Blood feeding tsetse flies as hosts and vectors of mammals-pre-adapted African Trypanosoma: current and expected research directions.

Research on the zoo-anthropophilic blood feeding tsetse flies' biology conducted, by different teams, in laboratory settings and at the level of the ecosystems- where also co-perpetuate African Trypanosoma- has allowed to unveil and characterize key features of tsetse flies' bacterial symbionts on which rely both (a) the perpetuation of the tsetse fly populations and (b) the completion of the developmental program of the African Trypanosoma. Transcriptomic analyses have already provided much information on tsetse fly genes as well as on genes of the fly symbiotic partners Sodalis glossinidius and Wigglesworthia, which account for the successful onset or not of the African Trypanosoma developmental program. In parallel, identification of the non- symbiotic bacterial communities hosted in the tsetse fly gut has recently been initiated: are briefly introduced those bacteria genera and species common to tsetse flies collected from distinct ecosystems, that could be further studied as potential biologicals preventing the onset of the African Trypanosoma developmental program. Finally, future work will need to concentrate on how to render tsetse flies refractory, and the best means to disseminate them in the field in order to establish an overall refractory fly population.

Bacterial diversity obtained by culturable approaches in the gut of Glossina pallidipes population from a non sleeping sickness focus in Tanzania: preliminary results.

BACKGROUND: Glossina pallidipes is a haematophagous insect that serves as a cyclic transmitter of trypanosomes causing African Trypanosomiasis (AT). To fully assess the role of G. pallidipes in the epidemiology of AT, especially the human form of the disease (HAT), it is essential to know the microbial diversity inhabiting the gut of natural fly populations. This study aimed to examine the diversity of G. pallidipes fly gut bacteria by culture-dependent approaches. RESULTS: 113 bacterial isolates were obtained from aerobic and anaerobic microorganisms originating from the gut of G. pallidipes. 16S rDNA of each isolate was PCR amplified and sequenced. The overall majority of identified bacteria belonged in descending order to the Firmicutes (86.6%), Actinobacteria (7.6%), Proteobacteria (5.5%)and Bacteroidetes (0.3%). Diversity of Firmicutes was found higher when enrichments and isolation were performed under anaerobic conditions than aerobic ones. Experiments conducted in the absence of oxygen (anaerobiosis) led to the isolation of bacteria pertaining to four phyla (83% Firmicutes, 15% Actinobacteria, 1% Proteobacteria and 0.5% Bacteroidetes, whereas those conducted in the presence of oxygen (aerobiosis) led to the isolation of bacteria affiliated to two phyla only (90% Firmicutes and 10% Proteobacteria). Phylogenetic analyses placed these isolates into 11 genera namely Bacillus, Acinetobacter, Mesorhizobium, Paracoccus, Microbacterium, Micrococcus, Arthrobacter, Corynobacterium, Curtobacterium, Vagococcus and Dietzia spp.which are known to be either facultative anaerobes, aerobes, or even microaerobes. CONCLUSION: This study shows that G. pallidipes fly gut is an environmental reservoir for a vast number of bacterial species, which are likely to be important for ecological microbial well being of the fly and possibly on differing vectorial competence and refractoriness against AT epidemiology.

Enhancing vector refractoriness to trypanosome infection: achievements, challenges and perspectives.

With the absence of effective prophylactic vaccines and drugs against African trypanosomosis, control of this group of zoonotic neglected tropical diseases depends the control of the tsetse fly vector. When applied in an area-wide insect pest management approach, the sterile insect technique (SIT) is effective in eliminating single tsetse species from isolated populations. The need to enhance the effectiveness of SIT led to the concept of investigating tsetse-trypanosome interactions by a consortium of researchers in a five-year (2013-2018) Coordinated Research Project (CRP) organized by the Joint Division of FAO/IAEA. The goal of this CRP was to elucidate tsetse-symbiome-pathogen molecular interactions to improve SIT and SIT-compatible interventions for trypanosomoses control by enhancing vector refractoriness. This would allow extension of SIT into areas with potential disease transmission. This paper highlights the CRP's major achievements and discusses the science-based perspectives for successful mitigation or eradication of African trypanosomosis.

