Citation
Phlebotomine sand flies (Diptera: Psychodidae) and diffuse cutaneous leishmaniasis in the Dominican Republic

Material Information

Title:
Phlebotomine sand flies (Diptera: Psychodidae) and diffuse cutaneous leishmaniasis in the Dominican Republic
Creator:
Johnson, Richard Nicholas, 1956- ( Dissertant )
Butler, Jerry F. ( Thesis advisor )
Hall, Donald W. ( Reviewer )
Young, David G. ( Reviewer )
Forrester, Donald J. ( Reviewer )
Greiner, Ellis C. ( Reviewer )
Fry, Jack L. ( Degree grantor )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1984
Language:
English

Subjects

Subjects / Keywords:
Coffee industry ( jstor )
Eggs ( jstor )
Female animals ( jstor )
Groves ( jstor )
Infections ( jstor )
Larvae ( jstor )
Leishmaniasis ( jstor )
Parasites ( jstor )
Rodents ( jstor )
Species ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis, Ph.D.
Sand flies
City of Gainesville ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )
Spatial Coverage:
Dominican Republic

Notes

Abstract:
A survey for phlebotomine sand flies (Diptera:Psycho- didae) in the Dominican Republic revealed that Lutzomyia cayennensis hispaniolae (Fairchild and Trapido) was widely distributed and fairly common. Lutzomyia christophei (Fairchild and Trapido) was more limited in geographic distribution. Specimens of the latter species were obtained by light traps, flight traps, and aspirator collection from human bait and resting sites. Laboratory colonies of both species were established and life-cycle data were obtained. Lutzomyia cayennensis females readily fed on Anolis lizards. Female Lu. christophei readily fed on rodents and were capable of experimentally transmitting a Dominican strain (Isabel-WR336) of Leishmania to BALB/c mice seven to ten days after biting infected mice. Development of the parasite occurred in the anterior midgut in both Lu. christophei and Lu. anthophora (Addis), a species that was also experimentally infected. The course of development in the sand fly was observed by dissecting 15 infected Lu. anthophora on days 1-7 post-feeding. Development in this species was parallel to that observed in the 17 Lu. christophei. Promastigotes from flies four and five days post-feeding were infective to hamsters, as determined by xenodiagnosis with sand flies and spleen culture. In culture medium, Leishmania-Isabel strain grew at a much slower rate than either of two strains of L. mexicana. Hamsters and TRC mice, experimentally inoculated from culture, showed no outward sign of infection until at least 2.5 months after inoculation with the Isabel strain. In the Dominican Republic, 10 of the 21 known case sites were visited. Coffee and cacao groves were characteristic of these sites. Two female Lu. christophei were captured while biting a patient. Four species of mammals (170 specimens) were trapped from five of the case sites and examined for leishmaniasis using various methods. None was found to be infected, though 4 of 44 Rattus rattus from one site were seropositive (1:16), as determined by indirect fluorescent antibody test. Lutzomyia christophei is most probably the vector of diffuse cutaneous leishmaniasis in the Dominican Republic. The identity of the reservoir remains unknown, but R. rattus is the most likely suspect.
Thesis:
Thesis (Ph.D.)--University of Florida, 1984.
Bibliography:
Include bibliographic references (leaves 119-125).
General Note:
Vita.

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University of Florida
Holding Location:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
ACN7945 ( LTUF )
11908433 ( OCLC )
0030497226 ( ALEPH )

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PHLEBOTOMINE SAND FLIES (DIPTERA:PSYCHODIDAE)
AND DIFFUSE CUTANEOUS LEISHMANIASIS
IN THE DOMINICAN REPUBLIC
















BY



RICHARD N. JOHNSON


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


I





































To my parents,

Robert and Mary Johnson


















ACKNOWLEDGEMENTS


This research could not have been done without the

advice, encouragement, and assistance of many people to whom

I am greatly indebted. My supervisory committee was helpful

in guiding my course of study: Dr. Jerry F. Butler, chair-

man; Drs. Donald W. Hall, Donald J. Forrester, and Ellis C.

Greiner; and especially Dr. David G. Young. Dr. Bryce C.

Walton, World Health Organization, first suggested the

project and was instrumental in securing financial support

under WHO grant #810314. Other support was generously given

by the Steffan Brown Foundation and the University of

Florida.

I am also indebted to the personnel of the Dominican

Dermatology Institute, including Dr. Huberto Bogaert-Diaz

(Director), Dr. Denis de Martinez, Lic. Margarita de

Quinones, Lic. Marvis Lebron, Tomas Castro, and particularly

Francisco Castillo, for his friendship and assistance. Gulf

and Western Americas Corporation graciously provided facili-

ties at the Pedro Sanchez field station. The staff and

their families at this ranch provided the friendship that

turned it into a home for more than a year. Dr. Rodrigo

Zeledon and Juan Murillo, Instituto Costarricense de

Investigation y Ensenanza en Nutricion y Salud, were of


L










great help during their brief visit to the Dominican

Republic in 1983.

I wish to thank Dr. Eskild Petersen, University of

Arizona, for his advice and assistance. I am indebted to

Dr. Sam Telford for the careful instruction on the identifi-

cation of lizard parasites. Dr. Robert Woodruff, Florida

Division of Plant Industry, provided valuable advice which

facilitated working in the Dominican Republic.

Personnel at Walter Reed Army Institute of Research

were of much help in realizing the goals of the project;

these included MAJ Peter Perkins, Dr. Edgar Rowton, Spec. 4

Pedro Quintero, LTC Donald Roberts (Entomology): CPT Patrick

McGreevy, LTC Jonathan Berman, Dr. Eileen Franke (Experi-

mental Therapeutics).

Dr. Charles Woods of the Florida State Museum provided

information on the Republic and its mammalian fauna. Rick

Sullivan, who concurrently resided in the country, provided

extra traps, friendship, and, at times, mutual commissera-

tion. Dr. Steven Zam was instructive in laboratory cultur-

ing of Leishmania. I thank Ms. Diana Simon and Mrs. Debra

Boyd who handled many of the concerns that arose during my

absences from Gainesville. Ms. Edna Mitchell helped in

laboratory rodent and sand fly maintenance. Drs. G.B.

Fairchild and R.C. Wilkerson gave freely of their knowledge

of Latin America. Drs. Richard Endris, Peter Perkins,

Andrew Beck, Mr. Eric Milstrey, MAJ Phillip Lawyer, and CPT

Terry Klein offered their advice, assistance, and friendship










throughout varying times of acquaintance. Finally, I

gratefully acknowledge the encouragement and assistance

provided by my family and other friends who have been

supportive for the past 28 years.















TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS . .

LIST OF TABLES . . .

LIST OF FIGURES . .

ABSTRACT . . .

CHAPTER

1 INTRODUCTION . .


Literature Review . . .
Geography of the Dominican Republic
Current Status of Diffuse Cutaneous
Leishmaniasis in the Dominican
Republic . . .
Study Site Selection . .
Study Site Descriptions . .
Objectives . . .


. iii

. viii

. x


xii


* 12


* 13
. 12


. 13
* 20
* 22
* 27


2 SURVEY FOR, AND COLONIZATION OF LUTZOMYIA
CAYENNENSIS HISPANIOLAE AND LUTZOMYIA
CHRISTOPHEI . . . .


Introduction . .
Methods and Materials .

Field Studies .
Laboratory Rearing .
Sand Fly Dissections

Results . . .

Field Studies .
Laboratory Studies .
Sand Fly Dissections .

Discussion . .


. . 30
S . 40
S . 46

S . 47


. 65









Chapter Page

3 GROWTH OF LEISHMANIA-ISABEL STRAIN IN CULTURE
MEDIUM, LABORATORY RODENTS, AND SAND
FLIES . . . . 73

Introduction . . . 73
Methods and Materials . . .. 74

Comparison of the Growth of Three
Strains of Leishmania . 74
Growth of Leishmania-Isabel Strain
in Laboratory Rodents . .. 75
Growth of Leishmania-Isabel Strain
in Sand Fly . . 77
Transmission of Leishmania-Isabel
Strain by Lutzomyia christophei 78

Results . . . ... 79

Comparison of the Growth of Three
Strains of Leishmania . 79
Growth of Leishmania-Isabel Strain
in Laboratory Rodents. . 81
Growth of Leishman-Isabel Strain
in the Sand Fly . .. 84
Transmission of Leishmania-Isabel
Strain by Lutzomyia christophei 89

Discussion . . . .. 90

4 SURVEY FOR RESERVOIR HOSTS OF HUMAN
LEISHMANIASIS IN THE DOMINICAN REPUBLIC 96

Introduction . . . 96
Methods and Materials . . .. 97
Results . . . 101
Discussion . . . .. 105

5 SUMMARY . . . . 111

APPENDICES
1 DOMINICAN LEISHMANIASIS PATIENT PARTIAL CASE
HISTORIES . . . .. 114

2 COLLECTION SITES AND DATES FOR LUTZOMYIA
CAYENNESIS HISPANIOLAE IN THE DOMINICAN
REPUBLIC, MAY 1981 AUAGUST 1983 ... .116

3 COLLECTION SITES AND DATES FOR LUTZOMYIA
CHRISTOPHEI IN THE DOMINICAN REPUBLIC,
MAY 1981 AUGUST 1983 . . .. 118

REFERENCES . . . .. . 119


BIOGRAPHICAL SKETCH . . . .


126


















LIST OF TABLES



Table Page

1-1 Sand fly species (Lutzomyia) which are
known or suspected vectors of leishmaniasis
in the New World, by country . . 5

1-2 Other mammalian hosts of Leishmania strains
which cause human leishmaniasis in the New
World, by country . . . 10

2-1 Location and dates for flight trap samples
in the Dominican Republic . ... 35

2-2 Location, trap type, and dates for CDC traps
in the Dominican Republic . ... 36

2-3 Sites and dates for Disney traps used for
phlebotomine sand fly sampling in the
Dominican Republic . . ... 39

2-4 Collection sites and dates for soil samples
examined for the presence of sand fly
larvae . . . . ... .41

2-5 Mean duration ( S.D.) in days of immature
stages of Lu. cayennensis hispaniolae at
three temperature regimes, according to sex
of sand fly . . . ... 57

2-6 Mean duration ( S.D.) in days of immature
stages of Lu. christophei at two temperature
regimes, according to sex of sand fly 63

2-7 Sites of collection for female Lu.
cayennensis dissected . . ... 66

3-1 Method of inoculation and number of
promastigotes used to infect laboratory
rodents with Leishmania-Isabel strain 76


viii










Table Page

3-2 Number of animals examined determined to be
infected with Leishmania-Isabel strain via
different isolation methods . ... 82

3-3 The course of development of Leishmania-
Isabel strain in the sand fly, Lutzomyia
anthophora, based on daily dissections,
Days 1 to 7 post feeding . ... 85

4-1 Mammal specimens collected at six
leishmaniasis case sites in the Dominican
Republic . . . . 99

4-2 Examination techniques used for mammals
collected during survey for reservoir hosts
of leishmaniasis in the Dominican Republic,
October 1981 to August 1983 . ... .104

















LIST OF FIGURES


Figure Page

1-1 The Caribbean region, showing the location
of the Dominican Republic, on the island of
Hispaniola . . . ... 15

1-2 The geographic regions of the Dominican
Republic . . . ... .16

1-3 Case sites for patients with diffuse
cutaneous leishmaniasis (DCL) in the
Dominican Republic . . ... 18

1-4 Typical terrain in the Cordillera Oriental,
Dominican Republic . . ... 19

1-5 A young Dominican DCL patient with healed
lesions on wrist and upper arm ...... 19

2-1 Field collecting equipment and rearing
containers for phlebotomine sand flies 31

2-2 Sand fly feeding cage a modified aquarium
with plaster of Paris bottom and back 32

2-3 The author in front of a flight trap in a
cacao grove at Altos de Peguero, El Seibo
Prov. . . . . ... 34

2-4 A CDC trap and modified traps . .. 34

2-5 A Disney trap, used for collecting rodent-
feeding sand flies ............ 38

2-6 Schematic diagram of laboratory techniques
for rearing of phlebotomine sand flies 43

2-7 A modified microtiter plate for rearing
individual sand fly larvae . .... 44

2-8 Collection sites for Lu. cayennensis
hispaniolae and Lu. christophei in the
Dominican Republic May 1981-August 1983 49










Figure Page

2-9 Weekly sample populations of Lu.
cayennensis at two study sites in the
Dominican Republic . . .. 51

2-10 Eclosion time, in days after oviposition,
for Lu. cayennensis reared under constant
conditions . . . ... 58

2-11 Eclosion time, in days after oviposition,
for Lu. cayennensis reared under ambient
conditions, August-September . ... 58

2-12 Eclosion time, in days after oviposition,
for Lu. cayennensis reared under ambient
conditions, January-February . ... 59

2-13 Female Lu. christophei feeding on BALB/c
mouse . . . . . 61

2-14 Eclosion time, in days after oviposition,
for F Lu. christophei reared under constant
condi ions . . . . 64

2-15 Eclosion time, in days after oviposition,
for F and F Lu. christophei reared under
constant conditions . . .. 64

3-1 Daily estimated mean population of three
strains of Leishmania grown in Schneider's
medium, at 250C . . ... 80

3-2 Leishmania-Isabel strain amastigotes in
spleen impression smear stained with Giemsa
(1000X) . . . ... 83

3-3 Promastigotes of Leishmania-Isabel strain
from the anterior midgut of the sand fly,
Lu. anthophora (1000X) . . ... 87

3-4 The course of Leishmania-Isabel strain
infection in the sand fly, Lutzomyia
anthopora . . . . 88

4-1 Sherman and Tomahawk live traps for small
mammals . . . ... .98

4-2 The three species of rodents trapped during
the survey . . . ... 102

















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


PHLEBOTOMINE SAND FLIES (DIPTERA:PSYCHODTDAE
AND DIFFUSE CUTANEOUS LEISHMANTASIS
IN THE DOMINICAN REPUBLIC


By

Richard N. Johnson

August 1984


Chairman: Dr. Jerry F. Butler
Major Department: Entomology and Nematology

A survey for phlebotomine sand flies (Diptera:Psycho-

didae) in the Dominican Republic revealed that Lutzomyia

cayennensis hispaniolae (Fairchild and Trapido) was widely

distributed and fairly common. Lutzomyia christophei

(Fairchild and Trapido) was more limited in geographic

distribution. Specimens of the latter species were obtained

by light traps, flight traps, and aspirator collection from

human bait and resting sites. Laboratory colonies of both

species were established and life-cycle data were obtained.

Lutzomyia cayennensis females readily fed on Anolis lizards.

Female Lu. christophei readily fed on rodents and were

capable of experimentally transmitting a Dominican strain

(Isabel-WR336) of Leishmania to BALB/c mice seven to

ten days after biting infected mice. Development of the


xii










parasite occurred in the anterior midgut in both Lu.

christophei and Lu. anthophora (Addis), a species that was

also experimentally infected. The course of development in

the sand fly was observed by dissecting 15 infected Lu.

anthophora on days 1-7 post-feeding. Development in this

species was parallel to that observed in the 17 Lu.

christophei. Promastigotes from flies four and five days

post-feeding were infective to hamsters, as determined by

xenodiagnosis with sand flies and spleen culture.

In culture medium, Leishmania-Isabel strain grew at a

much slower rate than either of two strains of L. mexicana.

Hamsters and TRC mice, experimentally inoculated from

culture, showed no outward sign of infection until at least

2.5 months after inoculation with the Isabel strain.

In the Dominican Republic, 10 of the 21 known case

sites were visited. Coffee and cacao groves were charac-

teristic of these sites. Two female Lu. christophei were

captured while biting a patient. Four species of mammals

(170 specimens) were trapped from five of the case sites and

examined for leishmaniasis using various methods. None was

found to be infected, though 4 of 44 Rattus rattus from one

site were seropositive (1:16), as determined by indirect

fluorescent antibody test.

Lutzomyia christophei is most probably the vector of

diffuse cutaneous leishmaniasis in the Dominican Republic.

The identity of the reservoir remains unknown, but R. rattus

is the most likely suspect.


xiii

















CHAPTER 1

INTRODUCTION



Literature Review

Phlebotomine sand flies* are biting members of the

dipteran family Psychodidae (Quate and Vockeroth, 1981).

They are suspected or confirmed vectors of various parasitic

agents including phleboviruses, such as sand fly fever

virus; bartonellosis (Adler and Theodor, 1957); saurian

malaria (Ayala and Lee, 1970); trypanosomes of amphibians,

lizards (Anderson and Ayala, 1968), and mammals (McConnell

and Correa, 1964); and leishmaniasis (Bray, 1974).

Leishmaniasis is a complex of diseases which is caused

by Leishmania spp. occurring in many parts of the world. In

1981, the World Health Organization estimated that there

were 400,000 new cases of leishmaniasis in the world,

annually. The disease occurs in the Americas, Europe,

Africa, and Asia. It has been considered the second most

important protozoan disease of man after malaria (Anonymous,

1981). The only known biological vectors are phlebotomine

sand flies (Bray, 1974). In the Americas, leishmaniasis




* In this presentation, sand fly will refer to members of
Diptera: Psychodidae: Phlebotominae.










occurs mainly among persons living in rural or forested

areas (Herrer et al., 1966). Leishmaniasis appears in three

basic clinical forms-visceral, mucocutaneous, and cutaneous.

In 1948, a subform of cutaneous leishmaniasis was

reported independently in Venezuela (Convit and Lapenta,

1948) and in Bolivia (Barrientos, 1948). At first, it was

considered a new form of the disease because of its charac-

teristic features (Convit et al., 1962; Convit and Kerdel-

Vegas, 1965). Bryceson (1969, p. 709) summarizes these

features as follows:

1. There is an initial lesion which spreads

locally, and from which the disease dissem-

inates to other parts of the skin, often

involving large areas.

2. The lesions are nodules and do not ulcerate.

3. There is a superabundance of parasites in the

lesion.

4. The histology is characteristic in that

macrophages full of amastigotes predominate

in the lesion.

5. Internal organs are not invaded and there is

no history of visceral leishmaniasis.

6. The leishmanin (Montenegro) test is negative.

7. The disease progresses slowly and becomes

chronic.

8. Treatment with antimony produces only slight

and temporary improvement.










By these criteria, other cases have been reported from

the USA in Texas (Simpson et al., 1968), Brazil, Ecuador,

Mexico, Ethiopia, and Tanzania (Bryceson, 1969). The

appearance of the lesions gave rise to the name diffuse (or

disseminated) cutaneous leishmaniasis (DCL). The causative

agent of the Venezuelan cases was first named Leishmania

pifanoi (Medina and Romero, 1962). Areas where cases

occurred were endemic for cutaneous leishmaniasis, but not

the visceral disease (Convit and Kerdel-Vegas, 1965).

Further work determined that rather than being a new para-

site, DCL was the result of a deficiency in the host's

cell-mediated immunity (Bryceson, 1970b; Convit et al.,

1971). The disease is caused by the same species of

Leishmania that causes ulcerative cutaneous leishmaniasis in

the same area, e.g., L. aethiopica in Ethiopia (Lemma et

al., 1970; Bray et al., 1973) and L. mexicana mexicana, L.

m. amazonensis, and L. m. pifanoi in Central and South

America (Lainson and Shaw, 1978).

In 1975, a new focus of DCL was reported in the Domin-

ican Republic (Bogaert-Diaz et al., 1975). Three humans,

siblings, were found infected. There have been 22 addi-

tional cases, none of which had ulcerating lesions, sup-

porting the concept that DCL is determined both by parasite

characteristics and a defect in host immunocompetence

(Walton, pers. comm.). Studies of some of the patients from

the Dominican Republic showed that this defect is a specific










inhibition of lymphocyte-proliferation responses by adherent

suppressor T-cells, which has a genetic basis (Petersen et

al., 1982). Many of the Dominican DCL patients have been

symptomatically cured using hot (450C) water treatment

(Neva, pers. comm.). The Leishmania causing DCL in the

Dominican Republic remains unnamed, but it appears to be

different from strains in the L. braziliensis complex and in

the L. mexicana complex based on enzyme electrophoretic

mobility assays (Kreutzer et al., 1983), excreted factor

serotype, growth in artificial media, and infectivity and

pathogenicity in laboratory rodents (Schnur et al., 1983).

Although leishmanaisis may be mechanically transmitted

under lab conditions by Stomoxys calcitrans, stable flies

(Lainson and Southgate, 1965), Rhipicephalus sanguineus,

brown dogs ticks (Sherlock, 1964), and Glossina morsitans,

tsetse flies (Lightner and Roberts, 1984), sand flies are

the only known biological vectors (Lainson and Shaw, 1978).

In the New World, 21 species of sand flies have been

reported as natural hosts of Leishmania spp. infecting man,

but only 6 species have been determined, at present, to be

natural vectors (Table 1-1).

Experimentally, New World Leishmania are capable of

infecting a number of sand fly species, most of which are

then capable of transmitting the infection to a susceptible

mammalian species (Killick-Kendrick, 1979). The amastigote

stage is parasitic in vertebrate macrophages (Bray, 1974).










Table 1-1. Sand fly species (Lutzomyia) which are known or
suspected vectors of leishmaniasis in the New
World, by country.




Suspected or Proven
Country Lutzomyia Vectors Leishmania Strain



Belize olmeca2 L.m.m.



Bolivia longipalpis3 L.d.c.



Brazil longipalpis L.d.c.

amazonensis L.b.b.

intermedia2 L.b.b.
.3
migonei L.b.b.

paraensis3 L.b.b.
.3
pessoai L.b.b.

wellcomei2 L.b.b.
.3
whitmani L.b.b.

anduzei3 L.b. .

umbratilis2 L.b.q.

flaviscutellata3 L.m.



Colombia longipalpis3 L.d.c.

trapidoi3 L.b.











Table 1-1. Continued


Suspected or Proven
Country Lutzomyia Vectors Leishmania Strain


Costa Rica





El Salvador



French Guiana



Guatemala





Honduras



Mexico





Nicaragua


Panama


shannoni3

ylephiletor3



longipalpis3



3
umbratilis



longipalpis

olmeca



longipalpis3



longipalpis3

olmeca2



longipalpis



gomezi

panamensis3

trapidoi2

ylephiletor3


L.b.

L.b.


L.d.c.


L.d.c.

L.m.



L.d.c.


L.d.c.

L.m.


L.d.c.




L.b.P.

L.b.p.
L.b.p.

L.b.E.










Table 1-1. Continued


Suspected or Proven
Country Lutzomyia Vectors Leishmania Strain



Paraguay longipalpis L.d.c.



Peru peruensis3 L.p.

verrucarum L.p.



Surinam umbratilis L.b._.



USA diabolica L.m.



Venezuela longipalpis3 L.d.c.

flaviscutellata3 L.m.a.

townsendi3 L.m.q.


Source: World Health Organization,


1984, pp.


Leishmania strains: L.m.m. = L. mexicana mexicana,
L.d.c. = L. donovani chagasi, L.b.b. = L. braziliensis
braziliensis, L.b._. = L.b. guyanensis, L.b.2. =
panamensis, L.p. = L. peruviana, L.m.a. = L.m.
amazonensis, L.m.q. = L.m. garnhami.
2
Proven vector.

Suspected vector.


56-61.









Following ingestion by the sand fly, a telmophagic feeder,

the parasite exsheathes its flagellum to become the promas-

tigote, which multiplies initially in the hind or midgut,

depending on the parasite species. Members of the

L. braziliensis complex (Section Peripylaria) develop in the

posterior midgut and anterior hindgut before moving anter-

iorly to effect transmission. Leishmania donovani and

subspecies in the L. mexicana complex (Section Suprapylaria)

multiply initially in the anterior midgut (Killick-Kendrick,

1979). Transmission to a vertebrete occurs during the sand

fly bite, but the actual mechanism is unknown (Killick-

Kendrick, 1978).

Two extant species of sand flies are known from the

Dominican Republic (Fairchild and Trapido, 1950). Two fos-

sil species have been discovered recently in Dominican amber

(Johnson and Young, in preparation), reported to be from the

Oligocene Period, 40 to 60 million years old (Sanderson and

Farr, 1960). Prior to this study, practically nothing was

known abut the distribution or biology of the living spe-

cies. One of these, Lutzomyia christophei (Fairchild and

Trapido, 1950), belongs to the Lu. verrucarum species group

(Lewis, 1968), which also contains a number of man-biting

species (Young, 1979) and at least one vector of leishmania-

sis in Peru (Lainson and Shaw, 1979). Contrary to Lainson's

(1983) statement, nothing was known about the feeding habits

of Lu. christophei, prior to the author's study. The other

species, Lu. cayennensis hispaniolae (Fairchild and Trapido,










1950), is an endemic subspecies of a species that occurs

from Mexico south to Ecuador and French Guiana (Young,

1979). This species is known to feed on poikilothermic

vertebrates, though there is one report of females feeding

on bats in Venezuela (Deane et al., 1978). The specific

feeding habits of Lu. c. hispaniolae were not known prior to

this study.

Laboratory rearing of sand flies is a necessary part of

studying disease transmission, because it is an assured

method of obtaining uninfected flies. Colonization can also

lead to a better understanding of the life cycle of the

vector and parasites (Killick-Kendrick, 1978). Successful

rearing has been accomplished using various techniques and

several different formulations of larval food (Chaniotis,

1967; Endris et al., 1982; Gemetchu, 1976; Young et al.,

1981).

In the New World, leishmaniasis is a zoonotic disease,

with rodents serving as reservoir hosts for the L. mexicana

complex, canids for L. donovani, a variety of rodents,

procyonids, sloths, dogs, and primates for the L. brazilien-

sis complex (Table 1-2) (Lainson and Shaw, 1978). In the

Dominican Republic, ten species of rodents, two primates,

four to five insectivores, and four to six sloths are known

from fossils (Varona, 1974; Woods, pers. comm.). Some

survived until the time of Columbus (1492 AD) but only

two endemic terrestrial species exist today: an insecti-

vore, Solenodon paradoxus Brandt; and a capromyid rodent,











Table 1-2. Other Mammalian hosts of Leishmania strains
which cause human leishmaniasis in the New
World, by country.




Leishman a Nonhuman
Country Strain Mammalian Hosts


L.m.



L.d.c.



L.b.b.


L.m.


rodents: Heteromys, Nyctomys,

Ototylomys, Sigmodon(R)2

dog(R), foxes: Cerdocyon(R),

Lyalopex

(rodents: Akodon, Oryzomys,

Proechimys?)

sloth: Choelopus; anteater(R),


tamandua(R)

rodents: Proechimys(R),


Colombia

Costa Rica

French Guiana

Guatemala

Mexico


L.d.c.

L.b.

L.b.g.

L.m.

L.m.


(Dasyprocta, Heteromys, Neacomys,

Nectomys, Oryzomys, oppossums,

Marmosa, Caluromys, Metachirus?)

dog(R)

sloths: Bradypus(R), Choelopus

sloths: Choelopus

Ototylomys

rodents: Heteromys, Nyctomys,

Ototylomys, Sigmodon


Belize



Brazil










Table 1-2. Continued


Leishmania Nonhuman
Country Strain Mammalian Hosts


Panama L.b.p. sloths: Bradypus(R), Choelopus;

(primates: Aotus, Sanguinus;

procyonids: Bassaricyon, Nasua,

Potos?)

Peru L.R. (dog?)

Venezuela L.m. (rodents: Heteromys, Proechimys,

Zygodontomys?)



Source: World Health Organization, 1984, pp. 56-61.


Leishmania strains: L.m.m. = L. mexicana mexicana,
L.d.c. = L. donovani chagasi, L.b.b = L. braziliensis
braziliensis, L.b.g. = L.b. guyanensis, L.b.p. = L.b.
panamensis, L.p. = L. peruviana.

R = Proven reservoir.

(Mammal?)--animal found infected in nature, extent of
infection not determined.










Plagiodontia aedium Cuvier, both of which are extremely

uncommon. Both animals are secretive and are found in

relatively undisturbed habitat (Woods, 1981). Introduced

mammals have replaced the endemic mammals in most areas.

Three species of Old World rodents now occur throughout the

island in cities, tropical rain forests, and deserts. These

are Rattus rattus alexandrinus (Geoffroy), the roof or black

rat; R. norvegicus (Berkenhout), the Norway or brown rat;

and Mus musculus brevirostris Waterhouse, the house mouse.



