Glossina dynamics in and around the sleeping sickness endemic Serengeti ecosystem of northwestern Tanzania

Transcription

1 Vol. 32, no. 2 Journal of Vector Ecology 263 Glossina dynamics in and around the sleeping sickness endemic Serengeti ecosystem of northwestern Tanzania I. I. Malele 1, S. M. Kinung'hi 2, H. S. Nyingilili 1, L. E. Matemba 3, J. K. Sahani 3, T. D. K. Mlengeya 4, M. Wambura 2, and S. N. Kibona 3 1 Tsetse & Trypanosomiasis Research Institute (TTRI), P. O. Box 1026, Tanga, Tanzania 2 National Institute for Medical Research, Mwanza Centre, P. O. Box 1462, Mwanza, Tanzania 3 National Institute for Medical Research, Tabora Centre, P. O. Box 482 Tabora, Tanzania 4 Veterinary Unit, Tanzania National Parks (TANAPA), P. O. Box 3134 Arusha, Tanzania Received 16 March 2007; Accepted 13 June 2007 ABSTRACT: We investigated the dynamics of Glossina spp. and their role in the transmission of trypanosomiasis in the sleeping sickness endemic Serengeti ecosystem, northwestern Tanzania. The study investigated Glossina species composition, trap density, trypanosome infection rates, and the diversity of trypanosomes infecting the species. Tsetse were trapped using monopyramidal traps in the mornings between 06:00 to 11:00 and transported to the veterinary laboratory in Serengeti National Park where they were sorted into species and sex, and dissected microscopically to determine trypanosome infection rates. Age estimation of dissected flies was also conducted concurrently. Tsetse samples positive for trypanosomes were subjected to PCR to determine the identity of the detected trypanosomes. Out of 2,519 tsetse trapped, 1,522 (60.42%) were G. swynnertoni, 993 (39.42%) were G. pallidipes, three (0.12%) were G. m. morsitans, and one (0.04%) was G. brevipalpis. The trap density for G. swynnertoni was between 1.40 and while that of G. pallidipes was between 0.23 and Out of 677 dissected G. swynnertoni, 63 flies (9.3%) were infected, of which 62 (98.4%) were females. A total of 199 G. pallidipes was also dissected but none was infected. There was no significant difference between the apparent densities of G. swynnertoni compared to that of G. pallidipes (t = 1.42, p = 0.18). Molecular characterization of the 63 infected G. swynnertoni midguts showed that 19 (30.2%) were trypanosomes associated with suid animals while nine (14.3%) were trypanosomes associated with bovid animals and five samples (7.9%) had T. brucei s.l genomic DNA. Thirty (47.6%) tsetse samples could not be identified. Subsequent PCR to differentiate between T. b. brucei and T. b. rhodesiense showed that all five samples that contained the T. brucei s.l genomic DNA were positive for the SRA molecular marker indicating that they were T. b. rhodesiense. These results indicate that G. swynnertoni plays a major role in the transmission of trypaniosomiasis in the area and that deliberate and sustainable control measures should be initiated and scaled up. Journal of Vector Ecology 32 (2): Keyword Index: Glossina, trypanosome, tsetse density, infection rate, Serengeti ecosystem. INTRODUCTION Trypanosoma brucei, a protozoan parasite transmitted by the bite of the tsetse fly (Glossina spp) is of great medical and economic interest in Africa. It causes human sleeping sickness, and nagana, a cattle disease that prevents cattle farming and the use of work animals over huge areas of sub-saharan Africa (Leak 1999). Only the Trypanosoma brucei rhodesiense form of human disease occurs in Tanzania (Kibona et al. 2006). The epidemiology of T. b. rhodesiense is subject to interactions among human beings, tsetse flies (number of infected tsetse flies), and domestic and wild animals in a specific area. Knowledge of the dynamics of tsetse flies is essential in order to understand the relationship between tsetse (as a vector) and its hosts, which in turn clarifies the role that tsetse play in disease transmission, which may be useful for determination of