Hindawi Journal of Parasitology Research Volume 2022, Article ID 2606871, 7 pages https://doi.org/10.1155/2022/2606871 Research Article Investigation on Prevalence of Canine Trypanosomiasis in the Conservation Areas of Bwindi-Mgahinga and Queen Elizabeth in Western Uganda James Robert Ochieng ,1 Marta Planellas Bachs,2 Anthony Nsubuga,3 Innocent B. Rwego,4 John Joseph M. Kisakye,1 Laura Muro Riba,5 and Jesus Muro Figueres5 1 Department of Zoology, Entomology and Fisheries Sciences, College of Natural Sciences, Makerere University, Kampala, Uganda 2 Departament de Medicina I Cirurgia Animals. Universitat Autònoma de Barcelona, Bellaterra, Spain 3 Department of Plant Sciences and Biotechnology, College of Natural Sciences, Makerere University, Kampala, Uganda 4 Department of Biosecurity, Ecosystem and Veterinary Public Health, College of Veterinary Medicine, Animal Resources and Biosafety (COVAB), Makerere University, Kampala, Uganda 5 Daktari East Africa, WARM Department, Makerere University, Kampala, Uganda Correspondence should be addressed to James Robert Ochieng; jjamesro@gmail.com Received 15 May 2022; Accepted 18 August 2022; Published 10 September 2022 Academic Editor: John Kagira Copyright © 2022 James Robert Ochieng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nowadays, despite the instauration of several control strategies, animal trypanosomiasis continues to be reported all over Uganda. Few canine African trypanosomiasis (CAT) studies have been carried out, yet dogs are known Trypanosoma reservoirs that share identical home ranges with livestock and serve as parasite link between livestock and humans. This study evaluates the prevalence of CAT in dogs in the Bwindi-Mgahinga and Queen Elizabeth conservation areas. This information will be useful to evaluate the possible role of dogs in the transmission cycle of Trypanosoma species in livestock and wild animals. Trypanosome tests using microhematocrit centrifugation/dark ground microscopy technique (MHCT) followed by conventional polymerase chain reaction (cPCR) were performed in blood samples collected from identified indigenous dogs (n = 124). Four (3.23%) out of 124 dogs were positive for CAT. One dog was positive with Trypanosoma congolense and three with T. vivax. There was no significant statistical difference in CAT prevalence rate in relation to dog’s age, sex, and site (P > 0:05). This study reports what we believe is the first time detection of T. congolense and T. vivax in the indigenous dogs found in the Bwindi-Mgahinga and Queen Elizabeth conservation areas in western Uganda. The noticed T. congolense and T. vivax could be responsible for both canine and animal trypanosomiasis and represent a serious threat to the livestock industry. Therefore, there is a need for continuous trypanosomiasis surveillance and integrated management in contiguity to wildlife reserves. 1. Introduction straint to livestock health and development [3, 6–9]. AAT annually affects hundreds of thousands of livestock espe- The African animal trypanosomiasis (AAT), commonly cially cattle in sub-Saharan Africa, leading to premature known as nagana, caused by protozoan parasites of the abortion, infertility, and a decline in meat, milk production, genus Trypanosoma, transmitted by Glossina spp. (tsetse and draught power [6, 7, 9–13]. The AAT dominant vectors fly) and mechanically by hematophagous flies in the genus include Glossina morsitans, G. pallidipes, G. f. fuscipes, G. Stomoxys (stable fly) and Tabanus (horse flies), is prevalent brevipalpis, G. m. morsitans, and G. p. palpalis [1, 8, 9]. in sub-Saharan Africa [1–3]. The reservoir hosts include Canine trypanosomiasis has been reported in the UK wild and domestic animals [1–5]. AAT caused by Trypano- [14], Brazil [15], the USA [16, 17], Senegal [18], Zambia soma congolense, T. vivax, and T. simiae is a significant con- [4], Nigeria [19], Kenya [20], and Uganda [9, 21, 22]. Studies 2 Journal of Parasitology Research by Abenga and Lawal [23] and Matete [20] stated that some including several habituated groups, which can indigenous dogs infected by trypanosomes in tsetse-infested be tracked regions of sub-Saharan Africa did not exhibit any clinical signs of the infection. Those dogs seem to be tolerant to (2) Mgahinga Gorilla National Park (MGNP), created in the trypanosomes, increasing difficulties in detecting or sus- 1991, is small, occupying 3,390 ha (33.9 km2) in pecting the disease. In Uganda, numerous bovine, caprine, southwestern Uganda, bordering Rwanda to the and porcine trypanosomiasis research have been conducted south and the Democratic Republic of Congo in several parts of the country [6, 7, 9, 12, 24–28]. In all these (DRC) to the west. MGNP is contiguous to Virunga studies, reservoir hosts