A survey of livestock to identify the presence of human infective trypanosomes in the Trypanosoma brucei gambiense endemic districts of north-west Uganda

A survey of livestock to identify the presence of human infective trypanosomes in the Trypanosoma brucei gambiense endemic districts of north-west Uganda | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search A survey of livestock to identify the presence of human infective trypanosomes in the Trypanosoma brucei gambiense endemic districts of north-west Uganda Enock Matovu , Annah Kitibwa , Darwin Bella Okot , Alex Boobo , Charles Wamboga , Steve J Torr , Sylvain Biéler , View ORCID Profile Paul R. Bessell , Joseph M. Ndung’u doi: https://doi.org/10.1101/2025.11.27.25341151 Enock Matovu 1 College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University , Kampala, Uganda Find this author on Google Scholar Find this author on PubMed Search for this author on this site Annah Kitibwa 1 College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University , Kampala, Uganda Find this author on Google Scholar Find this author on PubMed Search for this author on this site Darwin Bella Okot 2 Production Department, Amuru District Local Government , Uganda Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alex Boobo 1 College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University , Kampala, Uganda Find this author on Google Scholar Find this author on PubMed Search for this author on this site Charles Wamboga 3 Ministry of Health , Kampala, Uganda Find this author on Google Scholar Find this author on PubMed Search for this author on this site Steve J Torr 4 Liverpool School of Tropical Medicine (LSTM) , Liverpool, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sylvain Biéler 5 FIND, Global Health Campus Chemin du Pommier 40 1218 Le Grand-Saconnex , Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paul R. Bessell 6 Independent consultant , Edinburgh, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paul R. Bessell For correspondence: prbessell{at}gmail.com Joseph M. Ndung’u 7 FIND, c/o Kenya Medical Research Institute , Off Mbagathi Road, P. O. Box 54840 – 00200, Nairobi, Kenya Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Data/Code Preview PDF Abstract Background Gambian human African trypanosomiasis (gHAT) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by tsetse flies ( Glossina ). Humans are the major host and mass screening and treatment of the human population has contributed to the elimination of gHAT as a public health problem in many countries, including Uganda. There is some evidence that animals may act as hosts for T. b. gambiense which may undermine efforts to eliminate transmission. We undertook a study to determine whether cattle and pigs were harbouring T. b. gambiense in a region of north-western Uganda where efforts to eliminate transmission of gHAT were ongoing. Methodology Blood was collected from 2,775 cattle and 571 pigs and examined for the presence of trypanosomes by the haematocrit centrifugation technique (HCT). In addition, polymerase chain reaction (PCR) was used to amplify the trypanosomal internal transcribed spacer (ITS1). Those whose ITS-1 results indicated the Trypanozoon group ( T. brucei ) were further tested by sub-species specific PCRs for Trypanosoma brucei gambiense and T. b. rhodesiense . The results were analysed by species of infecting parasite, geographic location and presence or absence of vector control activities in the area. Principal Findings No samples were found positive for potentially human infective trypanosomes. Among the cattle samples, 2.2% were positive by HCT (95% CI = 1.7% - 2.8%), while 5% were positive by PCR (95% CI = 4.3% - 5.9%), but this varied depending on location with up to 26.5% positive in one village. Among pigs, 3 samples (0.6%; 95% CI 0.2% - 1.6%) were positive by HCT and none were positive by PCR. There was no significant association between trypanosome species and vector control activities (p > 0.05). Based on these results it was concluded with 95% confidence that human infective trypanosomes are not circulating in livestock at a prevalence greater than 1/1000, and when this is augmented with results of previous surveys the parasite is not circulating at a prevalence greater than 1/2734. At the level of the trypanosome species, there is no significant difference between the frequencies of species in vector controlled versus non-vector controlled areas (p > 0.05). Conclusions These results support the evidence base that livestock are not an important reservoir and therefore not a threat to the elimination of