Detection of Leishmania donovani status in dogs (Canis familiaris, Linnaeus, 1758) in the Karamoja sub-region of Uganda.

Detection of Leishmania donovani status in dogs (Canis familiaris, Linnaeus, 1758) in the Karamoja sub-region of Uganda. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Detection of Leishmania donovani status in dogs ( Canis familiaris, Linnaeus, 1758) in the Karamoja sub-region of Uganda. Charles D Kato, Angella Musewa, Tequiero A Okumu, Margaret Mbuchi, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6424553/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To date, the reservoir host for visceral leishmaniasis, a neglected tropical disease caused by Leishmania donovani , is unknown, although studies pointing to dogs, domestic animals, and rodents are emerging. We aimed to investigate whether the dog ( Canis familiaris , Linnaeus, 1758) is a potential reservoir for L . donovan i in the Karamoja sub-region of Uganda. Blood and lymph node aspirates were collected from dogs (n = 139) in disease endemic villages of Amudat and Moroto districts. An indirect enzyme-linked immunosorbent assay was used to detect anti- Leishmania IgG antibodies in serum. DNA extracted from lymph node aspirates was subjected to a polymerase chain reaction (PCR) targeting the rRNA internal transcribed spacer region of Leishmania species. All sera from 139 dogs did not demonstrate any evidence of circulating antibodies against Leishmania , as optical density (OD) values were below 0.25. Similarly, all the dog lymph node DNAs (n = 139) were negative for Leishmania parasites. Although our results indicate that dogs may not be reservoirs for L . donovani , studies utilizing larger sample sizes are recommended. Furthermore, the presence of L . donovani in sand flies and other suspected reservoirs, such as domestic animals and wild rodents, needs to be investigated. Animal Science Dog Leishmania donovani Karamoja sub-region Uganda Figures Figure 1 Figure 2 Introduction Leishmaniasis is a neglected tropical disease that globally affects about 700,000 to 1 million people, with an estimated death of around 40,000 annually [ 1 – 3 ]. Visceral leishmaniasis (VL) is the most serious, almost always fatal without treatment [ 2 , 3 ]. Around 90% of VL global cases are dominated by Kenya, Uganda, India, Brazil, Bangladesh, Ethiopia, Sudan, and South Sudan [ 3 , 4 ]. Despite the instauration of several control strategies, VL has remained endemic in rural hard-to-reach communities of the Karamoja sub-region, Uganda, Turkana, West Pokot, Baringo, Rift Valley, and western region in Kenya [ 1 , 4 ]. In 2022 and 2023, Uganda reported 264 and 195 cases, and Kenya reported 1573 and 1252 cases, respectively [ 5 ]. The two main parasite species responsible for human infection are L . donovani whose transmission is mostly anthroponotic, from human to human, and L . infantum , whose transmission is mostly zoonotic, with dogs, and rodents serving as reservoir hosts [ 1 ]. Recently epidemiological reports from the Indian subcontinent have identified evidence of L. donovani infection or exposure to multiple domestic mammals [ 6 ]. In Ethiopia, L. donovani has been isolated from wild rodent samples [ 7 ], and serological evidence of L. donovani infection was detected in healthy dogs [ 8 ]. Similarly, in neighboring Sudan, antibodies to L. donovani were detected in rats, goats, and cows [ 9 ]. In Kenya isolates of Leishmania parasites from dogs were reported, with one of the cases adjacent to VL endemic foci in Uganda [ 10 ], however, no other studies were conducted in the region to follow it up. In the Karamoja sub-region, Uganda most of the population are semi-nomadic pastoralists who keep dogs mostly for guarding property including livestock, herding, and hunting. These dogs roam freely in communities and if they are reservoirs for Leishmania species, they could increase the risk of parasite transmission among wildlife, livestock, and humans [ 11 ]. Despite dogs being known as reservoirs in other regions, scant information exists on the possibility of a zoonotic component of L . donovani in the Karamoja sub-region of Uganda. This study aimed to investigate whether the dog ( Canis familiaris, Linnaeus , 1758) is a potential reservoir for L . donovani in the Karamoja sub-region of Uganda. Materials and methods Study design We conducted a cross-sectional study that involved sampling domestic dogs