind the
D'Hanis, Medina County, Texas.


home of patient D in


the rural


~r;L-C'
~,. *` 'h




-41-


Figure 2-11.


Figure 2-12.


Wood pile located behind the home of patient D in D'Hanis,
Medina County, Texas. Six female and one male Lutzomyia
diabolica were collected in the CO2-baited CDC light trap
hanging in the foreground.


Hog pen located about 80 m west of the home of patient D
in D'Hanis, Medina County, Texas. Rock pile in foreground
offered many potential resting places for sand flies.




-42-


Figure 2-13.


Romer ranch, near Devine, Medina County,
the paternal grandparents of patient B.


Texas, home of


Figure 2-14.


Neighborhood of patient B in northwest San Antonio, Bexar
County, Texas.


ir~ m _.: o, ;.: ..
~ ri .. .. '" -.-" .,.. "





-43-


and is in a typical suburban neighborhood on the northwest limits of

San Antonio. About three blocks south of the residence are some open

fields, heavily overgrown with weeds and small shrubs. An equal

distance to the north is a small creek basin lined with a variety of

large shade trees and shrubs.

Site H. Kelly Residence, San Antonio, Bexar County, Texas (23 and

30 September, 1983; Fig. 2-15). Similar to site G, this site is in a

suburban neighborhood in southeast San Antonio. Three large pecan

trees shade the backyard. Adjoining the property to the southeast is

a large vacant lot, overgrown with tall weeds.

San Antonio climate is about the same as that of Medina County

(US Department of Commerce, 1982b) with hot humid summers and

pleasantly mild, dry winters. Average annual precipitation is

74.6 cm. Average daily maximum and minimum temperatures during September

1983 were 320C and 200C, respectively, with an overall mean of 260C.

Precipitation during September, 1983, measured 13 cm (US Department

of Commerce, 1983a).



Materials and Methods


Determination of Sand Fly Fauna


Several useful field methods for sampling sand flies have been

used with varying degrees of success and are reviewed by Young (1979)

and Killick-Kendrick (1981a). The diversity of potential sand fly

habitats at each study site dictated the selection of methods that

would sample the broadest cross section. As far as practicable, all

representative habitats within a 200 m radius of each site were




-44-


Figure 2-15. Neighborhood of patient
County, Texas.


A in southeast San Antonio, Bexar





-45-


surveyed to determine species diversity and relative abundance of sand

flies. Collection methods employed included the following:

Resting collections. An extensive search was made at each site

for diurnal and nocturnal resting places of adult sand flies.

Particular attention was directed to dark protected cavities in rocks,

trees, buildings and other man-made structures, as well as in animal

burrows, ground nests and brush piles where sand flies might seek

shelter. A flashlight was used to enhance visibility. Live specimens

were captured with a simple tube aspirator and placed in a 120 ml

feeding/rearing chamber (Endris et al., 1982).

Biting collections. Biting collections (Young, 1979) were made

at night, usually between 2100 and 2400 hrs. The collector sat in an

open area under an incandescent light or Coleman lantern and captured

sand flies with an aspirator as they attempted to feed.

Light traps. Battery powered or CDC miniature light traps (Sudia

and Chamberlain, 1962) were secured to tree branches about 2 m above

the ground at edges of clearings and near human habitations. Care was

taken to insure their placement was away from competing light. In

some instances they were placed adjacent to or directly over suspected

resting sites such as wood or rock piles (Fig. 2-11).

A New Jersey mechanical light trap (Mulhern, 1942), powered by

110-volt house current, was placed in the back yard, adjacent to the

dog kennel at the D'Hanis site. The trap will be operated there 2-4

nights per week for the next year.

A Shannon trap (Shannon, 1939; Fig. 2-16), made from white cotton

bedsheets, was erected in open areas near human and animal dwellings.

This apparatus does not actually trap insects, but the lantern or




-46-


Figure 2-16. Shannon trap erected behind the home of patient D, D'Hanis,
Medina County, Texas.


4rk
kte*Q r-lii' r





-47-


incandescent light inside provides a light source, enabling the

collector to aspirate flies which land on the illuminated cloth.

Flies are attracted to the light, the collector, or a combination or

both (Young, 1979).

Baited traps. Pieces of dry ice wrapped in newspaper and

suspended next to the CDC light traps provided CO2 as an adjunct

attractant for hematophagus insects (Fig. 2-11). A caged hamster,

suspended in a similar manner, also served as an attractant.

A Disney trap (Disney, 1966) baited with a hamster or wild cotton

rat (Sigmodon sp.) was also used. This trap consists of a shallow

tray filled with mineral oil and a caged animal supported on slats

just above the surface of the oil. After feeding, blood engorged sand

flies do not fly away, but hop to the "ground" to rest, becoming

entrapped in the mineral oil.

Sticky traps. Sheets of card stock, coated on one side with oil,

were placed directly in front of or over burrow entrances and crevices

in rocks and trees to catch sand flies emerging from such places at

night (BUtticker, 1979).


Processing and Maintenance of Wild-caught Sand Flies
and Recovery of Eggs

Wild-caught female sand flies showing evidence of a blood meal

(abdomen distended and dark-red or black in color) were transferred to

individual 7-dram oviposition vials fitted with screen lids (Endris et

al., 1982). Water was added to the plaster of Paris in the bottom of

each vial to maintain a high relative humidity, and a drop of Karo

syrup in water (1:1) was placed on the screen lid to serve as an




-48-


energy source. Unfed flies were offered a blood meal, through the

screen lid of the holding vial (Fig. 3-1, p. 98, Chap. 3) on a human

forearm, a hamster or a lizard. Those that fed immediately were

transferred to individual oviposition vials, as previously described,

and held until they oviposited and/or died. Females that did not

accept the initial offer of a blood meal were placed in a holding cage

(Endris et al., 1982) with males and offered a blood meal each

succeeding day until they either fed or died. Those that fed were

retained in oviposition vials until they oviposited and/or died. Dead

females were removed and, if they had deposited eggs in the vials, the

screen lids were replaced with solid plastic lids that had been

perforated with a dissecting needle to permit limited gas exchange.

