Tsetse flies vectors of trypanosomiasis

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1 Published by the International Laboratory for Research on Animal Diseases Volume 2 Number 3 Tsetse flies vectors of trypanosomiasis Tsetse colonies at ILRAD Some research results Tsetse characteristics and behaviour Control methods Assistance to other research institutes Annual meeting of the board of directors Recent articles by ILRAD scientists First N'Dama calves born at ILRAD Tsetse flies vectors of trypanosomiasis 'Tsetse' is a Setswana word it means 'fly destructive to cattle'. Tsetse flies are brown insects, 1 to 2 cm long, which feed solely on the blood of vertebrate animals. They infest a variety of habitats in 38 African countries, ranging from wooded savannas to forests and vegetation along the banks of rivers and lakes. Generally, they do not survive in the African highlands above 1,800 metres or in very dry areas. Thirty species and subspecies of tsetse flies belong to the genus Glossina. They have evolved through the millennia, feeding on a wide range of African wild animals. Tsetse show flexibility in their feeding preferences: one species might feed on a particular type of host animal in one area, but on another type of animal somewhere else. In areas of human settlement, tsetse may feed on cattle, sheep, goats, pigs, horses, donkeys, camels, and man. Tsetse flies pose a serious constraint on livestock production and agricultural development in Africa because they transmit the protozoan parasites which cause trypanosomiasis. All tsetse species are capable of transmitting trypanosomes, though some are more efficient disease vectors than others. The wild animals which are the tsetse fly's natural hosts do not seem to be seriously affected by trypanosome infection. However, when susceptible livestock are bitten by infected tsetse flies, they develop severe parasitaemia and their growth and productivity are substantially reduced. Many infected animals die. It is impossible to assess, even approximately, the direct losses caused by animal trypanosomiasis in Africa, but the fact that livestock on the continent are treated with more than 25 million doses of trypanocidal drugs a year gives some indication of the magnitude of the problem. Adult tsetse feed every 2 or 3 days. When they bite an animal, they create a pool of blood at the site of the bite. They pump saliva into this blood pool through the hypopharynx, a long tube in the insect's proboscis. The salivary secretion contains a powerful anticoagulant which keeps the blood fluid so the fly can continue feeding.

2 Diagram of the internal organs of an adult female tsetse fly. The three major trypanosome species develop in different parts of the fly. T brucei develops in the tsetse gut, proventriculus and salivary gland. Metacyclic forms which are infective to mammals are produced in the salivary gland. Tsetse infection rates with this parasite species are only about 0.1% in the field, but thousands of metacyclic parasites are found in each feeding probe of an infected fly. T congolense parasites develop in the tsetse gut, proventriculus and mouth parts, where infective metacyclic forms are produced. Infection rates average about 2% in the field; and 50 to 100 parasites are found in the feeding probe of an infected fly. T vivax parasites develop entirely in the tsetse mouth parts. Infection rates in the field average 10 to 20%, but apparently relatively few metacyclic parasites are produced. When a tsetse fly feeds on an animal infected with trypanosomes, bloodstream forms of the parasite are ingested along with the bloodmeal and infection may become established in the fly. The saliva of an infected fly contains metacyclic trypanosomes, highly infective forms of the parasite, which are injected into the blood pool every time the fly feeds. Once infected, tsetse continue to transmit trypanosomes for the rest of their lives.

3 Tsetse larvae (light coloured) and puparia (dark). In the field a larva burrows into soft earth or crawls on the surface until the surrounding cuticle hardens to form a puparial shell. An adult fly emerges from the puparium after about 30 days. Tsetse are not as prolific as many other insects. The females normally mate only once and then produce a single larva every 9 to 10 days. Depending on climatic conditions, adult tsetse survive in the field from 2 weeks up to 6 months. Tsetse colonies at ILRAD In searching for more effective methods to control trypanosomiasis, ILRAD scientists concentrate primarily on the parasites which cause the disease. It is possible to maintain trypanosomes in vitro and in laboratory animals without tsetse flies, and livestock can be infected experimentally by injecting the parasites into their bloodstream or tissues. However, trypanosomes normally pass through several stages of their lifecycle within the tsetse fly, and trypanosomes maintained and transmitted artificially are different in some respects from parasites transmitted through the bite of an infected fly. Thus, much of ILRAD's research and experimentation must be conducted with parasites which have developed in tsetse flies. In the search for better control methods, ILRAD scientists are also studying the transmission process which occurs when an animal is bitten by an infected fly, focusing on the development of the parasites and the first responses of an animal when it becomes infected. At an altitude of 1,800 metres, the Kabete area where ILRAD is located is not a natural habitat for tsetse flies. However, a modern tsetse laboratory has been constructed at ILRAD where tsetse breeding colonies are maintained in artificial climatic conditions and