in the Moroto and Amudat districts within the Karamoja sub-region of Uganda. The Karamoja sub-region is semi-arid, and is economically the poorest region in Uganda, with the population living on less than US $ 1.3 and one or no meal per day, increasing vulnerability to leishmaniasis and other diseases [ 12 , 13 ]. The Karamoja subregion communities are tribal, semi-nomadic pastoralists [ 14 , 15 ]. In this current study, we carried out active dog screening in five villages, Rupa and Boma ground in Moroto district, and in Murut, Napodo, and Angataret villages in Amudat district (Fig. 1 ). Sample collection from dogs Through the help of the Moroto and Amudat district veterinary offices and village health teams (VHTs), dog owners were sensitized and mobilized to bring their dogs to the respective sites on scheduled days. Following the recommended veterinary procedures [ 16 ], and with the help of a rope and Baskerville muzzle tied around the dog’s mouth, under the legs of the owner, 139 dogs were sampled. A 5 ml blood sample was collected from the cephalic vein of each dog into a plain vacutainer for ELISA screening. Next, the prescapular lymph node area was quickly shaved and disinfected. The lymph node cytological sample cells were obtained by puncture, and then suspended with serum in an EDTA tube [ 17 ]. The aliquots for ELISA were spun immediately after every field collection at 3500 rpm for 15 minutes and kept in 1.8 ml cryotubes, then stored at -196℃ in liquid nitrogen until laboratory diagnosis. Detection of Leishmania antibodies. Leishmaniasis antibodies in the collected dog sera were examined using an indirect ELISA, INgezim Leishmania (Gold Standard Diagnostics, Spain) as described previously by Ledesma et al. (2017) [ 18 ]. Briefly, positive, serum-free negative control, and dog sera samples were added accordingly. Microtiter immunoassay plates (Greiner, Frickenhausen, Germany) were coated with total soluble L . infantum promastigote antigens in 100 µl of coating buffer for 18 hours at 4°C in the dark. Free binding sites were blocked with a 2% casein solution for 1 h at 37°C. After three washes with 1XPBS-Tween (0.05%), plates were incubated with 100 µl of canine sera for 1 h at 37°C. Plates were washed three times with 1XPBS and incubated with 1:1000-fold diluted anti-canine IgG antibody (Sigma, USA) horseradish peroxidase-conjugate. The optical density (OD) was measured at 492 nm in an ELISA microplate spectrophotometer (SpectraMax Plus, USA). DNA extraction from dog lymph node samples Dog lymph node DNA was extracted using the PureLink Spin Column Kit (Thermo Fisher Scientific, USA) as described by the manufacturer. This involved adding 200µl lymph node samples to a sterile 2 ml microcentrifuge tube followed by 20 µl of Proteinase K and 20 µl of Rnase A. 200 µl PureLink® Genomic Lysis/Binding Buffer was also added, and vortexed briefly for homogenous mixing. The sample mixture was incubated at 55°C for 30 minutes with brief vertexing at 10-minute intervals to encourage complete lysis of cells. Next, 200 µl 96% ethanol and 200 µl of PureLinkR Genomic Lysis/Binding were added to the lysate. The lysate (about 640 µl) was vortexed briefly and loaded on the PurelinkR spin column. The spin column was centrifuged at 10,000 × g for 1 minute, and the collection tube and flow-through content were discarded. The spin column was transferred into a clean collecting tube and washed twice with appropriate wash buffers. Lastly, DNA elution was performed using 100 µl of DNA Elution Buffer, and the extracted DNA samples were kept at -20℃ until Polymerase Chain Reaction (PCR) analysis. Detection of Leishmania spp. in dog lymph node DNA by PCR The extracted DNA was subjected to PCR primers targeting the rRNA internal transcribed Spacer 2 region of Leishmania spp. as described by De Almeida et al. (2011) [ 19 ]. Briefly, the PCR was run in a reaction mixture of 50 µl, that included 1 µl of template DNA, 0.2 mM of each deoxynucleoside triphosphate, 0.2 µM each primer, 2.5 U of polymerase, 1.5 mM MgCl 2 . PCR conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and a final extension of 72°C for 7 min. The PCR included a negative control (DNA/RNA free water) and six positive controls (DNA samples of L . infantum ). Electrophoresis in 1.2% agarose gel at 100V was run to assess the PCR amplification. Results General characteristics of the sampled dogs. A total of 139 dogs (88 males, and 