MORPHOLOGY, TAXONOMY AND LIFE
CYCLES OF SOME SAURIAN HAEMATOZOA
by
Keith Robert Wallbanks B.Sc. (Lond.) A.R.C.S.
1982
A thesis submitted for the Degree of Doctor
of Philosophy of the University of London
Department of Pure and Applied Biology Imperial College Silwood Park Ascot Berkshire ii
TO MY MOTHER AND
FATHER WITH GRATITUDE AND LOVE iii
Abstract
The trypanosomes and Leishmania parasites of lizards are reviewed. The
development of Trypanosoma platydactyli in two sandfly species, Sergentomyia minuta and Phlehotomus papatasi and in in vitro culture was followed. In
sandflies the blood trypomastigotes passed through amastigote, epimastigote
and promastigote phases in the midgut of the fly before developing into short,
slender, non-dividing trypomastigotes in the mid- and hind-gut. These short trypomastigotes are presumed to be the infective metatrypomastigotes.
In axenic culture T. platydactyli passed through amastigote and epimastigote phases into a promastigote phase. The promastigote phase was very stable and attempts to stimulate -the differentiation of promastigotes to epi- or trypo-mastigotes, by changing culture media, pH values and temperature failed.
The trypanosome origin of the promastigotes was proved by the growth of promastigotes in cultures from a cloned blood trypomastigote. The resultant promastigote cultures were identical in general morphology, ultrastructure and the electrophoretic mobility of 8 enzymes to those previously considered to be Leishmania tarentolae. T. platydactyli and L. tarentolae are synonymised and the present status of saurian Leishmania parasites is discussed.
Promastigote cultures of T. platydactyli formed intracellular amastigotes. in mouse macrophages, lizard monocytes and lizard kidney cells in vitro. The parasites were rapidly destroyed by mouse macrophages jlii vivo and in vitro at 37°C.
Intestinal infections of trypanosomatine parasites were followed for up to 6 months in 3 Chamaeleo dilepis. The flagellates were not infective to
Musca domestica or Tenebrio molitor and failed to grow in in vitro culture, iv
probably because of fungal contamination of the cultures. The classification of trypanosomatine parasites of lizard intestine is discussed. The transfer of these parasites from the genus Leishmania to the genera Leptomonas,
Herpetomonas and Crithidia and the necessary redefinition of these genera to include a wider host range is suggested.
An attempt to discover the vector of Plasmodium agamae in the Gambia is described. Sandflies fed on lizards infected with P. agamae showed no signs of parasite gametogenesis or sporogenesis when dissected. Possible reasons for this failure including gametocyte immaturity or senility and other insects as the natural vectors, are discussed. V
Acknowledgement s
I am most grateful to the many people who made this work possible,
both known and unknown. My supervisor, Professor E.U. Canning, and adviser
Dr. R.R. Killick-Kendrick, provided advice and encouragement throughout the
project.
Drs. R.S. Bray, R.E. Sinden, R.J. Barker, D.P. Turner and J. Alexander
provided help and advice when asked. Mrs. A. Mendis and Mr. A.J. Leaney
taught me aTl I know about sandfly colonisation. L. Malone, D. Pinero, 0. .
and R. Rossell and especially N. Anez gave me ideas and companionship when I
needed them most. Technical assistance came from Messrs. J.P. Nicholas,
A.M. Page, R. Hartley andA. Righton. Mr. T.J. Wilkes helped with the
entomological side of the project and also with useful introductions in the
Gambia. Mrs. J. Pugh was an efficient source of stores and amusement and
Mrs. M. Wilmot managed to track down many obscure references. Professor
P.C.C. Garnham offered suggestions, interest, and new names for the author.
I am indebted to Professor J.A. Rioux, Drs. G. Lannotte and R. Maazoun,
Madame M. Bailly and their colleagues for their generosity, hospitality,
patience and assistance during my visits to Southern France.
My stay in the Gambia was made enjoyable by the many people who came to my aid and who were so friendly and generous in terms of their time. Dr. and
Mrs. B.M. Greenwood helped to make the necessary arrangements for my visit prior to and after my arrival. Dr. and Mrs. H.A. Wilkins, Dr. and Mrs. P.
Hagan, Dr. J.H. Bryan and especially Mr. P.J. Moore, went to great lengths to make me feel welcome and helped me whenever they could.
I am grateful to Father H. Fagan and the Peace Corps volunteers, especially Mr. and Mrs. B. Trimble who offered hospitality at Basse and helped broadcast my need for lizards.
Unpublished results, advice and information were generously given by vi
Professor P. Ranque, Drs. E.N. Arnold, J.R. Baker, P. Desjeux, D.A. Evans,
U. Joger, D.M. Minter, W.F. Snow and S.R. Telford and Mr. D.J. Ball.
Dr. Telford deserves a special mention for his long letters on saurian haemoflagellates, his permission to use data and the donation of slides of several parasite infections. Drs. M.L. Chance, S.L. Croft and C.J. Young are also to be thanked for donating parasite cultures.
Mrs. Joan Shepherd typed my thesis with speed and efficiency and a quiet tolerance of my handwriting.
The generous financial support of the Medical Research Council, London, and the Department of Pure and Applied Biology, Imperial College, London is gratefully acknowledged.
Finally I must thank my family, particularly my parents and my brother
Michael, for their constant support and encouragement over the years. vii
CONTENTS
PART A x Page
LIZARD HAEMOFLAGELLATES 1
General Introduction 2
The Morphology and taxonomy of the kinetoplastid parasites of lizard blood 2.1 Lizard trypanosomes 6 2.2 List of lizard trypanosomes 6 2.3 Lizard Leishmania parasites 24 2.4 List of lizard Leishmania parasites 24 2.5 A note on reports of Leishmania parasites from some Kenyan lizards 32
An examination of the haemoflagellates of Tarentola mauritanica (L. 1758) 33 3.1 Introduction 33 3.2 Materials and methods 34 3.2.1. Collection of lizards and their maintenance 34 3.2.2. Examination of blood and tissues 34 3.2.3. Culture of parasites in axenic culture 35 3.2.3.1. Routine culture and culture "en masse" 35 3.2.3.2. Culture in a variety of media 35 3.2.3.3. Culture at different temperatures and pH values 37 3.2.3.4. Culture in medium with stimulants of differentiation 37 3.2.3.5. The use of antibiotics and routine examination of cultures 37 3.2.4. Interaction of parasites with vertebrate cells 38 3.2.4.1. Lizard blood cells (i) in vitro 38 (ii) in vivo 38 3.2.4.2. Lizard tissue cells 39 3.2.4.3. Murine macrophage cells in vitro 39 3.2.4.4. Murine macrophage cells in vivo 40 3.2.5. Cloning of blood trypomastigotes 41 3.2.6. Comparison of stock morphology and biochemistry 42 3.2.6.1. Morphological comparison (i) by light microscopy 43 (ii) by electron microscopy 43 3.2.6.2. Biochemical comparison by isoenzyme electrophoresis 44 3.2.7. Origin, collection and maintenance of sandflies 44 3.2.7.1. Phlebotomus papatasi 44 3.2.7.2. Sergentomyia minuta minuta 46 3.2.8. Infection and dissection of sandflies 47 3.2.8.1. Infection of sandflies from a gecko 47 3.2.8.2. The observation of flagellate inrections by light and electron microscopy 47 3.2.8.3. Attempt to infect sandflies by membrane feeding 48 .3. Results 49 3.3.1. Collection of lizards 49 3.3.2. Examination of blood 49 3.3.2.1. Description of parasites 49 3.3.3. Culture of parasites in axenic culture 56 3.3.4. Interaction of parasites with vertebrate cells 60 3.3.4.1. Lizard blood cells (i) in vitro 60 (ii) jLn vivo 60 3.3.4.2. Lizard tissue cells 63 viii
Page
3.3.4.3. Murine macrophage cells in vitro 63 3.3.4.4. Murine cells in vivo 65 3.3.5. Cloning of blood trypomastigotes 68 3.3.6. Comparison of strain morphology and biochemistry 68 3.3.6.1. Comparison of strain morphology 68 3.3.6.2. " " " biochemistry 71 3.3.7. Origin collection and maintenance of sandflies 71 3.3.7.1. S .m.minuta 71 3.3.8. Infection of sandflies and dissection 73 3.3.8.1. Development of T. platydactyli in P. papatasi 73 3.3.8.2. " " " S. minuta 75 3.3.8.3. Electron microscopic examination of the sandfly infections 78 3.4. Discussion 87 3.4.1. The gecko haemoflagellates in vivo 87 3.4.1.1. The trypomastigotes 87 3.4.1.2. The amastigotes 87 3.4.1.3. The role of the intracellular amastigotes 90 3.4.2. The relationship between the promastigotes and trypomastigotes from the gecko 93 3.4.3. The morphological and biochemical comparison of stocks and strains of gecko haemoflagellates in culture 94 3.4.4. The development of T. platydactyli in sandflies 96 3.4.4.1. Previous observations of sandfly infections 96 3.4.4.2. Recent observations of sandfly infections 98 3.4.5. Comparison of S. minuta and P. papatasi as invertebrate hosts of T. platydactyli 100 3.4.6. The ultrastructure of T. platydactyli 100 3.4.6.1. Parasites in the sandfly 100 3.4.6.2. Parasites in in vitro culture 103 3.4.7. The interaction of promastigote cultures with vertebrate cells in vivo and iji vitro 104 3.4.7.1. The interaction of parasites with mouse cells 104 3.4.7.2. " " " " " lizard " 106 3.4.8. Promastigotes in the genus Trypanosoma and trypanosomatid phylogeny 108 3.4.9. The status of saurian leishmaniasis 109 ix
PART B
TRYPANOSOMATINE INFECTIONS OF LIZARD INTESTINE
Introduction
A study of intestinal trypanosomatine parasite infections of some Zambian Chamaeleo dilepis ,1. Materials and Methods 5.1.1. Collection and origin of chamaeleon 5.1.2. Maintenance and identification of the lizards 5.1.3. Examination of lizard blood, faeces and tissues for parasites 5.1.3.1. Blood smears 5.1.3.2. Faecal smears 5.1.3.3. Gut sections 5.1.4. Attempt to infect sandflies with promastigotes seen in the blood of one chamaeleon 5.1.5. Attempt to infect adult Musca domestica and Tenebrio molitor with the intestinal parasite 5.1.5.1. M. domestica 5.1.5.2. T. molitor 5.1.6. Attempts to culture the intestinal flagellates in vitro 2. Results 5.2.1. Lizard maintenance 5.2.2. Blood parasites 5.2.3. Intestinal parasites 5.2.3.1. Faecal smears 5.2.3.2. Gut sections 5.2.4. Attempts to infect sandflies 5.2.5. Attempts to culture the intestinal flagellates 5.2.5.1. Direct culture of faecal mucus 5.2.5.2. Culture of the blood and tissues of mice inoculated with a parasite suspension 5.2.6. Attempts to infect M. domestica and T. molitor 3. Discussion 5.3.1. Discussion of the present study 5.3.2. The classification of trypanosomatine flagellates of lizard intestine X
PART C Page MALARIA PARASITES OF LIZARDS 134
6. Introduction 135 6.1. General introduction 135 6.2. Description of site, vertebrate host and parasites 137 6.2.1. The site 137 6.2.2. The host 142 6.2.3. The parasites 142
7. Materials and methods 145 7.1'. Reptile collection, marking, maintenance and observation 145 7.1.1. Collection 145 7.1.2. Marking 145 7.1.3. Maintenance ' 145 7.1.4. Observation 145 7.2. Sandfly collection 148 7.2.1. Oral aspirator 148 7.2.2. Miniature "CDC" light traps and suction traps 148 7.2.3. Oiled paper traps 149 7.2.4. Emergence traps 149 7.2.5. Baited trap 150 7.3. The dissection and identification of sandflies and attempts to infect them with P. agamae 150 7.4. Other haematophagous insects offered bloodmeals 151 7.5. Attempts to observe the exflagellation of P. agamae microgametocytes 152
8. Results 154 8.1. Reptile collection and maintenance 154 8.2. Parasite infections observed 154 8.2.1. Plasmodium agamae, 154 8.2.2. Eimeriine coccidian parasites 157 8.2.3. P irhaemocyt on 15 7 8.3. Sandfly collection and dissection 157 8.4. Other haematophagous Diptera offered bloodmeals 162 8.5. Attempts to observe exflagellation 163
9. Discussion 164 9.1. The lizards and their infections 164 9.2. The collection and dissection of sandflies and attempts to infect them 165 9.3. The capture methods 166 9.4. Sandflies feeding on lizards 168 9.5. The Plasmodium agamae vector-potential of Gamhian insects 169 9.5.1. Sandflies - Phlehotominae 169 9.5.2. Culicidae 170 9.5.3. Glossinidae 171 9.5.4. Ceratopogonidae 172 9.5.5. Tabaniidae 172 9.5.6. Simuliidae 172 9.5.7. Acarina 173 9.5.8. Summary 173 9.6. Attempts to stimulate exflagellation 173 9.7. Attempts to infect sandflies with the coccidian parasites 175 xi
Page
APPENDICES 176 Appendix A. Morphometric data on saurian trypanosomes 177 Appendix B. Preparation of geckonid kidney cell cultures 179 Appendix C. Phosphate buffered saline 180 Appendix D. Food for sandfly larvae 180 Appendix E. Nesbitt's solution 180 REFERENCES 181 xii
List of Figures Page
Figure 1. Geographical distribution of Tarentola mauritanica ' ' \ 5 " 2. Trypanosomes of lizards A 9 " 3. Trypanosomes of lizards B 18 Figures 4-13. The Moorish gecko Tarentola mauritanica and haematozoa which infect it 52 " 14-15. Camera lucida drawings of 50 Trypanosoma platydactyli from blood smears of one gecko 53 Figure 16. Morphogenesis of T. platydactyli in culture in vitro 54 " 17. Appearance of promastigotes in a culture of T. platydactyli 55 " 18. The effect of temperature on the growth of TPCL2 in culture 59 " 19. The effect of temperature on the growth of G13/77 in culture 59 " 20. The effect of initial pH on the growth of TPCL2 in culture 61 " 21. The effect of pH on the morphology of TPCL2 in culture^ 61 Figures 22-24. Morphogenesis of TPCL2 in culture with 0.002yg/cm Conconavilin A, 3mM Lidocaine hydrochloride and 3% Dimethylsulfoxide 62 11 25-32. Interaction of TPCL2 promastigotes and vertebrate cells jri vivo and in vitro 64 11 33-41. Interaction of TPCL2 and mouse peritoneal macrophages in vitro after 24,48 and 72h at 25°C, 28°C and 37 C 66 Figure 42. Effect of temperature on the phagocytosis and lysis of TPCL2 promastigotes by mouse peritoneal macrophages 67 " 43. Growth of TPCL2 promastigotes in M199 with 5% FCS at 25,28 and 37°C 67 " 44. Dice-Leraas diagrams of promastigote body length for 5 strains of parasite 69 " 45. Dice-Leraas diagrams of the number of subpellicular microtubules in 5 strains of parasite 69 " 46. Dice-Leraas diagrams of the subpellicular microtubule interval in 5 strains of parasite 69 Figures 47-49. Electron micrographs of promastigotes of TPCL2 from
in vitro culture r. 70 Figure 50. Diagrammatic representations of iso-enzyme bands for 12 cultures of gecko haemoflagellates 72 " 51. Life cycle of T. platydactyli of Tarentola mauritanica and sandflies. Camera lucida drawings 74 Figures 52-57. The development of T. platydactyli in S. minuta and P. papatasi 77 Figure 58. Parasite-midgut cell interaction in Phlebotomus papatasi 79 " 59. Parasite-midgut cell interaction in Sergentomyia minuta 79 Figures 60-62. Parasite-midgut cell interactions in S. minuta 81 " 63 & 64. Groups of metacyclic trypanosomes in S. -minuta 7 days post-feed 82 " 65-68. Change in kinetoplast morphology during the development of T. platydactyli in S. minuta 84 " 69 & 70. The hindgut (Pylorus 69 and ileum 70) of a S. minuta 7 days post-feed to show absence of attached parasites 86 Figure 71. Parasite in the midgut lumen of a S. minuta 5 days post-feed 86 " 72. Camera lucida drawings of T. platydactyli from various sources 89 Figures 73-84. Kinetoplastid amastigotes in blood smears from 3 lizard species 92 xiii
Page
Figures 85-92. The chamaeleon Chamaeleo dilepis and blood and faecal promastigotes 122 Figure 93. Camera lucida drawings of promastigotes in a blood smear from C2/80 123 " 94. Camera lucida drawings of promastigotes from the faeces of C2/80,C3/80 and C4/80 123 " 95. Section of the hindgut of a C. dilepis to show fungal spores (arrows) within epidermal cells 125 Figures 96-98. Promastigotes in the cloacal wall of a C. dilepis 125 Figure 99. Lizard collection sites in the Gambia 138 Figures 100-106. Lizard and sandfly habitats in the Gambia 140,141 Figure 107. Light trap for sandflies 147 " 108. Lizard cage 147 Figures 109-110. Emergence trap dismantled and situ 147 11 111-113. Parasitemias in the 3 P-agamae infected Agama agama 156 11 114-123. Agama agama and the haematozoa which infect it 160 Figure 124. Sandfly catches from light traps 161 " 125. Wind speed records for Basse 161 11 126. Rainfall records for Basse 161
List of Tables 1. Morphometric comparison of amastigotes from lizards and mammals 25 2. Enzyme nomenclature 45 3. Growth of two flagellate cultures in various media 58 4. Trypanosome species with a promastigote stage 91 5. Previous reports of trypanosomatine parasites of lizard intestine 115 6. Combinations of Gentamycin with Nystatin, Fungizone or Natamycin used in attempts to grow promastigotes from faecal mucus 120 7. Measurements of promastigotes in the blood and faecal mucus of C. dilepis from Zambia 120 8. Previous attempts to find the vector or observe the sporogenesis of malaria parasites of lizards 136 9. Comparison of malaria parasites of agamid lizards as seen in stained blood smears 144 10. The number and sex of lizards caught in 6 areas of the Gambia 155 11. Sandfly species caught 155 12. Sandfly species taking lizard blood meals 155 1
Part A
LIZARD HAEMOFLAGELLATES 2
1. GENERAL INTRODUCTION
The kinetoplastid parasites of lizard blood fall into two genera,
Trypanosoma and Leishmania, which now include blood parasites previously
attributed to Herpetomonas or Leptomonas. At present the latter genera
are comprised of monoxenous species parasitic in invertebrates (Vickerman,
1976).
Trypanosomes and Leishmania parasites are well known as the causative,
agents of disease in mammals. Mostly, they are only lightly pathogenic in
lizards though experimental infections have been known to be fatal (Brygoo,
1963). The harmful effects of parasite infection are thought to diminish as
parasite and host evolve together. There is a selection pressure against such
effects which, by reducing the average life span of the host, reduce the
chance of transmission and survival of the parasite species. The association
between kinetoplastid parasites and reptiles is thought to be an old one, the
homoeotherm parasites having evolved from poikilotherm parasites only
relatively recently. Interest in the lizard parasites stems partly from
their assumed similarity to the parasites now causing much suffering to man
and domestic animals. It has been suggested that they are more primitive
and have less complex nutritional requirements and simpler metabolic
pathways than the parasites of mammals (Trager, 1957). It is therefore not
surprising that some species, particularly those that grow readily in in vitro
culture, have been used extensively as biochemical laboratory models.
The parasites may also be important in the field of medical epidemiology.
There is some concern that the cross-immunity between human and lizard
parasites may cause false-positive leishmanin tests in man inoculated with
lizard flagellates by sandflies. (Southgate and Manson Bahr 1967; Southgate
1967; Belova 1971 ; Ranque 1977; Fuller et_ al 1980; Mutinga and Ngoka, 1981). 3
Sandflies are known which feed on both mammals and reptiles. However
the choice of blood meal source in such species may be based on availability
or opportunity rather than "preference", since feeding behaviour may differ with location, e.g. Sergentomyia Clydei appears to bite man as well as
lizards, regularly in Kenya (Southgate, 1977) but rarely in Ethiopia
(Gemetchuj1977). Lizard parasites may gain entry into human bloodstreams by contamination, probably after a feeding sandfly has been squashed into the
site of the bite. True "anterior station" development and subsequent
transmission by the sandfly bite itself has yet to be proved. (Most
infections of sandfly heads are probably a result of overspills from massive midgut infections). The importance of lizard flagellates in human epidemiology may be overestimated. Some authors consider the chance of such parasites reaching the human bloodstream to be minimal (Bray, 1977; Gemetchuyl977;
Sergiev >1977).
There has also been some concern that lizard parasites seen in their vectors or in culture may have been misidentified as parasites of mammals including man (Anderson and Ayala, 1968; Ayala, 1971). The production of disease in susceptible hosts may be the only way to check for mammalian origin of a parasite in such a situation (Hertig et_ aT, 1968). Apart from descriptions of new species there has been relatively little research on the parasites in their own right. The development of the parasites in the vertebrate host after the initial infection is not known for any species. Extrinsic development in the suspected vector has been observed for only 6 of the 28 named trypanosome species (Molyneux, 1977A) and 4, possibly 5, of the 9 species of Leishmania reported from lizard blood (Wilson and Southgate, 1979). There are only two reports, and these are equivocal, of vector-mediated transmission in laboratory conditions; both of these were transmitted per os (Shortt and
Swaminath, 1931; Adler and Theodor, 1935). There is, therefore, still much 4
to be learned about the species of Trypanosoma and Leishmania which parasitise
lizards.
2.The Morphology and Taxonomy of the Kinetoplastid parasites of lizard blood.
The morphological similarity of trypanosomes and of Leishmania parasites,
their pleomorphism and unknown levels of host specificity create major
taxonomical problems.
Species have often been separated on the basis of assumptions, for which
little evidence exists. It has been thought, for instance, that parasites
from lizards of different families or from those collected from distant
localities must be separate species. However successful cross-infection
experiments have shown that trypanosomes and Leishmania parasites of lizards
can infect hosts from different families (Grewal, 1957; Mohiuddin, 1959;
Dollahon and Janovy, 1974; Belova, 1971). Furthermore parasite species are
known which exist in hosts over a wide area of land: Trypanosoma platydactyli,
for instance, probably occurs throughout the range of its host, the gecko
Tarentola mauritanica mauritanica. This includes the Iberian Peninsula,
South-Eastern France, Italy, Malta, Tunisia and Algeria. (Fig. 1).
All the trypanosomes of lizards probably undergo growth from relatively
small metacyclics to the large, often enormous, "adult" haematozoic trypomastigotes, although this has never been observed. Species descriptions based on single smears, or even, as in the case of T. chamaeleonis, on single parasites, must therefore only reflect a small part of a much larger size range.
It is therefore probable that many of the lizard parasites have been given more than one species name. Increasing application of biochemical techniques is beginning to clear the taxonomical confusion caused by the use of morphological comparison alone. However, in many instances, parasite species 01
Figure 1. Geographical distribution of Tarentola mauritanica and Trypanosoma platydactyli.
Key:- ililliilljl Tarentola m. mauritanica (after Arnold et_ al_, 1978; Bruno, 1980; U. Joger-, pers.comm.) • Trypanosoma platydactyli (see p. 11 for references) ? Approximate location, exact site unknown 6
have only been seen on one occasion and attempts to relocate them so that
they can be examined biochemically, may prove difficult or impossible.
At present there remain 28 species of Trypanosoma and 12 of
Leishmania known from lizards. Until their biochemistry or extrinsic
development are further examined or they are used in cross-infection
experiments, little of the necessary taxonomical revision can be made.
(One major problem with cross-infection experiments, as with vector-mediated
transmission experiments, is the availability of lizards which are known,
to be free of haemoflageHates. In general the Reptilia are the hardest
of the Chordata to breed in captivity (U.F.A.W., 1972) and the normal use
of laboratory reared animals in infection experiments is often impossible
with lizards).
2.1. Lizard Trypanosomes
There has been only one major attempt to revise lizard trypanosome
taxonomy (Bray, 1964). This was based on host rather than parasite similarity
except in one instance when a trypanosome from an agamid lizard was assigned
to T. houeti, a trypanosome more often found in skinks. There is little
evidence against Bray's suggested synonymy and, considering trypanosome
pleomorphism, it may be close to the true situation. However the present
author has been less bold suggesting synonymy only where it is probable, not
possible.
2.2.List of lizard Trypanosomes
Where drawings or photographs of lizard trypanosomes exist, they have
been redrawn to the same scale to make comparison easier (Figs. 2 and 3).
The available morphometric data has also been assimilated. (Appendix A ).
The size of each trypanosome is given below, as body length plus free 7
flagellum length, either as means alone or ranges plus means when available.
This is followed by a brief description. "Slender" describes trypanosomes more than 3 times longer than wide. "Broad" describes broader trypanosomes.
Gekkonidae hosts
1. T. pertenue Robertson, 1908.
In the present work T. hemidactyli Mackie et al, 1923 is
considered to be a synonym of T. pertenue.
Hosts:- Hemidactylus triedi, H. leschenaultii, H. fernatus.
Distribution:- India and Sri Lanka.
Size:- 30-35 + 15-20ym, ^slender, often curved in smears.
Note: Unfortunately Iteckie et_ al (1923) gave no measurements for T. hemidactyli but it appears identical to T. pertenue. The much larger trypanosome from
Hemidactylus sp. from Assam, provisionally attributed to T. hemidactyli by
Short and Swaminath (1928) more closely resembles T. phlebotomi Shortt and
Swaminath, 1931.
2. T. leschenaultii Robertson, 1908.
Hosts:- Hemidactylus leschenault ii.
Distribution:- Sri Lanka (Robertson, 1908), possibly also
Vietnam. (Mathis and Leger ,1911)
Size:- 56-60 + 17-22ym, slender, often curved in smears.
Note: Found in the same host as T. pertenue but longer. It is possibly an older stage. Similar parasites were seen in the agamid, Acanthosaura fruhstorfrei, from Indo-China. (Mathis and Leger 1911).
3. T. phlebotomi (Mackie, 1914) Shortt and'Swaminath, 1931.
Hosts:- Hemidactylus fernatus
Distribution:- India.
Size:- 25-41 (x = 29.4) + Oym, broad ovoid. 8
Figure 2. Trypanosomes of lizards A (x 1500)
c T. leschenaultii
d T. garnhami
e T. hemidactyli (size unknown)
f T. pertenue
g T. platydactyli
h T. phlebotomi
i T. gonatodi
j-1 T. ryukyuense
m Trypanosoma sp. A. from Phelsuma lineatum (Brygoo 1963)
n Trypanosoma sp. B. from Phelsuma lineatum (Brygoo 1963)
o T. thecadactyli
p T. phylluri
q T. torrealbai
r T. grayi (= T. varani)
s-t T. "loricatum"
u Trypanosoma sp. from Chamaeleo nasutus and C. willsi
(Brygoo, 1963)
v T. chamaeleonis
w-x T. therezieni
10
Note: The reported "axostyle" is possibly an elongate nucleus and the
"trophonucleus" an area of dense chromatin within the nucleus, as seen in
T. serveti and T. thecadactyli. Similar to T. boueti (synonymy suggested
by Grewal (1957)) but development in sandfly differs (Grewal, loc. cit;
Ashford et al, 1973).
4. T. garnhami Grewal, 1957
Hosts:- Hemidactylus brookii angulatus and Lacerta viridis
(experimental).
Distr ibut ion:-Kenya.
Size:- 18-39 (x = 24.8) + 11-21 (x = 15.2)ym broad ovoid.
Note: Similar size to T. phlebotomi but has free flagellum.
5. T. gallayi Bouet, 1909
Hosts:- Psylodactylus caudicinctus
Distribution:- Ivory Coast
Size:- 50 + 9ym, slender, often curled around itself.
Note: Similar shape to T. leschenaultii but distinguished by juxtanuclear kinetoplast (KN = 1.8ym in T. gallayi and lOym in T. leschenault ii) and
shorter free flagellum.
6. T. petteri Brygoo, 1966a
Hosts:- Phelsuma madagascariensis
Distribution:- Madagascar
Size:- From 33 + 12 ym to 43 + 25 ym, varied shapes from
slender forms often coiled and with round nucleus to broad
forms with elongate nucleus.
Note: Slender, broad and intermediate forms of this species have been observed (Brygoo, 1966$ . The broad form is similar to T. betschi. The slender form is shorter than T. leschenault ii with a longer free flagellum. 11
7. T. platydactyli Catouillard, 1909.
Hosts:- Tarentola mauritanica (= Platydactylus muralis).
(Catouillard, 1909)
Tarentola annularis (Ranque, 1973)
Distribution:- Tunisia (Catouillard, 1909 ; Chatton and Blanc,1914)
Algeria (Sergent et^ £l, 1914)
Spain (Wood, 1937)
France (Rioux et_ al^, 1979)
Sicily (Adler and Theodor, 1931)
Italy (Laveran and Franchini, 1921).
Malta (Adler and Theodor, 1935)
Senegal (as T.p. senegalensis) (Ranque, 1973)
Size:- 44 + 12-19ym, slender to broad, often curved in smears.
Note: The pointed posterior end is sometimes hidden by cytoplasmic fold
giving a "beaver-tail" appearance (vide section3.3.2.1;). The nucleus and
juxtanuclear kinetoplast are in the posterior third of body. Very similar to
T. martini of skinks but the twisting of the posterior extremity is not a
constant feature. The untwisted extremity is pointed, not blunt.
8. T. phylluri Mackerras, 1961.
Hosts:- Phylluris platurus.
Distribution:- Australia
Size:- 36-48 + 2-6ym, broad, often curved in smears.
Note: Similar to T. platydactyli but broader with smaller nucleus and
longer free-flagellum.
9. T.ryykyyense Miyata, 1977
• Hosts:- Eublepharis kuroiwae kuroiwae
Distribution:- Okinawa Island, Japan.
Size:- From 25.5 +6.2 to 33.3 + 8.9ym, slender to broad.
Note: Slender, broad and intermediate forms of this species have been 12
observed. (Miyata,1977). Distinguished from all other lizard trypanosomes by
its distinct spiral ridging.
10. T. gonatodi Telford, 1979a
Hosts:- Gonatodes albogularis fuscus.
Distribution:- Panama.
Size:- 34-47 (x = 39) + 5-13 (x = 9) ym, broad ovoid.
Note: Unusual "tongue-shaped" appearance, and indistinct flagellum
distinguish T. gonatodi.
11. T. torrealbai Telford, 1979a
Hosts:- Gonatodes taniae, Phyllodactylus centralis
Distribution:- Venezuela.
Size:- 28-38 (x = 32.7) + 1.5-24 (x = 10.4)ym, broad ovoid,
often with short posterior projection.
Note: Similar to the larger forms of T.betschi from a Madagascan Cordylid
lizard but synonymy is unlikely considering the distance between the type
localities.
12. T. thecadactyli Christenson and Telford, 1972
Hosts:- Thecadactylus rapicaudus
— Distribution:- Panama.
Size:- 19-24 (x = 21.9) + 9-20 (x = 12.4)ym, broad ovoid or
roughly triangular.
Note: Similar to T. torrealbai but smaller with longer acuminate posterior projection - possibly a younger stage. Very basophilic. Chromatin gives the nucleus a "half-moon" appearance.
13. T. ocumarensis Scorza and Dagert, 1955.
Hosts:- Thecadactylus rapicaudus
Distribution:- Venezuela.
Size:- 17-24 + 42.5 (x = 20.0)ym, broad ovoid. 13
Note: Only briefly described by the authorities. Similar size to T. gonatodi
but said to have a pyriform, not a round, nucleus and to be like T. rotatorium
of frogs which T. gonatodi is not. Same host as T. thecadactyli but much
larger. Original smears have been lost (J.V. Scorza, pers. comm.).
14. Trypanosoma spp. Brygoo (1963) described two species of trypanosome from
Phelsuma lineatum.from Madagascar. Both stained in an unusual manner.
Trypanosoma sp. A was a slender trypomastigote (21 + Oym)of very unusual
appearance. A very basophilic and elongate central zone was surrounded by,
much paler staining cytoplasm. A small pale circular zone within the central
area was thought to represent the nucleus.
Trypanosoma sp. B was a large broad trypanosome (27 + 8ym). The nucleus
could not be distinguished although a small irregular mass of chromatin was
seen in a few parasites. It resembled T. petteri, T. betschi and T. domerguei
but these species show "normal" nuclear staining.
The parasite of a Gambian Hemidactylus fasciatus provisionally identified
as T. loricatum (Garnham and Duke, 1953) was probably not this amphibian
trypanosome. At the time T. loricatum was the only trypanosome reported from
Gambian lizards (Todd and Wolbach, 1912) but the earlier identification was
incorrect, Todd and Wolbach's paper showing trypanosomes much more like T. boueti
(vide Bray, 1964).
Gehrke (1903) was the first to report a trypanosome of geckos but neither
parasite nor lizard nor locality were described.
Another undescribed trypanosome was found in unnamed geckos in Kenya by
Heisch (1954). 14
Scincldae hosts.
15. T. boueti Martin, 1907. (= Syn. T. mabuiae, T. perroteti)
Hosts:- Mabu^a raddoni (Martin, 1907)
. M. perroteti (As T. perroteti, Franca, 1911)
M. quinquetaeniata (As T. mabuiae, Wenyon, 1909)
M. maculilabris (As T. martini, Schwetz, 1931)
M. striata (As T. martini, Hoare, 1932; as T. boueti,
Ashford et al, 1973)
Lygosoma fernandi (As T. sp., Schwetz, 1933) and
Lygosoma sp. (As T. loricatum, Todd and Wolbach, 1912)
"Lezard a ventre orange" (Leger and Leger, 1914) =
? M. perroteti.
Distribution:- Guinea (Martin 1907)
Guinea Bissau (Franca, 1911)
Sudan (Wenyon, 1909)
Congo (Schwetz, 1931, 1933)
Uganda (Hoare, 1932)
Ethiopia (Ashford et^ «al, 1973) possibly also
Gambia (Todd and Wolbach, 1912) and
Mali (Leger and Leger, 1914).
Size:- 20-40 + 0-12ym, broad ovoid or circular.
Note: A pleomorphic trypanosome varying from a large circular trypanosome with a long crescent-shaped nucleus to a similar but smaller form with a circular nuclear outline and a broad curved trypanosome with a free flagellum. Much smaller trypanosomes 20-25ym long and 2-2.5ym in width seen in the Sudan (Wenyon, 1909) may represent "young" forms, possibly infective metatrypomastigotes. Originally
T. boueti was described as a large broad ovoid or round trypanosome with an elongate nucleus. Slightly different parasites from skinks were separated into 15
different species but later it was thought that these were forms of one
pleomorphic species, for which the name T. boueti had precedence. (Laveran and
Mesnil, 1912; Wenyon, 1926; Garnham, 1950). Drawings of trypanosomes seen in
Lygosoma sp. from Gambia (Todd and Wolbach, 1912) resemble T. boueti, not
T. loricatum, the species to which they were attributed.
16. T. martini Bouet, 1909.
Hosts:- Mabuya perroteti (Bouet, 1909).
M. maculilabris (Bouet, 1909; Garnham, 1950)
Distribution:- Ivory Coast (Bouet, 1909)
Kenya (Garnham, 1950)
Size:- 53 + 20pm in Ivory Coast, 33 + 15pm in Kenya.
Slender to broad trypanosome, usually curved in smears.
Note: Though from the same hosts as T. boueti, T. martini is thought to be a
distinct species since it appears to be monomorphic. It has a constantly twisted
posterior extremity, which is blunt, not pointed as in T. platydactyli. The
trypanosomes described as T. martini by Schwetz (1931) and Hoare (1932) were
polymorphic and probably T. boueti. The Kenyan parasites were described as a
"small race" by Garnham (1950) but may be younger stages than .those seen in
Ivory-Xoast.
17. T. mochli. Berghe et^ al, 1957.
Hosts:- Mochlus fernandi.
Distribution:- Congo.
Size:- 33-36 + 6-8pm, broad trypanosome, often curved in smears.
Note: Smaller than T. boueti. Similar size to the "small race" of T. martini but no twisting of posterior extremity and nucleus sub-terminal.
18. T. egerniae. Mackerras, 1961.
Hosts:- Egernia striolata.
E. cunninghami 16
Distribution:- Australia.
Size:- 22-28 + ll-12ym, small slender trypanosome, often
curved in smears.
A very basophilic trypanosome, the kinetoplast and nucleus
being difficult to distinguish in stained smears.
19. T. rudolphi. Carini and Rudolph, 1912.
Hosts:- Mabug-a agilis.
Distribution:- Brazil. % Size:- 20ym + 'short1 free flagellum, small ovoid (15ym in width).
Note: Considered to be like T. perroteti (= T. boueti). The only haemo- flagellate known from New World skinks.
