New Trypanosoma (Duttonella) vivax genotypes from tsetse flies in East Africa.

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FFLB identified the trypanosomes in 65 of 105 (61.9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main clade of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.

A novel, high-throughput technique for species identification reveals a new species of tsetse-transmitted trypanosome related to the Trypanosoma brucei subgenus, Trypanozoon.

We describe a novel method of species identification, fluorescent fragment length barcoding, based on length variation in regions of the 18S and 28Salpha ribosomal DNA. Fluorescently tagged primers, designed in conserved regions of the 18S and 28Salpha ribosomal DNA, were used to amplify fragments with inter-species size variation, and sizes determined accurately using an automated DNA sequencer. By using multiple regions and different fluorochromes, a barcode unique to each species was generated. The technique was developed for the identification of African tsetse-transmitted trypanosomes and validated using DNA from laboratory isolates representing known species, subspecies and subgroups. To test the methodology, we examined 91 trypanosome samples from infected tsetse fly midguts from Tanzania, most of which had already been identified by species-specific and generic PCR tests. Identifications were mainly in agreement, but the presence of an unknown trypanosome in several samples was revealed by its unique barcode. Phylogenetic analyses based on 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase gene sequences confirmed that this trypanosome is a new species and it is within the Trypanosoma brucei clade, as a sister group of subgenus Trypanozoon. The overall identification rate of trypanosome-infected midgut samples increased from 78 to 96% using FFLB instead of currently available PCR tests. This was due to the high sensitivity of FFLB as well as its capacity to identify previously unrecognised species. FFLB also allowed the identification of multiple species in mixed infections. The method enabled high-throughput and accurate species identification and should be applicable to any group of organisms where there is length variation in regions of rDNA.

The identification, diversity and prevalence of trypanosomes in field caught tsetse in Tanzania using ITS-1 primers and fluorescent fragment length barcoding.

We report on the development of two generic, PCR-based methods, which replace the multiple species-specific PCR tests used previously to identify the trypanosome species carried by individual tsetse flies. The first method is based on interspecies size variation in the PCR product of the ITS-1 region of the ribosomal RNA (rRNA) locus. In the second approach, length variation of multiple fragments within the 18S and 28S rRNA genes is assayed by PCR amplification with fluorescent primers; products are subsequently sized accurately and rapidly by the use of an automated DNA sequencer. Both methods were used to identify samples collected during large-scale field studies of trypanosome-infected tsetse in Tanzania in the National Parks of Tarangire and Serengeti, and the coastal forest reserve of Msubugwe. The fluctuations of trypanosome prevalence over time and two different field seasons are discussed. As well as facilitating the identification of trypanosome species with increased speed, precision and sensitivity, these generic systems have enabled us to identify two new species of trypanosome.

Seasonal variation in human African trypanosomiasis in Tarangire National Park in Babati district, Tanzania.

A survey was carried out to determine seasonal epidemiological variation of human African trypanosomiasis (HAT) in Tarangire National Park and villages around it in Babati District, Tanzania. Concentration and Field's stain techniques were employed to examine the presence of trypanosomes in human blood samples. Tsetse flies were collected using traps and dissected under light microscope to examine for presence of trypanosomes. Retrospective data on HAT were sought from health facilities. Blood samples were collected from a total 509 individuals (306 during the dry and 203 during wet seasons). None of the individuals was infected with trypanosomes in the area. A total of 766 tsetse flies were collected. Of these, Glossina swynnertoni accounted for 94.6% and G. pallidipes for 5.4% of the total collection. The largest proportion (63.8%) of the tsetse flies was collected during the wet season. Glossina swynnertoni was most abundant tsetse species during both wet and dry seasons. Salivary gland examination revealed the presence of Trypanosoma brucei type of infection in 3.2% of tsetse flies collected. All infective trypanosomes were found during the dry season. This study concludes that the transmission and prevalence of HAT among human population in Tarangire National Pars and its surrounding villages is low despite the recent reports on tourists acquiring the infection during their visits to the Park. However, disease surveillance needs to be strengthened to monitor any impending epidemic.

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

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 14.17 while that of G. pallidipes was between 0.23 and 9.70. 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.

Trypanosome identification in wild tsetse populations in Tanzania using generic primers to amplify the ribosomal RNA ITS-1 region.

Tsetse flies transmit many species of trypanosomes in Africa, some of which are human and livestock pathogens of major medical and socio-economic impact. Identification of trypanosomes is essential to assess the disease risk posed by particular tsetse populations. We have developed a single generic PCR test to replace the multiple species-specific PCR tests used previously to identify the trypanosome species carried by individual tsetse flies. In the generic PCR test, inter-species size variation in the PCR product of the internal transcribed spacer (ITS-1) region of the ribosomal RNA repeat region enables species identification. The test was applied to identify trypanosomes in midgut samples stored on FTA cards from wild-caught flies in two regions of Tanzania. Identifications were verified by sequencing the amplified ITS-1 region and/or species-specific PCR tests. The method facilitated the identification of large numbers of field samples quickly and accurately. Whereas species-specific tests are incapable of recognising previously unknown species, the generic test enabled a new species to be identified by the unique size of the amplified product. Thus, even without access to any isolate of this new species, we could collect data on its distribution, prevalence and co-occurrence with other trypanosomes. The combined molecular and ecological profiles should facilitate the isolation and full biological characterization of this species in the future.