51 females) were sampled, 115 (83%) from Moroto, and 24 (17%) from Amudat district. All the sampled dogs were above 6 months old. They were categorized following the country and international veterinary age protocol [ 20 , 21 ], namely, juvenile (6–12 months), young adult (aged 1 year or 12–24 months), mature adult (2–6 years), senior adults (7–11 years), and geriatric (above 11 years). Demographic data, signalment (species, age, sex, breed, GPS, owner, and season), and dog owners’ information were captured [ 20 ], and other necessary data related to dog leishmaniasis, including clinical features (Table 1 ). Quality control was taken to exclude dogs that had not lived at least one transmission season in the VL endemic area, and those from which the required sample volume could not be collected during the study. Table 1 Overall dog demographic and other clinical parameters during the study. Category Number examined (n = 139) Age (months or years) Juvenile (6–12 months) 24 (17.3%) Young adult (aged 1 year or 12–24 months) 41(29.5%) Mature adult (2–6 years) 52 (37.4%) Senior adult (7–11 years) 22 (15.8%) Breed African shepherd 132 (95%) Unidentified breed 7 (5%) Clinical parameters Fever 15 (11%) Lymphadenopathy 5 (4%) Cachectic 40 (50%) Nasal or ocular lesion 2 (1%) Dermatitis 43 (31%) Epistaxis 2 (1%) Anemia 14 (10%) Lameness 5 (4%) Cutaneous ulceration 16 (12%) Detection of Leishmania antibodies by ELISA . All the ELISA test results from the 139 examined dog sera were negative for circulating Leishmania spp. antibodies with an OD of 0.8. Detection of Leishmania DNA . All the dog lymph node sample aspirates were analyzed by PCR targeting the rRNA internal transcribed spacer 2 region, and all were negative for Leishmania spp. Figure 2 shows a representative 1.2% agarose gel obtained from PCR amplification of an rRNA internal transcribed Spacer 2 region of Leishmania species. Discussion The Karamoja sub-region in Uganda has reported over 100 VL new cases annually since 2013 and this is likely to worsen with the current increase in human population, changes in land use and climate if no interventions are taken. The zoonotic reservoir(s) for VL in the Karamoja sub-region of Uganda have not been elucidated. To our knowledge, this is the first attempt to identify VL nonhuman reservoirs within this region. We investigated the possibility that dogs are potential reservoirs for VL within the Karamoja sub-region of Uganda. Based on our findings, we did not detect any circulating Leishmania spp. antibodies in dog sera or Leishmania spp. parasites from DNA extracted from lymph node aspirates. Previous related studies elsewhere including in Machakos District, Kenya [ 22 ], Sudan [ 23 ], the Middle East (such as Yemen, United Arab Emirates, Syria, Saudi Arabia), and North African countries including Morocco, Algeria, Tunisia, Libya, Egypt [ 11 ], Suriname in South America [ 24 ], and other countries in the Old World [ 25 ] have reported dogs Canis familiaris as the main nonhuman reservoir for VL parasites. In Machakos District, Kenya, two (0.7%) of the 288 examined dogs were VL positive with Leishmania amastigotes, enlarged and darkened livers and spleens [ 26 ]. In Sudan, three (6%) of the 51 examined dogs had Leishmania zymodemes known to be present in human VL and post-kala-azar dermal leishmaniasis cases [ 27 ]. Studies by Zijlstra and El-Hassan (2001) [ 28 ] in Sudan stated that Sudanese rodents and some carnivores serve as reservoirs of human visceral disease. Hoogstraal and Heyneman (1969) [ 29 ] detected L . donovani responsible for VL in rodents and a few wild mammals: grass rats ( Arvicanthis niloticus ), spiny mouse ( Acomys albigena ), serval (Felis serval ) and genet ( Genetta genetta ) in Sudan, while Sixl et al. (1987) [ 30 ] also detected L . donovani in jackal ( Canis sp.) in Sudan. The currently recognized high VL human cases in the Karamoja sub-region could be associated with socio-economic factors like pastoralism, the social-cultural lifestyle of living in “manyatta” houses poorly built using clay, and with holes that allow sand flies to pass in and/or through and breed. We observed several termite hills/mounds around domestic settings, kraals, and grazing grounds during the present study. The termite mounds serve as important breeding and resting sites for the sand flies in the study area. Also, the pastoralists sit and sleep on the raised termite mounds while