Determination of the prevalence of trypanosome species in cattle from Monduli district, northern Tanzania, by loop mediated isothermal amplification.

Bovine African trypanosomosis (BAT) remains one of the major vector-borne diseases with serious impediment to cattle production and economic advancement in sub-Saharan Africa. The present study evaluated the performance of the trypanosome-species-specific loop-mediated isothermal amplification (LAMP), using parasite DNA obtained from 295 indigenous Tanzanian short horn Zebu (TSHZ) and Boran crosses in Monduli district within northern Tanzania, against routine microscopy on Giemsa-stained blood films. Compared to parasitological data in which the prevalence of BAT was estimated at 2.4% (95% CI 0.7-4.1%), LAMP increased the prevalence to 27.8% (95% CI 22.3-32.5%), of which 11.9% (95% CI 8.2-15.6%) were monolytic infections with Trypanosoma vivax, while 13.6% (95% CI 9.7-17.5%) were coinfections of either T. vivax and Trypanosoma brucei subspecies or T. vivax and Trypanosoma congolense, respectively. Among the T. brucei subspecies detected, 0.7% (95% CI 0-1.7%) were human-infective Trypanosoma brucei rhodesiense. Our study is in concordance with previous reports and suggests that LAMP is a potential tool for routine diagnosis of trypanosomes in domestic animals in BAT endemic regions. According to LAMP, T. vivax seems to be the predominant trypanosome species circulating among the indigenous Monduli cattle. Importantly, the detection of T. b. rhodesiense in cattle in such wildlife-domestic-animal-human-interface areas poses a risk of contracting human African trypanosomiasis (HAT) by local communities and tourists. Continuous trypanosome surveillances in domestic animals, humans, and tsetse flies using sensitive and specific tests such as LAMP are recommended.

Prevalence of SGHV among tsetse species of economic importance in Tanzania and their implication for SIT application.

Sterile Insect technique is an important component in area-wide integrated tsetse control. The presence of the salivary glands hypertrophy virus (SGHV) in the wild tsetse, which are the seeds for colony adaptations in the laboratory has become a stumbling block in establishing and maintaining colonies in the laboratory. The virus is transmitted both vertically (in the wild) and horizontally (in the laboratory). However, its prevalence is magnified in the laboratory as a result of the use of in vitro membrane feeding regimen. Fly species of Glossina fuscipes fuscipes, G. pallidipes, G. morsitans and G. swynnertoni were collected from the coastal and inland areas of Tanzania and virus infection rates were assessed microscopically and by PCR. The data showed that in a period of 4years, the virus was present in all species tested irrespective of their ages, sex, and season of the year. However, infection levels differed among species and from one location to another. Symptomatic infection determined by dissection was 1.2% (25/2164) from the coast as compared to 0.4% (6/1725) for inland collected flies. PCR analysis indicated a higher infection rate of 19.81% (104/525) of asymptomatic flies. From these observations, we conclude that care should be taken when planning to initiate tsetse laboratory colonies for use in SIT eradication program. All efforts should be made to select non-infected flies when initiating laboratory colonies and to try to minimize the infection with SGHV. Also management of SGHV infection in the established colony should be applied.

Characterization of the Bacterial Profile from Natural and Laboratory Glossina Populations.