4 carefully controlled under tight security. Seven species and subspecies of tsetse are maintained: Glossina morsitans centralis originating from mainland Tanzania, G austeni from Zanzibar, G palpalis palpalis from Nigeria, G p gambiense from Upper Volta, G fuscipes fuscipes from the Central African Republic, G tachinoides from Chad and G brevipalpis from Kenya. They are all important vectors of trypanosomiasis. The G brevipalpis colony was established in 1982 from wild flies captured in the Kibwezi forest: this species has not been successfully colonized before. ILRAD's colonies represent the three main taxonomic groups of tsetse flies fusca, palpalis and morsitans. Most experimental work is conducted using G m centralis, the largest colony, maintained at 8200 breeding females. The other colonies comprise 2000 to 4000 breeding females each. In the tsetse holding rooms where the breeding colonies are maintained, light, temperature and humidity conditions are carefully controlled to create a favourable environment for the flies. All the tsetse are maintained at 25 o C. The savanna species are kept at 70% humidity; the forest and riverine species at 85%. Indirect lights are on dimmer switches, timed to provide a rhythm of 12 hours of light followed by 12 hours of darkness. The tsetse are allowed to feed on rabbits for 10 minutes 6 days a week. The tsetse laboratory and isolation units are completely fly proofed and surrounded by moats to prevent the entry of other insects. Precautions are also taken to avoid contamination with insecticides. Some research results The tsetse laboratory provides trypanosomes and infected tsetse flies for a variety of research projects, and staff of the laboratory work closely with other ILRAD scientists. In the course of this work, several interesting findings have emerged on the role of tsetse flies as trypanosomiasis vectors. Studies have also concentrated on possible approaches to trypanosomiasis control through interference with the transmission cycle.

5 Technician at ILRAD examines male and female tsetse flies. Newly emerged flies called tenerals are immobilized in a chiller at 2 to 3 C and separated according to sex. Tsetse characteristics and behaviour In some field situations, tsetse flies feed on many different types of animals, while in other cases they show a preference for a particular animal species. At ILRAD, tsetse have been maintained on the blood of buffalo, bushbuck, eland, oryx, warthog, waterbuck, wildebeest, cattle, goats and rabbits. Differences in diet do not appear to affect the flies' survival rates or reproductive performance. This suggests that the feeding preferences shown by tsetse in the field are probably based on the, behavioural patterns of the flies and their hosts, rather than on nutritional differences between the blood of different animals. An extensive study on the feeding habits of infected tsetse revealed that infection with trypanosomes does not have a significant effect on tsetse feeding ability or behaviour. Infection with Trypanosoma brucei, and probably with other trypanosome species, does not affect the ultrastructure or function of the tsetse gut, suggesting that tsetse flies are not seriously harmed by trypanosome infection. An anticoagulant was detected in tsetse salivary gland extracts over 60 years ago, but little was known about its physical or chemical characteristics. In collaboration with a colleague from Harvard Medical School, ILRAD scientists recently isolated a powerful thrombin inhibitor from tsetse saliva. This substance has been given the name 'tsetsin'. A second anticoagulant was identified in the tsetse gut and this is also being characterized. These two enzymes function as a two stage defence mechanism to prevent the potentially harmful effects of coagulation in the tsetse intestine.