Geography of the Dominican Republic

The Dominican Republic occupies the eastern two-thirds

of the island of Hispaniola, with Haiti occupying the

western side. The island is a member of the Greater

Antilles, lying between latitudes 17030' to 2000' with the

Atlantic Ocean to the north and the Caribbean Sea to the

south (Fig. 1-1). The Republic has an area of 43,230km2, of

which roughly two-thirds is mountainous. Geographically the

country is broken up into four regions, based, in part, on

the mountain ranges (sierras and cordilleras). The Eastern

region includes the Cordillera Oriental with the Llano

Oriental (Eastern Plains) to the south. Directly west lies

the Cibao with the Cordillera Septentrional to the north,

just inland from the coast, the Cibao Valley, and the

Cordillera Central to the west and south. To the northwest

lies the Linea Noroeste, bordering Haiti. El Sur is the

region to the south west of the Cordillera Central; it also









borders Haiti, and contains two smaller mountain chains

Sierra de Neiba to the north of Sierra de Baoruco

(Fig. 1-2). Most of the 6.2 million inhabitants (1982

census) live on the valley floors that separate the

cordilleras, or on the rolling Eastern Plains. About 60% of

the Dominicans live in rural and agricultural areas. The

majority are small land owners. Much of the land has been

cleared for agriculture. The main agricultural products

include sugar cane, rice, coffee, cacao, cotton, tobacco,

corn, and beef. The country has a tropical maritime

climate; in the lower elevations, temperature average 220 to

280C with a range during July 1981 to August 1982 at Pedro

Sanchez in El Seibo Province, of 160 to 330C. Annual

rainfall averages 1397 to 1524mm with a range of 508 to

2413mm, depending on location. The highest rainfall usually

occurs in the eastern portion where the rainy season lasts

from May to November (Anonymous, 1977; Sholdt and Manning,

1979).



Current Status of Diffuse Cutaneous Leishmaniasis
in the Dominican Republic

Since the discovery of the first three cases of DCL in

the Dominican Republic in 1974 (Bogaert-Diaz et al., 1975),

an additional 22 cases have been diagnosed (Bogaert-Diaz,

unpublished data). Precise life histories are known only

for a few of the patients who have not moved to different

localities during the presumed evolution of the disease.

Personal data, when recorded, often did not denote the exact




























Figure 1-1. The Caribbean region, showing the location of the Dominican Republic, on the
island of Hispaniola.


























Atlantic Ocean


Jamaica


0* D










































Figure 1-2. The geographic regions of the Dominican Republic
(Cordilleras and Sierras are mountain ranges).








location of residence. Many patients had moved from the

country to towns before being diagnosed. Others have lived

in several different areas for various periods of time;

thus, current place of residence may not have been where the

disease was contracted. Presumed site visitations and

interviews led to the confirmation of the probable locality

of infection for 15 patients. Brief descriptions of these

sites are given below and are shown as confirmed sites in

Figure 1-3; the remaining sites are shown as presumed. The

unknown incubation period and the relative benignancy of the

infection make it difficult to determine the probably date

of infection. Eight of the patients had had nodules or

plaques for four or more years before the disease was

diagnosed. At least 13 of the patients were under

four years of age when signs of the infection first

appeared, so epidemiological data must be based upon recol-

lections of parents or other relatives. The most out-

standing feature of the epidemiology of the disease is its

positive correlation to the Cordillera Oriental, as

19 patients (76%) resided in or near this region of the

country (Fig. 1-4). Most of the patients have been symptom-

atically cured of the disease with hot (46C) water treat-

ment (Neva, pers. comm.) (Fig. 1-5).

During the period March to September 1982, a serolog-

ical survey for leishmaniasis was undertaken by personnel of

the Instituto Dermatologico, in which the author assisted.

In the survey, blood samples were taken from residents









200


190


180


0- Presumed site 700 690


-- Confirmed site


0 51
, i a


L mkm
Figure 1-3. Case sites for patients with diffuse cutaneous leishmaniasis (DCL) in
the Dominican Republic (see Appendix 1-1 for name of numbered localities).


HAITI































Figure 1-4.


Typical terrain in the Cordillera Oriental,
Dominican Republic.


Figure 1-5. A young Dominican DCL patient with healed
lesions on wrist and upper arm.









living in the area of 7 of the 21 case sites (Fig. 1-3;

cases 1-3, 7, 10, 17, 19 and 20, 21, and 22) and two control

sites (sites in regions where no cases of leishmaniasis had

been diagnosed). Site selection was based on accessibility

of the site to vehicular transport and on the probability of

obtaining an adequate number of volunteers from the local

residents. Indirect fluorescent antibody test (IFAT) was

performed on the sera, revealing that 26.0-48.0% of the

samples from case sites were seropositive for leishmanial

antibody, at 1:8 or higher titer. At the two control

sites 0% and 14% were determined to be seropositive (de

Quinones, unpublished data). The pertinent patient history

data are given in Appendix 1. Cases 1-3 are siblings, cases

14 and 15 are son and father, and cases 19 and 20 are

neighbors, but unrelated.



Study Site Selection

Four principal study sites were visited regularly

during the study. One, Pedro Sanchez, was not a leish-

maniasis case site, but was used as a control site, i.e. a

site with sand fly habitat but no known cases of leishman-

iasis. Pedro Sanchez was selected as a typical nonagricul-

tural wooded area. Due to extensive land clearing for

agricultural purposes, pasture, sugar cane planting, and

coffee/cacao groves, very little natural forest habitat

exists in the Dominican Republic. The Pedro Sanchez site

was representative of wooded river bank habitat in the









leishmaniasis endemic region of the Cordillera Oriental.

The field station (and author's residence) for the study was

located less than 0.5km distant, thus the site was con-

venient at all times of year. The other three principal

sites were leishmaniasis case sites, in all instances the

DCL patients were living at or near the sites. All

four patients (patients 19 and 20 were neighbors) were young

and had not lived elsewhere at the time the disease was

diagnosed. Interviews with the patients' parents led the

author to believe that the disease was contracted at the

sites. Very few changes had occurred at the sites in the

intervening years between the appearance of the first signs

of the disease and the beginning of the author's research,

this was not the case with several other presumed case

sites.

The system of roads in the Dominican Republic is

generally poor, particularly in mountainous regions and the

Eastern region, thus accessibility of the site was an

important criterion in study site selection. Certainty of

the site of disease contraction was another important

criterion. Many of the older DCL patients have lived in

various localities throughout their lives and do not

remember clearly where they were living when signs of the

disease first appeared, 20 years or more previous. Another

important factor was whether or not personnel of the

Institute Dermatologico were familiar with the location of

the case site. In a few cases, the patients had visited









rural public clinics where they had been diagnosed, their

place of residence at time of infection was not known except

in rather general terms. Eleven of the 21 DCL case sites

were never visited by the author, primarily due to the

factors mentioned above.

The three principal study case sites were approximately

35km west (Loma Pena Alta), 18km north (Morro de Miches),

and 13km east (Trepada de Jabilla) of the field station and

control study site at Pedro Sanchez. All four sites were

visited in excess of 100 man-hours during the study. The

remaining sites listed below were DCL case sites and were

visited one or more times, depending on proximity and

accessibility. Due to the concentration of cases in the

Cordillera Oriental, this area received the greatest amount

of attention by the author.



Study Site Description

Pedro Sanchez, El Seibo Province

Altitude: 76m

This site consists of a gallery forest along the banks

of the Rio Seibo on the southern outskirts of the village of

Pedro Sanchez. The Rio Seibo is 0.5 to 1.5m deep, depending

on the exact location and time of year, and 4 to 6m wide in

this region. The wooded border on the northwest bank was

20 to 50m wide and extended for several kilometers. It con-

tained several trees with diameter up to im, a moderately

thick shrub understory. A section of forest, with a length










of 40m, was used for this study, through August 1982; when

the site was revisited in May 1983, it had been extensively

altered for agricultural use.

The remaining study sites, listed alphabetically, all

are DCL case sites where a patient is living, or was living

at the presumed time of infection.



Altos de Peguero (5km E by 5km N of El Seibo), El Seibo

Province (Fig. 1-3, #17)

Altitude: 72m

The case site is on the northern side of an isolated

ridge on the southern edge of the Cordillera Oriental.

Several coffee and cacao groves are present in the area, the

largest of which is a 2ha cacao grove about 50m from the

former residence of the DCL patient. Coffee and cacao

groves also contain scattered larger hardwood trees to

provide light shade. There are no major streams or rivers

in the immediate vicinity, the closest being 1.5km distant.



Carrasco (10km S of Rio San Juan), Maria Trinidad Sanchez

Province (Fig. 1-3, #23)

Altitude: 15m

The area surrounding the patient's former residence is

flat pasture for at lease 2km, with the exception of a 1.5ha

cacao grove, 400m distant from the residence. A small

stream runs next to the grove and forms a small pool (7m

diameter) where the children of the area are said to swim.










La Culatica (10km S of Nisibon), Altagracia Province

(Fig. 1-3) #1-3)

Altitude: 150m

The patients' (three siblings) residence was located on

a small hill above a small (0.5ha) coffee grove. Other

coffee and cacao groves are in proximity to the house site,

some of which are owned by the patients' father. A small

wooded stream runs below the site, through parts of the

coffee grove. This locality is situated in the northeast

portion of the Cordillera Oriental.



Iguana Arriba (23km NE of Bani), Peravia Province (Fig. 1-3,

#9)

Altitude: 152m

The exact locality of the DCL patient's residence was

not known, but the area consists of extensive coffee plant-

ings throughout low mountain terrain. A few small streams

are present in the low areas between ridges. Larger trees

are found along the streams and in the coffee groves,

providing shade.



Loma Pena Alta (13km NW of Hato Mayor), El Seibo Province

(Fig. 1-3, #21)

Atltitude: 442m

The DCL patient lived at the top of the ridge, adjacent

to a 0.5ha coffee grove. The eastern side of the ridge was

shrub-overgrown pasture. Some coffee groves were present on









the western side, which was mostly lightly forested with

numerous rocky outcroppings.



Monte Claro (16km NE of Cotui), Sanchez Ramirez Province

(Fig. 1-3, #24)

Altitude: 76m

A 0.5ha coffee grove is 10m distance from the site

where the DCL patient lived at the time of infection. The

area consists of rolling hill pasture, with the coffee grove

being the only wooded area in a radius of 1km from the

house. The Rio Chacuey has a lightly wooded border, and is

about 1km distant from the site.



Morro de Miches (10km S of Miches), El Seibo Province

(Fig. 1-3, #19, 20; Fig. 1-4)

Altitude: 305m

Two unrelated patients live at this site, approximately

100m apart and separated by the peak of the ridge.

One patient lived besided a 0.25ha coffee grove which

contained several large shade trees. The grove is separated

from nearby wooded areas by pasture and cultivated plots. A

small stream runs down the ridge, 100m from the house site.

The other patient's residence was located on the northern

edge of a rather extensive mixed planting of coffee and

cacao which had a length of about 0.5km and a width of

10-30m.









Najayo Arriba (20km NW of San Cristobal), San Cristobal

Province (Fig. 1-3, #5)

Altitude: 400m

The patient's residence is located on the side of a

ridge immediately above a small (0.5ha) coffee grove. A

small stream runs along the bottom of the ridge, about 75m

from the house. The site is located on the southern edge of

the Cordillera Central.



Rio Llano (10km W by 30km N of Higuey), Altagracia Province

(Fig. 1-3, #14 and 15)

Altitude: 250km

The patients (father and son) lived in a house 15m from

a small stream. A small (0.5ha) coffee grove is situated on

the opposite side of the stream. Rio Guancho is less than

0.5km distant. A gallery forest, 10-30m wide, runs along

the banks of the river. This site is in the eastern portion

of the Cordillera Oriental, about 10-15km (straight line

distance) south of La Culatica.



Trepada de Jabilla (2.8km N of Las Cuchillas), El Seibo

Province (Fig. 1-3, #7)

Altitude: 130m

The patient's residence was approximately 10m from the

edge of a rather extensive coffee grove ( 3ha). As with

other coffee groves, there are some larger shade trees

present. The terrain is rolling hills, primarily pasture,










south of the Cordillera Oriental. The Rio Soco borders on

side of the coffee grove and is 0.5 to 2.0m deep, depending

on location and season, and generally 10 to 12m wide.

Several other coffee groves exist in the area.



Objectives

In 1975, Bogaert-Diaz et al. reported the discovery of

three human cases of diffuse cutaneous leishmaniasis (DCL)

in the Dominican Republic, the first autochthonous cases of

cutaneous leishmaniasis known in the country or in the

entire West Indies, except Trinidad, a continental island.

Over the succeeding four years, a concentrated search for

additional cases, carried out under the National Leprosy

Program, revealed 15 more cases of DCL. As of March 1984,

25 cases of DCL have been diagnosed. Surprisingly, none of

the patients had ulcerating lesions. Interest in this

unique situation led to grant support from the World Health

Organization to the Instituto Dermatologico Dominicano for

epidemiological studies of DCL in the Dominican Republic,

beginning in 1981. Some objectives of the author's

research, listed below, were included in the project. Field

studies were performed in the Dominican Republic during the

periods 19-24 May 1981, 27 July 1981 to 2 August 1982, and

18 May to 3 August 1983. Laboratory studies were performed

at a field station (Pedro Sanchez) and at the Instituto

Dermatologico in Santo Domingo, the University of Florida,

and Walter Reed Army Institute of Research.










A. Field Studies:

1. To survey for the presence of phleboto-

mine sand flies at selected locations in

the Dominican Republic.

2. To study the ecology of sand flies

including host preference, resting

sites, population dynamics, and seasonal

and geographic distribution for species

located in the survey.

3. To identify the wild and/or peridomestic

reservoir host(s) of leishmaniasis.



B. Laboratory Studies:

1. To establish laboratory colonies of

indigenous phlebotomine sand flies.

2. To study the life cycle of Leishmania in

the sand fly.

3. To determine the vector potential of

these flies.

4. To further elucidate some of the biolog-

ical differences between the Dominican

Leishmania and other known Leishmania

spp.

















CHAPTER 2

SURVEY FOR, AND COLONIZATION OF,
LUTZOMYIA CAYENNENSIS HISPANIOLAE
AND LUTZOMYIA CHRISTOPHEI



Introduction

Phlebotomine sand flies, the only known biological

vectors of leishmaniasis, also transmit other parasitic

agents of man and other vertebrates (Adler and Theodor,

1957). A new focus of leishmaniasis was discovered in 1974

in the Dominican Republic (Bogaert-Diaz et al., 1975), the

eastern portion of the island of Hispaniola. Only two extant

species of sand flies are known from Hispaniola, Lutzomyia

cayennensis hispaniolae (Fairchild and Trapido, 1950) and

Lu. christophei (Fairchild and Trapido, 1950). Both species

were collected from tree trunks and buttresses, primarily,

but little was known about the habits and range of these

species. Lutzomyia cayennensis hispaniolae is an endemic

subspecies of Lu. cayennensis (Floch and Abonnenc) that

ranges from Ecuador and French Guiana north to Mexico. Most

members of the Lu. cayennensis species group are reported as

reptile feeders (Young, 1979). Lutzomyia christophei is a

member of the Lu. verrucarum species group that contains an

number of man-biting species (Lewis, 1968) including one

suspected vector of leishmaniasis (Lainson and Shaw, 1979).









This study was conducted to determine the geographic

range, the habits, and life cycle of the two species.



Methods and Materials



Field Studies

Chaniotis (1978) and Young (1979) reviewed techniques

used for sampling phlebotomine sand flies. The methods used

in the present study were aspirator collections at resting

sites, man-biting collections, sticky paper traps, flight

traps, CDC light traps (Sudia and Chamberlain, 1962), and

Disney traps (Disney, 1966). Specific equipment preparation

and field techniques for aspirator collections were given by

Endris et al. (1982) (Fig. 2-1). Aspirator collections were

made at various sites around the country, mostly from tree

trunks, but also from tree holes and rock crevices.

Cigarette smoke was occasionally used to disturb resting

sand flies from the latter two types of resting sites. Live

wild-caught sand flies were transported to the field station

at Pedro Sanchez where they were either held in a feeding

cage (Fig. 2-2) for host preference studies or kept

individually for oviposition and later dissected for

parasites.

The other techniques were used at five leishmaniasis

case sites or the study site at Pedro Sanchez (see Chap-

ter 1). Attempts to collect sand flies from human bait at





























PLASTER
OF PARIS


(A)
120 ML SPECIMEN CONTAINER

FULL SCALE


SCREEN LID




SOLID LID









1.^' '.-


(B)
7 DR VIAL

FULL SCALE


(C)
AS PIRATOR


ONE-THIRD SCALE


Figure 2-1.


Field collecting equipment and rearing
containers for phlebotomine sand flies (from
Endris et al., 1982). (A) Collecting con-
tainers. (B) Oviposition/larval rearing
vial. (C) Aspirator.





































INSET SIDE VIEW WITH SLEEVE


INSET SIDE VIEW WITH SLEEVE


Figure 2-2.


Sandy fly feeding cage--a modified aquarium with
plaster of paris bottom and back (from Endris et al.,
1982).










Trepada de Jabilla were made various times during the day

and at dusk during November 1981 to July 1982 and May 1983.

Similar attempts were also made at Loma Pena Alta at dusk on

various days in June and July 1983. A sticky-paper trap of

double-sided tape on a 25cm square wooden frame was used at

Pedro Sanchez from 21-23 May 1981. The trap was placed in

the crotch of a tree (Im high) known to harbor resting sand

flies. Flight traps (Fig. 2-3) and CDC light traps

(Fig. 2-4) were set at various sites (Tables 2-1, 2-2). Two

modified CDC light traps were also employed. In one, a UV

light source was substituted for the normal light source;

the second type had no light source, instead a cage

containing a hamster, Mesocricetus auretus, was suspended

above the trap (Fig. 2-4). The light traps were run on

nights when the moon was less than one-half full, or when

the sky was mostly overcast. CDC traps were turned on in

late afternoon and taken down the following morning. Flight

traps were set up in coffee or cacao groves, with one

exception (lightly wooded stream bank at Hato Mayor). The

flight traps were emptied every two to four days. The trap

collections were checked for sand flies with the aid of a

dissecting microscope (7-30X). Disney traps (Fig. 2-5) were

used at two sites with hamsters serving as bait (Table 2-3).

The bottom of cake pans (22.5 x 30.0cm) or cookie sheets

(27.0 x 38.0cm) were covered with a thin layer of mineral

oil or castor oil. The condition of the hamsters was






















K' m


Figure 2-3.


The author in front of a flight trap in a cacao
grove at Altos de Peguero, El Seibo Prov.


Figure 2-4. A CDC trap and modified traps (from left CDC
with UV light source, normal CDC, and hamster-
baited trap).
















Table 2-1.


Location and dates for flight trap samples in
the Dominican Republic.


Location


Date


Altos de Peguero cacao grove


Carrasco cacao grove


Hato Mayor wooded stream bank


Loma Pena Alta coffee grove


Monte


Morro


Claro coffee grove


de Miches coffee grove


Pedro Sanchez wooded river bank


Trepada de Jabilla coffee grove


9-25 Mar, 1-28 May 1982


3-10 Jul 1982


17 Aug 1981


24-27 Jul 1982
9 Jun-3 Aug 1983


19-26 June 1982


7-14 Aug 1981
5 Oct-3 Nov 1981


20-22 May 1981


6 Nov-14 Dec 1981
18 Jan-26 Jul 1981
23 May-18 Jun 1983













Table 2-2.


Location, trap type, and dates for CDC in the
Dominican Republic.


1 Date2
Location # And Trap Type Date


Altos de Peguero


Carrasco

La Culatica


Loma Pena Alta


Monte Claro


Morro de Miches


3 CDC


CDC

CDC
CDC-UV

CDC

CDC
CDC-UV
CDC-HB

CDC-UV
CDC-HB

CDC-UV

CDC-UV

CDC-UV

CDC-UV

CDC

CDC
CDC-HB

CDC

CDC

CDC
CDC-UV
CDC-HB


2-3 Apr 1982
27-28 May 1982

9-10 Jul 1982

7-8 Jun 1983


26-27 Jul 1982

2-3 Jun 1983



23-24 Jun 1983


9-10 Jul 1983

17-18 Jul 1983

23-24 Jul 1983

30-31 Jul 1983

25-26 Jul 1982

21-22 May 1983


22-23 Sep 1981

18-19 Jul 1982

29-30 May 1983










Table 2-2. Continued


Location # And Trap Typel Date2


Pedro Sanchez 2 CDC 20-21 May 1981

1 CDC 7-8 Aug 1981

2 CDC 5-6 Jun 1982

Trepada de Jabilla 2 CDC 18-19 Jan 1982

1 CDC 16-17 Apr 1982
21-22 May 1982
30-31 May 1982
9-10 Jun 1982
30 Jun-
1 Jul 1982
14-15 Jul 1982

2 CDC 26-27 May 1983
2 CDC-UV
2 CDC-HB

1 CDC 30-31 May 1983
1 CDC-UV

1 CDC 18-19, 19-20
Jul 1983


CDC-UV with blacklight
trap, no light (see Fig.


source, CDC-HB -
2-4).


hamster baited


2From approximately 1830 hrs Day 1 1000 hrs Day 2













































Figure 2-5. A Disney trap, used for collecting rodent-
feeding sand flies.











Table 2-3. The sites and dates for Disney traps used for
phlebotomine sand fly sampling in the Dominican
Republic.



Site # traps Date


Loma Pena Alta 2 8 July 3 August 1983



Trepada de Jabilla 2 28 May 15 June 1982

4 23 May 2 June 1983

2 7 9 June 1983









checked twice a day by local personnel and food and water

were provided at all times. Hamsters were exposed for three

to four days and then replaced by others.

Soil samples (dirt, humus, and leaf litter) were

collected from tree buttresses and tree holes at five sites

where sand flies were common (Table 2-4). Samples

(1-4 liters in volume) were placed in plastic bags for

transport back to the field station. For observation, a

sample was transferred to a white plastic tray, and the

material was examined with the aid of a dissecting micro-

scope (7-30X). Larger leaf matter was checked for the

presence of larvae or pupae and then discarded. Recovered

larvae were held in larval rearing vials (Fig. 2-1b) with a

small amount of larval food (Young et al., 1981), and held

until adult emergence for species identification. The rest

of the sample was returned to the plastic bag and periodi-

cally checked for emerged adults or immature stages. The

bags were held at ambient temperature (19-30C) for up to

two months.



Laboratory Rearing

Techniques used for laboratory rearing of Dominican

sand flies (Fig. 2-6) were those described by Endris et al.

(1982). Individuals from an egg batch were raised together

or individually, as described by Perkins (1982), in a

modified tissue culture plate (Fig. 2-7) Newly hatched

(within 6 hrs) first instar larvae were transferred from










Table 2-4.


Collection sites and dates for soil samples
examined for the presence of sand fly larvae.


Site Date # Samples Microhabitat


Altos de Peguero


leaf matter
at tree base-
cacao grove


Loma Pena Alta




Monte Claro

Pedro Sanchez


2 Jul 1982

20 Jul 1982




25 Jul 1982

15 Aug 1981


as above


leaves and
humus at tree
base-coffee
grove

as above

leaves and
humus at tree
base-gallery
forest

as above

as above

leaves and
humus at tree
base-upland
woods


27 Aug 1981

22 Sep 1981

30 Oct 1981


Trepada de Jabilla


25 May 1982

15 Jun 1982

2 Dec 1981


as above

as above


leaves and
humus at tree
base-coffee
grove

leaves and
humus at tree
base and in
tree hole-
coffee grove


18 May 1982


8 Jun 1982 3


28 May 1982


as above






















Figure 2-6.


Schematic diagram of laboratory techniques for rearing of phlebotomine
sand flies. (1) Plaster of Paris at bottom of rearing vial in moistened
with tap water. (2) Wild-caught or lab-reared gravid females are placed
individually in vials and drop of sugar solution is placed on each screen
lid. (3) A solid lid replaces the screen lid after eggs are deposited.
(4) Larval food is sprinkled on the plaster of Paris anytime before the
larvae hatch. (5) Additional larval food is added as larvae grow.
(6) Lidless vials containing pupae are placed in the feeding cage; fruit
slices provide a sugar source for emerging adults. (7) For a bloodmeal
source, a vertebrate is placed in the feeding cage (Endris et al., 1982).









NOT TO SCALE


I


SLEEVE REMOVED


J


"'SI..


""Io


Tl-n


:


400














































Figure 2-7.


A modified microtiter plate for rearing
individual sand fly larvae (approximately
0.5cm layer of plaster of Paris in each well).









oviposition vials to wells in the tissue culture plate

(one larva per well). The larvae were checked at approxi-

mately the same time every day. Individual rearing was

performed under different temperature-humidity regimens for

both species of sand flies. Lutzomyia christophei larvae

were held in a Hotpack chamber (Hotpack, Inc., Philadelphia,

PA) at 23C or 28C. High humidity was maintained by

placing the plates with moistened plaster of Paris in

tightly sealed plastic boxes lined with moist paper towels.

Lutzomyia cayennensis were reared under three sets of

conditions. The first group was reared in a Hotpack chamber

at 28C, as above. The second and third groups were held at

ambient conditions in the Dominican Republic of 24 to 330C

and 60 to 95%RH (August-September), and 16 to 280C with 30

to 90%RH (January-February), respectively.

Eclosion was checked in the vials or plates once per

day. Adults were released into the feeding chamber. At the

field station, various types of locally available fruit were

provided as a sugar source. These included grapefruit,

orange, or sweet lemon sections (skin removed); cashew

fruit; and peeled mango skin. Lutzomyia christophei reared

at Walter Reed Army Institute of Research (WRAIR) were

provided with apple slices. For Lu. christophei females, an

anesthetized Rattus rattus, Syrian hamster (Mesocricetus

auretus), or BALB/c mouse (Mus musculus) was provided as a

blood source. An Anolis sp. lizard was regularly provided

as a blood source for female Lu. cayennensis; occasionally a









human hand or hamster was offered. The lizard was either

restrained in a small cylinder of hardware cloth or was

allowed to be free. If free, the mouth was taped shut to

prevent the ingestion of sand flies.

To determine the age at first feeding for either

species, all flies emerging in a six hour period were held

separate by species. For Lu. cayennensis, an Anolis lizard

was provided until all females were fed or dead. If neces-

sary, the lizard was exchanged every two to three days for a

similar sized, conspecific lizard. The lizards were not

restrained. For Lu. christophei females, an anesthetized

hamster was provided for one hour two times per day, approx-

imately 0903 hrs and 1530 hrs.



Sand Fly Dissections

A sample of both male and female sand flies from

various survey sites was routinely dissected for species

determination, using the methods given by Young (1979), or

by simply cutting off the head and the last few abdominal

segments and mounting them in a drop of Hoyer's mounting

medium (Young, pers. comm.). In addition, wild-caught and

lab-fed sand flies were dissected upon death to examine the

digestive tract for parasites. The dissections were done in

normal saline or in Medium 199 (GIBCO, Grand Island, NY) on

a microscope slide and observed with the aid of a compound

microscope (200X or 450x). Permanent slides were made by

removing the cover slip and allowing the liquid to dry, then









fixing with absolute methanol and staining with Giemsa for

20 minutes. After drying, a drop of Euparal mounting medium

was added and a cover slip placed over it.



Results



Field Studies

Adult Lu. cayennensis were aspirator-collected from

tree trunks (10 cm diameter and larger) at many localities

in the Dominican Republic (Fig. 2-8, Appendix 2). At all

sites, there was sufficient tree and shrub vegetation to

provide a "forest-floor" type habitat, with little ground

cover vegetation. The sites were shaded, to varying

degrees. Many sites in El Seibo Province were visited more

than once. This species was also recovered from flight trap

samples at three sites (Appendix 2). None was recovered by

any other method. Adult Lu. christophei were recovered at

seven sites by flight trap, CDC trap, or aspirator

collection from rock and tree crevices, primarily of ceiba

trees (Ceiba pentandra) (Fig. 2-8, Appendix 2). The use of

cigarette smoke facilitated their capture from deeper

crevices. Six of these sites were leishmaniasis case sites;

the seventh was about 5km distance from a case site. No Lu.

christophei were collected by hamster-baited CDC traps,

Disney traps, or man-biting collections. At Loma Pena Alta,

both Disney traps were within lm of resting sites of Lu.

christophei females. The man-biting collections attempted





























Figure 2-8. Collection sites for Lu. cayennensis hispaniolae and Lu. christophei
in the Dominican Repuglic from May 1981 August 1983 (See Appendix 2-1,
2-2).
















































t- no Lutzomyia recovered

* Lu. cayennensis only

* Lu. cayennensis and
Lu. christnphei










at this site were performed within 2 to 3m of tree crevices

known to harbor resting sand flies. Two female sand flies

were recovered while biting DCL patient #25, in September

1983; these were later identified as Lu. christophei, by the

author.

Populations of Lu. cayennensis at Pedro Sanchez varied

throughout the year and the population at Trepada de Jabilla

followed the same trends from January through July 1982

(Fig. 2-9). The population sample was based on the total

number of flies aspirator-collected from 10 marked trees.

The sample from Pedro Sanchez was always larger than the

same week's sample from Trepada. At Pedro Sanchez, the

samples varied from a high of 67 flies to a low of 2 flies;

the total for the 39 samples was 986 flies (536 males and

449 females). At Trepada, the high was 39 flies and the low

was 0 flies; the total for the 22 samples was 269 flies

(156 males and 113 females). Fewer flies were collected

during the period February to mid-May 1982, which corres-

ponds to the dry season and the first two weeks of the rainy

season. The population level began to rise approximately

two and one-half weeks after the beginning of the rainy

season (3 May 1982). The male:female ratio, on dates

when females were collected, ranged from 0.90 to 3.00

(x = 1.90) males/female (8 out of 22 samples had no

females). Blood-fed or gravid females comprised 0 to 100%

(x = 31.3%) of the females at Pedro Sanchez and 0 to 75%



















--------- Trepada de Jabilla
Pedro Sanchez


I a
\ ",
!