Tsetse flies (Glossina spp.; Diptera: Glossinidae) are viviparous flies that feed on blood and are found exclusively in sub-Saharan Africa. They are the only cyclic vectors of African trypanosomes, responsible for human African trypanosomiasis (HAT) and animal African trypanosomiasis (AAT). In this study, we employed high throughput sequencing of the 16S rRNA gene to unravel the diversity of symbiotic bacteria in five wild and three laboratory populations of tsetse species (Glossina pallidipes, G. morsitans, G. swynnertoni, and G. austeni). The aim was to assess the dynamics of bacterial diversity both within each laboratory and wild population in relation to the developmental stage, insect age, gender, and location. Our results indicated that the bacterial communities associated with the four studied Glossina species were significantly influenced by their region of origin, with wild samples being more diverse compared to the laboratory samples. We also observed that the larval microbiota was significantly different than the adults. Furthermore, the sex and the species did not significantly influence the formation of the bacterial profile of the laboratory colonies once these populations were kept under the same rearing conditions. In addition, Wigglesworthia, Acinetobacter, and Sodalis were the most abundant bacterial genera in all the samples, while Wolbachia was significantly abundant in G. morsitans compared to the other studied species. The operational taxonomic unit (OTU) co-occurrence network for each location (VVBD insectary, Doma, Makao, and Msubugwe) indicated a high variability between G. pallidipes and the other species in terms of the number of mutual exclusion and copresence interactions. In particular, some bacterial genera, like Wigglesworthia and Sodalis, with high relative abundance, were also characterized by a high degree of interactions.

Prevalence of Trypanosoma sp. in cattle from Tanzania estimated by conventional PCR and loop-mediated isothermal amplification (LAMP).

This study compared the prevalence of trypanosome infections estimated by PFR-loop-mediated isothermal amplification (LAMP) with conventional polymerase chain reaction (PCR) tests. One hundred forty eight cattle blood samples were collected from Robanda village, Mara region, Tanzania in April 2008. In conventional PCR, four sets of primers, specific for the detection of Trypanosoma sp., Trypanosoma brucei rhodesiense, Trypanosoma vivax, and Trypanozoon, as well as a modified LAMP were used. Conventional PCR detected no infection or up to 8, 1, and 3 infections with Trypanosoma congolense savannah, Trypanozoon, and T. vivax, respectively, whereas LAMP detected additional 44 Trypanozoon positive cases. Our results clearly indicate that the prevalence of Trypanozoon spp. in cattle in Robanda village estimated by PFR-LAMP (30.4%) was significantly higher than the estimates by PCR assays (0.6-2%). As such, future studies should target epidemiological surveys of Trypanozoon and T. brucei rhodesiense infections in possible reservoir animals by LAMP to further elucidate the actual prevalence of these parasites.

A machine learning approach to integrating genetic and ecological data in tsetse flies (Glossina pallidipes) for spatially explicit vector control planning.

Vector control is an effective strategy for reducing vector-borne disease transmission, but requires knowledge of vector habitat use and dispersal patterns. Our goal was to improve this knowledge for the tsetse species Glossina pallidipes, a vector of human and animal African trypanosomiasis, which are diseases that pose serious health and socioeconomic burdens across sub-Saharan Africa. We used random forest regression to (i) build and integrate models of G. pallidipes habitat suitability and genetic connectivity across Kenya and northern Tanzania and (ii) provide novel vector control recommendations. Inputs for the models included field survey records from 349 trap locations, genetic data from 11 microsatellite loci from 659 flies and 29 sampling sites, and remotely sensed environmental data. The suitability and connectivity models explained approximately 80% and 67% of the variance in the occurrence and genetic data and exhibited high accuracy based on cross-validation. The bivariate map showed that suitability and connectivity vary independently across the landscape and was used to inform our vector control recommendations. Post hoc analyses show spatial variation in the correlations between the most important environmental predictors from our models and each response variable (e.g., suitability and connectivity) as well as heterogeneity in expected future climatic change of these predictors. The bivariate map suggests that vector control is most likely to be successful in the Lake Victoria Basin and supports the previous recommendation that G. pallidipes from most of eastern Kenya should be managed as a single unit. We further recommend that future monitoring efforts should focus on tracking potential changes in vector presence and dispersal around the Serengeti and the Lake Victoria Basin based on projected local climatic shifts. The strong performance of the spatial models suggests potential for our integrative methodology to be used to understand future impacts of climate change in this and other vector systems.