20. Trypanos oma. s pp.
There are five reports of unnamed trypanosomes from skinks.
One from New Guinea, from Lygosoma (= Leiolopisma) fuscum, was large and broad (24-35ym). It was said to resemble T. loricatum or T. mabuiae (= T. boueti).
The kinetoplast was posterior and close to the nucleus (Thompson and Hart,1946)
Undescribed trypanosomes have been seen in blood smears from Sphenomorphus taeriolatus in Eidvold, Australia (Johnston and Cleland, 1912) and near Sydney
(Mackerras, 1961), from Mabuya sp. in Ethiopia (Fuller et al, 1980) and in
Mabuya carinata in India (Sinha, 1980).
Chamaeleontidae hosts.
There are only 3 reports of trypanosomes from Chamaeleons.
21. T. chamaeleonis Wenyon, 1909.
Hosts:- Chamaeleo gracilis
Distribution:- Sudan
Size:- 40 + lOym, slender.
Note: Described from a single overstained parasite, in which the kinetoplast and nucleus could not be seen. Best considered a nomen dubium. 17
Figure 3. Trypanosomes of lizards B (x 1500)
a T. gerronhoti
b-c T. betschi
d T. superciliosae
/ e-f T. sceloperi
g T. domerguei
h T. serveti
i T. plicae
j-k T. martini ("small Kenyan race")
1 T. mochli
m T. boueti (Ashford et al, 197 )
n T. mar t ini
o T. boueti (Martin, 1907)
p-r T. mabuiae
s T. egerniae
19
22. T. therezieni Brygoo, 1963.
Hosts:- C. brevicornis (Brygoo, 1963)
C. lateralis (experimental]
C. oustaleti ( "
C. verrucosus( "
C. parsonii (
C. wilsi ( (Fromentin, 1967)
Size:- 28-33 (x = 32) + 15-21 (x = 17)ym slender often curlpd
around itself.
Note: Similar to slender form of T. petteri but apparently monomorphic with granular, paler staining posterior end not seen in T. petteri.
23. Trypanosoma sp. Brygoo, 1963.
Hosts:- C. nasutus
C. willsi.
Distribution:- Madagascar.
Size:- 30-37 + 10-20ym, slender to broad rectangular or trapezoid.
Note: This parasite described by Brygoo (1963) has the same granular and paler staining posterior end seen in T. therezieni and may be a form of this parasite (Brygoo, 1965). Never seen in chamaeleons infected naturally or experimentally with T. therezieni but hosts short-lived in the laboratory (Brygoo,
1963) and these forms may develop late in infections. Some forms are also similar in outline to T.chamaeleonis.
Agamidae hosts.
Trypanosomes have been reported twice from agamid blood smears.
24. Trypanosoma sp. Todd and Wolbach, 1912.
Hosts:- Agama agama (= A. colononum).
Distribution:- Gambia.
Size:- 33 + 3ym and 23 + 5ym. 20
Note; Two forms of trypanosomes were seen, one slender, one broad. They were thought to be T. locicatum but both were possibly T. boueti, the large
form in particular closely resembles the original description of this parasite.
25. Trypanosoma sp. Mathis and Leger, 1911.
Hosts:- Acanthosaura fruhstorfrei.
Distribution:- Vietnam.
Size:- 56 + 22ym, long slender.
Note: Possibly T. leschenaultii of geckos which it closely resembles though
the kinetoplast of the Acanthosaura parasite is slightly closer to the nucleus.
Ctirdylidae hosts.
26. T. betschi Brygoo, 1966.
Hosts:- Zonosaurus madagascariensis.
Distribution:- Madagascar.
Size:- 20-30 (x = 26) + 3-26 (x = 10) broad.
Note: The posterior and anterior ends of this trypanosome are hard to define because of its globulous shape. The nucleus is spherical or elliptical and
indistinct. Some forms are similar to T. torrealbai of the Neotropics and
T. domerguei from a Madagascan iguanid lizard.
Varanidae hosts.
27. T. grdyi (= T. kochi, T. varani) Novy, 1906.
Hosts:- (Saurian only) Varanus niloticus (As T. grayi and
T. varani, Lloyd et_ al, 1924; as T. varani, Wenyon, 1909)
Varanus exanthematicus (As T. grayi, Lloyd et al, loc. cit;
as T. varani, Taylor, 1929; Ranque, 1973).
Distribution:- Nigeria (Lloyd e£ al, loc. cit).
Sudan (Wenyon, loc. cit.).
Senegal (Ranque, loc. cit).
Also Liberia and probably Congo and Uganda, as 21
"crocodile parasite" (vide Brygoo, 1966).
Size:- 35-40 + 5-6ym (as T. varani, Wenyon, 1909)
64 + 23ym (as T. sp. of Crocodilus niloticus, Bruce
et_ al^, 1911) Slender, usually curved in smears.
Note: The trypanosomes discovered in crocodiles (Osteolaemus tetraspis,
Crocodylus cataphractus, C. niloticus) were originally divided into two species:- T. kochi and T. grayi. A similar parasite, found in Varanufi spp., was named T. varani (Wenyon, 1909). T. grayi and T. kochi were later found to be synonyms (Hoare, 1929). T. varani also appears to be the same parasite, its development in Glossina tachinoides being identical to that of T. grayi
(Lloyd et al, loc. cit.).. Always seen as a slender parasite but variable in length.
Iguanidae hosts.
28. T. superciliosae Walliker, 1965.
Hosts:- Uranoscodon superciliosa
Distribution:- Brazil
Size:- 96.2 + 5-25 (x = 16.1)ym, slender.
Note: Easily identified because of its enormous size. Elongate nucleus.
The posterior end stains less intensely; as seen in T. thereziensi.
29. T. sceloperi Ayala, 1970.
Hosts:- Sceloporus occidentalis occidentalis (Ayala, 1970)
and possibly also Ctenosaura hemilopha (Mahrt , 1979).
Distribution:- California, U.S.A. (Ayala, 1970) and possibly also
Isla San Pedro Nolasco, Gulf of California, Mexico
( Mahrt,loc .cit.)
Size:- 42-67 (x = 54.7) + 7-17 (x = 13)ym, slender, often
curved in smears.
Note: Similar to T. grayi but broader, with free flagellum. Much smaller 22
than T. superciliosae, with a spherical, not elongate, nucleus. Mahr.t'.
(loc. cit.) saw a single trypanosome which resembled T. sceloperi but only
37ym long.
30. T. plicae Lainson et^ al, 1975.
Hosts:- Plica umbra.
Distribution:- Brazil.
Size:- 40.5 + 12-28 (x = 16.4)ym, broad ovoid.
Note: A broad trypanosome with rounded posterior end and tapered pointed anterior end. Similar to T. serveti but smaller and more slender without distinct chromatin mass and with a longer free flagellum.
31. T. serveti Pelaez and Streber, 1955.
Hosts:- Sceloporus teapensis (Pelaez and Streber, 1955) Anolis capito (as Trypanosoma sp., Telford, 1977a; 1979$. A. poecilopus 11
A. biporcatus "
A. tropidogaster "
A. le_mur iniis "
A. limifirons (as Trypanosoma sp., Telford, 1977a; 1979b*
as T. serveti, Guerrero et al, 1977).
A. frenatus "
A. fuscoauratus (as T. serveti, Guerrero and Ayala, 1977).
Anolis sp. "
Corytophares cristalus (as Trypanosoma sp., Telford, 1977q 1979$. Distribution:- Mexico (Pelaez and Streber, 1955)
Panama (Telford, 1977*; Guerrero et_ al_, 1977)
Belize (Telford, 1977a)
Peru (Guerrero and Ayala, 1977)
Colombia 11 23
Size:- 27-35 (x = 30.8) + 6-11 (x = 8.4)Mm, broad ovoid. Note; Often with a dense chromatin mass at the anterior end of the
elongate nucleus. Otherwise similar to T. plicae.
32. T. domerguei Brygoo, 1965.
Hosts:- Hoplurus sebae.
Distribution:- Madagascar.
Size:- 19.6-29.2 (x = 23.3)ym + 4.9-11.0 (x = 6.7)ym, broad ovoid.
Note: T. domerguei is very similar to T. betschi and is possibly a synonym;
both parasites were found in lizards from Madagascar. T. domerguei also
resembles an unnamed trypanosome of the gecko Phelsuma lineatum except for
the nuclear staining which is "normal" in T. domerguei and abnormal in the
unnamed parasite.
Anguidae hosts.
33. T. gerrhonoti Ayala and McKay, 1971.
Hosts:- Gerrhonotus multicarinatus.
Distribution:- California, U.S.A.
Size:- 56.8 + 0.5-6 (x = 3)ym, slender, usually twisted in a
spiral or extended "S".
Note: Very like T. grayi. Similar to T. scelopori in morphology and
development in sandflies and possibly a synonym, as the authorities suggested.
Experimental cross-infections are necessary to check if an identical parasite
or not. 24
2.3.Lizard Leishmania Parasites
Lizard leishmaniasis has nearly always been detected by culture of blood
in biphasic media, usually NNN (Wenyon, 1926). Infections in the vertebrate
are usually cryptic and intracellular amastigotes have only been reported from
32 or 33 lizards. (Chatton and Blanc, 1914; Telford, 19791; Rioux et_ al 1979).
Cells containing amastigotes are usually very rare and include erythrocytes
(Telford, 1979k) monocytes (David, 1929; Rioux et_ al, 1969) and thrombocytes
(Telford, 1979b). An unusually high amastigote cell infection rate has
recently been seen in Hemidactylus mabouia in Tanzania (S.R. Telford, Pers.
Comm.). However Telford (Pers. Comm.) believes that the amastigotes, which
are in proerythrocytes and mature erythrocytes, are of trypanosome origin.
Considerable confusion has resulted from Wenyon*s (1921) reclassification
of parasite lizard blood and lizard intestine from Herpetomonas and Leptomonas
into the genus Leishmania. Though promastigotes from each site have similar
morphology the ecological niches they occupy are very different and it has
been suggested that they should be split into 2 genera (Vickerman, 1976).
The two groups have therefore been separated in the present work. Haemo-
flagellates are discussed below and intestinal parasites in Part B.
24.List of lizard Leishmania parasites.
The Leishmania parasites of lizard blood are listed below. The sizes
given are for body length only for parasites in culture. The sizes of
haematozoic amastigotes are given in Table 1 where known. 25
Table 1 . Morphometric comparison of amastigotes
from lizards and mammals
Name of parasite Mean dimensions Ratio of Reference (ym) length to width Parasites of lizards Leishmania sp. from Hemi- dactylus mabouia 2.16 x 0.85 2.54 Telford per.comm. L. agamae 1.5-2.5x1.0-1.5 David, 1929 L. tarentolae 2.4 x 0.8 3.00 Chatton and Blanc,1914 2.4 x 1 2.40 Wallbanks, present study L. hemidactyli 2.7 x 1.9 1.42 Shortt & Swaminath,1928 Leishmania sp. from Tetrascincus scincus 2.96 x 1.47 2.01 Telford, 1979fc Leishmania sp. from Agama agilis 3.02 x 1.92 1.57 Parasites of mammals L. donovani 2.1 x 1.8 1.17 Christophers, 1926 L.braziliensis braziliensis 2.25 x 1.64 1.37 Shaw and Lainson, 1976 L.mexicana amazonensis 3.3. x 2.13 1.55 ii L. tropica 3.33 x 1.99 1.67 Kellina, 1962 L. major 4.48 x 3.33 1.35 it L. mexicana 5 x 2.5 2.00 Garnham and Lewis, 1959 L. enrietti 5.2 x 2.5 2.08 Muniz and Medina, 1948 26
Gekkonidae hosts.
1. L. tarentolae Wenyon, 1921.
Hosts:- Tarentola mauritanica
T. annularis (as L.t. senegalensis, Ranque, 1973)-
Distribution:- Algeria (as Leptomonas sp. Sergent et_ ai, 1914)
" (as L. tarentolae, Parrot, 1928;1931;
1934;1935)
Tunisia (as amastigotes, Chatton and Blanc, JL914)
11 (as Leptomonas sp. Chatton and Blanc, 1918
Nicolle, 1920; Nicolle £t al, 1920)
Malta (as L. tarentolae, Adler and Theodor, 1935)
Italy (as Herpetomonas tarentolae, Laveran and
Franchini, 1921)
France (as L. tarentolae, Rioux et al, 1969;1979)
Senegal (as L.t. senegalensis, Ranque, 1973).
Size:- Amastigotes 2.5-5ym; promastigotes 9-26ym, with longer
thinner forms. (Laveran and Franchini, loc. cit.).
Note: This is the first Leishmania parasite discovered in a lizard. Infected lizards were once considered as possible reservoirs of the parasites causing human disease (Sergent et^ al, 1914). The good development of L. tarentolae in in vitro culture has led to its use in many biochemical studies (Trager, 1957;
1969; Krassner and Flory, 1971; Simpson 1968a; 1968b; Simpson and Braly 1970;
Simpson and Simpson 1978; Krassner, 1965; 1968). Intracellular amastigotes have been seen on three occasions (Chatton and Blanc 1914; Rioux et al, 1969;
1979). Young and Hertig (1927) produced visceral, followed by cutaneous lesions in the Chinese hamster (Cricetus griseus) after inoculating so-called
"L. tarentolae" culture forms. 27
2. L. hemidactyli Mackie et al, 1923.
Hosts:- Hemidactylus gleadorii (Mackie et_ al^, 1923)
Hemidactylus sp. (Mackie et al, loc. cit., Shortt
and Swaminath, 1928)
Distribution:- India
Size:- Not given.
Note: This parasite was originally named Herpetomonas hemidactyli, a
name already occupied for a species with promastigotes in the intestine of,
Indian Hemidactylus brooki (Mello and Suctangar, 1922). Although the
intestinal and blood parasites may be the same species this seems unlikely.
Since only promastigotes were seen in the intestinal infection (Mello and
Suctangar, loc. cit.) it is perhaps best to transfer this parasite to the
genus Leptomonas and transfer the blood parasite to Leishmania.
Short and Swaminath (1928) saw amastigotes in cells they described as
"mononuclear cells" but which were probably erythrocytes (Telford, 1979$.
3. L. hoogstraali McMillan, 1965.
Hosts:- Hemidactylus turcicus
Distribution:- Sudan.
Size:- Amastigotes 2.5-7ym. Promastigotes 5.7-26ym.
4. L. ceramodactyli Adler and Theodor, 1929.
Hosts:- Ceramodactylus doriae
Distribution:- Iraq.
Israel.
Size:- Amastigotes and promastigotes 4 .5-18ym with longer
thin promastigotes (22-35ym) after 6th subculture.
Other Leishmania parasites of geckos.
Note: Popov (1941) reported promastigotes from blood smears from 3 Caspian geckos. Telford (1979b) saw intracellular amastigotes in blood smears of 28
Teratoscincus scincus from Pakistan. Promastigotes were cultured from the blood of T. scincus and Gymnodactylus caspius from Turkmenian SSR (vide
Belova, 1971) and from a Hemidactylus sp. from Kenya (Ngoka and Mutinga, 1978).
Lacertidae hosts.
5. L. adleri Heisch, 1958.
Hosts:- Latastia longicaudata revoili
Mabuj^a striata (Experimental Mohiuddin, 1959)
Agama mutablis "
Lacerta viridis "
Basiliscus vittatus (Experimental, Dollahon and Janovy,
Ameiva quadrilineata "
Dipsosaurus dorsalis "
Distribution:- Kenya
Size:- Amastigotes 3-4ym, promastigotes 6.5-21ym.
Note: L. adleri is of some considerable interest since it appears to cause transient infection in man for up to 7 days (Manson-Bahr and Heisch, 1961) and longer (5 week) infections in hamsters and mice (Adler, 1964). However attempts to repeat these experiments with other reptilian Leishmania parasites have not resulted in infections .(Khodukin and Sofiev, 1940; Belova, 1971).
Dollahon and Janovy (1974) successfully infected New;World lizards with this Old World parasite, though the parasites only survived for 1 to 56 days.
Ngoka and Mutinga (1978) cultured leptomonads from 3 Kenyan L. longicaudata these parasites were probably L. adleri.
6. L. zmeevi Andrusko and Markov, 1955.
Hosts:- Eremias intermedia, Egrammica.
Distribution:- Turkmenian SSR.
Size:- Haematozoic promastigotes 9.2-19.3ym. 29
The inadequate description of this species, combined with the apparent loss of the type material (Killick-Kendrick, pers. comm.) means that L. zmeevi should be considered a nomen dubium. The promastigotes, seen in the blood of several lizards, may have been contaminants from the lizard intestine
(see Part B).
Other Leishmania parasites of lacertids
Promastigotes have been reported from several lizards from the Turkmenian
% SSR. These include promastigotes seen in one tissue smear of E. lineolata
(Zmeyetf, 1936) and in cultures of blood from E. intermedia, E. grammica,
E. velox, E. lineolata and E. guttulata guttulata (Belova, 1971).
Agamidae hosts.
7. L. agamae. David, 1929.
Hosts: Agama stellio (David, 1929; Edeson and Himo, 1973).
A. sanguinolenta . " "
Cnemidophorus sexlineatus (Experimental, Dollahon and Janovy,
1979)
Distribution:- Israel.
Lebanon (Edeson and Himo, 1973)
Size:- Amastigotes 2-3ym, promastigotes 2-17ym, longer thin
promastigotes (up to 28ym) after 4th subculture.
Note: The experimental infection of C. sexlineatus lasted only 10 days.
8. L. gymnodactyli Hodukin and Sofiev, 1940
Hosts:- Agama sanguinolenta
Phrynocephalus mystaceus
P. heliscopus.
Distribution:- Turkmenian SSR.
Size:- Not known. 30
The original paper describing L. gymnodactyli is difficult to obtain and most details about the parasite are from Belova (1971). The parasite was not infective for Gymnodactylus caspius nor for several mammals inoculated with cultures.
Other Leishmania parasites of Agamidae
Cultures of blood from Iranian Agama agilis, A. melaneura and A. caucasia
(Nadim et al, 1968; Seyedi Rashti et_ al^, 1971) and Turkmenian A. sanguinolenta,
Phrynocephalus interscapularis, P. mystaceus, P. raddei raddei and P. heliscopus
(Hodukin and Sofiev, 1940; Belova, 1971) contained promastigotes after a few days. Promastigotes were actually seen in blood smears of P. interscapularis
(Andrusko and Markov, 1955) and in tissue smears of this lizard and A. sanguinolenta
(Belova, 1971) in the Turkmenian SSR. Telford (1979b) described intracellular amastigotes in blood smears of Pakistani Agama agilis (Figs.81-84).Ngoka and
Mutinga (1978) cultured "leptomonads" from.16 Kenyan A. agama.
Scincidae hosts.
There are only two reports of leishmaniasis from skinks. Franchini (1921) reported rare amastigotes in blood and liver smears of 2 Chalcides ocellatus from Sicily. "Leptomonads" were cultured from the blood of 33 Mabuya sp. from
Kenya (Ngoka and Mutinga, 1978).
Iguanidae hosts.
9. L. henrici Leger, 1918
Hosts:- Anolis sp.
Distribution:- Martinique.
Size:- Promastigotes 15-16ym.
Note: Considerable doubt exists about the status of this parasite in the genus Leishmania (Dollahon and Janovy, 1974; Lainson and Shaw, 1979). It is 31
the only Leishmania parasite of lizard blood reported from the New World in
spite of the fact that many thousands of lizards have been examined in that
area. The Anolis from which it was isolated had intestinal infections of
promastigotes and it is possible these parasites contaminated the blood.
Promastigotes were not seen in fresh blood smears, only in fixed smears of
blood taken from the heart after the abdominal cavity had been opened.
Therefore L. henrici is best considered, like L. zmeeyj a nomen dubium,
until the type host can be reexamined in Martinique. It does not appear
that any lizard Leishmania parasites of blood and tissues have been
adequately demonstrated in New World lizards. (Dollahon and Janovy, 1971;1974).
Chamaeleontidae hosts.
Though Chamaeleons have frequently been found to have intestinal
infections of promastigotes ( vide Part B ) there is only one report of
promastigotes in their blood (Killick-Kendrick and Wallbanks, 1981).
Promastigotes were found in one blood smear from a Chamaeleo dilepis and
rapidly disappeared from the circulation. Though they may have been
contaminants from a concurrent heavy intestinal infection the blood and
intestinal promastigotes were of different sizes. Considering their short
stay in the blood, it is unlikely they were true chamaeleon parasites and may
have been a transient infection of a parasite inoculated by a vector of a
Leishmania from some other animal.
Varanidae hosts.
Leishmania "leptomonads" were reported from a culture of the blood of a
Varanus sp. from Kenya (Ngoka and Mutinga (1978)). 32
2.5. A note on reports of Leishmania parasites from some Kenyan lizards.
In reports of their studies of visceral leishmaniasis of animal
reservoirs in Kenya, Ngoka and Mutinga (1978) and Mutinga and Ngoka (1981)
stated that they had found Leishmania "leptomonads" in Agama agama,
Latastia longicaudata, Mabuya sp., Hemidactylus sp. and Varanus sp. These
observations have been included in the list of Leishmania parasites for the
sake of completeness. However considerable doubt is cast on the accuracy
of these observations because Ngoka and Mutinga went on to report in the sajne
paper that similar leptomonads were present in cultures of blood from one owl,
Bubo africa, and two unidentified hawks. It seems likely that some of the
cultures contained Trypanosoma species and that these either grew as pro-
mastigotes or that epi- or trypo-mastigotes were taken to be promastigotes. 33
3.An examination of the haemoflagellates of Tarentola mauritanica (L.1758)
3.1. Introduction.
Of all the lizard haematozoa, the kinetoplastid parasites of the
Moorish gecko, Tarentola mauritanica, have perhaps received the most
attention. The gecko Leishmania parasite, Leishmania tarentolae Wenyon,
1921, was the first lizard Leishmania to be discovered (Sergent al_» 1914).
The trypanosome found in the gecko, Trypanosoma platydactyli Catouillard,
1909, was only the seventh known from reptiles.
The Leishmania parasite has been of particular interest over the years.
At one time the gecko was considered to be a possible reservoir of the
organism causing Mediterranean kala-azar (Sergent et al, 1914; Chatton and
Blanc, 1914). Interest in the parasite waned when this hypothesis was
disproved (Adler and Theodor, 1931) but grew again when the parasite was chosen
as a laboratory model for the genus Leishmania. The parasite grew better in
in vitro culture than the mammalian parasites and it was hoped had a simpler
metabolism (Trager, 1957).
The life cycle in sandflies (Sergentomyia spp.) of both trypanosome
(Adler and Theodor, 1935) and Leishmania parasite (Parrot, 1934;1935; Adler,
1933; Adler and Theodor, 1929;1930;1935) has been observed. However studies
on the Leishmania parasite gave conflicting results, some infections appearing
to be in the "anterior station" while others were in the "posterior station"
(vide Killick-Kendrick 1979). The discovery of both parasites in one locality
in South-Eastern France (Rioux et al, 1969;1979) has allowed a reexamination of
their development in the vector and in in vitro culture and a comparison of new
strains of known derivation with old stocks. 34
3.2. MATERIALS AND METHODS.
3.2.1. Collect ion of lizards and their maintenance.
Lizards were collected in September 1979 and on 10th and 11th of August
1981 in the town of Banyuls-sur-Mer, Pyrenee Orientales, France on the
Mediterranean coast and around the wine cellars 4km west of the town. They
were also caught 5km west of the town, at the Centre d'ecologie Mediterraneene,
a field station of the University of Paris. In 1981 all the geckos were
collected at night when they came towards street lights to feed off the inspects
which the lights attracted. They were directed towards ground level with a
long (7m) bamboo pole and then caught by hand. In 1979 all catches were made
by hand alone during the day by disturbing their resting places.
The geckoes were brought to England and kept separately in plastic boxes
measuring 17 x 11 x 6cm with a piece of folded tissue paper covering the base.
The boxes were kept on a dishwarmer (Photax, England) to protect the lizards
from extreme cold, the temperature varied from 10-27°C.
The lizards were sprayed with water and fed adult Musea domestica,
larval Tenebrio molitor or larval Galleria mellonella from laboratory colonies
twice weekly.
3.2.2.Examination of blood and tissues.
Blood samples for making thin smears were taken from the retro-orbital
sinuses of the lizards, using Pasteur pipettes drawn out to a width of 0.1-0.2mm.
The smears were air dried, fixed in methanol for one minute and stained in a
10% solution of Giemsa's stain (Traditional formula, "British Drug Houses,
Poole, Dorset), in Sorenson's buffer pH 7.2 for 40 minutes. Blood smears were
made as soon after capture as possible and then routinely at 2-3 week intervals.
Post-mortem tissue impression smears were made of tongue, brain, heart, lungs,
liver, kidneys, spleen, oesophagus, femoral bone marrow, skeletal muscle and 35
ventral skin from the thorax. They were fixed and stained as the blood smears
though staining time was reduced to 25 minutes. The parasites seen were
recorded and described.
3.2.3.Culture of parasites in axenic culture.
3.2.3.1.Routine culture and culture en masse
Sterile blood samples were taken from the geckos by cardiac puncture.
The geckos were held on their backs against a bench, and the ventral skin was
surface sterilised by wiping twice with 70% ethanol. A sterile Pasteur pipette was drawn to a short and fairly stout point about 0.2-0.4mm in diameter. This was pressed into the thorax at a midventral point on a line between the brachial
axes, directly above the heart (Parrot, 1927). The pipette began to fill with 3 blood, either immediately or after gentle rotation. About 0.2-0.3cm of blood
could be taken quickly in this manner. The blood was expelled into a tube
containing the culture medium and the gecko returned to a box. Only one of
39 geckos died as a result of cardiac puncture.
A biphasic culture medium was used routinely. One volume of tissue culture medium 199 (Flow, Irvine) was used as overlay for a slope of five volumes of
4% nutrient blood agar (4N, Baker, 1966). Rabbit blood was used initially until culture en masse required large volumes of blood agar. Horse blood
(Oxoid, Basingstoke) was then substituted. Both types of blood were used at
10-20% in blood agar number 2 (Oxoid). Stocks were maintained at 25 - 2°C in 3 3 3 25cm McCartney bottles with 5cm of blood agar. 500cm "medical flat" 3 bottles were used with 100cm of agar for culture en masse.
3«23.2.Culture in a variety of media
The development of one of the promastigote cultures (GB/79) and the double- cloned trypanosome strain (TPCL2, c.f. sect ion 3.2.5.) was investigated in a wide variety of media. Each of the two parasite strains was grown in 36
duplicate in each of 20 media.
Media included:-
1. 4% Blood base agar number 2 (Oxoid) with 10% defibrinated sheep blood
(Oxoid) with no overlay or 2% Glucose-peptone or medium 199 (M199,
Flow) with 5% foetal calf serum (FCS, Flow) or chick serum as overlay.
2. 4% plain New Zealand agar (British Drug Houses) with 10% sheep blood
and either no overlay or 2% Glucose-peptone or M199 with 5% FCS, or
chick serum overlay.
3. 4% Blood base agar number 2 with 10% defibrinated rabbit blood.
4. 4% Blood base agar number 2 with 10% Tarentola mauritanica blood and
no overlay.
5. 4% Brain heart infusion agar (BHIA, Oxoid) with 10% defibrinated
sheep blood and no overlay or M199 with 5% FCS as overlay.
6. Horse serum (Oxoid), coagulated as a slope by heating to 60°C for
30 min, with 2% Glucose peptone as overlay.
7. Schneider's medium (Flow) with 30% FCS.
8. M199 with>30% FCS.
9. EBLB (Evans, 1978).
10. A 1 to 1 mixture of EBLB and M199.
11. Matteis medium (Mattei et_ al^, 1977).
12. L.I.T. medium (Castellani et_ al, 1967).
13. Trager's medium C (Trager, 1957). 3 . 3 Cultures were grown in 25cm McCartney bottles with either 1cm of 3 3 monophasic medium or 5cm - of agar or coagulated serum with no overlay or 1cm 4 of overlay. Each culture was inoculated with 10 parasites from a 10 day old culture in Trager's Medium C.
The cultures were examined and stained smears were made weekly for 5 weeks.
Maximum growth of each parasite in each medium was estimated using an arbitrary 37
scale of pluses. Haemocytometer counts of a sample of cultures indicated that 4 3 5 cultures scored "+" had approximately 10 parasites/cm , those scored "++" 10 3 6 3 7 3 parasites/cm "+++" 10 parasites/cm and "++++" 10 parasites/cm . Three
hundred parasites from each smear were examined and their morphology recorded.
3.2.3.3.Culture at different temperatures and pH values
One parasite strain, TPCL2, was grown in Trager's medium C (Trager, 1957)
at 3 different temperatures (25 ,28 and 37 C) and in a separate experiment at % 8 different pH values (pH values of 5.0,6.0,6.5,7.0,7.2,7.4,7.6,7.8,8.0 and 9.0). 3 All experiments were made in triplicate in Bijou bottles, each with 1cm of 4 medium and 10 parasites from a 10 day culture in Trager's medium. Varying
amounts of 1MK0H or 1MHC1 were added to concentrated medium, which was then
adjusted to the correct dilution with distilled water, to give the different
pH values. Parasite division and morphogenesis were followed in each culture
by haemocytometer counts and stained smears made daily for 8 days.
3.243.4.Culture in medium containing stimulants for differentiation
One parasite strain, TPCL2, was grown in Trager's medium C containing 3mM -3 lidocaine hydrochloride or 3% dimethylsulfoxide or Conconavilin A at 0.002yg cm 4 Experiments were made in triplicate using Bijou bottles, each with 10 3
parasites in 1cm of medium (c.f. section 3.2.3.3). Stained smears were made of each culture every 3 days for 15 days. These were examined and the morphology of 30o parasites from each smear was recorded.
3,2.35.The use of antibiotics
The routine use of antibiotics was avoided. However they were found to be necessary for the primary isolation of parasites, for cloning experiments and experiments involving coverslip cultures in petri-plates, when Gentamycin was used at lOOyg/ml. 38
Cultures were examined for parasites by removal of a small sample of
overlay with a flamed platinum loop. This sample was then examined on a
microscope slide with phase-contrast illumination. Parasite densities were
estimated using an Improved Neubauer haemocytometer. Cultures with high 7 3 parasite densities (> 10 parasites/cm ) were first diluted with 0.9% saline
in a "red cell" haematology pipette (1 part culture to a 100 parts saline).
3.2.4. Interaction of parasites with vertebrate cells. % 3,2>4.1.Lizard blood cells
(i) In vitro
Blood from a Banyuls T. mauritanica and an Agama sanguinolenta, collected
in Kanshinskaya Steppe, Usbek SSR by Dr R Killick-Kendrick, was mixed with
promastigote cultures in 2 separate experiments. 30yl of blood from each of
the haemoflageHate free lizards (checked by fortnightly blood smears over
4 months and 2 blood cultures) was mixed on a sterile microscope slide with 6 3 30yl of overlay from a 10 day old culture containing 5 x 10 promastigotes/cm .
The drops were then covered with sterile 18mm square coverslips which were sealed
with molten beeswax. The slides were incubated at 25°C and examined under the
microscope each day for 5 days when the coverslips were removed. The coverslips
and the slides were air dried, fixed and stained as for the blood smears.
(ii) 111 vivo 3 6 3 lcm of overlay from a culture containing 5 x 10 promastigotes/cm was 3 placed m a sterile centrifuge tube and washed twice in 5cm 0.9% sterile NaCl
solution. The parasites were spun down by centrifugation at 500g for 1Q min 3 and finally resuspended in 0.1cm of the saline solution. This suspension was
injected into the heart of a haemoflagellate-free gecko, the same one as used
in part (i), using a hypodermic syringe with a 23G x 25mm needle. A smear was > made of blood from the geckofs retro-orbital sinus after one minute', and after 39
3,6,12,20,24 and 36 hours. After 40 hours another cardiac puncture was made.
The blood taken was transferred to culture medium which was incubated and
examined weekly for 5 weeks.
3.2.4.2.Lizard tissue cells
An attempt was made to create a gecko cell culture using kidney cells
from a specimen of the largest gekkonid lizard, namely the Asian Tokay gecko
Gekko gecko. Details of the production of cell monolayers in flasks are given o *
in Appendix B . Cells from one flask were suspended by incubation at 30 C in 0.05%
Trypsin0.02% EDTA solution (Flow, Irvine), washed once in cold minimum essential
medium (MEM) with Eagles salts (Flow), and centrifuged at 200g for 10 minutes 3 to spin down the cells. The cells were resuspended in 5cm of MEM, counted m 4 an Improved Neubauer haemocytometer and dispensed at 7 x 10 cells per well into
a 25 well petri-plate (Flow). Each well contained a 16mm coverslip which had
been rinsed twice in absolute ethanol and once in distilled water to clean it 3 before it was sterilised by dry heat. A further 1cm of tissue culture medium
was added to each well before the plate was incubated for a day at 25°C. 30yl
aliquots of overlay from a 10 day promastigote culture, each containing 1.5 x 10"*
promastigotes, were then added to each well. The promastigotes were left to
interact with the cells for 4 hours at 25°C and then the cell layers were
washed twice in fresh medium to remove unattached promastigotes. The plate was
then incubated for a further 4 days and examined with an inverted microscope
each day. On the fifth day the cell layers were drained, air dried, fixed in
methanol and stained as for blood smears.
3.2.4.3.Murine macrophage cells iri vitro
Peritoneal macrophages were obtained from 6 adult Tuck's Original (TO)
strain mice using the technique of Wasley and John (1972). The mice were killed by rapid dislocation of the neck and then their abdominal skin was 'stripped. 3 3 1.5cm of sterile air and 3.5cm of chilled Medium 199 with 10% newborn calf 40
serum (Flow) were injected into the peritoneum of each mouse. Their abdomens were then massaged to loosen the macrophages which were withdrawn as a suspension in the medium. The cells were then counted in a haemocytometer and, allowing for the fact that only about a third of the cells were macro- phages, dispensed at 7 x 10^ macrophages per well into 3 petri-plates (Flow).
Each well contained a coverslip cleaned and sterilised as those for the geckonid kidney cells (p 59 ). The macrophages were given 2 hours at 37°C to adhere to the coverslip. The cells were then rinsed once in culture medium previously warmed to 37°C. This rinse freed the cultures of most of the contaminating lymphocytes, eosinophils and mast cells. Promastigotes were added to all except
3 of the wells containing cells in exactly the same manner as to the geckonid cells; incubation was at 37°C not 25°C. 3 wells on each plate were left as controls without promastigotes. Once the unattached promastigotes had been rinsed off one plate was placed at 25°C, one at 28°C and the last at 37°C.
Seven coverslips, 6 with parasites and one control, were removed from each plate, after 24,48 and 72 hours and air dried, fixed and stained. The number of parasites within 250 macrophages from each coverslip was counted and the mean number of parasites per macrophage was calculated.
Mixtures of promastigotes and medium 199, with 10% newborn calf serum equivalent to those used with the macrophages, were kept concurrently with the macrophage cultures in Bijou bottles at 25,28 and 37°C, three bottles at each temperature. The parasite division in each bottle was estimated by daily haemocytometer counts for 8 days.
3.24.4.Murine cells jri vivo
"T.O." strain mice were inoculated subcutaneously with the overlay from a
7-day old culture of a gecko parasite, stock G7/79. 20yl of the overlay, 4 containing about 4 x 10 promastigotes, were inoculated in one ear hnd the base
of the tail of 5 mice using a 19G x 17mm needle and syringe. 41
The mice were killed 1,2,3,4 and 7 days after the injections. The
inoculation sites were surface sterilised with ethanol, allowed to dry and
then dissected out with sterile instruments. Half of the tissue from each
site was dropped into culture tubes containing blood agar slopes. The other
half was first used to make tissue impression smears and then dropped into
alcoholic Bouin's solution. These tissues were to be kept for sectioning if
parasites were found in the smears. Thin blood smears were also made.
The culture tubes were examined weekly for 5 weeks. The impression smears
were stained and examined under an oil-immersion lens (x 1000).
3.2.5.Cloning of blood trypomastigotes.
Cloning was attempted from the blood of 10 geckos. All cloning operations
were made in a laminar flow cabinet.