I
I'
'U
I
!
r
!l


So Oc
1981


De Ja Fe Mr
1982
Month


Je Jy


Figure 2-9.


Weekly sample populations of Lu. cayennensis at two study sites in the
Dominican Republic (based on tree trunk resting collections).


60
43

2 50
-o
t 40
4)

8 30

S20

10










(x = 48.9%) at Trepada. Due to the scarcity of Lu. christ-

ophei adults, seasonal abundance could not be monitored for

this species.

During the wettest months (May to December), Lu.

cayennensis adults could be found on tree trunks up to a

height of 2m. Normally when disturbed, the sand flies would

fly only a short distance (less than 10cm); however, on

rain-wetted tree trunks the sand flies readily flew off the

trees. During the dry season, the flies were generally

found at the tree base, often where the ground had separated

from the tree trunk in fissures, or in the moss at the base.

Smaller tree holes also harbored Lu. cayennensis at this

time of year. Female Lu. cayennensis were commonly observed

feeding on Anolis lizards during the day, principally on

Anolis distichus, but also on A. cybotes. Lizard identifi-

cation was based on Cochran (1941).

Adult Lu. christophei were usually recovered from deep

tree crevices and ground level tree holes in large shade

trees in coffee groves. Two types of trees provide crevices

which could serve as resting sites for Lu. christophei, the

ceiba and the strangler fig (Ficus spp.) The former may

have very large buttresses and the latter usually has

numerous crevices of various sizes as it entwines its host

tree. Cigarette smoke puffed into the crevices forced the

sand flies to the entrance, where they were more easily

captured. Most of the Lu. christophei collected were

obtained at Loma Pena Alta, where a total of 81 flies










(51 males and 22 females) were captured by aspirator,

7 flies (2 males and 5 females) were captured in a flight

trap, and 23 flies (10 males and 13 females) were captured

in CDC or CDC-UV traps. In one tree hole, 14 sand flies

were found in association with a rat nest constructed of

leaves. Of eight females, three were blood-fed or gravid

and were the only fed Lu. christophei females recovered in

aspirator collections. A common characteristic of the other

resting sites was the presence of land snails and millipede

feces. On rare occasions at Loma Pena Alta, male Lu.

christophei were found resting on tree buttresses near the

entrance to tree crevices, in association with Lu.

cayennensis. Lutzomyia christophei was the more active of

the two species.

Five fourth-instar larvae were recovered from a soil

sample at Monte Claro. The microhabitat was humus and leaf

litter which had accumulated in the buttress of a large

shade tree in a coffee grove. The larvae were reared to the

adult stage and were Lu. cayennensis. No other phlebotomine

larvae were seen or recovered from the other soil samples

(Table 2-3).



Laboratory Studies

Wild-caught and lab-reared Lu. cayennensis adults

behaved similarly. Females fed readily on Anolis lizards in

the feeding chamber, but showed no interest in feeding on

human, hamster, or rat (R. rattus). Over 50 wild-caught










females were exposed to a skink, Mabuya mabouya, in a

feeding chamber, but none fed or was seen probing. Twenty

lab-reared females were exposed to tree frogs, Hyla sp., and

toads, Bufo marinus, but the flies showed no interest in

feeding. The preferred feeding site on lizards was on the

mid-dorsal region to the tail base. Some females were

occasionally observed feeding on the top of the head and the

shoulder region. Females rarely probed more than one spot

before feeding to repletion. Feeding time ranged from

62.5min to 82.5min (n = 26, x = 73.Omin6.25min (S.D.)).

Feeding only occurred under lighted conditions. Females

would feed at any time of day in the feeding chamber,

provided that the chamber was left in a well lighted room.

Generally, females first fed at age 3.0 to 4.5 days (n = 50)

posteclosion; however, on various occasions lab-reared

flies, less than 48hrs old, fed on lizards. Mating was

observed before, during, or after bloodfeeding, with the

pair remaining in copula for up to 21min. Females began

oviposition four to five days post-bloodfeeding. Complete

oviposition usually required less than one day. Wild-caught

females generally died within 24hrs after laying eggs.

Holding the flies in a Hotpack environmental chamber helped

to increase the survivorship of lab-reared females.

Approximately 17 to 33% of the females of every generation

survived to take a second blood meal; 50 to 75% of these

survivors subsequently laid a second batch of eggs. No

males or unfed females lived more than six days.










Female Lu. cayennensis were anautogenous, each female

laying up to 60 eggs. Most unmated bloodfed females died

without ovipositing (20 out of 23 flies), but three laid

partial egg batches of 5 to 9 eggs. The size of a full egg

batch for wild-caught females was 38 to 60 eggs (n = 50,

x = 47.76.6 (S.D.)), based on females collected with a full

blood meal that had no eggs retained at death. For lab-

reared females, a full egg batch contained 39 to 60 eggs

(n = 50, x = 46.66.3 (S.D.)). Of 103 F1 females held

singly in vials, only 19 laid full egg batches, 61 females

laid partial egg batches of 7 to 29 eggs, and 23 females

died without laying eggs. The percent egg hatch, for full

egg batches from wild-caught and lab-reared females ranged

from 20.4-100% (n = 100, x = 68.4%29.7% (S.D.)). Percent

egg hatch for partial egg batches ranged from 0 to 100% (n =

50, x = 61.2%33.2 (S.D.)). Percent egg hatch was not

statistically different between the two groups (t-test, p =

0.05). When three or fewer eggs were laid by a female, none

hatched.

All hatching from an egg batch occurred within a 12hr

period. Under ambient conditions at the field station, the

incubation time for eggs was 8 to 12 days, depending on time

of year (ambient temperature). Longer incubation times were

associated with cooler temperatures, especially in January

and February (Table 2-5).

Larvae of Lu. cayennensis exhibited burrowing behavior.

Larvae tended to stay under their food, except when the food










was very damp. When individually reared, male sand fly

larvae developed faster than did females, under all three

temperature-humidity regimes (t-test, p = 0.05) (Table 2-5).

Males developed faster under ambient conditions in August-

September (24-33C, 60-95%RH) than males under constant

conditions (270C, 85%RH). Both groups developed faster than

those under ambient conditions in January-February in the

Dominican Republic (16-280C, 30-90%RH) (t-test, p = 0.05).

The time from oviposition to adult eclosion, for males and

females, is presented in Table 2-5 and Figures 2-10, 2-11,

2-12.

A closed colony of Lu. cayennensis was maintained for

six generations, or for almost one year.

Wild-caught and lab-reared Lu. christophei behaved

similarly in the laboratory. Females readily fed on

anesthetized rodents, including a wild-caught R. rattus,

laboratory mice and hamsters. They readily probed on a

human hand. The females showed no feeding site preference,

feeding equally well on the ears, paws, or eyelids of the

offered rodent host. Females often probed more than one

spot and after initiating feeding, many females moved to a

second site to complete feeding. Females of this species

fed equally well under light or dark conditions. Feeding

time ranged from 2.75min to 4.95min (n = 25, x = 3.35min

0.50min (S.D.)). Some lab-reared females fed as early as

24hrs after emergence, though most did not feed until 48 to

72hrs after eclosion. Females took a very large bloodmeal











Table 2-5. Mean duration ( S.D.) in days of immature stages of Lu. cayennensis
hispaniolae at three temperature regimes, according to sex of sand fly.




Larval Instars
Total
Condition Sex Egg 1 2 3 4 Pupa (egg-adult) n


Constant male 8 5.11.0 3.60.8 3.00.4 6.5+1.6 7.10.3 33.71.9 12
(280C)
female 8 5.01.0 3.20.8 4.41.3 6.5+2.3 7.50.8 36.12.5 11

(48) (31) (28) (28) (25) (23)


Ambient male 8 4.70.5 2.30.5 2.70.5 6.0+0.7 6.70.6 30.3+1.3 23
(24-330C)
female 8 5.00.4 2.40.5 3.10.3 6.4+0.7 7.70.7 32.51.0 24

(48) (48) (48) (48) (48) (47)


Ambient male 12 5.00.0 3.60.6 3.10.7 8.30.6 9.1+0.3 43.6+1.6 17
(16-280C)
female 12 5.41.0 3.3+0.8 3.60.7 8.90.8 9.40.5 45.51.6 24

(48) (47) (46) (46) (42) (41)


Note: Number in ( ) indicates individuals surviving each stage.















X, 343+1 .7


0


i ~


x =37


B

IhL


0 31 32 33 34 35 36 37 38
Oays after ovioosit on


Figure 2-10.


Eclosion time in days after egg deposition for
Lu. cayennensis reared under constant condi-
tions (280C, 90%RH).


Days after oviposition


Figure 2-11.


Eclosion time in days after egg deposition for
Lu. cayennensis reared under ambient condi-
tions August-September (24-330C, 60-90%RH).


1 5
ai
"3
:2

3


I .


39 40


m m


I


~I


M


.0+2.7





59












x = 45.5+1.6

10
9 x 43 6+1 .6
8
7
40 to 85%RH)6
S5
4
J 3
2
1 I ],
40 41 42 43 44 45 46 47 48 49
Days after ovioosition



Figure 2-12. Eclosion time, in days, after egg deposition,
for Lu. cayennensis reared under ambient
conditions, January-February (16 to 280C,
40 to 85%RH).









(Fig. 2-13), but were very active afterwards. Mating, only

rarely observed, occurred before or after feeding, with

coupling lasting up to 28.5min. Oviposition began at least

five days after blood-feeding, but for a few females, it was

delayed up to ten days post-feeding; it was usually com-

pleted in less than one day. Approximately 17% of the

females that survived five or more days post-feeding (8 of

46 F1 and F2 females) did not exhibit ovarian development

after their first blood meal. By seven days however, they

had excreted the blood meal remnants in their feces and were

capable of refeeding and developing eggs. In the F3 genera-

tion, 8 of 52 females (15.8%) developed partial egg batches

of eight or fewer eggs before refeeding. All eight died

without ovipositing.

Only 2 of the 11 lab-fed wild-caught females laid full

egg batches of 39 and 49 eggs. The nine other flies laid 0

to 35 eggs (x = 7.311.3 (S.D.)). The size of a full egg

batch for lab-reared females was 35 to 87 eggs (n = 10, x =

50.712.9 (S.D.)). Of 64 Fl to F3 females which survived

five or more days post-feeding, only 14 (21.9%) laid full

egg batches, 24 females (37.5%) laid partial egg batches of

5 to 27 eggs, and 26 females (40.6%) died without ovipos-

iting. The number of eggs laid, plus the number retained at

death (a measure of reproductive potential in the above

64 females) ranged 36-88 eggs and/or ovarioles/female (x =

64.1%24.3% (S.D.)). The percent egg hatch for full egg

batches ranged from 12.6% (11/87 eggs) to 100% (36/36 eggs)






61











































Figure 2-13. Female Lu. christophei feeding on BALB/c
mouse.









(n = 12, x = 64.4%31.1% (S.D.)). Percent egg hatch for

partial egg batches ranged from 0% (0/18 eggs) to 100%

(28/28 eggs) (n = 20, x = 56.8%38.1% (S.D.)). Percent egg

hatch was not statistically different between the two groups

(t-test, p = 0.05). All hatching from an egg batch occurred

within a 24hr period. The time period for egg incubation

was 10 to 17 days, depending, in part on the temperature

maintained (Table 2-6).

Lutzomyia christophei larvae exhibited burrowing

behavior, prefering to remain under the food in the larval

rearing containers. In the individual and group rearing

experiments, development time from egg to adult ranged from

51 to 69 days for the F1 generation (27C) and from 57 to

73 days for the F2 and F3 generations (230C). Males devel-

oped at a faster rate than did females (t-test, p = 0.05).

The data from these rearing experiments are presented in

Table 2-6 and Figures 2-14 and 2-15.

The closed colony of Lu. christophei is being main-

tained at Walter Reed Army Institute of Research, Washing-

ton, D.C.



Sand Fly Dissections

Dissections of 319 wild-caught parous Lu. cayennensis

were made. The flies came principally from five sites, of

which 46.4% (148 flies) of the total were from leishmaniasis

case sites. Most females had a blood meal evident at

capture and did not die, or were not killed, until the blood






63



G: tr D co in
N (( 3-


4- m (N 0
4- .
4J r-4l
cr +1 +1 +1 +1
-H a r- In L.o r-
SO
E-i U7 [- M (N in
1 CD 1.0 m a
0 U


OI *- o








H 14- 4
I I 01
UI +1 +1 0 +I +1







C v f -1 x I Ir
Q : ca m m-








S4-- H I I
r0 n ( 1
rn Im I C



1 I I r-
S* I I *

C ( N I I *v C
EU M +1 +1 I I
*t (0 ui o oo *- *roj



-0 Nl I I O
S*m >

0 I I
OCd I I V

I> I n

(a s 0 c N I I N m

N I I I
C I I 04



a V C-
* 0) 4 4N (N o *
0 4 I I 0
4l+1 +1 < I I
+1 M in a% I s
a, i- I* I (U

S* *3 -
O 5 H- C 0
-H W +1 + -l

40 4 O Cd *N d ) C
102 2 ) o o 2 4

C> 1. 4-)'
'0 1:

I X r-- I z

44 4-4-Q



M 4-j c Z fa Z *

S-4 4-1 u d0 0( ( O)*

C: .C C co co NC N co 041
) 0 0 ON [ O 0O
E- ( U L' 2 z















H | x = 57.7+6.3
S57.6.3 x 63.5+6.5
3





51 53 5 57 659 63 5 7 69 71 73
Days after oviposition


Figure 2-14.


Eclosion time, in days after oviposition, for
F1 Lu. christophei reared under constant
conditions.


Days after oviposition


Figure 2-15.


Eclosion time, in days after oviposition, for
F and F Lu. christophei reared under con-
stant coAditions.









had been digested and the residue excreted. The totals for

the sites, the percent blood-fed or gravid, and the time of

year collected are presented in Table 2-7.

Dissections were examined of 23 wild-caught Lu. christ-

ophei from Loma Pena Alta. Nine of these took bloodmeals in

the laboratory, two on a R. rattus captured at the site and

seven on a hamster. None of the 23 lab-fed females was

positive for parasites. The results of feeding lab-reared

Lu. christophei on leishmanial-infected BALB/c mice are

presented in Chapter 3.



Discussion

Lutzomyia cayennensis hispaniolae has a widespread

geographic distribution in the Dominican Republic. It

occurs in wooded areas within 100m of the coast and in

coffee groves in the interior (Loma Pena Alta, elevation

427m). Throughout the sugarcane growing regions, Lu.

cayennensis was found along streams and rivers wherever

there was a sufficient number and density of trees to

support a "forest-floor" type habitat (i.e. leaf and other

organic matter present). This species was found at or near

eight of the DCL case sites visited during the survey. In

the wild, Lu. cayennensis appears to be very sedentary, as

very few were collected by the flight traps, often despite

close proximity of the trap to known resting sites. At

Monte Claro, where 13 flies were recovered from the flight











Table 2-7. Site of collection for female Lu. cayennensis dissected.


# Blood-Fed
Site or Gravid #Parous Total Time of year


Pedro Sanchez 93 34 127 May 1981,
Aug 1981-Jul 1982
Trepada de Jabilla 50 21 71 Nov 1981-Jul 1982

Altos de Peguero 25 9 34 Mar-Jul 1982

Loma Pena Alta 13 7 20 Jul 1982

Monte Claro 12 8 20 June 1982

Morro de Miches 1 2 3 Aug 1981

Miscellaneous 37 7 44 Aug 1981-Jul 1982









trap collection, the trap was set so that the side of one of

the end panels was in contact with a tree on which were

found over 70 resting sand flies. The absence of Lu.

cayennensis in light trap collections suggests that this

species was not attracted to light. Both wild-caught and

lab-reared flies exhibited a positive phototaxic response in

the feeding chamber, which was noted by changing the

position of a desk lamp used to illuminate the chamber.

Later, as sand flies were collected from small tree holes,

it became clear that this was a probable response to light

as an escape attempt, with the path towards the light

replacing the path out of a tree hole. In the lab and in

the wild, this species was only diurnally active, as were

its hosts, Anolis lizards. Because these lizards are very

common, there is no great need for extensive host-seeking

behavior.

Although there may be some wind-borne dispersal of

adults, most of the distance an individual female travels

probably occurs during the 60+min that it is feeding on a

lizard. This length of time is much longer than that of

similar-sized mammalian feeding species, such as Lu.

anthophora (Endris, 1982) and Lu. christophei; however, the

lizards are not capable of reaching the sand flies to

dislodge them. Lutzomyia vexator (Coq.), also a lizard-

feeder that occurs in the USA and is about twice the size of

Lu. cayennensis, feeds to repletion in only 10min (Perkins,









unpublished data). Chaniotis (1967) reported 60+min feeding

times for some lizard-feeding sand flies in California.

The collection of larvae at Monte Claro represents the

first recovery of larvae of Lu. cayennensis in the field.

Hanson (1968) was unsuccessful in locating Lu. cayennensis

larvae in Panama. Monte Claro supported a very large

population of sand flies in a very small area. Many of the

trees sampled in the coffee grove had 50+ sand flies resting

on them; thus it does not seem surprising to find the

larvae in such a situation. Hanson (1968) noted that larvae

burrowed in culture, but to what depth they occur in nature

is unknown. In the current study, the soil samples were

jostled in transport back to the field station, so the depth

of the five recovered larvae was not known. The microsite

where they were collected had an unusually deep layer of

humus, 4 to 6cm deep. An injury to the tree trunk may have

been the cause of the ooze that started 0.5m above ground

level and continued to the ground, perhaps enriching the

soil at this spot. The soil samples taken from both Pedron

Sanchez and Trepada had very little humus.

The population levels of Lu. cayennensis are related to

wet and dry seasonal periods. Ambient temperature probably

influences the population level as well. In the eastern

region, the dry season usually lasts from late February to

the beginning of May. May is the wettest month, but from

June through December the ground remains fairly well satura-

ted. The population level of Lu. cayennensis, although










somewhat erratic on the weekly basis, remained fairly high

from August to December 1981 and from late May to July 1982

(Fig. 2-9).

The short lifespan of wild caught and lab-reared flies

at the field station was probably due to low humidity.

Other sand flies reared in the laboratory, and kept in the

environmental chamber at 85%RH, often lived 13 to 15 days

after eclosion. Early female death was the probable cause

when only partial egg batches were laid. Sugar feeding was

never observed in nature and only rarely in captivity. Lack

of carbohydrate may have been a contributing cause to early

mortality. As Lu. cayennensis is a very sedentary species,

the natural sugar source must be very close to the resting

sites, perhaps secretions from the trees.

The rearing times observed for Lu. cayennensis were

similar to those reported for Lu. anthopora, a similar sized

species (Endris, 1982), but much shorter than those for the

sympatric Lu. christophei. The individual rearing gave

somewhat misleading results, in that most flies emerged

within a seven day period. When larvae from a single egg

batch were reared together in a vial, emergence of adults

occurred during a period of up to 22 days. This delay could

be the effect of overcrowding or interference, as has been

reported among mosquito larvae (Ikeshoji and Mulla, 1970),

but not previously for sand flies.

Lutzomyia christophei appears to have a more limited

distribution than Lu. c. hispaniolae, as Lu. christophei










were recovered at seven sites, all of which were leishmania-

sis case sites, or in close proximity to such sites. As

resting Lu. christophei were secreted in tree crevices,

their apparent scarcity may have been partially an artifact

of the amount of time spent at the survey sites and the

collection methods used. It was not until cigarette smoke

was used to flush the flies from the crevices, that speci-

mens of this species were collected with any regularity.

With the exception of various locations in the Pedro Sanchez

area, including the study site and a small 15h woods, few

non-case sites received more than 10hrs of searching during

the study period. The habitat of this species, crevices and

ground-level tree holes in large shade trees primarilyy Ceiba

pentandra) in coffee groves and rock crevices in forested

areas, also may be a factor limiting its distribution. In

the Dominican Republic, very few virgin rain forests remain,

coffee and cacao groves, however, may simulate forest

conditions, as heavy leaf litter and much shade are charac-

teristic of these areas. Typically, land snails and milli-

pede feces were observed in the Lu. christophei-inhibited

crevices, it is quite likely that snail and millipede

byproducts enrich the soil of these crevices and possibly

provide sand fly larvae with an adequate diet. Lutzomyia

christophei may have evolved as a nest inhabiting species

with one or more of the tree hole-inhabiting mammals that

were present before the arrival of the Spaniards (1492 AD),

much the way Lu. anthophora is associated with woodrat









(Neotoma) nests in the USA (Young, 1972). Plagiodontia

aedium, the only endemic rodent in the D.R., is a tree hole

inhabitant (Woods, 1981). As the introduced rats replaced

the endemic rodents, the sand fly may have moved into the

tree hole nest of R. rattus or Mus musculus. Along with

this move may have been the development of a new reservoir

host for an endemic Leishmania; although there is discussion

as to whether the Dominican Leishmania is native to the

country or introduced (Walton, pers. comm., Zeledon, pers.

comm.).

Although sand flies were never encountered in large

numbers in crevice resting sites, residents at several case

and non-case sites reported them to be a major annoyance at

times. On Cuba, man-biting sand flies have been reported

from caves (Avila et al., 1969); although the species was

not determined, these flies were probably Lu. orestes

(Young, pers. comm.), a very close relative of Lu.

christophei. In the Dominican Republic, very few of the

visited DCL case sites had caves or rock outcroppings.

"Erisos" were reported to be common in June and July, at

Altos de Peguero, Loma Pena Alta, and Trepada de Jabilla,

though much more so in past years than in the three summers

(1981-1983) that the author was present. "Erisos" were

originally described to the author as pale-colored flies,

smaller than a mosquito, which start biting around dusk and

continue into the late evening, inside the house as well as

outside. The bite was described as being as painful as a









mosquito's. "Erisos" also hold their wings aloft, and tend

to hop around on the person before biting. All descriptions

were similar and all described typical sand fly behavior.

In September 1983, two "erisos" were collected while

feeding, on one of the leishmaniasis patients. These were

confirmed to be female Lu. christophei by the author and

Dr. D. G. Young.

Thus Lu. christophei is regarded as the probable vector

of leishmaniasis in the Dominican Republic. It fulfills the

requirements of readily feeing on man and rodents, the

probably reservior hosts, and is capable of experimentally

transmitting the Dominican Leishmania (see Chapter 3). One

of the difficulties of in studying this sand fly is based on

its long life cycle of more than two months. Futher work

needs to be done on determining the relationship of the long

cycle to the natural habitat. Much also remains to be

determined on the bionomics of this species. The epidemio-

logical data, such as vector efficiency and infection rates

in the field, need to be studied further. The sample of

23 wild-caught female flies available for examination during

this study was insufficient. A long-range program, per-

formed by personnel who could visit the sites at various

times over a two or three year period, is needed.
















CHAPTER 3

GROWTH OF LEISHMANIA-ISABEL STRAIN IN CULTURE MEDIUM,
LABORATORY RODENTS, AND SAND FLIES



Introduction

New World cutaneous leishmaniasis is caused by members

of the Leishmania braziliensis and L. mexicana complexes

(Bray, 1974), but diffuse cutaneous leishmaniasis (DCL) has

been associated only with the L. mexicana complex in the

Americas (Schnur et al., 1983) and with L. aethiopica in

Africa (Bray and Bryceson, 1969). Current knowledge of DCL

is based primarily on studies of L. aethopica in Ethiopia

(Bryceson, 1969, 1970a, b, c) and L. mexicana pifanoi in

Venezuela (Convit et al., 1971). The focus of DCL in the

Dominican Republic is unique owing to the complete absence

of human cases with ulcerating lesions (Bogaert-Diaz,

unpublished data). This is one of the features that has

sparked interest in the identity of this Leishmania.

Schnur et al. (1983) and Kreutzer et al. (1983) believe that

the Dominican parasite differs from other known subspecies

in the L. braziliensis and L. mexicana complexes, but

appears to be closer to strains in the L. mexicana complex.

Lainson (1983) reported that the Dominican parasite

developed in the









anterior midgut (Suprapylaria) of experimentally infected

Lutzomyia longipalpis from Brazil. Schnur et al. (1983)

described some of the biological characters of the parasite

in culture and lab animals, but only in subjective terms.

Since the identity of the Dominican Leishmania remains

undetermined, the commonly used strain, isolated from a

14-year-old female patient from the Dominican Republic, is

designated Leishmania-Isabel strain (Petersen et al., 1982).

The purpose of this study was to quantitate the growth

of the Dominican parasite in comparison to other strains of

Leishmania, to determine the course of infection in suscep-

tible laboratory rodents, to describe the course of infec-

tion in susceptible sand flies, and to effect transmission

with the probable natural vector species.



Methods and Materials



Comparison of the Growth of Three Strains of Leishmania

Stock culture of L. mexicana amazonensis was obtained

from Dr. K. P. Chang, Rockefeller University, inoculated

into a Syrian hamster, Mesocricetus auretus, and later

reisolated and passage one time on Schneider's Drosophila

Medium (GIBCO Laboratories, Grand Island, New York) supple-

mented with 20% (v/v) heat-inactivated (560C, 30min) fetal

bovine serum (FBS) (Hendricks and Wright, 1979). The stock

culture of L. mexicana-Texas (WR-411) was provided by

Dr. Larry Hendricks, Walter Reed Army Institute of Research









(WRAIR) and was handled as above. The culture of Leish-

mania-Isabel strain (WR-336) was provided by Dr. Eileen

Franke, WRAIR, but was not passage through animals. For

each strain, 15 tubes of enriched Schneider's medium were

inoculated so that the Day 0 populations were approximately

2.50 x 105 log phase promastigotes/ml. The tubes were held

in a Hotpack environmental chamber (Hotpack, Inc., Philadel-

phia, PA) at 24.000.50C. The populations were checked

daily for 16 days using a hemacytometer with the aid of a

compound microscope (200X)



Growth of Leishmania-Isabel Strain in Laboratory Rodents

Prior to inoculation in rodents, the Isabel strain was

passage one time on NNN medium (Mansour et al., 1974).

Subadult or young Syrian hamsters and IRC strain mice

(5-7 weeks old) were inoculated via intracardial (IC),

intraperitoneal (IP), or subcutaneous (sub Q) routes. the

dosages used are given in Table 3-1. The animals were

maintained by inoculation group. Two animals from each

species/inoculation group were examined at 30 and 60 days

post-inoculation. Four subcutaneously inoculated hamsters

were examined via xenodiagnosis with sand flies, Lutzomyia

anthophora, at 3, 7, and 11 months post-inoculation; at

15 months, they were killed and assayed, as outlined below,

using Schneider's medium for cultures.

Before killing the animal, 0.2-0.5ml of blood was taken

via heart puncture and added to 4ml RPMI medium (80% RPMI









Table 3-1 Method of inoculation and number of promastigotes
used to infect laboratory rodents with
Leishmania-Isabel strain.



Species Method of Inoculation Size of Inoculum


Hamster subcutaneous 9.00 x 104 promastigotes

Hamster intraperitoneal 2.25 x 105 promastigotes

Hamster intracardial 9.00 x 104 promastigotes

Mouse subcutaneous 1.13 x 105 promastigotes

Mouse intraperitoneal 5.65 x 105 promastigotes


Medium 1640 (GIBCO) + 20% FBS (v/v)). A subcutaneous

aspirate was taken from the left hind paw and placed in

another tube of RPMI. After death, tissue samples were

taken of hind paw skin, liver, and spleen. Impression

smears were made of each on microscope slides. The tissue

samples were then placed in a Petri dish of normal saline

which contained 4000U Penicillin G Sodium (U.S. Biochemical

Corporation, Cleveland, OH) and 1.5mg/ml Streptomycin

Sulfate (U.S. Biochemical Corp.), with one Petri dish/

animal. The Petri dishes were left in a refrigerator (40C)

for 24hrs. The method was adapted from Herrer and Christen-

sen (1975). The following day the Petri dishes were removed

and uncovered in a biological hood. Each tissue sample was

washed twice with normal saline. A small portion of tissue









(9mm2 skin, 27mm3 liver or spleen) was then placed in 2ml

sterile normal saline in a sterile mortar and ground by

pestle until macerated. The solution was allowed to settle

for 1 min then lml of supernant was drawn off and added to a

tube of RPMI Medium. The culture tubes were held at room

temperature for eight days then checked for the presence of

promastigotes, with the aid of a compound microscope (200X).

Impression smears were fixed in absolute methanol, stained

with Geimsa for 30 minutes, then observed with a microscope

(1000X).