implicated include wildlife species National Park (VNP) in the DRC and Volcanoes such as nonhuman primates, warthogs, hippopotamus, National Park in Rwanda. MGNP was established crocodiles, and monitor lizards with little or no suspicion mainly to protect mountain gorillas (Gorilla gorilla for dogs. beringei), vulnerable populations of indigenous In southwestern Uganda, including Kasese, Kabale, and plants and animals endemic to the area, and other Kisoro districts, traditional livestock farmers keep dogs ecological resources [32] mostly for security (guarding property including livestock), (3) Queen Elizabeth National Park (QENP), gazetted in herding, hunting, and scaring off vermin from the nearby 1950, is located within the Albertine Rift in Western protected areas. These dogs roam freely in communities, in Uganda, covers a total area of 1,978 km2, comprised and around Bwindi-Mgahinga and Queen Elizabeth national of hills, plains, forest, and swamp, and abuts the parks, increasing their chances of serving as a link for para- border of the Democratic Republic of Congo. QENP site exchange among wildlife, livestock, and humans. This is is the second largest National Park in Uganda, cover- critical given the fact that Bwindi-Mgahinga and Queen ing the districts of Kanungu, Bushenyi, Kasese, Elizabeth national parks are the largest contiguous and inter- Rukungiri, Kamwenge, and Ibanda. QENP includes linked conservation areas [29, 30] with diverse populations more than half the Uganda shoreline of the great of protected and tourist-attractive animals including the lakes Edward and George, as well as the 20-mile- mountain gorillas. Up-to-date, little is known about the long Kazinga Channel which connects the two, and Trypanosoma species parasites responsible for canine is listed as a world biosphere reserve [33, 34] African trypanosomiasis (CAT) in indigenous dogs in Uganda. The objective of the current study was to define the profile of CAT infection burden in dogs that live in the 2.2. Sample Collection. Clearance for sampling was obtained communities contiguous to Bwindi-Mgahinga and Queen from Uganda National Council for Science and Technology, Elizabeth conservation areas. This information can increase dog owners, and district veterinary offices, in order to obtain our awareness on health and transmission risk among dogs, canine blood samples during Daktari East Africa NGO activ- livestock, wild animals, farmers, and the tourists that flock ities in the conservation areas studied. Daktari East Africa the area from all over the world. On the other hand, this (http://www.daktariandorra.org) is a veterinary NGO that, study will increase the knowledge of canine trypanosomiasis among other activities, provides rabies vaccination, deworm- and the species involved in Uganda. ing, treatment, and surgical sterilizations in African domes- tic animals including dogs. Samples were collected between the 7 and 22 July of 2018. Demographic data, signalment 2. Materials and Methods (species, age, sex, breed, and season), and clinical signs were recorded for each dog. According to owners’ information, 2.1. Study Sites. Surveys were carried out in and around any dog that has never received trypanocidal treatment was Bwindi-Mgahinga and Queen Elizabeth conservation area included in the study. Blood samples were conveniently in western Uganda (Figure 1). collected from identified indigenous dogs (Canis familiaris; n = 124), living in the conservation areas of Queen Elizabeth (1) Bwindi Impenetrable National Park (331 km2; 0°53– (13 females, 29 males), Bwindi Impenetrable (14 females, 28 1°08′N; 29°35′–29°50′E) in southwestern Uganda males), and Mgahinga Gorilla National Park (13 females, 27 on the eastern edge of the Albertine Rift Valley is males). The dogs were aged 6 months and above and were an afro-montane moist, evergreen rainforest with ranked following the international veterinary age categories an altitude range of 1,160 to 2,607 m, characterized [35], namely, juvenile (6-12 months), young adult (aged 1 by steep-sided hills, peaks, narrow valleys, and aver- year or 12–24 months), mature adult (2-6 years), senior age daily rainfall of 1.25 to 3.88 ml and temperature adults (7-11 years), and geriatric (above 11 years). Two to range of 19.8 to 27.7°C [31]. This “impenetrable rain three milliliters of blood was obtained from the cephalic or forest” is one of the most diverse forest ecosystems in jugular vein of each dog into a 5-ml ethylenediamine tetra East Africa, with at least 223 known tree species of acetic (EDTA) vial. Of this blood, the portion was used for approximately 53% of Uganda’s tree flora, and pro- the preparation of microhematocrit tubes for subsequent vides shelter to several birds including 23 Albertine