trypanosomiasis hot spots. Determined vector hot spot sites and infection status can be instrumental in making decisions with regard to the formulation of sustainable tsetse control strategies. Tsetse flies are the only known transmitters of trypanosomes. The presence of large concentrations of wild animals surrounded by big herds of domestic animals in the northern circuit of Tanzania ensures the possibilities for transmission of the disease to humans, as domestic animals (cattle and pigs) have been known to be reservoir hosts of T. b. rhodesiense in the Rhodesian endemic areas (Welburn et al. 2001, Waiswa et al. 2003). The study was conducted in Serengeti National Park (SENAPA) following a recent upsurge of sleeping sickness among tourists visiting Serengeti National Park when nine tourists contracted the disease on different occasions (Jelinek et al. 2002). This raised concern at national and international levels on the safety of Serengeti National Parks as a famous tourist destination (Ripamonti et al. 2002, Jenelik et al. 2002). The study was conducted in order to understand the dynamics of tsetse flies through the determination of tsetse species composition, trap density, infection rates, and the identity of trypanosomes infecting the vectors in and around SENAPA.

2 264 Journal of Vector Ecology December 2007 MATERIALS AND METHODS Study area and population The study was conducted in five selected sites within SENAPA and two villages adjacent to the park. Within SENAPA, tsetse flies were sampled in sites which are frequently visited by tourists. These include Retima Hippo Pool, Death Valley, Sopa Lodge, Simiyu Post, and Kilimafedha. Villages outside the park included Robanda/ Ikoma gate in Serengeti district and Makao Meatu district. These two villages/settlements, though outside the park, share the same ecological system with SENAPA. The study population consisted of tsetse flies in the study area. Each fly trapping site was geo-referenced. Within SENAPA, wild animals frequently seen around the trapping sites at the time of study were zebras, giraffes, elephants, gazelle, warthogs, buffaloes, and bush bucks. Domestic animals seen in villages surrounding the park included cattle, sheep, goats, donkeys, pigs, and dogs. Wild animals like zebras, gazelle, and bush bucks were commonly seen roaming in the nearby villages. Tsetse fly sampling Tsetse flies were sampled using the modified monopyramidal traps (Gouteux and Lancien 1986) which have shown to be effective in trapping different tsetse species in different habitats of Tanzania. The sampling study was conducted during the onset of the dry seasons of 2005 and 2006 in the months of August-September and November Monopyramidal traps were deployed for a total of 15 days (five days each time the study was conducted) at a distance of 150 m apart and baited with attractants (acetone and 3-n-propylphenol 4-methyl-phenol and octenol at a ratio of 1:8:4) in order to enhance trap efficiency. Two traps were used per site and were deployed in a shaded area in order to avoid fly mortality (Hargrove and Langley 1990). Flies were harvested at about 11:00 local time daily and taken to the veterinary laboratory in SENAPA where they were sorted out into respective species and dissected microscopically. Determination of tsetse trap density and age The tsetse trap density was calculated as indicated in the FAO Tsetse Training Manual (1979) as the number of tsetse flies trapped per trap per day. Age estimation for both male and female flies were conducted as indicated in the FAO Tsetse Training Manual (1979) and Saunders (1962). Male fly ages were estimated based on the wing fray analysis, whereas for females, age was estimated based on the configuration of the ovary. Determination of infection rates Dissection was conducted to determine the infection in tsetse fly mouth parts, midguts, and salivary glands as described in the FAO Tsetse Training Manual (1979). This