grazing and guarding livestock, this increases the chance of human-sand fly interaction/ bites. On another related risk aspect, the Karamojong environmental settings possess lots of acacia-balanites woodland and black cotton soils, these are known to be good habitats for sand flies including Phlebotomus orientalis , the L . donovani vector and the causative agent for VL [ 31 ]. Limitations Our study has limitations that might have affected the results. The first one relates to the sample size of the dogs (n = 139), we might likely have missed positive dogs as we sampled around 5% of dogs per village. Secondly, dogs were sampled from mass vaccination campaigns instead of considering dogs from or near homesteads reporting VL cases. Thirdly, we did not sample other reservoir hosts like livestock, and wild animals like rodents known to be reservoirs for Leishmania parasites, nor did we monitor the status and feeding trends of the phlebotomine sand fly vectors. Future studies could benefit from sampling more dogs within VL hotspot villages and from homesteads reporting a significant number of human cases and examining the status of Leishmania parasites in sand flies, and their feeding trends. Conclusions The current study has demonstrated that dogs might not be reservoirs for L . donovani in the Karamoja sub-region of Uganda. Other domestic animals (like cattle, goats, rabbits, camels, and sheep), and wild rodents could be the potential reservoirs for this disease. Studies investigating the presence of L . donovani in domestic animals and wild rodents are required to address this gap. Abbreviations VL Visceral leishmaniasis DO Optical density PCR Polymerase chain reaction rRNA Ribosomal ribonucleic acid Declarations Ethical approval This study experimental plan was reviewed and approved by the Institutional Review Board (or Ethics Committee) of the Vector Control Division Research and Ethics Committee, and the Ministry of Health Uganda under protocol number (VCDREC167). Consent to participate In all cases, dog owners were informed about the study's purpose and provided consent to sample their dogs. Consent to publish Not applicable. Competing interests The authors declare no competing interests. Funding This investigation received financial support funding from the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), with grant number P22-00833. Author contributions CDK, AM, IA, TAO, EA, MM, JMF, and JRO conceived and designed this study. EM, BM, MPB, JMF, and JRO carried out field data collection and dog sampling. CDK, SCT, GPF, JRO, EA, and JRO conducted laboratory ELISA, DNA extraction, and PCR tests. CDK, AM, IA, TAO, MM, JMF, and JRO conducted statistical analyses. CDK, AM, IA, TAO, EA, MM, JMF, and JRO wrote the first draft of the manuscript. All authors have read and approved the manuscript to be submitted for publication. Acknowledgments We would also like to thank the District Veterinary Officers of Moroto and Amudat districts, the Village Health Teams, and the dog owners for their voluntary assistance during the sample collection exercise. Data availability The data supporting this study's findings are available from the corresponding author, upon request. References Jones CM, Welburn SC (2021) Leishmaniasis Beyond East Africa. Front Vet Sci 8(2):1–10. 10.3389/fvets.2021.618766 Ready PD (2014) Epidemiology of visceral leishmaniasis. Clin Epidemiol, pp. 147–154 World Health Organization (2010) Control of the leishmaniases. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6424553","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441474127,"identity":"168c6b1a-025f-4ea9-bb0c-9469778d6cb1","order_by":0,"name":"Charles D Kato","email":"","orcid":"https://orcid.org/0000-0003-3160-6657","institution":"College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"D","lastName":"Kato","suffix":""},{"id":441477906,"identity":"b8261a08-4521-47a2-97a4-399660abb498","order_by":1,"name":"Angella 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Ochieng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACxoYDEAY/iEgoIEWLZANIiwEp1hmAtRKjhbnx8NMNH/7ckzM+vzrxwwMDBnl+sQOEHHbM7ObMtmJjsxtvN0sAHWY4c3YCIS0HzG7zNiQkbrtxdgNIS4LBbYJajn+7/edPQuLmGWc3/yBSyxmz2wxsCYkb+Hu3EWvLmbKbvW0JxhI3eLdZJBhIEPaL4Yzj2278+JMgx99/dvPNHxU28vzSBLUcgLIkwCol8CsHAXn+BiiL/wAeZaNgFIyCUTCiAQAnh01rD9XZlAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0765-4958","institution":"College of Natural