During June 1982, the field laboratory consisted of a permanent,

screened camping shelter. To protect the captured flies from the heat

of the Texas summer, their containers were kept in a polystyrene

cooler. By placing ice in the cooler, removing or replacing the lid

as necessary, the temperature within the chambers and vials could be

maintained at or near 270C. During the September, 1983, trip the

field laboratory was in an air-conditioned motel room where the

temperature was maintained at 230C.

Preliminary identification of live, wild-caught specimens was

performed macroscopically. At death they were more reliably

identified by microscopic examination and comparison with appropriate

taxonomic keys (Young and Perkins, 1984). Field identifications were

later confirmed as necessary in the laboratory by Dr. D. G. Young,

after consulting appropriate keys and available type materials.

Specimens were prepared and cleared for permanent slide mounts

according to the technique of Young (1979).




-49-

Post mortem dissections of female sand flies were performed as

soon after death as possible (usually within 12 hours) according to

the technique of Johnson et al. (1963). Data recorded for each

specimen included information regarding collection, sex,

identification, blood meals, oviposition, longevity, condition of

accessory glands, and presence or absence of natural parasites.



Results


Species of Sand Flies Present


An estimated 2400 sand fly specimens representing five species of

Lutzomyia, including one new species, were collected and eight new

county records established during the two years of the study (Table

2-1). Sand flies were taken from five of eight case sites; none were

collected at sites F, G, and H (San Antonio and Devine).

Of the sampling methods used, only the Disney trap and the oil

traps failed to collect sand flies. Approximately 2000 flies, mostly

females, were taken in resting collections at Garner State Park and

Fawcett Boy Scout Camp. Other trapping methods also yielded mostly

females (Table 2-2). The ratio of male to female Lu. diabolica in

resting, biting and light trap collections ranged from a high of about

6:10 in resting collections at Fawcett Boy Scout Camp, to around 4:10

in resting collections at Garner State Park. More than 350 female and

no male Lu. diabolica were taken in biting collections at Garner State

Park. This method was not successful at other sites. At locations

where flies were not taken resting, CDC light traps were most

effective.






-50-


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-52-


Lutzomyia diabolica. Lu. diabolica (Fig. 2-17), the only

anthropophilic sand fly found, was collected at all positive sites and

accounted for 99% of the total catch (Table 2-1). They were collected

in greatest abundance at Garner State Park where they were captured

while resting on the tile walls of the public latrines (Fig. 2-6).

There were a total of 12 latrines at the park, 8 of which were

situated in the upland meadow areas and 4 along the Frio River flood

plain, shaded by large pecan, oak, and cypress trees. Up to 150 Lu.

diabolica per day (nights and mornings) were taken in resting

collections from the 8 latrines in the more elevated portions of the

park. Many were engorged with fresh blood, possibly from the

unprotected parts of unsuspecting campers, or from deer and jack

rabbits that frequent the surrounding meadows by night. No sand flies

were collected from the latrines near the river. Although a few sand

flies could usually be found in positive latrines at all hours of the

day, they typically appeared about one hour after sunset (2200 hrs in

June, 2100 hrs in September) and remained until mid morning (about

1000 hrs), their activity coinciding with the daily bathing ritual of

the campers. They were observed resting on the interior tile walls

between the level of the floor and a height of about 2.5 m and seemed

to prefer dark humid corners, such as in shower stalls and under sink

counters. Their resting attitude was almost always vertical, with

head pointing up. They were frequently observed hopping deliberately

toward the collector, presumably seeking a blood meal. If by chance

the lights in a latrine had not been turned on, the catch was greatly

reduced. Greatest numbers appeared on hot, humid nights (270C and 80%

or higher RH) with little or no air movement. On such nights sand flies




-53-


Figure 2-17. Lutzomyia diabolica female (approx. magn. x 10).





-54-


were also seen resting on the exterior walls of the latrines under the

outside lights and around the entrance. The number of flies collected

decreased dramatically with increased wind velocity. At Fawcett Boy

Scout Camp, sand flies were also taken in resting collections in a

lighted, open-air latrine (Fig. 2-8).

An extensive search for natural diurnal and nocturnal resting

sites of Lu. diabolica in rock crevices, under bark of trees, in

animal nests and burrows, in leaf litter, and other potential hiding

places was fruitless at all case sites.

Female Lu. diabolica were often taken in biting collections during

the evening hours at Garner State Park as they came in search of a

blood meal. Routine biting collections netted from one to about

twenty female sand flies per hour. One biting collection, however,

was worthy of special note: The night of 9 June, 1982, was hot and

humid (270C and 80-90% RH) and air movement was about nil. The author

and his son had returned to the field laboratory (a screen tent) about

2330 hrs after searching the latrines for resting sand flies. They

sat under an incandescent light at a table inside the tent and began

to count and feed the evening's catch. Shortly the author's son began

to complain of being bitten by sand flies, and five were collected

that were biting him on the legs. Lu. diabolica females flew

unobstructed through the screen sides of the tent in waves of ten or

twenty to feed, biting the occupants on the face, neck, hands, ankles

and other exposed skin. These foraging sorties were of about five

minutes' duration, with a brief respite interval between. They

continued for several hours and about 150 female Lu. diabolica were

collected before 0100 hrs, when the author finally retired for the





-55-


night. Even with the light turned out, the flies continued their

attack, but in lesser numbers.