6 Experience through the years has provided evidence on the vectorial capacity of the different tsetse species maintained at ILRAD. G m morsitans and G m centrali are both highly efficient vectors of the three main trypanosome species affecting livestock and people in Africa Trypanosoma vivax, T congolense and T brucei. These tsetse transmit the three parasite species more efficiently than G austeni or G tachinoides. It has also been demonstrated that G m morsitans and G m centralis can be infected with different trypanosome species at the same time and can transmit these mixed infections to animals. Electron micrograph of a tsetse proboscis. The tip of the proboscis is well armed with teeth and rasps which pierce the skin of the host and sever the subcutaneous capillaries, producing a pool of blood. Blood is sucked up through the proboscis by the action of a muscular pump. Control methods One approach to tsetse control which has been used experimentally in Nigeria, Tanzania, Upper Volta and Zimbabwe is based on the release of large numbers of male flies sterilized by gamma irradiation or chemosterilant. Because female tsetse normally mate only once, if most or all females in an area mate with sterile males the next generation of tsetse will be substantially reduced. Research was initiated at ILRAD in 1980 on trypanosome infection rates and transmission characteristics of sterile male tsetse, partly funded by the International Atomic Energy Agency. These studies have shown that all three important trypanosome species can develop normally in sterile male tsetse flies. Sterile males also transmit the parasites as efficiently as normal flies. This suggests that the release of large numbers of sterile males as part of a tsetse control program could increase the immediate risk of trypanosomiasis. Because of the risk involved, this approach should be used in combination with other disease control measures.

7 ILRAD technician collects tsetse larvae from breeding units. Treatment with trypanocidal drugs has been an important method of trypanosomiasis control for over 30 years, but little is known about the impact of regular drug treatment on the disease transmission cycle. Researchers at ILRAD have investigated the trypanosome infection and transmission rates of tsetse flies which feed on animals treated with trypanocidal drugs. When infected tsetse were maintained on goats treated with trypanocides, no significant differences were detected in their survival rates or reproductive performance, but their infection rates were significantly reduced. These results were consistent for the three major trypanosome species. They suggest that a carefully managed drug treatment program could reduce the transmission of trypanosomiasis in field situations where tsetse are feeding primarily on treated livestock. In another study, goats were immunized experimentally with trypanosome forms which normally develop in tsetse flies. The goats produced antibodies against this stage of parasite development, but were still fully susceptible to parasite forms which are normally infective to mammals. However, when infected tsetse flies were fed on the immunized goats, the antibodies contained in the bloodmeal sharply reduced trypanosome infection rates in the flies. ILRAD scientists are also investigating whether antibodies directed against tsetse salivary proteins might disrupt the feeding behaviour of the flies. Antibodies against trypanosomes or tsetse saliva might be stimulated to reduce the transmission of trypanosomiasis in the field. In collaboration with the Swiss Tropical Institute, ILRAD scientists have studied changes in the tsetse uterine gland at different stages of pregnancy. The goal is to identify possible approaches to control of tsetse populations by interfering with the reproductive process.

8 Assistance to other research institutes Over the past few years, the ILRAD tsetse laboratory has supplied thousands of puparia and adult tsetse flies to collaborating institutes and projects in several countries. In Kenya, these include the International Centre of Insect Physiology and Ecology (ICIPE), the Kenya Trypanosomiasis Research Institute, the University of Nairobi and a Commonwealth Institute of Biological Control project conducted at the Kenya Agricultural Research Institute. Tsetse have also been provided to the Centre de recherche et d'elevage in Togo and the National Council for Scientific Research in Zambia. In Europe, ILRAD has provided tsetse to the International Atomic Energy Agency in Austria, the Institut de médecine tropicale in Belgium and the Swiss Tropical Institute. A trainee from Tanzania and an ILRAD technician feed tsetse flies on defibrinated cow's blood through a silicone membrane. Tsetse are fed artificially on cultures of trypanosomes or infected or uninfected blood. Infected flies are then used to initiate experimental infections in laboratory animals and livestock. A serological testing system has been developed to identify the source of tsetse bloodmeals, using small quantities of residual blood obtained from flies. Antisera have been prepared against 42 species of mammals and 4 species of birds, and these have