The Growth of Leishmania-Isabel Strain in the Sand Fly

Four-month-old BALB/c mice were inoculted in the hind

foot pads with approximately 2.5 x 105 promastigotes of

Leishmania-Isabel strain. The mice were held for four to

six weeks to allow development of the histiocytoma, during

which time the foot pad grew to two to three times normal

size. Four to seven-day-old laboratory-reared Lutzomyia

anthophora, a species from Texas and Mexico, were allowed to

feed on the infected hind foot pads of the mice. The body

and tail of the mouse was covered so that only the hind feet

were exposed to the sand flies. After feeding, the sand

flies were held in groups of 20 to 50 flies, in 40dr rearing

vials, and held in a Hotpack environment chamber at

23.00.50C and 705%RH. A sample of flies were dissected on

each day (Day 1 to 7 post-feeding) so that a total of

15 infected flies were observed for each day. After









dissecting out the digestive tract in Medium 199 (GIBCO),

the mouthparts, head, and digestive tract were examined for

the presence of leishmanial promastigotes, with the aid of a

microscope (100X, 200X). The number of infected flies was

noted along with location, number and shape of the para-

sites. To determine if the parasites observed were infec-

tive, a sample (pooled by for Day 3, 4, and 5) was

inoculated into the hind foot pads of two six-week-old

hamsters.



Transmission of Leishmania-Isabel Strain by Lutzomyia
christophei

Female F3 generation Lu. christophei were allowed to

feed on the swollen hind foot pads of Leishmania-Isabel

(WR-336) infected BALB/c mice. The sand flies fed on the

mice five to seven weeks post-inoculation. The flies were

then held individually in 7dr vials in a Hotpack chamber,

23.000.50C and 805%RH, until death or until they were

ready to refeed, usually seven or more days after the first

bloodmeal. Females that died one to seven days post-feeding

were dissected and examined, to correlate the infection in

Lu. christophei with that in Lu. anthophora, performed as

above. For their second bloodmeal, the flies were released

into a feeding chamber with an anesthetized noninfected

BALB/c mouse, covered with a cloth sleeve except for the

hind feet and tail. The refed flies were then recaptured

and held individually in 7dr vials until death. Upon death,

the flies were dissected to determine the state of









infection. The mice used for refeeding the sand flies were

maintained in the laboratory for three weeks before attempt-

ing to xenodiagnose leishmanial infection with Lu. antho-

phora and/or diagnosing through culturing of spleen and

liver tissue sample and subcutaneous aspirate in Schneider's

medium, performed as above. Female Lu. anthophora were used

for xenodiagnosis due to the unavailability of female Lu.

christophei at the time.



Results



Comparison of the Growth of Three Strains of Leishmania

The two strains of Leishmania mexicana, L. mexicana-

Texas and L. m. amazonensis grew at rates that were not

statistically different, but their growth rates were much

faster than that of the Leishmania-Isabel (t-test, p = 0.05)

(Fig. 3-1). The L. mexicana strains maintained log phase

growth until Day 6, stayed in a stationary phase until

Day 10, and then decreased rapidly. Cultures of the Isabel

strain did not achieve peak population growth until Day 12,

after which they declined slowly. Post-peak populations

were difficult to estimate due to the large number of dead

or inactive promastigotes present in the samples, only

motile promastigotes were counted.






























0




*- Leishmania-Isabel
Sa- L. mexicana-Texas
4* L. m. amazonensis



0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Days












Figure 3-1. Daily estimated mean population of three
strains of Leishmania grown in Schneider's
medium, at 25.00C.








Growth of Leishmania-Isabel Strain in Laboratory Rodents

Leishmania-Isabel-inoculated hamsters and IRC strain

mice examined at 30 and 60 days post-inoculation showed no

externally visible signs of infection. Four hamsters were

inoculated via subcutaneous route and maintained for over

60 days, by 75 days post-inoculation, only one hamster

exhibited a very slight swelling of the hind paw. All four

were xenodiagnosed using lab-reared Lu. anthophora and were

infected at this time. By 7 months post-inoculation, the

cutaneous infection had apparently self-cured because none

of 30 sand flies feeding on the "infected" foot of each

hamster developed promastigote infections. Aspirate

cultures taken at seven months were also negative. When the

four hamsters were killed at 15 months, no parasites were

isolated by aspirate, liver, or spleen culture.

The most sensitive method for determination of infec-

tion, besides xenodiagnosis, was by culturing of splenic

tissue. No parasites were observed in the cultures of heart

blood or in skin impression smears. Active infections were

recovered from all animals killed at 30 days, by spleen

culture and from all, but one mouse, via liver culture. The

results were much more variable at 60 days (Table 3-2). At

60 days, no leishmaniae were observed or isolated from one

mouse (IP) and two hamsters (1IP, 1IC). Animals inoculated

via subcutaneous injection were the most readily confirmed

as infected by the different methods of culturing and

culturing and impression smears (Table 3-2, Fig. 3-2).











Table 3-2 Number of animals examined determined to be infected with Leishmania-
Isabel strain via different isolation methods.


Impression
Cultures Smears
Inoculation Day Heart Foot
Species Method Examined Blood Aspirate Skin Spleen Liver Skin Spleen Liver


Mouse sub Q 30 0 0 0 2 1 0 1 0

60 0 0 1 1 1 0 1 1

IP 30 0 0 0 2 0 0 1 1

60 0 0 0 2 0 0 2 0

Hamster sub Q 30 0 0 1 2 2 0 2 2

60 0 1 1 2 2 0 2 2

IP 30 0 0 0 2 2 0 2 2

60 0 0 0 1 0 0 0 0

IC 30 0 0 0 2 2 0 2 1

60 0 0 0 1 0 0 1 1


Note: n = 2, except Mouse-sub Q-60 days where n = 1.































ItM


p













Figure 3-2. Leishmania-Isabel strain amastigotes in a
spleen impression smear stained with Giemsa
(1000X).









Growth of Leishmania-Isabel Strain in the Sand Fly

Promastigotes were observed in some of the Lu.

anthophora females in each sample from Day 1 to Day 7

post-feeding on a Leishmania-Isabel infected BALB/c mouse.

The percent infected increased with time, but the percent

with bacterial contamination (Leishmania infection undeter-

minable) decreased with time, because of the high death rate

in these flies. In the sample from Day 1, only one promas-

tiogote was observed in 30 fly dissections; however, a few

amastigote-infected macrophages were observed in the blood

meals in five of the dissections which were later stained

and examined with the aid of microscope (1000X). For the

remaining days, infected flies represented 53.6% to 75.0% of

the sample dissected each day. The infections were of

several hundred to several thousand promastigotes/fly

(Table 3-3). Promastigotes were first observed only in the

anterior midgut, (Fig. 3-3) near the stomodael valve in

Day 2 and Day 3 flies. By Day 4, parasites were observed in

the posterior pharynx, as well. On Day 5, promastigotes

were observed in the mouthparts in 4 of the 15 infected

flies. Mouthpart infections were observed 12 of 15 and 13

of 15 infected Day 6 and Day 7 flies, respectively

(Fig. 3-4). The shape of the promastigotes was highly

variable in young infections (Day 2 and 3); however, only

elongate forms were observed anterior of the stomodael valve











Table 3-3. The course of development of Leishmania-Isabel strain in the sand fly,
Lutzomyia anthophora, based on daily dissections, Days 1 to 7 post-
feeding.



Day post- Status of Ovarian # infected Location, number &
feeding blood meal status flies/sample shape of parasites


RBC's intact,
meal dark red





RBC's intact
meal black



Blood remnants
present



Meal totally
excreted in most
flies

Meal excreted
in all flies


undeveloped






undeveloped




slightly
developed



well
developed


eggs fully
developed


*/30






15/28




15/27




15/25



15/22


anterior MG+ w/ blood meal,
only 1 promastigote ob-
served in dissections,
stumpy form, amastigotes
observed in macrophages in
5 dissections

anterior MG w/ blood meal,
100's observed in all
infected flies, primarily
stumpy forms

anterior MG, forward to
stomodaeal valve, 100's
observed, stumpy and some
elongate forms

1000 in anterior MG, 50-
100 in pharynx, elongate
forms

1000's in anterior MG, 50-
100 in pharynx, 10-20 in
cibarial region, 5-10 in
mouthparts, elongate forms











Table 3-3. Continued.


Day post- Status of Ovarian # infected Location, number &
feeding blood meal status flies/sample shape of parasites


6 as Day 5 commenced 15/21 as Day 5
oviposition

7 as Day 5 finished 15/20 1000's in anterior MG,
oviposition 100's in pharynx, 20-50 in
mouthparts, elongate forms


* parasites probably still in amastigote stage, infection not determinable

+ MG = midgut
















































Figure 3-3. Promastigotes of Leishmania-Isabel strain from
the anterior midgut of the sand fly, Lu.
anthophora (1000X).




Full Text

PAGE 1

PHLEBOTOMINE SAND FLIES (DIPTERA:PSYCHODIDAE) AND DIFFUSE CUTANEOUS LEISHMANIASIS IN THE DOMINICAN REPUBLIC BY RICHARD N. JOHNSON 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 198 4

PAGE 2

To my parents, Robert and Mary Johnson

PAGE 3

ACKNOWLEDGEMENTS This research could not ha v e been done without the advice, encouragement, and assistance of man y people to whom I am greatly indebted. M y super v isory committee was helpful in guiding m y course of stud y : Dr. Jerr y F. Butler, chairman; Drs. Donald W. Hall, Donald J. Forrester, and Ellis C. Greiner; and especiall y Dr. Da v id G. Walton, World Health Organization, Y oung. Dr. Br y ce C. first suggested the project and was instrumental in securing financial support under WHO grant # 810314. Other support was generousl y gi v en b y the Steffan Brown Foundation and the U ni v ersit y of Florida. I am also indebted to the personnel of the Dominican Dermatolog y Institute, including Dr. Huberto Bogaert-Diaz (Director) Dr. Denis de Martinez, Lie. M argarita d e Quinones, Lie. Mar v is Lebron, Tomas Castro, and particularl y Francisco Castillo, for his friendship and assistance. Gulf and Western Americas Corporation graciousl y pro v ided facili ties at the Pedro Sanchez field station. The staff and their families at this ranch pro v ided the friendship that turned it into a home for more than a y ear. Dr. Rodrigo Zeledon and Juan Murillo, Ins ti tuto Costarricense de In v estigacion y Ensenanza en N utricion y Salud, were of

PAGE 4

great help during their brief visit to the Dominican Republic in 1983. I wish to thank Dr. Eskild Petersen, University of Arizona, for his advice and assistance. I am indebted to Dr. Sam Telford for the careful instruction on the identification of lizard parasites. Dr. Robert Woodruff, Florida Division of Plant Industry, provided valuable advice which facilitated working in the Dominican Republic. Personnel at Walter Reed Army Institute of Research were of much help in realizing the goals of the project; these included MAJ Peter Perkins, Dr. Edgar Rowton, Spec. 4 Pedro Quintero, LTC Donald Roberts (Entomology): CPT Patrick McGreevy, LTC Jonathan Berman, Dr. Eileen Franke (Experi mental Therapeutics). Dr. Charles Woods of the Florida State Museum provided information on the Republic and its mammalian fauna. Rick Sullivan, who concurrently resided in the country, provided extra traps, friendship, and, at times, mutual cornmisseration. Dr. Steven Zarn was instructive in laboratory culturing of Leishmania. I thank Ms. Diana Simon and Mrs. Debra Boyd who handled many of the concerns that arose during my absences from Gainesville. Ms. Edna Mitchell helped in laboratory rodent and sand fly maintenance. Drs. G.B. Fairchild and R.C. Wilkerson gave freely of their knowledge of Latin America. Ors. Richard Endris, Peter Perkins, Andrew Beck, Mr. Eric Milstrey, MAJ Phillip Lawyer, and CPT Terry Klein offered their advice, assistance, and friendship

PAGE 5

throughout gratefully provided by varying times of acquaintance. acknowledge the encouragement and my family and other friends who supportive for the past 28 years. V Finally, I assistance have been

PAGE 6

TABLE OF C O NTENTS ACKNOWLEDGEMENTS LIST OF TABLES .. LIST OF FIGURES ABSTRACT CHAPTER 1 2 INTRODUCTION ..... Literature Review Geography of the Dominican Republic Current Status of Diffuse Cutaneous Leishmaniasis in the Dominican Republic ...... Study Site Selection Study Site Descriptions Objectives ...... SURVEY FOR, AND COLONIZATION OF LUTZOMYIA CAYENNENSIS HISPANIOLAE AND L U TZO.MYIA CHRISTOPHEI .. Introduction Methods and Materials Field Studies Laboratory Rearing Sand Fly Dissections Results Field Studies Laboratory Studies Sand Fly Dissections Discussion v i iii . viii X x ii 1 1 12 13 20 22 27 29 29 30 30 40 46 47 47 53 6 2 65

PAGE 7

Chapter 3 GROWTH OF LEISHMANIA-ISABEL STRAIN IN CULTURE MEDIUM, LABORATORY RODENTS, AND SAND 4 5 FLIES . . . 73 73 74 Introduction Methods and Materials Comparison of the Growth of Three Strains of Leishmania . . . 74 Growth of Leishmania-Isabel Strain in Laboratory Rodents . . . 75 Growth of Leishmania-Isabel Strain in Sand Fly . . . . . 77 Transmission of Leishmania-Isabel Strain by Lutzomyia christophei 78 Results . . 79 Comparison of the Growth of Three Strains of Leishmania . . . 79 Growth of Leishrnania-Isabel Strain in Laboratory Rodents 81 Growth of Leishman-Isabel Strain in the Sand Fly . . . . 84 Transmission of Leishrnania-Isabel Strain by Lutzomyia christophei 89 Discussion SURVEY FOR RESERVOIR HOSTS OF HUMAN LEISHMANIASIS IN THE DOMINICAN REPUBLIC Introduction ..... Methods and Materials Results ... Discussion SUMMARY 90 96 96 97 101 105 111 APPENDICES 1 DOMINICAN LEISHMANIASIS PATIENT PARTIAL CASE HISTORIES . . . . . . . 114 2 COLLECTION SITES AND DATES FOR LUTZOMYIA CAYENNESIS HISPANIOLAE IN THE DOMINICAN REPUBLIC, MAY 1981 AUAGUST 1983 . 116 3 COLLECTION SITES AND DATES FOR LUTZOMYIA CHRISTOPHEI IN THE DOMINICAN REPUBLIC, MAY 1981 AUGUST 1983 118 REFERENCES BIOGRAPHICAL SKETCH 119 126

PAGE 8

Table 1-1 1-2 2-1 2-2 2-3 2-4 2-5 2-6 2-7 3-1 LIST OF TABLES Sand fly species (Lutzomyia) which are known or suspected vectors of leishmaniasis in the New World, by country ... Other mammalian hosts of Leishmania strains which cause human leishmaniasis in the New World, by country ....... Location and dates for flight trap samples in the Dominican Republic Location, trap type, and dates for CDC traps in the Dominican Republic Sites and dates for Disney traps used for phlebotomine sand fly sampling in the Dominican Republic Collection sites and dates for soil samples examined for the presence of sand fly larvae Mean duration S.D.) in days of immature stages of Lu. cayennensis hispaniolae at three temperature regimes, according to sex of sand fly . . Mean duration S.D.) in days of immature stages of Lu. christophei at two temperature regimes, according to sex of sand fly Sites of collection for female Lu. cayennensis dissected ... Method of inoculation and number of promastigotes used to infect laboratory rodents with Leishmania-Isabel strain viii 5 10 35 36 39 41 57 63 66 76

PAGE 9

Table 3-2 3-3 4-1 4-2 Number of animals examined determined to be infected with Leishmania-Isabel strain via different isolation methods ... The course of development of Leishmania Isabel strain in the sand fly, Lutzomvia anthophora, based on daily dissections, Days 1 to 7 post feeding .. Mammal specimens collected at six leishmaniasis case sites in the Dominican Republic. . .... Examination techniques used for mammals collected during survey for reservoir hosts of leishmaniasis in the Dominican Republic, October 1981 to August 1983 ..... ix 82 85 99 104

PAGE 10

Figure 1-1 1-2 1-3 1-4 1-5 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 LIST OF FIGURES The Caribbean region, showing the location of the Dominican Republic, on the island of Hispaniola. . ...... The geographic regions of the Dominican Republic Case sites for patients with diffuse cutaneous leishmaniasis (DCL) in the Dominican Republic . .... Typical terrain in the Cordillera Oriental, Dominican Republic ..... A young Dominican DCL patient with healed lesions on wrist and upper arm .... Field collecting equipment and rearing containers for phlebotomine sand flies Sand fly feeding cage a modified aquarium with plaster of Paris bottom and back The author in front of a flight trap in a cacao grove at Altos de Peguero, El Seibo Prov. . . . . A CDC trap and modified traps A Disney trap, used for collecting rodent feeding sand flies .... Schematic diagram of laboratory techniques for rearing of phlebotomine sand flies .. A modified rnicrotiter plate for rearing individual sand fly larvae .. Collection sites for Lu. cayennensis hispaniolae and Lu. christophei in the Dominican Republic May 1981-August 1983 X 15 16 18 19 19 31 32 34 34 38 43 44 49

PAGE 11

Figure 2-9 2-10 2-11 2-12 2-13 2-14 2-15 3-1 3-2 3-3 3-4 4-1 4-2 Weekly sample populations of Lu. cayennensis at two study sitesin the Dominican Republic ..... Eclosion time, in days after oviposition, for Lu. cayennensis reared under constant conditions . . . Eclosion time, in days after oviposition, for Lu. cayennensis reared under ambient conditions, August-September ..... Eclosion time, in days after oviposition, for Lu. cayennensis reared under ambient conditions, January-February .. Female Lu. christophei feeding on BALB/c mouse Eclosion time, in days after oviposition, for F 1 Lu. christophei reared under constant conditions . . . .... Eclosion time, in days after oviposition, for F 2 and F 1 _L~. christophei reared under constant conaitions ........... Daily estimated mean population of three strains of Leishrnania grown in Schneider's medium, at 25C ..... Leishrnania-Isabel strain amastigotes in spleen impression smear stained with Giemsa (lOOOX) ........... Promastigotes of Leishmania-Isabel strain from the anterior midgut of the sand fly, Lu. anthophora (lOOOX) . . . .. The course of Leishrnania-Isabel strain infection in the sand fly, Lutzomyia anthopora . . Sherman and Tomahawk live traps for small mammals The three species of rodents trapped during the survey. . ....... xi 51 58 58 59 61 64 64 80 83 87 88 98 102

PAGE 12

,-------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 PHLEBOTOMINE SAND FLIES (DIPTERA:PSYCHODIDAE AND DIFFUSE CUTANEOUS LEISHMANIASIS IN THE DOMINICAN REPUBLIC By Richard N. Johnson August 1984 Chairman: Dr. Jerry F. Butler Major Department: Entomology and Nematology A survey for phlebotomine sand flies (Diptera: Psycho didae) in the Dominican Republic revealed that Lutzomyia cayennensis hispaniolae (Fairchild and Trapido) was widely distributed and fairly common. Lutzomyia christophei (Fairchild and Trapido) was more limited in geographic distribution. Specimens of the latter species were obtained by light traps, flight traps, and aspirator collection from human bait and resting sites. Laboratory colonies of both species were established and life-cycle data were obtained. Lutzornyia cayennensis females readily fed on Anolis lizards. Female Lu. christophei readily fed on rodents and were capable of experimentally transmitting a Dominican strain (Isabel-WR336) of Leishmania to BALB/c mice seven to ten days after bi ting infected mice. Development of the xii

PAGE 13

parasite occurred in the anterior midgut in both Lu. christophei and Lu. anthophora (Addis), a species that was also experimentally infected. The course of development in the sand fly was observed by dissecting 15 infected Lu. anthophora on days 1-7 post-feeding. Development in this species was parallel to that observed in the 17 Lu. christophei. Promastigotes from flies four and five days post-feeding were infective to hamsters, as determined by xenodiagnosis with sand flies and spleen culture. Tn culture medium, Leishmania-Tsabel strain grew at a much slower rate than either of two strains of L. mexicana. Hamsters and TRC mice, experimentally inoculated from culture, showed no outward sign of infection until at least 2.5 months after inoculation with the Isabel strain. Tn the Dominican Republic, 10 of the 21 known case sites were visited. Coffee and cacao groves were charac teristic of these sites. Two female Lu. christophei were captured while bi ting a patient. Four species of mammals (170 specimens) were trapped from five of the case sites and examined for leishmaniasis using various methods. None was found to be infected, though 4 of 44 Rattus rattus from one site were seroposi ti ve ( 1: 16) as determined by indirect fluorescent antibody test. Lutzomyia christophei is most probably the vector of di fuse cutaneous leishmaniasis in the Dominican Republic. The identity of the reservoir remains unknown, but R. rattus is the most likely suspect. xiii

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CHAPTER 1 INTRODUCTION Literature Review Phlebotomine sand flies* are biting members of the dipteran family Psychodidae (Quate and Vockeroth, 1981). They are suspected or confirmed vectors of various parasitic agents including phleboviruses, such as sand fly fever virus; bartonellosis (Adler and Theodor, 1957); saurian malaria (Ayala and Lee, 1970); trypanosomes of amphibians, lizards (Anderson and Ayala, 196 8) and mammals (McConnell and Correa, 1964); and leishrnaniasis (Bray, 1974). Leishmaniasis is a complex of diseases which is caused by Leishrnania spp. occurring in many parts of the world. In 1981, the World Health Organization estimated that there were 400,000 new cases of leishrnaniasis in the world, annually. The disease occurs in the Americas, Europe, Africa, and Asia. It has been considered the second most important protozoan disease of man after malaria (Anonymous, 1981). The only known biological vectors are phlebotomine sand flies (Bray, 1974). In the Americas, leishmaniasis In this presentation, sand fly will refer to members of Diptera: Psychodidae: Phlebotominae. 1

PAGE 15

2 occurs mainly among persons living in rural or forested areas (Herrer et al., 1966). Leishmaniasis appears in three basic clinical forms-visceral, mucocutaneous, and cutaneous. In 1948, a subform of cutaneous leishmaniasis was reported independently in Venezuela (Convit and Lapenta, 1948) and in Bolivia (Barrientos, 1948). At first, it was considered a new form of the disease because of its charac teristic features (Convit et al., 1962; Convit and Kerdel Vegas, 1965). Bryceson (1969, p. 709) summarizes these features as follows: 1. There is an initial lesion which spreads locally, and from which the disease dissem inates to other parts of the skin, often involving large areas. 2. The lesions are nodules and do not ulcerate. 3. There is a superabundance of parasites in the lesion. 4. The histology is characteristic in that macrophages full of amastigotes predominate in the lesion. 5. Internal organs are not invaded and there is no history of visceral leishmaniasis. 6. The leishmanin (Montenegro) test is negative. 7. The disease progresses slowly and becomes chronic. 8. Treatment with antimony produces only slight and temporary improvement.

PAGE 16

, 3 By these criteria, other cases have been reported from the USA in Texas (Simpson et al., 1968), Brazil, Ecuador, Mexico, Ethiopia, and Tanzania (Bryceson, 1969). The appearance of the lesions gave rise to the name diffuse (or disseminated) cutaneous leishmaniasis (DCL). The causative agent of the Venezuelan cases was first named Leishmania pifanoi (Medina and Romero, 1962). Areas where cases occurred were endemic for cutaneous leishmaniasis, but not the visceral disease (Convit and Kerdel-Vegas, 1965). Further work determined that rather than being a new para site, DCL was the result of a deficiency in the host's cell-mediated immunity (Bryceson, 1970b; Convit et al., 1971). The disease is caused by the same species of Leishmania that causes ulcerative cutaneous leishmaniasis in the same area, e.g. L. aethiopica in Ethiopia (Lemma et al., 1970; Bray et al., 1973) and L. mexicana mexicana, L. m. amazonensis, and L. m. pifanoi in Central and South America (Lainson and Shaw, 1978). In 1975, a new focus of DCL was reported in the Domin ican Republic (Bogaert-Diaz et al. 19 7 5) Three humans, siblings, were found infected. There have been 2 2 addi tional cases, none of which had ulcerating lesions, sup porting the concept that DCL is determined both by parasite characteristics and a defect in host immunocompetence (Walton, pers. comm.). Studies of some of the patients from the Dominican Republic showed that this defect is a specific

PAGE 17

4 inhibition of lymphocyte-proliferation responses by adherent suppressor T-cells, which has a genetic basis (Petersen et al., 1982). Many of the Dominican DCL patients have been symptomatically cured using hot (45C) water treatment (Neva, pers. comm.) The Leishmania causing DCL in the Dominican Republic remains unnamed, but it appears to be different from strains in the L. braziliensis complex and in the L. mexicana complex based on enzyme electrophoretic mobility assays (Kreutzer et al., 198 3) excreted factor serotype, growth in artificial media, and infecti vi ty and pathogenicity in laboratory rodents (Schnur et al., 1983). Although leishmanaisis may be mechanically transmitted under lab conditions by Stomoxys ca lei trans, stable flies (Lainson and Southgate, 1965), Rhipicephalus sanguineus, brown dogs ticks (Sherlock, 196 4) and Glossina morsi tans, tsetse flies (Lightner and Roberts, 19 8 4) sand flies are the only known biological vectors (Lainson and Shaw, 1978). In the New World, 21 species of sand flies have been reported as natural hosts of Leishmania spp. infecting man, but only 6 species have been determined, at present, to be natural vectors (Table 1-1). Experimentally, New World Leishrnania are capable of infecting a number of sand fly species, most of which are then capable of transmitting the infection to a susceptible mammalian species (Killick-Kendrick, 1979). The amastigote stage is parasitic in vertebrate macrophages (Bray, 1974).

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5 Table 1-1. Sand fly species (Lutzomyia) which are known or suspected vectors of leishrnaniasis in the New World, by country. Country Belize Bolivia Brazil Colombia Suspected or Proven Lutzomyia Vectors 2 olmeca 1 1 3 ongipa pis 1 l 2 ongipa pis 3 amazonensis t d. 2 in erme 1a . 3 m1gone1 3 paraensis 3 pessoa1 wellcomei 2 h t 3 w i mani d .3 an uzei mb 1 2 u rati 1s flaviscutellata 3 l l 3 ongipa pis a 3 trap1 01 L h S l eis mania train L.m.m. L.d.c. L.d.c. L.b.b. L.b.b. L.b.b. L.b.b. L.b.b. L.b.b. L.b.b. ~ Q:l L.m. L.d.c. L.b.

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Table 1-1. Continued Country Costa Rica El Salvador French Guiana Guatemala Honduras Mexico Nicaragua Panama Suspected or Proven Lutzornyia Vectors h 3 s annoni ylephiletor 3 1 1 3 ongipa pis mb 1 3 u rati is 1 1 3 ongipa pis olrneca 3 1 1 3 ongipa pis 1 1 3 ongipa pis olrneca 2 longipalpis 3 .3 gornezi panarnensis 3 a .2 trapi oi ylephiletor 3 6 Leishrnania Strain 1 L.b. L.b L.d.c. L.d.c. L. rn. L.d.c. L.d.c. L. rn. L.d.c. 1-~-:e1-~-:e1-~-:e!!-~E

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7 Table 1-1. Continued Country Suspected or Proven Lutzomyia Vectors L h S l eis mania train Paraguay Peru Surinam USA Venezuela longipalpis 3 peruensis 3 verrucarum umbratilis diabolica 1 1 3 ongipa pis flaviscutellata 3 townsendi 3 L.d.c. I:!-.e.9: L.m. L.d.c. L.m.a. Source: World Health Organization, 1984, pp. 56-61. 1 Leishmania strains: L.m.m. = L. mexicana me x icana, L.d.c. = L. donovani chagasi, 1._e._e. = ~braziliensis braziliensis, ~._e.g. = I:!-.eguyanensis, I:!-.e.E = panamensis, ~-E = ~peruviana, 1-~-~= L.m. amazonensis, ~-~S = L.m. garnhami. 2 Proven vector. 3 Suspected vector.