On several occasions, foraging females were seen flying through

the standard mesh screen of the permanent camping shelter (field

laboratory) to attack the author and his eight-year-old son. They

seemed to exhibit a preference for the son, biting him even after the

lights had been turned off. Although most of this biting activity

occurred at night, one female Lu. diabolica bit him at about 1400

hours in the heat of the day and in full sunlight (temperature about

380C, RH about 60%, air calm).

Lu. diabolica were collected in unbaited CDC light traps at Garner

State Park, Rio Frio, and Seminole Canyon State Park in June 1982.

They were also collected in CO2 baited CDC light traps (with light on)

and in a New Jersey light trap at the site in D'Hanis in September,

1983. Unbaited CDC light traps (light only) were unsuccessful at the

D'Hanis site.

One female Lu. diabolica was collected in a Shannon trap at Garner

State Park. The trap was set up near the camping shelter about an

hour before dark (1800 hrs), but was only operational for about three

hours due to inclement weather. Other attempts to attract sand flies

to a Shannon trap at D'Hanis and Devine failed.

Disney traps set out near rodent burrows at Garner State Park

failed to collect sand flies as did oil traps set out in similar

places at D'Hanis and Devine.

Lutzomyia anthophora. Lutzomyia anthophora were taken with Lu.

diabolica in resting collections in latrines at Garner State Park and

Fawcett Boy Scout Camp. They were also taken from armadillo (Dasypus

novemcinctus) burrows at the former site.





-56-


One male Lu. anthophora was taken from an active, carefully

dismantled woodrat (Neotoma) nest located approximately 75 m east of

the farm house at the D'Hanis site (Fig. 2-18). Four other Lu.

anthophora (males and females) were seen in the same nest, but escaped

capture.

Lutzomyia texana. Lutzomyia texana, a rather large sand fly that

inhabits mammal burrows, was taken in resting collections from

latrines at Garner State Park and Fawcett Boy Scout Camp. They were

also collected from the entrances of armadillo burrows at the former

site and in CO2 baited CDC light traps and in an unbaited New Jersey

light trap at the farm in D'Hanis. They were not found in armadillo

burrows at the latter site. Efforts to feed Lu. texana on a human, a

hamster and a lizard were unsuccessful.

Lutzomyia vexator. This species, which feeds on cold-blooded

vertebrates, was collected on two occasions while resting on latrine

walls at Garner State Park.

Lutzomyia new species. In September, 1983, a single female of an

undescribed species was taken in a resting collection from a latrine

at Garner State Park. Efforts to feed the fly on human, hamster and

amphibian (toad) blood were futile and she died without depositing

eggs. The fly was prepared and examined by Dr. D. G. Young who stated

that it belongs in the Cruciata group, but was unlike any other

species collected in Texas.



Processing and Maintenance of Wild-caught Sand Flies
and Recovery of Eggs


An estimated 1925 female Lu. diabolica were segregated and

maintained in individual or group oviposition containers and 7940 ova




-57-


V r~A


p~ c~~- :~fj;r ~ *L.
.4;,,-. *, I *'4 ~ C% 'V


Figure 2-18.


Dismantled woodrat (Neotoma) nest near the home of patient
D in D'Hanis, Medina County, Texas, where specimens of
Lutzomyia anthophora were found.





-58-


were recovered for laboratory colony stock. Six hundred and one

females were dissected post mortem and examined for retention of eggs,

condition of accessory glands and natural parasite infections.

Table 2-3 summarizes fecundity for 360 wild-caught females (263

from the first trip and 97 from the second). Regardless of efforts to

perform dissections within 24 hrs after death, many flies were in such

poor condition that dissection provided little usable information.

These and 54 females that died from overheating in a locked car in

June 1982 were excluded from the table. The mean number of eggs

deposited and the gross number of eggs produced (the number deposited

plus the number retained) remained fairly constant from one year to

the next. From 31 to 37% of the females retained some or all of their

eggs. Females neither depositing nor retaining eggs (in other words

maturing none) ranged from 3% in June 1982 to 10% in September 1983.

No evidence of autogenous behavior was observed. Females that were

denied a blood meal neither laid nor developed mature eggs.

Significant differences were noted between year groups in

preoviposition interval, postcapture longevity and postoviposition

longevity, all three being longer for flies captured in the fall

(Table 2-4). A smaller percentage of females survived oviposition in

the fall than in the spring.



Accessory Glands and Parity


Sixty-four wild-caught female Lu. diabolica, which had not been

offered a blood meal, were dissected to study the paired accessory

glands (Fig. 2-19). The glands of only 36 flies were clearly seen.