9 been used to analyse tsetse bloodmeals collected from Gabon, Togo and Zaire under the trypanotolerance research network organized in collaboration with the International Livestock Centre for Africa. Analyses are also being carried out as requested by ICIPE. Over the past 8 years, 95 scientists, technicians and field personnel have received training in ILRAD's tsetse laboratory. These specialists have come to ILRAD from research centres, universities and government departments in 28 African countries. Adult Glossina morsitans centralis. Tsetse flies are recognized by the long proboscis extending horizontally from under their heads and by the habit of resting with their wings folded scissor like over their backs. Annual meeting of the board of directors ILRAD's Directors held their annual meeting in Nairobi from 28 to 30 March The Program, Training, Finance and Executive Committees of the Board all met before the full Board meeting. Current members of the Board are: Dr P. Atang (Cameroon), Dr L.L. Callow (Australia), Dr D.M. Chavunduka (Zimbabwe), Prof Dr K. Eichmann(Germany), Dr A.R Gray (ILRAD), Dr G.L. Kazyumba (Zaire), Mr J.S. Mburu (Kenya), Dr W.K. Ngulo (Kenya), Prof E.N.W. Oppong (Ghana); Dr W.R. Pritchard (USA), Dr K.S. Warren (USA) and Dr K.F. Wells (Canada). Dr. Atang and Dr Eichmann were unable to attend and sent their apologies. Observers from several of ILRAD's donors attended the open sessions of the Board meeting, representing the United Nations Development Program and the Governments of Belgium, Holland, Switzerland, Italy, Canada, the UK and the USA. ILRAD was also pleased to welcome Prof J. Mortelmans, a member of the Technical Advisory Committee of the Consultative Group on International Agricultural Research (CGIAR), Mr C. Farrar, Executive Secretary of the CGIAR, and Dr A.K. Diallo, a member of the Board of the

10 International Livestock Centre for Africa. During the 3 day meeting, the Board considered reports presented by ILRAD's Director General, Dr Gray, the Director of Administration, Dr P.R. Rowe, and the Director of Research, Dr J.J. Doyle, as well as reports from the Board's four committees. Agenda items also included consideration of a report prepared in 1983, 'ILRAD a plan for the second decade', and the development of ILRAD's policy on patents and related matters. An important item for discussion was the 'Program and budget: mid term report'. The Board of Directors approved ILRAD's budget request to the CGIAR for 1985 and the budget projections for ILRAD's Financial Controller, Mr D.M. Sickelmore, also presented the auditors' report for Dr Wells resigned as Chairman of the Board and Dr Pritchard was elected as his successor. Dr Kazyumba and Dr Warren were re elected for their second 3 year terms as Board members. Dr Wells has made a valuable contribution to ILRAD's development for many years. He has been Chairman of ILRAD's Board of Directors since March 1981, and from November 1980 to March 1981 he served as acting Director General. He will now continue as a Board member for another 2 years, and is serving as Chairman of the Program Committee and a member of the Executive Committee. The new Chairman, Dr Pritchard, joined ILRAD's Board of Directors in Dr Pritchard has had a distinguished career in veterinary education and research. He is a Professor in the School of Veterinary Medicine at the University of California at Davis, and has served as Dean. He was a member of the study group which originally advised on the establishment of ILRAD. Recent articles by ILRAD scientists Some articles published by ILRAD staff in the first half of 1984 are listed here, while all articles published in 1983 are listed in the 1983 annual report. For reprints or photocopies, contact the authors directly or send requests to ILRAD's Information Services. Please mention the ILRAD publication number, listed at the end of each reference. Copies of annual reports, publications lists and brochures describing ILRAD's research and training activities may also be obtained from Information Services. Single copies are sent without charge. Barry, J.D. and Emery, D.L. (1984). Parasite development and host responses during the establishment of Trypanosoma brucei infection transmitted by tsetse fly. Parasitology. 88: (304). Doyle, J.J., Moloo, S.K. and Borowy, N.K. (1984). Development of improved control methods for animal trypanosomiasis: a review. Preventive Veterinary Medicine. 2: (253). Gray, A.R. (1984). Activities of the International Laboratory for Research on Animal Diseases. Preventive Veterinary Medicine. 2: (245). Jack, R.M., Black, S.J., Reed, S.L. and Davis, C.E. (1984). Indomethacin promotes differentiation of Trypanosoma brucei. Infection and Immunity. 43: (192). Massamba, N.N. and Williams, R.O. (1984). Distinction of African trypanosome species using nucleic acid hybridization. Parasitology. 88: (270). Moloo, S.K. (1984). Trypanosoma vivax, T. congolense or T. brucei infection rates in Glossina morsitans when maintained in vitro on the blood of goat or calf. Acta Tropica. 41: (255). Morrison, W.I. (1984). Immune responses involved in immunity against Theileria parva.