Blood taken from a gecko infected with Trypanosoma platydactyli by cardiac
puncture was used to fill sterile lOyl "Microcap" capillary tubes (Scientific
Supplies, London). These were sealed at one end with dental wax (Taab, Reading) . . . 3 and centnfuged at 200 g for 10 mm in centrifuge tubes containing 1cm of
dental wax to protect the tubes. After centrifugation the tubes were scored.
with a diamond and broken just below the "buffy layer" of white cells. The 3 white cell layer and overlying plasma were expelled into 1cm of medium 199. The technique of Soldo and Brickson (1981) was then used to clone the blood 3 3 trypomastigotes in this suspension. 0.2cm of the suspension was added to 4cm
of silicone fluid of 10 centipoise viscosity (Dow Corning 200/10cs, Hopkin and
Williams, Essex) in a 120x16mm tissue culture tube (Flow). The fluid had been
sterilised in the tube by autoclaving. An emulsion of the aqueous suspension
in the fluid was made by vortexing the tube for 15 seconds on aWhirlimixer
(Jencons, Hemel Hempstead). The emulsion was then poured quickly into a
sterile 6cm petri dish, covered and allowed to stand for 30 min. The microdrops 42
of tissue culture medium which settled and flattened out on the base of the
dish under the silicone fluid were then examined using an inverted microscope
at xlOO or x400 magnification. After a drop was found to contain one trypo-
mastigote, the isolated parasite was checked by 3 independent observers. If
no more parasites were seen in the drop, it was transferred to a blood agar
slope using sterile Pasteur pipettes drawn to a diameter of less than 150ym. 3 These were pre-filled with 0.5cm of culture medium so that the contents of
the drop could be more easily washed out. The cultures were examined after,
10 days at 25°C.
, The whole cloning procedure was repeated using parasites derived from one
blood trypomastigote 15 days after its isolation.
3-2.6.Comparison of stock morphology and biochemistry.
At first 20 parasite cultures were available for comparison. 17 of
these came from Banyuls geckos, 12 isolated in 1977-78 by Dr. R. Killick-Kendrick
(G6/77,G13/77,G16/77,G17/77,G18/77,G19/77,G20/77,G26/77,G27/77,G42/77,G58/77,
G26/78), 2 isolated in 1979 (G7/79,G13/79) and 3 isolated by the author in
1981 (G104/81,G116/81 = TP? and TPCL2). There were two other cultures, one
of parasites found in a sandfly Sergentomyia minuta at Banyuls (S/F2004,
Killick-Kendrick, 1979) and one old strain (LV414) descended from TARVI a
stock isolated from Algerian T. mauritanica by Parrot (1949).
During isoenzyme comparison a further 7 cultures were used. Five of these
came from Italian geckos from the Adriatic coast, four from T. mauritanica
(LEM321,LEM322,LEM323,LEM324) and one from Cyrtodactylus kotschyi (LEM325)
(M. Grammicia, Pers. Comm.). Another culture (LV108) came from T. annularis
from Senegal and was named L. tarentolae senegalensis. (Ranque 1973).
A culture of L. infantum (LEM75) isolated from a child in the Cevennes, France, / (Maazoun et al, 1981) was also used. 43
TPCL2 was the only stock known to be of trypanosome origin. It was a
double-cloned strain derived from a single blood trypomastigote.
3.2.6.1 .Morphological comparison
(i) Light microscopy
The body length of 200 promastigotes from 10 day old cultures of 5 / strains (S/F2004,G27/73,G13/79,LIV414 and TPCL2) was measured from Giemsa
stained parasites using a calibrated eyepiece micrometer. All the parasites,
except TPCL2 were grown at the same time in the same batches of culture media.
50 promastigotes from each strain were measured at random from each of 4
subsequent subcultures.
(ii) Electron microscopy
The overlays from 10-day old cultures of 5 strains (S/F2004,G27/77,
G13/79.LIV414 and TPCL2) were transferred to centrifuge tubes. The pro-
mastigotes were then washed twice in phosphate buffered saline pH^ ^
(Appendix C ), and fixed in 3% Glutaraldehyde in 0.1M cacodylate buffer for
5 min at room temperature and an hour at 4°C. After fixation the suspensions
were washed 3 times in 0.15M sucrose in 0.1M cacodylate, centrifuging at 200g
for 5 min each time to bring down the promastigotes. After post-fixation in
1% osmium tetroxide in buffer the parasites were dehydrated in an ethanol
series and embedded in Spurr's low viscosity resin. Sections were cut on an
LKB ultramicrotome, mounted on uncoated copper grids and stained with lead
citrate and uranyl acetate. They were examined on a Phillips EM300 electron
microscope at 60kV.
The number of subpellicular microtubules in 25 transverse sections of
promastigotes of each culture was counted. Calibrated dividers were used to
measure the circumference of these sections from photographic prints. The
average microtubule interval for each section add each culture was then
calculated. 44
3.2.6.2.Biochemical comparison
(i) Isoenzyme electrophoresis g
Lysates were prepared from approximately 10 parasites of each of 11
strains from lizards and 1 strain (LEM75) of Leishmania infantum (LEM321,
LEM323,LEM324,LEM325,LEM75 ,S/F2004,LV414,LV108,TPCL2,G17/77,G42/77,G116/81)
grown in culture "en masse". After 10 days in culture the overlays were
transferred to centrifuge tubes, so that the promastigotes could be washed
three times in 0.9% NaCl solution and the contaminating erythrocytes lysed »
by a brief rinse in 0.3% NaCl solution. Parasites were spun down each time
at 200g for 10 min. After a final wash in 0.9% NaCl solution the supernatant
was discarded and a volume of 5% Triton-X-100 equivalent to that of the pro-
mastigote pellet was added. The pellet and surfactant were mixed with a ~
Pasteur pipette, left for 2 min and then dropped from the pipette into liquid 3 nitrogen to form 25yl "pearls". These were transferred to 2cm plastic
ampoules (Flow), where they were stored in.liquid nitrogen.
When used for the electrophoresis the pearls (2 or 3 per gel) were thawed
in the ampoules on ice, then centrifuged at 4°C for 20 min at 10,000 rpm. The
ampoules were then left on ice while 0.7 x 0.8cm rectangles of Whatman No. 3
paper were added to absorb the supernatant. These papers were transferred to
slots made in thick (1cm) gels of 10.0 or 10.5% starch made up in the appropriate
buffer. Details of the buffers and staining methods are given by Maazoun (1982).
The stock of Leishmania infantum (LEM75) was used as a marker during the
electrophoresis. Details of the enzymes stained are given in Table 2 .
3-2.7.0rigin collection and maintenance of sandflies.
3.2.7.1.Phlebotomus papatasi
The P. papatasi used in the experiments came from a laboratory colony which
had originally been established and maintained by Dr. R. Killick-Ke'ndrick. 45
Table 2 . Enzyme nomenclature
Enzyme Abbreviation Enzyme number*
Malate dehydrogenase MDH E.C.I.1.1.37.
Malate dehydrogenase (decarboxylating NADP) "Malic enzyme" ME E.C.I.1.1.40.
Isocitrate dehydrogenase ICD E.C.I.1.1.42.
6-Phosphogluconate dehydrogenase 6PGD E.C.I.1.1.44.
Glucose-6-phosphate dehydrogenase G6PD E.C.I.1.1.49.
Glutamic-oxaloacetate transaminase GOT E.C.2.6.1.1..
Phosphoglucomutase PGM E.C.2.7.5.1.
Glucose phosphate isomerase GPI E.C.5.3.1.9.
*Enzyme numbers according to the Commission on Enzyme
Nomenclature (1972).
> 46
Rearing was continued according to the techniques developed for Lutzomyia
longipalp is (Killick-Kendrick et al 1977) except that the flies were kept at
30°C not 25°C and larvae were given food based on rabbit faeces not liver
powder (c.f. Appendix D ).
The colony was derived from flies collected near Auvangabad, Mahareshta
State, India on several occasions in 1972 by Dr. G.B. Modi.
3.2.7»2.Sergentomyia minuta minuta
The Sergentomyia were caught in August 1981 from the drainage holes or
"barbacanes" in walls at Banyuls-sur-Mer and throughout the valley of the
Tech in the Pyrenees Orientales, France. An oral aspirator was used and flies
were held in the aspirators, in plastic bags, with vz.et cotton wool to maintain
a high humidity, until they could be transferred to suspended cages at 25°C.
These cages were also kept in plastic bags with wet cotton wool. Some of the
female flies were dissected to check for parasite infection, others were fed
on a gecko and were then kept, as were the fed P. papatasi, in small vials with
filter paper. The filter paper was kept dry for 3 days, then moistened. The
flies were provided with balls of cotton wool moistened with 30% sucrose
solution and were kept in sealed plastic boxes with wet cotton wool. The
females were removed as soon as they had oviposited and, after one day to
allow the eggs to harden, the eggs were transferred to plaster pots. The colony
was then treated in the same way as the P. papatasi, except that it was kept
at 25°C not 30 C, it was given 15 hours of light per day, not total darkness
and, of course, the flies were fed on geckos, not hamsters. The time taken
for each stage to develop was recorded. 47
3.2.8.Infection and dissection of sandflies
3.2.8.1 .Infection of sandflies from a gecko.
Geckos, seen to be infected with a haemoflagellate, were immobilised by
having their limbs fastened to a cork board with adhesive PVC tape. The
geckos were held on their backs.
The gecko and board were then placed in a suspended cage with 2-to 5-
day-old adult female flies. The caged gecko was then returned to a plastic
bag with wet cotton wool and left at 25°C in the dark overnight. In the
morning the gecko was removed and released back into a box. Female flies,
which had fed, were removed using the vial in which they were to be kept.
The vials were covered with gauze and sucrose solution was provided as for
the colony but the filter paper in the vial was never moistened. The flies
were dissected at intervals from 6 hours to 11 days after the infective meal.
A few gravid flies were offered a second blood meal from an uninfected gecko
8 days after the first meal.
Flies to be dissected were anaesthetized with carbon dioxide and
decapitated. Their guts were then teased out into a drop of sterile 0.&5Z. salihe
solution by pulling on the last abdominal segments while holding the thorax.
i
3 28 2 The observation of flagellate infections by light and electron microscopy,
(i) Light microscopy
Sandfly guts were examined intact in a drop of saline solution with pha6e
contrast illumination, under 100,250 and 400x magnification. If seen or
suspected to be infected the gilts were either teased apart, air dried and fixed
and stained as for blood smears or fixed embedded and sectioned for electron
microscopy (see section3;2.8~.2.ii) When flies had fed more than 2 days previously
their heads were teased apart so that the pharynges and proboscides could be
examined. 48
Paraffin wax sections of 5ym thickness were made from a few whole flies
fixed in Carnoy's fluid for 2h. The flies were embedded first in celloidin
and then in paraffin, a technique created by Peterfi (see Pantin, 1969).
The sections were stained by the Giemsa Colophonium method (Bray and Garnham,
1962).
(ii) Electron microscopy
A few heavily infected sandfly guts and one gut from a S. minuta and
P. papatasi fed on an uninfected gecko 3 days previously were sectioned for
electron microscopy. The guts were picked out of the drops of saline and
dropped in 3% Glutaraldehyde in 0.1M cacodylate buffer. The details of fixation,
embedding and sectioning are the same as for parasite cultures (section 3.2.6.1.) but
centrifugation was not necessary.
3-2.8-3.Attempt to infect sandflies by membrane feeding.
2 to 4-day old adult female S. minuta were anaesthetized with carbon dioxide
and introduced into a 6cm pill box fitted with a nylon stocking top. They were
allowed to recover in a plastic bag with wet cotton wool. The gauze top was 3 then placed against the feeding unit (Mellor, 1971) which contained 0.5cm of 3 gecko blood and 0.5cm of overlay from a 10 day old parasite culture containing
3x10** promastigotes. The suspension was kept at 37°C - 1°C*using an immersion
thermostat
water pump. The unit was covered with the skin of a day old chick,
epidermis towards the flies. The human observer breathed into the box 3 times
to activate the flies. The apparatus was then left at room temperature
(24 - 2 C)
in subdued and indirect light for one hour. Details of the feeding technique
are given by Ready (1978). 49
3.3. RESULTS
3.31.Collection of lizards
14 T. mauritanica were caught in 1979 by Professor E.U. Canning and
colleagues and a further 29 were caught by the author and Dr. R. Maazoun
in 1981. All fed readily in laboratory conditions and most survived for at
least one year.
3.3.2.Examination of the blood » Large trypomastigotes were seen in blood smears from seven geckos
(G5/79,G7/79,G12/79,Gl3/79,G104/81,G126/81,G127/81)• This form was the only
one seen in tissue impression smears, always>apparently, from contaminating
blood.
Three of these geckos (G7/79,G12/79,G104/81) had Haemocystidium sp. and
one of these also had the virus Pirhaemocyton (G7/79).
Three other geckos had Pirhaemocyton alone (G6/79,G10/79,G104/81) and one
had kinetoplastid amastigotes in some thrombocytes (G103/81).
3.3.21.Description of parasites
The trypomastigotes.
The trypomastigotes were monomorphic.
In fresh preparations the trypanosomes appeared less broad than in fixed
smears. Undulations of the flagellum travelled towards the anterior for a few
seconds, then towards the posterior for a few seconds, then back. The free
flagellum beat vigorously from side to side, often pulling away from the body
near the pointed anterior end. The body was often seen flexed, with posterior
and anterior ends adjacent. Occasionally the posterior end appeared to be
fixed to the slide, the body turning around it. Though in constant motion the / trypanosome makes little progress through the blood. The pointed posterior end 50
was often covered with a cytoplasmic fold to give a "beaver-tail" appearance,seen ^ particularly in rather thick thin blood smears.
In fixed and stained smears.
When fixed and stained the trypanosome measured 32 to 49ym long
(x =40.7 - 3.9ym, N = 50) by 9.2 to 15.2ym wide (x = 11.6 m ±.l.lym) with a free flagellum of 5 to 17ym (x = 10.6 - 2.6ym). The nucleus is on average
21.6ym from the anterior end and 8.5ym from the posterior. It is usually round to oval and 2-3ym long. It occasionally appears curved around*the punctiform kinetoplast which is adjacent to it. The flagellum arises near the kinetoplast and in 88% (N = 200) of trypanospmes it crosses over the nucleus. (Figs/5-7 )•
No signs of division have T>een seen in any trypanosome.
Camera lucida drawings of 50 trypanosomes, 25 from each of two smears made from one gecko (G5/79) are shown in Figs. 14 and 15.
Amastigotes
Small ovoid amastigotes were seen in clumps lying free in the plasma and within thrombocytes (20 cells) and occasionally within erythrocytes (2 cells)
of one gecko (G103/81). The infection was observed for 5 months from 4.9.1981.
Infected cells were always rare and only 1 or 2 were usually found on a smear.
The 22 infected cells seen contained 3 to 11 amastigotes (x = 8.5). No division
stages were seen (Fig. 9 and Figs. 73. to 76 ).
Intracellular amastigotes usually appeared isolated within the host cell cytoplasm. In 4 cells however the amastigotes seemed to be present within a
paristophorous vacuole, giving the appearance of a pseudocyst.
The amastigotes measured 2.0-2.7ym x 0.8-1.2ym (x = 2.4 x 1.0, N =50).
The round nucleus filled one end of the parasite. The kinetoplast was round or
oval, the longer axis always being perpendicular to the long axis of the amastigote. 51
Figures 4-13. The Moorish gecko Tarentola mauritanica and haematozoa
which infect it.
Figure 4 The Moorish gecko T. mauritanica (length 13cm)
Figures 5-7 Trypanosoma platydactyli (x 1500)
Figure 8 Pirhaemocyton "
" 9 Kinetoplastid amastigotes "
" 10 Haemocystidium sp. Microgametocyte "
" 11 " Microgametocyte "
" 12 " Macrogametocyte 11
" 13 " Macrogametocyte " 52 53
Figures 14-15. Camera lucida drawings of 50 Trypanosoma platydactyli from blood smears of one gecko (G13 /77). Figure 14. T. platydactyli from a Figure 15. T. platydactyli from relatively thick "thin" a thin blood smear blood smear 54
PRIMARY CULTUREPRIMARY CULTURE (l4d) PRIMARY CULTURE (28d)
1° SUBCULTURE (!4d)
2° SUBCULTURE(14d)
10pm 3° SUBCULTURE (I4d)
Figure 16. Morphogenesis of T. platydactyli in culture in vitro 1. Camera lucida drawings 55
DAYS IN 14 28 CULTURE
SUB f
SUB f PROMASTIGOTES
AMASTIGOTES
EPI-TRYPO- AND SUB SPHAEROMASTIGOTES
SUB t
FIG. 17 APPEARANCE OF PROMASTIGOTES W A CULTURE OF T. PLATYDACTYLI 56
It was situated next to the nucleus* A small amount of pale—staining
cytoplasm occupied the rest of the space within the amastigote membranes. The
darkly staining nucleus and kinetoplast made the parasites obvious when
examining a smear, even under low power (x 100).
Haemocystidium sp.
Both macro- and microgametocytes of a Haemocystidium species were seen
in blood smears from 3 geckos. The infected cells were rare (less than one in
104 erythrocytes). The parasites more closely resembled those described from
Tarentola annularis from Khartoum (Riding, 1930) than those from Algerian
T. mauritanica (Parrot, 1927), in that they were large (c 14 x 8ym) often
kidney or halter idium-shap'ed and the microgametocytes were pink rather than
colourless in stained smears (Figs.10-13 ). 30 S. minuta fed on geckos with
Haemocystidium and other haematozoa showed no sign of haemoproteid development
when dissected 1 to 10 days later. Microgametocytes failed to exflagellate
within lOh of removal from the gecko and examination between slide and
coverslip, even with the addition of 2% glucose peptone which stimulated
exflagellation in Sudanese parasites (Riding, 1930).
Pirhaemocyton
Four geckos had infections of the virus Pirhaemocyton (Fig. 8 ).
Erythrocyte infection rates were 12,15,62 and 79% (N = 500' ). Virus infection
was seen either as a small acidophilic area within the cytoplasm, a similar area
accompanied by a vacuole up to 10ym in diameter or as disruption of the host
cell nucleus into small fractions surrounded by purpie-staining cytoplasm. The
morphology, ultrastructure and distribution of Pirhaemocyton have been studied
by Stehbens and Johnston (1969) and Johnston (1975).
3.3.3.Culture of parasites in axenic culture / Flagellates developed in cultures from 8 geckos (G5/79,G7/79,G12/79,G13/79, 57
G104/81,G12/81,G127/81 and G105/81). No parasites were seen in cultures made from the amastigote carrier G103/81. Stained smears of 7-day-old cultures showed that most of the parasites were amastigotes with a few epi- and trypomastigotes. After 2 weeks in culture most of the parasites were epimastigotes but a few were promastigotes. After 4 weeks many of the parasites in the primary culture were promastigotes.
The positive primary cultures were subcultured after 2 weeks and then
% at 2-weekly intervals. In all 8 cultures the proportion of promastigotes in the parasite population gradually increased until by the 3rd subculture only promastigotes and amastigotes remained. Further subcultures contained only these
2 forms. A subculture made from the 4-week old primary culture had lost all epi- and trypo-mastigote forms 10 days later. Only promastigotes and amastigotes remained.
The morphogenesis of the gecko haemoflagellates in culture is illustrated in Fig. 16 by camera lucida drawings from fixed and stained cultures and in
Fig. 17 by pie charts showing the changing proportion of the various forms in the primary culture and subcultures.
The growth of 2 of the promastigote cultures, G13/79 and the double-cloned i Trypanosoma species, TPCL2, when subcultured into various biphasic and mono- phasic media and the morphological composition of a 14-day-old culture in each medium are summarised in Table 3 - Promastigotes and amastigotes were the only forms seen in any culture.
The growth curves for G13/79 and TPCL2 in Trager's medium C are plotted in Figs. 18 and 19 for the 3 temperatures tested. The maximum density of parasites was recorded at 28°C with slightly fewer parasites being produced at 25°C. Few parasites divided at 37°C and the parasites at this temperature died within
4 days. The two strains produced similar growth.curves. 58
Table 3 . Growth of two flagellate cultures in various media G13/77 TPCL2 Medium Maximum Maximum** Maximum Maximum density* Proportion density Proportion of pro- of pro- mastigotes mastigotes (%) (%)
MONOPHASIC
Schneiders + 30% FCS ++++ 98 ++++ 84 + M199 + 30% FCS 87 + 93 EBLB ++++ 85 +++ 86 +++ ++++ EBLB/M199 92 89 +++ +++ 81 84 Matteis +++ +++ LIT ++++ 93 ++++ 99 Trager's Medium C 96 88
BIPHASIC Agar Blood Overlay Base No. 2 Sheep ++++ 71 ++++ 74 2% Glucose +++ +++ peptone, 62 56 M199+5% FCS +++ 65 ++++ 80 Chick serum ++++ 70 +++ 67 Rabbit M199+5% FCS ++++ 94 ++++ 89 Gecko ++++ 83 ++++ 86 Plain (New ++ +++ Zealand) Sheep 89 88 it 2% Glucose ++ ++ peptone +++ 42 +++ 50 M199+5% FCS 96 ++++ 100 ++++ BHI Sheep 97 91 ++++ ++++ it M199+5% FCS 88 85 +++ +++ Coagulated 2% Glucose Horse serum peptone 78 72
* See p37 for details of density scale ** Remainder of parasite population formed by amastigotes in all media Figure 18. The effect of temperature on the growth of Figure 19. The effect of temperature on the growth of TPCL2 in culture. Vertical bars represent range of G13/77 in culture. Vertical bars represent range of counts for replicates (*37°C, • 28 C,«25°C) counts for replicates (±37°C,b 28°C, • 25°C) Number of parasites/cm3 (x107) 60
The development of TPCL2 in Trager's medium C at 25°C in a pH range from
5 to 9 is shown in Fig.20 • Maximum growth occurred at pH- c. No parasite / .o
division occurred at pH_ to pHA and parasites slowly degenerated in acidic D 0 .0 media. All parasites seen were pro- or amastigotesC^ig-21)
The morphogenesis of TPCL2 in medium with Lidocaine, Conconavilin A and
dimethylsulfoxide is shown in Figs.22-24.Only promastigotes and amastigotes
were seen. Amastigotes formed a larger proportion of the parasite population
% as the culture aged.
3.3-4.Interaction of parasites with vertebrate cells
3.3.4.1.Lizard blood cells * (i) in vitro
Blood cells from both lizard species behaved in the same manner jLn vitro.
Many promastigotes were seen to be attached to phagocytic monocytes ("macrophages")
within 15 minutes of being mixed with the lizard blood-93% of these promastigotes
were attached flagellum first (N = 300). Four hours later the majority of the
promastigotes had been phagocytised, though their elongate shape and flagella
were still apparent. A day after the blood and promastigotes were mixed all
the parasites had been phagocytised and most had begun to round up. A few
appeared to be in division and had two nuclei and/or kinetoplasts. The
parasites continued to round up until they were fixed and stained after 5 days,
when the blood cells had begun to vacuolate and develop pycnotic nuclei (Figp.25-26) •
The fixed parasites measured approximately 3 x 4ym with very pale staining
cytoplasm and dense, probably pycnotic, nuclei.
(ii) iri vivo
The blood smears from the gecko inoculated with culture promastigotes
showed identical monocyte-parasite interactions to those seen in vitro, but these / were more rapid. The blood taken directly after the inoculation already contained 61
24-
20i o O
CO E 16- . o
Q») w 2 12- co a
O- 6 8 Initial pH Figure 20 The effect of initial pH on the growth of TPCL2 in culture £ 60- w -»—(D> 0 .5? w 1 40 o .2 —oi a o 0« Or n 6 7 8 9 Initial pH Figure 21 The effect of pH on the morphology of TPCL2 in culture 62 Figures 22-24. Morphogenesis of TPCL2 in culture with 0.002yg/cm Conconavalin A. (22) 3mM Lidocaine hydrochloride (23) and 3% Dimethylsulfoxide (24) lOOi 80 60 Figure 22 40J CON A 20 O 6 9 12 15 Days in culture P lOOi UJ o 80 GC LU CL 6a Figure 23r CD LU I— 4a o LIDOCAINE (3 20 CD< DC 6 9 12 15 CL Days in culture 100- LL O 80 60 Figure 24 O 40 DMSO o CL O 20 QD_C O 6 9 12 15 Days in culture 63 monocytes with attached promastigotes, most of them attached flagellum first. There were no free parasites in smears made at 3 h, most having been phago- cytised (Fig. 27 ) • By 6 h the parasites had rounded up within the monocytes and many appeared unhealthy with pycnotic nuclei and rather pale- staining cytoplasm. (Fig. 28). Between 12 and 24 h the parasites were lysed. They became vacuolated and eventually broke open. The remains of kinetoplasts and nuclei were visible in the monocyte cytoplasm (Fig. 29)- At 36 h most signs of the parasites had disappeared though the monocytes still had frothy or "angry" cytoplasm. (Fig. 30). Cultures of blood taken 40 h after the initial inoculation remained negative over 5 weeks. 3.3.4.2.Lizard tissue cells' Unfortunately the gekkonid kidney cells grew poorly after their passage into the petri-plate wells. Many of the cells failed even to attach to the coverslips and most were washed away with the unattached promastigotes. Promastigotes were seen attached to the remaining cells, (Fig. 31) all of them flagellum first. One day after addition of the parasites 2 parasites were seen inside cells and the rest of the parasites were still attached but immobile or moving slowly. Three more intracellular amastigotes were found in the next 3 days but none appeared to be dividing. Most of the amastigotes were in pseudopodial outgrowths of the kidney cells (Fig. 32)- The gekkonid cells continued to round up and detach themselves from the coverslip and the experiment was terminated on day 5 by fixation of the cell layers. The stained cells showed a few, apparently healthy and intracellular amastigotes and more attached promastigotes. No parasites were seen in division. 3.3.4.3.Murine macrophage cells in vitro Figures 25-32. Interaction of TPCL2 promastigotes and vertebrate cells in vivo and jm vitro (x 1800) Figure 25. Monocyte of Agama sanguinolenta with amastigotes after infection for 5 days in vitro Figure 26. Monocyte of Tarentola mauritanica with amastigotes after infection for 5 days in vitro. Figures 27-30. Monocytes of Tarentola mauritanica with amastigotes after infection for 3h (27), 6h (28) -24 h (29) and 36h (30) in vivo Figures 31-32. Pseudopodial outgrowths of Gekko gecko kidney cells with attached (31) and intracellular (32) parasites in vitro. 65 At 24h more parasites were present in macrophages at 37°C than at 28°C or 25°C. At 48h however, more intracellular parasites were seen at the lower temperatures and at 72h all the parasites at 37°C were lysed while many macrophages at 25 and 28°C were packed with apparently normal amastigotes many of them in division with two nuclei and two kinetoplasts. The heavily infected macrophages were beginning to detach from the coverslips after 72h. Examples of infected cells are shown in Figs. 33-41. Mean values for the number of intracellular parasites per macrophage at 24,48 and 72h are shown in Fig%. 42 for each of the three temperatures. Parasite-free macrophages remained attached to the coverslips at all three temperatures though the cells at 25 and 28°C were less flattened than those at 37°C. Mean values for the parasite densities seen daily for 8 days in the macrophage-free cultures at each of the three temperatures are shown in Fig. 43. The results were similar to those seen in Trager*s Medium C (Section 3.3.3- ). 7 3 o 7 3 Maximum parasite densities were 3.6 x 10 parasites/cm at 28 C, 2.9 x 10 /cm at 25°C and only 8.5 x 106/cm3 at 37°C. Most of the parasites at 25 and 28°C were active promastigotes during the 8-day observation. The parasites at 37°C became increasingly inactive and though mostly (82%) promastigote on the second day they rapidly became more stumpy and many (61%) were amastigote by day 5. 3.3.4.4.Murine cells iri vivo Parasites were seen only once in smears and cultures from inoculated mice. Three sphaeromastigotes and four stumpy promastigotes were seen in a tissue impression smear of an ear one day after inoculation. The parasites seen were all vacuolated but otherwise of normal appearance. The culture from this ear and all other cultures remained negative. (Inflammation at the inoculation sites was only noticed in the tails of 2 mice on the second day. In these mice the base of the tail was slightly swollen and erythrematous). No bacteria were present in the impression smears from the inoculation sites. 66 ® : V ® 41. Interaction of TPCL2 and mouse peritoneal macrophages in vitro after 24,48 and 72h at 25°C (33-35)7"28^cT36-38) and 37°C (39-41) Figure 42. Effect of temperature on the phagocytosis and lysis of TPCL2 promastigotes by mouse peritoneal macrophages. Vertical bars represent range of means for each re plicate. (a37 C, • 28C,m 25 C) Figure 43. Growth of TPCL2 promastigotes in M199 with 5% FCS at 25, 28 and 37°C.X Vertical bars represent range of counts for replicates (A37°C, • 28°C, • 25°C). 68 3.3.5.Cloning of blood trypomastigotes Three of the 10 culture tubes inoculated with single trypanosomes contained epimastigotes and amastigotes when examined 10 days later. Each tube contained approximately 2 x 10^ parasites. On the 11th day, while these were mostly epimastigote, one of the cultures was cloned again 10 times. Ten days later 1 of the 10 tubes contained parasites. Some of them (12%) were promastigotes and most of the rest were epimastigotes. This strain, derived from a single blood trypomastigote, was designated TPCL2. 3.3.6.Comparison of strain morphology and biochemistry 3.3.61Comparison of strain morphology Although the mean body lengths of the stocks tested varied, the differences were not significant. This is illustrated in Fig. 44 by Dice-Leraas diagrams (Dice and Leraas, 1936) in which all the white boxes (means - standard error) overlap. The 5 parasite cultures examined by electron microscopy also appeared identical. The promastigote ultrastructure was the same as Lewis (1975) described for other promastigote cultures of lizard parasites. Promastigotes had flagella with the usual 9 pairs of peripheral tubules surrounding a central pair alongside a paraxial rod (Fig. 49 ). The central tubules terminate in the body of the promastigote at a transverse basal plate, posterior to the flagellar pocket or reservoir. The kinetoplast is a disc of 130 nm depth identical to that seen in parasites in the early stage infection of sandflies (p.78 ). The mitochondrion is branched with 2 or 3 arms one of which often extends anteriorly from one side of the kinetoplast. Multivesiculate and pigment bodies were seen in culture promastigotes. Mean numbers of subpellicular tubules and the mean separation of the tubules are shown in Figs>.45-46 for the 5 cultures 69 Figure 44. Dice-Leraas diagrams of promastigote body length for 5 strains of parasite Body Length (yum) 6 8 10 12 14 SF2004 G13 79 G27 77 LV414 TPCL2 Figure 45. Dice-Leraas diagrams of the number of subpellicular microtubules in 5 strains of parasite Microtubule Number 60 80 90 100 110 120 -r— 70 S F2004 G13 79 G27 77 L.V414 TPCL2 Figure 46. Dice-Leraas diagrams of the subpellicular microtubule interval in 5 strains of parasite Microtubule Interval (nm) 30 40 50 60 70 80 90 100 70 Figures 47-49. Electron micrographs of promastigotes of TPCL2 from in vitro culture. Figure 47. Longitudinal section (x 32900) Figures 48-49. Transverse sections to show subpellicular microtubules (x 39400 and x 46000) 71 as Dice-Leraas diagrams. Differences in these parameters for the 5 strains are not significant at the 5% level. 3.3.6.2.Comparison of strain biochemistry Diagrammatic representatives of the bands which resulted from the electrophoresis of lysates of 12 parasite stocks are shown in Fig. 50 . The gels were stained to reveal enzymes which occur in the Kreb's cycle. The migration of all 8 enzymes was identical for 10 of the stocks. Two stocks, LV108, a parasite isolated in Senegal (Ranque, 1973) and LEM75 (Leishmania infantum) had some enzymes of different mobility to those of the French, Italian and Algerian gecko parasites. 3.3.7.0rigin, collection and maintenance of sandflies 3.3.7.1 ,S.m. minuta Thirty female and 32 male S. minuta were caught at Banyuls and 14 females and 26 males from the Valley of the Tech. All of the females from the Valley of the Tech and 20 from Banyuls were dissected within 3 days of capture. No parasites were found. The remaining 10 females were offered a blood meal from a gecko (G101/81). Eight fed, including 3 gravid flies. Three flies survived transport to England and oviposited a total of 115 eggs, which formed the basis of a small laboratory colony. In 3 generations 31 colony females laid 454 eggs and retained 1388, an average of 59.4 eggs per female. The development time for each stage was as follows:- Egg 4-9 days First larval stage (L^) 3-8 days L^ 3-4 days Lg 3-5 days L^ 3-5 days Pupae 4 days. The complete life cycle from new-laid egg to adult took 23 to 35 days and V CD + G6PD E2 GPI GOT PGM ME + i MDH + 6PGD 5 2 5 5 ll := ^ V r- 2 5 5 5 Figure 50. Diagrammatic representations of iso-enzyme bands for 12 cultures of gecko haemoflagellates (See Table 2 p.45 for explanation of abbreviations) 73 205 flies (96?? and 109 were produced. The 4th generation was destroyed by an incubator failure. 3.3.8.Infection of sandflies and dissection Of 39 S. minuta and 187 P. papatasi fed on geckos known to be infected with T. platydactyli 19 (49%) of the S. minuta and 50 (27%) of the P. papatasi became infected. Attempts to feed S. minuta through membranes failed, though the flies readily fed off geckos shortly after the experiment. » 3-3.8.1.Development of T. platydactyli in P. papatasi The blood trypomastigotes (Fig.5la ) began to transform in the midgut after 6 h, rounding up and losing their flagella (Fig51b,c). Division began after about 12 h, the roughly spherical bodies cleaving repeatedly to give amastigotes shaped like sectors of spheres by 18 h. (Fig51d,e). One day after the infective feed each trypanosome had produced a mass of 20-30 spherical and sector-shaped amastigotes (Fig .52,53) .. These produced flagella within 8 h, becoming stumpy epi- and trypomastigotes (Fig.5If ) and then began to elongate to long slender forms (Fig.51g). Approximately 40 h after the feed these slender parasites spilled out from the posterior end of the peritrophic membrane. They attached to the gut wall in the posterior ectoperitrophic space to form a regular palisade of epi- and promastigotes. Many of the parasites seemed to be i lost with the blood residue as it passed from the midgut 3 days after the feed. Only a few parasites, mostly attached sphaero- and stumpy promastigotes remained after loss of the residue. However the parasite population rapidly recovered by means of a second division phase and 4^ days after the feed the midgut was again packed with parasites, mostly large vacuolated amastigotes and sphaero- mastigotes (Fig.