method makes use of trypanosome developmental sites in the tsetse flies since it is difficult to identify trypanosomes morphologically. Dissection was conducted on a 0.9% saline solution for live flies only. Each organ was examined using a compound microscope at 400x maginification. Parasites found in mouth parts and midguts were recorded as Nannomonas, those found in the mouth parts, midgut, and salivary gland were considered as Trypanozoon, and those found in the mouth parts only were regarded as Duttonella. Molecular characterization of trypanosomes detected in tsetse midguts Blood meals of infected tsetse flies were smeared onto the Whatmann filter paper No. 1, sun dried, and washed in acetone for five min and then air dried and stored at -20 C according to Boid et al. (1999). Only blood meals from infected tsetse (determined by dissection of organs like mouthparts and salivary glands) were collected, as it would have been too costly to analyze all blood meals randomly and not based on the infection of other organs. At the Tsetse and Trypanosomiasis Research Institute laboratory, hot elution was carried out as described by Boid et al. (1999) and elutes were used for PCR analyses of the presence of T. brucei s.l. DNA. PCR analyses were carried out using primers described by Moser et al. (1989). A 25µ PCR reaction contained a 1 Units BIOTAQ Red TM DNA polymerase (Bioline), 1 NH 4 buffer with a final concentration of 1.5 mm MgCl 2, 0.2 µm of each dntp and 0.4 µm of each primer, with 1 µl of hot eluted solution. PCR reactions were followed as indicted by Picozzi et al. (2002). The denaturation step of 95 C for 3 min was followed by 35 cycles of 94 C for 20 s, 55 C for 30 s, 72 C for 30 s, plus a final extension step of 5 min. Samples confirmed as T. brucei s.l. were subsequently analyzed by PCR to differentiate between T. b. brucei and T. b. rhodesiense infections (Welburn et al. 2001). PCR products were visualized through electrophoresis on 1.5% (w/v) agarose containing 0.5 µg/ml Ethidium bromide. Positive signals were confirmed by Southern blotting as in Gibson et al. (2002). RESULTS Tsetse species composition in and around the Serengeti ecosystem The composition of tsetse fly species in and around SENAPA is as shown in Table 1. A total of 2,519 tsetse flies was trapped from the seven sites of which 1,522 (60.42%) were G. swynnertoni, 993 (39.42%) were G. pallidipes, three (0.12%) were G. m. morsitans and one (0.04%) was G. brevipalpis. The numbers of G. m. morsitans and G. brevipalpis were very low and were excluded from further analysis. More males of the two species G. swynnertoni and G. pallidipes were trapped (58.9% and 57.2%) than females (41.1% and 42.8%), respectively. Tsetse trap densities (tsetse/trap/day) in different sites Tsetse trap densities from different study sites are presented in Table 2. There was no significant difference between the tsetse trap densities of G. swynnertoni compared to that of G. pallidipes (t = 1.42, p = 0.18). The trap densities of G. swynnertoni was the lowest at Sopa Lodge (1.40) followed by Simiyu Post (3.57). Trap density was highest at

3 Vol. 32, no. 2 Journal of Vector Ecology 265 Table 1. Tsetse fly population composition by species and sex in SENAPA and surrounding areas (n = 2,515). Trapping site G. swynnertoni G. pallidipes Females Males Total Females Males Total Retima Hippo Pool Robanda/Ikoma Gate Sopa Lodge Kilimafedha Simiyu post Death Valley Makao Total 625 (41.06%) 897 (58.94%) 1, (42.80%) 568 (57.20%) 993 Kilimafedha (11.20) within SENAPA. Outside SENAPA, the trap density of G. swynnertoni was higher at Makao (14.17) than at Robanda/Ikoma gate (8.53). For G. pallidipes, the trap density was lowest at Kilimafedha (2.70) and highest at Retima Hippo Pool (9.70). Outside SENAPA, the trap density was lowest at Makao (0.23). Age composition of the tsetse species trapped in SENAPA and surrounding areas The average age of the trapped male