Development of loop-mediated isothermal amplification (LAMP) assays for rapid detection of Ehrlichia ruminantium.

BACKGROUND: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium. RESULTS: Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries. CONCLUSIONS: Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.

Phylogeography and population structure of the tsetse fly Glossina pallidipes in Kenya and the Serengeti ecosystem.

Glossina pallidipes is the main vector of animal African trypanosomiasis and a potential vector of human African trypanosomiasis in eastern Africa where it poses a large economic burden and public health threat. Vector control efforts have succeeded in reducing infection rates, but recent resurgence in tsetse fly population density raises concerns that vector control programs require improved strategic planning over larger geographic and temporal scales. Detailed knowledge of population structure and dispersal patterns can provide the required information to improve planning. To this end, we investigated the phylogeography and population structure of G. pallidipes over a large spatial scale in Kenya and northern Tanzania using 11 microsatellite loci genotyped in 600 individuals. Our results indicate distinct genetic clusters east and west of the Great Rift Valley, and less distinct clustering of the northwest separate from the southwest (Serengeti ecosystem). Estimates of genetic differentiation and first-generation migration indicated high genetic connectivity within genetic clusters even across large geographic distances of more than 300 km in the east, but only occasional migration among clusters. Patterns of connectivity suggest isolation by distance across genetic breaks but not within genetic clusters, and imply a major role for river basins in facilitating gene flow in G. pallidipes. Effective population size (Ne) estimates and results from Approximate Bayesian Computation further support that there has been recent G. pallidipes population size fluctuations in the Serengeti ecosystem and the northwest during the last century, but also suggest that the full extent of differences in genetic diversity and population dynamics between the east and the west was established over evolutionary time periods (tentatively on the order of millions of years). Findings provide further support that the Serengeti ecosystem and northwestern Kenya represent independent tsetse populations. Additionally, we present evidence that three previously recognized populations (the Mbeere-Meru, Central Kenya and Coastal "fly belts") act as a single population and should be considered as a single unit in vector control.

Genetic diversity of Glossina fuscipes fuscipes along the shores of Lake Victoria in Tanzania and Kenya: implications for management.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are sole vectors for trypanosomiasis, which affect human health and livestock productivity in Africa. Little is known about the genetic diversity of Glossina fuscipes fuscipes, which is an important species in Tanzania and Kenya. The main objective of the study was to provide baseline data to determine the genetic variability and divergence of G. f. fuscipes in the Lake Victoria basin of Tanzania and Kenya in order to guide future vector control efforts in the region. FINDINGS: Two hundred and seventy five G. f. fuscipes from 8 sites along the shores of Lake Victoria were screened for genetic polymorphisms at 19 microsatellite loci. Samples were collected from two sites in Kenya and six sites in Tanzania. Four of the Tanzanian sites were located in the Rorya district, on the eastern shores of Lake Victoria, while the other two sites were from Ukerewe and Bukoba districts from the southern and western Lake Victoria shores, respectively. Four genetically distinct allopatric clusters were revealed by microsatellite analysis, which sorted the sampling sites according to geography, with sites separated by as little as ~65 km belonging to distinct genetic clusters, while samples located within ~35 km from each other group in the same cluster. CONCLUSION: Our results suggest that there is ongoing genetic admixture within sampling sites located ~35 km from each other, while sites located ~65 km apart are genetically isolated from each other. Similar patterns emerged from a parallel study on G. f. fuscipes analyzed from the Lake Victoria Uganda shores. From a control perspective these results suggest that for sites within the same genetic cluster, control efforts should be carried out in a coordinated fashion in order to avoid re-invasions. Future work should focus on better quantifying the extent and spatial patterns of the observed genetic discontinuities of the G. f. fuscipes populations along the Tanzanian shores. This will aid in their control by providing guidelines on the geographical extent of the area to be treated at the same time.