~511). These transformed, elongating to give a population of long slender trypo- and epi-mastigotes remarkably similar to the population 2k days previously. These forms reattached to the gut wall, carpeting the entire midgut and filling the lumen in heavy infections. From 6 to 7 days post-feed 74 Figure 51. Life cycle of T. platydactyli of Tarentola mauritanica and sandflies. Camera lucida drawings"! (The division phase L-n only occurred in Phlebotomus papatasi) LOJUM 75 some of the slender forms shortened and the resultant stumpy epi- and para- mastigotes divided and/or transformed into short, slender, very active trypo- mastigotes as they passed into the hind gut triangle at 7k days post-feed (Fig .51j ). As the infection aged the proportion of these short trypomastigotes fell but the midgut slender forms persisted till the flies died, about 10^ days after the feed. 3.3.8-2.Development of T. platydactyli in S. minuta The trypanosome infection of S. minuta was similar to that in P. papatasi. However very few parasites were lost with the blood residue in S. minuta and so colonization of the entire midgut occurred earlier. Some midguts were so packed with parasites 3 to 4 days after the bloodmeal that they appeared swollen (Fig. 55 ). The change from attached stumpy pro-^epi- and para-mastigotes to free slender forms occurred without the intermediate form of amastigotes and sphaeromastigotes seen in P. papatasi. Short slender trypomastigotes appeared after 5 days in large aggregates in the fly lumen, especially next to the basement membrane in the gaps left by degenerating epithelial cells (Fig. 54 ). A few parasites were seen in the pharynges of flies with massive midgut infections 6 or 7 days post-feed (Fig. 56 ) when many of the short trypo- mastigotes were moving back into the hindgut causing distension of the pylorus and rectum (Fig. 57 ). Many of the parasites appeared attached to the hindgut wall. Three syncytia containing up to 7 nuclei and kinetoplasts with short free flagella were seen in one fly 5 days post-feed. Four infected flies were given a second bloodmeal 8 days after the infective meal. All the parasites seen 2 days later appeared to be within the new peri- trophic membrane. A day later a dense palisade of promastigotes had formed in the posterior ectoperitrophic space and by 4 days post-feed the short, slender 76 Figures 52-57. The development of T. platydactyli in S. minuta and P. papatasi \ Figure 52. Longitudinal section of infected P. papatasi 24h post-feed. Groups of amastigotes (arrows) are visible in the bloodmeal. . Paraffin wax section. Giemsa staining (x 150) " 53. Detail of one of the groups of amastigotes seen in Fig. 52. (x 1400) " 54. The'midgut of a S. minuta 7 days post-feed. A palisade of attached parasites (P) and groups of metacyclic trypomastigotes (arrows) are visible. Semi-thin resin section. (x 150). " 55. The distended thoracic (TMG) and abdominal (AMG) midgut of a S. minuta infected 3k days previously. Phase contrast illumination (x 150) " 56. The foregut of a S. minuta 6 days post-feed. Promastigotes are attached around the everted oesophageal valve (0V) and one parasite is visible in the pharynx (arrow). Phase-contrast illumination (x 500) " 57. The hindgut of a S. minuta infected 7 days previously. The pylorus and region around the rectal ampullae are distended with parasites. The ileum and rectum appear normal. Phase contrast illumination (x 110). 77 78 trypomastigotes were already in evidence. 3.3.8.3.Electron microscopic examination of the sandfly infections. Flagellates were seen attached to the sandfly midgut wall by two methods. In P. papatasi the long flagella of parasites were seen interdigitated with the long midgut microvilli and there was no evidence of hemidesmosome formation in any section (Fig. 58 ). In S. minuta attached parasites had much shorter free flagella which were expanded to form rather amorphous digitate masses which extended into the midgut epithelial cells, although the plasmalemma of these cells was intact. The junction between parasite'and midgut cell was electron dense (Fig. 59 ). Many of the parasites attached to S. minuta cells had 1-3 large (l-2.5ym) granular or multivesiculate bodies posterior to the nucleus (Figej50-62). These bodies were membrane bound. The midgut microvilli were absent or unusually small close to attached parasites. Most of the attached parasites in both flies were promastigotes though a few para- and epimastigotes were also seen. Lumen-dwelling parasites included pro-, para-, epi-, trypo- and amastigotes. Most parasites seen in division with two nuclei and/or two kinetoplasts were epi or amastigotes. Trypomastigotes were rare until the production of the presumed metatrypomastigotes 5 to 7 days post-feed. These short slender trypomastigotes, apparently formed by division of stumpy epimastigotes, were seen in large aggregates in which they appeared joined by thin bridges of membrane bound cytoplasm. (Fig&63-64) • Small vacuoles containing electron dense spheres were also seen in all parasites, except the presumed metatrypomastigotes, in both sandfly species. (Fig. 60 ). All the parasites had the kinetoplast-mitochondrion complex typical of the Kinetoplastidae. The mitochondrion was branched with elongate arms 79 Figure 58. Parasite-midgut cell interaction in Phlebotomus papatasi (x 10300) 59. Parasite-midgut cell interaction in Sergentomyia minuta (x 23500) 80 Figures 60-62. Paras it e-midgut cell interactions in S. minuta. Figure 60. Four days post-feed (x 11600) Figure 61. Six days post-feed showing electron dense zone of attachment (arrow) (x 20600) Figure 62. Six days post-feed show electron dense zones of an attachment (arrows) and parasites with multivesiculate bodies (MVB) mitochondria (M) kinetoplasts (K) and basal bodies (B) (x 12900) 82 Figures 63 and 64 Groups of metacyclie trypanosomes in S. minuta 7 days post-feed. Figure 63 Longitudinal section (x 19500) 64 Transverse section (x 10100) 83 travelling posterior and anterior of the kinetoplast (Fig. 62 ). The arrangement of the kinetoplast DNA differed between the short slender trypomastigotes and other parasites. In the trypomastigotes the nucleoid had a double-layer appearance in vertical section and the kinetoplast was ovoid or spherical (Fig. 68 ). in the other parasites the kinetoplast was a slightly concave disc I'Wnm in depth with a single layer of anisotropically arranged DNA fibrils (Fig^65-6^. The kinetoplast increased in width, not depth, prior to parasite division (Fig. 65 ). A surface coat was not present on any of the parasites seen. No attached parasites were found in extensive searches of infected hindguts (Figs. 69 and 70 ). Sections of the uninfected S. minuta and P. papatasi show that the midgut cell microvilli of S. minuta are short (1.1-2.1 ym) and sparse while those of P. papatasi are relatively long (2.3-3.9 ym) and densely packed 3 days post-feed. 84 iigures 65-68 . Change in kinotoplast morphology during the development ol T. platydactyli in S. minuta Figure 65. The wide kinetoplast of a dividing promastigote (x 42900) 66. The single band of fibres in a parasite kinetoplast (x 35300) " 67. Double banding in a kinetoplast of a parasite 6 days post-feed (x 36900) 11 68. Double banding in a metacyclic trypomastigote 7 days post-feed (x 46700) 85 Figures 69 and 70. The hindgut (Pylorus 69 and ileum 70) of a S. minuta 7 days post-feed to show absence of attached parasites (x 4200, x 14600) Figure 71. Parasite in the midgut lumen of a S. minuta 5 days post-feed. The flagellar swelling characteristic of attached parasites is visible (x 21500) 86 87 3.4.DISCUSSION 3.4.1.The gecko haemoflagellates iji vivo. 3.4.1'l.The trypomast igotes The blood trypanosome found in T. mauritanica at Banyuls in shape, size and in the presence of a posterior cytoplasmic fold giving a "beaver tail" appearance closely resembled T. platydactyli, described by Catouillard (1909) from T. mauritanica in Tunisia. The nucleus of the Tunisian trypanosome was slightly more anterior and further from the kinetoplast than in the Fretich parasite but the differences are small. The flagellum crossing the nucleus was an almost constant feature of the French trypanosomes which has not been recorded previously for T., platydactyli. However drawings of T. platydactyli from different areas show some variation in the size and position of the nucleus (Fig. 72 ) an(j the author does not consider the differences in the French parasite sufficient to create a new species. 3.4.12The amastigotes The observation of intracellular amastigotes in lizard blood smears has been taken as confirmation of the presence of a Leishmania parasite. The amastigotes discovered in Banyuls geckos in 1969 and 1979 (Rioux et_ al, 1969; 1979) were also thought to be Leishmania species and were attributed to the Leishmania already described from T. mauritanica viz L. tarentolae. As a result of the present study it is proposed that L. tarentolae is a synonym of T. platydactyli (Section 2.2 ). The amastigotes seen in blood smears from the geckos appear to be a stage of this lizard trypanosome and this may be true for amastigotes in other lizards. Lizard trypanosome life cycles are poorly understood. Intracellular amastigotes have only been seen 9 times (Telford 1971 and pers. comm.) including 3 times in hosts of named species of Leishmania parasites of lizard blood viz L. hemidactyli (Shortt and Swaminath, 1928) L. tarentolae (Rioux et^ Figure 72. Camera lucida' drawings of T. platydactyli from various sources (x 2000)• a Spain (Wood, 1937) b Senegal (as T.p. senegalensis, Ranque, 1973) c Tunisia (Catoullard, 1909) d Tunisia (Chatton and Blanc, 1914) e France (Wallbanks, present study) f France •» 89 90 Himo, 1973). In 2 of these 3 reports concurrent trypanosome infections were observed (Shortt and Swaminath loc. cit.; Rioux et al loc. cit.) . The amastigotes differ from those in mammals in morphology (Table 1 ) and host cell type. Most are small elongate ovoids, with a juxtanuclear kinetoplast. They occur in cells which are rarely (e.g. thrombocytes, Hartman, 1925) or never phagocytic (e.g. erythrocytes) even when immature (Figs . 73-84 ) . In contrast, Leishmania parasites of mammals are generally rounder and larger and always occur in phagocytic cells. The presence of amastigotes in non-phagocytic cells suggests active invasion by the parasite. This is typical of some trypanosomes, especially T. cruzi (Dvorak and Schmunis, 1972). Leishmania parasites of mammals rely on induced phagocytosis to enter vertebrate cells (Miller and Tw.ohy, 1967). 3'4.1.3.The role of the intracellular amastigotes The role of the amastigotes, if they are of trypanosome origin is unclear. They possibly represent a reproductive phase in the life cycle. This may occur only once, between the infective metacyclic parasites and the adult haematozoic trypomastigotes, which do not appear to divide (Davis, 1952; Molyneux, 1969k 1976; Mello, 1982) or at regular intervals interspersed with an extracellular phase as seen in T. cruzi (Hoare, 1972). Telford (1979® has provided evidence for amastigote division of cells of Teratascincus scincus but unfortunately only had blood smears from a 38-day period of a natural infection. His blood smears from . Hemidactylus mabouia show some cells containing single large amastigotes and other cells which appear to contain parasites dividing into groups of smaller amastigotes within parisotophorous vacuoles (Figs.77-80 slide kindly donated by Dr. S.R. Telford). Such acute infections may become chronic with time. Trypanosome division forms have rarely been seen in poikilotherms. 91 Table 4 . Trypanosome species with a promastigote stage Trypanosome Vertebrate host Location of Reference promastigotes w n p < c < rt> Rt Mrr Mrt rIt on>* nC recr pH re rt Pi-i re rert PISCES T.danilewskyi Camp (Cyprinus carpio) Smolikova e£ al,1977 Trypanosoma sp. Loach (Misgurnus anguillo caudatus Tanabe, 1924 Trypanosoma spp. "Several fish species" Becker, 1977 AMPHIBIA T. bocagei Toad (Bufo bufo garganizans) Feng and Chas,1943 T .buf ophlebot omi Toad (Bufo boreas) Anderson and Ayala,1968 T.diemyctyli Newt (Triturus viridescens) Barrow, 1953 T.rotatorium Various frogs and Creemers and toads (Anura) Jadin, 1966 REPTILIA T. garnhami Gecko (Hemidactylus brookii angulatus) Grewal,1957a AVES T. numidae Guinea fowl (Numida meleagris) Fallis et_ al, 1973 Trypanosoma sp. Sparrow (Passer domesticus) Nair and David,1956 Trypanosoma avium Various species of bird Bennett,1961;1970 MAMMALIA T. conorhini Rat (Rattus rattus) Lambrecht,1965 T.cruzi Various wild and domestic mammals, including man Baker and Price,1973 T .dionisii Bat (Pipistrellus pipistrellus) Baker £t al,1980 T.helogalei Mongoose (Herpestes ichneumon) Grewal,1960 T.ichneumoni Mongoose (Helogale parvula) Grewal,1961 T.lambrechti Monkey (Cebus albifrons) Marinkelie,1968 T.microti Vole (Microtus agrestis) Molyneux, 1969c- T.minasense Monkey (Callithrix penicillata) and other Brumpt,1909 monkeys / T.nabiasi Rabbit (Oryctolagus cuniculusl » Grewal,1957b T.otospermophili Ground squirrel (Citellus spp) Hilton,1972 T.theileri Cow (Bos taurus), other bovids and antelope Carpano,1932 T.zapi Jumping mouse (Zapus princeps) Davis,1952 92 Figures 73-84. Kinetoplastid amastigotes in blood smears from 3 lizard species (x 1700). Figures 73- 76. Amastigotes from Tarentola mauritanica from France . " 77- 8Q Amastigotes from Hemidactylus mabouia from Tanzania. " 81 - 84. Amastigotes from Agama agilis from Pakistan (vide Telford, 1979& 93 Division is thought to occur in the prepatent period which is observed in fish infected with trypanosomes, but it has never been recorded. The blood trypanosomes of fish do divide by binary fission in heavy infections (Lom 1979). Binary and multiple fission have been seen several times in trypanosomes of amphibians (Bardsley andHarmsen, 1973) though only once intracellularly: T. inopinatum was seen as amastigotes in monocytes from the liver and bone marrow of a frog (Buttner and Bourcar.t, 1955). These infected cells appear identical to the gecko monocytes seen in the present study from lizards inotulated with T..platydactyli promastigotes which suggests the amastigotes may have been in the process of lysis. There is a single report of erythrocytic infection by trypanosomes, in a Brazilian frog (Carini, 1910). However the intracellular "amastigotes" developed "kinetoplasts" half way through the life cycle and it is thought that Carini was confused by a concurrent infection of a trypanosome and sporozoan (Bardsley and Harmsen, 1973). The adoption of the intracellular habit by lizard trypanosomes may also be considered as a device for escaping host responses. Movement into immunologically privileged sites as a means of survival appears to be rare in protozoa (Wakelin, 1976) but the development of amastigotes of Trypanosoma brucei in the choroid ple.x^us may represent such an escape mechanism (Omerod and Venkatesan, 1971). 3.4-2.The Relationship between the promastigotes and trypomast igotes from the gecko Seven trypanosome infections T. platydactyli, were revealed in the initial examination of blood smears from 43 T. mauritanica from Banyuls. Parasites developed in blood cultures from these seven gecko hosts and from one other: trypanosomes were later found in smears from the latter gecko. This suggested that the parasites seen in culture were probably Trypanosoma platydactyli. However, only promastigotes, forms more typical of the genus Leishmania, were seen in old cultures and subcultures. In order to investigate the possibility 94 that the promastigotes were not of trypanosome origin but were derived from concurrent cryptic infections of a Leishmania species, the blood trypomastigotes were cloned and allowed to reach the epimastigote phase when they were cloned again. The double-cloned strain derived from a single trypomastigote (TPCL2) developed into a pure promastigote culture demonstrating that the promastigotes were of trypanosome origin. 3.4.3.The morphological and biochemical comparison of stocks and strains of gecko haemoflagellates in culture. This cloned culture was morphologically and biochemically indistinguishable from all the stocks, thought to be Leishmania, isolated in culture from Banyuls geckos, from a Banyuls S.'minuta, from a gecko caught in the type locality of L. tarentolae (Parrot, 1949) and from geckos of two species caught on the Italian Adriatic coast. The biochemical similarity in particular renders it unlikely that these stocks and strains belong to different genera and, thus, the very existence of Leishmania tarentolae is put in doubt. It seems likely that Parrot's strain of "L. tarentolae" is in fact T. platydactyli. This strain, also known as TAR VI (Parrot, 1949), the "Rockefeller" strain (Dollahon and Janovy, 1974) and Lt-S, which was cloned to give LtC-1 (Simpson and Braly, 1970) has been used in most of the biochemical studies on L. tarentolae over the last 25 years. Other research has been done using strain TAR II also isolated in Algeria and probably the same parasite species (Parrot, 1949). The present experiments in which the trypanosome strain was subjected to different culture conditions indicate that, once formed, the promastigote stage of the trypanosome is very stable. The differentiation of promastigotes back into epi- or trypomastigotes could not be stimulated by changing the temperature or pH or composition of the culture medium nor by adding gecko serum or chemicals known to stimulate 95 differentiation in Herpetomonas (Castellanos £t al_ 1981). Further geckos inoculated with the promastigotes did not become infected. Maximum parasite growth occurred at 28°C as Krassner (1965) also found and at a pH of 7.8. Parasites only survived for a very short period at 37°C in Trager's medium C but growth in media with serum (present study, p. 65 ) or other blood components (Krassner, loc. cit.) is better at this temperature. The promastigotes grew much better in alkaline media than in acidic media. Most trypanosomatids are grown in media with a pH between 7.2 and 7.5 (Evans, 1978) and T. platydactyli is slightly unusual in showing a pH optimum as high as 7.8. However other parasites including Crithidia spp. (Edwards and Lloyd, 1973) and Trypanosoma brucei (Brun and Jenni, 1977) are grown in media with a pH between 8.0 and 8.2). There were no apparent differences in the ultrastructure of promastigotes from the 5 cultures examined. It is possible to distinguish some species of trypanosomat id by the number of subpellicular microtubules they possess and the microtubule interval (Gardener eit £l 1977; Chance, 1979). Avakjan (see Garnham, 1971) noted that some promastigote forms of Leishmania from reptiles had a longer microtubule interval (58-67mm) than those from mammals (35-45mm) a general trend confirmed by Lewis (1975). Promastigotes of so-called "Leishmania tarentolae" have a microtubule interval of about 52mm and therefore lie between Avakjan1s ranges for Leishmania parasites of reptiles and mammals (Avakjan, loc. cit.; Strauss, 1971; present study) but it is interesting to note that Trypanosoma congolense and intracellular amastigotes of some mammalian parasites have similar microtubule intervals to those "typical"of promastigotes in cultures of reptilian haemoflagellates (Vickerman 1969; Gardener et al^ 1977). The stability of the promastigote explains the general acceptance of 96 L. tarentolae as a separate species even though it had been extensively studied. There is either a constant selection pressure causing the trypanosome to maintain a promastigote form, perhaps due to the artificiality of the culture media, or there has been a movement of genes?perhaps causing gene suppression. Irreversible gene suppression may be responsible for the senility which develops in some Plasmodium gametocytes in vivo (Vandenberg and Gwadz, 1980) in in vitro culture (R.E. Sinden pers. comm.). It is a constant feature of some strains, just as the change to promastigotes appears to be in some trypanosomes. If the change in form is indeed irreversible the only hope of checking whether promastigote cultures are of Leishmania or Trypanosoma origin will be by bio- chemical methods, comparing them with cultures of known derivation. 3.4.4.The development of T. platydactyli in sandflies. 3.4-4.1 .Previous observations of sandfly infections. Studies on the development of "L. tarentolae" and T. platydactyli in sandflies have produced conflicting results (Killick-Kendrick, 1979). After developing in the sandfly midgut "L. tarentolae" from Algerian geckos (found to be infected by blood culture) began to degenerate in the hindgut and was lost when infected Sergentomyia antennata defaecated (Parrot, 1934; 1935). The S. antennata dissected by Parrot were possibly infected with T. platydactyli for which the sandfly species was not a suitable host. Parrot would only have seen the early stages in the development of the trypanosome which could have been easily confused with a Leishmania parasite. Adler (1933) thought L. tarentolae had "anterior station" development, after observing it in the pharynges and proboscides of several P. papatasi fed on promastigote cultures although he did not find head infections in S. minuta fed directly on an infected gecko. (Adler and Theodor, 1929; 1930). If, as now seems likely, L. tarentolae is a synonym of T. platydactyli the foregut infections seen by Adler and Theodor (loc. cit.) in flies fed on promastigote cultures though 97 appearing more healthy than those seen by Parrot (loc. cit.) can have little relevance to the natural situation. Flies are infected when they ingest blood trypomastigotes not promastigotes, which must occur rarely if at all, in the gecko. S. minuta appears to be a suitable host for the trypanosome and is probably the natural vector in South Eastern France (Rioux et^ al, 1969 and present study). Adler and Theodor (1935) found infections in 43 of 46 % S. minuta fed on geckos naturally infected with T. platydactyli. Parasites were seen "reaching up to the oesophagus" but neither head nor hindgut infections were seen. One pharyngeal infection was seen in 14 P. papatasi fed on a gecko with the same trypanosome but the other 9 infected flies only had midgut infections which did not move after the flies took a second meal (Adler and Theodor, loc. cit). This apparent lack of hindgut infections is in contrast to observations made in the present study and may have been a result of most flies being dissected too early or differences in temperature or supplementary feeding (i.e. sugar solutions) of the flies (c.f. Ashford et^ ajL, 1973). Another conflicting observation is that of Killick-Kendrick (1979) who observed natural flagellate infections in S. minuta from Banyuls and Gard. One of those flies was fixed, sectioned and examined under the electron microscope. Parasites in this fly closely resembled those seen in the present study in terms of morphology and the peculiar mode of attachment to the hosts midgut cells (R. Killick-Kendrick and A.J. Leaney, pers. comm.). Four of the flies came from a site in Banyuls from which geckos infected with T. platydactyli have been caught in 1969, 1979 (Rioux et^ al, 1979) and 1981 (Present study). These flagellates might therefore be readily attributed to T. platydactyli but for the fact that all five flies had parasites in the Malpighian tubules. Parasites were never seen in the tubules of any fly in the present study. However considering the identity of the mode of attachment those parasites found by 98 Killick-Kendrick are provisionally attributed to T. platydactyli, although they could have been of a different species or even genus (Killick-Kendrick, 1979). Christensen and Telford (1972) apparently saw Malpighian tubule infections of T. thecadactyli in wild caught flies and not in laboratory infected flies. The difference may be due to extra nutrients which flies derive from plants or the honey dew of aphids or coccids in the wild (Killick-Kendrick, 1979) which are not provided in the laboratory. 3.4.4.2;Recent observations of sandfly infections In the present study of T. platydactyli, both mid-gut and hind-gut were involved in the developmept in S. minuta and in P. papatasi. Presumed metacyclic forms were found in both sites. The development from blood trypomastigote to long slender epimastigotes appears to be similar for all saurian trypanosomes in sandflies (Shortt and Swaminath, 1931; Christensen and Telford, 1972; Adler and Theodor, 1935; Ayala and Mackay, 1971; Ashford et_ Al, 1973). All appear to become amastigotes, then divide to form clumps of smaller amastigotes which develop flagella from apparently randomly placed kinetoplasts. The resultant mixture of stumpy epi- and trypomastigotes and occasionally promastigotes elongates to give a population of long slender epimastigotes. In most trypanosomes of lizards seen in sandflies (vide Molyneux 1977«> these migrate into the hindgut. One species, T. gerrhonoti appears to have anterior station development since head infections occur without massive midgut infections to cause overspill.(Ayala and McKay,1971) In T. phlebotomi the epimastigotes transform into pyriform or spherical amastigotes which were thought to be the metacyclic parasites (Shortt and Swaminath 1931). In T. boueti (Ashford et al, 1973) T. gerrhonoti, T. scelopori (Ayala and Mackay 1971) and possibly T. thecadactyli (Christensen and Telford, 1972) the epimastigotes themselves were considered to be the infective stage. 99 T. platydactyli is the only lizard trypanosome which has been seen to produce short slender, very active and non-dividing trypomastigotes, as the terminal stage of a sandfly infection. These resemble many metacyclic forms of mammalian trypanosomes and may be the normal infective stage of many lizard trypanosomes. In much of the previous work the observed sandfly infections were terminated relatively early possibly before the actual infective stages were produced. T. platydactyli is also unusual in producing so many pro- and paramastigote forms in the sandfly. Most of these are attached to the midgut but, before the production of the presumed metacyclic trypomastigotes, they form a large proportion of the unattached parasites. Epi- and trypomastigotes are quite rare in infections of 3-6 days age and the parasite populations then resemble Leishmania species. Moorish geckos are probably infected with T. platydactyli when they feed on infected sandflies. Though Sergent at a_l (1915) found no evidence of sandflies in the stomachs of several hundred geckos, Tarentola annularis have been seen feeding regularly on sandflies attracted to room lights in Dakar, Senegal (P.J. Desjeux, pers. comm.). Sandflies contain the presumed meta- trypomastigotes from 6 days post feed and appear to retain them through subsequent bloodmeals. The early development of a trypanosome in a lizard host has never been seen. This stage may possibly hold the clue to the amastigote enigma and it was unfortunate that the sandfly colonies died out before parasite transmission could be attempted. True "anterior station" development of flagellates has rarely been seen in flies fed on lizards. If the cross-immunity between haemoflagellates of lizards and man does lead to false positive immunological tests for human parasites (Southgate 1967, Southgate and Manson-Bahr 1967, Fuller et al 1980) the accidental introduction of lizard parasites into man is probably by contamination. (This is most likely to be the result of squashing the fly into the site of the 100 bite). Cross-immunity exists between Trypanosoma and Leishmania parasites (Depieds et_ £l, 1958; Shaw and Lainson, 1975; Le Ray et_ al, 1977) and so the observed reactions in no way exclude the possibility that the so-called "Leishmania" parasites of lizards are in fact Trypanosoma species (Section 3.4.9. )• 3.4.5.Comparison of S. minuta and P. papatasi as invertebrate hosts of T. platydactyli The vector capacity of P. papatasi would seem to be less than that of S. minuta, since infections were far less intense in the former species. However all the stages which occurred in S. minuta were also found in P. papatasi so P. papatasi could be a vector in some parts of the world, though perhaps a not very efficient one. The relatively poor infections in P. papatasi may have resulted from differences in the internal midgut surface. The long microvilli of P. papatasi may interfere with the attachment of parasites which appears to occur on cells with few if any microvilli. The virtual absence of microvilli near attached parasites in S. minuta may have been caused by the flagellates themselves. T. rotatorium probably induces similar changes in its leech vector (Desser, 1976). The morphogenesis of parasites in the two fly species was similar though the amastigote division phase was repeated in P. papatasi to compensate for parasites lost with the blood residue. Fewer parasites were seen attached in P. papatasi, and far fewer of the presumed metatrypomastigotes were produced than in S. minuta. Hindgut infections were restricted to the hindgut triangle in P. papatasi but spread to the pylorus and ileum in S. minuta, probably as a result of overcrowding or competition in the midgut. 3.4.6-The ultrastructure of T. platydactyli in the sandfly and in vitro culture 3.4.6-1.Parasites in the sandfly The parasites in the sandfly were of trypo-, epi-,para-,pro- and amastigote 101 form. Many of the promastigotes and a few epi- and paramastigotes were attached to midgut cells of S. minuta. These forms were stumpy with short expanded digitate flagella which distorted the plasmalemma of the cells to which they were attached. The junction of flagellum and midgut cell was electron dense and probably formed a long "Zonular" hemidesmosome. This mode of attachment appears to be unique at present. Most other reports of trypanosomatid attachment in invertebrates involve hemidesmosomal (Molyneux, 196915 Brooker, 1971; Vickerman, 1973; Killick-Kendrick, 1974; Brun, 1974; Tetley et_ al, 1981) and desmosomal (Molyneux and Ashford, 1975) junctions between flagella and chitin or flagella and other flagella (Brooker, 1970; Vickerman, 1973) not flagella and host cell membranes. Lewis and Ball (1979) reported that epimastigotes of the fish trypanosome, Trypanosoma cobitis had large swollen intra-flagellar areas which interdigitated with the microvilli of the leech crop wall. Similarly metacyclic stages of T. brucei have flagellar shafts with branched extensions which intertwine with the microvilli of the salivary epithelium cells (Vickerman and Tetley, 1981). However neither the flagellar extensions of T. cobitis nor T. brucei extend into the body of the host cell. The flagella of T. cobitis do not form hemidesmosomes with the host cells, those of T. brucei have been seen to form hemidesmosomes (Steiger, 1973) but this may not be a constant feature. (Vickerman and Tetley loc. cit.). Promastigotes of L. mexicana amazonensis (Killick-Kendrick et_ al, 1974) and epimastigotes of T. melophagium (Molyneux, 1975) also attach to the midgut of their vectors by interdigitation with the microvilli. They do not form hemidesmosomes and there is no obvious alteration in the flagella to become enlarged or moulded to fit the microvilli surface. Free promastigotes with expanded flagellar tips (Fig.71 , p.86 ) have been observed in the lumen of the midgut of S. minuta. It is possible that contraction of the tip is progressive as the promastigotes detach from the host 102 cells and transform into epimastigote and trypomastigote "nectomonads". These "nectomonads" are similar to the attached "haptomonads" but are longer and have long, unexpanded flagella which probably enable them to move through the midgut and colonise new areas of the midgut. Parasite division appears to take place mostly as large amastigotes or stumpy epimastigotes in the fly lumen. Multivesiculate bodies were seen in promastigotes attached to midgut cells of Sergentomyia minuta. Similar bodies have been reported from T. brucei (Steiger, 1973) T. cruzi (Wery and de Groodt-Lasseel, 1966) T. congolense (Vickerman, 1969) T. cobitis (Lewis and Ball, 1980) and Crithidia fasciculata (Brooker, 1971) and are generally thought to contain waste material to be eliminated into the flagellar pocket (Lewis and Ball, loc. cit). The bodies may be heterolysosomes formed by the fusion of a food vacuole with a primary lysosome. After some digestion of the food the parasites expel the residue by exocytosis (Brooker, loc. cit., Vickerman and Preston, 1976). The parasites attached to sandfly midgut cells may therefore represent a "feeding phase" allowing accumulation of nutrients for the subsequent division phase. Vickerman (1969) thought that the multivesiculate bodies contained a secretory product and that their fusion with lysosomes possibly led to partial digestion or activation of the product before its transport to the exterior. The surface coat seen in the bloodstream and metacyclic phase of some trypanosomes may be formed from such a product (Vickerman, loc. cit.). In the present study no parasites were seen with a surface coat but this may develop in the bloodstream phase of T. platydactyli. Surface coats on parasites in their invertebrate hosts are only known from a few more "advanced" salivarian trypanosomes of mammals (Vickerman, 1976). The small vacuoles containing electron dense spheres are possibly polyphosphate reserves. Similar bodies, described as "inclusion vacuoles" or "pigment bodies" have been seen in several trypanosomatids (vide Vickerman, 1976). 103 Heywood et_ al (1974) thought that the vacuoles contained electron dense pigment produced during digestion of haemoglobin, since Trypanosoma cobit is epimastigotes contained "pigment bodies" only when grown in media containing haemoglobin. The bodies have also been seen in cultures of gecko haemoflagellates grown in a medium containing haemin (Strauss, 1971; present study). However the occurrence of such bodies in a wide range of kinetoplastids casts doubt on the idea that they contain pigment (Vickerman, loc. cit.) and X-ray analysis of similar bodies in Chlorella have shown them to be formed of polyphosphates (Atkinson et^ al^, 1974). The structure of the aggregates of short, slender trypomastigotes seen in S. minuta suggest that these are produced either by multiple fission or incomplete binary fission since single parasites become a mass of linked trypomastigotes. These short trypomastigotes have a kinetoplast DNA arranged in a double layer which is thought to represent amplification of the DNA (Vickerman, 1976) T. cruzi and Herpetomonas muse arum show similar changes in kDNA arrangement during the life cycle (Brack, 1968; Delain and Riou , 1969; Brun, 1974). 3.4.6.2.Parasites in in vitro culture The promastigotes grown in in vitro appear similar in ultrastructure to those of L. adleri, with fewer subpellicular tubules than L. agamae and L. hoogstraali- (Lewis, 1965). They have flagella and kinetoplast-mitochondrion complexes similar to those of most other trypanosomatids (Vickerman and Preston, 1976). There were insufficient good sections of Golgi bodies to compare the morphology of these organelles. Lewis (loc. cit.) found differences in the degree of development of the Golgi bodies among the strains he examined. 