Gambian human African trypanosomiasis in Uganda. Author summary Two subspecies of the same parasite cause human African trypanosomiasis (HAT) known as sleeping sickness. The east African form has a reservoir in livestock and wildlife with spillover to human hosts, whilst the west African form circulates in humans. However there have been some reports of the west African parasite in animals. Uganda is nearing elimination of the west African form, however to ensure elimination it is essential to verify the absence of the parasite in livestock in the endemic area. In this study a comprehensive livestock survey with testing of blood samples did not find the parasite in livestock. Whilst it is practically impossible to verify the total absence of the parasite, this study combined with studies show that the prevalence of the parasite in cattle or pigs is less than 1/2734 making a very unlikely reservoir. Introduction Uganda is endemic for both the acute and chronic forms of human African trypanosomiasis (HAT) caused by Trypanosoma brucei rhodesiense and T. b. gambiense respectively. Control efforts by the WHO, several NGOs, as well as the national control program have resulted in a continued decline in incidence of both forms of the disease over the past decades [ 1 ]. HAT incidence in Uganda has continued to decline, and the past three years have not registered any T. b. gambiense (gHAT) cases, while just a handful of T. b. rhodesiense (rHAT) cases have been recorded ( Fig 1 ). In the T. b. gambiense endemic districts in north western Uganda, there were deliberate efforts to improve access to HAT screening and case detection [ 2 ]. This was alongside vector control activities that have been shown to be effective at reducing vector numbers and gHAT case numbers [ 3 – 6 ]. Thanks to this and other efforts, only 8 cases were reported between 2017 and 2020, and not one case of gHAT has been reported since then ( Fig 1 ). Download figure Open in new tab Fig 1. Reported HAT cases in Uganda. Top 1990-2023 and bottom 2010 – 2024 for the two species of human infective trypanosome – T.b.g = Trypanosoma brucei gambiense ; T.b.r = Trypanosoma brucei rhodesiense . Data from WHO Global Health Observatory https://www.who.int/data/gho/data/themes/topics/human-african-trypanosomiasis . As a consequence of these efforts, in May 2022 the WHO validated the elimination of gHAT as a public health problem in Uganda ( https://www.who.int/news/item/24-05-2022-benin--uganda-and-rwanda-eliminate-human-african-trypanosomiasis-as-a-public-health-problem ). A component of the validation dossier was understanding the role of domestic animals in transmission and in particular their role as potential reservoirs of the disease. In the rhodesiense areas of south-eastern Uganda, it has been well demonstrated that cattle, dogs and pigs are reservoirs of the acute disease [ 7 – 9 ]. In north western Uganda, there had previously been two surveys to investigate animal African trypanosomiasis (AAT) and the role of animals in chronic HAT transmission [ 10 , 11 ]. These surveys did not cover the entire area at risk and neither study was able to demonstrate any potentially infective trypanosomes in cattle, goats, sheep, dogs and pigs but did identify that these species form a large part of tsetse bloodmeals [ 11 ]. The number of livestock in that region is also not as high compared to those in rhodesiense endemic areas where some districts are more populated by pastoral communities. The national livestock census of 2008 put the figures in the study area at 643,658 cattle; 1,187,039 goats; 325,694 sheep and 82,668 pigs [ 12 ]. Assuming a 3% increase per annum, at the time of study the populations would be 0.8m cattle, 1.5m goats, 0.4m sheep and 100,000 pigs. However, these numbers could have been underestimates as over the past 5 years there had been an influx of animals brought by pastoralists from the southern districts to exploit the abundant pasture in the region. Secondly, the frequent periods of political instability in South Sudan leads to refugees who cross with animals. Given this gap in knowledge about the role of livestock as reservoirs of HAT in north western Uganda and the need for information to support elimination of HAT in the region, we undertook a study to determine whether livestock in the region harbour potentially human infective trypanosomes, which might threaten efforts to eliminate transmission of gHAT. The key objectives of the study were: To identify to species level, the trypanosomes circulating in domestic animals in T. b. gambiense endemic districts of north western Uganda To determine whether