Of these 36, only three showed evidence of having had a blood meal;





-59-


00

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-60-


Table 2-4. Summary of preoviposition intervals, postcapture and post-
oviposition longevities in wild-caught Lutzomyia diabolica
females from south central Texas (based on a sample of 100
females per year group) (4-28 June, 1982; 19-30 Sept., 1983)

June 1982 September 1983

Days Days
Category n/100 x 1 SD (range) n/100 x 1 SD (range)


Preoviposition Interval

Captured w/ 31/100 4.5 2.9 (1-10) 46/100 8.6 3.7 (1-16)
Blood Meals

Captured w/o 69/100 4.7 2.1 (1-11) 54/100 8.5 3.8 (1-16)
Blood Meals


Postcapture Longevity

Overall 5.8 3.0 (1-19) 9.7 4.2 (1-19)

Laying Some 56/100 6.5 6.5 (1-16) 75/100 9.6 3.79 (3-19)
Eggs
Maturing No 3/100 5.6 2.1 (3-9) 10/100 10.4 5.4 (1-19)
Eggs


Postoviposition Longevity

Taking One 56/100 2.2 1.7 (1-8) 29/100 3.2 3.2 (1-11)
Blood Meal

Taking Two 0/100 4/100 4.7 2.3 (2-6)
Blood Meals





-61-


all three had either mature or developing ova and the accessory glands

were full of dark granules. Of the remaining 33 females, 17 had

developing ova (most were about half developed) and all of these had

either full or partially full accessory glands. Sixteen had unde-

veloped ovaries,and of these, five had no granules in the accessory

glands; one had a very few granules; nine had accessory glands half

full of granules; and two had completely full accessory glands.

Observations on the condition of the accessory glands (granule

formation) in another 94 dissected wild-caught females are presented

in Fig. 2-20. All of these females were blood-fed at the time of

capture or were given a blood meal immediately after capture. These

were compared with dissections of laboratory-reared, fed and unfed

females of known ages. In general, granule formation in accessory

glands of wild-caught sand flies paralleled ovarian development, and

when the ova were mature, the accessory glands were dark and full of

granules. Partial or complete emptying of the accessory glands often

occurred as eggs were deposited, but many females that deposited most

or all of their eggs maintained full accessory glands. Blood-fed

laboratory-reared flies showed basically the same trend as blood-fed

wild-caught flies. Unfed laboratory-reared flies either did not

develop granules in the accessory glands or, if they did, they

developed them within 24 hrs, but to a lesser degree, even after six

days, than seen in blood-fed females.


Natural Parasite Infections


The incidence of natural parasite infections found in 341 female

sand flies taken from south central Texas, dissected during the two

years of the study, is presented in Table 2-5.




-62-


~-~2ZA7
d.

r9~qy -II


Figure 2-19.


Paired accessory glands of a gravid female Lutzomyia
diabolica showing dense granular material (magn. x 160).


'it)




It





-63-


.c



-C

3
10






6-0 0

-i0
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111
K 2c



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-64-


Table 2-5.


Incidence of natural parasite
Lutzomyia diabolica collected
(4-28 June, 1982; 19-30 Sept.,


infections in 341 female
in south central Texas
1983)


June 1982 (N = 243)1


September


1983 (N = 98)2


# infected


% infected


# infected


% infected


Small, round,
fast moving
flagellates

Epimastigote-
like
flagellates

Aseptate
gregarines

Microsporidians


146



1



12


<.05


Mites


1. Specimens collected primarily from Garner State Park, Uvalde County,
Texas, with small numbers collected from Fawcett Boy Scout Camp, Edwards
County, Texas, and Rio Frio, Real County, Texas.

2. All specimens collected from Garner State Park, Uvalde County, Texas,
with the exception of six specimens collected from D'Hanis, Medina
County, Texas.


Parasite




-65-


Flagellates. Small, rounded, highly motile flagellates were

found swimming free in the hemocoel of 27 to 60% of the specimens

dissected. They were most often seen in the hemocoel of the anterior

thoracic region and immediately posterior to the head. They numbered

from one to several, but rarely more than 25. Efforts to photograph

these tiny parasites were unsuccessful.

Two females collected in the fall of 1983 contained what appeared

to be small thin flagellates in their midguts. The organisms did not

move and appeared to be dead. They were stained with Giemsa and

mounted on microslides with Euparal, but were unidentifiable.

An infection of unknown epimastigote-like flagellates was

discovered in the midgut of one of the females dissected in the spring

of 1982 (Fig. 2-21). Several hundred of these promastigotes were

observed freely swimming in the lumen of the gut; some attached to the

midgut epithelium by their flagella; others formed rossettes with

their flagella directed toward the center much like L. mexicana

parasites seen in laboratory infections and in cultures in vitro.

Their general appearance, however, was not like Leishmania. The

infected fly was unfed when captured and was given a blood meal on the

author's forearm. Upon dissection (six days later), the blood meal

remnant was evident in the midgut. Midgut contents containing the

flagellates were inoculated subcutaneously into the foot pad of a

hamster. No lesions or other signs of infection developed in either

the author or in the hamster.

Gregarines. Aseptate gregarines, possibly Ascocystis chagasi

(Adler and Mayrink), were found in 7-9% of dissected females. Up to

five gamonts were observed in the abdominal cavity of some infected





-66-


flies (Fig. 2-22). These could be seen slowly changing shape and

moving in amoeboid fashion. When present, one to three gametocysts

could be seen attached to the accessory glands (Fig. 2-23). The

eliptical oocysts were most frequently seen spilling from the

accessory glands or from ruptured, mature gametocysts (Fig. 2-24).

Oocysts were seen glued by the accessory gland material to the chorion

of eggs deposited by gregarine infected females.

Microsporidians. Massive microsporidian infections were

discovered in the hemocoels of 3-8% of the dissected females (Fig.

2-25). Germination of the spores was stimulated in vitro by adding a

few drops of 0.2m KC1 (pH 9) to the dissecting medium (insect Ringers

solution) (Fig. 2-26). Ungerminated spores washed onto the larval

medium were fed to uninfected larvae. No infections were acquired.

Mites. Mites were found attached to the venter and dorsum of the

abdomen of 14 females collected in the spring of 1982. None were

found on females collected in the fall of 1983. The mites were

tentatively identified as Eustigmaeus sp. (Berlese) (Family

Stigmaeidae). Scanning electron micrographs of one mite specimen are

presented as Figures 2-27 and 2-28.