104 3.4.7.The interaction of promastigote cultures with vertebrate cells in vivo and vitro 3.4.7.1.The interaction of parasites with mouse cells. To check whether promastigote cultures from Banyuls geckos could cause infection in mammals, the cultures were mixed with mouse macrophages in vitro and also inoculated subcutaneously into neonate mice. Parasites were rapidly lysed by the macrophages in vitro at 37 C. At suboptimal temperatures the number of promastigotes phagocytised was reduced. No lysis was apparent in the intracellular parasites at low temperatures, and the parasites divided rapidly until the macrophages were ruptured or so distorted that they could no longer adheree to the coverslip surface. Parasite destruction at 37°C was not simply due to the supraoptimal temperature since promastigotes remained active for at least 3 days in axenic culture at this temperature. The retention of parasites in mouse macrophages at 25 and 28°C is probably a result of suppressed lysosomal activity in the macrophages caused by the lower temperatures. This suppression also probably allowed cultures of L. adleri, L. agamae, L. hoogstraali and L. tarentolae to grow in hamster peritoneal cells at 28 and 32°C (Mattock and Molyneux, 1973). However Scheiber (1972) observed hamster peritoneal macrophages lyse L. adleri promastigotes at 28°C, and also at 37°C. The lysis seen at 37°C is in conflict with the parasite"survival seen in hamsters by Adler (1964). Trypanosoma platydactyli promastigotes were also rapidly destroyed when inoculated into neonate mice. Presumably the mouse macrophages were as effective in vivo as they were seen to be In vitro. Leishmania parasites of mammals form larger amastigote infections of macrophages and retain them longer at 37°C than at lower temperatures (Akiyama and Taylor 1970). They are apparently able to do this by surviving in the hostile 105 environment of the phagolysosome, either by resistance to enzyme degradation or by inactivation of the hydrolytic enzymes. (Alexander and Vickerman, 1975). The promastigotes from lizards appear to lack this capability at least at 37°C. (Section 3.3.4.3). Murine macrophages efficiently lyse parasites from the Banyuls geckos and it would be unlikely for these parasites to cause any infection in mice and probably other mammals, other than a very transient one. Sergent et al (1915), Chatton and Blanc (1918) and Parrot (1928) also failed to infect mite with promastigote cultures derived from T. mauritanica blood, and Nicolle et al (1920) failed to infect men, monkeys or mice with the same parasites. Poor parasite survival was also observed in mice and gerbils by Gleyberman in similar experiments using promastigotes from Russian lizards (Belova 1971). However there are reports of transient infections (Manson-Bahr and Heisch, 1961) longer, cryptic infections (Adler, 1964) or even visceral and cutaneous lesions (Young and Hertig, 1927) in mammals caused by inoculated promastigote cultures derived from lizard haemoflagellates. Adler (loc. cit.) cultured promastigotes from the blood of hamsters and mice up to 5 weeks after they were inoculated with L. adleri. The other two reports of lizard parasite survival (Manson-Bahr and Heisch, loc. cit.; Young and Hertig, loc. cit.) are less convincing. Manson-Bahr and Heisch (loc. cit.) took the production of small transient skin nodules at the site of inoculation of L. adleri in human volunteers to indicate the parasite's close relation to Leishmania parasites of mammals. However a similar inflammatory response was seen in 2 mice inoculated with T. platydactyli promastigotes (present study). Young and Hertig (1927) produced visceral and cutaneous lesions in the Chinese hamster (Cricetus griseus) as a result of inoculating, intraperitoneally, promastigotes of "L. tarentolae". However, this experiment has been repeated and no lesions were produced (F. Pratlong, pers. comm.). Young and Hertig may have mixed the 106 stocks they were using, which included Leishmania parasites of mammals, during their culture or their transport from Tunisia to China. There is only one reported attempt to infect avian cells with lizard haemoflagellates: McGhee (1959) incubated L. tarentolae cultures on the chorioallantoic membrane of fertile duck eggs but, though promastigotes multiplied for a few days, no amastigotes were seen. 3«4.7.2The interaction of parasites with lizard cells Monocytes from lizard blood phagocytised T. platydactyli promastigotes in vivo and in vitro and lysed them in vivo. Parasite lysis in vitro was probably retarded by the lack of oxygen and/or nutrients which caused all the cells to deteriorate in the 5-day observation period. The processes of engulfment were identical to those observed by Dollahon and Janovy (1973) using "L. adleri" and leucocytes of New World lizards and similar to those seen with T. platydactyli promastigotes and mouse macrophages (present study). Though the promastigotes rounded up within the cells they never resembled the small elongate ovoids seen in thrombocytes and erythrocytes from some of the geckos. The failure to infect geckos by intracardiac inoculation of parasites and the destruction of the parasites in the blood stream suggests that the promastigotes are not infective to the vertebrate host. "L. tarentolae" promastigotes also failed to become established in New World lizards inoculated with the parasite, though transient infections of L. adleri and L. agamae were observed (Dollahon and Janovy, 1974). The present recent isolate of L. tarentolae did not infect geckos therefore the failure of Dollahon and Janovy's isolate to infect their lizards is not likely to be due to age (c.f. Miller and TW'ohy 1967) It would be useful to repeat these experiments using the short, slender trypomastigotes which develop in S. minuta infected with T. platydactyli. These are probably the infective stage of the trypanosome. 107 Amastigotes were seen in the gekkonid kidney cells grown in vitro but these were very rare and represented a very small fraction of the number of parasites used. They failed to divide and were confined to pseudopodial outgrowths of the cells (Fig. 32 ) suggesting that they had been engulfed and had not actively invaded the cells. Unfortunately too few kidney cells survived to allow any conclusions to be drawn about whether the promastigotes could enter the cells and divide as amastigotes. However promastigotes from culture of some reptilian parasites do enter or are taken up by turtle heart cells at 25°C in which they grow as amastigotes (Molyneux 19771). Promastigotes are probably not the infective form of T. platydactyli. The amastigotes of T. platydactyli seen in cell cultures inoculated with promastigotes are a result of phagocytosis by professional phagocytes or cells which have become phagocytic. Dedifferentiation of tissue cells in in vitro culture can lead to "non-professional" phagocytes developing phagocytic ability (Paul, 1975). It is therefore possible that promastigotes from lizards do not actively enter cells or induce phagocytosis in the manner of Leishmania parasites of mammals. The production of intracellular amastigotes by some lizard haemoflagellates cannot be taken as evidence that the parasite concerned belongs in the genus Leishmania. Although the present study appears to be the first report of trypanosomes of poikilotherms producing amastigotes in cells from homeotherms there are several reports of trypanosomes of mammals doing this. These try- panosomes most of which belong to the subgenus Schizotrypanum, survive and divide as amastigotes in mouse peritoneal macrophages and other vertebrate cells which are not "professional" phagocytes, provided the vertebrate cells are mixed with infective forms of the parasite. (Rodriguez and Marinkelle, 1970; Baker et al, 1980). This need for the correct, infective form of a parasite to be used in attempts to infect vertebrate cells will be discussed further in the next section. 108 3.4.8.Promastigotes in the genus Trypanosoma and trypanosomatid phytogeny It is generally thought that the trypanosomatids diverged into two main groups, the epimastigote and promastigote "stocks", while they were still monoxenous parasites of insects. When haematophagous insects transferred these parasites to vertebrates and they gained entry into the bloodstream some of them evolved to give rise to most of the genus Trypanosoma (from the epimastigotes) and the genus Leishmania and possibly the subgenus Schizotrypanum (from the promastigotes) (Baker, 1973). However the discovery of several trypanosomes with promastigote forms and sandfly vectors puts considerable doubt on this distant dichotomy. The author favours the idea that the genera Leishmania and Endotrypanum evolved from poly- morphic trypanosomes of poikilotherm vertebrates, probably lizards. Evidence for this comes from the common characteristics of the two genera of mammal parasites and trypanosomes of toads (T. bufophlebotomi, Anderson and Ayala, 1968, T. bocagei Feng and Chao 1943) and of lizards (T. platydactyli, present study, T. garnhami Grewal 1957), i.e. the promastigote form in culture and in the vectors; the sandfly vectors themselves and, possibly, an intracellular phase in the parasite life cycle. The monoxenous and heteroxenous flagellates may have evolved separately from a common but ancient ancestor. Grasse (1952) suggested how the heteroxenous parasites may have developed. He hypothesised that the trypanosomatidae were originally parasites of aquatic invertebrates, including leeches which transmitted the flagellates to fishes and amphibians. Blood sucking insects feeding on amphibians then acquired their parasites and eventually passed them on to terrestrial animals, the trypanosomes of which were taken up and subsequently transmitted by haematophagous arthropods. It is interesting to note that members from most of the families of haematophagous insects which transmit trypanosomatid parasites to vertebrates feed on reptiles, including Culicidae Phlebotominae (Present study) Triatominae (Zarate et^ al, 1980)Glossinidae (Molyneux 1973). 109 Lavier (1943) suggested that the earliest trypanosomes, those of fish, were rather uniform. Later they became more diversified in amphibians and reptiles. As they moved to more stable and species-specific environments in homeotherms (Bardsley and Harmsen 1973) they again became uniform, possibly losing some of their morphological forms to give the separate generas and subgenera of the present day. There may then have been a separate evolution of 'hnterior station" development in vectors of some Leishmania and Trypanosoma parasites, which is apparently happening now in triatomid vectors of T. ran^eli (Hoare 1972, Anez 1981). Urdaneta (1972) considered that T. cruzi may have evolved from a parasite of neotropical lizards. The subsequent adaptation to life in homeotherms may have reduced its infectivity to lizards, in which it now causes only transient infections when inoculated (Ryckman 1954,1965; Utdaneta 1972, Urdaneta-Morales and McLure 1981) and reduced its antigenic similarity to Leishmania species (Le Ray and Afchain 1980). The existence of intracellular amastigotes of lizard trypanosomes would support this hypothesis. It is interesting to note that Chagas (1909) reported seeing intraerythrocytic T. cruzi, though the parasites may have been over rather than inside the cells (Wenyon, 1926). The author is tempted to suggest that, since some salivarian trypanosomes are now known to have an extravascular (Luckins and Gray 1978) and perhaps even an intracellular phase of development in the vertebrate (Ormerod 1981), the Salivaria and SterOoraria may have had a more recent common ancestor than previously thought. This may have been a trypanosome of lizards. 3-4.9.The status of saurian leishmaniasis. The discovery of a gecko trypanosome which produces pure promastigote cultures in vitro puts doubt not only on the gecko Leishmariia but also on all other lizard Leishmania parasites. All such parasites have been described from 110 cultures. The subsequent discovery of intracellular amastigotes in blood smears from some of the hosts was taken as confirmation of Leishmania parasites. However the amastigotes were very rare compared with the frequency with which cultures of blood produced promastigotes and were in unusual, i.e. non-phagocytic, cells. The possibility of confusing Leishmania parasites of mammals and trypanosomes of poikilotherms was discussed by Anderson and Ayala (1968). They discovered a trypanosome of a toad, which grew, like T. platydactyli, as pure promastigote cultures in vitro and as "Leishmania-1 ike" infections in sandflies. Trypanosomes which grow as promastigotes, either in vitro or in vivo, are by no means rare but a proportion of trypo- or epimastigotes is usually present to indicate the genus to which they belong (Table 4 ). Trypanosomes were present in the lizards carrying L. tarentolae (Sergent et_ al 1914) and of L. hemidactyli infections (Mackie et^ al 1923) when these species were described and also in geckos and sandflies in the area where L. adleri was isolated (Heisch 1954,1958). Unfortunately the authorities for other Leishmania species of lizards fail to say which, if any, other haematozoa were encountered (Leger 1918, Adler and Theodor 1929, David 1929, Khodukin and Sofier 1940, Andrusko and Markov 1955). It would be surprising if none of the many thousands of lizards involved, especially those from Russia (Belova 1971), had trypanosome infections. The author sees no unequivocal evidence for any Leishmania parasite of lizard blood and considers that the generic status of all 9 species should be questioned until further research can be done. The production of human lesions by inoculation of L. adleri (Manson-Bahr and Heisch 1961) and of hamster lesions with L. tarentolae (Young and Hertigl927 ) may support their retention in the genus but, without studies on the effect of comparable inoculations of poiklotherm 112 trypanosomes the doubt remains. Humans, monkeys, hamsters, mice and gerbils inoculated with promastigotes from cultures of Russian lizard blood did not develop lesions (Hodukin and Sofiev 1940, Kryukova 1941, Belova 1971). Neonate mice inoculated with T. platydactyli promastigotes had destroyed all the parasites within 40 h, (present study) and other promastigote cultures from Banyuls geckos do not create lesions or survive when inoculated subcutaneously or intraperitoneally into hamsters (F. PratLong, pers. comm.). The large lesions produced by Young and Hertig (1927) have never been reproduced and may have resulted from the mistaken use of a mammalian Leishmania parasite. 113 PART B TRYPANOSOMATINE INFECTIONS OF LIZARD INTESTINE 114 4. INTRODUCTION There have been 18 reports of trypanosomatine parasites from lizard intestine and cloaca (Table 5 ). The generic status of these parasites is the subject of some controversy. Although no intracellular amastigotes were known from the intestinal infections, Wenyon (1921) transferred an intestinal parasite of a chamaeleon, previously named Herpetomonas sp. (Bayon, 1914-), to the genus Leishmania. Wenyon (loc. cit.) thought that the parasite probably had both an invertebrate and vertebrate host and that designating it Herpetomonas or Leptomonas sp. "would imply that it was a purely insect, or at least invertebrate, flagellate". Later, Curasson (1943) followed .Wenyon's example and transferred other intestinal parasites of lizards from Herpetomonas or Leptomonas to the genus Leishmania. However, the absence of a pronounced amastigote tissue phase excludes the flagellates from the genus Leishmania (Grasse*, 1952; Vickerman, 1976). It has been suggested that some, if not all, of these flagellate species are monoxenous parasites of insects, of the genera Leptomonas, Herpetomonas and Crithidia, which have been ingested while in their invertebrate hosts by lizards and have managed to survive in the lizard intestine (Brygoo, 1963; Vickerman, 1965; Dollahon and Janovy, 1971). Table 5 . Previous reports of trypanosomatinc parasites in lizard intestines Parasite Vertebrate host Distribution Site of Infection Forms of Parasite Reference -- U m xoj wire b w«- >re ci u bo ij o re e to .-j 01 JS E O mE ioj ) mE u oio o.c re o re ^ >> o vi e o> a u ^ Eu C W O H CJ Her petomonas. sp. Chamaeleo pumilus South Africa Cloaca + 4 - - - - Bayon, 191A Leptomonas henrici Anolis sp. Mart inique Rectum + 4 - - Leger, 1918 Le is_hmania chamae leonis. Chamaeleo vulgaris Egypt Cloaca and rectum + 4 - - Wenyon, 1921 t Herpetomonas sp. Chamaeleo vulgaris Libya Complete gut except stomach + 4 - - - - Franchini, 1921;1933 Chalcides ocellatus Sicily Cloaca and rectum 4 4 - - - - Franchini, 1921 Tarentoln maurit_anica_ Sard inia Cloaca and rectum 4 4 - - - - tt J lor petomon as ho mi da c_tylj. Uemidactvlus Jjxaokei. India Gut contents * 4 4 - - - - Mello & Suctangar,1922 Leptomonas davidi Cncmidophorus lemniscatus Colombia Rectum 4 Strong, 1924 gaigei Herpetomonas mansoni Chamaeleo dilepis) Zoo animals, Cloaca 4 4 Bayon, 1926 C. senegalensis ) probably African Herpetomonas zonuri Zonurus cordylus South Africa Cloaca and rectum 4 4 - - - - Fantham, 1926 ; 1931 tt Z. cataphractus ii 4 4 - - - - Fantham, 1931 Leishmania chamaeleonis Chamaeleo ellioti Uganda Cloaca and rectum 4 Hoare, 1932 Leishmania chamaeleonis Cham.aele.o. y.ul^arJLs_ Not given Cloaca and large intestine 4 4* - - - FrenVel, 1941 - 01 Leptomonas sp. Cnemidophorus sexlineatus Oklahoma, U.S.A. Cloaca and intestine) 4 4 - - Zimmerman & Brown,1952 Eumeces fasciatus Phrynosoma cornutum Scincella laterale Sceloporus undulatus Herpetomonas chamaelconis Chamneleo chamaeleon Zoo animal, probably African Complete gut +4 +- Kulda, 1958 JLoptornonas chamaeleonis Chamaeleo pardalis Ma d a g a s c a r Rectum) Brygoo, 1963 C. oustaleti,C.verrucosus " ) C . lateralis,C.brevicornis " ) **** Herpetomonas sp • Chamaeleo senegalens is Niger i a Cloaca and rectum + - + + - - Vickerman, 1965 + + - + - + Crithidia sp. and Herpeto- Amciva sp. Central America Faecal material and Dollahon 6 JanOvy;1971 jnonaji sp. or Leptomonas sp, Hasiliscus sp gut contents Cnemidophorus sexlineatus (exptl.) Anolis carolinensis (exptl.) Promastigotes (Lcisjimania Chamae leo d_ilepis Zamb ia Gut posterior of Killick-Kendrick & sp.?) stomach, especially Wallbanks, 1981 cloaca and rectum * The only teport of intracellular amast igotes in lizard intestine ** . Epi- and opistho-mastigotes were only seen in in vitro cultures of the faecal promastigotes 116 5.A study of intestinal trypanosomatine parasite infections of some Zambian Chamaeleo dilepis. 5.1.MATERIALS AND METHODS 5.1.1 .Col lection and origin of chamaeleons. Six Chamaeleo dilepis were collected by hand by Dr. R. Killick- Kendrick from 4 localities in Zambia. Three were found on 19.4.80 as follows: Cl/80 near Mkushi (U^l'S, 29O40fE), C2/80 near Chitambo (12°18,S, SO^^E) and C3/80 near Mulilima (13°49,S, 29°38,E). The other ' 3 lizards (C4/80, C5/80 and C6/80) were collected on 29.11.80 from Ndola (W^'S, 28°38fE). 5.1.2.Maintenance and identification of the. lizards. The chamaeleons were kept in aluminium boxes 60 x 50 x 40cm divided into 3 sections by chipboard partitions. A piece of bark and several twigs were put into each compartment as shelter and support for the lizards. A 40w light bulb over each compartment provided heat for the lizards (20-28°C). The lizards and twigs were sprayed daily with distilled water and given adult or larval Musea domestica, Tenebrio molitor or Locusta migratoria 3 times a week. All insects were from closed laboratory colonies and about 10% of them were dissected into 0.9% saline and checked for flagellate infection. The lizards were of sufficiently different sizes to make marking for identification purposes unnecessary. 5.1.3.Examination of lizard blood, faeces and tissues for parasites. 5.1.3.1.Blood smears. Blood was obtained from the lizards by clipping the end of their tails. 117 In Zambia Dr. R. Killick-Kendrick made blood smears from Cl/80, c2/80 and C3/80 4 days after their capture. (They had been kept in a refrigerator since capture). Thin smears were also made of the blood from all the lizards on their day of arrival in England, then 4,8,12 and 16 days later and then at wekly intervals until the lizards died. In England Cl/80, C2/80 and C3/80 were also bled after they had spent 18 and 48h in a refrigerator at 5°C, once immediately after their removal from the refrigerator and again 2h later. The blood smears were fixed in methanol and stained in Giemsa's stain (Section 3.2.2.). 5.1.3.2.Faecal smears Smears were made from fresh faecal pellets from all the lizards about once a week and these were fixed and stained as the blood smears. 5.13.3.Gut sections. The complete gut was removed from C2/80, less than 2h after it had died, and from C4/80 after it had been killed by over-anaesthetization with ether. Each gut was divided into 5 sections of equal length, which were then fixed separately in Carnoy's fluid for 5h. The fixed tissues were washed 3 times in ethanol (6-12h each), cleared in cedarwood oil (18h, changed after 4h) and xylene (Ih), embedded in paraffin wax and sectioned at 6ym. The sections were stained by the Giemsa-Colophonium technique (Bray and Garnham, 1962). 5.1-4-Attempt to infect sandflies with promastigotes seen in the blood of one chamaeleon. Twenty-four 2-4-day old laboratory bred female Phlebotomus papatasi 118 were fed on chamaeleon C2/80 4 to 5 weeks after it was caught. The unrestrained lizard was put in a gauze cage containing the flies, which was returned to a plastic bag containing damp cotton wool and left overnight in the dark at 30°C. The flies which fed were caught and maintained in small glass vials with dry filter paper (Section 3.2.7. ). They were dissected 1 to 10 days post-feed and their guts were examined fresh and also as fixed and stained smears. 5-1.5.Attempt to infect adult Musea domestica and Tenebrio molitor with the intestinal parasite. 5-1.5.1.Musca domestica Twenty-five adult M*. domestica from a laboratory colony were deprived of water for 3h. A petri dish containing fresh faecal pellets heavily infected with flagellates from C2/80 and C4/80 was placed in the fly cage and left in the light at room temperature (15-18 C) for 20 min. The flies were observed during this time and a record was kept of the number of flies feeding on the faecal mucus. After 20 min. the faeces were removed and replaced with a vial of 5% sucrose solution with a filter paper wick. The cage was placed in a plastic bag with some damp cotton wool and maintained in an incubator at 20°C. Five flies were removed after 1,6,12,24 and 30h. They were anaesthetised with carbon dioxide, their guts were dissected into 0.9% saline and examined under phase-contrast illumination (x250). 5-1.5-2.T. molitor In a separate experiment 20 adult T. molitor were enclosed in a 9cm petri dish with 2 fresh faecal pellets from the flagellate-infected lizards C2/80 and C3/80. They were left for 20 min and observed during this time. They were then returned to a box of wheat middlings, kept at 20°C and dissected and examined as the flies. 119 5.1.6.Attempts to culture the intestinal flagellates in vitro. Several attempts were made to culture flagellates from the intestinal mucus of 3 chamaeleons. Small samples of mucus were removed with sterile scissors from the top of fresh faecal pellets from C2/80, C3/80 and C4/80. Mucus was also obtained from the epithelium of the hindgut of C4/80 after this had been carefully dissected from its peritoneal surface to avoid faecal contamination. % The samples of mucus were transferred to sterile tubes containing BHI blood . agar, NNN with M199 ihedium overlay, EBLB, LIT (Section 3.2.3.2) Mansour 's medium (Dollahon and Janovy, 1971) or Trichomonas medium (Oxoid, Basingstoke). 3 Antibiotic concentrations varied from 0 to 500yg/cm Gentamycin, with 3 3 • 3 0-200yg/cm natamycin, or 0-50yg/cm , fungizone or O-lOOOyg/cm nystatin (Table 6 ). Cultures were incubated at 25°C. Samples, taken with a flamed platinum loop after 3,7 and 14 days, were examined under phase-contrast illumination (x250). 3 Fresh faecal pellets from C2/80 were carefully rolled in 5cm of EBLB medium in a sterile petri dish. A sample of the medium was examined under phase-contrast illumination (x250) and the parasite density was estimated 3 in an improved Neubauer haemocytometer. 0.5cm aliquots of the suspension were injected into the tail vein of each of 4 mice, using a 23G x 10mm needle. The mice were'killed by over-anaesthetization in ether fumes 10 min, 30 min, lh and 2h after the inoculation. The mice were bled by cardiac puncture using a syringe and 23G x 15mm needle immediately after death. The blood and also the heart and spleen, which were dissected out by sterile techniques, and macerated were each put into a culture tube of NNN with M199 overlay. The tubes were incubated at 25°C and examined weekly for 5 weeks. 120 Table 6 . Combinations of Gentamycin with Nystatin, Fungizone or Natamycin used in attempts to grow promastigotes from faecal mucus 3 Antibiotic Concentration (yg/cm ) Gentamycin Nystatin Fungizone Natamycin (0 0 0 ^ 50 0 0 (100 0 0 x 500 0 0 (1000 0 0 0 or 100 or 250 £ 0 5 0 or 500 (0 10 0 [0 25 0 ( 0 50 0 [0 0 25 ( 0 0 50 [0 0 75 (0 0 100 , 0 0 200 Table 7 . Measurement of promastigotes in the blood and faecal mucus of C. dilepis from Zambia chameleon material total flagellum body length length width C2/80 blood 24.91-4.12 14.23-2.69 1.71-0 .37 C2/80 faecal mucus 13.19-2.04 6.05-1.88 2.91-0 .69 C3/80 faecal mucus 16.70-3.22 7.05-1.59 1.89-0 .38 C4/80 faecal mucus 15.24-3.13 6.48-1.82 2.25-0 .56 n = 30 measurements inym 121 5-2.RESULTS 5.2.1 .Lizard maintenance Although the chamaeleons fed readily in the laboratory they all died after 4-6 months in England. No flagellates were seen in samples of the insects fed to the lizards. 5.22Blood parasites Haematozoa were seen once only, in one lizard, C2/80, in a smear made by Dr. Killick-Kendrick 4 days after he caught the reptile. (Parasites were not present in smears taken subsequently even from those lizards which, like the lizard with the blood infection, had been kept several days in a refrigerator). Thirty promastigotes were seen in the smear. These were small with a long free flagellum (Table 7 ); none of the parasites seen was in division (figs.86-89,93) 5.2.3.Intestinal parasites 5.243.1.Faecal smears Three chamaeleons C2/80, C3/80 and C4/80 had intestinal infections of promastigote parasites when first examined and retained them until their deaths (Figs .90-92,94) ..The majority of parasites, were "found in the mucus covering the faeces. A few fungal spores but no bacteria were seen in this mucus c The promastigotes were small, (see Table 7 ) most had unusually thick flagella and about a quarter had "squared off" posterior ends. Some were in division. This morphology was retained by the parasites throughout the period of their observation in the laboratory. Promastigotes were the only forms seen. 5.2.3.2.Gut sections Stained sections of the guts of C2/80 and C4/80 revealed a similar 122 Figures 85-92. The chamaeleon Chamaeleo dilepis and blood and faecal promast igotes. Figure 85. Chamaeleo dilepis (Length 20cm). Figures 86-89. Promastigotes in a blood smear from C2/80 (x 150Q) Figures 90-92. Promastigotes in faecal smears from C2/80 (x 1500) 123 smear from C2/80. Figure 94. Camera lucida drawings of promastigotes from the"faeces of C2/80,C3/80 and C4/80. 124 distribution of parasites. They were very common in the cloacae, recta and just posterior to the stomach (c60-200 parasites/section) but rare in mid hindgut (cl-35 parasites/section) and absent from the rest of the gut. In C2/80 a few parasites were found in the connective tissue and between muscle fibres of the cloacal wall (Fig^96-98)- All other parasites were found close to the gut epithelium between folds in the gut wall and cloacal glands Fungal spores were also seen in the intestinal mucus and within epithelial cells in the hindgut and cloacal wall (Fig.95 ). 5-24.Attempts to infect sandflies No parasites were seen in sandflies fed on C2/80 4 to 5 weeks after its capture. 5-2.5.Attempts to culture the intestinal flagellates All attempts to create axenic cultures of the intestinal flagellates failed, probably because of fungal contamination. 5.2.5.1.Direct culture of faecal mucus No bacterial contamination occurred in 14 of 15 cultures containing 3 500yg/cm Gentamycin nor in 8 of 15 cultures containing no Gentamycin. 3 Fungal contamination occurred in all cultures containing less than 1000yg/cm 3 3 nystatin or less than 50yg/cm fungizone or less than 75yg/cm natamycin. Active promastigotes were seen in cultures containing lower concentrations 3 3 3 of antimycotics (500]jg/cm nystatin, 10yg/cm fungizone or 25yg/cm natamycin) but were overgrown by fungi within 3 days. 5.2.52.Culture of the blood and tissues of mice inoculated with a parasite suspension. EBLB medium washings of fresh faecal pellets contained many active 125 Figure 95. Section of the hindgut of a C. dilepis to show fungal spores (arrows) within epidermal cells. Paraffin wax section. Giemsa staining (x 1700). Figures 96-98. Promastigotes in the cloacal wall of a C. dilepis. (x 1700) 126 5 3 promastigotes (1.8 x 10 parasites/cm ) but no flagellates were seen in cultures of the blood and tissues from any mouse inoculated with the parasite suspension. 2 of the 12 cultures were contaminated with a fungus similar in appearance to that seen in cultures inoculated with faecal mucus (section5.2.5.1.). 5.2.6Attempts to infect M. domestica and T. molitor. All the M. domestica and 19 of the T. molitor fed readily on the mucus surrounding the chamaeleon faeces. The insects dissected after lh contained many promastigotes but all were immobile. Five hours later the parasites were still immobile and most were vacuolated. Twelve hours after the infective feed few of the parasites remained intact in the T. molitor, while those in M. domestica were similar to those seen at 6h. At 24h and 30h no intact parasites were observed, though a few empty membrane ghosts with flagella remained. 127 5-3.DIS CUSS ION 5.3.1.Discussion of the present study 3 of the Zambian chamaeleons studied had a similar intestinal infection of promastigotes and one also had an apparently transient promastigote infection of the blood. The intestinal and blood parasites differed significantly in size and probably represent at least two species of trypanosomatid. The intestinal parasites were shorter and broader than the blood forms and had a thicker flagella. The blood parasites may have represented an earlier intestinal infection which had gained entry to the blood stream and which was being destroyed by the lizards' immune responses. Several other intestinal parasites including Trichomonas have been found in lizard blood (vide Brygoo, 1963) and probably result from damage of the gut epithelium rather than active invasion by. the protozoans. Contamination of lizard blood with intestinal parasites is particularly common in Zoo animals in poor condition (Labbe,1894). It is unlikely that the promastigotes seen in the blood of C2/80 are Leishmania parasites of other animals transmitted to the chamaeleon by the vector; the number of the parasites seen in one smear is too large to represent an infective dose inoculated by an invertebrate. The intestinal parasites seen in faecal mucus were smaller than those seen in C. vulgaris and C. pumilus from Egypt by Wenyon (1921) which he named Leishmania chamaeleonis and also smaller than similar parasites seen in C. dilepis and C. senegalensis (Bayon 1926) and C. ellioti (Hoare 1932). Their average body length is within the ranges given for Leptomonas chatnaeleonis (Brygoo, 1963) Herpetomonas sp. (Fantham, 1931) and Herpetomonas sp. (Franchini, 1921) but all of those parasites had much longer flagella ( > 20ym) than those seen in the present study (6.5ym).The unusually thick 128 flagella seen in flagellates from Zambian C. dilepis were also noted by Bayon (1914) in parasites from South African C. pumilus and from Central American lizards (Dollahon and Janovy, 1971). Bayon (loc. cit) suggested the flagella were surrounded by a 'feheath or slight membrane". We now know that all flagella are bounded by a membrane continuous with the parasite's plasmalemma (Vickerman and Preston, 1976). Thick flagella may indicate an unusual amount of cytoplasm surrounding the axoneme and paraxial rod. The parasites from Zambian C. dilepis do not accord with any of the previous descriptions of intestinal parasites of lizards but, owing to the variation seen in the parasites previously it is impossible to say whether or not the parasites belong to a new species. Unlike L. chamaeleonis (Wenyon, 1921) the parasites from C. dilepis only survived in Musea domestica (and T. molitor) for a few hours and rapidly became vacuolated and inactive. This suggests that the parasites have some invertebrate host specificity or direct transmission (see p. 132). They may produce heavy infections in other insects. All attempts to culture the parasites were thwarted by fungal contamination. The fungi which probably developed from the spores seen in faecal mucus and lizard gut epithelial cells could only bq controlled by levels of antimycotics which probably killed the flagellates. (It is possible but unlikely that none of the media used were suitable for culture of the parasites). Vickerman (1965) overcame this problem by spreading drops of a contaminated flagellate suspension on agar and collecting flagellates from the angle of contact. There have been recent successes using 5-Fluorocytosine as an antimycotic in flagellate cultures (Kimber et al, 1981) and this might have been effective in the present study but was not tried. An attempt was made to use the phagocytic cells of mice in vivo t:o destroy . 129 the fungi while allowing the flagellates to survive. However when contaminated parasite suspensions were injected into mice both the parasites and fungi appeared to be destroyed or rendered non-viable within a few minutes of inoculation. In fact the presence of fungal contamination in two cultures indicates that the flagellates were destroyed before the fungi. Flagellate parasites of lizard intestines have been successfully cultured 3 times (Franchini, 1921; Vickerman, 1965; Dollahon and Janovy, 1971). Cultures set up by Bayon (1914) were contaminated with bacteria, those set up by P.T. Gardener (pers. comm.) were spoilt by fungi while parasites from the faeces of lizards fed cultures of lizard haemoflagellates by Mohiuddin (1958) failed to' grow. Gut sections from infected lizards demonstrated that the parasites were distributed throughout the midgut of the lizards but were particularly numerous in the cloacae and recta , where, most of the intestinal mucus is secreted. Most other intestinal flagellate infections have been confined to the hindgut (Table 5 ) although Franchini (1921) and Kulda (1958) found flagellates along the entire gut of Chamaeleo spp. The foregut and stomach are probably unsuitable for the flagellates because of their pH, the presence of enzymes, or the absence of microvilli from thq gut's internal surface. The C. vulgaris found with parasites throughout its intestine may have recently fed on insects infected with flagellates. Two muscoid larvae were also found in its gut (Franchini loc. cit.). The promastigotes seen within corrective tissue and muscle fibres of the cloaca of one lizard were probably the result of post-mortem deterioration of the cloacal epithelium. None of the parasites were intracellular. Though most of the tissues appeared normal,bacteria were seen 130 within the gut wall in many sections and the gut epithelium was broken in several areas. 