Glossina swynnertoni (Diptera: Glossinidae): effective population size and breeding structure estimated by mitochondrial diversity.

Nucleotide diversity was examined at mitochondrial COI and r16S2 loci in eight Glossina swynnertoni Austen collections from northern Tanzania and from a culture maintained by the International Atomic Energy Agency. Eighteen composite haplotypes were observed among 149 flies, two of which were common to all samples and 10 were private. Mean haplotype diversity was 0.59 and nucleotide diversity was 0.0013. There were excess singular haplotypes and mutation-drift disequilibrium suggesting that populations had experienced an earlier bottleneck and subsequent expansion. Factorial correspondence analysis showed that haplotype frequencies varied much more temporally (G ST=0.18) than spatially (G ST=0.04). The estimate of effective population size N e in Tarangire was a harmonic mean approximately 50 reproductive flies averaged over approximately 47 generations. The mean rate of gene flow was estimated to be approximately 5+/-1 reproducing females per generation but inflated because of mutation-drift disequilibrium arising from likely earlier bottlenecks.

Knowledge, attitudes and practices on tsetse and sleeping sickness among communities living in and around Serengeti National Park, Tanzania.

A study was undertaken to investigate knowledge, attitudes and practices about sleeping sickness (human African trypanosomiasis) among communities living in and around Serengeti National Park (SENAPA). Structured questionnaires were administered to a total of 1490 consenting participants. Of the respondents, 924 (62%) knew sleeping sickness, and 807 (87.3%) knew the right place to seek healthcare. Of 924 who knew sleeping sickness, 386 (42%) said the disease was present in the areas they live. Most respondents (85.4%) knew that sleeping sickness infections were acquired in the bush and forest. The most common (69.3%) sources of information about sleeping sickness were relatives and friends. Symptoms of sleeping sickness mentioned included abnormal sleep (45.2%), fever (35.3%), body malaise (14.5%), headache (7.6%) and lymph node enlargement (6.1%). Of 1490 people interviewed 90.4% knew tsetse flies and 89.8% had been bitten by tsetse flies. The majority (86.6%) of the respondents knew that sleeping sickness is transmitted through a tsetse bite. Activities that exposed people to tsetse bites included working in tsetse infested bushes/forests, grazing livestock in tsetse infested areas and hunting game animals. In conclusion, communities living in and around SENAPA were knowledgeable about tsetse and sleeping sickness. The communities can thus understand and support community based tsetse and sleeping sickness control programmes to ensure success.

Human African Trypanosomiasis and challenges to its control in Urambo; Kasulu and Kibondo Districts; western Tanzania

A study was carried out to determine the prevalence and management of Human African Trypanosomiasis (HAT) in Urambo; Kasulu and Kibondo districts of western Tanzania. Parasitological surveys for trypanosome and other blood parasites were conducted in selected villages. Interviews with health workers were conducted to explore facility capacity to diagnose and manage HAT. Community knowledge on tsetse and availability of trypanocidal drugs was explored. Results showed that; although health facility records showed HAT is an important public health problem in the three districts; typanosomes were found in 0.6of the examined individuals in Urambo district only. Malaria parasites with a prevalence of 12.1; 19.7and 9.7; in Urambo; Kibondo and Kasulu; respectively were detected in blood samples from the same individuals examined for trypanosomes. There was poor capacity for most of the health facilities in the diagnosis; treatment and control of HAT. In both districts; communities were knowledgeable of the tsetse identity (82.4) and had experienced tsetse bites (94). The majority (91.4) of the community members knew that they were at risk of acquiring HAT. However; only 29of the respondents knew that anti-trypanocidal drugs were readily available free of charge from health care facilities. Late treatment seeking behaviour was common in Kasulu and Urambo districts. In conclusion; health facilities in western Tanzania are faced with problems of poor capacity to diagnose and manage HAT and that treatment seeking behaviour among the communities at risk is poor. Efforts should be made to strengthen the capacity of the health facility to handle HAT cases and health education to the population at risk

Mating age of Glossina austeni Newstead.

Before removal from the emergence cage, 12.8% of 141 newly emerged females of Glossina austeni Newstead less than 24 h old were found on dissection to have been inseminated. Likewise, dissection of a sample of sterilised females destined for release showed that 5.43% of 2487 females had already been inseminated while still in the emergence cages. It was decided therefore to put female and male flies together in production cages from the day of emergence at a ratio of 1 male to 5 females and leave them to mature and mate in the cages. The females produced viable pupae of acceptable mean weight and desired quality with the proportion of A-Class pupae less than 10%. The number of pupae per initial female (PPIF) did not differ from pupae produced by pre-aged parent flies. It is now clear that there is no need to age female and male flies of G. austeni before mating. Parent flies of less than 1 day old put together in production cages from the day of emergence have been used for mass rearing G. austeni in the Tsetse and Trypanosomosis Research Institute (TTRI) colony to produce males for the eradication programme in Zanzibar since December 1995. This has substantially reduced the labour of fly production by removing the need to age flies and the need to chill and separate flies after mating.