centrifugation and examination for trypanosomes as pre- Rift endemics, nonhuman primates, and other wild scribed by [36]. The rest of the blood samples were stored animals. Bwindi protects an estimated 400 mountain in liquid nitrogen until polymerase chain reaction was gorillas, roughly half of the world’s population, performed in the laboratory. Journal of Parasitology Research 3 Figure 1: Location of western Uganda showing Queen Elizabeth, Bwindi Impenetrable and Mgahinga Gorilla National Parks, and the conservation areas (marked in round red dots) where dogs were sampled from. 2.3. Ethical Clearance. This study was approved by the to include the uppermost layer of red blood cells and 3 cm Uganda National Council for Science and Technology above to include the plasma and then transferred to a glass (UNCST) on 10 June 2018. At the district level, this study slide and covered with a cover slip (22 × 22 mm). The wet was approved by the district veterinary officers (DVOs) from smear was observed for trypanosomes under the microscope Kasese, Kabale, and Kisoro. Dog owners were mobilized by at × 10 eyepieces in combination with a × 25 objective with holding village meetings in churches and centers through reduced illumination and classified into species basing on their the local council authorities who kept on reminding dog size and movement behavior, whereby T. vivax is large and owners to be alert of the sampling exercises on the agreed moves faster across the field, T. congolense is small in size com- date and time. pared to red blood cell diameter, sluggish/vibrates without progressing and its invariable attachment to red blood cells, 2.4. Detection of Trypanosomes with Microhematocrit and T. brucei is large and swims within a limited area [36]. Centrifugation/Dark Ground Microscopy Technique (MHCT). The capillary tubes were centrifuged at 12,000 rpm for 5 min, 2.5. DNA Extraction and Molecular Identification by PCR. and the trypanosomes present were detected by observation All the collected dog samples (males =84 and females =40) of parasite motion just above the buffy coat. In a sample where were analyzed. GenElute Blood Genomic DNA kit was used trypanosomes were detected, the microhematocrit tube was to extract DNA from 100 μl of each blood sample following cut with a diamond pointed pen 1 mm below the buffy coat the genomic DNA protocol (http://www.sigma-aldrich.com/ 4 Journal of Parasitology Research genomicdna). Small aliquots of extracted DNA were stored (X 2 = 3:494, df = 2, p = 0:174). There was no co-infection, at −20°C until required for use as template DNA for testing and no T. brucei group was detected despite using ITS 1 rDNA individual DNA extracts for infecting trypanosome species. speciation of trypanosomes targeting T. brucei inclusive. The extracted DNA samples were subjected to a conventional polymerase chain reaction (cPCR) in 25 μl 4. Discussion reaction volumes containing 4.375 μl double distilled water, 0.0625 μl ITS-CF primer, 0.0625 μl ITS-BR primer, 6.25 μl The current study reports for the first time canine African 2xmy Taq mix enzyme, 0.25 μl MgCl2, and 1.5 μl of the trypanosomiasis caused by T. congolense and T. vivax in template DNA. The primers (ITS-CF and ITS-BR) were free-ranging indigenous dogs that live in and around Bwindi designed targeting the internal transcribed spacer (ITS) and Queen Elizabeth conservation areas in Uganda. Animal region for efficient Trypanosoma species differentiation. trypanosomiasis is an important concern for livestock devel- The PCR cycling conditions were programmed following a opment allover Uganda. Trypanosomiasis is mainly reported protocol outlined by [37] including initial denaturation at in ruminants and pigs [9], but very little investigation has 94°C for 5 min, followed by 35 amplification cycles each con- been developed in dogs [22]. The lack of knowledge about sisting of 1 min denaturation at 94°C, 1 min primer anneal- CAT in southwestern Uganda may be due to the absence ing at 60°C, and 1 min polymerization at 72°C, with a final of concern of a possible role of dogs in trypanosomiasis extension at 72°C for 5 min. Positive controls: purified transmission, yet routine livestock treatment is carried out DNA of T. b. brucei, T. congolense, T. vivax and T. b. rhode- with different trypanocides such as diminazene aceturate siense, and negative controls: double distilled water were (Berenil®), isometamidium chloride (Samorin®), and homi- included in each set of reaction. Amplified products were dium chloride (Novidium®) in many areas [37, 38]. How- resolved by electrophoresis through 1.5% w/v agarose gels ever, the recently recognized trypanosomes’ resistance to containing 0.5 μg/ml ethidium bromide and flooded with isometamedium