5.32.The classification of trypanosomatine flagellates of lizard intestine. The author believes it to be incorrect to classify these intestinal parasites in the genus Leishmania. There are 4 reasons for this:- (a) The absence of intracellular amastigotes. There is a single unconvincing report of intracellular amastigotes seen in the intestine of a lizard (Frenkel, 1941). The sketch of the parasites shows rather amorphous amastigotes in unusually large vacuoles and these are probably either recently phagocytized flagellates (Dollahon and Janovy, 1971) or flagellates introduced into tissue damaged by the procedure of anal introduction of faeces used to infect the lizards. (b) The infection of non-haematophagous insects with faecal parasites. Musca domestica and Calliphora sp. have been temporarily infected with parasites from lizard intestines (Wenyon, 1921; Vickerman, 1965). Bayon (191.4-) thought Chamaeleo pumilus were probably infected when they ate Scatophaga hottentota carrying the parasite because lizards and flies carried similar parasites. (c) The infection of lizards fed on non-haematophagous insects with flagellate infections. Strong (1924) found parasites in Cnemidophorus sp. fed on the hemipteran, Chariestrus cuspidatus naturally infected with a flagellate parasite. Unfortunately these experiments were uncontrolled. Dollahon and Janovy (1971) fed flies, Megaselia scalaris naturally infected with flagellates (Crithidia and either LeptQmonas or Herpetomonas) to Cnemidophorus sp. and Anolis sp. Uninfected Tenebrio molitor were fed 131 as controls. Trypanosomatine flagellates were demonstrated in lizards fed the flies after 3 to 16 days but were never found in the control lizards. (d) The presence of opistho, epi- and choano-mastigotes, in the lizard infections or cultures derived from them. Parasites in the genus Leishmania by definition have only two forms: promastigote and amastigote. Although the promastigote is usually the most common form of flagellate observed in lizard intestines, other forms also occur. Kulda (1958) found that 50% of the parasites of a Chamaeleo chamaeleon were opisthomastigotes, 37% were promastigote, 13% epimastigote and trypomastigotes and amastigotes were occasionally observed. Though he only observed promastigotes in faeces of C. senegalensis Vickerman (1965) found pro-j epi- and opistho-mastigotes in cultures derived from these faecal parasites. Similarly Dollahon and Janovy (1971) saw a mixture of forms in cultures from Central American lizards including choano-jpara- and opisthomastigotes. It seems likely that most, if not all, of the parasites seen in lizard intestines are true parasites of insects which have entered into another suitable environment in the intestinal mucus of lizards. The intestinal infections are not necessarily transient and some have been followed for over 4 months (Kulda, 1958; present study). The apparent high frequency with which these parasites occur in lizards of the genus Chamaeleo (Brygoo, 1963) is probably a result of 2 factors: the large amount of intestinal mucus produced by the lizard which coats the faecal pellet and the slow movement of the lizard host which makes the chamaeleons very easy to collect in good condition. Faecal smears, sections of chamaeleon gut and culture of mucus in media without antibiotics show that the intestinal mucus is sterile apart from a few fungal spores and so the parasites are not growing 132 in the presence of bacteria as David (1929) supposed. The parasites have little in common with lizard haemoflagellates. Attempts to create intestinal infections with haemoflagellate cultures have failed (Mohiuddin, 1959) and no infections were seen in lizards inoculated intraperitoneally with intestinal flagellates (Brygoo, 1963). Franchini (1921) found rare amastigotes in the blood, liver and muscle of two mice fed promastigotes from lizard intestine but attempts to repeat this observation were unsuccessful (Brygoo, 1963; present study). Strong (1924) saw amastigotes in a monkey inoculated with flagellates from lizard intestine but thepe were confined to the inoculation site and transient. It is unlikely that haemoflagellates of lizards evolved from the intestinal flagellates by invasion of the blood as has been proposed (Minchin, 1908; Mesnil, 1918; Lavier, 1943; Adler, 1933). Lizards are probably infected when they ingest flies or other arthropods which either have gut infections of the parasites or have recently been feeding on faecal mucus from other lizards. Flies appear to be attracted to faecal mucus and lizards may be attracted to the concentrated food supply which results (present study). Although lizards can be infected experimentally by feeding them infected faecal mucus (Frenkel, 1941; Brygoo, 1963) this direct transmission is unlikely in the wild. Although resistant "cyst-like forms" are known in the genus Leptomonas (Vickerman, 1976; Abe, 1980; Molyneux and Croft, 1980) no such forms have been reported from intestinal infections of lizards. Rather than create new genera for the intestinal parasites the author considers it preferable to redefine the genera Leptomonas (for parasites only having promastigote and amastigote form) Herpetomonas (for parasites 133 having an opisthomastigote form) and Crithidia (for parasites having a choanomastigote form) to include the monoflagellate kinetoplastid parasites seen in the intestinal mucus of lizards and amphibia (Fantham 1931). The wider host range would have to be included in the definition. All the trypanosomatine parasites seen in lizard intestine become Leptomonas sp. except Herpetomonas chamaeleonis Kulda 1958 and the Herpetomonas sp and Crithidia sp. reported by Vickerman (1965) and Dollahon and Janovy (1971). (Kulda's synonymy of Leishmania chamaeleonis and Herpetomonas chamaeleonis * (Kulda 1958) lacks proof. Kulda (loc. cit.) thought that the parasite he found in C. chamaeleon was the same species as that found by Wenyon (1921) in C. pumilus or C. vulgaris, simply because the promastigotes were a similar size).- 134 PART C MALARIA PARASITES OF LIZARDS 135 6-INTRODUCTION 6«1 .General introduction Parasites of the genus Plasmodium have been found in lizards of all the major families in the Americas, tropical Africa, Indonesia, the Pacific Islands and Australia. They are rare or absent in lizards from Europe and Asia (Garnham, 1966). The Plasmodium species of lizards resemble those of birds and mammals but have been generally less well studied. However they have been recently listed and reviewed (Ayala, 1977; 1978). At present there are 62 named species and subspecies (Ayala, loc. cit.; Telford, 19771; 1978; 1980), including parasites originally placed in the genera Garnia, Fallisia and Saurocytozoon. Telford (1973), Hsu et al (1973) and Ayala (1977) considered that these genera should be synonymised with Plasmodium; in contrast to Lainson et al (1974) who thought they should be retained and placed in a separate family to avoid redefining the family Plasmodiidae to accommodate them. This controversy is still developing (Ayala, loc. cit). The first blood stages of a saurian malaria parasite were seen by Wenyon in 1907 (Wenyon, 1909) but another 34 years elapsed before experimental work on the parasite was done and an oocyst stage observed (Huff, 1941). Sporozoites were not seen for a further 29 years (Ayala and Lee, 1970). Many attempts to observe sporogony in invertebrates, mostly mosquitoes, have either failed or only partially succeeded (Table8 ). The observations of Ayala (1977) and Ayala and Lee (1970) of complete sporogony of P. mexicanum and P. floridense in sandflies led to speculation that the earlier failures were due to the incorrect assumption that mosquitoes transmitted the Table 8 Previous attempts to find the vector or observe the sporogenesis of malaria parasites of lizards Paras ite Vertebrate host Invertebrates fed on Stapes seen (and number where known) Reference infected lizards Micro- Ooki- Immature Mature Sporo- gametes netes oocyst oocyst zoites in sali- vary gland P.agamae Agama agama Aedes aegypti + (2) + (1) Bray,1959 Ae .africanus ii Culicoides sp. J.R.Baker pers. comm. Ae .apicoargenteus, Ae .simpsoni ,Ae: .aegypti, Ae .afr icanus P.basilisci Basiliscus vittatus Culex spp. Garnham, 1966 P.cnemi- Cnemidophorus dophori lemnisc£tus Culex pipiens +* Telford,1970 P.fischeri Chamaeleo fischeri Ae.aegypt i Ball & Pringle 1965 P.floridense Sceloporus undulatus ' Ae ."atlanticus tormentor , ) and Anolis carolin- Ae.tr iser iatus tPsorophora) confinnis, P.ferox, ) Jordan,1964 CO Mansonia pertubans ) Q Ae .aegypt i + (1) Culex territans + (42) Culex quinquefasciatus + (4) Culex sp. + (70) +* Culex pipiens +* Telford ,1970 + Lutzomyia vexatrix + Ayala,1977 P.giganteum A.agama Ae. aegypti,Ae.africanus Bray,1959 Ae .simpsoni,Culicoides sp. J.R.Baker pers. comm • Ae .aegypti + »» P.mexicanum S .occidentalis L .vexatrix,L.stewarti +(many) +(many) +(many) Ayala S Lee 1970 -•-(many) Hirstiella sp. Pelaez £t al_ 1948 P.(Garnia) Mabuya mabouya Culex pipiens fatigans (+ ?) Lainson et al morula +(many) 1974 P.rhadinunum Iguana iguana Ae . aegypti,Culex pipiens** Thompson & Huff,1944 P.robinsoni Chamaeleo brevicor.nis Culex fatigans Brygoo,1962 P. (Saurocyto- Tupinambus Culex +(many) +(many) +(few) Landau et^ al coon) tupinambi nigropunctatus Ae.aegypt i +(many) 1973 An .stephens i +(many) Culex fat igans P.sp, Sceloporus £_. Ae .aegypti + (1) Huff, 1941 ferraniperezi Culex pipiens * Tarshis thought these "parasites" were in fact mycetomes (see Ayala, 1977; Tarshis, 1953) ** All mos uitoes fed on infect d ' ... 137 malaria parasites of lizards, as well as those of birds and mammals. The sporogenesis of these two malaria species occurred in sandfy species which, like the parasites and lizard hosts, came from the New World. It seemed that sandflies were also likely to be vectors of Old World saurian malaria. Reports of malaria infections in the agamid lizards and synpatric sauriophilic sandflies in the Gambia indicated that investigation there might throw further light on this hypothesis. 6»2.Description of site, vertebrate host and parasite 6.2.1.The site The Gambia is the smallest African country comprised of a strip approximately 320 x 25km, bordering the western length of the River Gambia. It is bounded on the West by the North Atlantic Ocean and elsewhere by Senegal. It lies 13.5° North of the Equator, along the northern edge of the Upper Guinea Savanna district (vide Edwards, 1941) and in the southern Guinea Zone of the savanna vegetational region (Keay, 1953). The savanna is modified by Rhizophora and Av.icennia mangrove swamps and fragments of gallery forest. The country has a tropical climate with one well defined rainy season extending from June to October during which there is approximately 1000mm of rainfall (Murphy, 1960). Sandfly and lizard collections were based at Basse Santa Su (shortened to Basse), the headquarters of the Upper River Division (Fig.99 ). The town is on the southern bank of the river; surrounded by dry scrub (Fig.100 ) and irrigated rice (Fig.101 ). One kilometre to the east of the town is the Prufu Bolon or creek which runs through the large freshwater Prufu Swamp. During the dry season the swamp dries and the Bolon breaks down into small isolated pools (Fig. 104 ). Men, donkeys, cattle (Fig. 106), sheep, goats and chicken were abundant Figure 99. Lizard collection sites in the Gambia 139 Figures 100-106. Lizard and sandfly habitats in the Gambia. Figure 100. Dry scrub 11 101. Irrigated rice 11 102. Dry scrub with water erosion channels " 103. Termitaria with exposed air vents (V) 11 104. Isolated pool in Prufu Bolon " 105. Typical sandfly resting site between tree roots " 106. Cattle on river bank, Fatato. 140 141 142 in the area. Anuran amphibians, agamid (Agama agama) and geckonid lizards (Hemidactylus fasciatus) and a wide variety of wild birds, particularly weavers (Ploceidae), doves (Columbidae) and starlings (Sturnidae) were common. Monitor lizards (Varanus niloticus), snakes, including pythons (Python sebae), puff adders (Bitis arietans) and black cobras (Naja melanoleuca) and green vervet monkeys (Cercopithecus aethiops) were seen occasionally. About 100 bats (Taphlozous sp.) roosted in the loft of one house on the eastern outskirts of the town, near the sandfly collection sites. No other small mammals were seen but apparently occupied burrows were found and owl pellets from two resident Barn owls (Tyto alba) contained many rodent skulls. 6.2.2.The host There are about 280 species of lizard in the family Agamidae. These are mainly confined to Africa, Asia and Australia, being replaced in America, Madagascar and Fiji by the closely related Iguan.ids (Arnold et^ Only one species, Agama agama occurs in the Gambia. Agama agama is the most common lizard in the Gambia and is often seen on the ground feeding on insects. They are most active in the early morning and late evening and retreat into tree holes, water erosion channels, burrows and crevices during the hottest part of the day (1400-1600 hours) and the night. They occur in all dry habitats including human settlements. 6-2.3.The parasites There are only two malaria parasites known from Agama species: Plasmodium agamae (Wenyon 1909) and P. giganteum Theiler 1930. Though synonymy of the two species was once considered (vide: Bray, 1959) it is now clear that two separate parasites exist. 143 P. agamae is the "most widely distributed malaria parasite of lizards in Africa" (Garnham, 1966). It has been reported from West Africa (Gambia, Liberia, Nigeria and Congo), Kenya, Ethiopia and Sudan and possibly from South Africa (Ayala, 1978). It appears to be absent from Uganda, Tanzania, Egypt and Yemen, although the host lizard is common in these countries (Garnham, 1954). P. giganteum also occurs in West Africa (Mali, Sierra Leone and Liberia) Kenya and Tanzania, often in mixed infections with P. agamae (Ayala, loc. cit.). The young intraerythrocytic stages of both parasites are similar and difficult to separate. However the size of mature gametocytes and form of the schizonts differs, allowing easy distinction of these stages of the two parasites (Table 9 ). J.R. Baker (pers. comm.) saw ookinetes of P. giganteum in Aedes aegypti and of P. agamae in Culicoides sp. and 2 ookinetes and one P. agamae oocyst* were seenby Bray (1959) in Aedes aegypti. These are the only records of sporogonic stages of the two parasites (Table 8 ). Table 9. Comparison of malaria parasites of agamid lizards as seen in stained blood smears (after Garnham 1966) Parasite P. agamae P. giganteum Trophozoites and Similar Similar immature gametocytes Mature schizonts Fan-shaped or oval Sausage-shaped or spherical 6-12 merozoites 24-96 merozoites Mature macrogametocytes 10 x 4-5 ym 16-18 x 6-7 ym " microgametocytes 10 x 4-5 ym 12 x 8 ym Host cell type Mature erythrocytes Mostly immature erythrocytes Effect on host cell of None Cell distended and nucleus mature gametocytes and displaced. Pinkish granular schizonts discoloration of cytoplasm and rim of stain round the margin 145 7. MATERIALS AND METHODS 7.l.Reptile collection, marking, maintenance and observation 7.11.Collection Agama agama were collected from 6 towns or villages, mostly by local boys (Fig. 99 ). Most lizards were chased onto wicker fences and trapped against them with long thin sticks. Since only healthy lizards were bought the catchers ensured that very few were mutilated by this procedure. Other reptiles were also examined whenever possible. 7.1.2.Marking Lizards were marked as soon as possible after capture with a number written with a felt tip pen on their ventral skin. Later all lizards infected with haematozoa plus twenty uninfected lizards were more permanently marked with a band of adhesive plaster taped around the base of their tails which was also numbered. The snout to vent length of all lizards was recorded. The sex of all lizards in which this length exceeded 7cm was recorded.' (Male lizards of this size have distinctly enlarged preanal scales). These records allowed lizards losing their numbers to be identified and renumbered. 7- 1.3.Maintenance The agamids were kept in wooden boxes measuring 80x15x30cm, topped with nylon gauze, with up to 20 lizards per box. (Fig. 108 ). They were fed daily with adult mayflies (Ephemeroptera) and alate termites (Isoptera). Fresh water was provided daily in petri dishes. 7.1.4.0bservation Thin blood smears were made from lizards as soon as possible after capture and 2 and 4 days later. Blood was obtained by clipping a claw. Snakes were bled by clipping the end of the tail after inducing anaesthesia 147 Figure 107. Light trap for sandflies with battery unit (B) lamp (L) and fan (F). 11 108. Lizard cage. Figures. 109-110. Emergence trap dismantled (Fig. 109) and in situ (Fig. 110). Scale = 20cm. ~" ' 148 with ether. Each smear was fixed in methanol, stained in Giemsa's stain (Section 3.2.2. ) and scanned under an oil-immersion lens (x500) for 5 minutes. Most lizards free of haematozoa were released 4 days after capture. Those lizards infected with blood parasites and 20 uninfected lizards were kept and examined for parasites every 4 days. A record was kept of parasitaemias (parasites/104 erythrocytes) and the ratio of asexual to sexual stages• 8 lizards with haematozoic parasites resembling Schellackia $.p. were killed with ether a week after capture. Impression smears were made from several regions of the gut and from lung, liver and spleen. The smears were fixed in methanol and stained in Giemsa's stain (Section 3.2.2. )• 7-2.Sandfly collection Sandflies were collected by six methods:- 7.2.1 .Oral aspirator An oral aspirator, the "capturateur a bouche" described by Croset et^ al^ (1977), was used in collections from interior walls and ceilings of houses, from the buttresses of trees, tree holes and from the open vents of termitaria. Collections were made at night, using a torch to attract the sandflies. 7.2.2.Miniature "GDC" light traps and suction traps 5 "CDC" traps, which combine light and suction trapping methods, were used. Each was powered by 4 rechargeable Nickel-Cadmium batteries (Sanyo 1.3V, 4A/h, Radio Spares, London) held in series in a vertical tube (Fig. 107 ). The batteries were charged for 3-4 hours each day using a hand-built five- channel charger giving 0.25 Amps/channel. 149 Collections were made on alternate nights for three months in the late dry season (April to June). Once each month a trap was run throughout the night from 1800 to 0900 hours. The battery unit and attached cage were charged every 3 hours so that the period of maximum sandfly activity could be estimated. Routine nightly catches were then timed to include this period. Collection time was limited to 9 hours per night by the electricity supply to the charger which came from a diesel generator run for 4-6 hours per day. Two of the traps were hung from trees in the dry scrub near deep water erosion channels with many holes and crevices (Figs.102 and 105 ), one from a verandah oh the first floor of a concrete-built house close to the river and two from wire fencing surrounding the house (one of these close to a stand of silk cotton trees and one close to the main Basse to Dam fa Kunda road, opposite irrigated rice). On 3 nights the traps were run without their light bulbs so that they were acting solely as suction traps. 7.2.3.Oiled paper traps A sheet of thin white paper 25x30cm soaked in castor oil was placed in each of two tree holes in the area of dry scrub (Fig. 102 ) and in one open vent of a termite hill (Fig. 103 ) and left overnight (11.4.81-12.4.81). Flies adhering to the papers were removed using a small brush dipped in ethanol. 7.2.4.Emergence traps Old tin cans measuring 12x6cm were used to make emergence traps. Both ends of the tins were removed. A cone of cardboard 5cm high was taped into one end of the remaining cylinder with the point of the cone with a small (0.5cm) hole at the apex directed into the tin and the other end of the 150 cylinder was covered with nylon gauze (Fig. 109 ). The whole assembly was placed in a tree hole or rodent burrow with the gauze toward the light. The trap was then surrounded with dense polyurethane foam so that light only entered the hole or burrow through the hole in the cone (Fig. 110). The traps were left in position overnight and checked for insects each morning. 7.2.5.Baited trap Three adult Agama agama were left for 10 nights in a gauze cage 40x40x40cm. One side of the cage was formed into a large inwardly pointing cone which had a small (2cm) aperture at its tip. The cage was placed so that the cone formed its leeward side (i.e. towards the North-East). The trap was checked for insects each morning when the lizards were removed. 7.3.The dissection and identification of sandflies and attempts to infect them with P. agamae. All flies caught were counted and transferred as soon as possible after capture to cages held in black plastic bags, containing damp cotton wool to maintain a high humidity- They were given 60% (w/v) sucrose solution on cotton wool balls held in petri dishes and kept at laboratory temperatures (25-39°C). -sAll female sandflies were dissected to check for parasite infection (Section 3.2.8.1). Their heads and genitalia were transferred to a drop of Berlese fluid on a microscope slide and covered with a coverslip. Sandflies were identified using a simple visual key constructed from drawings of the spermathecae and the cibarial and pharyngeal teeth of all the sandfly species reported from West Africa (Senegal, Gambia, Guinea Bissau, Guinea, Sierra Leone, Mauritania and western Mali). Further identification involved the length of the third antexmal segment and the palpal formulae. The key was 151 used to identify all females. Male flies were counted and preserved in Nesbitt's Solution (Appendix E ) but not identified. All but 10 female flies caught in April were dissected 1 to 4 days after capture. 10 flies (9 Sergentomyia magna and 1 Sergentomyia sp) were 4 fed on a lizard with 56 "haemogregarines"/10 erythrocytes and dissected 1 to 3 days later. Female flies caught in May and June were offered a bloodmeal daily fqr 3 days after capture from an Agama agama infected with P. agamae. The infected lizard was placed in the cage containing the sandfly females with its legs taped together 1:o protect the cage and left there for 3 to 6 hours in the dark. Flies which had fed from the lizard were removed, using a short glass vial, transferred to another cage, and dissected 2 to 10 days later. 10 of these sandflies were kept in a box covered with a damp cloth to reduce the temperature to 24 to 28°C for 5 days, when they were dissected. Female flies which failed to feed were dissected 4 days after capture. Male flies were anaesthetised with carbon dioxide and plunged into Nesbitt's solution after the same period. 7.4.0ther haematophagous insects offered bloodmeals. Over 200 mosquitoes (Culicidae) and a 100 midges (Ceratopogonidae) were caught by,, oral aspirator and in light traps from the Basse area. These flies and 400 laboratory bred Culicoides nubeculosus (from the Animal Virus Research Institute, Pirbright) were offered bloodmeals from infected lizards restrained as section 7.3 . (Experiments with wild-caught flies were done in the Gambia. Experiments with C. nubeculosus were conducted in England with the imported lizards) Flies which had fed were maintained and dissected as the sandflies. 20 acarine mites, from lizards infected with P. agamae, were removed by brushing them with alcohol, smeared and stained in Giemsa's stain. 152 7-5.Attempts to observe the exflagellation of P. agamae microgametocytes Coverslips were placed on drops of fresh blood, containing apparently mature gametocytes of P. agamae. These wet films of blood were rapidly transferred to plastic boxes with damp cotton wool to keep them moist and subjected to temperatures of 6,15,20 and 26°C for periods of up to 12 hours. Microgametocytes were examined intermittently during this time under phase- contrast illumination (x 250) and all changes were noted. This experiment was repeated with more blood which was breathed on before being covered with a coverslip. 5 Sergentomyia dubia were dissected and their guts were smeared 2,20,40,60 and 600 minutes after they had fed on a lizard gametocyte carrier. The smears were first examined in saline and then after fixation and staining in Giemsa's stain. Drops of blood containing gametocytes were mixed with an equal volume of 2% glucose peptone, a solution which stimulated Haemocystidium tarentolae to exflagellate (Riding, 1930). The suspensions were examined in the same way as the wet blood films. 3 In England 1cm of infected blood was taken from an Agama agama by cardiaa. puncture (Section 3.2.3.1) and immediately mixed with Tris buffered saline(*(TBS; 166mMNaCl, lOmM glucose and lOmM Tris at pH7.4) at a dilution of 1 part to 5 parts of saline (Martin et^ al^, 1978). 0.5cm aliquots of this suspension were centrifuged at 500g for 30sec and the supernatant was removed. The cells were resuspended in various test solutions and centrifuged, the 3 supernatant was removed and the cells were resuspended in 0.2cm of the same test solution. Drops of the suspensions were then placed on microscope slides, covered with Vaseline-rimmed coverslips and examined under phase-contrast illumination (x250) at 10 minute intervals for 6 hours. Some of the test 153 solutions used had previously been found to stimulate exflagellation of other Plasmodium species. They were: (a) TBS with 50mM NaHC0o at pH0 r. (Carter and Nijhout, 1977) and 3 o .U at pH?#Q . (b) Foetal calf serum (Flow, Irvine), pH_ (Carter and Beach, 1977) o .U and at pH^ and pH^ q. (c) Chick serum (Flow, Irvine) pHg (d) TBS with 5mM 8-bromo-cyclic adenosine monophosphate (Sigma, London) at pH0 _ (Martin et al, 1978). o .U —— (e) TBS with 5mM caffeine (Sigma, London) at pH0 _ (Martin et al, O . U -— loc. cit.). (f) TBS with the macerated guts or heads of 4 Phlebotomus papatasi or 4 Lutzomyia longipalpis or 4 Culex pipiens or 20 Culicoides nubeculosus (all from laboratory colonies), (vide Nijhout, 1979). 154 8.RESULTS 8.1.Reptile collection and maintenance. 338 Agama agama were collected from 6 areas. Many more males (59.5%) were caught than females (25.1%) or juveniles (15.4%) (Table 10). The employment of local boys to catch lizards had the advantage that the lizards examined came from a much wider area than the towns where collections were based. % Six Banded geckos (Hemidactylus fasciatus) and one African python (Python sebae) from Basse, one Puff adder (Bitis arietans) from Mansajang and one Black cobra (Naja melanoleuca) from Dinguiri were also caught and examined. The lizards fed readily and survived well in captivity but large males fought and were kept in separate cages. 82.Parasite infections observed. 8.2.1. Plasmodium agamae 3 male A. agama were infected with P. agamae, two were from Misera and one from Mansajang. The changes in composition of these infections and the parasitemias are shown in Figs 111-113. All parasitemias were low with a maximum of 0.25% of erythrocytes infected. These parasitemias fell gradually and all infections were lost within 10 weeks of capture. Sexual stages of the parasites outnumbered the asexual stages. Gametocytes formed 40 to 98% of the parasites seen in blood smears and there were approximately 3 macro- gametocytes to every 2 microgametocytes. All infected cells appeared to be mature erythrocytes. The parasites resembled P. agamae as described by Garnham (1966), with very amoeboid trophozoites and oval or fan-shaped schizonts with 6 to 10 merozoites. (Figs. 115,119-121) 155 Table 10 . The number and sex of lizards caught in 6 areas of the Gambia Area Division Juveniles Males Females Total Bansang Maccarthy Island 5 32 12 49 Basse Upper River 17 32 18 67 Brikama Ba Maccarthy Island 2 46 5 53 Fajara Western 0 4 1 5 Mansajang Upper River 23 36 18 77 Misera Upper River 5 51 31 87 - Totals 52 201 85 338 /jf 156 Figures 111-113. Parasitemias in the 3 P.agamae-infected Agama agama Days after capture Figure 111. Parasitemias in lizard 104 from Misera. Days after capture Figure 112. Parasitemias in lizard 261 from Mansajang Figure 113. Parasitemias in lizard 241 from Mansajang 157 Mature microgametocytes measured 7-14 x 5-8 pm (x = 10.3x6.0, N=40) and macrogametocytes measured 10-15 x 5-8 pm (x = 12.5x5.8pm, N=40). (Figs 116-118) There was no abnormal staining of infected cells as is seen in P. giganteum infections (Garnham, loc. cit.). 8.2.2Eimeriine Coccidian parasites. 28 lizards had erythrocytes infected with monomorphic coccidian sporozoites, measuring approximately 8 x 3 pm (Fig.122-123) J.1 of these % lizards came from Mansajang, 6 from Brikama Ba, 4 from Basse, 4 from Bansang and 3 from Misera. Similar haematozoic sporozoites were the only stages seen in tissue impression smears from the infected lizards. 8.2-3.Pirhaemocyt on. Pirhaemocyton virus infections were seen in one banded gecko and in the python, both from Basse- 8-3.Sandfly collection and dissection A total of 1370 sandflies were caught in the three month collection period. The change in the number of sandflies caught in the light traps, each night they were used, is shown in Fig. 125-127 together with the "Hyd.romet" weather records for the period. Low numbers of flies were caught in the traps after rain and during windy nights. Indoor catches with oral aspirators., showed that sandfly activity was greatest between 2230 and 2330 hours. Results from the light traps examined every 3 hours throughout the night supported this observation in that most flies (78% of the catch) were collected between 2100 and 2400h. However 21% of the catch was collected later and routine catches using light traps were made between Z200fr-^and 070Qh. The flies caught belonged to 12 species in the genus Sergentomyia (Table 11 ). The oiled paper traps caught many more flies per night than any other method though all the flies were killed. The best live catches came from the light 158 Table 11. Sandfly species caught Type of trap Aspirator Light Suction. Emer- Bait Oiled Total gence paper (females) Number used 1 5 1 5 1 3 Number of collections 1 29 3 10 10 1 Sergentomyia dubia 28 138 2 3 1 204 376 " squamipleur is 9 104 9 - - 2 124 " ghesqueri 7 97 1 - " - 105 " magna 9 46 - - 1 56 " sp. - 22 - - - . 22 " bedfordi 6 18 - - 13 37 " schwetzi 2 17 - - 8 27 11 inermis - 16 - - 3 19 " buxtoni 1 15 - - 6 22 " adleri - 2 - - - 2 " antennata - 1 - - 5 6 " Clydei - 1 - - - 1 Total females 62 477 11 4 1 242 797 Total males 27 354 . 1 0 0 193 575 1372 Table 12 . Sandfly species taking lizard blood meals Species Number fed Number fed Number Proportion of on P.a^amae- on Schell- offered females offered infected ackia bloodmeals meals which lizard infected fed (%) lizard S.magna ~ 19 9 41 68 schwetzi 11 - 18 61 sp. 8 - 19 42 buxtoni 7 - 12 58 dubia 4 151 3 clydei - 1 - 100 159 Figures 114-123. Agama agama and the haematozoa which infect it. Figure 114. An adult male Agama agama (length 24cm) " 115. Plasmodium agamae trophozoites (x 1400) " 116. P. agamae Immature gametocyte " " 117. P. agamae Mature microgametocyte 11 " 118. P. agamae Mature micro- and macro-gametocytes (x 1400) " 119-121. P. agamae schizonts. " 122-123. Schellackia agamae sporozoites 11 160 O o 161 O o a) CNJ 00 X) CN cfl S E 6 o o 80 e u u o u-i •r4 a 4J M o x o O •h o a) o •u o o OS Or^ . Q) O 60 O o o •u 4-1 J3 o n iE 40 20 0 O O boooo o ooooooo bpQ 2 March April May Day of collection Figure 124 . Sandfly catches from light traps U4 rr In 5' 3 - March Apri May Day of collection Figure 125 . Wind speed records for Basse. 30- "E E 20- CO c 10 "co cr 1 1 - r—i—I t I I i » i i t N ' 1 < I 0< i , i i i i % i i i i i i i i .i i i i i • i » » » i ' ' |h..ri March April May Day of collection Figure 12'6 . Fainfall records for Basse 162 traps. Fewer flies were caught by oral aspirator and only 17 flies were found in the suction, emergence and baited traps combined. Most of the female flies caught from tree holes and the termitaria air vent were S. dubia (84% of the catch) . This fly was also the most common one to be caught in light traps (29%) but S. squamipl.euris (22%) and S. ghesqueri (20%) were only slightly less common in these traps. 21 of the female flies caught were probably of a new species or subspecies. They were % similar to S. clydei but had 9 or 10, not 12, cibarial teeth. No parasites were seen in 276 female flies dissected soon after capture nor in 51 flies fed on A. agama infected with P. agamae and dissected 2 to 10 days later, including those kept below room temperature for 5 days (Tableli! ). Many of the female flies caught were found dead and too dry to dissect. Female S. squamipleuris, S. ghesqueri and S. inermis did not feed on lizards but fed on a frog (Rana sp.) when this was placed in a cage shortly after a lizard was removed. One Sergentomyia sp. had several hundred sporocysts packing its ovaries, 3 days post-feed. Ovarian infection is typical of coccidia in the family Kanyolysidae. No parasitic development was seen in the 10 sandflies fed on a ~lizard with intraerythrocytic coccidian sporozoites. 8-4.Other haematophagous insects offered bloodmeals. Four sandflies, 25 mosquitoes (20 Anopheles gambiae, 4 Mansonia uniformis and 1 Culex decans) and 19 Culicoides sp. were caught in the emergence traps from lizard resting sites. Some of the mosquitoes caught in oral aspirators, light and emergence traps in the Gambia (28 of 30 Mansonia africans, 16 of 67 M. uniformis, 32 or 135 Anopheles gambiae and 45 of 109 Culicoides sp.). took blood when placed with -the A^agama infected. with P.agamae.No parasites were seen in any of these insects when they were dissected one to ten days 163 days post-feed. In England none of the Culicoides nubeculosus kept in a cage with an Agama agama fed on the reptile though many fed readily on man. Attempts were made to stimulate feeding by breathing on the flies, by anaesthetizing the flies with carbon dioxide and then offering the lizard . to them 10 minutes after they recovered, by prewarming the lizard to 37°C, by including a cage containing a mouse in the same plastic bag as the fly cage and by putting the cage in bright and dim light and total darkness. All these attempts failed. No parasites were seen in the smears of acar'ine mites. 8.5.Attempts to observe exflagellation No exflageHating gametocytes were seen in the gut contents of recently fed flies, in wet blood films or in any of the test solutions. However microgametocytes underwent some changes in the arrangement of their pigment granules, 15 to 20 minutes after the infected blood was taken from the lizard. The granules were agitated with a strong Brownian-type motion in the periphery of the cytoplasm. This movement gradually quickened until, at about 30 min after the blood was taken, the granules had aggregat-ed into a single clump near one -pole of the gametocyte. No further development occurred and the parasites remained intracellular. No changes were noted in macrogametocyte morphology. 164 9. DISCUSSION O.l.The lizards and their infections. Only 3 (0.9%) of the 338 Agama agama were infected with P. agamae. Very low parasitaemias may have been missed because of the short time spent scanning blood smears (a total of 15 min for each lizard). Both lizard infection rates and parasitaemias were disappointingly low in comparison with other reports. During the period of the present investigation (March to June) P. agamae was not as common as Garnham (1966) found it in the Gambia. About half the Agama agama examined in the Congo in 1931 were infected with Plasmodium spp. (Schwetz 1931). Prevalences in Liberia have varied from 14 to 50% (Theiler, 1930; Bray, 1959; J.R. Baker, pers. comm.) and in Kenya Ball (1967) found nearly 17% of lizards with P. agamae. It is not uncommon for malaria infection rates to surpass 30% in grassland and forest lizard communities (Ayala, 1975). The parasitaemias seen in lizards are generally higher than the 0.;25% observed in the present study. Garnham (1966) and Adler (1924) saw "heavy" infect ions of Plasmodium spp. in A. agama from West Africa yet the heaviest infection observed by Bray (1959) in Liberia was only 0.4%. Most reported parasitaemias of malaria parasites of other lizards are between 1 and 10% (Telford 1972, 1978; Ayala, 1977) with some Neotropical parasites such as P. floridense and P. mexicanum reaching 60 to 80% (Ayala loc. cit.). According to Garnham (1966) lizards infected with malaria parasites are often "very localised, being frequent in one community and entirely absent in another, only a few miles distant " It is unlikely that the localisation was the reason for the low prevalence found in the present investigation since collections came from a wide area. 16 5 A possible reason may be that acute infections are more common in the wet season. The only author to give figures for P. agamae parasitaemias and also record the season, Bray (1959), found that the parasitaemias were low, not exceeding 0.4%fin lizards caught, as those in the present study, in the dry season. The coccidian parasites were probably sporozoites of Schellackia agamae (Laveran and Petitt, 1909). This parasite was originally found in Senegal (Laveran and Petitt, loc. cit.) and was described in the Central African Republic in 1977 (Rogier, 1977). In the Gambian collections I found that the coccidia were more common than the Plasmodium and more widely distributed. It was surprising that nb intracellular tissue parasites were present in the impression smears of the intestinal epithelium, liver or lungs of infected lizards, since Rogier (loc. cit.) found many oocysts and sporozoites in these sites. It is likely that the phases of schizogony, gametogony and sporogony had been completed in the 8 lizards before they were caught and that the intraerythrocytic sporozoites are long-lived. Sporozoites of Schellakia spp. are thought to enter into long term "diapause" and remain in cells of the reticulo-endothelial system of the viscera (Lainson et^ al, 1976). These may have been present in the tissue smears but confused with the haematozoic sporozoites i 9.2.The collection and dissection of sandflies and attempts to infect them. After consideration of the synonymy proposed by Abonnenc (1972), the number of sandfly species reported from the Gambia is reduced to 13 (Bertram et al, 1958; Lewis and Murphy, 1965; Snow, 1979). This number is composed of 2 Phlebotomus species (P. rodhaini and P. duboscqi) and 11 Sergentomyia species. (S. antennata, S. bedfordi, S.buxtohi. J; S. clydei,*S. collarti, S. dubia, S. dureni, Si.nermis: i S. magna, S. schwetzi and S. squamipleuris). 