Other. Fungal and bacterial infections were commonly found in

dissections of wild-caught Lu. diabolica.



Discussion and Conclusions


Determination of Sand Fly Fauna


Many methods have been used for sampling sand fly populations.

Some were devised for sampling particular habitats (e.g., resting




-67-


Figure 2-21.


Unidentified flagellates in the midgut of a Lutzomyia
diabolica female collected at Garner State Park, Uvalde
County, Texas, June 1982 (approx. magn. 2100).


Figure 2-22.


Gamont
female
Uvalde


of aseptate gregarine in the abdominal cavity of a
Lutzomyia diabolica collected at Garner State Park,
County, Texas, June 1982 (approx. magn. x 850).


;$.~~
4''P~L~";1I iZ
i'*
r*,




-68-


Figure 2-23.


Figure 2-24.


Gametocyst of an aseptate gregarine (center) attached to the
accessory gland of a female Lutzomyia diabolica collected at
Garner State Park, Uvalde County, Texas, June 1982 (approx.
magn. x 850).


Gregarine oocysts spilling from the accessory glands of a
female Lutzomyia diabolica collected at Garner State Park,
Uvalde County, Texas, June 1982 (approx. magn. x 2100).




-69-


4',
Mill n"Ag&l


Figure 2-25.


Figure 2-26.


Microsporidian spores in the hemocoel of a female Lutzomyia
diabolica collected at Garner State Park, Uvalde County,
Texas, June 1982 (approx. magn. x 2100).


Microsporidian spore germinated in vitro by addition of
0.2 M KC1 (pH 9) to dissecting medium(insect Ringer's
solution, pH 7.2) (approx. magn. x 2640).





-70-


Figure 2-27.


U
* Hai.i


Figure 2-28.


Scanning electron micrograph of mite (Eustigmaeus sp.) found
on the abdomen of a female Lutzomyia diabolica collected at
Garner State Park, Uvalde County, Texas, June 1982 (dorsal
aspect; magn. x 1704).



;,, : ,i"









IOw









Scanning electron micrograph of mite (Eustigmaeus sp.) found
on the abdomen of a female Lutzomyia diabolica collected at
Garner State Park, Uvalde County, Texas. June 1982 (ventral
aspect; magn. x 1700).





-71-


collections, sticky traps, Disney traps) and are of little value

elsewhere; others have broader utility (light traps) and can be used

regardless of the availability of suitable habitat. From the data in

Table 2-1 it appears that the latrines at Garner State Park and at

Fawcett Boy Scout Camp were universally attractive structures. All

species taken at these two sites were taken in resting collections in

the latrines. In other localities, where resting sites were not

discovered, even after extensive searching, light traps proved most

effective. Biting collections were effective as a selective means of

sampling anthropophilic species at Garner State Park, and probably

would have been useful at other sites as well. Baiting of CDC light

traps with dry ice also selected for man biters and was the only way

Lu. diabolica were collected at the D'Hanis site.

Due to the success of collections in latrine resting stations at

Garner State Park and Fawcett Boy Scout Camp, other methods such as

light trapping and bait trapping were not routinely used.

Most sampling methods collected predominantly females, which is

consistent with findings of other authors (Young, 1972; Endris, 1982).

No males were taken in biting collections, although some would be

expected to mate with females on the host as she feeds. The figures in

Table 2-2 should not be interpreted to represent the true sex ratios

in the natural population. Most trapping methods, especially those

using light or bait attractants, select for females. This is

especially true if the female's movement is part of a hunting strategy

(Killick-Kendrick and Rioux, 1981). Chaniotis et al. (1972) stated

that the true sex ratio could only be properly assessed by studying





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populations from each habitat using a variety of sampling methods

(especially those that do not select one sex over the other).

Lutzomyia diabolica. This species was discovered in Uvalde, Texas,

in 1915 and was said to attack man freely (Parman, 1919). It was

subsequently collected by other workers in areas limited almost

exclusively to the south central part of the state (Young and Perkins,

1984; Fig. 2-29). Disney (1968) held that Lu. diabolica was

conspecific with Lu. cruciate, a species widely distributed through

Central America, and a suspected vector of leishmaniasis in Belize

(Williams, 1966a, 1966b). Young and Perkins (1984) showed that Lu.

diabolica from Texas represents a distinct species, separate from Lu.

cruciata.

Parman (1919) found Lu. diabolica hiding in dark places during the

day, one or two specimens to a place. He offered few clues as to

where these "dark hiding places" were, except to say there was evidence

that the breeding places are in neglected poultry houses, since the

flies were observed in abundance around such places in the late

twilight hours. Lindquist (1936) captured male and female Lu.

diabolica on walls and curtains in lighted rooms on first and second

floors of dwellings. Endris (1982) collected Lu. diabolica resting in

latrines at Garner State Park. All resting collections of Lu.

diabolica have been made in or on human dwellings or outbuildings. To

date, a "natural" resting habitat of this species has not been found,

indicating that it may be a peridomestic sand fly. This underscores

the need for future searches to locate natural resting places and

pinpoint breeding sites. Resting Lu. diabolica were collected in

latrines only in the upland portions of Garner State Park, an





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S= autochthonous human case of
cutaneous leishmaniasis
E= known distribution of
Lutzomvia diabolica


Figure 2-29.


Known geographical distribution of the sand fly Lutzomyia
diabolica in Texas.




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observation consistent with Parman's statement (1919) that the sand

fly was found in Uvalde in the more elevated parts of the city.