flies within SENAPA was 17 days, whereas outside SENAPA, the average age was 23 days. Most female flies trapped within SENAPA were aged between days, whereas at Makao and Robanda/Ikoma gate (outside SENAPA) most females were aged above 30 days. At Makao, females with an estimated age above 51 days were about 50% (Table 3). Trypanosome infection rates in tsetse flies The trypanosome infection rates for dissected tsetse flies are given in Table 4. A total of 876 tsetse flies were dissected, of which 677 (77.3%) were G. swynnertoni and 199 (22.7%) were G. pallidipes. Out of the 677 dissected G. swynnertoni, 63 (9.3%) were found infected, of which 62 were females and only one male G. swynnertoni. None of 199 dissected G. pallidipes flies were found to be infected. The overall infection rate in the northern zone was 9.3%. Within SENAPA the infection rate was 7.5%, whereas outside SENAPA at Makao and Robanda/Ikoma gate, the infection rate was 16.3% and 7.4%, respectively. Trypanosomes identified belonged to the Dutonella (vivaxtype) group (4.3%), Nannomonas (congolense-type) group (2.07%), and Trypanozoon (brucei-type) group (2.95%). The infection rate was highest at Makao settlement which is outside SENAPA. Diversity of trypanosomes detected in tsetse midguts Molecular characterization of trypanosomes found in the midgut of the 63 tsetse samples showed that nine (14.3%) had trypanosomes which are associated with bovid animals. These include T. vivax, T. congolense savannah, and Kilifi. In addition, 19 (30.2%) of the samples had trypanosomes associated with suid animals including T. simiae, T. simiae tsavo, and T. godfreyi. Only five (7.9%) of the samples had T. brucei s.l. genomic DNA. Thirty (47.6%) samples could not be identified. Subsequent PCR to differentiate between T. b. brucei and T. b. rhodesiense showed that all five samples were positive for the SRA molecular marker, indicating that the samples were T. b. rhodesiense. The SRA-PCR product on the five T. brucei s.l DNA positive samples were sequenced and aligned by the DNAstar and showed a homology of 100% with the published SRA gene deposited in the Gene bank with ascension numbers Z37159 and AJ (Gibson et al. 2002). DISCUSSION We report on the dynamics of Glossina spp and the role they play in the transmission of trypanosomiasis. The study was a follow-up to the recent increase in the number of tourists who contracted sleeping sickness when visiting the northern parks of Tanzania (Ripamonti et al. 2002, Jelinek et al. 2002). G.swynnertoni was found to be the most widespread and abundant of the tsetse species in the area followed by G. pallidipes. Our findings show that G. swynnertoni is a species to be considered in the epidemiology of sleeping sickness in the area. This is clearly shown by its higher proportion of species composition and trypanosome infection. Our results are contrary to the findings which were obtained by Rogers and Boreham (1973) that failed to establish role of the species as a vector in the transmission cycle of Trypanosoma brucei subgroup. In that study, a total number of 7,000 flies were dissected and none was found to have salivary gland mature infection. G. swynnertoni was also the most abundant species in areas surrounding SENAPA that shared the same ecosystem. The supremacy of this species has not changed even after three decades (Rogers

4 266 Journal of Vector Ecology December 2007 Table 2. Apparent density (AD) of tsetse fly species in SENAPA and surrounding areas (n = 2,515). Trapping site Total trapped Overall AD + SD AD by species + SD G. swynnertoni G. pallidipes Retima Hippo Pool Sopa Kilimafedha Simiyu post Death Valley Robanda/Ikoma Gate Makao Total/ mean 2, ± ± ± 3 Table 3. Age composition of G. swynnertoni trapped in sites within and outside SENAPA (n = 625). Number of Ovulations Estimated age in days Within SENAPA (%) Makau (Outside SENAPA) (%) (9.00) 10 (5.53) (19.82) 10 (5.53) (33.78) 15 (8.29) (22.52) 31 (17.12) (8.11) 25 (13.8) >5 >51 30 (6.76) 90 (49.72) Total Table 4. Trypanosome infection rates in G. swynnertoni (by microscopic examination) by site within SENAPA and surrounding area (n = 677). Trapping sites Total dissected Total infected (%) Trypanosome types vivax congolense brucei Retima H. P Sopa 42 4 (9.3) Kilimafedha (13.7) Simiyu post 19 1 (5.3) Death Valley 98 7 (7.1) Robanda/Ikoma Gate (7.4) Makao (16.3) Overall total (9.3%) 29 (4.3%) 14 (2.07%) 20 (2.95%)