9 of these Sergentomyia species were caught in the present study (No Phlebotomus spp, 166 S. collar.ti or S. dureni were collected). Three other species S. adleri, S. ghesqueri and S. sp. are new records for the Gambia. The Gambian sandfly fauna has not been well studied. One unidentified sandfly was caught at Bathurst (= Banjul) in 1902 (Simpson, 1911), 4 were caught in 1952 (Bertram et al, 1952), 1116 flies were caught during a mosquito survey between 1957 and 19£$ (Lewis and Murphy, 1965) and Snow (1979) found 3 P. duboscqi in Bansang in 1977. In 1981 the first Gambian sandfly survey was begun, after two cases of human leishmanias were discovered in the Western division (B.M. Greenwood, pers. comm.). The results of this survey (by Drs. J.H. Bryan and P. Desjeux) have yet to be published but will greatly increase the data on Gambian sandflies. S. adleri and S. ghesqueri have been reported from West Africa, both north and south of the Gambia (see Abonnenc, 1972) and it was therefore no surprise to find them in the present collections*Phlebotomus spp. are rare in the Gambia and only a few species are known from West Africa as a whole. It is thought they may have been restricted in their movement westwards by a climatio barrier which no longer exists. (Moreau, 1963; Lewis and Murphy, 1965). 9.3.The Capture methods Oiled paper traps were the most efficient in terms of the number of sandflies caught per night. However only 8 of the 12 species caught by light traps were found on the oiled paper and only two of these, S. dubia (84% of catch) and S. bedfordi (5%) in any numbers. Lewis and Murphy (1965) found a similar predominance of S. dubia in tree holes but they also found more S. buxtoni and S. antennata than in the present study. Though the traps gave a good indication of the flies which were nesting in tree holes and termitaria, they were of no use for transmission experiments. All flies caught were killed. 167 Most live flies came from light traps which also had the advantage of attracting all the species caught in the survey. As in oil traps S. dubia was the most common fly in light traps. S. squamipleuris, S. ghesqueri and S.inermis were attracted to light traps in large numbers but only a few of each of these flies were found on oiled paper. These three species belong to the subgenus Grassomyia and are only infrequent occupants of tree holes and other crevices. They rest on vegetation, particularly in areas of permanent moisture (Abonnenc, 1972). Flies caught in the light traps probably came frcm the nearby ricefields or river bank. Only 25 S. schwetzi and 56 S. magna were caught in the survey although these two species were the most abundant in collections made by Lewis and Murphy (1965). This difference is probably due to the season. S. schwetzi and S. magna appear to be much more common in the wet season (Lewis and Murphy, loc. cit.). Sandfly activity appears to be restricted by high winds and rain (Ashford, 1974) and light trap catches were low during and after such weather. Suction, emergence and baited traps only caught 17 flies in total. The low numbers of flies caught in suction traps indicates that most, if not all, of the sandfly species collected in light traps were phototropic and not merely flying in the vicinity of the traps. Catches in emergence traps placed in tree holes were disappointingly low in comparison to catches on oiled paper in similar habitats. Chaniotis and Anderson (1968) achieved much better results using similar traps in ground squirrel burrows in California. Sandflies entering the traps may have managed to return through the cardboard cone. Other haematophagous insects were caught but not in great numbers. A single female S. dubia was caught in the lizard-baited trap. Similar low collections were experienced by J.R. Baker (pers. comm.) using A. agama- baited traps in Liberia, by Chaniotis and Anderson (1968) and Christen and 168 Herrer (1980) using lizard- and snake-baited traps in California and Panama and by Ball and Pringle (1965) using chamaeleon-baited traps in Tanzania. Though haematophagous Diptera can detect mammals and birds from several tens of metres distance (Gillies and Wilkes, 1969, 1970; Snow and Boreham, 1973) lizards and other polkilotherms may not emit sufficient odour or carbon dioxide for the insects to detect from long range. The insects may only locate lizards from close range, possibly while insects and resting lizards share the same hole or crevice. 9-4.Sandflies feeding on lizards The number of females of each species feeding on lizards in the laboratory was not directly related to their abundance in the collections. Only 4 (2.6%) S. dubia, the most common species, took a bloodmeal from lizards. S. dubia will feed on geckos in the laboratory (Abonnenc et al, 1957). However geckos are soft skinned, unlike agamid lizards and the hard skin, with rows of tubercles and large scales, of A. agama may prevent S. dubia feeding, although few were seen even probing the lizard skin. None of the second and third most abundant species, S. squamipleuris and Sy~ghesqueri, nor S.inermis fed on the lizards. These species are thought to feed on amphibians and some snakes (Abonnenc, 1972). An amphibian source of-b-lo'dff would accord with the frequent discovery of these species near water. -'^The fact that flies of all three species fed on a frog (Rana sp.) when this was placed in a cage shortly after a lizard was removed adds weight to the belief that these species are amphibian feeders. None of the 24 live-caught female S. bedfordi fed from lizards, although the species feeds readily on skinks in Ethiopia (Ashford et^ al, 1973). Many of the flies were gravid and were not expected to feed. 169 Some of the S. magna, S. schwetzi, S. buxtoni and S. sp. fed on the lizards, S. magna being the most frequent feeder. Too few S. adleri or S. antennata were collected to deduce whether or not they are sauriophilic, although S. antennata has been reported to feed on geckos in captivity (Abonnenc et_ al, 1957). (S. schwetzi, S. adleri and S. clydei are known also to feed on mammals including man in some parts of Africa (see Abonnenc, 1972). There are no % reports of Sergentomyia spp. biting man in the Gambia. The 3 cases of human leishmaniasis (Walters, 1949, B.M. Greenwood pers. comm.) and one of canine leishmaniasis (P.J. Desjeux, pers. comm.) are thought to have been transmitted by P. duboscqi.) 9.5.The P. agamae vector potential of Gambian insects. There was no development of P. agamae in any of the sandflies studies nor in the mosquitoes, A. gambiae, M. uniformis, M. africanus nor in Gambian Culicoides sp. fed on infected lizards. Three possible reasons for this lack of success are suggested: the natural vector may not have been collected; it may have been among those which were given the opportunity to feed on lizards, but refused to do so under laboratory conditions; it may have been one of those which took a bloodmeal but the gametocytes may have been immature or senile at the time. 9.5JL. Sandflies - Phlebotominae. Although 4 species of sandfly, reported from the Gambia, were not collected they are unlikely to be the vectors of the malaria parasite. Two species are mammiophilic (P. rodhaini and P. dubosqi) and two, though sauriophilic (Wanson, 1942), only occur in humid conditions near the coast (S. collarti and S. dureni; Lewis and Murphy, 1965) (where the malaria parasite has not been reported). Of the 6 species caught which did not feed 170 on lizards 3 probably feed only on amphibians, 2 were caught in very low numbers (S. adleri and S. antennata) and most S. bedfordi were gravid when collected. Most of the Gambian sandfly species - which are possible vectors of P. agamae, were therefore fed on infected lizards without developing infections. 9-5.2.Culicidae There are 61 species of mosquito known from the Gambia (T.J. Wilkes, pers. comm.) but the biology of only a few species is known. Mosquitoes may yet prove to be the vectors of Old World saurian malaria parasites. Previous attempts to infect mosquitoes have mostly involved species which are normally anthropophilic (in particular Culex pipiens, Culex fatigans and Aedes aegypti). There are no reports of Gambian mosquitoes which feed entirely on lizards, like some neotropical Deinocerites spp. (Tempelis and Galindo, 1970). It would seem likely that these exist, considering the abundance of lizards, and they probably belong to the genus Culex - other genera are known or thought to feed on animals other than reptiles. Most of the Gambian Anopheles, Aedes and Mansonia species enter mammal- i baited^raps and many bite man although these mammophilic insects will often also luod on lizards in the laboratory (Table 8 and present study) . McClell- and .and Weitz (1963) recorded a very large proportion of reptile feeds from a wild population of A. aegypti in Uganda. Snow thinks that the dark "bush" form of Gambian A. aegypti (mostly the ssp. formosus), which rarely attacks man, possibly feeds on lizards or other poikilotherms (W.F. Snow, pers. comm.). Only microgametes and/or ookinetes of P. agamae were seen by Bray (1959) and J.R. Baker (pers. comm.) in A. aegypti which had fed on infected lizards but these and other experiments probably involved the pale 171 "domestic" form of the mosquito. The possibility that further sporogenesis occurs in the dark form should be investigated. Unfortunately A. aegypti are only common in the Gambia during the wet season and none were seen in the present study. The use of precipitin tests has shown that most Ficalbia and Uranotaenia spp. feed mainly on amphibia although one U. mashonaensis appeared to have fed on a reptile (Boreham et_ al^ 1975) and some Uranotaenia appeared to be % attracted to a lizard-baited trap (Corbet and Ssenkubuge, 1962). Similarly there is one report of a reptile feed from C. univittatus, although most Culex spp. and also Coquillettida spp, appear to be ornithophilic (Gillett, 1972; Gillies and Wilkes; 1974-, Snow and Boreham, 1973). 9.5.3 .Gloss inidae Reptiles form an important and preferred food source for Tiverine Glossina (Molyneux, 1980), particularly Glossina palpalis (Glasgow, 1963), the most common tsetse fly in the Gambia (Bertram et al, 1958). A Glossina sp. vector of P. agamae would explain the fall in infection rates from "common" (Garnham, 1966) to the 0.9% seen in the present study. The size of Glossina populations in the Gambia has declined dramatically in the past few years as a result of the felling of much of the gallery forest and the falling water table (Mann, 1975). A tsetse vector would also explain the absence of P. agamae in Egypt and Yemen (Garnham 1954) since tsetses are unknown in these areas. Unfortunately the absence of reports of sporozoites or oocysts in any of the many thousands of Glossina spp. dissected in the course of surveys of human trypanosomiasis makes it unlikely they are the vectors (Hutchinson, 1953; Mulligan, 1970). 172 9.5.4.Ceratopogonidae Too little is known about sauriophilic Culicoides to draw any conclusions about their potential as malaria vectors. Most Culicoides spp. are thought to feed on mammals or birds (Downes, 1958). Although anthropophilic Culicoides spp. are known in the Gambia (Bertram et^ al, 1958) none of the many Culicoides at Basse were biting man and most fed readily on lizards in the laboratory, suggesting that they may have been naturally sauriophilic. J.R. Baker (pers. comm.) also fed Culicoides spp. on Plasmodium-infected A. agama in Liberia and observed ookinetes in one fly. In Brazil Lainson et^ al (1974) found lizards infected with malaria in areas where sandflies did not occur and "strongly" suspected the ubiquitous Culicoides as the vector. It is possible that infections have been missed, especially if oocysts develop, like those of Hepatocystis, at some distance from the host gut (Garnham et_ al, 1961). However J.R. Baker (pers. comm.) sectioned Culicoides which had fed on infected lizards to check for such oocysts and failed to find .them. 9.5.5. Tabaniidae ATew species of Tabanus attack lizards (Downes, 1958) but these and other Tabaniidae probably occur in insufficient numbers in the Gambia (Bertram et al, 1958) to be possible vectors of saurian malaria. * v'y« 9.5.6.Simuliidae There are no records of simulids feeding on reptiles and amphibians (Downes, 1958) and these flies are rare in the Gambia, particularly in the west where the virtual absence of perennial fresh running water prevents them breeding (Bertram et al, 1958; McGregor and Smith, 1952). They are not likely to be vectors. 173 9.5-7. Ac ar ina Most lizards are infested with acarine mites but none have been found to harbour developmental stages of malaria parasites. Pelaez et^ al (1948) examined mites (Hirstiella sp.) feeding on lizards infected with P. mexicanum in Mexico. They found that the mites retained malaria parasites in their midguts for up to 3 months but no sporogenesis was observed. J.R. Baker (pers. comm.) saw no P. agamae in mites, which he removed from P. agamae- infected lizards and thought that the mites probably . , feed on tissue fluids other than blood. No parasites were seen in acarine mites fed on Gambian lizards. 9.5.8.Summary 1. The failure of the gametocytes to exflagellate and develop further in any of the insects which ingested them may be due to their immaturity or senility. 2. If however the gametocytes were infective sandflies are unlikely to be the vectors of saurian malaria in the Gambia. 3. Other insects, probably mosquitoes but possibly Culicoides spp., are potential vectors. If possible, lizards with much higher parasitaemias and more "active" infections should be used in future attempts to find the vector. Experiments should be conducted at different times of the year. 9.6.Attempts to stimulate exflagellation Attempts to stimulate exflagellation (and therefore check for gametocyte viability) failed, although morphological changes in microgametocytes, similar to those observed in P. tropiduri prior to exflagellation (Scorza, 1970) were observed. Only a few Plasmodium species from lizards have been seen to exflagellate, (Ayala, 1977). Wenyon (1915) could not find microgametes in wet blood film from a living lizard but both he and Pelaez (1967) saw 174 exflageHating parasites in blood taken from lizards several hours post-mortem. Autolytic processes in the dead lizards may have stimulated the change or perhaps even a fall in pH (in contrast to avian parasites which are stimulated by an increase in pH; Nijhout and Carter, 1978). Aragoa and Neira (1909) observed an intensification of the movement of pigment granules of P. diploglossi microgametocytes followed by a slowing down; a similar observation was made in the present study of P. agamae. Thompson and Huff (1944) saw exflagellation of P. mexicanum in vitro but gave no details. Scorza (1970) suggested that in general saurian malaria parasites take longer to exflagellate than those of mammals and birds. He saw that P. tropiduri microgametocytes took 40-70 minutes to exflagellate. In the present study gametocytes were observed for up to 6 hours without exflagellation being seen. Scorza (loc. cit.) also noted that in general exflagellation only takes place in gametocytes from infections with high parasitaemias (over 150 gametocytes/10^ erythrocytes) and suggested that lower infections were post-crisis, with non-viable gametocytes. In lizards, as in other animals, crises follow the peak of the growth curve in most infections which reach high parasitaemias. The crises are probably a result of the production of circulating anti-Plasmodium antibodies by the hosts which lead to parasite destruction (Thompson, 1944; Scorza, 1970; Ayala, 1977). It is possible that all three P. agamae parasitaemias were low and declining, having passed peak levels, rather than in ascendancy. Although a predominance of gametocytes over asexual stages, as seen in the Gambian lizards, typifies chronic infections in many malaria parasites (Ayala, 1977) it is also commonly found in acute infections of P. agamae (Garnham, 1966) e.g. in infections observed by Bray (1959), where gametocytes formed approximately 60% of the population gametocyte maturity was demonstrated by observation of microgametes. All the Gambian malaria parasites appeared normal 175 and none were vacuolated or pale-staining like the typical non-viable "crisis" forms described by Ayala (1977)* The author is unable to explain the total lack of success in attempts to stimulate exflagellation. Gametocytes of P. agamae and Haemoproteus sp. are very similar (Bray, 1959) and it is possible that the intracellular schizonts in the present study were of P. agamae while the gametocytes were of a Haemoproteus sp. but the similarity of all 3 infections makes this very unlikely. 9.7,Attempts to infect sandflies with the coccidian parasite No parasites were seen in the 10 sandflies fed on a lizard with 4 56 coccidian parasites/10 erythrocytes. If the parasite is a Schellackia spp. no development, other than entry of the sporozoites into midgut epithelial cells, would be expected (Lainson £l, 1976). No intracellular sporozoites were noticed in the gut smears but these would be few in number and were possibly missed. The oocysts seen in one fly fed on a lizard infected with P. agamae were not the same parasite as seen in lizard blood smears and probably came from some other animal on which the fly had fed. 176 APPENDICES 177 Appendix A Morphometric data on saurian trypanosomes Morphological parameter* Trypanosome BL F NA KN PN PK W ' N T.betschi X 26 (10)** - - — - - min 20 (3) ------max 30 (26) ------T.boueti X 40 0 (12) (11) (8) (7.5) (28) (22) min - - - - - — • - — max ------T.chamaeleonis X 40 ------min - - - - - — — — max ------— T.domerguei X 23.3 6.7 (10.6) (6.6) (12.2) 5.6 17.4 6.8 min 19.6 4.9 (8.7) (4.7) - 2.3 14.3 5.9 • max 29.2 11.0 (14.7) (9.7) - 10.0 21.6 14.6 T.egernia X ------— min 22 11 - - - 5 4 - max 28 12 - - - 8 5 - T.gallayi X (50) 9 32 1.8 (18) 16.2 5.4 - * min - - - - — — — max - - - - - — - - T.garnhami X 24.8 15.2 10.6 5.9 3.2 - - 4.1 min 18 11 6 0.5 1 - - 2 max 39 21 17 13 6 - - 8 T.gerrhonoti X 56.8 3.0 34.1 - 22.2 (21) 8.4 3.5 min - . 0.5 30 1 16 - 4 - max - 6.0 41 2 29 - 16 - T.gonatodi X 39.0 9.0 - 8.7 - 13.1 22.5 5.7 min 34 5 - 4 - 3 17 5 max 47 13 0 21 - 21 28 7 T.grayi X - - - (5) (20) (15) - - min 35 5 (15) - - - 6 - max 40 6 (20) - - - 8 - T.leschenaultii X - - (29) (10) (28) (18) - - min 56 17 ------ max 60 22 ------T.martini X (53) 20 (36) (1) (17) 16 - 2 min ------7 - max - - - - - 8 - T. mochli X - - 38 - 3.8 1.5 - 1.6 min 33 6 - - - - 9 - max 36 8 - - - - 11 - T. ocumarensis X 42.5 20.0 - - - 2.4 - - min - 17 ------ max - 24 ------T.pertenue X - - - - -' - - - min 30 15 - - - - - — max 35 20 ------T.petteri(type 1) X 43 25 16 (18) - 15 8 - min - - - — — — — — max ------— (type 2) X 50 0 16 (18) - 15 10 - min - - - - - — — — max ------— T.phlebotomi X 29.4 0.0 (21) (18) (29) (11) 14.2 - min 25 - - - - - 12 - max 41 - - - - 18 - T.phylluri X ------min 36 2 20 0 - 14 . 7 - max 48 6 25 1 - 21 15 - 178 T.platydactyli X (44) - (29) (3) (14.5) (10) - - min - 12 - - - 8 - max - 19 - - - - 14 - T.plicae X 40.5 16.4 27.1 9.3 11.9 2.4 15.9 14.4 min - 12.0 19.0 6.0 8.0 0.5 10.0 12 max - 28.0 35.0 11.5 15.0 4.5 21.0 17 T.rudolphi X 20 "short" - "short" - - 15 5 min ------max ------T.ryukyuense X 37.2 11.9 18.4 4.4 18.9 17.6 10.6 2.8 min 3.1 8 14 2 16 14 8 2 max 4.5 22 24 6 22 21 14 4 T.scelopori X 54.7 13 29.7 (8) 24.9 (23) 5 2.8 min 42 7 25 - 16 - 3 1.8 max 67 17 43 - 32 - 10 4.0 T.serveti X 30.8 8.4 12.7 (5.0) 18.1 (6.1) 20.1 9.9 min 27 6 7 (3.8) 15 (2.3) 15 8 max 35 11 15 (6.5) 22 (8.5) 25 11.5 T.superciliosae X 96.2 16.1 (58.0) (11.2) (42.2) - 14.2 16.8 min - 5 - - - - 9 9 max - 25 - - - - 19 24 T.thecadactyli X 21.9 12.4 14.4 4.2 (6.2) 2 - - min 19 9 11 3.5 - - - - max 24 20 17 5 - - - - T.therezieni X 32 • 17 (12) (10) (17) 7 5.5 - min 28 15 (11) (8) - 5 4.5 - max 33 21 (13) (11) - 11 6.5 - T.torrealbai X 32.7 10.4 - 12.5 - 8.1 18.7 5.8 min 28 1.5 - 9 - 6 13 5 max 38 24 - 16 - 13 23 7 Trypanosoma sp. X (56) 22 (28) (15) (28) 13 8 4 (of Acanthosaura) min ------max - — - - - - — - * The parameters are based on those of Hoare (1970) BL = body length PK = distance from posterior end to kinetoplast PN = " " posterior end to middle of nucleus KN = it ii kinetoplast to middle of nucleus NA = " " anterior end to middle of nucleus F = length of free flagellum. ** Parameters in parentheses represent approximate values based on the available data (e.g. PN ^ PK + KN)or on figures given in the original descriptions. All data comes from the original descriptions listed in Part A pp. 179 Appendix B Preparation of geckonid kidney cell cultures The kidneys were aseptically removed from a female Tokay gecko (Gekko gecko) obtained from commercial suppliers (Biopet, Richmond). (The gecko had been exsanguinated for production of anti-serum). The kidneys were transferred to a sterile syracuse watchglass and the major veins and ureters removed. The remaining tissue was macerated, and washed several times in Minimum Essential Medium with Eagle's salts (EMEM, Flow, Irvine) and 300 i.u. Penicillin and Streptomycin (Flow). The tissue was then transferred to a sterile Ehrlenmeyer flask containing 2cm Trypsin/EDTA (Flow) and a magnetic stirrer bar and incubated at 37°C for 3 min with continuous stirring. The supernatant was discarded and replaced with fresh Trypsin/EDTA solution. The supernatants from 2 further 5 min incubations were collected, pooled and centrifuged at 400g for 5min. The cell pellet was resuspended in cold (4°C) 3 5cm EMEM with 10% FCSTcentrifuged again and resuspended in the same medium. 3 The cell suspension was decanted into a 50cm Falcon flask (Flow) and incubated at 25°C. The overlying medium was changed after 5 days. Twenty days later the cells were resuspended by treatment with Trypsin/EDTA solution for 90 sees at 37°C, washed once in the culture medium and split 1:3 into 3 flasks. Lizard haemoflagellates were mixed with cells from one flask 15 days later. 180 Appendix C Phosphate buffered saline, pH^ 2 NaCl 8.OOg K.HPO. 1.21 KHoP0. 0.34 2 4 3 H20 1000cm Appendix D Food for sandfly larvae First stage larvae: 30g dried rabbit faeces lOOg dried washed sand. Second, third and fourth stage larvae: 30g dried rabbit faeces lOOg dried washed sand. 5g yeast tablets. Faeces, sand and where appropriate yeast tablets are ground together in a mortar and pestle, shaken through a sieve (mesh size 1mm) and used to 3 fill 25cm McCartney bottles. The food is sterilised by autoclavmg. A flamed spatula is used to dispense the food which is given to larvae about twice weekly. Appendix E Nesbitt's solution 2,2,2-Trichloro-1,1-ethanediol (Chloral hydrate) 24g HC1 (36% w/w) 15cm3 3 H20 150cm 181 REFERENCES ABE, Y. 1980. On the encystment of Leptomonas sp. (Kinetoplastida: Trypanosomatidae) a parasite of the Silkworm, Bombyx mori Linnaeus. J. Protozool. 27 (4) 372-4. ABONNENC, E. 1972. "Les phlebotomes de la region Ethiopienne (Diptera, Psychodidae)" ORSTOM, Paris. ABONNENC, E., LARIVIERE, M. and YVINEC, M.L. 1957. Observations sur la . biologie de quelques Phlebotomes de la region Ethiopienne en milieu experimental. Annls. Paras it. hum, comp. 32, 73-84 . AOLER, S. 1924. A note on Plasmodium agamae. Ann. trop .Med .Paras'it. 18 131-3. ADLER, S. 1933. Mode de transmission des Protozoaires sangiucoles et particulierement des leishmanioses. Bull.Soc.Path.exot. 26 207. ADLER, S. 1964. Leishmania. Adv.Parasitol. 2_, 35-96. ADLER, S. and THEODOR, 0. 1929. Observations on Leishmania ceramodactyli n.sp. Trans.R.Soc.trop.Med.Hyg. 22 (4) 343-56. ADLER, S. and THEODOR, 0. 1930. The behaviour of insect flagellates and leishmanias in Phlebotomus papatasii. Ann.trop.Med.Parasit. 24, 193-6. ADLER, S. and THEODOR, 0. 1931. Investigations on Mediterranean kala azar I-V. Proc.R.Soc. 108B 447-502. ADLER, S. and THEODOR, 0. 1935. Investigations on Mediterranean kala azar X. A note on Trypanosoma platydactyli and Leishmania tarentolae. Proc.R.Soc. 116B 543-4. AKIYAMA ', H.J. and TAYLOR, J.C. 1970. The effect of macrophage engulfment and temperature on the transformation process of Leishmania donovani. Am.J.Trop.Med.Hyg. 19_, 747-54. ALEXANDER, J. and VICKERMAN, K. 1975. Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana- infected macrophages. J.Protozool. 22, 502-8. ANDERSON, J.R. and AYALA, S.C. 1968. Growth pattern of Leishmania in Phlebotomine sandflies. Science 165, 1380-1. ANDRUSHKO, A.M. and MARKOV, G.S. 1955. Movye nakhodki leishmanii u presmykajushchiksya Sredrei Azii. Vestn.Leningr.Univ. _1, 55-59. ANEZ, N. 1981. Trypanosomatidae of Venezuela with special reference to Trypanosoma rangeli and Leishmania garnhami. Doctoral Thesis, University of London. ARAGOA, H. de B. and NEIVA, A. 1909. A contribution to the study of the intraglobular parasites of the lizards. Two new species of Plasmodium, PI. diploglossi n.sp. and PI. tropiduri. n.sp. Mems.Inst.Oswaldo Cruz. 1, 44-50. 182 ARNOLD, E.N., BURTON, J.A. and OVENDEN, D.W. 1978. "A field guide to the reptiles and amphibians of Britain and Europe". Collins, London. ASHFORD, R.W. 1974. Sandflies (Diptera: Phlebotomidae) from Ethiopia: Taxonomic and biological notes. J.med.Ent. 11, 605-16. ASHFORD, R.W., BRAY, M.A. and FOSTER, W.A. 1973. Observations on Trypanosoma boueti parasitic in the skink Mabuya striata and the sandfly Sergentomyia befordi in Ethiopia. J.Zool.Res. 171, 285-92. ATKINSON, A.W., JOHN, P.C.L. and GUNNING, B.E.S. 1974. The growth and • division of the single mitochondrion and other organelles during the cell cycle of Chlorella, studied by quantitative stereology and three-dimensional reconstruction. Protoplasma 81, 77-109. AYALA, S.C. 1970. Two new trypanosomes from California Toads and Lizards. J.Protozoal. 17 (3) 370-3. AYALA, S.C. 1971a. Trypanosomes in wild California Sandflies, and extrinsic stages of Trypanosoma bufophlebotomi. J.Protozool. 18 (3) 433-6. AYALA, S.C. 1971b. Sporogony and experimental transmission of Plasmodium mexicanum. J.Parasit. 57 (3) 598-602. AYALA, S.C. 1975. Malaria in reptile populations. Herpetol. Rev. 6_, 10. AYALA, S.C. 1977. Plasmodia of reptiles. In "Parasitic Protozoa" Vol. 3 (J.P. Kreier, ed.) Academic, New York pp 267-309. AYALA, S.C. 1978. Checklist, Host Index, and annotated Bibliography of Plasmodium from Reptiles. J.Protozool. 25 (1) 87-100. AYALA, S.C. and LEE, D. 1970. Saurian Malaria: Development of sporozoites in Two species of Phlebotomine Sandflies. Science, N.Y. 167, 891-2. AYALA, S.C. and McKAY, J.G. 1971. Trypanosoma gerrhonoti n.sp. and extrinsic development of lizard trypanosomes in California sandflies. J.Protozool. 18 (3) 430-3. BAKER, J.R. 1966. Studies on Trypanosoma avium. IV. The development of infective metacyclic trypanosomes in vitro. Parasitology. 56, 15-9. BAKER, J.R. 1973. "Parasitic Protozoa" Ed. 2. Hutchison University Library, London. BAKER, J.R. and PRICE, J. 1973. Growth in vitro of Trypanosoma cruzi as amastigotes at temperatures below 37 C. Int. J .Parasitol. 3^, 549-51. BAKER, J.R., GLAUERT, A.M. and SELDEN, L.F. 1980. Interactions between Trypanosoma (Schizotrypanum) dionisii and "buffalo" lung cells in vitro. J.Protozool. 27 (3) 244. BALL, G.H. 1967. Some blood sporozoans from East African reptiles. J.Protozool. 14, 198-210. 183 BALL, G.H. and PRINGLE, G. 1965. Plasmodium fischeri n.sp. from Chamaeleo fischeri. J.Protozool. 12 (4) 479-82. BARDSLEY, J.E. and HARMSEN, R. 1973. The Trypanosomes of Anura. Adv.Parasitol. 11, 1-73. BARROW, J.H. 1953. The biology of Trypanosoma diemyctyli (Tobey). I. Trypanosoma diemyctyli in the leech, Batrachobdella picta (Verrill). Trans.Am.microsc.Soc. 72, 197-216. BAYON, H. 1914. Herpetomonidae found in Scatophaga hottentota and • Chamaeleon pumilus. Trans.R.Soc.S.Afr. 5, 61-3. BAYON, H.P. 1926. Herpetomonas in the cloaca of African Chamaeleons. Parasitology. 18, 361-2. BECKER, C.D. 1977. Flagellate parasites of fish. In "Parasitic Protozoa" Vol. 1. (J.P. Kreier, ed.). Academic Press, New York, pp 357-416. BELOVA, E.M. 1971. Reptiles and their importance in the epidemiology of leishmaniasis. Bull.Wld.Hlth.Org. 44, 553-60. BENNETT, G.F. 1961. On the specificity and transmission of some avian trypanosomes. Can.J.Zool. 39, 17-33. BENNETT, G.F. 1970. Development of trypanosomes of the T. avium complex in the invertebrate host. Can.J.Zool. 48, 945-57. BERGHE, L.V.d., CHARDOME, M. and PEEL, E. 1964. Trypanosoma mochli, a trypanosome from an African lizard. Parasitology. 54, 451-2. BERTRAM, D.S., McGREGOR, I.A. and McFADZEAN, J.A. 1958. Some Diptera, other than mosquitoes, from the colony and protectorate of the Gambia. Trans.R.Soc.trop.Med.Hyg. 52, 217-22. BOREHAM, P.F.L., CHANDLER, J.A. and HIGHTON, R.B. 1975. Studies on the feeding patterns of mosquitoes of the genera Ficalbia, Mimomyia and Uranotaenia in the Kisumu area of Kenya. Bull.ent.Res. 65, 69-74. BOUET, G. 1909. Sur quelques trypanosomes de vertebres a sangfroid de l'Afrique Occidentale Francaise. C.r.Seanc.Soc.Biol. 66, 609-11. BRACK, C. 1968. Elektronmikroskopische Untersuchungen zum Lebenzyklus von Trypanosoma cruzi. Acta trop. 25 , 289-356. BRAY, R.S. 1959. On the parasitic protozoa of Liberia. II. The Malaria parasites of Agamid Lizards. J.Protozool. 6_ (1) 13-8. BRAY, R.S. 1964. A check-list of the parasitic protozoa of West Africa with some notes on classification. Bull.Inst.fr.Afr.noire. 26 (A) 238-315. BRAY, R.S. 1977. In the discussion of a paper entitled "Les leishmanioses au Senegal etude epid^miologique et ecologique" by P. Ranque. In "Ecologie des leishmanioses," Editions du CNRS. Paris, pp 226-32. 184 BRAY, R.S. and GARNHAM, P.C.C. 1962. The Giemsa-Colophonium method for staining protozoa in tissue sections. Indian J.Malar. 16, 153-5. BROOKER, B.E. 1970. Desmosomes and hemidesmosomes in the flagellate Crithidia fasciculata. Z.Zellforsch.mikrosk.Anat. 116, 532-63. BROOKER, B.E. 1971. Flagellar attachment and detachment of Crithidia fasciculata to the gut wall of Anopheles gambiae. Protoplasma 73, 191-202. BRUCE, D., HAMERTON, A.E., BATEMAN,. H.R., MACKIE, F.P. and LADY BRUCE. 1911. A trypanosome found in the blood of a crocodile. Rep.sleep.Sickn.Comm. . R.Soc. 11, 184. BRUMPT, E. 1909. Inoculation et culture du Trypanosoma vickensae Brumpt. Culture et essai d'inoculation du Trypanosoma minasense Chagas. Bull.Soc.Path.exot. 2, 395-7. BRUN, R. 1974. Ultrastruktur und Zyklus von Herpetomonas muscarum, "Herpetomonas mirablis" und Crithidia luciliae in Chrysomyia chloropyga. Acta Trop. 31 (3) 219-90. BRUN, R. and JENNI, L. 1977. A new semi-defined medium for Trypanosoma brucei sspp. Acta Trop. 34, 21-23. BRUNO, S. 1980. Considerazioni tassonomiche e biogeografiche sui "Gekkonidae" italiani. Atti Mus.civ.Stor.nat.Trieste 32, 111-34. BRYGOO, E.R. 1962. Un nouveau Plasmodium de cameleon Haemamoeba robinsoni n.sp. Archs.Inst.Pasteur Madagascar 30 (2) 161-9. BRYGOO, E.R. 1963. Contribution a la connaissance de la Parasitologie des Cameleons malgaches (2 partie). Annls.Parasit.hum.comp. 38 (4) 525-739. BRYGOO, E.R. 1965. Hematozoaires de reptiles malgaches III. Deux Trypanosomes nouveaux: Trypanosoma haranti n.sp., parasite d'Ophidien et Trypanosoma domerguei n.sp. parasite d'iguane. Archs.Inst.Pasteur Madagascar. 34_, 47-54. BRYGOO, E.R. 1966a. Hematozoaires•de reptiles Malgaches. V. Notes sur les h£moparasites des Gerrhosaurides de Madagascar avec description d'un trypanosome nouveau: Trypanosoma betschi n.sp. parasite de Zonosaurus. Archs.Inst.Pasteur Madagascar. 35, 165-70. BRYGOO, E.R. 1966b. Hematozoaires de reptiles Malgaches. VI. Trypanosoma petteri n.sp. parasite de Phelsuma. Liste des Trypanosomes de Reptiles. Archs.Inst.Pasteur Madagascar. 35, 171-84. BUTTNER, A. and BOURCART, N. 1955. Observations sur le cycle evolutif de Trypanosoma inopiriatum Sergent 1904. C.r .Seanc .Soc.Biol. 149 , 1146-52. CARINI, A. 1910. Stades endoglobulaires de trypanosomes. Annls.Inst. Pasteur, Paris. 24, 143-51. CARINI, A. and RUDOLPH, M. 1912. Sur quelques hematozoaires de lezards au Brlsil. Bull.Soc.Path.exot. 5, 592-5. 185 CARPANO, M. 1932. Localisations du Trypanosoma theileri dans les organes internes des bovins. Son cycle evolutif. Annls.Parasit.hum.comp. 10, 305. CARTER, R. and BEACH, R.F. 1977. Gametogenesis in culture by gametocytes of P. falciparum. Nature, Lond. 270, 240-1. CARTER, R. and NIJHOUT, M.M. 1977. Control of gamete formation (exflagellation) in malaria parasites. Science, N.Y. 195, 407-9. CASTELLANI, 0., RIBEIRO, L.V. and FERNANDES, J.F. 1967. Differentiation of . Trypanosoma cruzi in culture. J.Protozool. 14, 447-51. CASTELLANOS, G.B., ANGLUSTER, J. and DE SOUZA, W. 1981. Induction of differentiation in Herpetomonas samuelpessoai by dimethylsulfoxide. Acta trop. 38, 29-37. CATOUILLARD, G. 1909. Sur un trypanosome du Gecko commun de Tunisie (Platydactylus muralis) C.r.Seanc.Soc.Biol. 67, 804-5. CHAGAS, C. 1909. Uber eine neue Trypanosomiasis des Merschen Studien liber Morphologie und Entwicklungszyklus des Schizatrypanum cruzi n.gen.,n.sp. Mems.Inst.Oswaldo Cruz 1, 159. CHANCE, M.L. 1979. The identification of Leishmania. In "Problems in the identification of parasites and their vectors" (A.E.R. Taylor and R. Muller, eds.). Symp.Br. Soc. Parasit. 17, 55-74 . CHANIOTIS, B.N. and ANDERSON, J.R. 1968. Age structure, population dynamics and vector potential of Phlebotomus in Northern California. II. Field Population Dynamics and Natural Flagellate Infections in Parous Females. J.med.Ent. 5_ (3) 273-92. CHATTON, E. and BLANC, G. 1914. Existence de corps leishmaniformes dans les hematoblastes d'un gecko barbaresque. Tarentola mauritanica L. Gunth. C.r.Seanc.Soc.Biol. 77, 430-3. CHATTON, E. and BLANC, G. 1918. Culture du trypanosome du gecko chez la punaise des tits. Bull.Soc.Path.exot. 11, 387-91. CHRISTENSEN, H.A. and HERRER, A. 1976. Neotropical sandflies (Diptera Psychodidae) , invertebrate hosts of Endotrypanum schaudinni (Kinetoplastidae: Trypanosomatidae). J.med.Ent. 13 (3) 299-303. CHRISTENSEN, H.A. and HERRER, A. 1980. Panamanian Lutzomyia (Diptera: Psychodidae) host attraction profiles. J.med.Ent. 17, 522-8. CHRISTENSEN, H.A. and TELFORD, S.R. 1972. Trypanosoma thecadactyli sp.n. from Forest Geckoes in Panama, and its development in the Sandfly Lutzomyia trinidadensis. J.Protozool. 19 (3) 403-6. CHRISTOPHERS, S.R., SHORTT, H.E. and BARRAUD, P.J. 1926. The morphology and life-style of the parasite of Indian Kala-azar in culture. Indian med.Res.Mem. 4, 19-53. 186 COMMISSION ON ENZYME NOMENCLATURE. 1972. "Recommendations of the Inter- national Union of Pure and Applied Chemistry and International Union of Biochemistry. Elsevier Scientific Publishing Company, Amsterdam. CORBET, P.S. and SSENKUBUGE, Y. 1962. Mosquitoes attracted to various baits in forest. Rep.E.Afr.Virus Res.Inst. 1961-2, 49-56. CREEMERS, J. and JADIN, J.M. 1966. Ultrastructure et biologie de Trypanosoma rotatorium, Mayer 1843. Acta Zool. Pathol. Antwerp. 