Comparative performance of traps in catching tsetse flies (Diptera: Glossinidae) in Tanzania.

This study was conducted to determine the efficiency of different tsetse traps in 28 sites across Tanzania. The traps used were biconical, H, NGU, NZI, pyramidal, S3, mobile, and sticky panels. Stationary traps were deployed at a distance of 200 m apart and examined 72 h after deployment. The results showed that 117 (52.2%) out of the 224 traps deployed captured at least one Glossina species. A total of five Glossina species were captured, namely Glossina brevipalpis, Glossina pallidipes, Glossina swynnertoni, Glossina morsitans, and Glossina fuscipes martinii. Biconical traps caught tsetse flies in 27 sites, pyramidal in 26, sticky panel in 20, mobile in 19, S3 in 15, NGU in 7, H in 2 and NZI in 1. A total of 21 107 tsetse flies were trapped, with the most abundant species being G. swynnertoni (55.9%), followed by G. pallidipes (31.1%), G. fuscipes martinii (6.9%) and G. morsitans (6.0%). The least caught was G. brevipalpis (0.2%). The highest number of flies were caught by NGU traps (32.5%), followed by sticky panel (16%), mobile (15.4%), pyramidal (13.0%), biconical (11.3%) and S3 (10.2%). NZI traps managed to catch 0.9% of the total flies and H traps 0.7%. From this study, it can be concluded that the most efficient trap was NGU, followed by sticky panel and mobile, in that order. Therefore, for tsetse fly control programmes, NGU traps could be the better choice. Conversely, of the stationary traps, pyramidal and biconical traps captured tsetse flies in the majority of sites, covering all three ecosystems better than any other traps; therefore, they would be suitable for scouting for tsetse infestation in any given area, thus sparing the costs of making traps for each specific Glossina species.

Occurrence of Ticks in Cattle in the New Pastoral Farming Areas in Rufiji District, Tanzania.

Ticks and tick-borne diseases plus trypanosomosis are a constraint to cattle rearing in Tanzania. Rufiji district was not known for important ticks infesting cattle because inhabitants were not engaged in keeping livestock. Not only has settlement of pastoralists and cattle in Rufiji increased the number of cattle but also cattle have been the source of bringing in and spreading of ticks. This study investigated tick species that have been introduced and managed to establish themselves in the new livestock farming areas in cattle in Rufiji. Tick distribution study was undertaken in three villages of Chumbi ward seasonally in 2009, 2011, and 2012. The identified ticks were Amblyomma variegatum (56.10%), Rhipicephalus evertsi (10.25%), R. microplus (27.40%), and R. appendiculatus (6.19%) out of 12940 ticks. Results indicate that ticks are present in the new livestock settlement areas. The occurrence of ticks is correlated with the recent settlement of cattle in the district.

Quantifying Heterogeneity in Host-Vector Contact: Tsetse (Glossina swynnertoni and G. pallidipes) Host Choice in Serengeti National Park, Tanzania.