PAGE 21

8 Following ingestion by the sand fly, a telmophagic feeder, the parasite exsheathes its flagellum to become the promas tigote, which multiplies initially in the hind or midgut, depending on the parasite species. Members of the L. braziliensis complex (Section Peripylaria) develop in the posterior midgut and anterior hindgut before moving anter iorly to effect transmission. Leishrnania donovani and subspecies in the L. mexicana complex (Section Suprapylaria) multiply initially in the anterior midgut (Killick-Kendrick, 1979). Transmission to a vertebrete occurs during the sand fly bite, but the actual mechanism is unknown (Killick Kendrick, 1978). Two extant species of sand flies are known from the Dominican Republic (Fairchild and Trapido, 1950). Two fos sil species have been discovered recently in Dominican amber (Johnson and Young, in preparation), reported to be from the Oligocene Period, 40 to 60 million years old (Sanderson and Farr, 1960). Prior to this study, practically nothing was known abut the distribution or biology of the living spe cies. One of these, Lutzomyia christophei (Fairchild and Trapido, 1950), belongs to the Lu. verrucarum species group (Lewis, 19 6 8) which also contains a number of man-bi ting species (Young, 1979) and at least one vector of leishmania sis in Peru (Lainson and Shaw, 1979). Contrary to Lainson's (1983) statement, nothing was known about the feeding habits of Lu. christophei, prior to the author's study. The other species, Lu. cayennensis hispaniolae (Fairchild and Trapido,

PAGE 22

9 1950), is an endemic subspecies of a species that occurs from Mexico south to Ecuador and French Guiana (Young, 1979) This species is known to feed on poikilothermic vertebrates, though there is one report of females feeding on bats in Venezuela (Deane et al., 1978). The speci fie feeding habits of Lu. c. hispaniolae were not known prior to this study. Laboratory rearing of sand flies is a necessary part of studying disease transmission, because it is an assured method of obtaining uninfected flies. Colonization can also lead to a better understanding of the life cycle of the vector and parasites (Killick-Kendrick, 1978). Successful rearing has been accomplished using various techniques and several different formulations of larval food (Chaniotis, 1967; Endris et al., 1982; Gemetchu, 1976; Young et al., 1981). In the New World, leishmaniasis is a zoonotic disease, with rodents serving as reservoir hosts for the L. mexicana complex, canids for L. donovani, a variety of rodents, procyonids, sloths, dogs, and primates for the L. brazilien sis complex (Table 1-2) (Lainson and Shaw, 1978). In the Dominican Republic, ten species of rodents, two primates, four to five insectivores, and four to six sloths are known from fossils (Varona, 1974; Woods, pers. comm.). Some survived until the time of Columbus (1492 AD), but only two endemic terrestrial species exist today: an insecti vore, Solenodon paradoxus Brandt; and a capromyid rodent,

PAGE 23

10 Table 1-2. Other Mammalian hosts of Leishmania strains which cause human leishrnaniasis in the New World, by cotintry. Country Belize Brazil Colombia Costa Rica Leishmanta Strain L.m. L.d.c. L.b.b. L.m. L.d.c. L.b. French Guiana ~-!?..sf Guatemala L. m. Mexico L.m. Nonhuman Mammalian Hosts rodents: Heteromys, Nyctomys, Ototylomys, Sigmodon(R) 2 dog(R), foxes: Cerdocyon(R), Lyalopex (rodents: Akodon, Oryzornys, Proechimys?) 3 sloth: Choelopus; anteater(R), tamandua(R) rodents: Proechimys(R), (Dasyprocta, Heteromys, Neacornys, Nectornys, Oryzomys, oppossums, Marrnosa, Calurornys, Metachirus?) dog(R) sloths: Bradypus(R), Choelopus sloths: Choelopus Ototylornys rodents: Heterornys, Nyctornys, Ototylornys, Sigmodon

PAGE 24

Table 1-2. Continued Country Panama Leishman}a Strain Nonhuman Mammalian Hosts sloths: Bradypus(R), Choelopus; 11 (primates: proc y onids: Aotus, Sanguinus; Bassaricyon, Nasua, Potos?) Peru (dog?) Venezuela L.m. (rodents: Heteromys, Proechim y s, Zygodontom y s?) Source: World Health Organization, 1984, pp. 56-61. 1 Leishmania strains: L.m.m. = L. mexicana mexicana, L.d.c. = L. donovani chagasi, = ~braziliensis braziliensis, = L.b. gu y anensis, = panamensis, ~-E = ~peruviana. 2 R = Proven reservoir. 3 (Mammal?)--animal found infected in nature, extent of infection not determined.

PAGE 25

12 Plagiodontia aedium Cuvier, both of which are extremely uncommon. Both animals are secreti v e and are found in relativel y undisturbed habitat (Woods, 1981). Introduced mammals have replaced the endemic mammals in most areas. Three species of Old World rodents now occur througout the island in cities, tropical rain forests, and deserts. These are Rattus rattus ale x andrinus (Geoffro y ), the roof or black rat; R. norvegicus (Berkenhout), the Norwa y or brown rat; and Mus musculus brevirostris Waterhouse, the house mouse. Geograph y of the Dominican Republic The Dominican Republic occupies the eastern t w o-thirds of the island of Hispaniola, with Haiti occup y ing the western side. The island is a member of the Greater Antilles, l y ing between latitudes 17' to 20' with the Atlantic Ocean to the north and the Car i bbean Sea to t h e south (Fig. 1-1). 2 The Republic has an area of 43,230km of which roughl y two-thirds is mountainous. Geographicall y the countr y is broken up into four regions, based, in part, on the mountain ranges (sierras and cordilleras). The Eastern region includes the Cordillera Oriental with the Llano Oriental (Eastern Plains) to the south. Directl y west lies the Cibao with the Cordillera Septentrional to the north, just inland from the coast, the Cibao V alle y and the Cordillera Central to the west and south. lies the Linea Noroeste, bordering Hai ti. To the northwest El Sur is the region to the south west of the Cordillera Central; it also

PAGE 26

13 borders Haiti, and contains two smaller mountain chains Sierra de Neiba to the north of Sierra de Baoruco (Fig. 1-2). Most of the 6.2 million inhabitants (1982 census) live on the valley floors that separate the cordilleras, or on the rolling Eastern Plains. About 60% of the Dominicans live in rural and agricultural areas. The majority are small land owners. Much of the land has been cleared for agriculture. The main agricultural products include sugar cane, rice, coffee, cacao, cotton, tobacco, corn, and beef. The country has a tropical maritime climate; in the lower elevations, temperature average 22 to 28C with a range during July 1981 to August 1982 at Pedro Sanchez in El Seibo Province, of 16 to 33C. Annual rainfall averages 1397 to 1524mm with a range of 508 to 2413mm, depending on location. The highest rainfall usually occurs in the eastern portion where the rainy season lasts from May to November (Anonymous, 1977; Sholdt and Manning, 1979). Current Status of Diffuse Cutaneous Leishmaniasis in the Dominican Republic Since the discovery of the first three cases of DCL in the Dominican Republic in 1974 (Bogaert-Diaz et al., 1975), an additional 22 cases have been diagnosed (Bogaert-Diaz, unpublished data) Precise life histories are known only for a few of the patients who have not moved to different localities during the presumed evolution of the disease. Personal data, when recorded, often did not denote the exact

PAGE 27

Figure 1-1. The Cartbbean region, showing the location of the Dominican Republic, on the island of Hispaniola.

PAGE 28

Central America a1 . Jamaica .. .. 6 9 Atlantic Ocean South America Puerto Rico C76 3 . 0 () D ...... U1

PAGE 29

l-----+--------+-----t -20 Figure 1-2. I I I I I I I df/t A, Ye "I, Q CIBAO I I I Cordillera 0 rien1a/ E Slli I SU~---------', llano Orieolal I ------lf-----------+18 70 0 I 50 69 The geographic regions of the Dominican Republic (Cordill e ras and Sierras are mountain ranges).

PAGE 30

17 location of residence. Many patients had moved from the country to towns before being diagnosed. Others have lived in several different areas for various periods of time; thus, current place of residence may not have been where the disease was contracted. Presumed site visitations and interviews led to the confirmation of the probable locality of infection for 15 patients. Brief descriptions of these sites are given below and are shown as confirmed sites in Figure 1-3; the remaining sites are shown as presumed. The unknown incubation period and the relative benignancy of the infection make it difficult to determine the probably date of infection. Eight of the patients had had nodules or plaques for four or more years before the disease was diagnosed. At least 13 of the patients were under four years of age when signs of the infection first appeared, so epidemiological data must be based upon recol lections of parents or other relatives. The most out standing feature of the epidemiology of the disease is its positive correlation to the Cordillera Oriental, as 19 patients (76%) resided in or near this region of the country (Fig. 1-4). Most of the patients have been symptom atically cured of the disease with hot (46C) water treat ment (Neva, pers. comm.) (Fig. 1-5) During the period March to September 1982, a serolog ical survey for leishmaniasis was undertaken by personnel of the Ins ti tuto Dermatologico, in which the author assisted. In the survey, blood samples were taken from residents

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HAI Tl -;-------------+------------+------------l-20 7 2 ;----.----------+----------_J.._18 71 Presumed site 70 )tConfirmed site 0 I 50 I I km N Figure 1-3. Case sites for patients with diffuse cutaneous leishmaniasis (DCL) in the Dominican Republic (see Appendix 1-1 for name of numbered localities).

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Figure 1-4. Typical terrain in the Cordillera Oriental, Dominican Republic. Figure 1-5. A young Dominican DCL patient with healed lesions on wrist and upper arm. 19

PAGE 33

20 living in the area of 7 of the 21 case sites (Fig. 1-3; cases 1-3, 7, 10, 17, 19 and 20, 21, and 22) and two control sites (sites in regions where no cases of leishmaniasis had been diagnosed). Site selection was based on accessibility of the site to vehicular transport and on the probability of obtaining an adequate number of volunteers from the local residents. Indirect fluorescent antibody test ( IFAT) was performed on the sera, revealing that 26.0-48.0% of the samples from antibody, at case sites were seroposi ti ve for titer. At the leishmanial two control 1:8 or higher sites 0% and 14% were determined to be seropositive (de Quinones, unpublished data). The pertinent patient history data are given in Appendix 1. Cases 1-3 are siblings, cases 14 and 15 are son and father, and cases 19 and 20 are neighbors, but unrelated. Four principal during the study. Study Site Selection study sites were One, Pedro Sanchez visited regularly was not a leishmaniasis case site, but was used as a control site, i.e. a site with sand fly habitat but no known cases of leishman iasis. Pedro Sanchez was selected as a typical nonagricul tural wooded area. Due to extensive land clearing for agricultural purposes, pasture, sugar cane planting, and coffee/cacao groves, very little natural forest habitat exists in the Dominican Republic. The Pedro Sanchez site was representative of wooded river bank habitat in the

PAGE 34

21 leishmaniasis endemic region of the Cordillera Oriental. The field station (and author's residence) for the study was located less than O. 5km distant, thus the site was con venient at all times of year. The other three principal sites were leishmaniasis case sites, in all instances the DCL patients were living at or near the sites. All four patients (patients 19 and 20 were neighbors) were young and had not lived elsewhere at the time the disease was diagnosed. Interviews with the patients' parents led the author to believe that the disease was contracted at the sites. Very few changes had occurred at the sites in the intervening years between the appearance of the first signs of the disease and the beginning of the author's research, this was not the case with several other presumed case sites. The system of roads in the Dominican Republic is generally poor, particularly in mountainous regions and the Eastern region, thus accessibility of the site was an important criterion in study site selection. Certainty of the site of disease contraction was another important criterion. Many of the older DCL patients have lived in various localities throughout their lives and do not remember clearly where they were living when signs of the disease first appeared, 20 years or more previous. Another important factor was whether or not personnel of the Institute Dermatologico were familiar with the location of the case site. In a few cases, the patients had visited

PAGE 35

22 rural public clinics where they had been diagnosed, their place of residence at time of infection was not known except in rather general terms. Eleven of the 21 DCL case sites were never visited by the author, primarily due to the factors mentioned above. The three principal study case sites were approximately 35km west (Loma Pena Alta) 18km north (Morro de Miches), and 13km east (Trepada de Jabilla) of the field station and control study site at Pedro Sanchez. All four sites were visited in excess of 100 man-hours during the study. The remaining sites listed below were DCL case sites and were visited one or more times, depending on proximity and accessibility. Due to the concentration of cases in the Cordillera Oriental, this area received the greatest amount of attention by the author. Study Site Description Pedro Sanchez, El Seibo Province Altitude: 76m This site consists of a gallery forest along the banks of the Rio Seibo on the southern outskirts of the village of Pedro Sanchez. The Rio Seibo is 0.5 to l.Sm deep, depending on the exact location and time of year, and 4 to 6m wide in this region. The wooded border on the northwest bank was 20 to 50m wide and extended for several kilometers. It con tained several trees with diameter up to lm, a moderately thick shrub understory. A section of forest, with a length

PAGE 36

23 of 40m, was used for this study, through August 1982; when the site was revisited in May 1983, it had been extensively altered for agricultural use. The remaining study sites, listed alphabetically, all are DCL case sites where a patient is living, or was living at the presumed time of infection. Altos de Peguero (5km E by 5km N of El Seibo), El Seibo Province (Fig. 1-3, #17) Altitude: 7 2m The case site is on the northern side of an isolated ridge on the southern edge of the Cordillera Oriental. Several coffee and cacao groves are present in the area, the largest of which is a 2ha cacao grove about 50m from the former residence of the DCL patient. Coffee and cacao groves also contain scattered larger hardwood trees to provide light shade. There are no major streams or rivers in the immediate vicinity, the closest being 1.5km distant. Carrasco ( 10km S of Rio San Juan) Maria Trinidad Sanchez Province (Fig. 1-3, #23) Altitude: 15m The area surrounding the patient's former residence is flat pasture for at lease 2km, with the e x ception of a 1.Sha cacao grove, 400m distant from the residence. A small stream runs next to the grove and forms a small pool (7m diameter) where the children of the area are said to swim.

PAGE 37

24 La Culatica (10km S of Nisibon), Altagracia Province (Fig. 1-3), #1-3) Altitude: 150m The patients' (three siblings) residence was located on a small hill above a small (0. Sha) coffee grove. Other coffee and cacao groves are in proximity to the house site, some of which are owned by the patients' father. A small wooded stream runs below the site, through parts of the coffee grove. This locality is situated in the northeast portion of the Cordillera Oriental. Iguana Arriba (23km NE of Bani), Peravia Province (Fig. 1-3, ill Altitude: 152m The exact locality of the DCL patient's residence was not known, but the area consists of extensive coffee plantings throughout low mountain terrain. A few small streams are present in the low areas between ridges. Larger trees are found along the streams and in the coffee groves, providing shade. Loma Pena Alta (13km NW of Hato Mayor), El Seibo Province (Fig. 1-3, #21) Atltitude: 4 4 2m The DCL patient lived at the top of the ridge, adjacent to a 0.Sha coffee grove. The eastern side of the ridge was shrub-overgrown pasture. Some coffee groves were present on

PAGE 38

25 the western side, which was mostl y lightly forested with numerous rocky outcroppings. Monte Claro ( 16km NE of Cotui) Sanchez Ramirez Province (Fig. 1-3, #24) Altitude: 7 6m A O. Sha coffee grove is 10m distance from the site where the DCL patient lived at the time of infection. The area consists of rolling hill pasture, with the coffee grove being the onl y wooded area in a radius of 1km from the house. The Rio Chacuey has a lightly wooded border, and is about 1km distant from the site. Morro de Miches (10km S of Miches), El Seibo Province (Fig. 1-3, # 19, 20; Fig. 1-4) Altitude: 305m Two unrelated patients li v e at this site, approximately 100m apart and separated by the peak of the ridge. One patient lived besided a 0.25ha coffee grove which contained several large shade trees. The grove is separated from nearb y wooded areas b y pasture and cultivated plots. A small stream runs down the ridge, 100m from the house site. The other patient's residence was located on the northern edge of a rather extensive mixed planting of co::fee and cacao which had a length of about O. 5km and a width of 10-30rn.

PAGE 39

26 Najayo Arriba (20km NW of San Cristobal), San Cristobal Province (Fig. 1-3, #5) Altitude: 400m The patient's residence is located on the side of a ridge immediately above a small (0. Sha) coffee grove. A small stream runs along the bottom of the ridge, about 75m from the house. The site is located on the southern edge of the Cordillera Central. Rio Llano (10km W by 30km N of Higuey), Altagracia Province (Fig. 13 #14 and 15) Altitude: 250km The patients (father and son) lived in a house 15m from a small stream. A small (0.Sha) coffee grove is situated on the opposite side of the stream. Rio Guanche is less than 0. 5km distant. A gallery forest, 10-3 Orn wide, runs along the banks of the river. This site is in the eastern portion of the Cordillera Oriental, about 10-15 km ( straight line distance) south of La Culatica. Trepada de Jabilla (2. 8km N of Las Cuchillas), El Seibo Province (Fig. 1-3, #7) Altitude: 130m The patient's residence was approximately 10m from the edge of a rather extensive coffee grove 3ha). As with other coffee groves, there are some larger shade trees present. The terrain is rolling hills, primarily pasture,

PAGE 40

27 south of the Cordillera Oriental. The Rio Soco borders on side of the coffee grove and is 0.5 to 2.0m deep, depending on location and season, and generally 10 to 12m wide. Several other coffee groves exist in the area. Objectives In 1975, Bogaert-Diaz et al. reported the discovery of three human cases of diffuse cutaneous leishmaniasis (DCL) in the Dominican Republic, the first autochthonous cases of cutaneous leishmaniasis known in the country or in the entire West Indies, e x cept Trinidad, a continental island. Over the succeeding four years, a concentrated search for additional cases, carried out under the National Leprosy Program, revealed 15 more cases of DCL. As of March 1984, 25 cases of DCL have been diagnosed. Surprisingl y none of the patients had ulcerating lesions. Interest in this unique situation led to grant support from the World Health Organization to the Instituto Dermatologico Dominicano for epidemiological studies of DCL in the Dominican Republic, beginning in 1981. Some objectives of the author's research, listed below, were included in the project. Field studies were performed in the Dominican Republic during the periods 19-24 May 1981, 27 July 1981 to 2 August 198 2 and 18 May to 3 August 1983. Laboratory studies were performed at a field station (Pedro Sanchez) and at the Institute Dermatologico in Santo Domingo, the University of Florida, and Walter Reed Arm y Institute of Research.

PAGE 41

A. Field Studies: 1. To survey for the presence of phleboto mine sand flies at selected locations in the Dominican Republic. 2. To study the ecology of sand flies including host preference, resting sites, population dynamics, and seasonal and geographic distribution for species located in the surve y 3. To identif y the wild and/or peridomestic reservoir host(s) of leishmaniasis. B. Laboratory Studies: 1. To establish laboratory colonies of indigenous phlebotomine sand flies. 2. To stud y the life c y cle of Leishmania in the sand fly. 3. To determine the vector potential of these flies. 4. To further elucidate some of the biolog ical differences between the Dominican Leishmania and other known Leishmania spp. 28

PAGE 42

CHAPTER 2 SURVEY FOR, AND COLONIZATION OF, LUTZOMYIA CAYENNENSIS HISPANIOLAE AND LUTZOMYIA CHRISTOPHEI Introduction Phlebotomine sand flies, the only known biological vectors of leishmaniasis, also transmit other parasitic agents of man and other vertebrates (Adler and Theodor, 1957). A new focus of leishmaniasis was discovered in 1974 in the Dominican Republic (Bogaert-Diaz et al., 1975), the eastern portion of the islnd of Hispaniola. Only two extant species of sand flies are known from Hispaniola, Lutzomyia cayennensis hispaniolae (Fairchild and Trapido, 195 0) and Lu. christophei (Fairchild and Trapido, 1950). Both species were collected from tree trunks and buttresses, primarily, but little was known about the habits and range of these species. Lutzomyia cayennensis hispaniolae is an endemic subspecies of Lu. cayennensis (Floch and Abonnenc) that ranges from Ecuador and French Guiana north to Mexico. Most members of the Lu. cayennensis species group are reported as reptile feeders (Young, 19 79) Lutzomyia christophei is a member of the Lu. verrucarum species group that contains an number of man-bi ting species (Lewis, 19 6 8) including one suspected vector of leishmaniasis (Lainson and Shaw, 1979). 29

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30 This study was conducted to determine the geographic range, the habits, and life cycle of the two species. Methods and Materials Field Studies Chaniotis (1978) and Young (1979) reviewed techniques used for sampling phlebotomine sand flies. The methods used in the present study were aspirator collections at resting sites, man-bi ting collections, sticky paper traps, flight traps, CDC light traps ( Sudia and Chamberlain, 19 6 2) and Disney traps (Disney, 1966). Specific equipment preparation and field techniques for aspirator collections were given by Endris et al. (1982) (Fig. 2-1). Aspirator collections were made at various sites around the country, mostly from tree trunks, but also from tree holes and rock crevices. Cigarette smoke was occassionally used to disturb resting sand flies from the latter two types of resting sites. Live wild-caught sand flies were transported to the field station at Pedro Sanchez where they were either held in a feeding cage (Fig. 2-2) for host preference studies or kept individually for oviposition and later dissected for parasites. The other techniques were used at five leishmaniasis case sites or the study site at Pedro Sanchez (see Chap ter 1). Attempts to collect sand flies from human bait at

PAGE 44

(A) 120 ML SPEC! MEN CONTAINER FULL SCALE SCREEN LID SOLID LID PLASTER . OF PARIS --'. ::: (C) ASPIRATOR (B) 7 DR VIAL FULL SCALE ONE-THIRD SCALE Figure 2-1. Field collecting equipment and rearing containers for phlebotomine sand flies (from Endris et al., 1982). (A) Collecting con tainers. (B) Oviposition/larval rearing vial. (C) Aspirator. 31

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/ 0 0 Figure 2-2. 0 0 0 / 0 0 0 O NE TIURO SC ALE 0 0 / / '~/, /; / IN S ET S IDE V IEW WITH SLEEVE Sandy fly f e eding cage--a modified aquarium with plaster of paris bottom and back (from Endris et al., 1982). w N

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33 Trepada de Jabilla were made various times during the day and at dusk during November 1981 to July 1982 and May 1983. Similar attempts were also made at Loma Pena Alta at dusk on various days in June and July 1983. A sticky-paper trap of double-sided tape on a 25cm square wooden frame was used at Pedro Sanchez from 21-23 May 1981. The trap was placed in the crotch of a tree (lm high) known to harbor resting sand flies. Flight traps (Fig. 2-3) and CDC light traps (Fig. 2-4) were set at various sites (Tables 2-1, 2-2). Two modified CDC light traps were also employed. In one, a UV light source was the second type substituted for the normal light had no light source, instead source; a cage containing a hamster, Mesocricetus auretus, was suspended above the trap (Fig. 2-4) The light traps were run on nights when the moon was less than one-half full, or when the sky was mostly overcast. CDC traps were turned on in late afternoon and taken down the following morning. Flight traps were set up in coffee or cacao groves, with one exception (lightly wooded stream bank at Hato Mayor). The flight traps were emptied every two to four days. The trap collections were checked for sand flies with the aid of a dissecting microscope (7-30X). Disney traps (Fig. 2-5) were used at two sites with hamsters serving as bait (Table 2-3). The bottom of cake pans (22.5 x 30.0cm) or cookie sheets (27.0 x 38.0cm) were covered with a thin layer of mineral oil or castor oil. The condition of the hamsters was

PAGE 47

34 Figure 2-3. The author in front of a flight trap in a cacao grove at Altos de Peguero, El Seibo Prov. Figure 2-4. A CDC trap and modified traps (from left CDC with UV light source, normal CDC, and hamster baited trap).

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Table 2-1. Location and dates for flight trap samples in the Dominican Republic. Location Date 35 Altos de Peguero cacao grove 9-25 Mar, 1-28 May 1982 Carrasco cacao grove Hato Mayor wooded stream bank Loma Pena Alta coffee grove Monte Claro coffee grove Morro de Miches coffee grove Pedro Sanchez wooded river bank Trepada de Jabilla coffee grove 3-10 Jul 1982 17 Aug 1981 24-27 Jul 1982 9 Jun-3 Aug 1983 19-26 June 1982 7-14 Aug 1981 5 Oct-3 Nov 1981 20-22 May 1981 6 Nov-14 Dec 1981 18 Jan-26 Jul 1981 23 Ma y -18 Jun 1983

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36 Table 2-2. Location, trap type, and dates for CDC in the Dominican Republic. Location # And Trap Type 1 Date 2 Altos de Peguero 3 CDC 2-3 Apr 1982 27-28 May 1982 Carrasco 2 CDC 9-10 Jul 1982 La Culatica 1 CDC 7-8 Jun 1983 1 CDC-UV Loma Pena Alta 2 CDC 26-27 Jul 1982 2 CDC 2-3 Jun 1983 2 CDC-UV 2 CDC-HB 1 CDC-UV 23-24 Jun 1983 1 CDC-HE 1 CDC-UV 9-10 Jul 1983 2 CDC-UV 17-18 Jul 1983 1 CDC-UV 23-24 Jul 1983 3 CDC-UV 30-31 Jul 1983 Monte Claro 2 CDC 25-26 Jul 1982 4 CDC 21-22 May 1983 2 CDC-HE Morro de Miches 1 CDC 22-23 Sep 1981 2 CDC 18-19 Jul 1982 3 CDC 29-30 May 1983 2 CDC-UV 2 CDC-HB

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Table 2-2. Continued Location Pedro Sanchez Trepada de Jabilla 1 # And Trap T y pe 2 CDC 1 CDC 2 CDC 2 CDC 1 CDC 2 CDC 2 CDC-UV 2 CDC-HB 1 CDC 1 CDC-UV 1 CDC 2 Date 37 20-21 Ma y 1981 7-8 Aug 1981 5-6 Jun 1982 18-19 Jan 1982 16-17 Apr 1982 21-22 May 1982 30-31 Ma y 1982 9-10 Jun 1982 30 Jun1 Jul 1982 14-15 Jul 1982 26-27 May 1983 30-31 May 1983 18-19, 19-20 Jul 1983 1 cDC-UV with blacklight source, CDC-HB hamster baited trap, no light (see Fig. 2-4). 2 Frorn approximately 1s 30 hrs Da y 1 10 0 hrs Da y 2

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Figure 2-5. used flies. A Disney trap, feeding sand 38 for collecting rodent

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39 Table 2-3. The sites and dates for Disney traps used for phlebotomine sand fly sampling in the Dominican Republic. Site Loma Pena Alta Trepada de Jabilla # traps 2 2 4 2 Date 8 July 3 August 1983 28 May 15 June 1982 23 May 2 June 1983 7 9 June 1983

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40 checked twice a day by local personnel and food and water were provided at all times. Hamsters were exposed for three to four days and then replaced by others. Soil samples (dirt, humus, and leaf litter) were collected from tree buttresses and tree holes at five sites where sand flies were (1-4 liters in volume) common (Table were placed in 2-4) plastic Samples bags for transport back to the field station. For observation, a sample was transferred to a white plastic tray, and the material was examined with the aid of a dissecting micro scope (7-30X). Larger leaf matter was checked for the presence of larvae or pupae and then discarded. Recovered larvae were held in larval rearing vials (Fig. 2-lb) with a small amount of larval food (Young et al., 1981), and held until adult emergence for species identification. The rest of the sample was returned to the plastic bag and periodi cally checked for emerged adults or immature stages. The bags were held at ambient temperature (19-30C) for up to two months. Laboratory Rearing Techniques used for laboratory rearing of Dominican sand flies (Fig. 2-6) were those described by Endris et al. (1982). Individuals from an egg batch were raised together or individually, as described by Perkins (1982) in a modified tissue culture plate (Fig. 2-7) Newly hatched (within 6 hrs) first instar larvae were transferred from

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41 Table 2-4. Collection sites and dates for soil samples examined for the presence of sand fly larvae. Site Date # Samples Microhabitat Altos de Peguero 28 May 1982 3 leaf matter at tree basecacao grove 2 Jul 1982 2 as above Loma Pena Alta 20 Jul 1982 2 leaves and humus at tree base-coffee grove Monte Claro 25 Jul 1982 5 as above Pedro Sanchez 15 Aug 1981 3 leaves and humus at tree base-gallery forest 27 Aug 1981 4 as above 22 Sep 1981 3 as above 30 Oct 1981 4 leaves and humus at tree base-upland woods 25 May 1982 2 as above 15 Jun 1982 3 as above Trepada de Jabilla 2 Dec 1981 3 leaves and humus at tree base-coffee grove 18 May 1982 5 leaves and humus at tree base and in tree holecoffee grove 8 Jun 1982 3 as above

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Figure 2-6. Schematic diagram of laboratory techniques for rearing of phlebotomine sand flies. (1) Plaster of Paris at bottom of rearing vial in moistened with tap water. (2) Wild-caught or lab-reared gravid females are placed individually in vials and drop of sugar solution is placed on each screen lid. (3) A solid lid replaces the screen lid after eggs are deposited. (4) Larval food is sprinkled on the plaster of Paris anytime before the larvae hatch. (5) Additional larval food is added as larvae grow. (6) Lidless vials containing pupae are placed in the feeding cage; fruit slices provide a sugar source for emerging adults. (7) For a bloodmeal source, a vertebrat e is placed in the feeding cage (Endris et al., 1982).

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fll1lllb 11 I I I I I a l~ \.: \; :< I .: ... . .. : . .. / :~> \ f 1 NOT TOSCALE 7 6 3 5 SLEEVE REMOVED f

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Figure 2-7. 44 A modified microtiter plate for rearing individual sand fly larvae (approximately 0.5cm layer of plaster of Paris in each well).