Perhaps this is a clue as to where other resting sites are to be

found.

Parman (1919) reported that the earliest authentic record of

appearance of Lu. diabolica in Uvalde was 3 September and the latest

was 24 November. Lindquist (1936) reported collections between 3 May

and 16 November from Uvalde. Although no collecting was done in

Uvalde during this study, Lu. diabolica were taken in resting and

biting collections at nearby Garner State Park (50 km north of Uvalde)

as early as 4 June and in light trap collections at the D'Hanis site

(50 km east of Uvalde) as early as 5 May and as late as 4 December.

Specimens of this species were taken at Garner State Park in human

biting collections by Young (1972) as early as May 17. Adults are

probably present in these locations throughout the frost free season,

with populations increasing over the summer months and reaching a peak

in the fall when they reach a nuisance level and are noticed by the

public. Parman (1919) apparently based appearance records on biting

activity. If so, this is consistent with the idea of a gradually

expanding population over the summer months. Surveys throughout the

year in and around Uvalde will be essential to confirm this idea.

Parman (1919) believed that Lu. diabolica never venture out of

hiding until well after sunset and never attack earlier than one hour

after sunset. Wilkerson (pers. comm., 1984) collected a female Lu.

diabolica biting him at 1400 hrs in full sunlight in mid July at

Canyon Lake, Comal County, Texas. During the 1982 research trip one

female was collected while biting the author's son at 1400 hrs in full

sunlight (temperature 380C, RH approximately 60%). These may be





-75-


isolated instances, but they indicate that perhaps the daily feeding

activity of Lu. diabolica is not as restricted as previously reported.

Lindquist (1936) reported the feeding period to be from 2000 hrs to

2400 hrs and Endris (1982) reported Lu. diabolica feeding during all

hours of darkness. Williams (1966b), in studies of biting rhythms of

ten anthropophilic sand flies in Belize, found the greatest period of

activity was between 0600 and 0659 hrs. After this small burst of

activity, the number of flies decreased. He said that the flies were

least active between 1400 and 1459 hrs (the hottest part of the day),

but that biting activity increased gradually from 1500 hrs onward.

Flies were not collected in appreciable numbers, however, until dusk

(1800-1859 hrs). He noted that numbers increased still further during

the early hours of darkness, reaching the peak of greatest activity

between 2100 and 2159 hrs. Thereafter, biting density diminished.

Consistent with these later reports, the peak feeding period of Lu.

diabolica in June 1982 was observed between one hour after sunset and

midnight (2130 to 2400 hrs). Killick-Kendrick and Rioux (1981)

described similar activity for Phlebotomus ariasi Tonnoir in the

Cevennes, France, and said it was probably triggered, at least

partially, by the rise in relative humidity as the temperature falls

at sunset. Parman (1919) said Lu. diabolica would not bite in total

darkness or in full moonlight. Although most biting collections in

June 1982 were taken in the presence of artificial light, sand flies

were also collected in lesser numbers while biting in total darkness,

having no apparent difficulty finding their host. They were also

observed biting outdoors in full moonlight.





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Parman (1919) noted that the abundance of Lu. diabolica in the

fall was extremely variable, ranging from only one specimen attacking

in several nights, to 25 to 30 attacking each night for a short

period. This variability in numbers and peak time of attack was

observed both in the spring and fall during this study, and appears to

be strongly influenced by ambient temperatures, relative humidity and

air movement. Sand flies were observed in greatest numbers on hot

humid nights (270C or above and 70% or greater RH) with little or no

air movement. Not surprisingly, air movement seemed to affect their

presence the most, as numbers decreased dramatically with an increase

in wind velocity above 8 kph (5 mph). Foraging sorties or wave

attacks such as described previously did not occur except on hot,

humid and windless nights. Killick-Kendrick and Rioux (1981) reported

the peak biting activity of P. ariasi may be delayed because of wind

or suppressed by storms or a fall in temperature below about 160C.

Some workers have studied the effects of wind on the movement and

activity of sand flies but probably overestimated the wind speed at

which activity ceased (Killick-Kendrick and Rioux, 1981).

Blood-fed females have greater difficulty flying than unfed

females and apparently rest in protected sites for up to 24 hrs to

allow for diuresis and partial digestion of the blood-meal. This

accounts for the large number (31 to 46%) of blood-fed females taken

in latrine resting stations.

Both male and female Lu. diabolica are assumed to disperse

from their breeding sites, possibly in search of sugar or a blood-

meal. Their attraction to light draws them close to human dwellings

where other shorter range attractants, such as exhaled CO2





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or body warmth, may aid them in finding a blood meal. This may also

be a genetically selected, triggered-sequence response, i.e., lights =

people = food. For whatever the reason, their response to light

contributes to the success of light-trapping, and several workers have

collected Lu. diabolica by this method (Young and Perkins, 1984).

Indeed the lighted latrines at Garner State Park and Fawcett Boy Scout

Camp functioned as giant light traps. The exposed positions of the

positive latrines was somehow important, since those at lower

elevations in more protected positions yielded nothing. Perhaps they

were more visible and attracted flies from considerable distances. It

may also be that the sand flies were seeking something other than a

blood meal, such as shelter. As was the case at the farm in D'Hanis,

CDC light traps may not be bright enough to attract sand flies and

must be augmented with CO2 (dry ice). The brighter lights of the

Shannon trap and the New Jersey light traps were apparently sufficient

to attract foraging flies.