domestic animals in that region harbor potentially human infective T. brucei . To identify any association between tsetse control activities and circulating trypanosome species. Methods Study area The study area was the gHAT focus in north-western Uganda, which as of 2022 comprised 10 districts (Adjumani, Amuru, Arua, Koboko, Madi-Okollo, Maracha, Moyo, Obongi, Terego and Yumbe) with a population of 2.22 million, and a large and fluid population of refugees from neighbouring Republic of South Sudan [ 2 , 13 , 14 ]. The area has a large livestock population, dominated by ruminant farming and sporadic pig rearing. Between August and October 2019, we carried out field surveys targeting selected parishes across the study area covering the 7 districts of Adjumani, Amuru, Arua, Koboko, Maracha, Moyo and Yumbe (district boundaries have subsequently changed but here we use the boundaries as they were in 2019, as defined at the time of the study). Sample size estimation and site selection Focusing on cattle and pigs, we estimated a required sample size of 200 animals from each of 16 sampling sites (total of 3,200 animals). This sample size gives 80% confidence (power) of detecting at least one infected animal at a gHAT prevalence of 1/1,000. To estimate the required sample size we: Test a range of number of sampling sites from 1 to 20 (nSites) Randomly sample the prevalence of gHAT at each site from a beta (1, 1000) distribution (cPrev) equivalent to a median prevalence of 1/1000. Test at each sample site a number of cattle samples (nCattle) from 10 to 500 at increments of 10, based on the cPrev for that site and nCattle we use a binomial distribution to sample the number of positive cattle at that site Run each combination for 100,000 iterations. Calculate for each combination of nSites and nCattle the proportion of iterations with one or more positive cattle Find the smallest combination of nSite and nCattle which gives one or more positive cattle with a probability greater than 80%. For practical reasons, sampling was done at the parish level. Parishes were selected based on sampling parishes with greater cattle numbers, greater tsetse numbers (from reported tsetse survey data), greater levels of HAT RDT positivity in humans and the number of confirmed HAT cases. The following sites (parishes) were selected, including six where no tsetse control had been undertaken in the recent past ( Fig 2 ): Download figure Open in new tab Fig 2. Study area. Study parishes included in the livestock survey based on whether there was ongoing tsetse control activities in the area. The stars denote cases that were detected between 2016 and 2018. Boundary data are from GADM ( https://gadm.org/ ), rivers are derived by the authors from the NASA Shuttle Radar Topography Mission (SRTM) using the HydroSHEDS method [ 15 ]. Data are available from CC-BY compatible sources. Adjumani district: Okangai Amuru district: Bibia, Pogo Arua district: Aliba-Robu, Ayuri Koboko district: Godia, Lurujo, Asunga Maracha district: Mundru, Robu Moyo district: Dufile, Gweere, Ewafa, Yumbe district: Kerwa, Gojuru, Oluba Sample collection Farmers owning cattle and pigs were mobilized through the District Veterinary Officers in consultation with local leaders, to bring their animals at a central site selected per parish. Owners presented with cattle and pigs and where the owners consented animals were bled by venipuncture into EDTA tubes, and the blood subjected to the haematocrit centrifugation technique (HCT) (Woo, 1971) to estimate the parasitological prevalence per site. Aliquots were spotted on filter paper, while the remaining blood was centrifuged to separate out plasma. Both the plasma and blood cell pellets were frozen in liquid nitrogen; the former for future studies and the latter for DNA extraction and downstream analyses to identify the infecting trypanosome species (Cox et al., 2005; Njiru et al., 2005). In the laboratory, we started with the PCR targeting the Internal Transcribed Spacer (ITS-1) as previously described, first by the single step ITS-1 PCR (Njiru et al., 2005) and nested ITS-1 PCR (Cox et al., 2005) for those in which no signals could be picked by the single step PCR. Any samples that indicated presence of T. brucei were further subjected to the T. b. gambiense and T. b. rhodesiense specific PCRs targeting the Trypanosoma gambiense Surface Glycoprotein (TgSGP) and the Serum Resistance Associated (SRA) genes respectively, using nested PCR as previously described (Maina et al., 2007), the latter to rule out the spread of the acute rHAT with animal movement from the south-east. As an incentive to