5 Vol. 32, no. 2 Journal of Vector Ecology 267 and Boreham 1973), despite the fact that density differs from one area to the other depending on the vegetation cover. Molecular analysis of collected tsetse midguts showed that 7.9% had the T. brucei s.l genomic DNA which were all positive to the SRA gene molecular marker, an indication that the human infective trypanosomes are circulating in SENAPA and surrounding areas and that G. swynnertoni is the major vector. Studies by Kaare et al. (2007) in the same area indicated that warthogs were important reservoirs of T. b. rhodesiense, with a prevalence of 9.5%. It is known that G. swynnertoni feeds preferentially on warthogs (FAO 1979, Clausen et al. 1998). Rogers and Boreham (1973) reported that 40% of the 47 G. swynnertoni blood meals analyzed were obtained from warthogs, 36% from buffalo, 8.5% from the cat family, 2% each from giraffe and hartebeest, and 2% from avians. Although no blood meal analysis was conducted in this study, there is no doubt that the warthog is still the preferred host of G. swynnertoni in the area according to findings by Kaare et al. (2007). We further hypothesize that G. swynnertoni resorts to domestic animals like cattle as an alternative source of blood meal when warthogs are not available. This hypothesis is supported by the findings of Kaare et al. (2007), where the prevalence of T. brucei s.l in cattle at Robanda was 11.5%. During our study, it was common to see livestock grazing on the periphery of SENAPA and to find tsetse in the human settlement outside SENAPA. Wild animals act as reservoirs of the parasite while tsetse flies constitute the bridge to cattle and humans especially when cattle graze adjacent to protected areas. The existing interaction between wild animals and livestock in the presence of tsetse flies poses a risk to rural communities and park visitors. Trypanosomes that cause trypanosomiasis in domestic animals like T. vivax, T. congolense, T. simiae, and T. godfreyi were also detected in the midguts analyzed. About half of the samples of infected tsetse flies could not be identified, probably due to degradation of the DNA, or very little DNA was extracted from the filter paper so that there was no amplification, or these samples contained some unknown trypanosomes (Malele et al. 2003) of which primers are not available at the moment. A similar study by Adams et al. (2006) revealed the presence of T. godfreyi-like trypanosomes in the northern zone by making use of the generic primers. Hence there is a probability that the bulk of unidentified samples in this study could contain T. godfreyi -like trypanosomes (Adams et al. 2006). In this study, 62 (98.4 %) out of the 63 infected tsetse flies were females. A similar observation was made by Waiswa et al. (2006) in Uganda where only female flies were found to be infected. This could be attributed to the age of male and female tsetse flies at the time of the study. The trapped male flies were on average 11 days old or less, while infection takes between 5-53 days to mature. In SENAPA and surrounding areas, the average estimated age (in days) was 17 and 23, respectively. This could explain why all dissected male flies (except one) were not infected. On the other hand, most female flies in SENAPA were aged between days, whereas at Makao most females were aged above 30 days. At Makao, about 50% of the female flies were older than 51 days. The longer the fly survives, the higher