41, 119-36. CROSET, H., RIOUX, J.A., LEGER, N., HOUIN, M., CADI SOUSSI, M., BENMANSOUR, N. and MAISTRE, M. 1977. Les methodes d1echantillonnage des populations de phlebotomes en region mediterraneenne. In "Ecologie , des leishmanioses". Editions du CNRS Paris, pp. 139-51. CURASSON, G. 1943. "Traite de Protozoologie veterinaire et compatee Vigot, Paris. DAVID, A. 1929. Recherches experimentales sur un hematozoaire du genre Leishmania (L. agamae A. David). These Sciences, Paris. DAVIS, B.S. 1952. Studies on the trypanosomes of some California mammals. Univ. Calif. Pubis. Zool. 57, 145-250. DELAIN, E. and RIOU, G. 1969. Ultrastructure du DNA du kinetoplaste de Trypanosoma cruzi cult ire in vitro. C.r.hebd.Seanc.Acad.Sci.,Paris. 268, 1225-7. DEPIEDS, R., COLLOMB, H., MATHORIN, J. and RANQUE, J. 1958. L'intradermo- reaction a Trypanosoma equiperdum dans le bouton d'Orient. Bull.Soc. Path.exot. 51, 501-4. DESSER, S.S. 1976. The ultrastructure of the epimastigote stages of Trypanosoma rotatorium in the leech Batracobdella picta. Can.J.Zool. 54, 1712-23. DICE, L.R. and LERAAS, H.J. 1936. A graphic method for comparing several sets of measurements. Contr . Lab.vertebr.Genet.Univ.Mich. 31-3. DOLLAHON, N.R. and JANOVY, J. 1971. Insect flagellates from faeces and gut contents of four genera of lizards. J.Parasit. 57 (5) 1130-2. DOLLAHON, N.R. and JANOVY, J. 1973. Leishmania adleri: In vitro phagocytosis by lizard leukocytes and peritoneal exudate cells. Expl.Parasit. 34 56-61. DOLLAHON, N.R. and JANOVY, J. 1974. Experimental infection of New World lizards with Old World Leishmania species. Expl.Parasit. 36, 253-60. DOWNES, J.A. 1958. The feeding habits of biting flies and their significance in classification. A.Rev.Ent. _3, 249-66. DVORAK, J.A. and SCHMUNIS, G.A. 1972. Trypanosoma cruzi interaction with mouse peritoneal macrophages. Expl.Parasit. 32, 289-300. EDESON, J.F.B. and HIMO, J. 1973. Leishmania sp. in the blood of a lizard (Agama stellio) from Lebanon. Trans.R.Soc.trop.Med.Hyg. 67, (1) 27. 187 EDWARDS, C. and LLOYD, D. 1973. Terminal oxidases and carbon monoxide- reacting haemoproteins in the trypanosomatid, Crithidia fasciculata. J.gen.Microbiol. 79, 275-84. EDWARDS, F.W. 1941. "Mosquitoes of the Ethiopian Region III". British Museum (Nat.Hist.) London. EVANS, D.A. 1978. Kinetoplastida. In "Methods of cultivating parasites in vitro" (A.E.R. Taylor and J.R. Baker, eds.) Academic, London pp 55-88. FALLIS, A.M., JACOBSON, R.L. and RAYBOULD, J.N. 1973. Experimental . transmission of Trypanosoma numidae Wenyon to guinea fowl and chickens in Tanzania. J.Protozool. 20, 436-7. FANTHAM, H.B. 1926. Some parasitic protozoa found in,South Africa-X. S.Afric.J.Sci. 23, 561. FANTHAM, H.B. 1931. Some parasitic protozoa found in South Afric-XIV. S.Afric.J.Sci. 28, 323-33. FENG, L.C. and CHAO, C.S. 1943. The development of Trypanosoma bocagei in Phlebotomus squaminostris. China med.J. 62, 210-7. FRANCA, C. 1911. Notes sur les hematozoaires de la Guinee Portugaise. Archos.R.Inst.bact.Camara Pestana 3, 229. FRANCHINI, G. 1921. Sur les flagelles intestinaux du type Herpetomonas du Chamaeleon vulgaris et leur culture, et sur les flagellds du type Herpetomonas de Chalcides ocellatus et Tarentola mauritanica. Bull.Soc.Path.exot. 14, 641-5. FRANCHINI, G. 1933. Su di un Emogregarine del Camaeleonte "Chamaeleon vulgaris" n.sp. Arch.ital.Sci.med.col. 14, 201-6. FRENKEL, J. 1941. Note on an intracellular stage of Leishmania chameleonis, Wenyon 1921. Indian J.Med.Res. 29, 811-2. FROMENTIN, H. 1967. Mise en culture de Trypanosoma therezieni Brygoo, 1963. Archs.Inst.Pasteur Madagascar. 36, 51-62. FULLER, G.K., LEMMA, A. and HAILE, T. 1980. A comparison of skin-test responses using antigen from Leishmania donovani and a lizard trypanosome. Trans.R.Soc.trop.Med.Hyg. 74 (2) 205-8. GARDENER, P.J., SHCHORY, L. and CHANCE, M.L. 1977. Species differentiation in the genus Leishmania by morphometric studies with the electron microscope. Ann.trop.Med.Parasit. 71, 147-55. GARNHAM, P.C.C. 1950. Blood parasites of East African vertebrates, with a brief description of exo-erythrocytic schizogony in Plasmodium pitmani. Parasitology. 40, 328-37. GARNHAM, P.C.C. 1954. Distribution of blood protozoa in Africa. Proc.Linn.Soc.London. 165, 61-6. GARNHAM, P.C.C. 1966. "Malaria parasites and other Haemosporidia". Blackwell, Oxford. GARNHAM, P.C.C. 1971. The genus Leishmania. Bull.Wld.Hlth.Org. 44, 477-89. 188 GARNHAM, P.C.C. and DUKE, B.O.L. 1953. Certain parasitic protozoa from the Gambia. Trans.R.Soc.trop.Med.Hyg. 47, 7-8. GARNHAM, P.C.C., HEISCH, R.B. and MINTER, D.M. 1961. The vector of Hepatocystis (= Plasmodium) kochi; the successful conclusion of observations in many parts of tropical Africa. Trans.R.Soc.trop.Med.Hyg. 55_, 497-502. GARNHAM, P.C.C. and LEWIS, D.J. 1959. Parasites of British Honduras with special reference to leishmaniasis. Trans.R.Soc.trop.Med.Hyg. 53, 12-40 GEHRKE, H. 1903. Dt.med.Wschr. p. 402. GEMETCHU, T. 1977. In the discussion of a paper entitled "Les leishmanioses au Senegal etude epidemiologique et ecologique" by P. Ranque. In "Ecolog des leishmanioses, Editions du CNRS, Paris, pp 226-32. GILLETT, J.D. 1972. "Common African mosquitoes and their medical importance" William Heinemann Medical Books, London. GILLIES, M.T. and WILKES, T.J. 1969. A comparison of the range of attraction of animal baits and of carbon dioxide for some West African mosquitoes. Bull.ent.Res. 59, 441-56. GILLIES, M.T. and WILKES, T.J. 1970. The range of attractions of single baits for some West African mosquitoes. Bull.ent.Res. 60, 225-35. GILLIES, M.T. and WILKES, T.J. 1974. The range of attraction of birds as baits for some West African mosquitoes (Diptera, Culicidae). Bull.ent.Res. 63, 573-81. GLASGOW, J.P. 1963. "The distribution and abundance of tsetse". Pergamon, Oxford. GRASSE, P.P. 1952. Ordre des Trypanosomides. In "Traite de Zoologie" Vol. (1) Masson, Paris, pp 602-68. GREWAL, M.S. 1957a. On a new trypanosome from the blood of an African gecko, Hemidactylus brookii angulatus Gray, 1845. Res.Bull.Panjab Univ.Sci. 106, 269-81. GREWAL, M.S. 1957b. The life cycle of the British rabbit trypanosome Trypanosoma nabiasi Railliet 1895. Parasitology. 47, 100-18. GREWAL, M.S. 1960. On a new trypanosome, Trypanosoma helogalei Grewal, 1956, from.the blood of an African mongoose, Helogale undulata rufula Peters (Peters' pigmy mongoose). Indian J.med.Res. 48, 418. GREWAL, M.S. 1961. A new trypanosome, Trypanosoma ichneumoni Grewal, 1961 from the blood of an Egyptian mongoose (Ichneumon) Herpestes ichneumon Linnaeus. Res.Bull.Panjab Univ.Sci. 12, 39-55. GUERRERO, S. and AYALA, S.C. 1977. Hemoparasitos de algunos reptiles y anfibios de la selva Amazonica del Peru. Revta.Inst.Med.trop.S. Paulo. 19, 283-8. 189 GUERRERO, S., RODRIGUEZ, C. and AYALA, S.C. 1977. Prevalencia de hemoparasitos en lagartijas de la Isla Barro Colorado, Panama. Biotropica 8, 118-23. HARTMAN, 1925. BeitrHge zur Thrombozytengenese bei niederen Vertebraten, sowie zur Frage ihrer Stellung zum Megakaryozyten der SHuger. Fol.Haem. 32, 1-14. HEISCH, R.B. 1954. Studies in leishmaniasis in East Africa. Trans.R.Soc.trop.Med.Hyg. 48 (6) 449-64. HEISCH, R.B. 1958. On Leishmania adleri sp.nov. from Lacertid lizards (Latastia sp.) in Kenya. Ann.trop.Med.Parasit. 52, 68-71. HERTIG, M., JOHNSON, P.T. and McCONNELL, E. 1968. Growth pattern of Leishmania in Phlebotomine Sandflies. Science, N.Y. 165, 1379-80. HEYWOOD, P., WEINMAN, D. and LIPMAN, M. 1974. Fine structure of Trypanosoma eyelops in non-cellular cultures. J.Protozool. 21, 232-8. HILTON, D.F.J. 1972. In vitro culture of Trypanosoma (Herpetosoma) otospermophili. Trans.R.Soc.trop.Med.Hyg. 66 (1) 189-90. HOARE, C.A. 1929. Studies on Trypanosoma grayi. 2. Experimental transmission to the crocodile. Trans.R.Soc.trop.Med.Hyg. 23, 39. HOARE, C.A. 1932. On protozoal blood parasites collected in Uganda. Parasitology. 24, 210-24. HOARE, C.A. 1972. "The Trypanosomes of Mammals. A Zoological Monograph". Blackwell, Oxford. HSU, C.K., CAMPBELL, G.R. and LEVINE, N.D. 1973. A check-list of the species of the genus Leucocytozoon (Apicomplexa, Plasmodiidae). J.Protozool. 20, 195-203. HUFF, C.G. 1941. Saurian malaria. J.Parasit. 27, 29 (Abstr.). HUTCHINSON, M.P. 1953. The epidemiology of human trypanosomiasis in British West Africa. I and II: The Gambia. Ann.trop.Med.Parasit. 47, 156. JOHNSTON, M.R.L. 1975. Distribution of Pirhemocyton Chatton and Blanc and other, possibly related, infections of poikilotherms. J.Protozool. 22 (4) 529-35. JOHNSTON, T.H. and CLELAND, J.B. 1912. The Haematozoa of Australian Reptilia. No. 2. Proc.Linn.Soc.N.S.W. 36, 479-91. JORDAN, H. 1964. Lizard malaria in Georgia. J. Protozool. 11 (4) 562-6. KEAY, R.W.J. 1953. "An outline of Nigerian vegetation". Government Printer, Lagos. KELLINA, 1962. On the dimensions of Leishmania tropica major and Leishmania tropica minor. Medskaya Parazit. 31, 716-8. KHODUKIN, N.I. and SOFIEV, M.S. 1940. Leishmaniae in some Central Asian lizards and their epidemiological significance. Problemy Subtropiceskoj Patalogii. 4, 218-28. 190 KILLICK-KENDRICK, R. 1979. Biology of Leishmania in phlebotomine sandflies In "Biology of the Kinetoplastida". Vol. 2 (W.H.R. Lumsden and D.A. Evans, eds.). Academic, London, pp 395-460. KILLICK-KENDRICK, R. LEANEY, A.J. and READY, P.D. 1977. The establishment, maintenance and productivity of a laboratory colony of Lutzomyia longipalpis (Diptera: Psychodidae). J.med.Ent. 13, 429-40. KILLICK-KENDRICK, R., MOLYNEUX, D.H. and ASHFORD, R.W. 1974. Leishmania in phlebotomid sandflies- 1. Modifications of the flagellum associated with attachment to the midgut and oesophageal valve of the sandfly. Proc.R.Soc.(B). 187, 409-19. KILLICK-KENDRICK, R. and WALLBANKS, K.R. 1981. The identity of Leishmania chamaeleonis Wenyon, 1921. Trans.R.Soc.trop.Med.Hyg. 75, 326. KIMBER, C.D., EVANS, D.A., ROBINSON, B.L. and PETERS, W. 1981. Control of yeast contamination with 5-fluorocytosine in the in vitro cultivation of Leishmania spp. Ann.trop.Med.Parasit. 75 (4) 453-4. KRASSNER, S.M. 1965. Effect of temperature on growth and nutritional requirements of Leishmania tarentolae in a defined medium. J.Protozool. 12, 73-8. KRASSNER, S.M. 1968. Isozymes in the culture forms of Leishmania tarentolae. J.Protozool. 15 (3) 523-8. KRASSNER, S.M. and FLORY, B. 1971. Essential amino acids in the culture of Leishmania tarentolae. J.Parasit. 57, 917-20. KRYUKOVA, A.P. 1941. Experimental cutaneous leishmaniasis in wide rodents of Turkmenia. In "Problemy Koznogo lejsmanioza, Ashkhabad", pp 241-8. KULDA, J. 1958. Prisperek k poznani druhu Herpetomonas chamaeleonis (Wenyon 1921) Trypanosomidae, Protozoa. Folia Parasitol. 5_, 133-45. • LABBE, A. 1894. Recherches zoologiques et biologiques sur les parasites endoglobulaires du sang des Vertebres. Archs .Zool.exp.gen. 2, 550r.58. LAWSON, R., LANDAU, I. and SHAW, J.J. 1974. Observations on non-pigmented haemosporidia of Brazilian lizards, including a new species of Saurocytozoon in Mabuya mabouya (Scincidae). Parasitology. 69, 215-23. LAINSON, R. and SHAW, J.J. 1979. The role of animals in the epidemiology of South American leishmaniasis. In "Biology of the Kinetoplastida". Vol. 2. (W.H.R. Lumsden and D.A. Evans, eds.). Academic, London pp 1-116. LAINSON, R., SHAW, J.J. and LANDAU, I. 1975. Some blood parasites of the Brazilian lizards Plica umbra and Uranoscodon superciliosa (Iguanidae). Parasitology. 70, 119-41. LAINSON, R., SHAW, J.J. and WARD, R.D. 1976. Schellackia landauae sp.nov. in the Brazilian lizard Polychrus marmoratus: experimental transmission by Culex pipiens fatigans. Parasitology. 72, 225-43. LAMBRECHT, F.L. 1965. An unusual trypanosome in £ebus griseus F. Cuvier 1819, from Colombia, South America. Revta.Inst.Med.trop.S. Paulo. 7 (2) 89-98. 191 LANDAU, I., LAINSON, R., BOULARD, Y., MICHEL, J.C. and SHAW, J.J. 1973. Developpement chez Culex pipiens de Saurocytozoon tupinambi, parasite de lezards bresiliers. C.r.hebd.Seanc.Acad.Sci.,Par is. 276, 2499-52. LAVERAN, A. and FRANCHINI, G. 1921. Des hematozoaires du gecko et specialement de Herpetomonas tarentolae. Procede simple de culture des Herpetomonas. Bull.Soc.Path.exot. 14, 323-6. LAVERAN, A. and MESNIL, F. 1912. "Trypanosomes et trypanosomiases". Ed. 2. Masson, Paris. LAVERAN, A. and PETITT, A. 1909. Contribution a l'etude des Hemogregarines de quelques sauriens d'Afrique. 3 Hemogregarine d1Agama colonorum. Bull.Soc.Path.exot. 2^, 511-2. LAVIER, G. 1943. L'evolution de la morphologie dans le genre Trypanosoma. Annls.Parasit.hum.comp. 19, 168-200. LEGER, M. 1918. Infection sanguine par Leptomonas chez un saurien. C.r.Seanc.Soc.Biol. 81, 772-4. LEGER, M. and LEGER, A. 1914. Hematozoaires des Reptiles du Haut-Senegal Niger. Bull.Soc .Path.exot. 7_, 488-93. LE RAY, D. and AFCHAIN, D. 1980. Immunotaxonomie du genre Leishmania. J.Protozool. 27 (3) 84A. LE RAY, D., AFCHAIN, D. and CAPRON, A. 1977. Contribution de la connaissance des antigenes de Leishmania "h 1*immunologic des Trypanosomatidae. In "Ecologie des Leishmanioses". Editions du CNRS, Paris, pp 63-73. LEWIS, D.H. 1975. Ultrastructural study of promastigotes of Leishmania from Reptiles. J.Protozool. 22(3) 344-52. LEWIS, D.J. and MURPHY, D.H. 1965. The sandflies of the Gambia. (Diptera: Phlebotominae). J .med .Ent. JL, 371-6. LEWIS, J.W. and BALL, S.J. 1979. Attachment of the epimastigotes of Trypanosoma cobitis (Mitrophanow, 1883) to the crop wall of the Leech Vector Hemiclepsis marginata. Z.Parasit k.de. 60, 29-36. LLOYD, L., JOHNSON, W.B., YOUNG, W.A. and MORRISON, A. 1924. Second report of the tse-tse fly investigation in the northern provinces of Nigeria. Bull.ent.Res. 15, 1-28. LOM, J. 1979. Biology of the trypanosomes and trypanoplasms of fish. In "Biology of Kinetoplastida". Vol. 2. (W.H.R. Lumsden and D.A. Evans). Academic, London, pp 269-337. LUCKINS, A.G. and GRAY, A.R. 1978. An extravascular site of development of Trypanosoma congolense. Nature, Lond. 272, 613-4. MAAZOUN, R. 1982. Identification des leishmaines dans 11ancien-monde: Signification de la variation isozymique. These Sciences, Montpellier. 192 MAAZOUN, R., LANOTTE, G., RIOUX-J-A., PASTEUR, N., KILLICK-KENDRICK, R. and PRATLONG, F. 1981. Signification du polymorphisms enzymatique chez les leishmaines. Annls.Parasit.hum.comp. 56, (5) 467-75. MACKERRAS, M.J. 1961. The haematozoa of Australian reptiles. Aust.J.Zool. 61-122. MACKIE, F.P. 1914. A flagellate infection of sandflies. Indian J.med.Res. 11, (1) 1-3. MACKIE, F.P., DAS GUPTA, B.M. and SWAMINATH, C.S. 1923. Progress report on Kala-azar . Indian J.med.Res. 11, 591-9. MAHRT, J. 1979. Hematozoa of lizards from southeastern Arizona and Isla San Pedro Nolasco, Gulf of California, Mexico. J.Parasit. 65, 972-5. MANN, R.D. 1975. The Gambia: land and vegetation degradation survey: the need for land reclamation by comprehensive ecological methods. Gambian Dept. of Agriculture. C/GAM/LRP/75. MANSON-BAHR, P.E.C. and HEISCH, R.B. 1961. Transient infection of man with a Leishmania (L. adleri) of lizards. Ann.trop.Med.Parasit. 55, 381-2. MARINKELLE, C.J. 1968. Trypanosoma lambrechti n.sp. aislado de micos. - (Cebus albifrons) de Colombia. Caldasia. 10, 155-64. MARTIN, G. 1907. Sur un Trypanosome de Saurien (Trypan, boueti n.sp.). C.r.Seanc.Soc.Biol. 62, 594-6. MARTIN, S.K., MILLER, L.H., NIJHOUT, M.M. and CARTER, R. 1978. Plasmodium gallinaceum: Induction of Male Gametocyte exflagellation by Phosphodiesterase Inhibitors. Expl.Parasit. 44, 239-42. MATHIS, C. and LEGER, M. 1911. "Recherches de parasitologie et de pathologie humaines et animales au Tonkin". Masson et C * Paris. MATTEI, D.M., GOLDBERG, S., MOREL, C., AZEVEDO, H.P. and ROITMAN, I. 1977. Biochemical strain characterisation of Trypanosoma cruzi by restriction endonuclease cleavage of kinetoplast DNA. Fed.Europ.Biol.Soc.Letters. 74, 264-8. MATTOCK, N.M. and MOLYNEUX, D.H. 1973. Susceptibility of dog sarcoma and hamster peritoneal cells to Leishmania. Trans.R.Soc.trop.Med.Hyg. 67_, 18. McCLELLAND, G.A.SH. and WEITZ, B. 1963. Serological identification of the natural hosts of Aedes aegypti (L.) and some other mosquitoes (Diptera, Culicidae) caught resting in vegetation in Kenya and Uganda. Ann.trop.Med.Parasit. 57, 214-224. McGHEE, R.B. 1959. The infection of avian embryos with Crithidia species and Leishmania tarentola.• J.infect.Pis. 105, 18-25. McGREGOR, I.A. and SMITH, D.E. 1952. A health, nutrition and parasitological survey in a rural village (Keneba) in West Kiang, Gambia. Trans.R.Soc. trop.Med.Hyg. 46, 403-27. 193 McMILLAN, B. 1965. Leishmaniasis in the Sudan Republic. 22. Leishmania hoogstraali sp.n. in the gecko. J.Parasit. 51 (3) 336-9. MELLO, D.A. 1982. Morphological and biological features of Trypanosoma (Herpetosoma) mariae Mello, 1978 in experimentally infected Calomys callosus. Annls.Parasit.hum.comp. 57, 111-9. MELLO, F. de and SUCTAN6AR, C. 1922. Morphologie et cycle evolutif d'un Herpetomonas de l'intestin d'Hemidactylus brookei. Bull.Soc.Path. exot. 15, 795-7. MELLOR, P.S. 1971. A membrane feeding technique for the infection of Culicoides nubeculosus Mg. and Culicoides variipennis sonorensis Coq with Onchocerca cervicalis Rail, and Henry. Trans.R.Soc.trop.Med.Hyg. 65, 199-201. * > MESNIL, F. 1918. (Review of paper by C. franqa entitled "Quelques considerations sur la classification des hematozoaires"). Bull.Inst. Pasteur, Paris. 16, 536. MILLER, H.C. and TUOHY, D.W. 1967. Infection of macrophages in culture by leptomonads of Leishmania donovani. J.Protozool. 14, 781-9. MINCHIN, E.A. 1908. Investigations on the development of trypanosomes in tsetse flies and other Diptera. Quart.J.micro.Sci. 52, 159. MIYATA, A. 1977. Trypanosoma nyukyuense n.sp. Detected from Eublepharis kuroiwae kuroiwae in Okinawa Island. Trop.Med. 19, 157-67. MOHIUDDIN, A. 1959. The behaviour of Leishmania adleri in various lizards. E.Afr.med.J. 36 (3) 171-6. MOLYNEUX, D.H. 1969a. The fine structure of the epimastigote forms of Trypanosoma lewisi in the rectum of the flea Nosopsyllus fasciatus. Parasitology. 59, 55-66. MOLYNEUX, D.H. 1969b. The morphology and biology of Trypanosoma (Herpetosoma) evotomys of the bank-vole, Clethrionomys glaneolus. Parasitology 59, 843-57. MOLYNEUX, D.H. 1969c. The morphology and life-history of Trypanosoma (Herpetosoma) microti, of the field-vole Microtus agttestis. Ann.trop.Med.Parasit. 63, 229-44. MOLYNEUX, D.H. 1973. Animal reservoirs and Gambian trypanosomiasis. Annls.Soc.beige.Med.trop. 53, 605-18. MOLYNEUX, D.H. 1975. Trypanosoma (Megatrypanum) melophagium: modes of attachment of parasites to midgut, hindgut and rectum of the sheep ked, Melophagus ovinus. Acta trop. 32, 65-74. MOLYNEUX, D.H. 1977«. Vector relationships in the Trypanosomatidae. Adv.Paras itol. 15, 1-82. MOLYNEUX, D.H. 1977b. In the discussion of a paper entitled "Mixed experimental infection of Phlebotomus papatasi (Sc.) with different species of Leishmania" by V.M. Safyanova and A.N. Alexeiev. In "Ecologie des leishmanioses". Editions du C.N.R.S. Paris, pp. 153-6. 194 MOLYNEUX, D.H. and CROFT, S.L. 1980a. Studies on the Ultrastructure of Candidate "Cysts" in Leptomonas species of Siphonaptera. Z.Parasitkde. 63, 233-9. MOLYNEUX, D.H. 1980b. Animal reservoirs and residual "foci" of Trypanosoma brucei gambiense sleeping sickness in West Afica. Insect Sci. Application I, 59-63. MOLYNEUX, D.H., and ASHFORD, R.W. 1975. Observations on a trypanosomatid flagellate in a flea Peromyscopsylla silvatica spectablis. Ann.trop.Med.Parasit. 50, 265-74. MOREAU, R.E. 1963. Vicissitudes of the African biomes in the late Pleistocene. Proc.zool.Soc.Lond. 141, 395-421. MULLIGAN, H.W.* (Ed.) 1970. "The African trypanosomiases". George Allen and Unwin, London. MUNIZ, J. and MEDINA, H. 1948. Leishmaniose tegumentar do cobaio Leishmania enrietti n.sp. Hospital, Rio de J. 33, 7-25. MURPHY, D.H. 1960. Collembola Symphypleona from the Gambia, with a note on the biogeography of some characteristic savanna forms. Proc.zool.Soc. Lond. 134, 557-94. MUTINGA, M.J. and NGOKA, J.M. 1981. Suspected vectors of lizard leishmaniasis in Kenya and their possible role in partial immunization of the human population against Leishmania donovani in Kala-Azar endemic areas. Insect Sci.Application. 1_, 207-10. NADIM, A., SEYEDI RASHTI, M.A. and MESGHALI, A. 1968. On the nature of leptomonads found in Sergentomyia sintoni in Khorassan, Iran and their relation to lizard leishmanias. J.trop.Med.Hyg. 71 (9) 240. NAIR, C.P. and DAVID, A. 1956. Observation on a natural (cryptic) infection of trypanosomes in sparrows (Passer domesticus Linnaeus). Part II. Attempts at experimental transmission and determination of the sites of infection in birds and mosquitoes. Indian J.Malar. 10, 137-47. NGOKA, J.M. and MUTINGA, M.J. 1978. Visceral leishmaniasis animal reservoirs in Kenya. E.Afr.med.J. 5b (7) 332-6. NICOLLE, C. 1920. La question du reservoir de virus du bouton d'Orient Hypoth^se du Gecko. Hypothese du Charneau. Bull.Soc.Path.exot. 13_, 511-5. NICOLLE, C., BLANC, G. and LANGERON, M. 1920. Recherches experiment a les sur le role du Gecko (Tarentola mauritanica) dans l'etiologie du bouton d'Orient. Mission de Tamerza (Octobre 1919).Note preliminaire. Bull.Soc.Path.exot. 13, 508-11. NIJHOUT, M.M. 1979. Plasmodium gallinaceum: Exflagellation stimulated by a mosquito factor. Expl.Parasit. 48, 75-80. NIJHOUT, M.M. and CARTER, R. 1978. Gamete development in malaria parasites: bicarbonate-dependant stimulation by pH in vitro. Parasitology. 76 (1) 39-53. 195 NOVY, F.G. 1906. The trypanosomes of tsetse flies. J.infect.Pis. 3, 394. ORMEROD, W.E. 1981. The life cycle of the sleeping sickness trypanosome compared with the Malaria life cycle. In "Parasitological Topics" (E.U. Canning, Ed.) Society of Protozoologists, Special Publication No. 1 pp 191-9. ORMEROD, W.E. and VENKATESAN, S. 1971. The occult visceral phase of mammalian trypanosomes with special reference to the life cycle of Trypanosoma (Trypanozoon) brucei. Trans.R.Soc.trop.Med.Hyg. 65, 722-35. PANTIN, C.F.A. 1969. "Notes on microscopical technique for zoologists" Cambridge University Press, Cambridge. PARROT, L. 1927. Sur un parasite intraglobulaire pigmente de Tarentola mauritanica (L.). Archs .Inst .Pasteur Alger. 5_, 1-8. PARROT, L. 1931. Evolution d'un Hematozoaire du Gecko (Leishmania tarentolae chez un moucheron piqueur, du groupe des Phlebotomes (Phlebotomus minutus) C.r.hebd.Seanc.Acad.Sci.,Paris. 199, 1073-4. PARROT, L. 1934. L'evolut ion de Leishmania tarentolae Wenyon chez Phlebotomus minutus Rond. Bull.Soc.Path.exot. 27, 839-43. PARROT, L. 1935. Nouvelles recherches sur l'evolution de Leishmania tarentolae chez Phlebotomus minutus Rondani. Bull.Soc.Path.exot. 28, 958-60. PARROT, L. and FOLEY, H. 1939. Sur la frequence de la leishmaniose du gecko dans le sud oranais. Archs.Inst.Pasteur Alger. 17, 231-2. PARROT, L. 1949. Sur quelques souches de Leishmania. Archs.Inst.Pasteur Alger. 2_7, 106-9. PAUL, J. 1975. "Cell and Tissue Culture" ed. 5. Livingstone, Edinburgh. PELAEZ, D. 1967. Estudios sobre hematozoarios XIII. Un nuevo Plasmodium de Ameiva en Mexico. Ciencia, Mex. 25, 121-29. PELAEZ, D., REYES, R.P. and BARRERA,-A. 1948. Plasmodium mexicanum Thompson and Huff, 1944 en sus huespedes naturales. Ann Escuela nac. Cienc.biol. 5_, 197-202. PELAEZ, D. and STREBER, F. 1955. Estudios sobre hematozoarios V. Trypanosoma serveti nov. sp., parasito de un Sceloporus de Mexico. An.Esc.nac.Cienc. biol., Mex. 8, 147-52. v POPOV, P.P. 1941. Cutaneous leishmaniasis in Azerbardzhan. Tri ""Problemy Koznogo Lejsmanioza, Ashkahabad". pp 107-12. RANQUE, P. 1973. Etude morphologique et biologique de quelques trypanosomatides recoltes au Senegal. These Sciences, Marseille. RANQUE, P. 1977. Les leishmanioses au Senegal etude epidemiologique et ecologique. In "Ecologie des leishmanioses" Editions du C.N.R.S. Paris, pp 225-232. READY, P.D. 1978. The feeding habits of Laboratory-bred Lutzomyia longipalpis (Diptera: Psychodidae). J.med.Ent. 14 (5) 545-52. . 196 RIDING, D. 1930. Haemoproteus of Tarentola annularis. Trans.R.Soc.trop.Med.Hyg. 23, 635-7. RIOUX, J.A., KILLICK-KENDRICK, R. and GARNHAM, P.C.C. 1979. Leishmania tarentolae and other blood parasites of geckoes at Banyuls in the south of France. Trans.R.Soc.trop.Med.Hyg. 73 (3) 319. RIOUX, J.A., KNOEPFLER, L.P. and MARTINI, A. 1969. Presence en France de Leishmania tarentolae Wenyon, 1921. Parasite du gecko Tarentola mauritanica (L. 1758). Annls .Parasit .hum.comp. _1, 115-6. ROBERTSON, M . 1908. A preliminary note on haematozoa from some Ceylon reptiles. Spolia Zeylan. 5_, 178-85 . RODRIGUEZ, E. and MARINKELLE, C.J. 1970. Trypanosoma cruzi; Development in tissue culture. Expl.Parasit. 27, 78-87. ROGIER, E. 1977. Description et cycle biologique de Schellackia agamae (Laveran et Petitt 1909), Lankesterellidae parasite d'agames de republique centre africaine. Protistologica. 13 (1) 9-13. RYCKMAN, R.E. 1954. Lizards: a laboratory host for Triatominae and Trypanosoma cruzi: Chagas (Hemiptera, Reduviidae) (Protomonadida Trypanosomidae). Trans.Am.microsc.Soc. 73, 215-8. RYCKMAN, R.E. 1965. Epizootiology of Trypanosoma cruzi in Southwestern North America. J.med.Ent. 2, 93-5. SCHWETZ, J. 1931. Sur quelques hematozoaires des lezards de Stanleyville et du Lac Albert. Annls .Parasit .hum.comp. 9_, 193-201, SCHWETZ, J. 1933. Trypanosomes rares de la region de Stanleyville (Congo beige). Annls.Parasit.hum.comp. 11, 287-96. SC0RZA, J.V. 1970. Lizard malaria. Doctoral Thesis, University of London. SCORZA, J.V. and DAGERT, C. 1955. Trypanosoma mega Dutton and Todd 1903 y Trypanosoma rotatorium Mayer 1843 en batracios y una nueva especie en un saurio de Venezuela. Boln.Soc.venez.Cienc.nat. 16, 205-8. SERGENT, EiD. and E.T., LEMAIRE, G. and SENEVET, G. 1914. Insecte transmetteur et Reservoir de virus du Clou de Biskra. Hypothese et experiences preliminaires . Bull .Soc .Path .exot. 1_, 577-9. SERGIEV, V.P. 1977. In the discussion of a paper entitled "Mixed experimental infection of Phlebotomus papatasi (Sc.) with different species of Leishmania" by V.M. Safyanova and A.N. Alexeiev. In "Ecologie des leishmanioses". Editions du C.N.R.S., Paris, pp. 153-6. SEYEDI, RASHTI, M.A., NADIM, A. and NAFICY, A. 1971. Further report on lizard leishmaniasis in the northern part of Iran. J.trop.Med.Hyg. 72, 70-1. SHAW, J.J. and LAINS0N, R. 1975. Leishmaniasis in Brazil: X. Some observations on intradermal reactions to different trypanosomatid antigens of patients suffering from cutaneous and muco-cutaneous leishmaniosis. Trans.R.Soc. trop.Med.Hyg. 69, 323-35. 197 SHAW, J.J. and LAINSON, R. 1976. Leishmaniasis in Brazil XI. Observations on the morphology of Leishmania of the mexicana and braziliensis complexes. J.trop.Med.Hyg. 79, 9-13. SHORTT, H.E. and SWAMINATH, C.S. 1928. Preliminary note on three species of Trypanosomidae. Indian J.med.Res. 16, 241-4. SHORTT, H.E. and SWAMINATH, C.S. 1931. Life-history and morphology of Trypanosoma phlebotomi (Mackie, 1914). Ind.J.med.Res. 19, 541-71. SIMPSON, J.J. 1911. Entomological research in British West Africa. Bull.ent.Res. 2, 187-239. SIMPSON, L. 1968a. Behaviour of the kinetoplast of Leishmania tarentolae upon cell rupture. J.Protozool. 15, 132-6. SIMPSON, L. 1968b. Effect of acriflavin on the kinetoplast of Leishmania tarentolae. Mode of action and physiological correlates of the loss of kinetoplast DNA. J.biophys.biochem.Cytol. 37, 660-82. SIMPSON, L. and BRALY, P. 1970. Synchronization of Leishmania tarentolae by Hdroxyurea. J.Protozool. 17 (4) 511-7. SIMPSON, L. and SIMPSON, A.M. 1978. Kinetoplast RNA of Leishmania tarentolae. Cell 14, 169-78. SINHA, C.K. 1980. Trypanosoma infection in a fish Lepidocephalus guntea and in a skink Mabuya carinata. Indian J.Parasitol. 3^, 38. SMOLIKOVA, V., LOM, J. and SUCHANKOVAE. 1977. Growth of the Canp Trypanosome T. danilowskyi in fish tissue culture. J.Protozool. 24 (4) 54A. SNOW, W.F. 1979. Records of Phlebotomus duboscqi Neven-Lemaire from the Gambia. Trans .R.Soc .trop .Med .Hyg. 73 (2) 24~5-6 . SNOW, W.F. and BOREHAM, P.F.L. 1973. The feeding habits of some West African Culex (Dipt. Culicidae) mosquitoes. Bull.ent.Res. 62, 517-26. SOLDO, A.T. and BRICKSON, S.A. 1980. A simple method for plating and cloning ciliates and other protozoa. J.Protozool. 27 (3) 328-31. SOUTHGATE, B.A. 1967. Studies in the epidemiology of East African leishmaniasis. 5. Leishmania adleri and natural immunity. J.trop.Med.Hyg. 70, 33-36. SOUTHGATE, B.A. 1977. In the discussion of a paper entitled "Les leishmanioses au Senegal etude epidemiologique et ecologique" by P. Ranque. In "Ecologie des leishmanioses". Editions du C.N.R.S., PaTis. pp 226-32. SOUTHGATE, B.A. and MANSON-BAHR, P.E.C. 1967. Studies in the epidemiology of East African leishmaniasis. 4. The significance of the positive leishmanin test. J.trop.Med.Hyg. 70, 29-33. STEHNENS, W.E. and JOHNSTON, M.R.L. 1969. The viral nature of Pirhemocyton tarentolae. J.Ultrastruct.Res. 15, 543-53. STEIGER, R.F. 1973. On the ultrastructure of Trypanosoma (Trypanozoon) brucei in the course of its life cycle and some related aspects. Acta trop. 30, 64-168. 198 STRAUSS, P.R. 1971. The effect of homologous Rabbit Antiserum on the growth of Leishmania tarentolae - a fine structure study. J.Protozool. 18 (1) 147-56. STRONG, R.P. 1924. Investigations upon flagellate infections. Amer .J.trop.Med.Hyg. 4_,l-56. TANABE, M. 1924. Studies on the hemoflagellata of the loach. Misgurnus anguillocaudatus. Kitasato Archs.exp.Med. 6_9 121-38. TARSHIS, I.B. 1953. Oocyst-like bodies on the midgut of Stilbometopa impressa (Bigot) (Diptera: Hippoboscidae). Science, N.Y. 118, 199-202. TAYLOR, A.W. 1929. Note on the occurrence of Crithidia (Trypanosoma varani) in Phlebotomus minutus var. africanus in Northern Nigeria. Ann.trop. Med.Parasit. 23, 33-4. TELFORD, S.R. 1970Comments on the vector relationships of saurian malaria. J.Parasit. 56, 340. TELFORD, S.R. 1970> Plasmodium chiricahuae sp.nov. from Arizona lizards. J.Protozool. 17 (3) 400-5. TELFORD, S.R. 1972. Malarial parasites of the "Jesu Cristo" lizard Basiliscus basiliscus (Iguanidae) in Panama. J.Protozool. 19 (1) 77-81. TELFORD, S.R. 1973. Saurian malarial parasites from Guyana: their effect upon the validity of the family Garniidae and the genus Garnia with descriptions of two new species. Int.J.Parasitol. 3^, 829-42. TELFORD, S.R. 1977. The distribution, incidence and general ecology of Saurian malaria in Middle America. Int .J.Parasitol. 7_9 299-314. TELFORD, S.R. 1978. The saurian malarias of Venezuela: Haemosporidian parasites of gekkonid lizards. Int .J.Parasitol. 8_9 341-53. TELFORD, S.R. 1979a. Two new trypanosomes from neotropical gekkonid lizards. J.Parasit. 65_ (6) 898-901. TELFORD, S.R. 1979b. Evolutionary implications of Leishmania amastigotes in circulating blood cells of lizards. Parasitology. 79, 317-24. TELFORD, S.R. 1980. The saurian malarias of Venezuela: Plasmodium species from iguanid and teiid hosts. Int.J.Parasitol. 10, 365-74. TEMPELIS, C.H. and GALINDO, P. 1970. Feeding habits of fi^e species of Deinocer *.tes mosquitoes collected in Panama. J.med .Ent. 7, 175-9. TETLEY, L., VICKERMAN, K. and M0L00, S.K. 1981. Absence of a surface coat from metacyclic Trypanosoma vivax: possible implications for vaccination against vivax trypanosomiasis. Trans.R.Soc.trop.Med.Hyg. 75, 409-14. THEILER, M. 1930. Special protozoological studies of the blood; Protozoological studies of smaller animals. In "The African Republic of Liberia and the Belgian Congo. Harvard African Expedition", Vol. 1. (R.P. Strong, ed.). Harvar University, Cambridge, pp 490-8. 199 THOMPSON, P.E. 1944. Changes associated with acquired immunity during initial infections in saurian malaria. J.infect.Pis. 75, 139-49. THOMPSON, P.E. and HART, T.A. 1946. Plasmodium lacentiliae n.sp. and other saurian blood parasites from the New Guinea area. J.Parasit. 32, 79-82. THOMPSON, P.E. and HUFF, C.G. 1944a. A saurian malarial parasite Plasmodium mexicanum n.sp. with both elongatum- and gallinaceum-types of exoerythrocytic stages. J.infect.Pis. 74, 48-67. TODD, J.L. and WOLBACH, S.B. 1912. Parasitic protozoa from the Gambia. J.med.Res. 2£ (2) 195-218. TRAGER, W. 1957. Nutrition of a hemoflagellate (Leishmania tarentolae) having an interchangeable requirement for Choline or Pyridoxal. J.Protozool. 4_, 269-76. TRAGER, W. 1969. Pteridine requirement of the haemoflagellate Leishmania tarentolae. J.Protozool. 16, 372-5. U.F.A.W. 1972. "The UFAW handbook on the care and management of laboratory animals". 4th Ed. Churchill Livingstone, London. URDANETA-MORALES, S. and McLURE 1981. Experimental infections in Venezuelan lizards by Trypanosoma cruzi. Acta Trop. 38, 99-105. VANDERBERG, J.P. and GWADZ, R.W. 1980. The transmission by mosquitoes of Plasmodia in the laboratory. In "Malaria" Vol. 2; Pathology, vector studies and culture. (J.P. Kreier, ed.) Academic, London, pp. 154-234. VICKERMAN, K. 1965. The identity of Leishmania chamaeleonis Wenyon, 1921. Trans.R.Soc.trop.Med.Hyg. 59, 372. VICKERMAN, K. 1969. The fine structure of Trypanosoma congolense in its bloodstream phase. J.Protozool. 16 (1) 54-69. VICKERMAN, K. 1973. The mode of attachment of Trypanosoma vivax in the proboscis of the tsetse fly Glossina fuscipes. J.Protozool. 20, 394-404. VICKERMAN, K. 1976. The diversity of the kinetoplastid flagellates. In "Biology of the Kinetoplastida" Vol. 1. (W.H.R. Lumsden and D.A. Evans, eds.). Academic, London pp 1-34. VICKERMAN, K. and PRESTON, T.M. 1976. Comparative cell biology of the kinetoplastid flagellates. In "Biology of the Kinetoplastida" Vol. 1. (W.H.R, Lumsden and D.A. Evans, eds.). Academic, London pp 35-130. VICKERMAN, K. and TETLEY, L. 1981. Differentiation of the metacyclic stage in the life cycle of Trypanosoma brucei. Trans.R.Soc.trop.Med.Hyg. 75 (6) 894. WAKELIN, D. 1976. Host responses. In "Ecological Aspects of Parasitology" (C.R. Kennedy, ed.). North Holland Publishing, Amsterdam pp 115-35. WALLIKER, D. 1965. Trypanosoma superciliosae sp.nov. from the lizard Uranoscodon superciliosa L. Parasitology. 55, 601-6. WALTERS, J.H. 1949. A case of indigenous kala-azar in the Gambia. Trans.R.Soc.trop.Med.Hyg. 43, 287-92. 200 WANSON, M. 1942. Sur la biologies des Phlebotomes congolais. Rev.Trav. Sci .med .CongoBelg. 19 23-43. WASLEY, G.R. and JOHN, R. 1972. The cultivation of mammalian macrophages in vitro. In "Animal tissue culture: Advances in technique" (G.D. Wasley, ed.) Butterworths, London pp 101-37. WENYON, C.M. 1909. Report of travelling pathologist and protozoologist. 3rd Rep.Wellcome Trop.Res.La.,Khartoum 121-68. WENYON, C.M. 1915. The pigmented parasites of cold-blooded animals, with some . notes on a Plasmodium of the Trinidad Iguana. J.trop.Med.Hyg. 12, 133-40. WENYON, C.M. 1921. Observations on the intestinal protozoa of three Egyptian lizards, with a note on a cell-invading fungus. Parasitology. 12, 350-65. WENYON, C.M. 1926. "Protozoology" Vol. 1. Bailliere, Tindall and Cox, London. WERY, M. and DE GROODT-LASSEEL, M. 1966. Ultrastructure de Trypanosoma cruzi en culture sur milieu semisynthetique. Annls.Soc.beige.Med.trop. 46, 337-48. WILSON, V.C.L.C. and SOUTHGATE, B.A. 1979. Lizard Leishmania. In "Biology of the Kinetoplastida" Vol. 2. (W.H.R. Lumsden and D.A. Evans) Academic, London, pp 241-68. WOOD, S.F. 1937. Variations in the cytology of the blood of geckoes (Tarentola mauritanica) infected with Haemogregarina platydactyli, Trypanosoma platy- dactyli and Pirhemocyton tarentolae. Univ.Calif.Pubis.Zool. 41, 9-21. YOUNG, C.W. and HERTIG, M. 1927. Peripheral lesions produced by L^donovani and allied leishmaniae. Proc.Soc.exp.Biol.Med. 25, 196-7. ZARATE, L.G., ZARATE, R.J.,TEMPELIS, C.H. and GOLDSMITH, R.S. 1980. The biology and behaviour of Triatoma barberi in Mexicol. Bloodmeal sources and infection with T. cruzi. J.med.Ent. 17 (2) 103-16. ZIMMERMAN, R.S. and BROWN, H.P. 1952. Observations on some intestinal protozoa in Oklahoma lizards with the description of a New Genus, Biflagella. Proc.Okla.Acad.Sci. 33, 103-11. ZMEYEV, G.J. 1936. Haemoparasite fauna of wild vertebrates in some southern areas of Tadzhikistan. In "Trudy Tadzikskoj bazy AN USSR" 6 249-66.