farmers, their trypanosome positive cattle were treated with diminazene aceturate. This drug is also used for treatment of the tick-borne disease babesiosis, thus treatment of the animals was likely to lead to an improved general herd health. Data analysis In the event of the pathogen not being found, we evaluated the likelihood of absence of the pathogen by applying a negative binomial distribution to the actual sample size. We simulated a range of prevalences from 1/(100…5000) and used the rnbinom() function in the R statistical environment [ 16 ] to give the probability of finding at least one positive sample at a given prevalence and the size of the livestock population that was sampled. To add further weight to the analysis, we augmented our results with data form three previous studies [ 10 , 11 , 17 ] ( Table 1 ). This brought in an additional 5,109 cattle and 902 pig samples. View this table: View inline View popup Download powerpoint Table 1. Additional livestock testing data used to augment the survey results from surveys that also found absence of human infective trypanosomes in the area. To evaluate the difference between tests in vector controlled and non-vector controlled areas, we used a mixed effects logistic regression model with the test result the outcome and the clustering at village level as a random effect. This was implemented in the lme4 [ 18 ] package for R [ 16 ]. 95% confidence intervals for binary data were calculated using the Hmisc package for R [ 16 ]. Ethics statement The study protocol was reviewed and approved by the Ministry of Health (Vector control Division Research Ethics Committee; VCDREC) and final clearance was provided by the Uganda National Council for Science and Technology as study number A604. Results Samples were taken from 2,775 cattle from 16 villages and 531 pigs from 10 villages ( Table 2 and Fig 3 ). By HCT, 60 (2.2%) samples were positive among cattle (95% CI = 1.7% - 2.8%) and three samples from pigs were positive (0.6%; 95% CI 0.2% - 1.6%). By PCR, 5.0% (95% CI = 4.2% - 5.9%) were positive from cattle, but this varied hugely from 26.5% (n=155) in one village (Mindrabe) to 0% in the villages of Esia (n = 116) ( Table 2 ). No pigs were PCR positive ( Table 2 ). Download figure Open in new tab Fig 3. Sampling sites. Map of livestock sampling sites that were used in these analyses. The boundary and waterbody layers were obtained from CC-BY License compatible sources: GADM ( http://www.gadm.org ), and EnergyData ( https://energydata.info/ ).Other layers were digitised by the authors. View this table: View inline View popup Download powerpoint Table 2. Summary of the key results by parasitology and PCR. Considering the whole sample collection (3,306 samples from 2,775 cattle and 531 pigs), PCR yielded many more positive results (from 140 samples; 4.2%) than the HCT (63 samples; 1.9%). The species composition among the PCR positive samples was mainly T. congolense (47.1%), followed by T. vivax (38.6%) and the benign T. theileri (29.3%), while T. brucei was least represented (12.9%). No samples were positive by T.b. gambiense or T.b. rhodesiense specific PCR. Whilst it is impossible to measure zero in such an instance, we can conclude with 95% certainty that if human infective trypanosomes had been present among cattle then it would have been at a prevalence below 0.11% and among pigs below 0.56%, and overall between both species below 0.09%. Based on the number of animals tested, if the pathogen had been circulating at a prevalence of 1/1,000, the probability of picking an infected animal would have been 96.3%. Based on this, we can say with 95% confidence that if human infective trypanosomes were circulating, then the prevalence among all livestock was less than 1/1,104; among cattle, the corresponding prevalence was less than 1/927 and among pigs less than 1/178 ( Fig 4 ). Download figure Open in new tab Fig 4. Probability of finding trypanosomes. Cumulative distribution function of the changes in the probability of finding at least one trypanosome infected animal as the prevalence varies given the sample sizes for cattle, pigs and all livestock. Updating the results in Fig 4 by bringing in results from previous studies we can say with 95% confidence that if human infective trypanosomes were circulating, then the prevalence among all livestock was less than 1 / 2848; among cattle, the corresponding prevalence was less than 1 / 2369 and among pigs less than 1 / 479 ( Fig 5 ). Download figure Open in new tab Fig 5. Combined probability of finding trypanosomes. Updating the results of Fig 4 to include results of sampling from