BACKGROUND: Identifying hosts of blood-feeding insect vectors is crucial in understanding their role in disease transmission. Rhodesian human African trypanosomiasis (rHAT), also known as acute sleeping sickness is caused by Trypanosoma brucei rhodesiense and transmitted by tsetse flies. The disease is commonly associated with wilderness areas of east and southern Africa. Such areas hold a diverse range of species which form communities of hosts for disease maintenance. The relative importance of different wildlife hosts remains unclear. This study quantified tsetse feeding preferences in a wilderness area of great host species richness, Serengeti National Park, Tanzania, assessing tsetse feeding and host density contemporaneously. METHODS: Glossina swynnertoni and G. pallidipes were collected from six study sites. Bloodmeal sources were identified through matching Cytochrome B sequences amplified from bloodmeals from recently fed flies to published sequences. Densities of large mammal species in each site were quantified, and feeding indices calculated to assess the relative selection or avoidance of each host species by tsetse. RESULTS: The host species most commonly identified in G. swynnertoni bloodmeals, warthog (94/220), buffalo (48/220) and giraffe (46/220), were found at relatively low densities (3-11/km2) and fed on up to 15 times more frequently than expected by their relative density. Wildebeest, zebra, impala and Thomson's gazelle, found at the highest densities, were never identified in bloodmeals. Commonly identified hosts for G. pallidipes were buffalo (26/46), giraffe (9/46) and elephant (5/46). CONCLUSIONS: This study is the first to quantify tsetse host range by molecular analysis of tsetse diet with simultaneous assessment of host density in a wilderness area. Although G. swynnertoni and G. pallidipes can feed on a range of species, they are highly selective. Many host species are rarely fed on, despite being present in areas where tsetse are abundant. These feeding patterns, along with the ability of key host species to maintain and transmit T. b. rhodesiense, drive the epidemiology of rHAT in wilderness areas.

The role of domestic animals in the epidemiology of human African trypanosomiasis in Ngorongoro conservation area, Tanzania.

BACKGROUND: Trypanosomiasis is a neglected tropical disease caused by the trypanosome parasite and transmitted by the tsetse fly vector. In Sub-saharan Africa, both the human and animal variants of the disease are a great obstacle towards agriculture, development, and health. In order to better understand and therefore combat Trypanosomiasis, characterizing disease hotspots across species is critical. METHODS: In this study, 193 samples from cattle, sheep, and goats were collected from eight sites. Samples were taken from animals belonging mostly to Maasai herdsmen in the Ngorongoro Crater Conservation Area (NCA) and analysed for the presence of trypanosomiasis infection using PCR techniques. Those that tested positive for T. brucei parasite were further tested using SRA LAMP technique to check for T. brucei rhodesiense, the human infective subspecies of parasite. RESULTS: Our study found a high incidence of Trypanosoma brucei infections across species. Of animals tested, 47 % of cattle, 91.7 % of sheep, and 60.8 % of goats were infected. Most of the infections were of the T. brucei species. We also identified sheep and goats as carriers of the T. brucei rhodesiense subspecies, which causes acute human trypanosomiasis. CONCLUSIONS: Together, these results point toward the need for stricter control strategies in the area to prevent disease outbreak.

The use of specific and generic primers to identify trypanosome infections of wild tsetse flies in Tanzania by PCR.

The accurate identification of trypanosome species and subspecies remains a challenging task in the epidemiology of human and animal trypanosomiasis in tropical Africa. Currently, there are specific PCR tests to identify about 10 different species, subspecies or subgroups of African tsetse-transmitted trypanosomes. These PCR tests have been used here to identify trypanosomes in four species of tsetse (Glossina brevipalpis, G. pallidipes, G. swynnertoni, G. morsitans morsitans) from two areas of Tanzania. PCR using species-specific primers was performed on 1041 dissection-positive proboscides, giving an overall positive identification in 254 (24%). Of these, 61 proboscides (24%) contained two or more trypanosomes. The trypanosome with the greatest overall prevalence at both field sites was Trypanosoma simiae Tsavo, which was identified in a total of 118 infected tsetse proboscides (46%). At Pangani, T. godfreyi was found in G. pallidipes but not in G. brevipalpis, suggesting that these flies might have different susceptibility to this trypanosome or might have fed on a different range of hosts. A high proportion (about 75%) of trypanosome infections remained unidentified. To investigate the identity of these unidentified samples, we used primers complementary to the conserved regions of trypanosomal small subunit ribosomal RNA (ssu rRNA) genes to amplify variable segments of the gene. Amplified DNA fragments were cloned, sequenced and compared with ssu rRNA genes on database of known trypanosome species. In this way, we have tentatively identified two new trypanosomes: a trypanosome related to Trypanosoma vivax and a trypanosome related to T. godfreyi. The T. godfreyi-related trypanosome occurred frequently in the Tanzanian field samples and appears to be widespread. Molecular identification of these two new trypanosomes should now facilitate their isolation and full biological characterisation.

