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.

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.

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.

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.

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.

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.

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.

Tracking the feeding patterns of tsetse flies (Glossina genus) by analysis of bloodmeals using mitochondrial cytochromes genes.

Tsetse flies are notoriously difficult to observe in nature, particularly when populations densities are low. It is therefore difficult to observe them on their hosts in nature; hence their vertebrate species can very often only be determined indirectly by analysis of their gut contents. This knowledge is a critical component of the information on which control tactics can be developed. The objective of this study was to determine the sources of tsetse bloodmeals, hence investigate their feeding preferences. We used mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) gene sequences for identification of tsetse fly blood meals, in order to provide a foundation for rational decisions to guide control of trypanosomiasis, and their vectors. Glossina swynnertoni were sampled from Serengeti (Tanzania) and G. pallidipes from Kenya (Nguruman and Busia), and Uganda. Sequences were used to query public databases, and the percentage identities obtained used to identify hosts. An initial assay showed that the feeds were from single sources. Hosts identified from blood fed flies collected in Serengeti ecosystem, included buffaloes (25/40), giraffes (8/40), warthogs (3/40), elephants (3/40) and one spotted hyena. In Nguruman, where G. pallidipes flies were analyzed, the feeds were from elephants (6/13) and warthogs (5/13), while buffaloes and baboons accounted for one bloodmeal each. Only cattle blood was detected in flies caught in Busia and Uganda. Out of four flies tested in Mbita Point, Suba District in western Kenya, one had fed on cattle, the other three on the Nile monitor lizard. These results demonstrate that cattle will form an integral part of a control strategy for trypanosomiasis in Busia and Uganda, while different approaches are required for Serengeti and Nguruman ecosystems, where wildlife abound and are the major component of the tsetse fly food source.

Multiple Trypanosoma infections are common amongst Glossina species in the new farming areas of Rufiji district, Tanzania.

BACKGROUND: Tsetse flies and trypanosomiasis are among several factors that constrain livestock development in Tanzania. Over the years Rufiji District was excluded from livestock production owing to tsetse fly infestation, however, a few years ago there was an influx of livestock following evictions aimed at conserving the Usangu wetlands. METHODS: A study was conducted to determine the efficiency of available traps for catching tsetse flies, Glossina species infesting the area, their infection rates and Trypanosoma species circulating in the area. Trapping was conducted during the semi dry season for a total of 30 days (ten days each month) during the onset of the dry season of May - July 2009. Harvested flies after every 24 hours were dissected and examined under a light microscope for trypanosome infections and whole fly DNA was extracted from 82 flies and analyzed for trypanosomes by polymerase chain reaction (PCR) using different sets of primers. RESULTS: The proportions of total tsetse catches per trap were in the following decreasing order S3 (33%), H-Trap (27%), Pyramidal (19%), sticky panel (11%) and biconical trap (10%). Of the 1200 trapped flies, 75.6% were identified as Glossina pallidipes, 11.7% as G. brevipalpis, 9.6% as G. austeni and 3.0% G. morsitans morsitans. Dissections revealed the overall infection rate of 6.6% (13/197). Whole DNA was extracted from 82 tsetse flies and the prevalence of trypanosomes circulating in the area in descending order was 92.7% (76/82) for T. simiae; 70.7% (58/82) for T. brucei types; 48.8% (40/82) for the T. vivax types and 32.9% (27/82) for the T. congolense types as determined by PCR. All trypanosome types were found in all tsetse species analysed except for the T. congolense types, which were absent in G. m. morsitans. None of the T. brucei positive samples contained human infective trypanosomes by SRA - PCR test CONCLUSION: All tsetse species found in Rufiji are biologically important in the transmission of animal trypanosomiasis and the absence of T. congolense in G. m. morsitans could be a matter of chance only. Therefore, plans for control should consider all tsetse species.

Landmarks in the evolution of technologies for identifying trypanosomes in tsetse flies.

Understanding what the trypanosome pathogens are, their vectors and mode of transmission underpin efforts to control the disease they cause in both humans and livestock. The risk of transmission is estimated by determining what proportion of the vector population is carrying the infectious pathogens. This risk also depends on the infectivity of the trypanosomes to humans and livestock. Most livestock pathogens are not infective to humans, whereas the two sub-species that infect humans also infect livestock. As with other infectious diseases, we can therefore trace the foundation of many continuing disease control programs for trypanosomiasis to the discovery of the pathogens and their vectors more than a century ago. Over this period, methods for detecting and identifying trypanosomes have evolved through various landmark discoveries. This review describes the evolution of methods for identifying African trypanosomes in their tsetse fly vectors.