the probability of being infected. The differences in age of tsetse flies between SENAPA and Makao might have been caused by rigorous tsetse control measures which were implemented in SENAPA by deployment of insecticide-impregnated targets following the flare-up of sleeping sickness among tourists (Jelinek et al. 2002). No tsetse control campaigns were implemented in Makao, which in turn might explain the high percentage of flies aged above 30 days. G. swynnertoni is a species of concern in SENAPA and surrounding areas. The species plays a major role in the transmission of African trypanosomiasis. Apart from harboring the human infective trypanosomes, the species also carries trypanosomes that cause animal Trypanosomiasis in bovid animals like T. vivax and T. congolense and in suids like T. simiae and T. godfreyi (Lehane et al. 2000). This species was the most dominant in terms of overall tsetse density within and outside SENAPA. Furthermore, all infected tsetse flies were G. swynnertoni and not G. pallidipes, despite the fact that the latter species was dissected in large numbers. To break the transmission cycle of trypanosomes to both humans and livestock in the area, there is an urgent need for tsetse control activities in the Serengeti ecosystem. The strategy of choice to control G. swynnertoni and other tsetse species is deployment of insecticide-impregnated targets in all important sites. Furthermore, since G. swynnertoni are low flyers and easily attracted to moving objects, the lower parts of all vehicles in and around SENAPA should be sprayed with insecticides to control flies attempting to land on the vehicles. All preferred ranger posts, such as Simiyu, and walls surrounding houses should also be sprayed with insecticides to kill all flies to reduce humanfly contact. To reduce or eliminate the number of domestic animal reservoirs, control measures should be extended to livestock in communities around SENAPA by periodic prophylactic treatments using trypanocides. Awareness campaigns focusing on sleeping sickness transmission, signs and symptoms as well as treatment, prevention, and control should be launched particularly in communities surrounding SENAPA. Further studies focusing on trypanosomiasis risk assessment and blood meal analysis need to be carried out to collect updated information on the epidemiology of the disease and on the feeding preferences of G. swynnertoni and G. pallidipes in order to establish the role played by preferred hosts in disease transmission. Acknowledgments We acknowledge the logistic support provided by the Directors for NIMR-Mwanza Centre and TTRI-Tanga during the planning and implementation phase of this study. We also thank the Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Austria, for training and all support related to PCR protocols for TTRI staff. Our sincere gratitude also goes to the Tanzania National Park Authority (TANAPA) and particularly the staff of Serengeti National Park Veterinary Laboratory for providing a space and assistance

6 268 Journal of Vector Ecology December 2007 during preliminary analysis of tsetse samples. We also thank the District Agriculture and Livestock Office (DALDO) for Meatu district in Shinyanga region for assistance during fieldwork. This study received financial support from the Tanzania Ministry of Health and Social Welfare (MoHSW) through the National Institute for Medical Research (NMIR), Mwanza Centre. REFERENCES CITED Adams, E.R., I.I. Malele, A.R. Msangi, and W.C. Gibson Novel species of Trypanosoma from wild tsetse populations in Tanzania identified using generic primers to amplify the ITS-1 region. Acta Trop. 100: Boid, R., T.W. Jones, and A. Munro A simple procedure for the extraction of trypanosome DNA and host protein from dried blood meal residues of haematophagous. Diptera.Vet. Parasitol. 85: Clausen, P.H., I. Adeyemi, B. Bauer, M. Breloeer, F. Salchow, and C. Staak Host preferences of tsetse (Diptera: Glossinidae) based on blood meal identifications. Med. Vet. Entomol. 