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45 oviposition vials to wells in the tissue culture plate (one larva per well). The larvae were checked at approxi mately the same time every day. Individual rearing was performed under different temperature-humidity regimens for both species of sand flies. Lutzomyia christophei larvae were held in a Hotpack chamber (Hotpack, Inc., Philadelphia, PA) at 23C or 28C. High humidity was maintained by placing the plates with moistened plaster of Paris in tightly sealed plastic boxes lined with moist paper towels. Lutzomvia cayennensis were reared under three sets of conditions. The first group was reared in a Hotpack chamber at 28C, as above. The second and third groups were held at ambient conditions in the Dominican Republic of 24 to 33C and 60 to 95%RH (August-September), and 16 to 28C with 30 to 90%RH (January-February), respectively. Eclosion was checked in the vials or plates once per day. Adults were released into the feeding chamber. At the field station, various types of locally available fruit were provided as a sugar source. These included grapefruit, orange, or sweet lemon sections (skin removed); cashew fruit; and peeled mango skin. Lutzomyia christophei reared at Walter Reed Army Institute of Research (WRAIR) were provided with apple slices. For Lu. christophei females, an anesthetized Rattus rattus, Syrian hamster (Mesocricetus auretus), or BALB/c mouse (Mus musculus) was provided as a blood source. An Anolis sp. lizard was regularly provided as a blood source for female Lu. cayennensis; occasionally a

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46 human hand or hamster was offered. The lizard was either restrained in a small cylinder of hardware cloth or was allowed to be free. If free, the mouth was taped shut to prevent the ingestion of sand flies. To determine the age at first feeding for either species, all flies emerging in a six hour period were held separate by species. For Lu. cayennensis, an Anolis lizard was provided until all females were fed or dead. If neces sary, the lizard was exchanged every two to three days for a similar sized, conspecific lizard. The lizards were not restrained. For Lu. christophei females, an anesthetized hamster was provided for one hour two times per day, approx imately 0903 hrs and 1530 hrs. Sand Fly Dissections A sample of both male and female sand flies from various survey sites was routinely dissected for species determination, using the methods given by Young (1979), or by simply cutting off the head and the last few abdominal segments and mounting them medium (Young, pers. comm.). in a drop of Hoyer' s mounting In addition, wild-caught and lab-fed sand flies were dissected upon death to examine the digestive tract for parasites. The dissections were done in normal saline or in Medium 199 (GIBCO, Grand Island, NY) on a microscope slide and observed with the aid of a compound microscope (200X or 450x). Permanent slides were made by removing the cover slip and allowing the liquid to dry, then

PAGE 60

47 fixing with absolute methanol and staining with Giemsa for 20 minutes. After drying, a drop of Euparal mounting medium was added and a cover slip placed over it. Results Field Studies Adult Lu. cayennensis were aspirator-collected from tree trunks (10 cm diameter and larger) at many localities in the Dominican Republic (Fig. 2-8, Appendix 2) At all sites, there was sufficient tree and shrub vegetation to provide a "forest-floor" type habitat, with little ground cover vegetation. The sites were shaded, to varying degrees. Many sites in El Seibo Province were visited more than once. This species was also recovered from flight trap samples at three sites (Appendix 2). None was recovered b y an y other method. Adult Lu. christophei were recovered at seven sites by flight trap, CDC trap, or aspirator collection from rock and tree crevices, primarily of ceiba trees (Ceiba pentandra) (Fig. 2-8, Appendix 2) The use of cigarette smoke facilitated their capture from deeper crevices. Six of these sites were leishmaniasis case sites; the seventh was about 5km distance from a case site. No Lu. christophei were collected by hamster-baited CDC traps, Disney traps, or man-biting collections. At Loma Pena Alta, both Disney traps were within lm of resting sites of Lu. christophei females. The man-biting collections attempted

PAGE 61

Figure 2-8. Collection sites for Lu. cayennensis hispaniolae and Lu. christophei in the Dominican Repuglic from May 1981 August 1983 (See Appendix 2-1, 2-2)

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HAI Tl 72 ~--1------------t--------------t--18 no Lutzomy i a recovered Lu. cayennensis only Lu. cayennens is and Lu chi:istophei 0 I 50 I I km

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50 at this site were performed within 2 to 3m of tree crevices known to harbor resting sand flies. Two female sand flies were recovered while biting DCL patient #25, in September 1983; these were later identified as Lu. christophei, by the author. Populations of Lu. cayennensis at Pedro Sanchez varied throughout the year and the population at Trepada de Jabilla followed the same trends from January through July 19 8 2 (Fig. 2-9). The population sample was based on the total number of flies aspirator-collected from 10 marked trees. The sample from Pedro Sanchez was always larger than the same week's sample from Trepada. At Pedro Sanchez, the samples varied from a high of 67 flies to a low of 2 flies; the total for the 39 samples was 986 flies (536 males and 449 females). At Trepada, the high was 39 flies and the low was O flies; the total for the 22 samples was 269 flies (156 males and 113 females). Fewer flies were collected during the period February to mid-May 19 8 2, which corres ponds to the dry season and the first two weeks of the rainy season. The population level began to rise approximatel y two and one-half weeks after the beginning of the rainy season (3 May 1982). The male:female ratio, on dates when females were collected, (x = 1.90) males/female (8 ranged out of from 0.90 22 samples to had 3.00 no females). Blood-fed or gravid females comprised O to 100 % (x = 31.3 % ) of the females at Pedro Sanchez and Oto 75 %

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70 VI 60 Ill Ill L 0 50 ...___ -0 IU ..., 40 u Ill 0 u 30 VI Ill .._ 20 "'10 Au Figure 2-9. Oc 19 81 No De Jo Fe Month .--------Trepada de Jabl I la ----Pedro Sanchez Mr 1982 Ap My Jy Weekly sample populations of Lu. cayennensis at two study sites in the Dominican Republic (bas ed on tree trunk resting collections).

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52 (x = 48.9 % ) at Trepada. Due to the scarcit y of Lu. christophei adults, seasonal abundance could not be monitored for this species. During the wettest months (Ma y to December), Lu. cayennensis adults could be found on tr e e trunks up to a height of 2m. Normally when disturbed, the sand flies would fl y onl y a short distance ( less than 10cm) ; however, on rain-wetted tree trunks the sand flies readil y flew off the trees. During the dr y season, the f lies were generall y found at the tree base, often where the ground had separated from the tree trunk in fissures, or in t h e moss at the base. Smaller tree holes also harbored Lu. cayennensis at this time of y ear. Female Lu. cayennensis were commonl y observed feeding on Anolis lizards during the da y principall y on Anolis distichus, but also on~cybotes. Lizard identifi cati o n was based on Cochran (1941). Adult Lu. christophei were usuall y reco v ered from deep tree crevices and ground level tree holes in large shade trees in coffee groves. Two t y pes of trees provide crevices which could ser v e as resting sites for Lu. christophei, the ceiba and the strangler fig (Ficus spp.) The former ma y ha v e ver y large buttresses and the latter usuall y has numerous cre v ices of various sizes as it entwines its host tree. Cigarette smoke puffed into the cre v ices forced the sand flies to the entrance, w here the y were more easil y captured. Most of the Lu. christophei collected were obtained at Loma Pena Alta, where a total of 81 flies

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53 (51 males and 22 females) were captured by aspirator, 7 flies ( 2 males and 5 females) were captured in a flight trap, and 2 3 flies (10 males and 13 females) were captured in CDC or CDC-UV traps. In one tree hole, 14 sand flies were found in association with a rat nest constructed of leaves. Of eight females, three were blood-fed or gravid and were the only fed Lu. christophei females recovered in aspirator collections. A common characteristic of the other resting sites was the presence of land snails and millipede feces. On rare occasions at Lorna Pena Alta, male Lu. christophei were found resting on tree buttresses near the entrance to tree crevices, in association with Lu. cayennensis. Lutzomyia christophei was the more active of the two species. Five fourth-instar larvae were recovered from a soil sample at Monte Claro. The microhabitat was humus and leaf litter which had accumulated in the buttress of a large shade tree in a coffee grove. The larvae were reared to the adult stage and were Lu. cayennensis. No other phlebotornine larvae were seen or recovered from the other soil samples (Table 2-3) Laboratory Studies Wild-caught and lab-reared Lu. cayennensis adults behaved similarly. Females fed readily on Anolis lizards in the feeding chamber, but showed no interest in feeding on human, hamster, or rat (B. rattus). Over 50 wild-caught

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54 females were exposed to a skink, Mabuya mabouya, in a feeding chamber, but none fed or was seen probing. Twenty lab-reared females were exposed to tree frogs, Hyla sp., and toads, Bufo marinus, but the flies showed no interest in feeding. The preferred feeding site on lizards was on the mid-dorsal region to the tail base. Some females were occasionally observed feeding on the top of the head and the shoulder region. Females rarely probed more than one spot before feeding to repletion. Feeding time ranged from 62.5min to 82.5min (n = 26, x = 73.0min.25min (S.D.)). Feeding only occurred under lighted conditions. Females would feed at any time of day in the feeding chamber, provided that the chamber was left in a well lighted room. Generally, females first fed at age 3.0 to 4.5 days (n = 50) posteclosion; however, on various occasions lab-reared flies, less than 48hrs old, fed on lizards. Mating was observed before, during, or after bloodfeeding, with the pair remaining in copula for up to 2 lmin. Females began oviposi tion four to five days post-bloodfeeding. Complete oviposition usually required less than one day. Wild-caught females generally died within 24hrs after laying eggs. Holding the flies in a Hotpack environmental chamber helped to increase the survivorship of lab-reared females. Approximately 17 to 33% of the females of every generation survived to take a second blood meal; 5 0 to 7 5 % of these survi vars subsequently laid a second batch of eggs. No males or unfed females lived more than six days.

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55 Female Lu. cayennensis were anautogenous, each female laying up to 60 eggs. Most unmated bloodfed females died without ovipositing (20 out of 23 flies), but three laid partial egg batches of 5 to 9 eggs. The size of a full egg batch for wild-caught females was 38 to 60 eggs (n = 50, x = 47.7.6 (S.D.)), based on females collected with a full blood meal that had no eggs retained at death. For labreared females, a full egg batch contained 39 to 60 eggs (n = 50, X = 46.6.3 (S.D.)). Of 103 F 1 females held singly in vials, only 19 laid full egg batches, 61 females laid partial egg batches of 7 to 29 eggs, and 23 females died without laying eggs. The percent egg hatch, for full egg batches from wild-caught and lab-reared females ranged from 2 0 4-10 0 % ( n == 10 0 x = 6 8 4 % 2 9 7 % ( S. D. ) ) Percent egg hatch for partial egg batches ranged from Oto 100% (n = 50, X = 61.2%.2 (S.D.)). Percent egg hatch was not statistically different between the two groups (t-test, p = 0.05). When three or fewer eggs were laid by a female, none hatched. All hatching from an egg batch occurred within a 12hr period. Under ambient conditions at the field station, the incubation time for eggs was 8 to 12 days, depending on time of year (ambient temperature). Longer incubation times were associated with cooler temperatures, especially in January and February (Table 2-5). Larvae of Lu. cayennensis exhibited burrowing behavior. Larvae tended to stay under their food, except when the food

PAGE 69

56 was very damp. When individually reared, male sand fly larvae developed faster than did females, under all three temperature-humidity regimes ( t-test, p = 0. 0 5) (Table 2-5) Males developed faster under ambient conditions in August September (24-33C, 60-95%RH) than males under constant conditions (27C, 85%RH). Both groups developed faster than those under ambient conditions in January-February in the Dominican Republic (16-28C, 30-90%RH) (t-test, p = 0.05). The time from oviposition to adult eclosion, for males and females, is presented in Table 2-5 and Figures 2-10, 2-11, 2-12. A closed colony of Lu. cayennensis was maintained for six generations, or for almost one year. Wild-caught and lab-reared Lu. christophei behaved similarly in the laboratory. Females readily fed on anesthetized rodents, including a wild-caught R. rattus, laboratory mice and hamsters. They readily probed on a human hand. The females showed no feeding site preference, feeding equally well on the ears, paws, or eyelids of the offered rodent host. Females often probed more than one spot and after initiating feeding, many females moved to a second site to complete feeding. Females of this species fed equally wel 1 under light or dark conditions. Feeding time ranged from 2.75min to 4.9Smin (n = 25, x = 3.35min .SOmin (S.D.)). Some lab-reared females fed as early as 24hrs after emergence, though most did not feed until 48 to 72hrs after eclosion. Females took a very large bloodmeal

PAGE 70

Table 2-5. Mean duration S.D.) in days of immature stages of Lu. cayennensis hispaniolae at three temperature regimes, according to sex of sand fly. Larval Instars Total Condition Sex Egg 1 2 3 4 Pupa (egg-adult) Constant male 8 5.1.0 3.6.8 3.0.4 6.5.6 7.1.3 33.7.9 (28C) female 8 5.0.0 3.2.8 4.4.3 6.5.3 7.5.8 36.1.5 (48) ( 31) ( 2 8) ( 2 8) ( 2 5) ( 2 3) Ambient male 8 4.7.5 2.3.5 2.7.5 6.0.7 6.7.6 30.3.3 (24-33C) female 8 5.0.4 2.4.5 3.1.3 6.4.7 7.7.7 32.5.0 (48) ( 4 8) ( 4 8) ( 4 8) ( 4 8) ( 4 7) Ambient male 12 5.0.0 3.6.6 3.1.7 8.3.6 9.1.3 43.6.6 (16-28C) female 12 5.4.0 3.3.8 3.6.7 8.9.8 9.4.5 45.5.6 ( 4 8) ( 4 7) ( 4 6) ( 4 6) ( 4 2) ( 41) Note: Number in ( ) indicates individuals surviving each stage. n 12 11 23 24 17 24 lJl --.J

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58 x rf : 3 4 3 :_ l.7 -0 5 X 3 37.0 :_ 2 7 u ,, :J 4 J d 0 u 3 .,. ,, 2 :, <::::::; "" '.:'--J 30 31 35 36 37 38 39 40 Day s after o v io osit io n Figure 2-10. Eclosion time in days after egg depo siti on for Lu. cayennensis re ared under constant condi tions ( 28C 90 % RH). xd = 31. 3+1. 3 X = 33. 5+ I. 0 8 7 6 rJ i <1J
PAGE 72

"
PAGE 73

60 (Fig. 2-13), but were very active afterwards. Mating, only rarely observed, occurred before or after feeding, with coupling lasting up to 28.5rnin. Oviposition began at least five days after blood-feeding, but for a few females, it was delayed up to ten days post-feeding; it was usually cornpleted in less than one day. Approximately 17 % of the females that survived five or more days post-feeding (8 of 46 F 1 and F 2 females) did not exhibit ovarian development after their first blood meal. By seven days however, they had excreted the blood meal remnants in their feces and were capable of refeeding and developing eggs. In the F 3 genera tion, 8 of 52 females (15.8 % ) developed partial egg batches of eight or fewer eggs before re feeding. without ovipositing. All eight died Only 2 of the 11 lab-fed wild-caught females laid full egg batches of 39 and 49 eggs. The nine other flies laid 0 to 35 eggs (x = 7.3.3 (S.D.)). The size of a full egg batch for lab-reared females was 35 to 87 eggs (n = 10, x = 50.7.9 (S.D.)). Of 6 4 F 1 to F 3 females which survived five or more days post-feeding, onl y 14 (21.9 % ) laid full egg batches, 24 females (37.5 % ) laid partial egg batches of 5 to 27 eggs, and 26 females (40.6 % ) died without ovipos iting. The number of eggs laid, plus the number retained at death (a measure of reproducti v e potential in the abo v e 6 4 females) ranged 3 6-8 8 eggs and / or ovarioles / female (x = 64.1 % .3 % (S.D.)). The percent egg hatch for full egg batches ranged from 12.6 % (11/8 7 eggs) to 100 % ( 3 6 / 36 eggs)

PAGE 74

Figure 2-13. Female Lu. christophei feeding on BALB/c mouse. 61

PAGE 75

62 (n = 12, x = 64.4%.1% (S.D.)). Percent egg hatch for partial egg batches ranged from 0% (0/18 eggs) to 100% (28/28 eggs) (n = 20, x = 56.8%.1% (S.D.)). Percent egg hatch was not statistically different between the two groups (t-test, p = 0.05). All hatching from an egg batch occurred within a 24hr period. The time period for egg incubation was 10 to 1 7 days, depending, in part on the temperature maintained (Table 2-6). Lutzomyia christophei larvae exhibited burrowing behavior, prefering to remain under the food in the larval rearing containers. In the individual and group rearing experiments, development time from egg to adult ranged from 51 to 69 days for the F 1 generation (27C) and from 57 to 73 days for the F 2 and F 3 generations (23C). Males devel oped at a faster rate than did females (t-test, p = 0.05). The data from these rearing experiments are presented in Table 2-6 and Figures 2-14 and 2-15. The closed colony of Lu. christophei is being main tained at Walter Reed Army Institute of Research, Washing ton, D. C. Sand Fly Dissections Dissections of 319 wild-caught parous Lu. cayennensis were made. The flies came principally from five sites, of which 46.4 % (148 flies) of the total were from leishmaniasis case sites. Most females had a blood meal evident at capture and did not die, or were not killed, until the blood

PAGE 76

Table 2-6. Mean duration (S.D.), in days, of immature stages of Lu. christophei at constant temperature and humidity, according to sex of sand fly. Generation/ Condition F Constant (28C, 85%RH) F2 & F1 Constant: ( 2 3C, 85%RH) Larval Instars Sex Egg 1 2 3 4 male 10 9.5.9 6.1.3 5.3.1 13.4.1 female 10 9.9.2 6.2.3 6.2.4 17.5.3 ( 4 6) ( 4 6) (43) ( 4 2) ( 41) male 15.2.7-------------N.D.------------------female 15.2.7-------------N.D.------------------( 20 3) Note: Numbers in ( ) indicate individuals surviving each stage. N.D. = Not Determined Pupa 13.2.8 13.4.1 ( 4 0) 15.3.4 16.7.2 (173) Total (egg-adult) 57.7.3 63.5.5 62.6.2 65.7.0 n 14 26 78 95

PAGE 77

tl I u 3 "' rf 0 u 2
PAGE 78

65 had been digested and the residue excreted. The totals for the sites, the percent blood-fed or gravid, and the time of year collected are presented in Table 2-7. Dissections were examined of 23 wild-caught Lu. christ ophei from Loma Pena Alta. Nine of these took bloodmeals in the laboratory, two on a R. rattus captured at the site and seven on a hamster. None of the 23 lab-fed females was positive for parasites. The results of feeding lab-reared Lu. christophei on leishmanial-infected BALB/c mice are presented in Chapter 3. Discussion Lutzomvia cayennensis hispaniolae has geographic distribution in the Dominican a widespread Republic. It occurs in wooded areas within 100m of the coast and in coffee groves 1.n the interior (Loma Pena Alta, elevation 427m). Throughout the sugarcane cayennensis was found along streams growing regions, Lu. and rivers wherever there was a sufficient number and density of trees to support a "forest-floor" type habitat (i.e. leaf and other organic matter present). This species was found at or near eight of the DCL case sites visited during the survey. In the wild, Lu. cayennensis appears to be very sedentary, as very few were collected by the flight traps, often despite close proximity of the trap to known resting sites. At Monte Claro, where 13 flies were recovered from the flight

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Table 2-7. Site of collection for female Lu. cayennensis dissected. # Blood-Fed Site or Gravid #Parous Total Time of year I Pedro Sanchez 93 34 127 May 1981, Aug 1981-Jul 1982 Trepada de Jabilla 50 21 71 Nov 1981-Jul 1982 Altos de Peguero 25 9 34 Mar-Jul 1982 Loma Pena Alta 13 7 20 Jul 1982 Monte Claro 12 8 20 June 1982 Morro de Miches 1 2 3 Aug 1981 Miscellaneous 37 7 44 Aug 1981-Jul 1982

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67 trap collection, the trap was set so that the side of one of the end panels was in contact with a tree on which were found over 70 resting sand flies. The absence of Lu. cayennensis in light trap collections suggests that this species was not attracted to light. Both wild-caught and lab-reared flies exhibited a positive phototaxic response in the feeding chamber, which was noted b y changing the position of a desk lamp used to illuminate the chamber. Later, as sand flies were collected from small tree holes, it became clear that this was a probable response to light as an escape attempt, with the path towards the light replacing the path out of a tree hole. In the lab and in the wild, this species was only diurnally active, as were its hosts, Anolis lizards. Because these lizards are very common, there is no great need for extensive host-seeking behavior. Although there may be some wind-borne dispersal of adults, most of the distance an individual female travels probably occurs during the 60+rnin that it is feeding on a lizard. This length of time is much longer than that of similar-sized mammalian anthophora (Endris, 1982) lizards are not capable feeding species, such as Lu. and Lu. christophei; however, the of reaching the sand flies to dislodge them. Lutzomyia vexator (Coq.), also a lizard feeder that occurs in the USA and is about twice the size of Lu. cayennensis, feeds to repletion in only lOrnin (Perkins,

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68 unpublished data). Chaniotis (1967) reported 60+min feeding times for some lizard-feeding sand flies in California. The collection of larvae at Monte Claro represents the first recovery of larvae of Lu. cayennensis in the field. Hanson (1968) was unsuccessful in locating Lu. cayennensis larvae in Panama. Monte Claro supported a very large population of sand flies in a very small area. Many of the trees sampled in the coffee grove had 50+ sand flies resting on them; thus it does not seem surprising to find the larvae in such a situation. Hanson (1968) noted that larvae burrowed in culture, but to what depth they occur in nature is unknown. In the current study, the soil samples were jostled in transport back to the field station, so the depth of the five recovered larvae was not known. The microsi te where they were collected had an unusually deep layer of humus, 4 to 6cm deep. An injury to the tree trunk may have been the cause of the ooze that started O.Sm above ground level and continued to the ground, perhaps enriching the soil at this spot. The soil samples taken from both Pedron Sanchez and Trepada had very little humus. The population levels of Lu. cayennensis are related to wet and dry seasonal periods. Ambient temperature probably influences the population level as well. In the eastern region, the dry season usually lasts from late February to the beginning of May. May is the wettest month, but from June through December the ground remains fairly well satura ted. The population level of Lu. cayennensis, although

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69 somewhat erratic on the weekly basis, remained fairly high from August to December 1981 and from late May to July 1982 (Fig. 2-9). The short lifespan of wild caught and lab-reared flies at the field station was probably due to low humidity. Other sand flies reared in the laboratory, and kept in the environmental chamber at % RH, often lived 13 to 15 days after eclosion. Early female death was the probable cause when only partial egg batches were laid. Sugar feeding was never observed in nature and only rarely in captivity. Lack of carbohydrate may have been a contributing cause to early mortality. As Lu. cayennensis is a very sedentary species, the natural sugar source must be very close to the resting sites, perhaps secretions from the trees. The rearing times observed for Lu. cayennensis were similar to those reported for Lu. anthopora, a similar sized species (Endris, 1982), but much shorter than those for the sympatric Lu. christophei. The individual rearing gave somewhat misleading results, in that most flies emerged within a seven day period. When larvae from a single egg batch were reared together in a vial, emergence of adults occurred during a period of up to 22 days. This delay could be the effect of overcrowding or interference, as has been reported among mosquito larvae (Ikeshoji and Mulla, 1970), but not previously for sand flies. Lu tzomyia christophei appears to have a more limited distribution than Lu. c. hispaniolae, as Lu. christophei

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70 were recovered at seven sites, all of which were leishmania sis case sites, or in close proximity to such sites. As resting Lu. christophei were secreted in tree crevices, their apparent scarcity may have been partially an artifact of the amount of time spent at the survey sites and the collection methods used. It was not until cigarette smoke was used to flush the flies from the crevices, that speci mens of this species were collected with any regularity. With the exception of various locations in the Pedro Sanchez area, including the study site and a small 15h woods, few non-case sites received more than lOhrs of searching during the study period. The habitat of this species, crevices and ground-level tree holes in large shade trees (primarly Ceiba pentandra) in coffee groves and rock crevices in forested areas, also may be a factor limiting its distribution. In the Dominican Republic, very few v irgin rain forests remain; coffee and cacao groves, however, ma y simulate forest conditions, as heavy leaf litter and much shade are charac teristic of these areas. Typically, land snails and milli pede feces were observed in the Lu. christophei-inhibi ted crevices, it is quite likely that snail and millipede byproducts enrich the soil of these crevices and possibly provide sand fly larvae with an adequate diet. Lutzornyia christophei ma y have evolved as a nest inhabiting species with one or more of the tree hole-inhabiting mammals that were present before the arrival of the Spaniards (1492 AD), much the way Lu. anthophora is associated with woodrat

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71 (Neotoma) nests in the USA (Young, 1972). Plagiodontia aedium, the only endemic rodent in the D.R., is a tree hole inhabitant (Woods, 1981). As the introduced rats replaced the endemic rodents, the sand fly may have moved into the tree hole nest of R. rattus or Mus musculus. Along with this move may have been the development of a new reservoir host for an endemic Leishmania; although there is discussion as to whether the Dominican Leishmania is native to the country or introduced (Walton, pers. comm., Zeledon, pers. comm.) Al though sand flies were never encountered in large numbers in crevice resting sites, residents at several case and non-case sites reported them to be a major annoyance at times. On Cuba, man-bi ting sand flies have been reported from caves (Avila et al. 19 6 9} ; al though the species was not determined, these flies were probably Lu. orestes (Young, pers. comm.), a very close relative of Lu. christophei. In the Dominican Republic, very few of the visited DCL case sites had caves or rock outcroppings. "Erisos" were reported to be common in June and July, at Altos de Peguero, Lorna Pena Al ta, and Trepada de Jabil la, though much more so in past years than in the three summers (1981-1983) that the author was present. "Erisos" were originally described to the author as pale-colored flies, smaller than a mosquito, which start biting around dusk and continue into the late evening, inside the house as well as outside. The bite was described as being as painful as a

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72 mosquito's. "Erisos" also hold their wings aloft, and tend to hop around on the person before biting. All descriptions were similar and all described typical sand fly behavior. In September 1983, two "erisos" were collected while feeding, on one of the leishmaniasis patients. These were confirmed to be female Lu. christophei b y the author and Dr. D. G. Young. Thus Lu. christophei is regarded as the probable vector of leishmaniasis in the Dominican Republic. It fulfills the requirements of readil y feeing on man and rodents, the probabl y reservior hosts, and is capable of experimentally transmitting the Dominican Leishmania (see Chapter 3). One of the difficulties of in studying this sand fly is based on its long life cycle of more than two months. Futher work needs to be done on determining the relationship of the long cycle to the natural habitat. Much also remains to be determined on the bionomics of this speci e s. The epidemio logical data, such as vector efficienc y and infection rates in the field, need to be studied further. The sample of 23 wild-caught female flies available for e x amination during this study was insufficient. A long-range program, per formed b y personnel who could visit the sites at various times over a two or three y ear period, is needed.