chloride and/or diminazene aceturate 1× TBE buffer. Electrophoresis conditions were 55 V and [39–41] remains a big threat to animal industry and calls 250 mA and run for 30 min. Trypanosome species determi- for immediate satisfactory investigation. The present study nation by cPCR analysis involved comparing individual offers knowledge about CAT prevalence and its possible role sample PCR amplicons with the corresponding band size in transmission of trypanosomiasis among livestock, dogs, of the positive control bands. The presence of two or more wildlife, and humans. On the other hand, the dogs studied bands indicates mixed trypanosome infections, while single did not manifest any clinical signs suggestive of CAT as bands signify single/monolithic infections. was the same scenario in a previous related study [19]; there- fore, clinical suspicion of CAT disease remains difficult. For 2.6. Statistical Analysis. Microsoft Excel spreadsheets were this reason, MHCT and PCR detection can aid to define used for raw data entry and management. The prevalence CAT incidence in an area where dogs live in close contact rate was defined as the proportion of trypanosome infected with livestock, wildlife, and humans. Studies in other areas dogs among all the dogs examined during the study. All evaluating canine trypanosomiasis describe clinical signs analyses were carried out using IBM SPSS Statistics 23 pack- associated with the infection such as anemia, fever, body age. Pearson Chi-square test was used to assess any statistical weakness, corneal opacity leading to blindness, arrhythmia, difference in the prevalence rate of CAT among the three lymphadenopathy, weight loss, infertility, abortion, and conservation sites, age groups, and between the dog’s sex, death if not treated [19, 42]. One limitation of our current and p value of 0.05 was considered significant. study was that neither thorough clinical history, nor a com- plete blood count and biochemistry profile of each dog was 3. Results included to help define if infected dogs were clinically healthy. All the infected dogs were mature adults which are active The buffy coat dark ground illuminated preparations and may roam freely within communities and to the neigh- detected trypanosomes in four samples, which were also boring national parks without restriction. This could be positive with the PCR. One sample had a separate band of because mature adult dogs prioritize regular exercise and/ 800 base pair (bp) for T. congolense, and three samples had or stimulating activities including running, security, herding, separate bands of 250 bp for T. vivax, unlike for T. brucei and hunting, which, together with their free-roaming behav- of approximately 480 bp. Therefore, we conclude that four iors, exposes them to risks of diseases including CAT. How- dogs out of 124 presented CAT detected by MHCT and ever, further studies with large dog population of different PCR, indicating a prevalence rate of 3.23% (Table 1). The age groups are still needed to define accurately the impact four positive dogs were three mature adult females and a of age on CAT prevalence. mature adult male from Bwindi and Queen Elizabeth con- The 3.23% CAT prevalence rate in dogs in the current servation areas, respectively (Table 2). They did not show study does not vary from the 3.3% and 4.7% recorded in evident clinical signs apart from poor body conditions and dogs in Buvuma Islands in Lake Victoria, Uganda [21], the presence of ecto-parasites mainly ticks of Rhipichepalus and Eastern Uganda [22], respectively. However, this preva- species and fleas, such as the other dogs included in the lence rate is relatively low compared with the 14% and 6.42% study. There was no significant statistical difference in reported in bovines in Uganda [9, 38]. This could indicate a CAT prevalence rate in relation to sex (X 2 = 3:456, df = 1, canine resistance to infection compared to other animal spe- p = 0:063), age (X 2 = 8:680, df = 4, p = 0:07), and site cies, making dogs serve as an important reservoir, providing Journal of Parasitology Research 5 Table 1: Summary showing the total number of dogs examined and their infection status in the three conservation areas. No. of trypanosome infected dogs detected by both MHCT and PCR rDNA Conservation area Sample size (n) T. vivax T. congolense Prevalence rate (%) Queen Elizabeth 42 0 1 2.38 Bwindi 42 3 0 7.14 Mgahinga 40 0 0 0 Total 124 3 1 3.23 Table 2: CAT prevalence rate (%) in relation to age in the three conservation areas. Conservation area Juvenile (n) Young adult (n) Mature adult (n) Senior adult (n) Geriatric (n) Prevalence rate (%) Queen Elizabeth 8 14 12 7 1 2.38 Queen Elizabeth CAT status 0 0 1 (T. c) 0 0 Bwindi 10 11 11 10 0 7.14 Bwindi CAT status 0 0 3 (T. v) 0 0 Mgahinga 13 9 7 9 2 0 Mgahinga CAT status 0 0 0 0 0 Total 31 34 30 26 3 3.23 Key: n: number of dogs sampled; T.c: T. congolense; T.v: T. vivax. a link for tsetse fly infection, and onward transmission to should be important to consider the dogs’ significant role livestock and wild animals. Therefore, further studies are in the epidemiology of CAT. Trypanosomiasis is a big con- required in order to define a CAT prevalence and its clinical straint in the livestock industry, and the Ugandan national effects in all trypanosomiasis endemic and suspected areas in policy on control of tsetse and trypanosomiasis mainly con- Uganda and Africa. The recognized T. congolense and T. siders 5% prevalence rate of infection as the minimum for an vivax in the conservation areas could be due to livestock area to be considered for appropriate intervention [39]. In since farming is their main economic activity and the sur- the case of CAT, the recognized 3.23% prevalence rate is rounding dense vegetative forests that harbor Glossina spp. worrying, suggesting that careful monitoring of epidemio- and reservoir hosts all need to be investigated. Apparently, logical data should be carried out for one to be aware of there are no relevant and latest entomological Glossina spp. the future requirements of the interventions. The fact that data for Bwindi-Mgahinga and Queen Elizabeth conserva- even the CAT-positive dogs did not show any significant tion areas, and this should be looked at immediately. The clinical signs related to trypanosomiasis infection calls for perceived T. vivax may also reflect mechanical transmission further investigation to define if there is canine resistance by hematophagus Tabanus and Stomoxys flies which makes to this disease. There is a need for enough financial support its control difficult even with integrated vector management for regular monitoring of both domestic and wild animals as has been noticed elsewhere [3, 8, 43]. with molecular diagnosis and routine investigation on tsetse Lastly, climate change can be an important variable for fly spp. and mechanical transmitters in all endemic and sus- CAT prevalence, but further studies are required to define pected areas in Uganda. This study increases awareness of its impact. The current increase in human population and CAT in Uganda and its relation with cattle and wild animal changes in land use, massive penetration, and destruction trypanosomiasis. It is required that further studies should of Glossina spp. natural conducive habitat, including wet- screen for CAT in a larger canine Ugandan population in lands, and the establishment of new contact zones might order to define its role and the appropriate management. change the parasite-host interactions over time (personal observation). These factors together with global warming may trigger Glossina spp. and CAT mechanical transmitters Data Availability to respond by survival adaptation, increasing chances of The data supporting the findings of this study are available parasite-host interactions and risks of parasite spread to from the corresponding author upon request. domestic animals. 5. Conclusion and Recommendation Ethical Approval The scarcity of CAT reports in Ugandan dogs including the Permission to use animals in this research was granted by studied sites can be attributed to insufficient epidemiological the Research and Higher Degrees Committee of the Faculty surveillance and trypanosomiasis control exercises on dogs of Veterinary Medicine, Makerere University, Uganda, and unlike livestock which are routinely monitored and treated the Uganda National Council for Science and Technology. with trypanocides (personal observations). Therefore, it This study adhered to Ugandan legal requirements. 6 Journal of Parasitology Research Consent [5] N. N. Nongo, M. T. Tion, T. T. Apaa, and B. Ogunro, “A case of canine trypanosomosis with epistaxis in a two- year old The Research and Higher Degrees Committee of the Faculty Alsatian dog,” IOSR Journal of Agriculture and Veterinary Sci- of Veterinary Medicine, Makerere University, and the ence, vol. 8, no. 11, pp. 2319–2372, 2015. Uganda National Council for Science and Technology [6] D. Muhanguzi, K. Picozzi, J. Hatendorf et al., “Collateral ben- granted consent to participate in this study. The consent efits of restricted insecticide application for control of African for publication of this study was granted by the Research trypanosomiasis on Theileria parva in cattle: a randomized and Higher Degrees Committee of the Faculty of Veterinary controlled trial,” Parasites & Vectors, vol. 7, no. 1, p. 432, 2014. Medicine, Makerere University, and the Uganda National [7] D. Muhanguzi, K. Picozzi, J. 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