Occurrence of haemoparasites in cattle in Monduli district, northern Tanzania.

Haemoparasite infections are among the most economically important cattle diseases in sub-Saharan Africa. The present study investigated the occurrence of haemoparasites in 295 indigenous cattle from five villages (Mswakini, Lake Manyara, Naitolia, Makuyuni and Nanja) of the Monduli district, a wildlife-domestic animal-human interface area in northern Tanzania. The data showed that the overall occurrence of haemoparasites in the sampled cattle was 12.5% (95% CI: 8.7% - 16.3%), involving single and mixed infections with Theileria parva, Anaplasma marginale, Babesia bovis, Trypanosoma vivax and Trypanosoma brucei. The highest haemoparasite occurrence was recorded in Lake Manyara (18.3%; 95% CI: 8.5% - 28.1%), and the lowest was recorded in Nanja (6.5%; 95% CI: 0.4% - 12.6%). This preliminary study, furthermore, provided evidence of the possible arthropod vectors (ticks and tsetse flies) that may be involved in the transmission of haemoparasites to cattle in the Monduli district. It is envisaged that this survey will stimulate more studies to determine the prevalence of haemoparasites in livestock by using more sensitive molecular techniques.

Comparative diagnostic and analytical performance of PCR and LAMP-based trypanosome detection methods estimated using pooled whole tsetse flies and midguts.

Detection of trypanosomes that cause disease in human beings and livestock within their tsetse fly hosts is an essential component of vector and disease control programmes. Several molecular-based diagnostic tests have been developed for this purpose. Many of these tests, while sensitive, require analysis of trypanosome DNA extracted from single flies, or from pooled tsetse fly heads and amplified trypanosome DNA. In this study, we evaluated the relative analytical and diagnostic sensitivities of two PCR-based tests (ITS and TBR) and a Trypanozoon specific LAMP assay using pooled whole tsetse flies and midguts spiked with serially diluted procyclics of a laboratory strain of Trypanosoma brucei brucei (KETRI 3386). Test sensitivity was also evaluated using experimentally infected tsetse flies. The aim was to determine the most appropriate pooling strategy for whole tsetse and midguts. RIME-LAMP had the highest diagnostic sensitivity (100%) followed by TBR-PCR (95%) and ITS-PCR (50%) in detecting trypanosome DNA from pooled tsetse midguts. RIME-LAMP also had the best diagnostic specificity (75%) followed by ITS-PCR (68%) and TBR-PCR (50%). The relative detection limit determined by serial dilution of procyclics was below 10(-6) (equivalent to 1parasite/ml). Using TBR-PCR, ITS-PCR and RIME-LAMP, it was possible to detect trypanosome DNA in single flies or in pools of 2, 3, 4, 5, 10, or 15 flies/midguts. The proportion of positive pools declined by up to 60% when testing pools of 15 whole flies as opposed to testing pools of 5-10 flies. Additionally, it was possible to detect DNA in a single infected tsetse fly in the background of 4, 9, or 14 uninfected tsetse flies. Averaged across pool sizes and tsetse species, RIME-LAMP detected the highest proportion of positive pools in spiked whole tsetse and midguts (86.6% and 87.2%) followed by TBR-PCR (78. 6% and 79.2%) and ITS-PCR (34.3% and 40.2%). There were no significant differences between the proportions of positive pools detected in whole flies and midguts. We conclude that pooling of whole tsetse/midguts is an effective strategy to reduce hands-on-time and hence has potential application in large scale xenomonitoring to generate epidemiological data for decision making. RIME-LAMP offers the best diagnostic sensitivity and specificity on pooled tsetse midguts, thus demonstrating its superior diagnostic performance when compared with TBR-PCR and ITS-PCR. Using pools of whole tsetse or midguts as source of DNA does not have any significant effect on test results and is more representative of the field conditions where the proportion of flies with infected midguts tends to be higher than flies with infected salivary glands. Therefore to save time and minimize costs, pooling of whole tsetse flies is recommended.