12: FAO Training Manual for Tsetse Control Personnel. Food and Agriculture Organisation of the United Nations (FAO), Rome. Gibson, W., T. Backhouse, and A. Griffiths The human serum resistance associated gene is ubiquitous and conserved in Trypanosoma brucei rhodesiense throughout East Africa. Inform. Genom. Evol. 1: Gouteux, J.P. and J. Lancien Le piege pyramidal a tsetse (Diptera, Glossinidae) pour la capture et la lutte. Essaias comparatives et description de nouveaux systems de capture. Trop. Med. Parasitol. 37: Hargrove, J.W. and P.A. Langley Sterilizing tsetse in the field: a successful field trial: Bull. Entomol. Res. 80: Jelinek, T., Z. Bisoffi, L. Bonazzi, P. Van Thiel, U. Bronner, A. Frey, S. Gundersen, P. McWhinney, and D. Ripamonti Cluster of African trypanosomiasis in travelers to Tanzanian national parks. Emerg. Infect. Dis. 8: Kaare, M. T., K. Picozzi, E. Fèvre, S. Cleaveland, M. Mtambo, L. Mellau, T. Mlengeya, and S. Welburn Sleeping sickness a re-emerging disease in the Serengeti? Trav. Med. Infect. Dis. 5: Kibona, S.N., K. Picozzi, L. Matemba, and G.W. Lubega Characterization of the Trypanosoma brucei rhodesiense isolates using serum resistance associated (SRA) gene as molecular marker. Proceedings of the 8 th Annual EANETT Conference, Kampala Uganda, 18 th - 20 th September, 2006, Africana Hotel, Kampala, Uganda. Leak, S.G.A Tsetse Biology and Ecology. Their Role in the Epidemiology and Control of Trypanosomosis. CAB International, Oxford, pp Lehane, M. J., A. R. Msangi, C. J. Whitaker, and S. M. Lehane Grouping of trypanosome species in mixed infections in Glossina pallidipes: Parasitology 120: Lloyd, L. and W.B. Johnson The trypanosome infections of tsetse flies in northern Nigeria and a new method of estimation: Bull. Entomol. Res. 14: Malele, I., L. Craske, C. Knight, V. Ferris, Z. Njiru, P. Hamilton, S. Lehane, M. Lehane, and W. Gibson The use of specific and generic primers to identify trypanosome infections of wild tsetse flies in Tanzania by PCR. Infect. Genet. Evol. 3: Masiga, D.K., J.J. McNamara, and W.C. Gibson A repetitive DNA sequence specific for Trypanosoma (Nannomonas) godfreyi: Vet. Parasitol. 62: Moser, D.R., G.A. Cook, D.E. Ochs, C.P. Bailey, M.R. McKane, and J.E. Donelson Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain-reaction. Parasitology 99: Picozzi, K., A. Tilley, E.M. F`evre, P.G. Coleman, J.W. Magona, M. Odiit, M.C. Eisler, and S. Welburn The diagnosis of trypanosome infections: applications of novel technology for reducing disease risk. Afr. J. Biotechnol. 1: Ripamonti, D., M. Massari, C. Arici, E. Gabbi, C. Farina, M. Brini, C. Capatti, and F. Suter African sleeping sickness in tourists returning from Tanzania: the first 2 Italian cases from a small outbreak among European travelers. Clin. Infect. Dis. 34: E Rogers, D. and P.F.L Boreham Sleeping sickness in the Serengeti area Tanzania 1971, Part 2. The vector role of Glossina swynnertoni. Acta Trop. 30: Saunders, D.S Age determination for female tsetse flies and the age composition of samples of Glossina pallidepes Aust., G.palpalis fuscipes Newst. and G. brevipalpis Newst. Bull. Entomol. Res. 53: 579. Waiswa, C., W. Olaho-Mukani, and E. Katunguka- Rwakishaya Domestic animals as reservoirs for sleeping sickness in three endemic foci in south-eastern Uganda. Ann. Trop. Med. Parasitol. 97: Waiswa, C., K. Picozzi, E. Katunguka-Rwakishaya, W. Olaho-Mukani, R.A. Musoke, and S.C. Welburn Glossina fuscipes fuscipes in the trypanosomiasis endemic areas of south eastern Uganda: Apparent density, trypanosome infection rates and host feeding preferences. Acta Trop. 99: Welburn, S.C., K. Picozzi, E.M. Fevre, P.G. Coleman, M. Odiit, M. Carrington, and I. Maudlin Identification of human-infective trypanosomes in animal reservoir of sleeping sickness in Uganda by means of serum-resistance-associated (SRA) gene. Lancet: 358: