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Investigates wild host (Varanus niloticus) as a source of blood meal for tsetse based on a study conducted in south-eastern Uganda. Treatment of livestock with trypanocidal drugs; Significance of monitor lizards in the persistence of sleeping sickness; Analysis of the patterns of tsetse flies.

Abstract Background Diagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR. Results A comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%). Conclusions Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach.

Abstract Background In East Africa, animal trypanosomiasis is caused by many tsetse transmitted protozoan parasites including Trypanosoma vivax, T. congolense and subspecies of T. brucei s.l. (T. b. brucei and zoonotic human infective T. b. rhodesiense) that may co-circulate in domestic and wild animals. Accurate species-specific prevalence measurements of these parasites in animal populations are complicated by mixed infections of trypanosomes within individual hosts, low parasite densities and difficulties in conducting field studies. Many Polymerase Chain Reaction (PCR) based diagnostic tools are available to characterise and quantify infection in animals. These are important for assessing the contribution of infections in animal reservoirs and the risk posed to humans from zoonotic trypanosome species. New matrices for DNA capture have simplified large scale field PCR analyses but few studies have examined the impact of these techniques on prevalence estimations. Results The Whatman FTA matrix has been evaluated using a random sample of 35 village zebu cattle from a population naturally exposed to trypanosome infection. Using a generic trypanosome-specific PCR, prevalence was systematically evaluated. Multiple PCR samples taken from single FTA cards demonstrated that a single punch from an FTA card is not sufficient to confirm the infectivity status of an individual animal as parasite DNA is unevenly distributed across the card. At low parasite densities in the host, this stochastic sampling effect results in underestimation of prevalence based on single punch PCR testing. Repeated testing increased the estimated prevalence of all Trypanosoma spp. from 9.7% to 86%. Using repeat testing, a very high prevalence of pathogenic trypanosomes was detected in these local village cattle: T. brucei (34.3%), T. congolense (42.9%) and T. vivax (22.9%). Conclusions These results show that, despite the convenience of Whatman FTA cards and specific PCR based detection tools, the chronically low parasitaemias in indigenous African zebu cattle make it difficult to establish true prevalence. Although this study specifically applies to FTA cards, a similar effect would be experienced with other approaches using blood samples containing low parasite densities. For example, using blood film microscopy or PCR detection from liquid samples where the probability of detecting a parasite or DNA molecule, in the required number of fields of view or PCR reaction, is less than one.

Abstract Background In a mammalian host, the cell surface of African trypanosomes is protected by a monolayer of a single variant surface glycoprotein (VSG). The VSG is central to antigenic variation; one VSG gene is expressed at any one time and there is a low frequency stochastic switch to expression of a different VSG gene. The genome of Trypanosoma brucei contains a repertoire of > 1000 VSG sequences. The degree of conservation of the genomic VSG repertoire in different strains has not been investigated in detail. Results Eighteen expressed VSGs from Ugandan isolates were compared with homologues (> 40 % sequence identity) in the two available T. brucei genome sequences. Fourteen homologues were present in the genome of Trypanosoma brucei brucei TREU927 from Kenya and fourteen in the genome of T. b. gambiense Dal972 from Cote d'Ivoire. The Ugandan VSGs averaged 71% and 73 % identity to homologues in T. b. brucei and T. b. gambiense respectively. The sequence divergence between homologous VSGs from the three different strains was not random but was more prevalent in the parts of the VSG believed to interact with the host immune system on the living trypanosome. Conclusion It is probable that the VSG repertoires in the different isolates contain many common VSG genes. The location of divergence between VSGs is consistent with selection for strain-specific VSG repertoires, possibly to allow superinfection of an animal by a second strain. A consequence of strain-specific VSG repertoires is that any vaccine based on large numbers of VSGs from a single strain will only provide partial protection against other strains.

Background: Malaria is a leading cause of mortality in children aged 5 years and below in Nigeria. Treatment guidelines stipulate among other recommendations, testing by microscopy or a rapid diagnostic test (RDT) before treatment. Non-adherence to these guidelines portends a challenge, especially among vulnerable under-five children. This study explored the factors influencing Nigerian public health workers' (HWs) adherence to these guidelines in under-five children., Methods: A review of literature published between 2011- 2023 was conducted on Web of Science, Ovid Embase, Medline, Global Health, CAB Abstracts, Scopus, and Global Index Medicus. Data was extracted and analyzed under 4 themes: diagnosis, compliance with test results, use of recommended treatment, post-treatment counselling and severe malaria management., Results: Nineteen (19) studies were included for review. Training and supervision, RDT and antimalarial availability, good knowledge of, and positive perception of RDTs promoted adherence to mRDT use. A lack of confidence in RDTs and age (≥ 40 years) fuelled presumptive treatment, especially among clinicians. mRDT and artemisinin-based combination therapy (ACT) stockouts dissuaded HWs from adhering to case management guidelines. Caregiver pressure for treatment was identified as a barrier to compliance with test results., Conclusions: It is important to design context-specific strategies to improve adherence to guidelines for malaria case management, especially in under-five children. Training on the guidelines should be tailored, needs-based, and continuous, and HWs should be supportively supervised in implementing case management. Maintaining an adequate supply of quality-assured mRDTs and antimalarials can facilitate adherence to the guidelines., Competing Interests: The authors declare no competing interests., (Copyright © 2024 Ezenyi et al.)

The control of African trypanosomiasis (AT) in Eastern and Southern Africa, including Zambia, faces huge challenges due to the involvement of wild and domestic animal reservoirs. Free-roaming dogs in wildlife-populated and tsetse-infested villages of Zambia's Mambwe district are exposed to infectious tsetse bites. Consuming fresh raw game meat and bones further exacerbates their risk of contracting AT. We focus on the reservoir role of such dogs in maintaining and transmitting diverse species of trypanosomes that are infective to humans and livestock in Zambia's Mambwe district. A cohort of 162 dogs was enrolled for follow-up at 3 different time points from June to December 2018 in selected villages of Malama, Mnkhanya, and Nsefu chiefdoms of Mambwe district, eastern Zambia. Blood and serum were screened for AT by microscopy, GM6 ELISA, PCR (ITS1 and SRA), and Sanger sequencing. Out of the 162 dogs in the cohort, 40 were lost to follow-up and only 122 remained traceable at the end of the study. GM6 ELISA detected Trypanosoma antibodies in 121 dogs (74.7%) and ITS1-PCR detected DNA involving single and mixed infections of T. congolense, T. brucei, and suspected T. simiae or T. godfreyi in 115 dogs (70.9%). The human-infective T. b. rhodesiense was detected by SRA PCR in 67 dogs (41.4%), and some sequence data that support the findings of this study have been deposited in the GenBank under accession numbers OL961811, OL961812, and OL961813. Our study demonstrates that the Trypanosoma reservoir community in Zambia is wider than was thought and includes domesticated dogs. As dogs are active carriers of human and livestock-infective trypanosomes, they pose a risk of transmitting AT in endemic villages of Mambwe district as they are neglected and left untreated. To fully bring AT under control, countries such as Zambia where the role of animal reservoirs is important, should not limit their prevention and treatment efforts to livestock (especially cattle) but also include dogs that play an integral part in most rural communities., (© 2024. The Author(s).)

Good dog-keeping practices and access to veterinary care are essential for the well-being of dogs. As the main causes of morbidity and mortality in the rural canine population in Zambia are poorly understood, we followed a cohort of 162 indigenous dogs for six months in wildlife-populated and tsetse-infested villages of Mambwe district, eastern Zambia to gain deeper insights. Dogs lacked basic home and veterinary care, they were often starved and burdened with ticks, and some passed live adult worms in their stool. The frequent exposure of dogs to tsetse bites and consumption of fresh raw game meat and bones puts them at greater risk of acquiring African trypanosomiasis. Nearly 20 % of dogs were lost to follow-up, with the main causes being poor health (58.1 %), predation by wild carnivores (29 %), and owner culling or euthanasia (12.9 %). We observed that indigenous dogs' general well-being and survival were largely influenced by their environment, infectious diseases, injuries sustained during interaction with conspecifics and wildlife, and community attitudes and practices associated with dog ownership., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

A longitudinal study was conducted within a cattle ranch in Gabon to determine the diminazene aceturate (Berenil) index (DAI) in a group of Zebu, raised under low tsetse density; this measure providing an assessment of trypanosomiasis risk. The objective was to evaluate the trypanosomiasis pressure thus informing trypanosomiasis control methods and cattle management. Twenty female adult Zebu were monitored for 24 weeks during the dry season. Blood samples were collected on a weekly basis and subjected to parasitological and haematological analysis (n = 480), using the buffy-coat method and the packed cell volume value (PCV), respectively, infected animals were treated with a single intramuscular injection of diminazene aceturate (8 mg/kg). Twenty-nine single infectious events were recorded and a DAI of 1.45 was calculated. Two trypanosome species were identified: Trypanosoma congolense (96.2%) and Trypanosoma vivax (3.8%). The mean PCV value of the infected animals was lower (26.6) compared to non-infected animals (32.0). This study shows that DAI may be a useful tool to assess trypanosomiasis. However, this is a time-consuming method that may be improved by using randomly selected sentinel animals to adapt the chemoprophylactic schemes, hence decreasing the costs and the drug resistance risk.

Background: Uganda has suffered from a series of epidemics of Human African Trypanosomiasis (HAT), a tsetse transmitted disease, also known as sleeping sickness. The area affected by acute Trypanosoma brucei rhodesiense HAT (rHAT) has been expanding, driven by importation of infected cattle into regions previously free of the disease. These regions are also affected by African Animal Trypanosomiasis (AAT) demanding a strategy for integrated disease control., Methods: In 2008, the Public Private Partnership, Stamp Out Sleeping Sickness (SOS) administered a single dose of trypanocide to 31 486 head of cattle in 29 parishes in Dokolo and Kaberamaido districts. This study examines the impact of this intervention on the prevalence of rHAT and AAT trypanosomes in cattle from villages that had (HAT +ve ) or had not (HAT -ve ) experienced a recent case of rHAT. Cattle herds from 20 villages were sampled and screened by PCR, pre-intervention and 6-months post-intervention, for the presence or absence of: Trypanosoma brucei s.l.; human infective T. b. rhodesiense; Trypanosoma vivax; and Trypanosoma congolense savannah., Results: Post-intervention, there was a significant decrease in the prevalence of T. brucei s.l. and the human infective sub-species T. b. rhodesiense in village cattle across all 20 villages. The prevalence of T. b. rhodesiense was reduced from 2.4% to 0.74% (P < 0.0001), with the intervention showing greater impact in HAT -ve villages. The number of villages containing cattle harbouring human infective parasites decreased from 15/20 to 8/20, with T. b. rhodesiense infection mainly persisting within cattle in HAT +ve villages (six/eight). The proportion of T. brucei s.l. infections identified as human infective T. b. rhodesiense decreased after the intervention from 8.3% (95% CI = 11.1-5.9%) to 4.1% (95% CI = 6.8-2.3%). Villages that had experienced a recent human case (HAT +ve villages) showed a significantly higher prevalence for AAT both pre- and post-intervention. For AAT the prevalence of T. vivax was significantly reduced from 5.9% to 0.05% post-intervention while the prevalence of T. congolense increased from 8.0% to 12.2%., Conclusions: The intervention resulted in a significant decrease in the prevalence of T. brucei s.l., human infective T. b. rhodesiense and T. vivax infection in village cattle herds. The proportion of T. brucei s.l. that were human infective, decreased from 1:12 T. brucei s.l. infections before the intervention to 1:33 post-intervention. It is clearly more difficult to eliminate T. b. rhodesiense from cattle in villages that have experienced a human case. Evidence of elevated levels of AAT in livestock within village herds is a useful indicator of risk for rHAT in Uganda. Integrated veterinary and medical surveillance is key to successful control of zoonotic rHAT.

Introduction: Human African trypanosomiasis (HAT) comprises two fatal parasitic diseases. Uganda is home to both chronic T. b. gambiense (gHAT) and the acute zoonotic form T. b. rhodesiense (rHAT) which occur in two large but discrete geographical foci. The area affected by rHAT has been rapidly expanding due to importation of T. b. rhodesiense infected cattle into tsetse infested but previously HAT free districts. Migration of rHAT has resulted in a considerable human health burden in these newly affected districts. Here, we examined the impact of a single, district-wide, mass chemotherapeutic livestock intervention, on T. b. rhodesiense prevalence in cattle and on incidence and distribution of human rHAT cases in Kamuli and Soroti districts in eastern Uganda., Methods: A single mass intervention in domestic cattle (n=30,900) using trypanocidal drugs was undertaken in November and December 2002 under the EU funded Farming in Tsetse Controlled Areas (FITCA) programme. The intervention targeted removal of the reservoir of infection i.e. human infective T. b. rhodesiense parasites in cattle, in the absence of tsetse control. Interventions were applied in high-risk sub-counties of Kamuli district (endemic for rHAT) and Soroti district (where rHAT has been recently introduced). The prevalence of T. brucei s.l. and the human infective subspecies, T. b. rhodesiense in cattle (n=1833) was assessed before and 3 and 12 months after intervention using PCR-based methods. A combination of descriptive statistical analysis and spatial scan statistics were applied to analyse rHAT cases reported over a 5-year period (January 2000-July 2005)., Results: A single intervention was highly effective at removing human infective T. b. rhodesiense parasites from the cattle reservoir and contributed to a significant decrease in human rHAT cases. Intervention coverage was higher in Kamuli (81.1%) than in Soroti (47.3%) district but despite differences in coverage both districts showed a reduction in prevalence of T. b. brucei s.l. and T. b. rhodesiense. In Kamuli, the prevalence of T. brucei s.l. decreased by 54%, from 6.75% to 3.11%, 3, months post-intervention, rising to 4.7% at 12 months. The prevalence of T. b. rhodesiense was 3% pre-intervention and no T. b. rhodesiense infections were detected 3 and 12, months post-treatment. In Soroti, the prevalence of T. brucei s.l. in cattle decreased by 38% (from 21% to 13%) 3 months after intervention decreasing to less than 10% at 12 months. The prevalence of T. b. rhodesiense was reduced by 50% at 12-months post-intervention (6%-3%). Most notably, was the impact of the intervention on the population dynamics between T. b. brucei and human infective T. b. rhodesiense. Before intervention in Kamuli district 56% of T. b. brucei s.l. circulating in cattle were T. b. rhodesiense; at both 3 and 12 months after intervention none of the re-infecting T. b. brucei s.l. were human infective, T. rhodesiense. For human rHAT cases, there was a seven-fold decrease in rHAT incidence after intervention in Kamuli district (5.54 cases/1,000 head of population 2000-2002 to 0.76 cases/1,000, 2003-2005). Incidence data suggests that the intervention had minimal impact on the number of rHAT cases in Soroti overall, but showed a significant decrease in the seasonal peak of cases in the year following treatment., Conclusion: A single intervention, targeted at cattle, introduced at district level, in the absence of tsetse control, was highly effective at removing human infective rHAT parasites from the cattle reservoir and contributed to a significant decrease in human rHAT cases. The differential impacts observed between the two districts are related to both the different stages of rHAT endemicity in the districts, and levels of intervention coverage achieved in the cattle population. Treatment of cattle to remove the reservoir of rHAT infection offers a promising and cost effective approach for the control of rHAT. It is important that cattle are treated before relocation to prevent possible merger of the two HAT foci, which would complicate diagnosis and treatment of both gHAT and rHAT., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Sleeping sickness or Human African Trypanosomiasis (HAT) is a neglected tropical disease of public health importance across much of Sub-Saharan Africa. In Uganda, chronic T. b. gambiense HAT (gHAT) and acute T. b. rhodesiense HAT (rHAT) occur in two large but discrete geographical foci. Both forms are difficult to diagnose, expensive to treat and ultimately fatal in the absence of treatment. The area affected by zoonotic rHAT has been steadily expanding, placing a high burden on local health systems. HAT is a disease of neglected populations and is notorious for being under-reported. Here we examine the factors that influence passive rHAT surveillance within the district health system in four Ugandan districts into which the disease had recently been introduced, focusing on staff knowledge, infrastructure and data management., Methods: A mixed methods study was undertaken between 2011 and 2013 in Dokolo, Kaberamaido, Soroti and Serere districts to explore health facility capacity and clinical service provision, diagnostic capacity, HAT knowledge and case reporting. Structured interviews were undertaken with 86 medical personnel, including clinicians, nurses, midwives and technicians across 65 HC-II and HC-III medical facilities, where the health infrastructure was also directly observed. Eleven semi-structured interviews were undertaken with medical staff in each of the three designated HAT treatment facilities (Dokolo, Lwala and Serere HC-IV) in the area. HAT treatment centre case records, collected between 2009 and 2012, were analyzed., Results: Most medical staff in HC-II and HC-III facilities had been made aware of HAT from radio broadcasts, newspapers and by word of mouth, suggestive of a lack of formal training. Key knowledge as regards the causative agent, clinical signs and that HAT drugs are provided free of charge was lower amongst HC-II than HC-III staff. Many respondents did not know whether HAT was endemic in their district. In rHAT specialist treatment centres, staff were knowledgeable of HAT and were confident in their ability to diagnose and manage cases. Between 2009-2012, 342 people were diagnosed in the area, 54% in the late stage of the disease. Over the period of this study the proportion of rHAT cases identified in early stage fell and by 2012 the majority of cases identified were diagnosed in the late stage., Conclusion: This study illustrates the critical role of the district health system in HAT management. The increasing proportion of cases identified at a late stage in this study indicates a major gap in lower tier levels in patient referral, diagnosis and reporting that urgently needs to be addressed. Integrating HAT diagnosis into national primary healthcare programs and providing training to medical workers at all levels is central to the new 2030 WHO HAT elimination goal. Given the zoonotic nature of rHAT, joined up active surveillance in human and animal populations in Uganda is also needed. The role of the Coordinating Office for Control of Trypanosomiasis in Uganda in implementing a One Health approach will be key to sustainable management of zoonotic HAT., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

In April 2008, whole blood samples were collected from 36 dromedary camels in Sokoto, North-western Nigeria. Following PCR and reverse line blotting, twenty-two samples (61%) resulted positive for Ehrlichia/Anaplasma spp. and three (8%) for Theileria/Babesia spp., with three (8%) cases of co-infections being found. Both sequence and BLAST analyses identified Ehrlichia/Anaplasma spp. and Theileria/Babesia spp. positive cases as Anaplasma platys and Theileria ovis, respectively. This is the first report of the detection of A. platys and T. ovis in camels from sub-Saharan Africa. The epidemiological relevance of this finding is enhanced by the close living of these animals with both dogs and small ruminants. The high prevalence detected for A. platys suggests a possible role of camels as carriers of this infection., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Ticks and tick-borne diseases undermine cattle fitness and productivity in the whole of sub-Saharan Africa, including Nigeria. In this West African country, cattle are challenged by numerous tick species, especially during the wet season. Consequently, several TBDs are known to be endemic in Nigerian cattle, including anaplasmosis, babesiosis, cowdriosis and theilerioris (by Theileria mutans and Theileria velifera). To date, all investigations on cattle TBDs in Nigeria have been based on cytological examinations and/or on serological methods. This study aimed to ascertain the occurrence of tick-borne pathogens of veterinary and zoonotic importance in cattle in Nigeria using molecular approaches., Methods: In October 2008, 704 whole blood samples were collected from indigenous cattle in the Plateau State, Nigeria. Analysis for tick-borne pathogens was conducted by means of PCR-based reverse line blotting (RLB) and sequencing targeting a panel of five genera of microorganisms (i.e. Babesia, Theileria, Anaplasma, Ehrlichia and Rickettsia spp.)., Results: In total, 561/704 (82.6%) animals were found infected, with 465 (69.6%) of them being infected by two or more microorganisms, with up to 77 possible combinations of pathogens detected. Theileria mutans was the most prevalent microorganism (66.3%), followed by Theileria velifera (52.4%), Theileria taurotragi (39.5%), Anaplasma marginale (39.1%), Anaplasma sp. (Omatjenne) (34.7%), Babesia bigemina (7.9%), Anaplasma centrale (6.3%), Anaplasma platys (3.9%), Rickettsia massiliae (3.5%), Babesia bovis (2.0%) and Ehrlichia ruminantium (1.1%). Calves were found significantly less infected than juvenile and adult cattle., Conclusions: This study provides updated, molecular-based information on cattle TBDs in Nigeria. The molecular approach employed allowed the diagnosis of numerous positive cases including carrier statuses, multiple infections and novel pathogen detections within the indigenous cattle population. Moreover, the RLB method here described enabled the detection of veterinary agents not only pertaining to bovine health, including also those of zoonotic importance. The high prevalence recorded for T. mutans, T. velifera, A. marginale, T. taurotragi and Anaplasma sp. (Omatjenne), suggests they may be endemically established in Nigeria, whereas the lower prevalence recorded for other microorganisms (i.e. A. centrale and B. bovis) highlights a less stable epidemiological scenario, requiring further investigations.

Tsetse flies are able to acquire mixed infections naturally or experimentally either simultaneously or sequentially. Traditionally, natural infection rates in tsetse flies are estimated by microscopic examination of different parts of the fly after dissection, together with the isolation of the parasite in vivo. However, until the advent of molecular techniques it was difficult to speciate trypanosomes infections and to quantify trypanosome numbers within tsetse flies. Although more expensive, qPCR allows the quantification of DNA and is less time consuming due to real time visualization and validation of the results. The current study evaluated the application of qPCR to quantify the infection load of tsetse flies with T. b. brucei and T. congolense savannah and to study the possibility of competition between the two species. The results revealed that the two qPCR reactions are of acceptable efficiency (99.1% and 95.6%, respectively), sensitivity and specificity and can be used for quantification of infection load with trypanosomes in experimentally infected Glossina morsitans morsitans. The mixed infection of laboratory Glossina species and quantification of the infection suggests the possibility that a form of competition exists between the isolates of T. b. brucei and T. congolense savannah that we used when they co-exist in the fly midgut.

Background: African animal trypanosomiasis (AAT) is considered to be one of the greatest constraints to livestock production and livestock-crop integration in most African countries. South-eastern Uganda has suffered for more than two decades from outbreaks of zoonotic Human African Trypanosomiasis (HAT), adding to the burden faced by communities from AAT. There is insufficient AAT and HAT data available (in the animal reservoir) to guide and prioritize AAT control programs that has been generated using contemporary, sensitive and specific molecular techniques. This study was undertaken to evaluate the burden that AAT presents to the small-scale cattle production systems in south-eastern Uganda., Methods: Randomised cluster sampling was used to select 14% (57/401) of all cattle containing villages across Tororo District. Blood samples were taken from all cattle in the selected villages between September-December 2011; preserved on FTA cards and analysed for different trypanosomes using a suite of molecular techniques. Generalized estimating equation and Rogen-Gladen estimator models were used to calculate apparent and true prevalences of different trypanosomes while intra cluster correlations were estimated using a 1-way mixed effect analysis of variance (ANOVA) in R statistical software version 3.0.2., Results: The prevalence of all trypanosome species in cattle was 15.3% (95% CI; 12.2-19.1) while herd level trypanosome species prevalence varied greatly between 0-43%. Trypanosoma vivax (17.4%, 95% CI; 10.6-16.8) and Trypanosoma brucei rhodesiense (0.03%) were respectively, the most, and least prevalent trypanosome species identified., Conclusions: The prevalence of bovine trypanosomes in this study indicates that AAT remains a significant constraint to livestock health and livestock production. There is need to implement tsetse and trypanosomiasis control efforts across Tororo District by employing effective, cheap and sustainable tsetse and trypanosomiasis control methods that could be integrated in the control of other endemic vector borne diseases like tick-borne diseases.

Background: African trypanosomes constrain livestock and human health in Sub-Saharan Africa, and aggravate poverty and hunger of these otherwise largely livestock-keeping communities. To solve this, there is need to develop and use effective and cheap tsetse control methods. To this end, we aimed at determining the smallest proportion of a cattle herd that needs to be sprayed on the legs, bellies and ears (RAP) for effective Human and Animal African Trypanosomiasis (HAT/AAT) control., Methodology/principal Finding: Cattle in 20 villages were ear-tagged and injected with two doses of diminazene diaceturate (DA) forty days apart, and randomly allocated to one of five treatment regimens namely; no treatment, 25%, 50%, 75% monthly RAP and every 3 month Albendazole drench. Cattle trypanosome re-infection rate was determined by molecular techniques. ArcMap V10.3 was used to map apparent tsetse density (FTD) from trap catches. The effect of graded RAP on incidence risk ratios and trypanosome prevalence was determined using Poisson and logistic random effect models in R and STATA V12.1 respectively. Incidence was estimated at 9.8/100 years in RAP regimens, significantly lower compared to 25.7/100 years in the non-RAP regimens (incidence rate ratio: 0.37; 95% CI: 0.22-0.65; P<0.001). Likewise, trypanosome prevalence after one year of follow up was significantly lower in RAP animals than in non-RAP animals (4% vs 15%, OR: 0.20, 95% CI: 0.08-0.44; P<0.001). Contrary to our expectation, level of protection did not increase with increasing proportion of animals treated., Conclusions/significance: Reduction in RAP coverage did not significantly affect efficacy of treatment. This is envisaged to improve RAP adaptability to low income livestock keepers but needs further evaluation in different tsetse challenge, HAT/AAT transmission rates and management systems before adopting it for routine tsetse control programs.

Background: Tick and tsetse-borne diseases (TTBDs) constrain livestock production in tropical and subtropical regions of the world. Of this community of endemic diseases, East coast fever (T. parva) is the most important tick-borne disease (TBD) accounting for 70% of all losses due to TBDS in this region where control efforts target either tsetse or TBDs and seldom both. In those instances where simultaneous pyrethroid insecticide TTBD control is implemented, collateral benefits of tsetse control on TBD control have not been quantified. In the interest of guiding future TTBD control efforts, the effect of restricting pyrethroid insecticides to the legs, belly and ears (RAP) of cattle for tsetse and trypanosomiasis control on T. parva prevalence in crop-livestock production systems in Tororo district, south-eastern Uganda was determined., Methods: We randomly allocated 16 villages to diminazene diaceturate (DA) and 3 graded RAP (25%, 50% and 75% of village herd sprayed respectively) treatment regimens. All cattle were ear-tagged, treated with diminazene diaceturate (DA) and those in regimens 2-4 received monthly graded RAP. Blood samples taken fourteen days post DA treatment and once three monthly were analysed by molecular techniques for T. parva., Results: In total, 8,975 samples from 3,084 animals were analysed. Prevalence of T. parva varied between 1-3% in different treatment regimens. RAP regimens were associated with slightly lower average risk of infection compared to DA. However, the confidence interval was broad and the result was not statistically significant. There was no evidence of a dose response relationship between graded RAP and T. parva prevalence. These findings are discussed herein with regard to endemic stability development to different TBDs., Conclusions: We found only a slight effect of RAP on T. parva infection. Since sample size determination was based on trypanosomes incidence, the study was underpowered given the low T. parva prevalence. While the findings need to be confirmed in future studies, the observed slight reduction in the risk of infection with T. parva might not compromise endemic stability.

Background: Years of advocacy for the neglected tropical diseases (NTDs) have focused the world's attention on these diseases of the poor, resulting most recently in the 2012 "London Declaration" and the recent World Health Assembly Resolution WHA66.12 on NTDs in May 2013. Control of the endemic neglected zoonotic diseases (NZDs) would benefit from a similar campaign, which needs the support of a global community., Methodology/principal Findings: The resolutions from all 66 World Health Assembly (WHA) meetings held between 1948 and 2013 were examined to determine how many contain a specific focus on any of the following eight NZDs as defined by the World Health Organisation (WHO): anthrax, bovine tuberculosis (TB), brucellosis, Taenia solium cysticercosis, cystic echinococcosis (hydatidosis), leishmaniasis, rabies, and zoonotic human African trypanosomiasis (HAT or sleeping sickness). Twenty-one resolutions adopted in the 16 assemblies between 1948 and 2013 targeted one or more of these eight NZDs, representing 4% of the total resolutions on infectious diseases passed to date. The 2013 adoption of Resolution WHA66.12 targeting all 17 NTDs marks a change in approach by the WHA. Whereas previous resolutions have targeted the NTDs as separate entities, the new approach of the combined resolution will help increase the overall momentum to target these ancient diseases as coendemic clusters in endemic countries. However, three major NZDs remain outside this recent resolution: anthrax, brucellosis, and bovine TB., Conclusions and Significance: The recent adoption of a specific resolution at the WHA in 2013 that emphasises a One Health approach for the successful control of 17 NTDs is a major development in advocacy. However, recognition of the importance of three major NZDs to public health in endemic countries-anthrax, brucellosis, and bovine tuberculosis-is still lacking despite being prioritised by the WHA as early as the 1950s. Global advocacy for control of the NZDs as a whole would similarly benefit from adoption of a One Health approach as is promoted for the NTDs under WHA66.12.

Background: Uganda has active foci of both chronic and acute HAT with the acute zoonotic form of disease classically considered to be restricted to southeast Uganda, while the focus of the chronic form of HAT was confined to the northwest of the country. Acute HAT has however been migrating from its traditional disease focus, spreading rapidly to new districts, a spread linked to movement of infected cattle following restocking. Cattle act as long-term reservoirs of human infective T. b. rhodesiense showing few signs of morbidity, yet posing a significant risk to human health. It is important to understand the relationship between infected cattle and infected individuals so that an appropriate response can be made to the risk posed to the community from animals infected with human pathogens in a village setting., Methodology/principal Findings: This paper examines the relationship between human T. b. rhodesiense infection and human infective and non-human T. brucei s.l. circulating in cattle at village level in Kaberamaido and Dokolo Districts, Uganda. The study was undertaken in villages that had reported a case of sleeping sickness in the six months prior to sample collection and those villages that had never reported a case of sleeping sickness., Conclusions and Significance: The sleeping sickness status of the villages had a significant effect with higher odds of infection in cattle from case than from non-case villages for T. brucei s.l. (OR: 2.94, 95%CI: 1.38-6.24). Cattle age had a significant effect (p<0.001) on the likelihood of T. brucei s.l. infection within cattle: cattle between 18-36 months (OR: 3.51, 95%CI: 1.63-7.51) and cattle over 36 months (OR: 4.20, 95%CI: 2.08-8.67) had significantly higher odds of T. brucei s. l. infection than cattle under 18 months of age. Furthermore, village human sleeping sickness status had a significant effect (p<0.05) on the detection of T. b. rhodesiense in the village cattle herd, with significantly higher likelihood of T. b. rhodesiense in the village cattle of case villages (OR: 25, 95%CI: 1.2-520.71). Overall a higher than average T. brucei s.l. prevalence (>16.3%) in a village herd over was associated with significantly higher likelihood of T. b. rhodesiense being detected in a herd (OR: 25, 95%CI: 1.2-520.71).

Background: Tick-borne diseases (TBDs) present a major economic burden to communities across East Africa. Farmers in East Africa must use acaracides to target ticks and prevent transmission of tick-borne diseases such as anaplasmosis, babesiosis, cowdriosis and theileriosis; the major causes of cattle mortality and morbidity. The costs of controlling East Coast Fever (ECF), caused by Theileria parva, in Uganda are significant and measures taken to control ticks, to be cost-effective, should take into account the burden of disease. The aim of the present work was to estimate the burden presented by T. parva and its spatial distribution in a crop-livestock production system in Eastern Uganda., Methods: A cross sectional study was carried out to determine the prevalence and spatial distribution of T. parva in Tororo District, Uganda. Blood samples were taken from all cattle (n: 2,658) in 22 randomly selected villages across Tororo District from September to December 2011. Samples were analysed by PCR and T. parva prevalence and spatial distribution determined., Results: The overall prevalence of T. parva was found to be 5.3%. Herd level prevalence ranged from 0% to 21% with majority of the infections located in the North, North-Eastern and South-Eastern parts of Tororo District. No statistically significant differences in risk of infection were found between age classes, sex and cattle breed., Conclusions: T. parva infection is widely distributed in Tororo District, Uganda. The prevalence and distribution of T. parva is most likely determined by spatial distribution of R. appendiculatus, restricted grazing of calves and preferential tick control targeting draft animals.

Leptospirosis, caused by the spirochete bacterium Leptospira spp. is a zoonosis, distributed worldwide and classified as an emerging infectious disease. Fatal outcomes to leptospiral infection do occur and the disease can cause abortion and other reproductive problems in cattle, goats, and pigs. In humans the symptoms range from subclinical infection to acute febrile illness, pulmonary hemorrhage and renal failure. Leptospirosis has never been officially reported to the World Health Organization (WHO) or the World Animal Health Organization in animals or humans in Uganda. However, favorable ecological conditions and suitable animal hosts can be found within the country. A commercially available enzyme-linked immunosorbent (ELISA) kit was used to screen sera samples from domesticated cattle and African buffalo (Syncerus caffer) at two locations in southwestern Uganda, collected over a 4-year period. Positive samples were found in both cattle and African buffalo samples, from both locations and across the sampling period. Overall seroprevalence was 42.39% in African buffalo and 29.35% in cattle.

Background: Pig keeping is becoming increasingly common across sub-Saharan Africa. Domestic pigs from the Arusha region of northern Tanzania were screened for trypanosomes using PCR-based methods to examine the role of pigs as a reservoir of human and animal trypanosomiasis., Methods: A total of 168 blood samples were obtained from domestic pigs opportunistically sampled across four districts in Tanzania (Babati, Mbulu, Arumeru and Dodoma) during December 2004. A suite of PCR-based methods was used to identify the species and sub-species of trypanosomes including: Internally Transcribed Sequence to identify multiple species; species specific PCR to identify T. brucei s. l. and T. godfreyi and a multiplex PCR reaction to distinguish T. b. rhodesiense from T. brucei s. l., Results: Of the 168 domestic pigs screened for animal and human infective trypanosome DNA, 28 (16.7%) were infected with one or more species of trypanosome; these included: six pigs infected with Trypanosoma vivax (3.6%); three with Trypanosoma simiae (1.8%); two with Trypanosoma congolense (Forest) (1%) and four with Trypanosoma godfreyi (2.4%). Nineteen pigs were infected with Trypanosoma brucei s. l. (10.1%) of which eight were identified as carrying the human infective sub-species Trypanosoma brucei rhodesiense (4.8%)., Conclusion: These results show that in Tanzania domestic pigs may act as a significant reservoir for animal trypanosomiasis including the cattle pathogens T. vivax and T. congolense, the pig pathogen T. simiae, and provide a significant reservoir for T. b. rhodesiense, the causative agent of acute Rhodesian sleeping sickness.

Background: In recent years, PCR has been become widely applied for the detection of trypanosomes overcoming many of the constraints of parasitological and serological techniques, being highly sensitive and specific for trypanosome detection. Individual species-specific multi-copy trypanosome DNA sequences can be targeted to identify parasites. Highly conserved ribosomal RNA (rRNA) genes are also useful for comparisons between closely related species. The internal transcribed spacer regions (ITS) in particular are relatively small, show variability among related species and are flanked by highly conserved segments to which PCR primers can be designed. Individual variations in inter-species length makes the ITS region a useful marker for identification of multiple trypanosome species within a sample., Methods: Six hundred blood samples from cattle collected in Uganda on FTA cards were screened using individual species-specific primers for Trypanosoma congolense, Trypanosoma brucei and Trypanosoma vivax and compared to a modified (using eluate extracted using chelex) ITS-PCR reaction., Results: The comparative analysis showed that the species-specific primer sets showed poor agreement with the ITS primer set. Using species-specific PCR for Trypanozoon, a prevalence of 10.5% was observed as compared to 0.2% using ITS PCR (Kappa = 0.03). For Trypanosoma congolense, the species-specific PCR reaction indicated a prevalence of 0% compared to 2.2% using ITS PCR (Kappa = 0). For T. vivax, species-specific PCR detected prevalence of 5.7% compared to 2.8% for ITS PCR (Kappa = 0.29)., Conclusions: When selecting PCR based tools to apply to epidemiological surveys for generation of prevalence data for animal trypanosomiasis, it is recommended that species-specific primers are used, being the most sensitive diagnostic tool for screening samples to identify members of Trypanozoon (T. b. brucei s.l). While ITS primers are useful for studying the prevalence of trypanosomes causing nagana (in this study the species-specific primers did not detect the presence of T. congolense) there were discrepancies between both the species-specific primers and ITS for the detection of T. vivax.

Background: African Animal Trypanosomiasis (AAT) is a widespread disease of livestock in Nigeria and presents a major constraint to rural economic development. The Jos Plateau was considered free from tsetse flies and the trypanosomes they transmit due to its high altitude and this trypanosomiasis free status attracted large numbers of cattle-keeping pastoralists to the area. The Jos Plateau now plays a major role in the national cattle industry in Nigeria, accommodating approximately 7% of the national herd, supporting 300,000 pastoralists and over one million cattle. During the past two decades tsetse flies have invaded the Jos Plateau and animal trypanosomiasis has become a significant problem for livestock keepers. Here we investigate the epidemiology of trypanosomiasis as a re-emerging disease on the Plateau, examining the social factors that influence prevalence and seasonal variation of bovine trypanosomiasis., Methods: In 2008 a longitudinal two-stage cluster survey was undertaken on the Jos Plateau. Cattle were sampled in the dry, early wet and late wet seasons. Parasite identification was undertaken using species-specific polymerase chain reactions to determine the prevalence and distribution of bovine trypanosomiasis. Participatory rural appraisal was also conducted to determine knowledge, attitudes and practices concerning animal husbandry and disease control., Results: Significant seasonal variation between the dry season and late wet season was recorded across the Jos Plateau, consistent with expected variation in tsetse populations. However, marked seasonal variations were also observed at village level to create 3 distinct groups: Group 1 in which 50% of villages followed the general pattern of low prevalence in the dry season and high prevalence in the wet season; Group 2 in which 16.7% of villages showed no seasonal variation and Group 3 in which 33.3% of villages showed greater disease prevalence in the dry season than in the wet season., Conclusions: There was high seasonal variation at the village level determined by management as well as climatic factors. The growing influence of management factors on the epidemiology of trypanosomiasis highlights the impact of recent changes in land use and natural resource competition on animal husbandry decisions in the extensive pastoral production system.

Background: The northwards spread of acute T. b. rhodesiense sleeping sickness in Uganda has been linked to cattle movements associated with restocking following the end to military conflict in 2006. This study examined the number of cattle traded from T. b. rhodesiense endemic districts, the prevalence of the parasite in cattle being traded and the level of trypanocidal treatment at livestock markets., Methods: Between 2008 and 2009 interviews were carried out with government veterinarians from 20 districts in Uganda, 18 restocking organisations and numerous livestock traders and veterinarians. Direct observations, a review of movement permit records (2006 to 2008) and blood sampling of cattle (n = 1758) for detection of parasites were also conducted at 10 livestock markets in T. b. rhodesiense endemic districts., Results: Records available from 8 out of 47 identified markets showed that 39.5% (5,238/13,267) of the inter-district cattle trade between mid-2006 and mid-2008 involved movement from endemic areas to pathogen-free districts. PCR analysis showed a prevalence of 17.5% T. brucei s.l. (n = 307/1758 [95% CI: 15.7-19.2]) and 1.5% T. b. rhodesiense (n = 26/1758 [95% CI: 0.9-2.0]) from these same markets. In a two-year period, between late-2006 to late-2008, an estimated 72,321 to 86,785 cattle (57, 857 by 18 restocking organisations and 10,214 to 24,679 by private traders) were imported into seven pathogen-free northern districts, including districts that were endemic for T. b. gambiense. Between 281 and 1,302 of these cattle were likely to have carried T. b. rhodesiense. While governmental organisations predominantly adhered to trypanocidal treatment, most Non-Governmental Organisations (NGOs) and private traders did not. Inadequate market infrastructure, poor awareness, the need for payment for drug treatments, and the difficulty in enforcing a policy of treatment at point of sale contributed to non-compliance., Conclusion: With increasing private trade, preventing the spread of Rhodesian sleeping sickness in Uganda requires government support to ensure mandatory trypanocidal treatment at livestock markets, investment in market infrastructure and possible drug subsidy. Mapping the northern reaches of T. b. rhodesiense in livestock and preparation of risk assessments for cattle trading could mitigate future outbreaks.

Background: Trypanosomiasis is a widespread disease of livestock in Nigeria and a major constraint to the rural economy. The Jos Plateau, Nigeria was free from tsetse flies and the trypanosomes they transmit due to its high altitude and the absence of animal trypanosomiasis attracted large numbers of cattle-keeping pastoralists to inhabit the plateau. The Jos Plateau now plays a significant role in the national cattle industry, accommodating approximately 7% of the national herd and supporting 300,000 pastoralists and over one million cattle. However, during the past two decades tsetse flies have invaded the Jos Plateau and animal trypanosomiasis has become a significant problem for livestock keepers., Methods: In 2008 a longitudinal two-stage cluster survey on the Jos Plateau. Cattle were sampled in the dry, early wet and late wet seasons. Parasite identification was undertaken using species-specific polymerase chain reactions to determine the prevalence and distribution bovine trypanosomiasis. Logistic regression was performed to determine risk factors for disease., Results: The prevalence of bovine trypanosomiasis (Trypanosoma brucei brucei, Trypanosoma congolense savannah, Trypanosoma vivax) across the Jos Plateau was found to be high at 46.8% (39.0 - 54.5%) and significant, seasonal variation was observed between the dry season and the end of the wet season. T. b. brucei was observed at a prevalence of 3.2% (1% - 5.5%); T. congolense at 27.7% (21.8% - 33.6%) and T. vivax at 26.7% (18.2% - 35.3%). High individual variation was observed in trypanosomiasis prevalence between individual villages on the Plateau, ranging from 8.8% to 95.6%. Altitude was found to be a significant risk factor for trypanosomiasis whilst migration also influenced risk for animal trypanosomiasis., Conclusions: Trypanosomiasis is now endemic on the Jos Plateau showing high prevalence in cattle and is influenced by seasonality, altitude and migration practices. Attempts to successfully control animal trypanosomiasis on the Plateau will need to take into account the large variability in trypanosomiasis infection rates between villages, the influence of land use, and husbandry and management practices of the pastoralists, all of which affect the epidemiology of the disease.

Background: Ticks and tick-borne diseases (TBDs) undermine cattle fitness and productivity in the whole of sub-Saharan Africa, including Nigeria. The aim of this study was to document the composition of tick species, assessing the burden of infestation, in traditionally managed cattle in an area of central Nigeria where acaricides have not been used historically., Methods: The study was carried out in September 2010 in 9 villages belonging to three neighbouring local government areas in Plateau State, Nigeria. In each village all visible adult ticks were collected from at least 15 cattle (mean number = 25). Collected ticks were preserved in 70% ethanol to be counted and morphologically identified to the species level., Results: A total of 5011 ixodid ticks (1935 males and 3076 females) were collected from 228 cattle, comprising 14 calves, 33 juveniles, and 181 adults. Three tick genera (i.e., Amblyomma, Hyalomma, and Rhipicephalus, including the Boophilus sub-genus) and 11 species were identified. The most prevalent species was Rhipicephalus (Boophilus) decoloratus (41.4%), followed by Rhipicephalus (Boophilus) annulatus (15.4%), Rhipicephalus guilhoni (12.0%), Rhipicephalus (Boophilus) geigyi (7.6%), Hyalomma truncatum (7.4%), Amblyomma variegatum (6.3%), Rhipicephalus simus Group (4.0%), Rhipicephalus turanicus (1.2%), Rhipicephalus sanguineus (0.3%), Hyalomma rufipes (0.2%), and Rhipicephalus lunulatus (n = 1). Mean tick loads recorded were relatively high (22 ± 1.4), in spite of the practice of hand removal of ticks traditionally undertaken by the Fulani pastoralists in the area. Calves bore a significantly lower tick burden than adults (p = 0.004). Rhipicephalus (Boophilus) microplus was not found in the area, suggesting that the eastbound expansion of this tick species in West Africa, has not yet reached central Nigeria., Conclusions: This study ascertained the presence of a broad variety of cattle tick species, most of which are of veterinary importance. The presence of each tick species is correlated with the potential occurrence of tick-borne pathogens and suggestions for tick control in the area are considered. Results should assist the diagnosis of related TBDs in cattle as well as the strategic planning of cost-effective tick control.

Background: The importance of wildlife as reservoirs of African trypanosomes pathogenic to man and livestock is well recognised. While new species of trypanosomes and their variants have been identified in tsetse populations, our knowledge of trypanosome species that are circulating in wildlife populations and their genetic diversity is limited., Methodology/principal Findings: molecular phylogenetic methods were used to examine the genetic diversity and species composition of trypanosomes circulating in wildlife from two ecosystems that exhibit high host species diversity: the Serengeti in Tanzania and the Luangwa Valley in Zambia. Phylogenetic relationships were assessed by alignment of partial 18S, 5.8S and 28S trypanosomal nuclear ribosomal DNA array sequences within the Trypanosomatidae and using ITS1, 5.8S and ITS2 for more detailed analysis of the T. vivax clade. In addition to Trypanosoma brucei, T. congolense, T. simiae, T. simiae (Tsavo), T. godfreyi and T. theileri, three variants of T. vivax were identified from three different wildlife species within one ecosystem, including sequences from trypanosomes from a giraffe and a waterbuck that differed from all published sequences and from each other, and did not amplify with conventional primers for T. vivax., Conclusions/significance: Wildlife carries a wide range of trypanosome species. The failure of the diverse T. vivax in this study to amplify with conventional primers suggests that T. vivax may have been under-diagnosed in Tanzania. Since conventional species-specific primers may not amplify all trypanosomes of interest, the use of ITS PCR primers followed by sequencing is a valuable approach to investigate diversity of trypanosome infections in wildlife; amplification of sequences outside the T. brucei clade raises concerns regarding ITS primer specificity for wildlife samples if sequence confirmation is not also undertaken.

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.