previous studies in the area. Note the different scale compared to Fig 4 . A total of 140 samples were positive by PCR (prevalence 4.2%), and of these 40 were also positive by HCT out of a total of 63 positive by HCT (prevalence 1.9%). Among cattle samples, 60 were positive by HCT; 40 were also positive by PCR; while 20 negative by PCR. Of the 40 positive by PCR, 5 were positive for T. vivax , 14 for T. congolense ; 4 for brucei and 19 for T. theileri, a common but benign species in cattle transmitted by Tabanids. Noteworthy is that by PCR, 34 were positive for T. theileri , so for samples that were PCR positive for T. theileri, the majority (83%) were positive by HCT; this is in contrast to T. brucei (22.2% positive by HCT), T. congolense (21.2% positive) and T. vivax (9.3% positive). Results in areas with vector control activities Comparing the results from the livestock samples to the distribution of vector control activities, 60.4% of samples were from areas undergoing vector control, comprising a greater proportion of porcine samples, with 64.6% of pig samples and 59.6% of bovine samples from areas undergoing vector control. Evaluating the results of different PCR tests by trypanosome species and the vector control status, there was no significant difference in any of the tests (p > 0.05). once clustering at the village level was accounted for in a mixed model. Discussion This study set out to investigate possible existence of potentially human infective trypanosomes circulating within domestic animals in the T. b. gambiense endemic districts of Northwestern Uganda, as a basis to inform gHAT elimination activities in the region. Presence of an undetected animal reservoir would frustrate elimination efforts as it would provide a source of infection from which the existing tsetse flies would continue transmitting gHAT. Previous studies conducted in West Africa had indicated that several animals could act as reservoirs of T. b. gambiense in countries such as Cameroon, Guinea, and Ivory coast, among others (Reviewed by Buscher et al, 2018). The results of this study were surprising in that 20 of the samples in which trypanosomes had been detected by the HCT did not yield any signals from PCR, which is supposed to be highly sensitive, yet it detected signals in samples in which trypanosomes were not detected by the HCT. This could have been due to failure of primers to adequately anneal at the target templates, or to limiting parasite numbers much as they had been detected by parasitology; the parasiteamia was generally low, in many cases sighting just a single trypanosome wriggling in the buffy coat area. The very few detectable trypanosomes could have been lost during sample processing, leading to negative PCR results. In a separate study, Ilboudo et al (2023) observed that some parasitology positive samples from experimentally infected pigs could turn out negative by PCR at some time points, mirroring our observations in field collected samples. According to the ITS-1 PCR, the majority of infections were T. congolense followed by T. vivax and T. brucei (Trypanozoon), while none of the latter had any signals for T. b. gambiense or T. b. rhodesiense . The district veterinarians and farmers were sensitized about the need to deliberately control trypanosomiasis and tsetse flies even as a measure against the important animal disease affecting livestock productivity, while securing health by clearing any possible animal reservoir of HAT. Presence of the benign T. theileri indicates that the Tabanids are in existence alongside tsetse flies, with the associated impact on livestock, especially as nuisance flies in addition to driving mechanical transmission of T. vivax alongside biological transmission by tsetse flies. Twenty of the infections comprised more than one infecting species (see supplementary data table), which could be expected in the natural setting in which tsetse flies obtain their blood meals from multiple hosts that could be carrying different trypanosome species. In the gHAT focus of north-western Uganda, case numbers have been in decline, driven by integrated medical and vector control activities [ 2 , 5 , 6 , 19 ]. As a result of this reduction in incidence, the WHO in 2022 validated Uganda as having eliminated gHAT as a public health problem. As well as demonstrating low incidence in the human population, it was also necessary to demonstrate a reduction in numbers of vectors and the absence of human infective trypanosomes in the sizeable livestock population in this area. As we were not able to detect human infective trypanosomes despite a relatively large sample size we concluded that if the pathogens were circulating then this would not have been above a very low prevalence. Whilst we cannot rule out a risk to humans, this is very unlikely when one considers the very poor vectorial capacity of tsetse flies and relatively low risk of onward transmission to humans, as demonstrated in modelling studies [ 20 ]. This is in agreement with findings from a focus in Côte d’Ivoire, where much higher rates of infections with Trypanosome species were found in all animals (30% pig samples positive by PCR), but also no samples were positive for T. b. gambiense [ 21 ]. However, in the same study 27.6% of pig samples were positive by immunological testing using the trypanolysis test [ 21 ]. Noteworthy is a recent study in pigs which indicated that the trypanolysis test may be reactive even for non- T. b. gambiense infections (Ilboudo et al. 2022). In this study, trypanolysis was not done as it would not have given unequivocal evidence for presence of T. b. gambiense in the animals. Strengths and limitations One of the challenges of such analyses is reasonably measuring zero in a very large population. It is only possible to survey a small proportion of the livestock in the area. Hence, the approach that we adopted here was to demonstrate that the pathogen was circulating below a certain prevalence. The baseline rate that we have adopted here was 1/1,000 in that we have shown that the pathogen, if circulating, was circulating at a prevalence below 1/1,000. When augmented with previous studies the corresponding prevalence dropped to 1 / 2,848. Even if the pathogen were circulating at this very low prevalence the low number of flies and low vectorial capacity would give very little scope for livestock to contribute to transmission to human hosts [ 22 ]. Conclusions Whilst this is the third study to measure levels of human infective trypanosomes in this focus it is a innovative study in that 1) we covered each of the districts in the focus and 2) it was conducted following the implementation of vector control in the area which should have reduced transmission. However, we demonstrated that there was no observable impact of vector control on infection rates, which is consistent with a reduction in tsetse across the area [ 23 ]. Whilst the number of animals positive by PCR seems to have declined since 2008 [ 10 ], there has been no measurable decline since the more recent studies [ 11 ]. This suggests that there is no strong signature of vector control activities on detection of trypanosome species in this focus. In conclusion, this study has not demonstrated the presence of T. b. gambiense circulating in cattle and pigs from the endemic districts of Northwestern Uganda. However, as a requirement of post elimination surveillance prescribed by the NTD elimination roadmap, surveillance should be continued, at least at the sentinel sites within the region, to pre-empt any posible re-emergence of gHAT as a public health problem. Data Availability All data produced in the present work are contained in the manuscript Funding statement This study was financed by the Bill and Melinda Gates Foundation INV-001785. Author contributions Conceptualization . Enock Matovu, Charles Wamboga, Steve Torr, Sylvain Biéler, Paul Bessell Joseph Ndung’u Data Curation . Enock Matovu, Annah Kitibwa, Darwin Bella Okot, Alex Boobo Formal analysis . Paul Bessell Funding acquisition . Steve Torr Sylvain Biéler, Joseph Ndung’u Investigation . Enock Matovu, Annah Kitibwa, Darwin Bella Okot, Alex Boobo Methodology Enock Matovu, Charles Wamboga, Steve Torr Sylvain Biéler, Paul Bessell, Joseph Ndung’u Supervision . Enock Matovu, Charles Wamboga Visualisation. Paul Bessell Writing – original draft preparation. Enock Matovu, Sylvain Biéler, Paul Bessell, Joseph Ndung’u Writing – review & editing. Enock Matovu, Annah Kitibwa, Darwin Bella Okot, Alex Boobo, Charles Wamboga, Steve J Torr Sylvain Biéler, Paul Bessell, Joseph Ndung’u Acknowledgements We wish to thank the District Veterinary and Health Offices in the entire region for their support, the farmers who presented their animals for the surveys as well as all the technical staff for their various inputs. References 1. ↵ Franco JR , Cecchi G , Priotto G , Paone M , Diarra A , Grout L , et al. Monitoring the elimination of human African trypanosomiasis at continental and country level: Update to 2018 . Animal Production and Health Division . 2020 . doi: 10.1371/journal.pntd.0008261 OpenUrl CrossRef 2. ↵ Wamboga C , Matovu E , Bessell PR , Picado A , Biéler S , Ndung’u JM . 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