The distance traveled by a sand fly to its host may depend upon

the habitat and the species. Estimates by other workers of distance

traveled to light ranges from 200 m in a neotropical forest (Chaniotis

et al., 1974), to as far as 2300 m in open habitat (Killick-Kendrick

and Rioux, 1981). These authors also suggested that the movement of

females is a hunting strategy and hence the difference in dispersal

distance between males and females. Since Lu. diabolica occurs in a

rather open habitat, it is likely that they disperse a considerable

distance (1000 m or more) from the unknown breeding site. Males

probably have a more limited flight range. The greater number of

males found in resting collections at Fawcett Boy Scout Camp may




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have been an indication that the breeding site was close by. Mark-

release and recapture experiments will be necessary to determine the

actual extent of dispersal from the breeding site.

Lutzomyia anthophora. This species was first collected

while feeding on rabbits in Uvalde, Texas, in the type locality of Lu.

diabolica (Addis, 1945a). Easton (1968) collected them in Malaise

traps in Kinney and Presidio Counties, Texas. Young (1972) found Lu.

anthophora in the nest of the plains woodrat (Neotoma micropus) in San

Antonio, along the Rio Grande near Brownsville, Texas, and at Welder

Wildlife Refuge near Sinton, Texas. Endris (1982) also collected Lu.

anthophora from woodrat nests near Brownsville, Texas. Finding this

species at Garner State Park, Fawcett Boy Scout Camp, and D'Hanis

expands its known geographic distribution (Fig. 2-30). The close

association of Lu. anthophora with the woodrat, and the finding of

recently engorged females in the soft inner nest of the main Neotoma

den strongly suggest the preferred host to be the woodrat (Young,

1972). Endris (1982) fed females of this species on the following

anesthetized animals: woodrat, white footed mouse (Peromyscus

leucopus), Syrian hamster (Mesocricetus auretus), grey squirrel

(Sciurus carolinensis), white mouse (Mus musculus), guinea pig (Cavia

porcellus), domestic rabbit (Oryctolagus cuniculus), and opossum

(Didelphis marsupialus). Rodent reservoirs of leishmaniasis in the

Neotropics, but not in Texas, have been reported by several authors

(Bray, 1974a; Lainson and Shaw, 1979). Still, this possibility must

not be overlooked and should be investigated in future studies. Lu.

anthophora is not known to be anthropophilic, but Perkins (pers.





-79-


comm., 1984) reported that females in flourishing laboratory colonies

will occasionally bite humans.

Lutzomyia texana. Lutzomyia texana was described from

specimens collected in the nest of the leaf-cutting ant, Atta texana

(Buckley), in San Antonio, Texas. This species has been collected in

light traps at several sites throughout south central Texas (Fig.

2-31). Young (1972) reported that Lu. texana frequently inhabit

mammal burrows, especially those dug by armadillos, and that they were

collected from such places throughout most of the year. Efforts

to feed specimens on a variety of hosts in June 1982 and September

were unsuccessful. Young (pers. comm., 1984) believes that armadillos

may be the principal hosts. Lainson et al. (1979) isolated Leishmania

from armadillos in Brazil. There is no evidence of armadillo

reservoirs of cutaneous leishmaniasis in Texas; however, the

matter has never been investigated and should be given further

attention.

Lutzomyia vexator (Coquillet). Lutzomyia vexator is the most

widely distributed sand fly in the USA (Young and Perkins, 1984). It

was collected previously by Young (1972) at Garner State Park and

Fredricksburg, Texas, in light traps. These flies are reptile feeders

and are of no known medical or economic importance.

Lutzomyia new species. This new species, known from a single

female, raises to seven the number of sand fly species collected from

Texas. Future field surveys will be essential to collect more

specimens on which to base a valid description of the species and to

study its biology and host associations.





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*= autochonous human case I
of cutaneous leishmaniasis!
0= known distribution of
Lutzomyia anthophora


Figure 2-30.


Known geographical distribution of the sand fly
anthophora in Texas.


*= autochthonous human case
of cutaneous leishmaniasi
0= known distribution of
Lutzomyia texana


Figure 2-31.


Known geographical distribution of the sand fly Lutzomyia
texana in Texas.


Lutzomyia




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Potential Vectors


Based on the results of this field study and the records of other

workers, it can be said that Lu. diabolica is an abundant, widely

distributed sand fly species in south central Texas. As the only

known anthropophilic species known in the state, with peridomestic

habits, and having a distribution that roughly coincides with areas of

Leishmania endemicity, it is strongly implicated as the probable

vector of human cutaneous lesishmaniasis in Texas (Fig. 2-29).

Further evidence to support this is provided by experiments in which

laboratory-fed Lu. diabolica were shown capable of transmitting

L. mexicana from infected to uninfected hamsters by bite. These

experiments are discussed in detail in Chapter 4.

Endris et al. (1984) transmitted L. mexicana from infected to

uninfected hamsters with laboratory-bred Lu. anthophora. This led

them to suggest that L. mexicana could be maintained in wild rodent

populations by a non-anthropophilic species, such as Lu. anthophora,

and secondarily transmitted to man by a sympatric anthropophilic sand

fly, such as Lu. diabolica, thus implicating Lu. anthophora as a

possible accomplice in the transmission of human cutaneous

leishmaniasis in Texas. A further possibility that can not be ruled

out is that Lu. texana may likewise be implicated as an accomplice

vector when knowledge of its hosts and feeding behavior are revealed.



Processing and Maintenance of Wild-Caught Sand Flies
and Recovery of Eggs


The advantage of recovering eggs from wild-caught females in the

field is clear. They are much easier to handle than adults and




-82-


without significant mortality, allowing for field transportation of

F1 genetic strains for laboratory studies.