Using molecular data for epidemiological inference: assessing the prevalence of Trypanosoma brucei rhodesiense in tsetse in Serengeti, Tanzania.

BACKGROUND: Measuring the prevalence of transmissible Trypanosoma brucei rhodesiense in tsetse populations is essential for understanding transmission dynamics, assessing human disease risk and monitoring spatio-temporal trends and the impact of control interventions. Although an important epidemiological variable, identifying flies which carry transmissible infections is difficult, with challenges including low prevalence, presence of other trypanosome species in the same fly, and concurrent detection of immature non-transmissible infections. Diagnostic tests to measure the prevalence of T. b. rhodesiense in tsetse are applied and interpreted inconsistently, and discrepancies between studies suggest this value is not consistently estimated even to within an order of magnitude. METHODOLOGY/PRINCIPAL FINDINGS: Three approaches were used to estimate the prevalence of transmissible Trypanosoma brucei s.l. and T. b. rhodesiense in Glossina swynnertoni and G. pallidipes in Serengeti National Park, Tanzania: (i) dissection/microscopy; (ii) PCR on infected tsetse midguts; and (iii) inference from a mathematical model. Using dissection/microscopy the prevalence of transmissible T. brucei s.l. was 0% (95% CI 0-0.085) for G. swynnertoni and 0% (0-0.18) G. pallidipes; using PCR the prevalence of transmissible T. b. rhodesiense was 0.010% (0-0.054) and 0.0089% (0-0.059) respectively, and by model inference 0.0064% and 0.00085% respectively. CONCLUSIONS/SIGNIFICANCE: The zero prevalence result by dissection/microscopy (likely really greater than zero given the results of other approaches) is not unusual by this technique, often ascribed to poor sensitivity. The application of additional techniques confirmed the very low prevalence of T. brucei suggesting the zero prevalence result was attributable to insufficient sample size (despite examination of 6000 tsetse). Given the prohibitively high sample sizes required to obtain meaningful results by dissection/microscopy, PCR-based approaches offer the current best option for assessing trypanosome prevalence in tsetse but inconsistencies in relating PCR results to transmissibility highlight the need for a consensus approach to generate meaningful and comparable data.

Fifty years of tsetse control in Tanzania: challenges and prospects for the future.

Tsetse flies are the vectors of trypanosomes, the causative organisms of trypanosomiasis, nagana, in animals and sleeping sickness in man. In Tanzania, tsetse transmitted trypanosomiasis is one of the most important disease affecting both animals and humans. About 40% of land suitable for grazing and areas with high agricultural potential are currently tsetse infested. It is estimated that about 4.4 million livestock and 4 million people are at risk of contracting tsetse borne trypanosomiasis. African animal trypanosomiasis (AAT) causes loss in animals due to mortality and reduced milk yield, which is estimated at US$ 7.98 million annually. Even after 50 years of independence, Human African Trypanosomiasis (HAT) or Sleeping Sickness is still one of the major public health problems with about 300 cases being reported annually. Tsetse control has been sporadic and uncoordinated hence no tangible results have been accrued since independence despite the fact that technologies which have facilitated tsetse control in other places are available. Fifty years of independence have seen shrinkage of tsetse belt to 43% in 16 surveyed regions. Opportunities for future are wide open if tsetse control will involve all stakeholders, who are directly or indirectly affected by the tsetse problem; if tsetse and trypanosomiasis eradication will adopt an area wide and participatory approach with emphasis on environmentally and user friendly techniques for expanded livestock sector; improved food security and livelihood in affected communities, for achievement of the millennium development goals.