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CHAPTER 3 GROWTH OF LEISHMANIA-ISABEL STRAIN IN CULTURE MEDIUM, LABORATORY RODENTS, AND SAND FLIES Introduction New World cutaneous leishmaniasis is caused by members of the Leishmania braziliensis and L. mexicana complexes (Bray, 1974), but diffuse cutaneous leishmaniasis (DCL) has been associated only with the L. rnexicana complex in the Americas (Schnur et al., 1983) and with L. aethiopica in Africa (Bray and Bryceson, 1969). Current knowledge of DCL is based primarily on studies of L. aethopica in Ethiopia (Bryceson, 1969, 1970a, b, c) and L. mexicana pifanoi in Venezuela (Convi t et al. 19 71) The focus of DCL in the Dominican Republic is unique owing to the complete absence of human cases with ulcerating lesions (Bogaert-Diaz, unpublished data) This is one of the features that has sparked interest in the indentity of this Leishmania. Schnur et al. (1983) and Kreutzer et al. (1983) believe that the Dominican parasite differs from other known subspecies in the L. braziliensis and L. mexicana complexes, but appears to be closer to strains in the L. mexicana complex. Lainson ( 1983) reported that the Dominican parasite developed in the 73

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74 anterior midgut (Suprapylaria) of experimentally infected Lutzomyia longipalpis from Brazil. Schnur et al. ( 1983) described some of the biological characters of the parasite in culture and lab animals, but only in subjective terms. Since the identity of the Dominican Leishmania remains undetermined, the commonly used strain, isolated from a 14-year-old female patient from the Dominican Republic, is designated Leishmania-Isabel strain (Petersen et al., 1982). The purpose of this study was to quantitate the growth of the Dominican parasite in comparison to other strains of Leishmania, to determine the course of infection in suscep tible laboratory rodents, to describe the course of infec tion in susceptible sand flies, and to effect transmission with the probable natural vector species. Methods and Materials Comparison of the Growth of Three Strains of Leishmania Stock culture of L. mexicana amazonensis was obtained from Dr. K. P. Chang, Rockefeller University, inoculated into a Syrian hamster, Mesocricetus auretus, and later reisolated and passaged one time on Schneider's Drosophila Medium (GIBCO Laboratories, Grand Island, New York) supple mented with 20% (v/v) heat-inactivated (56C, 30min) fetal bovine serum (FBS) (Hendricks and Wright, 19 79) The stock culture of L. rnexicana-Texas (WR-411) was provided by Dr. Larry Hendricks, Walter Reed Army Institute of Research

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75 (WRAIR) and was handled as above. The culture of Leishmania-Isabel strain (WR-336) was provided by Dr. Eileen Franke, WRAIR, but was not passaged through animals. For each strain, 15 tubes of enriched Schneider's medium were inoculated so that the Day 0 populations were approximately 2.50 x 10 5 log phase promastigotes/rnl. The tubes were held in a Hotpack environmental chamber (Hotpack, Inc., Philadelphia, PA) at 24.0.5C. The populations were checked daily for 16 days using a hernacytorneter with the aid of a compound microscope (200X) Growth of Leishrnania-Isabel Strain in Laboratory Rodents Prior to inoculation in rodents, the Isabel strain was passaged one time on NNN medium (Mansour et al., 19 7 4) Subadult or young Syrian hamsters and IRC strain mice (5-7 weeks old) were inoculated via intracardial (IC) intraperitoneal (IP), or subcutaneous (.sub Q) routes. the dosages used are given in Table 3-1. The animals were maintained by inoculation group. Two animals from each species/ inoculation group were examined at 30 and 60 days post-inoculation. Four subcutaneously inoculated hamsters were examined via xenodiagnosis with sand flies, Lutzornyia anthophora, at 3, 7, and 11 months post-inoculation; at 15 months, they were killed and assayed, as outlined below, using Schneider's medium for cultures. Before killing the animal, 0.2-0.Sml of blood was taken via heart puncture and added to 4ml RPMI medium (80% RPMI

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76 Table 3-1 Method of inoculation and number of promastigotes used to infect laboratory rodents with Leishmania-Isabel strain. Species Method of Inoculation Size of Inoculum Hamster subcutaneous 9.00 X 10 4 promastigotes Hamster intraperitoneal 2.25 X 10 5 promastigotes Hamster intracardial 9.00 X 10 4 promastigotes Mouse subcutaneous 1.13 V 10 5 promastigotes ~Mouse intraperitoneal 5.65 X 10 5 promastigotes Medium 1640 (GIBCO) + 20% FBS (v/v)). A subcutaneous aspirate was taken from the left hind paw and placed in another tube of RPMI. After death, tissue samples were taken of hind paw skin, liver, and spleen. smears were made of each on microscope slides. Impression The tissue samples were then placed in a Petri dish of normal saline which contained 40000 Penicillin G Sodium (U.S. Biochemical Corporation, Cleveland, OH) and l.5rng/ml Streptomycin Sulfate (U.S. Biochemical Corp.), with one Petri dish/ animal. The Petri dishes were left in a refrigerator (4C) for 24hrs. The method was adapted from Herrer and Christen sen (1975). The following day the Petri dishes were removed and uncovered in a biological hood. Each tissue sample was washed twice with normal saline. A small portion of tissue

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77 ( 9rnm 2 skin, 2 7mm 3 liver or spleen) was then placed in 2ml sterile normal saline in a sterile mortar and ground by pestle until macerated. The solution was allowed to settle for 1 min then 1ml of supernant was drawn off and added to a tube of RPMI Medium. The culture tubes were held at room temperature for eight days then checked for the presence of promastigotes, with the aid of a compound microscope (200X). Impression smears were fixed in absolute methanol, stained with Geimsa for 30 minutes, then observed with a microscope (l000X). The Growth of Leishmania-Isabel Strain in the Sand Fly Four-month-old BALB/c mice were inoculted in the hind foot pads with approximately 2.5 x 10 5 promastigotes of Leishmania-Isabel strain. The mice were held for four to six weeks to allow development of the histiocytoma, during which time the foot pad grew to two to three times normal size. Four to seven-day-old laboratory-reared Lutzomyia anthophora, a species from Texas and Mexico, were allowed to feed on the infected hind foot pads of the mice. The body and tail of the mouse was covered so that only the hind feet were exposed to the sand flies. After feeding, the sand flies were held in groups of 20 to 50 flies, in 40dr rearing vials, and held in a Hotpack environment chamber at 23. 0 .5 C and 70 % RH. A sample of flies were dissected on each day (Day 1 to 7 post-feeding) so that a total of 15 infected flies were observed for each day. After

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78 dissecting out the digestive tract in Medium 199 (GIBCO), the mouthparts, head, and digestive tract were e x amined for the presence of leishmanial promastigotes, with the aid of a microscope (l00X, 200X). The number of infected flies w a s noted along with location, number and shape of the parasites. To determine if the parasites observed were infective, a sample (pooled by for Day 3, 4, and 5) was inoculated into the hind foot pads of two six-week-old hamsters. Transmission of Leishmania-Isabel Strain by Lutzomyia christophei Female F 3 generation Lu. christophei were allowed to feed on the swollen hind foot pads of Leishmania-Isabel (WR-336) infected BALB/ c mice. The sand flies fed on the mice fi v e to seven weeks post-inoculation. The flies were then held indi v idually in 7dr vials in a Hot pack chamber, 23.0.5C and 80 % RH, until death or unti l they were ready to refeed, usually seven or more days after the first bloodmeal. Females that died one to seven da y s post-feeding were dissected and examined, to correlate the infection in Lu. christophei with that in Lu. anthophora, performed a s abo v e. For their second bloodmeal, the flies were released into a feeding chamber with an anesthetized noninfected BALE/ c mouse, covered with a cloth sleeve e x cept for the hind feet and tail. The re fed flies were then recaptured and held individually in 7dr vials until death. Upon death, the flies were dissected to determine the state of

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79 infection. The mice used for refeeding the sand flies were maintained in the laboratory for three weeks before attempt ing to xenodiagnose leishmanial infection with Lu. antho phora and/or diagnosing through culturing of spleen and liver tissue sample and subcutaneous aspirate in Schneider's medium, performed as above. Female Lu. anthophora were used or xenodiagnosis due to the unavailability of female Lu. christophei at the time. Results Comparison of the Growth of Three Strains of Leishmania The two strains of Leishmania mexicana, L. mexicana Texas and L. m. amazonensis grew at rates that were not statistically different, but their growth rates were much faster than that of the Leishmania-Isabel (t-test, p = 0.05) (Fig. 3-1) The L. mexicana strains maintained log phase growth until Day 6, stayed in a stationary phase until Day 10, and then decreased rapidly. Cultures of the Isabel strain did not achieve peak population growth until Day 12, after which they declined slowly. Post-peak populations were difficult to estimate due to the large number of dead or inactive promastigotes present in the samples, only motile promastigotes were counted.

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7 "' "' 6 w 0 "' w "' 5 '-""0 :n 0 ...J 0 1 Figure 3-1. 1 Leishma n ial sabel a L. mexicana -Te x as L m. amazonensis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Days Daily estimated mean population of three strains of Leishmania grown in Schneider's medium, at 25.0C. 80

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81 Growth of Leishmania-Isabel Strain in Laboratory Rodents Leishmania-Isabel-inoculated hamsters and IRC strain mice examined at 30 and 60 days post-inoculation showed no externally visible signs of infection. Four hamsters were inoculated via subcutaneous route and maintained for over 60 days, by 75 days post-inoculation, only one hamster exhibited a very slight swelling of the hind paw. All four were xenodiagnosed using lab-reared Lu. anthophora and were infected at this time. By 7 months post-inoculation, the cutaneous infection had apparently self-cured because none of 3 0 sand flies feeding on the "infected" foot of each hamster developed promastigote infections. Aspirate cultures taken at seven months were also negative. When the four hamsters were killed at 15 months, no parasites were isolated by aspirate, liver, or spleen culture. The most sensitive method for determination of infec tion, besides xenodiagnosis, was by culturing of splenic tissue. No parasites were observed in the cultures of heart blood or in skin impression smears. Active infections were recovered from all animals killed at 30 days, by spleen culture and from all, but one mouse, via liver culture. The results were much more variable at 60 days (Table 3-2). At 60 days, no leishmaniae were observed or isolated from one mouse (IP) and two hamsters (lIP, lIC). Animals inoculated via subcutaneous injection were the most readily confirmed as infected by the different methods of culturing and culturing and impression smears (Table 3-2, Fig. 3-2).

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Table 3-2 Number of animals examined determined to be infected Isabel strain via different isolation methods. Cultures Inoculation Day Heart Foot Species Method Examined Blood Aspirate Skin Spleen Liver Mouse sub Q 30 0 0 0 2 1 60 0 0 1 1 1 IP 30 0 0 0 2 0 60 0 0 0 2 0 Hamster sub Q 30 0 0 1 2 2 60 0 1 1 2 2 IP 30 0 0 0 2 2 60 0 0 0 1 0 IC 30 0 0 0 2 2 60 0 0 0 1 0 Note: n = 2, except Mouse-sub Q-60 days where n = 1. with LeishmaniaImpression Smears Skin Spleen Liver 0 1 0 0 1 1 0 1 1 0 2 0 0 2 2 0 2 2 0 2 2 0 0 0 0 2 1 0 1 1 00 N

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Figure 3-2. Leishrnania-Isabel strain amastigotes in a spleen impression smear stained with Giemsa (l000X). 83

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84 Growth of Leishmania-Isabel Strain in the Sand Fly Promastigotes were observed in some of the Lu. anthophora females in each sample from Day 1 to Day 7 post-feeding on a Leishrnania-Isabel infected BALB/c mouse. The percent infected increased with time, but the percent with bacterial contamination (Leishrnania infection undeter minable) decreased with time, because of the high death rate in these flies. In the sample from Day 1, only one promastiogote was observed in 30 fly dissections; however, a few arnastigote-infected macrophages were observed in the blood meals in five of the dissections which were later stained and examined with the aid of microscope (lOOOX). For the remaining days, infected flies represented 53.6% to 75.0% of the sample dissected each da y The infections were of several hundred to several thousand promastigotes/ fly (Table 3-3). Promastigotes were first observed only in the anterior midgut, (Fig. 3-3) near the stomodael valve in Da y 2 and Day 3 flies. By Da y 4, parasites were observed in the posterior pharynx, as well. On Da y 5, promastigotes were observed in the mouthparts in 4 of the 15 infected flies. Mouthpart infections were observed 12 of 15 and 13 of 15 infected Day 6 and Day 7 flies, respectively (Fig. 3-4). The shape of the promastigotes was highly variable in young infections (Da y 2 and 3) ; tiowever, onl y elongate forms were observed anterior of the stomodael valve

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Table 3-3. The course of development of Leishmania-Isabel strain in the sand fly, Lutzomyia anthophora, based on daily dissections, Days 1 to 7 post feeding. Day post feeding 1 2 3 4 5 Status of blood meal RBC's intact, meal dark red RBC's intact meal black Blood remnants present Meal totally excreted in most flies Meal excreted in all flies Ovarian status undeveloped undeveloped slightly developed well developed eggs fully developed # infected flies/sample */30 15/28 15/27 15/25 15/22 Location, number & shape of parasites anterior MG+ w/ blood meal, only 1 promastigote ob served in dissections, stumpy form, amastigotes observed in macrophages in 5 dissections anterior MG w/ blood meal, l00's observed in all infected flies, primarily stumpy forms anterior MG, forward to stomodaeal valve, l00's observed, stumpy and some elongate forms in anterior MG, 50100 in pharynx, elongate forms l000's in anterior MG, 50100 in pharynx, 10-20 in cibarial region, 5-10 in mouthparts, elongate forms CX) Ul

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Table 3-3. Continued. Day postStatus of Ovarian # infected Location, number & feeding blood meal st a tus flies/sample shape of parasites 6 as Day 5 commenced 15/21 as Day 5 ovip o sition 7 as Day 5 finished 15/20 l000's in anterior MG, oviposition lOO's in pharynx, 20-50 in mouthparts, elongate forms + parasites probably still in amastigote stage, infection not determinable MG= midgut 00 0\

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8 7 Figure 3-3. Promastigotes of Leishmania-Isabel strain from the anterior midgut of the sand fly, Lu. anthophora (lOOOX).

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Mout Figure 3-4. DAY 1 DAY 2 ~.,...,~~~'-; ~~i~Ii~{%\~f m: L~ Blood m ea l DAY 4 -----I Blood meal remnants DAY 5 DAY 6 DAY The course infection in anthophora. Leishmania-Isabel strain sand fl y Lutzomyia of the 88

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89 in Day 4-7 flies. Parasite development in 17 Lu. christophei, dissected between Days 3-6 post-feeding, was parallel to that observed in Lu. anthophora. These data are summarized in Table 3-3. Promastigotes from flies, both four and five days post-feeding, were infective to hamsters, as determined b y xenodiagnosis using Lu. anthophora, three weeks after inoculation and/ or by spleen and skin tis sue culture five weeks after inoculation. Day 3 promastigotes were not infective, as determined b y the same methods. The estimated inoculation dose for the two hamsters in each of the three groups was 2.0 x 10 4 promastigotes / hind food pad. Transmission of Leishmania-Isabel Strain by Lutzomyia christophei A total of 8 out of 52 (15.4 % ) F 3 Lu. christophei females sur v ived seven to twelve da y s post-feeding to refeed on noninfected BALB/c mice. All of the flies subsequentl y died within 24hrs of their second bloodrneal. One fly each fed on each hind foot and tail on two mice ( # 1 and 3); one fl y fed on a hind foot, another on the tail of a third mouse ( # 2) All eight flies had leishrnanial infections when e x amined. All three mice were accidently killed, b y overdose of anesthetic, approximately four weeks after being fed upon. Only one mouse had any externally obvious signs of leishrnaniasis. Mouse #2 had a slight, but noticeable, swelling of the sand fly-bitten hind foot. X enodiagnosis with Lu. anthophora and culturing of a subcutaneous aspirate

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90 revealed that the swelling was due to leishmanial infection. Spleen cultures from all three mice were positive by seven days, cultures of liver tissue were negative for all the mice. A subcutaneous aspirate from the tail of mouse #3 was positive; however, aspirate cultures from the hind feet of mice #1 and #3 were negative, as was the aspirate from the tail of #1. A few amastigote-infected macrophages were observed in a spleen impression smear from mouse #2. Discussion Due to the recent discovery of leishmaniasis in the Dominican Republic, relatively little comparative work has been done to characterize the parasite. The Leishmania still does not have a specific name; therefore, the most commonly used strain is referred to as Leishmania-Isabel (WR-336) after the patient from whom the primary isolate was made in the Dominican Republic. Strain determination must be made on the basis of a variety of biological and biochemical characteristics, as compared to those character istics of other known strains. The differences between L. mexicana and L. braziliensis complexes are often matters of degrees, rather than absolute contrasts. In laboratory cultures, the Dominican parasite did not grow as rapidly as the two~mexicana ssp., although it did achieve a maximum concentration (promastigotes/ml) very near that of the L. mexicana strains. Hendricks et al. (1978) and Childs et al. (1978) reported similar results with various L.

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91 mexicana strains, though the maximum yields for~brazil iensis strains were much lower. After discovering the slower growth rate of the Dominican parasite, cultures of tissues from potential reservoir hosts (Chapter 4) were checked on Day 7, rather than Day 3 or 4, as would have been the case with 1979) Other a L. mexicana strain (Hendricks and Wright, biochemical characters of the Dominican Leishmania are more typical of L. mexicana strains (Schnur et al., 1983). The almost inapparent lesions produced in hamsters were not typical of the growth exhibited by most strains of Leishmania in this species of rodent (Zeledon et al., 1982), nor were hamsters known to self-cure leishmanial infections. The low pathogenici ty of DCL-producing para sites in laboratory rodents has also been noted for L. aethiopica, the causative agent of DCL in Ethiopia. Inap parent lesions are also characteristic in wild reservoir hosts for cutaneous leishmaniasis in the Americas (Herrer and Christensen, 1975). Perhaps the most outstanding result from the animal inoculations was determining the relative sensitivities of the methods for detecting infection. The purpose of this was to use the infection of laboratory rodents as a model for determining which techniques might be best applicable to the survey for the reservoir host in the Dominican Republic (see Chapter 4). The two most successful methods for detecting infection were the culturing of liver tissue and

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92 of spleen tissue. These two methods require the use of sterile facilities and were not used in the reservoir study until June 1983. They should be used in any further studies. Liver and spleen impression smears also often confirmed infection, but may be adequate for laboratory studies only, as these may result in a much greater number of amastigote parasitized macrophages than would be found in a wild host (Hoogstraal and Heyneman, 1969). The cutaneous mode of inoculation, either subcutaneous or intraderrnal, would be the normal route when the promastigotes are transmi tted by the sand fly. In laboratory animals, this also proved to be the most successful in establishing the infection. Intracardial and intraperitoneal inoculation were successful in establishing leishmanial infections in some animals. It is unknown if these resulted 1.n inapparent cutaneous infections. Other strains of Leishrnania causing DCL have not been found to infect laboratory rodents via IP or IC inoculation (Bray et al. 19 7 3; Convi t and KerdelVegas, 1965). Both hamsters and mice were susceptible to the Isabel strain, which is not true of L. aethiopica, to which mice are refractory (Bray et al., 1973). These results might have been due to the strain of mouse used rather than the parasite itself. The BALB/c mice obtained for infecting the sand flies developed very large lesions in less than four weeks after inoculation ( 2. 5 X 5 10 promastigotes/foot pad) which worsened to virtual amputation

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93 by six to eight weeks post-inoculation (Berman, pers.comm.). The IRC strain mice used by the author did not develop obvious infections, despite an inoculum half as large as that used for the BALB/c mice. Hamsters fit in between the two for mice strains, the Dominican in terms of acceptable Leishmania. The loss laboratory hosts of infection, by seven months post-inoculation, was a startling discovery as hamsters are not known to be able to self-cure from leishmanial infections. This further suggests the low pathogenicity of Leishmania-Isabel strain. Growth of the parasite in sand flies can also be used to assist in species determination of the Leishmania. In the classification of the New World Leishmania, those that develop in the anterior midgut are considered to belong in the L. mexicana complex or L. donovani ( Section Suprapylaria) Members of the~braziliensis complex develop in the posterior midgut and subsequently move forward (Section Peripylaria) (Killick-Kendrick, 19 79) On the basis of site of development, the Dominican Leishmania would be classified as L. mexicana ssp., although it differs from other members of the group in other respects. Strains of L. mexicana develop rapidly in the sand fly Lu. anthophora, producing infections of promastigotes which often number in the tends of thousands (Young, pers. comm.). The Isabel strain developed fewer parasites in both Lu. anthophora and Lu. christophei; however, the sand fly promastigotes appeared to be highly infective, as the inoculation and transmission

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94 experiments indicated. Sacks and Perkins ( 19 8 4) recently reported the phenomenon of increased promastigote infec tivity with time. In the author's study, promastigotes were infective as early as four days after ingestion by the sand fly. Both Lu. anthophora and Lu. christophei lay their eggs as early as five Chapter 2). Thus, days post-feeding (Endris, 1982; see if the first bloodmeal was from an infected host, the sand fly could transmit infective pro mastigotes as soon as it refeeds, after oviposition on Day 5. While Old World leishmaniases are very vector species specific, normal development (Killick-Kendrick those of the New World usually exhibit in a wide range of sand fly species and Ward, 1981). Thus, it should be possible to extrapolate between development of Leishmania Isabel in Lu. anthophora to that in Lu. christophei. The 17 infected Lu. christophei females that died three to six days post-feeding, exhibited parallel parasite develop ment to that observed in detail in Lu. anthophora. The Dominican sand good host for fly, the Lu. christophe i, proved to be a very Dominican Leishrnania, being capable of maintaining an infection at least 15 days post-feeding. Further work should be done to determine the effect of the promastigote infection on this species of sand fly once the laboratory colony becomes more firmly established and adapted to captivity. Transmission of the parasite by the sand fly is further evidence that it may be the natural vector in the Dominican Republic.

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95 Field studies must be continued so as to give further support to the laboratory studies with the sand fly. The laboratory models established in this work must now be extended to field studies, using the appropriate post-mortem Leishmania-isolation techniques on potential reservoir hosts, taking into account that longer incubation periods may be necessary for cultures. The question of the identity of the Dominican Leishmania also remains to be answered, whether it is a new species or a new subspecies of L. mexicana.

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CHAPTER 4 SURVEY FOR RESERVOIR HOSTS OF HUMAN LEISHMANIASIS IN THE DOMINICAN REPUBLIC Introduction American cutaneous leishmaniasis is a zoonotic disease caused by the members of the Leishmania braziliensis and L. mexicana complexes. A variety of wild mammals have been recorded as hosts for these parasites, primarily rodents for the L. mexicana complex; however, members of the L. brazil iensis complex are known from a more diverse grouping which includes rodents, edentates, procyonids, marsupials, and can ids ( Lainson and Shaw, 19 7 9) The potential host fauna for leishmaniasis in the Dominican Republic is very limited. Only two native terrestrial mammal species are known: a capromyid rodent, Plagiodontia aedium Cuvier, and a large insectivore, Solenodon paradoxus Brandt. Both species are rare and inhabit only relatively undisturbed areas (Woods, 1981). Introduced species have replaced the native fauna and include the murid rodents Rattus rattus alexandrinus (Geoffroy), R. norvegicus (Berkenhout), and Mus musculus brevirostris Waterhouse; the mongoose, Herpestes auropunctatus Hodgson; and feral cats, Felis catus L. (Woods, pers. comm.; Garcia M., pers. comm.). Many people keep dogs which 96

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97 are somewhat free-ranging. The reservoir host of human diffuse cutaneous leishmaniasis in the Dominican Republic is unknown, but as the parasite is more similar to members of the L. mexicana comple x (Schnur et al., 1983), the murid rodents are the most likely suspects. The American leishmaniases very rarely produce obvious lesions in most of their wild hosts (Herrer and Christensen, 1975). However, the parasite may be recovered from culture of skin, liver, or spleen biopsies (Herrer et al., 1966) or from subcutaneous aspirates of infected animals. Serodi agnosis, which is routinely performed for detecting visceral leishmaniasis, and occasionally cutaneous leishmaniasis, in man (Wal ton et al., 197 2) is generally not used for the detection of wild hosts of cutaneous leishmaniasis. No other tr y panosomatid parasites of mammals, including man, ar e known to occur in the Dominican Republic (Walt o n, pers. comm.). The prevalence of subclinical human leishmaniasis is not known, although this could be an important factor in maintenance of the disease and its distribution. Materials and Methods Tomahawk live traps and Sherman collapsible traps (Fig. 4-1) for mammals were set at six leishmaniasis case sites (T a ble 4-1). Traps were baited with various foods such as: peanut butter-corn flour mi x ture, avocado pieces, fried plantain, banana, coconut, or sausage for rodents and

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Figure 4-1. Sherman (on left) and Tomahawk live traps for small mammals. 98

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Table 4-1. Mammal specimens collected at six leishmaniasis case sites in the Dominican Republic. # TrapSite Nights Rattus rattus R. norveg:icus Mus musculus Heq~estes Total Carrasco 30 0 0 0 0 0 La Culatica 4 3 0 0 0 3 Loma Pena Alta 757 44 1 6 3 54 Monte Claro 15 0 0 2 0 2 Morro de Miches 730 10 0 6 0 16 Trepada de Jabilla 5689 63 (38+25)* 5 (5+0)* 27 (22+5)* 0 95 'l'otal 7266 120 6 41 3 170 # collected 1981-82 and# collected 1983. \.0 \.0

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100 raw egg in shell or chicken leg for mongoose. When poss ible, local personnel checked the traps daily and were paid on a per animal basis. If known in advance that the case site would not be visited according to schedule, then trapping was only done on the three nights preceding the next visit. Captured animals were removed from the field to nearby residences and later to the field station; there they were given water and food. They were transported to the Dominican Dermatology Institute (Institute Dermatologico) laboratory in Santo Domingo for examination, usually once per week during April to July 1982 and June to August 1983. Animals that died at the station, and all animals trapped prior to April 1982, were examined at the station. Various methods were used to check for potentially infected animals. These included visual examination for suspicious lesions; culturing of subcutaneous aspirates, liver, splien, and skin tissue (Herrer et al., 1966) in NNN medium (Mansour et al., 1983) or Schneider's Drosophila medium (Grand Island Biological Co. (GIBCO), Grand Island, NY) (Hendricks and Wright, 1979); indirect fluorescent antibody test (IFAT) with fluoresce in (FITC)-labelled conjugate (SIGMA Chemical Co., St. Lousi, MO); impression slides of skin, liver, and spleen tissue; and histological section slides of liver and spleen tissue. Animals alive at time of examination were humanely killed with ether or by suffocation through samples for IFAT were compression of taken through the thorax. Blood cardiac puncture

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101 technique prior to killing. Aspirates were taken by inject ing 0. 03 to 0. l0ml of normal saline of Medium 1640 (GIBCO) subcutaneously in the nose, foot, or tail base with a tuberculin syringe and 26 gauge needle. The liquid was then withdrawn, along with any blood that appeared, and injected into a culture tube. Prior to injection, the skin surface was cleaned with 100% ethanol or 95 % isopropanol and allowed to dry. The culture tubes were maintained at ambient temperature and checked Contaminated tubes were for growth at seven to checked on the first ten days. day that contamination was noted. Impression and section slides were stained with Giemsa and hematoxylin, respectively, for 20min. The slides were later examined with the aid of a compound microscope amastigote-infected (400X, l000X) for macrophages. Other the presence of domestic or feral mammals were visually examined live, when available. Results A total of 167 specimens of three species of rodents (Fig. 4-2) and three Herpestes (mongoose) were collected. Mammals were collected during two time periods: October 1981 to July 1982 (5721 trap-nights producing 83 rodents (0.015 animals/trap-night)) and May to August 1983 (1545 trap-nights producing 87 animals). (During the latter period animals/trap-night was not applicable as an unknown number were collected by hand.) Seventy-two (42.6 % ) rodents were trapped at Morro de Miches or Trepada de Jabilla from

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102 Figure 4-2. The three species of rodents trapped during the survey (from topRattus norvegicus, R. rattus, and Mus musculus).

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103 April to July 1982. Thirty (17.8%) rodents came from Trepada during May and June 1983. Fifty-four (32.0%) were collected at Lorna Pena Alta in July 1983 (Table 4-1). The six Rattus norvegicus (four females and two males, range of body length 175 to 225mm, x = 214.2mm) were examined by various methods (Table 4-2), but were not found to be infected with Leishmania. Rattus rattus was the most commonly collected mammal species. However, no Leishrnania were isolated from the 120 specimens examined (61 females, 56 males, and 3 not identified due to degree of decomposi tion, body length range 105 to 22mm, x = 180.0mm). Aspir ates or tissue samples of some specimens were combined in culture tubes according to sex and tissue type. Tubes for the 21 specimens examined at the field station were con taminated by bacteria and/or mold. The IFAT showed that 4 (9.1 % ) of the 44 R. rattus from Lorna Pena Alta examined were seropositive for antibodies against the Dominican Leishrnania. One rat had severe edema of the feet, but only bacteria were recovered f rorn the affected limbs. Another had a large (20mm diameter) spot on the hind dorsal region which was almost hairless, resembling a leishmanial lesion, but no parasites were revealed by skin impression or cul tured subcutaneous aspirate. Forty-one Mus rnusculus (house mouse) were trapped. There were 25 females and 16 males; body length ranged 71-93mm (x = 81.0mm). As above, some aspirates or tissue

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Table 4-2. Examination techniques used for mammals collected during survey for reservoir hosts of leishmaniasis in the Dominican Republic, October 1981 to August 1983. Species # Collected Rattus rattus 120 R. norve9:icus 6 Mus musculus 41 Her:eestes auro12unctatus 3 # Visually Examined Only 18 0 4 0 # Examined by Aspirate Culture* 41 5 22 3 Tissue Culture 78 5 33 3 Tissue-skin (ear, foot, and/or tail), spleen, and liver. Post-Mortem Technique Histo Impression logical IFAT Smears Sections 44 39 90 0 5 6 0 28 21 0 0 3

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105 samples were combined according to sex and tissue type. No mice were found to be infected. The three Herpestes collected were females; body length ranged 270-320mm (x = 300mm). None was infected. The examination data for the above four species are summarized in Table 4-2. In addition, one aspirate was taken from a wild-caught Plagiodontia aedium female being held at the University of Florida. This animal was assumed to be approximately six years old when captured in 1974 from a locality in the Cordillera Oriental. It showed no suspicious lesions and the culture was also negative. One feral cat was trapped at Morro de Miches, but no lesions were observed. all less than one year old, were visually Trepada; none showed any suspicious lesions. Five dogs, examined at Contamination with bacteria and/or mold was a frequent problem in culture tubes during the first survey period, October 1981 to July 1982. During the second period, contamination affected only the dermal samples. Discussion Leishmanial infections were not detected in any of the 170 mammals trapped; however, it is possible that some infections were not revealed by the methods used. Impression smears may be positive only at very high infection levels, as observed in laboratory infections (Hoogstraal and Heyneman, 1969). The aspirate and tissue

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samples cultured at the field station, bacterial and/or fungal contamination 106 invariably developed due to a lack of sterile facilities. The tissue ( spleen and liver) samples cultured at the Institute were not contaminated, but demonstrated no Leishmania as well. Due to the lack of standardized treatment and contamination of samples, it is not known if some infection were missed, however few in numbers. The animals from Loma Pena Alta were collected in the same time period as the Lu. chri stophei from that site. Unfortunately, it is not known exactly how many of the animals were from the forests and coffee groves. At this site, leaf nests, 2 to 3m high, were common in the shrubs in the pasture lands surrounding the coffee groves. Many of the 18 subadult R. rattus from the site were probably collected by hand from these nests. Contact with sand flies was probably limited for these young rats. The serological analysis indicated that some of the R. rattus from Pena Alta had been exposed to the leishmanial parasite; however, the status of the infection was not known, recovered. Research by Zovein et al. as no parasite were (1984) suggested a high correlation between seropositive results and infection for Old World rodent leishmaniasis. It is also possible that a seropositive individual in the Dominican study might not have had a current infection ( Zuckerman and Lainson, 1977).