11 Preventive Veterinary Medicine. 2: (242). Murray, Max, Trail, J.C.M., Davis, C.E. and Black, S.J. (1984). Genetic resistance to African trypanosomiasis. Journal of Infectious Diseases. 149: (257). Murray, Max and Gray, A.R. (1984). The current situation on animal trypanosomiasis in Africa. Preventive Veterinary Medicine. 2: (246). Murray, Max, Trail, J.C.M. and Grootenhuis, I.G. (1984). Trypanotolerant livestock: potential and future exploitation. Outlook on Agriculture. 13: (276). Nantulya, V.M., Musoke, A.J., Rurangirwa, F.R. and Moloo, S.K. (1984). Resistance of cattle to tsetse transmitted challenge with Trypanosoma brucei or Trypanosoma congolense after spontaneous recovery from syringe passaged infections. Infection and Immunity. 43: (247). First N'Dama calves born at ILRAD Scientists embarked on an embryo transfer project at the end of 1982, aimed at bringing a small number of trypanotolerant N'Dama cattle to ILRAD. The project has also provided information on the applicability of embryo transfer technology for the movement of trypanotolerant cattle across Africa. It was conceived primarily as a support activity to facilitate ILRAD's trypanosomiasis research program. According to ILRAD's Director General, 'it turned out to be a research project in its own right'. Some of ILRAD's earliest research, carried out between 1976 and 1979, emphasized the importance of the resistance to trypanosomiasis exhibited by N'Dama cattle in The Gambia. Since that time, ILRAD scientists have been involved in collaborative field studies with colleagues from the International Livestock Centre for Africa, concentrating primarily on the indigenous taurine cattle breeds of West and Central Africa the N'Dama and West African Shorthorn which are significantly more resistant to trypanosomiasis than Bos indicus or European Bos taurus breeds. While field studies are important, detailed research on the mechanisms of trypanotolerance requires a full complement of scientific staff and sophisticated equipment which is not available in the field. For this reason, scientists have been hoping for many years to have trypanotolerant cattle accessible for experiments at ILRAD. However, the movement of live animals from West Africa to Kenya is restricted by international regulations and is also extremely expensive. Purebred N'Dama cows and bulls selected by the Livestock Marketing Board and the Veterinary Department in The Gambia were mated and the embryos obtained were deep frozen for shipment to East Africa. These were surgically transferred into Boran heifers at ILRAD's ranch on the Kapiti plains in June The first N'Dama calf, a heifer, was born at ILRAD at the end of March In April, 4 more heifers and 5 bull calves were born. These are being raised at Kabete under the supervision of staff veterinarians. Scientists will be studying these animals to determine the mechanisms involved in resistance to trypanosomiasis both immunological and nonimmunological. Comparative studies will also be undertaken using resistant N'Dama and susceptible East African Zebu cattle. One goal is to find genetic markers which can be used to identify relatively resistant animals for livestock improvement programs. A better understanding of how some animals resist trypanosomiasis may also suggest promising methods for increasing the resistance of others.

12 ILRAD's first N'Dama calf, one day old, with her Boran foster mother. ILRAD Reports International Laboratory for Research on Animal Diseases P O Box 30709, Nairobi, Kenya telephone: Nairobi telex: 'ILRAD'