It was found that gravid females could be transported long

distances in oviposition vials in a vehicle when they were protected

from excessive heat and provided moisture periodically. One batch of

about 40 adult females was hand carried 1,000 miles on an airplane

with no mortality.

Oviposition records (Table 2-3) indicate that most of the females

did not deposit all their eggs and that the number of eggs laid per

female was quite variable (1-84 in June 1982; 15-76 in September

1983). Less variability in number of eggs deposited was observed in

1982. The higher percentage of females depositing ova during the fall

1983 trip as compared to spring 1982 (Table 2-3) is probably a

reflection of improved handling techniques and "laboratory"

facilities, and cooler temperatures. During June, 1982, the

"laboratory" was a screened camping shelter in which temperatures

remained about ambient. Mean temperatures at Garner State Park and

surrounding areas during that month were about 280C with average daily

maximum temperatures of about 350C. Relative humidity ranged between

about 50 and 95%. Special care such as adding ice to the polystyrene

cooler and addition of water to the plaster in the vials kept the

specimens alive until oviposition. In September, 1983, ambient

temperatures were lower (mean, 250C; maximum, 320C; RH about 65%) and

the "laboratory" facility consisted of an air-conditioned room that

was maintained at approximately 240C.

The mean gross egg production (eggs laid plus eggs retained) of

captured females was about the same for both trips. The high





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percentage of females retaining eggs may simply reflect the stress of

captivity. The number of females retaining eggs in the laboratory

colony (after 13 generations) was much less and presumably

approximates what would be found in nature. Females that neither

deposited nor retained ova amounted to 3% of the catch in 1982 and 10%

in 1983. Whether this is evidence of possible gonotrophic

disassociation is only conjecture. Occurrence of the phenomenon to a

higher degree in the fall of the year is consistent with what has been

observed in other insects.

Differences in preoviposition interval, postcapture longevity, and

postoviposition longevity were also probably due to improved handling

techniques in the second year when it was cooler (Table 2-4).

Comparison of preoviposition longevity of females captured with and

without blood meals (Table 2-4) reveals no significant differences,

suggesting that females captured with blood meals had recently fed and

that blood-fed females do not linger at the latrine resting stations

more than a few hours.

At least 57% of the egg batches laid by wild-caught, blood-fed

females hatched. Since these females were not placed with males after

capture, it is obvious that at least this percentage of females were

inseminated prior to capture. Sperm were observed in the spermathecae

of many of the dissected females, but spermathecae were not always

visible in dissections.



Accessory Glands and Parity


According to Chapman (1971), accessory glands in most insects

arise from the genital chamber or vagina. Their function varies in





-84-


different insects, but they commonly produce a substance for attaching

the eggs to the substrate. This material may also serve as a

protective covering for the eggs. There is considerable confusion,

even controversy, in the literature over the value of accessory gland

examination as an indicator for determine parity in sand fly

populations. Adler and Theodor (1935) stated that it was possible to

distinguish between blood-fed and unfed females of the palearctic sand

fly, P. perniciosis Newstead, by examining the accessory glands since

granules never appeared in the accessory glands unless the female had

had a blood meal. A few days after a small blood meal they were full

of granules. They further stated that during egg laying most of the

granules were passed out with the eggs, but some granules remained and

could be seen in dissections. The presence of granules in the

accessory glands of female P. perniciosus with an empty alimentary

tract was the only morphological feature that distinguished parous

females from newly hatched ones. They also stated that the granules

were formed independent of copulation, depending only on a blood-meal

in P. perniciosus.

Adler and Theodor (1957) referred to the indicative value of the

accessory glands for sand flies in general. Garnham and Lewis (1959)

noted high proportions of dissected sand flies with granules in the

accessory glands and concluded that some of the flies might be

nullipars secreting granules. They suggested studying the value of

these organs as indicators of nulliparous flies in Belize. Lewis and

Minter (1960) examined ovaries and accessory glands of some tropical

African sand flies, in Kenya, and found that when the ovaries were

small, residual secretions in the accessory glands were useful in





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recognizing most parous females. Johnson and Hertig (1961) found that

some Panamanian sand flies secreted accessory gland granules before

taking blood and discharged them at various times afterwards. Adler

and Mayrink (1961) observed dark brown fluid in the accessory glands

of blood-fed Lu. longipalpis (Lutz and Neiva) and noted changes in

glands of five other species. Johnson et al. (1963) found that in

laboratory-reared Panamanian sand flies, granules may be present or

absent without regard to parous or nulliparous condition of the

female. They concluded that examination of the accessory glands does

not aid in determining whether females of Panamanian sand flies are

parous or nulliparous. Minter (1964) believed that the accessory

glands were useful in estimating parous rates in Kenya. Lewis (1965)

examined accessory glands of five species of Lutzomyia in Belize and

concluded that the glands were unreliable for indicating whether or

not a fly was parous. Chaniotis and Anderson (1968) studied

laboratory-bred females of three California species and found no

granules in accessory glands of 150 nullipars. Scorza et al. (1968),

on the other hand, concluded that accessory gland granules were

unreliable for recognizing parous females in Venezuela. Lewis et al.

(1970) reconsidered a statement made earlier by Lewis (1965) regarding

the indicative value of accessory glands by stating that the glands

are quite useful for recognizing parous females of many Old World and

some New World species. They reported that errors in interpretation

are caused by irregular secretions, loss of secretion, the effect of

blood-meals, and the parasite Monocystis (Ascocystis). Ward (1974)

found that 82.05% of laboratory-reared Lu. longipalpis developed

granules, but no eggs when kept for seven days without blood or sugar.

He questioned the value of accessory glands in determining parity in