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107 The native mammalian fauna of the Dominican Republic is very depauperate today consisting of 13 species of bats, 1 species of rodent, and 1 species of insectivore (Woods, 1981). The latter two exist in low numbers and definitely were not present at several of the DCL case sites, as they require relatively undisturbed habitat (Woods, 1981). The hutia, Plagiodontia aedium, makes its dens in partially decayed large trees or rock crevices. It is more prevalent in areas where Rattus is less prevalent (Sullivan, 1983). Most of the case sites visited had neither rock outcroppings or large trees with crevices, the exception being Loma Pena Alta. Introduced species have replaced the native mammals and the four most common are Rattus rattus R. norvegicus, Mus musculus and Herpestes auropunctatus, all of which occur throughout the island (Woods, pers. comm.). As rodents are the known reservoir hosts for members of the L. mexicana complex (Lainson and Shaw, 1979), it is possible that one or more of the introduced rodents is acting in that capacity in the Dominican Republic. Due to the presumed intimate relationship between~rattus and the probably vector, Lu. christophei (see Chapter 2), this species of rodent is the most likely reservoir suspect, with M. musculus the second most likely due to its abundance and habitat. Rattus norvegicus has different habits from R. rattus and may be much less common in wooded areas. Of the nine species of endemic rodents (Varona, 1974) existent on Hispaniola at the arrival of the Spaniards, all

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108 were tree cavity or ground hole dwellers (Woods, pers. comm.). As the Rattus and Mus invaded these habitats, they might have come to represent an increasingly important role in the epidemiology of leishrnaniasis in the Dominican Republic. Other species of mammals, including dog, cat and mongoose, are present at many of the case sites and may have some role in the epidemiology of the disease. Several dogs were visually examined at Trepada de Jabilla, but none showed any suspicious lesions; however these dogs were all young, less than one year old, and may not have been around during a period of abundance of sand flies. There were no dogs present at several of the case sites. Cats live primarily in the coffee groves and forested areas where they were present only as feral animals. They might serve as an additional blood source to a potential vector, but felids are not know to support infections by Leishrnania spp. in the Americas. The mongoose live in more open areas and the possibility of sand fly contact is much less, viverids are also not known to support Leishrnania infections in the New World. The three mongoose obtained in the study were very carefully examined, due to difficulty of capture, but were uninfected. These three larger species are probably not present in the high populations that would be necessary to maintain the disease. Another possibility is that man could be serving as the reservoir, as is the case with 1 tropica and L. donovani in India (Bray, 1974). A serological survey performed by

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109 that the percent seropositive at seven leishmaniasis case sites ranged from 26.0 to 48.0% of the individuals tested. The observation that children as young as one year old were demonstrated as seropositives (de Quinones, unpublished data) may indicate that transmission was actively occurring during the author's study period. It has been suggested that the seropositive individuals may have subclinical leishmaniasis (Zeledon, pers. comm.) or perhaps uncompli cated skin lesions, as is suspected with L. mexicana pifanoi infections in Venezuela (Lainson, 1983); but man is probably not the reservoir in the Dominican Republic (Zeledon, pers. comm.). The high prevalence of seroposi ti ves at the various case sites suggests that the leishmanial parasite should be abundant in the reservoir host as well. Of all the suspect species, only the murid rodents are present at high population levels, as would be necessary to maintain the parasite in the absence of man. It is curious as to how the Dominican Leishmania came to occur in the Dominican Republic (and possibly Haiti), whether endemic for millenia, imported with the Indians of the Caribbean basin, or imported with the Europeans and Africans. Determining the reservoir host fauna might shed some light on this perplexity. It is important to determine the relationship between seropositive (human or rat) and the state of infection. Xenodiagnosis, using laboratory-reared Lu. christophei would be an excellent way to determine the prevalence of subclinical

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110 infections in man and other mammals. Only very intensive trapping of all possible wild reservoir hosts will lead to a more definitive answer as to whether leishrnaniasis is indeed a zoonotic disease in the Dominican Republic.

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CHAPTER 5 SUr,,'1'.MARY During the field and laboratory investigations, the following were achieved or determined: 1. A survey of phlebotomine sand flies was conducted in the Dominican Republic between May 1981 and August 2. 3 1983. Two species, Lutzomyia christophei and Lu. cayennensis hispaniolae were recovered. Laboratory iolae and colonies of Lutzomyia Lu. christophei were cayennensis hispanestablished. Of 319 wild caught Lu. cayennensis and 23 Lu. christophei dissected, none was found infected with Leishmania or other vertebrate parasites. Leishmania-Isabel strain developed in the anterior midgut of both Lu. christophei and Lu. anthophora. 4. Leishmanial promastigotes from experimentally-infected sand flies were infective to hamsters four days post-blood meal. Lutzomyia christophei females were 111

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112 capable of supporting a hamster-infective leishmanial infection up to at least 15 days post-feeding. 5. Laboratory-reared Lu. christophei were capable of transmitting Leishmania-Isabel strain from infected BALB/c mice to uninfected mice. 6. In culture medium, Leishmania-Isabel strain grew at slower rate than did two strains of L. mexicana. 7. Of 1 70 wild mammals examined for leishmaniasis, none was positive; however 4 of 47 Rattus rattus from Loma Pena Alta were positive for anti-Leishmania antibodies.

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APPENDICES

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APPENDIX 1. DOMINICAN LEISHMANIASIS PATIENT PARTIAL CASE HISTORIES. Age at Time of Presumed DiagEvoluAge at Site of Site nosis tion Infection Lesions 1. Nisibon 9y 6y 3y arms, face 2. Nisibon 4y 3y ly face, arm, leg 3 Nisibon 8y 3y S y extremities 4 El Valle 40y ly 39y ear 5 San Cristobal 4y 3y ly cheek, arm 6 Higuey 8y 4mo 7.Sy arm 7. Las Cuchillas 12 y 4y By arms, cheek 8 Nisibon Sy 2y 3y cheek, arm 9 Bani 13y 2y lly thigh 10. El Valle 29y ? young ears 11. El Seibo 57y 6mo 56.Sy leg, arms 12. Miches 32y 8y 24 y forearm 13. Santiago 6y 14. Higuey llmo 3mo Brno face, arm 15. Higuey 32y Brno 3ly knee, hand 16. Navarrete 42y 7y 35y thigh 17. El Cue y 60y arm, leg 114

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Site 18. El Seibo 19. Miches 20. Miches 21. Yerba Buena 22. Santa Lucia 23. Carrasco 24. Monte Claro 25. Las Cuchillas Age at Diag nosis 43y Sy 4y l.Sy 4y 20y 3y 40y Time of Presumed EvoluAge at tion Infection 15y 28y 9mo 4y ly 3mo 3. Sy 6mo 4.Sy 5. Sy 2.Sy 3mo 20y 20y Source: Bogaert-Diaz, unpublished data. Note: y = year, mo= months. 115 Site of Lesions arm, leg arm, face, thigh arm cheek face, forearm arm, leg cheek, legs arms, shoulders, knees

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APPENDIX 2. COLLECTION SITES AND DATES FOR LUTZOMYIA CAYENNESIS HISPANIOLAE IN THE DOMINICAN REPUBLIC, MAY 1981 AUGUST 1983. Altagracia Province. Bayahibe: 29 Aug 81, 6km W, 2 females, 8km W, 1 male, 14km W, 2 males. Boca de Yuma: 15 Jun 82, 1km W, 2 females, 3km N, female, 2 males. Higuey: 30 Aug 81, Rio Yuma 2km S, 1 female, 1 male, Rio Sanate 8km S, 1 male, 13 km W, 1 female, 23km W 1 male, 28km W, 1 male; 5 Oct 81, 10km N, 3 females, 3 males; 26 May 82, 15km N, 2 females, 3 males. Nisibon: 5 Oct 81, 1km S, 1 female, 3 males. Barahona Province. Cienaga: 7 Jul 8 2, 1 km S, 11 flies. Polo: 7 Jul 82, 8km E, 6 flies; 4km N, 8 flies. Duarte Province. Castillo: 10 Jul 82, 5km E, 3 flies. El Seibo Province. El Cuay: 5km S (Altos de Peguero), Mar 82, 5 visits, May 82, 4 visits, Jun 82, 3 visits, Jul 82, 3 visits. El Seibo: 28 Aug 81, 10km W, 1 ma 1 e 1 4 km W 1 ma 1 e 1 8 km W 1 f ema 1 e 3 5 km N 1 female, 5.2km N, 1 male; 16 Oct 81, 8km N; 15 Dec 81, 11km W. Hato Mayor: 23 Sep 81, 11km N, 5 flies; 28 Sep 81, 11km N, 6 females, 15 males, 16km N, 3 females, 5 males; 20 Oct 81, 8km N, 1 female, 4 males, 11km N, 5 females, 9 males; 21 Nov 81, 11km. N. Las Cuchillas: 6 Nov 81, 5km S, 10km S, 16km S; 2.8km N (Trepada de Jabilla) Nov 81, 7 visits, Dec 81, 5 visits, Jan 82, 5 visits, Feb 82, 5 visits, Mar 82, 6 visits, Apr 82, 5 visits, May 82, 6 visits, Jun 82, 9 visits, Jul 82, 8 visits. Loma Pena Alta: 22 Jun 82, 24 July 82. Miches: 28 Aug 81, 9km S (Morro de Miches, 2 females, 2 males; 4 Nov 81, 3km W, 2 males. Pedro Sanchez: 19-21 May 81, Aug 81, 10 visits, Sep 81, 6 visits, Oct 81, 6 visits, Nov 81, 5 visits, Dec 81, 4 visits, Jan 82, 5 visits, Feb 82, 7 visits, Mar 82, 6 visits, Apr 82, 6 visits, May 82, 9 visits, 116

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117 Jun 82, 10 visits, Jun 82, 9 visits, 25 Sep 81, 7km s, 1 male. Sabana de la Mar: 4 Nov 81, 6km S, 5km E. Santa Lucia: 17 Aug 81; 31 Aug 81, 47 flies, 12km N, 3 flies; 19 Sep 81, 7 females, 5 males; 22 Oct 81 20km N, 1 male; 4 Dec 81. Yerba Buena: 22 Jun 82, 1km S, 3km NW, 7km NW. La Vega Province: Bonao: 10 Jul 82, 1km SW, 2 males. La Romana Province. Guerrero: 28 Aug 81, 1km N, 1 female, 1 male. La Romana: 29 Aug 81, 10km E (Rio Chavon), 1 male. Rio Cumayasa: 28 Aug 81, 2 males. Maria Trinidad Sanchez Province. Carrasco (Rio San Juan): 3 July 82, 1 female, 1 male. Pera via Province. Iguana Arriba ( 20 km NE Bani) : 82, 13 flies. 8 July San Cristobal Province. 10 km NW, 5 flies. Villa Altagracia: 19 June 82, San Pedro de Macoris Province. San Pedro de Macoris: 14 Sep 82, 16 km N, 4 females, 3 males. Sanchez Ramirez Province. Monte Claro (Cotui): 19, 16 Jun 82. Pimentel: 27 Apr 82, 5 km S, 1 female, 1 male.

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APPENDIX 3. COLLECTION SITES AND DATES FOR LUTZOMYIA CHRISTOPHEI IN THE DOMINICAN REPUBLIC, MAY 1981 AUGUST 1983. Altagracia Province. (CDC-UV) Nisibon: 4 males. La Culatica: 7-8 June 83, 7 June 83, 1 female 5 km SW, 2 females, El Seibo Privince. Altos de Peguero: 22-25 Mar 82, 1 female (flight trap). Loma Pena Alta: 31 May 83, 5 females, 2 males (CDC, CDC-UV); Jun 83, 4 visits; Jul 83, 10 visits (CDC-UV, flight trap, aspirator); 3 Aug 8 3, 1 female ( flight trap) Morro de Miches: 19 May 81, 1 male. Trepada de Jabilla: 15-20 Nov 81, 1 female ( flight trap) ; 20 Nov 81, 2 males; 14 Dec 81, 2 males; 1-5 Feb 82, 1 male (flight trap); 5-10 Feb 82, 1 male (flight trap). Sanchez Ramirez Province. Monte Claro: 9 Jun 83, 1 male. Note: Aspirator was used as the method of collection unless otherwise specified. 118

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REFERENCES Adler, S., and O. Theodor. 1957. Transmission of disease agents by phlebotomine sand flies. Ann. Rev. Entomol. 2:203-223. Anderson, J.R., and S.C. Ayala. 1968. Trypanosome trans mitted by Phlebotornus: First report from the Americas. Science. 161:1023-1025. Anonymous. 1977. Official guide to the Dominican Republic. Turismo Dorninicano, C.por A. 56 pp. Anonymous. 1981. Annual Report: Special programme for research and training in tropical diseases (UNDP/World Bank/WHO) Chapter Seven: Leishrnaniases. TDR/AR(5)81.7-LEISH: 143-165. Avila, I.G., A.V. Gutsevich, and R.G. Broche. 1969. Neuvos datos sobre la familia Phlebotomidae en Cuba. Torreia. 14:3-7. Ayala, S.C., and D. Lee. ment of sporozoites sandflies. Science. 1970. Saurian malaria: develop in two species of phlebotomine 167:891-892. Barrientos, L.P. cutaneo-mucosa 46:425-418. 194 8. Un caso a tipico (espundia). Mem. Inst. de leishmaniose Oswaldo Cruz. Bettini, S., L. Gradoni, and E. Pozlo. 1978. Isolation of Leishmania strains from Rattus Rattus in Italy. Trans. R. Soc. Trop. Med. Hyg. 72:441. Bogaert-Diaz, H. Dermatologico Republic. Unpublished Dominicano. data. Santo 1984. Domingo, Institute Dominican Bogaert-Diaz, H., R.F. Rojas, A. deLeon, D. de Martinez, and M. de Quinoes. 19 7 5. Leishmaniasis tegumen taria arnericana: Reporte de los primeros tres casos des cubiertos en R.D. Rev. Domin. Derrnatol. 9:19-31. Bray, R.S. 1974. Leishmania. Ann. Rev. Microbial. 28:189-217. 119

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120 Bray, R.S., R.W. Ashford, and M.A. Bray. 1973. The para site causing leishmaniasis in Ethiopia. Trans. R. Soc. Trop. Med. Hyg. 67:345-348. Bray, R.S., and A.D.M. Bryceson. 1969. Studies on the immunology and serology of leishmaniasis. VIII. The identity of the strains of Leishmania from Ethiopian diffuse cutaneous leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 63:524-527. Bryceson, A.D.M. 1969. Diffuse cutaneous leishmaniasis in Ethiopia. I. The clinical and histological features of the disease. Trans. R. Soc. Trop. Med. Hyg. 63:708-737. Bryceson, A.D.M. 1970a. Diffuse cutaneous leishmaniasis in Ethiopia. II. Treatment. Trans. R. Soc. Trop. Med. Hyg. 64:369-379. Bryceson, A.D.M. 1970b. Diffuse cutaneous leishmaniasis in Ethiopia. III, IV, III. Immunological studies. Trans. R. Soc. Trop. Med. Hyg. 64:380-393. Bryceson, A.D.M. 1970c. Immunological aspects of clinical leishrnaniasis. Proc. R. Soc. Med. 63:1056-1060. Chaniotis, B.N. mus under 4:221-223. 1967. The biology of California Phleboto laborator y conditions. J. Med. Entomol. Chaniotis, B. N 1978. Phlebotomine sandflies (Famil y Psychodidae). pp. 19-30. In R.A. Bram [ed.]. Sur veillance and collection of arthropods of veterinary importance. USDA Handbook No. 518. U.S. Gov Printing Office, Washington, D.C. Childs, G.E., K.A. Foster, and M.J. McRoberts. 1978. Insect cell culture media for cultivation of New World Leishmania. Int. J. Parasitol. 8:255-258. Cochran, D.M. 1941. The herpetolog y of Smithsonian Inst. U.S.N.M. Bull. 1977. Printing Office, Washington, D.C. 351 pp. Hispaniola. U.S. Gov. Convit, J., and F. Kerdel-Vegas. cutaneous leishmaniasis. Arch. 1965. Disseminated Dermatol. 91:439-447. Conv it. J. F. seminated Dermatol. Kerdel-Vegas, and anergic cutaneous 7 4:132-135. B. Gordon. 1962. Dis leishmaniasis. Brit. J.

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121 Convit, J., and P. Lapenta. 1948. Sobre un caso de leishmaniasis tegumentaria de forma diseminada. Rev. Policlin. Caracas. 17:153-158. Convit. J., M.E. Pinardi, and A.J. Rondon. 1971. Diffuse cutaneous leishrnaniasis: a disease due to an immuno logical defect of the host. Trans. R. Soc. Trap. Med. Hyg. 60:526-532. Deane, L.M., S. Sarjeant C., and E. Fernandez. 1978. Hallazgo de Trypanosoma (Megatrypanum) pessoai Deane and Sugary, 1963, en murcielagos de Venezuela Bol. Dir. Malarial. San. Amb. 18:321-327. de Quinones, M.R. Unpublished data. 1983. Instituto Derrnatologico, Santa Domingo, Dominican Republic. Disney, R.H.L. 1966. attracted to rats. A trap for phlebotomine sandflies Bull. Entomol. Res. 56:445-451. Endris, R.G. 1982. Studies of Lutzornyia anthophora (Addis) (Diptera: Psychodidae) and other potential vectors of Rio Grande virus. Ph.D. dissertation. University of Florida, Gainesville, FL. 90 pp. Endris, R.G., P.V. Perkins, D.G. Young, and R.N. Johnson. 1982. Techniques for laboratory rearing of sand flies (Diptera:Psychodidae). Mosq. News. 42:400-407. Fairchild, G.B., and H. Trapido. 1950. The West species of Phlebotomus (Diptera:Psychodidae). Entomol. Soc. Arner. 43:405-417. Indian Ann. Garcia M., N. Personal communication. 1983. Museo Nacional de Historia Natural. Santo Domingo, Dominican Republic. Garnham, P. C. C. 1966. Malaria parasites and other Haemosporidia. Oxford. 1114 pp. Blackwell Scientific Publications. Gemetchu, T. 1976. The biology of a laboratory colony of Phlebotomus longipes Parrot and Martin (Diptera:Psycho didae). J. Med. Entomol. 12:661-671. Hanson, W.J. 1968. The immature stages of the subfamily Phlebotominae in Panama (Diptera:Psychodidae). Ph.D. dissertation. University of Kansas. 160 pp. Hendricks, L., and N. Wright. 1979. Diagnosis of cutaneous leishmaniasis by in vitro cultivation of saline aspir ates in Schneider's Drosophila medium. Arner. J. Trap. Med. Hyg. 28:962-964.

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122 Hendricks, L.D., D.E. Wood, and M.E. Hajduk. 1978. Haemo flagellates: commercially available liquid media for rapid cultivation. Parasitology. 76:309-316. Herrer. A., and H.A. Christensen. 1975. The infrequency of gross skin lesions among Panamanian forest mammals with cutaneous leishmaniasis. Parasitology. 71:87-92. Herrer, A., V.E. Thatcher, and C.M. Johnson. 1966. Natural infections of Leishmania and trypanosomes demonstrated by skin culture. J. Parasitol. 52:954-957. Hoogstraal, H., and D. Heyneman. 1969. Leishmaniasis in the Sudan Republic. 30. Final epidemiological report. Arner. J. Trap. Med. Hyg. 18:1089-1210. Ikeshoji, T., and M.S. Mulla. 1970. Overcrowding factors of mosquito larvae. J. Econ. Entomol. 63:90-96. Johnson, R.N., and D.G. Young. In preparation. Two fossil sand fly species (Diptera: Psychodidae) in amber, from the Dominican Republic. Killick-Kendrick, R. 1978. Recent advances and outstanding problems in the biology of phlebotomine sand flies. Acta Trop. 35:297-313. Killick-Kendrick, R. 1979. phlebotomine sand flies. Evans [eds.]. Biology 2:395-460. Academic Press, Killick-Kendrick, Leishmania. R., and R. D. Parasitology. of Leishmania in Lumsden and D. A. Biology In W.H.R. of the New York. Kinetoplastida. Ward. 1981. 82:143-152. Ecolog y of Kreutzer, R.D., M.E. Semke, L.D. Hendricks, and N. Wright. 1983. Identification of Leishrnania spp. by multiple isozyrne analysis. Arner. J. Trap. Med. Hyg. 32:703-715. Lainson, R. 1983 The American leishrnaniases: some observations on their ecology and epidemiology. Trans. R. Soc. Trap. Med. Hyg. 77:569-596. Lainson, R., and J.J. Shaw. 1978. Epidemiolog y and ecology of leishrnaniasis in Latin-America. Nature. 273:595-600. Lainson, R., and J.J. Shaw. 1979. The role of animals in the epidemiology of South American leishmaniasis. In W.H.R. Lumsden and D.A. Evans [eds.]. Biolog y of the Kinetoplastida. 2:1-116. Academic Press, New York.

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123 Lainson, R., and B. Southgate. 1965. Mechanical transmis sion of Leishmania mexicana by Stomoxys calcitrans. Trans. R. Soc. Trop. Med. Hyg. 59:716. Lemma, A., T. Haile, and W.A. Foster. 1970. Epidemio logical investigation on diffuse and localized cutan eous leishmaniasis in Ethiopia. 2nd Internat. Congr. Parasitoll. J. Parasitol. 56:439-440. Lewis, D.H. 1968. Phlebotomine sand-flies from Cayman Brae Island (Diptera:Psychodidae). J. Nat. Hist. 2:73-83. Lightner, L., and L.W. Roberts. 1984. Mechanical transmis sion of Leishmania major by Glossina morsitans morsi tans (Diptera:Glossinidae). J. Med. Entomol. 21:243. Mansour, N.S., J. Hady, and E. McConnell. liquid medium for Leishmania. 59:1088-1090. 1973. A modified J. Parasi tol. McConnell, E., and M. Correa. 1964. microorganisms from Panamanian J. Parasitol. 50:523-528. Trypanosomes and other Phlebotomus sandflies. Medina, R., and J. Romero. El agente casual de difusa. Arch. Venez. 4:349-353. 1962. Leishmania pifanoi n. sp. la leishmaniasis tegumentaria Med. Trop. Parasiotol. Med. Moya Pons, F. 1981. Manual de historia dominicana. Industrias Graficas, Barcelona, Spain. 666 pp. Neva, F. Personal communication. 1982. National Institutes of Health, Bethesda, MD. Perkins, P.V. Unpublished data. 1982. University of Florida, Gainesville, FL. Perkins, P.V. 1982. The identification and distribution of phlebotomine sand flies in the United States with notes on the biology of two species from Florida (Dipera:Psy chodidae). PhD. dissertation. University of Florida, Gainesville, FL. 195 pp. Peterson, E.A., F.A. Neva, C.M. Oster, and H. Bogaert-Diaz. 1982. Specific inhibtion of lymphocyte proliferation responses by adherent suppressor cells in diffuse cutaneous leishmaniasis. N.E. J. Med. 305:387-392.

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124 Quate, L.W., and J.R. Vockeroth. 1981. Psychodidae. pp. 392-300. In. J.F. Alpine, B.V. Peterson, G.E. Shewell, M.J. Teskey, J.R. Vockeroth, and D.M. Wood [eds.]. Manual of nearctic Diptera Vol. 1. Research Branch Agric. Canada Monograph No. 27. Canadian Gov. Pub. Centre, Hull, Quebec. Sacks, D.L., and P.V. Perkins. 1984. Identification of an infective stage of Leishmania promastigotes. Science. 223:1417-1419. Sanderson, M.W., and T.H. and plant inclusions Science. 131:1313. Farr. from 19 6 0 Amber with insect the Dominican Republic. Schnur, L.F., B.C. Walton, and H. Bogaert-Diaz. 1983. On the identity of the parastie causing diffuse cutaneous leishmaniasis in the Dominican Republic Trans. R. Soc. Trop. Med. Hyg. 77:756-762. Sherlock. I .A. 1964. Notas sobre a transmissao da leish maniose visceral no Brasil. Rev. Bras. Malariol. Doenc. Trop. 16:19-26. Sholdt, L.L., and J.F. Manning. 1979. Vector surveillance activities in the Dominican Republic following Hurri cana David-1979. Tech. Rep. 1/79. Dis. Vector Ecol. Con tr. Center, Naval Air Station, Jacksonville, FL. 94 pp. Simpson, M.H., serninated Dermatol. J.F. Mullins, and O.J. Stone. 1968. Disanergic cutaneous leishmaniasis. Arch. 97:301-303. Sudia, W.D., trap, an and R.W. Chamberlain. 1962. Battery light improved model. Mosq. News. 22:126-129. Sullivan, C.P. 1983. Plagiodontia aedium thesis. University 60 pp. Status and distribution of in the Dominican Republic. M. S. of Florida, Gainesville, Florida. Varona, L.S. 1974. Catalogo de extinguidos de las Antillas. Cuba. La Habana. 139 pp. las mamiferos v ivientes y Academia de Ciencias de Vaughan, T.A. 1978. Philadelphia, PA. Mammalogy. 522 pp. Walton, B.C. Personal communication. EPILEISH Steering Committee. Organization, Geneva, Switzerland. W.E. Saunders Co., 1981. Secretar y World Health

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125 Walton, B.C., W.H. Brooks, Serodiagnosis of American flurescene antibody test. 21:296-299. and I. Arjona. 1972. leishmaniasis by indirect Amer. J. Trop. Med. Hyg. Woods, C .A. Personal communication. 19 8 4. Department of Natural Science, Florida State Museum, Gainesville, FL. Woods, C.A. 1981. Last endemic mammals in Hispaniola. Oryx. 16:146-152. World Health Organization. 1984. The leishmaniases. WHO Tech. Rep. Ser. 139 pp. Young, D.G. Personal communication. 1983. Nematology Department, University Gainesville, FL. Entomology and of Florida, Young, D.G. 1972. Phlebotomine sand flies from Texas and Florida 55:61-64. (Diptera:Psychodidae). Fla. Entomol. Young, D.G. 1979. A review flies of Colombia Sycorinae). IFAS Tech. Florida, Gainesville, FL. of the bloodsucking psychodid (Diptera:Phlebotominae and Bull. 806. University of 266 pp. Young, D.G., P.V. Perkins, and R.G. Endris. 1981. A larval diet for the rearing of phlebotomine sand flies (Diptera:Psychodidae). J. Med. Entomol 18:466. Zahar, A.R. [ed.]. 1979. Studies on vector/reservoirs and their control in World Health Organization document Geneva, Switzerland. 68 pp. leishmaniasis the Old World. WHO/VBC/79.749. Zeldon, R. Personal communication. 1983. Escuela de Medicina Veterinaria, Universidad Costa Rica. Nacional, Heredia, Zeledon, R., R. Soto, and G. Gonzalez, 1982. Experimental superimposed infection of the hamster with Leishmania mexicana and L. braziliensis. Acta Trop. 39:367-372. Zovein, A., Gh. H. Edrissian, and A. Nadim. 1984. Application of the indirect fluorescent antibody test in serodiagnosis of cutaneous leishmaniasis in experimentally infected mice and naturally infected Rhombomys opimus. Trans. R. Soc. Trop. Med. Hyg. 78:73-77. Zuckerman, A., and R. Lainson. 1977. Leishmania. In J.P. Kreier [ed.]. Parasitic Protozoa Vol. 1:57-133. Academic Press, New York.

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BIOGRAPHICAL SKETCH Richard N. Johnson entered into the world on June 30, 1956, in Wilmington, Delaware. He attended Wilmington Friends School and graduated from there in 1974. He began his college career as an undergraduate at the University of Delaware in that same year. He received his B. S. with a major in entomology and applied ecolog y in 1977. In the fall of that year, he travelled to Gainesville, Florida, to enroll at the University of Florida and was subsequentl y awarded a research assistantship in medical-veterinar y degree in December After receiving his M. S. entomolog y 1979, Richard continued on at the university in a Ph.D. program the following Januar y research for the doctoral In the process of performing dissertation, he spent over 14 months in the Dominican Republic. Richard is currently a student member of the Entomo logical Societ y of America, the Florida Entomological Society, the American Society of Tropical Medicine and Hygiene, and the Wildlife Disease Association. 1 2 6

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ) ~ -rJ.', -~4~ Jerry~utler, Chairman Professor of Entomology and / Nematology .., I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Donald W. Hall Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. David G. Yng Associate Professor of Entomology and Nernatology

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Donald J. Vorrester Professor of Veterinary Medicine I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Phil_osoph. I I / / / ) l, <..: l ---.\~t_ -" Ellis C. Greiner Associate Profes s or of Veterinary Medicine This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August 1984 Dean, I ollege V Dean for Graduate Studies and Research

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