Sciences, Makerere University","correspondingAuthor":true,"prefix":"","firstName":"James","middleName":"Robert","lastName":"Ochieng","suffix":""}],"badges":[],"createdAt":"2025-04-11 04:08:36","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6424553/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6424553/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80520610,"identity":"a76310aa-f2a2-4116-a587-0580346456e8","added_by":"auto","created_at":"2025-04-14 09:03:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3260071,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing the dog \u003cem\u003eLeishmania \u003c/em\u003eactive screening sites in the Karamoja sub-region, Uganda. Dog sampling was conducted in five villages, Rupa, Boma, Murut, Napodo, and Angataret.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6424553/v1/e1a3e372c83380da1ce653b8.png"},{"id":80519999,"identity":"8a6d516d-6e48-427e-bcb6-cdeb5d764f72","added_by":"auto","created_at":"2025-04-14 08:55:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":636424,"visible":true,"origin":"","legend":"\u003cp\u003eA 1.2 % agarose gel obtained from PCR amplification. “M” is a 100 bp marker, “P” is the positive control \u003cem\u003eLeishmania infantum,\u003c/em\u003e and “1” is the negative control (DNA/RNA free water).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6424553/v1/cb03ce6aaef0e7ea9cf89252.png"},{"id":80520644,"identity":"11a11721-7aa1-4d44-9340-4c89fe6295c9","added_by":"auto","created_at":"2025-04-14 09:03:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4429968,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6424553/v1/af8b9429-2d3c-4c4d-9813-757b8669c7a1.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eDetection of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLeishmania donovani\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e status in dogs (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCanis familiaris,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Linnaeus, 1758) in the Karamoja sub-region of Uganda.\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLeishmaniasis is a neglected tropical disease that globally affects about 700,000 to 1\u0026nbsp;million people, with an estimated death of around 40,000 annually [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Visceral leishmaniasis (VL) is the most serious, almost always fatal without treatment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Around 90% of VL global cases are dominated by Kenya, Uganda, India, Brazil, Bangladesh, Ethiopia, Sudan, and South Sudan [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite the instauration of several control strategies, VL has remained endemic in rural hard-to-reach communities of the Karamoja sub-region, Uganda, Turkana, West Pokot, Baringo, Rift Valley, and western region in Kenya [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In 2022 and 2023, Uganda reported 264 and 195 cases, and Kenya reported 1573 and 1252 cases, respectively [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The two main parasite species responsible for human infection are \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e whose transmission is mostly anthroponotic, from human to human, and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003einfantum\u003c/em\u003e, whose transmission is mostly zoonotic, with dogs, and rodents serving as reservoir hosts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently epidemiological reports from the Indian subcontinent have identified evidence of \u003cem\u003eL. donovani\u003c/em\u003e infection or exposure to multiple domestic mammals [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In Ethiopia, \u003cem\u003eL. donovani\u003c/em\u003e has been isolated from wild rodent samples [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and serological evidence of \u003cem\u003eL. donovani\u003c/em\u003e infection was detected in healthy dogs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similarly, in neighboring Sudan, antibodies to \u003cem\u003eL. donovani\u003c/em\u003e were detected in rats, goats, and cows [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In Kenya isolates of \u003cem\u003eLeishmania\u003c/em\u003e parasites from dogs were reported, with one of the cases adjacent to VL endemic foci in Uganda [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], however, no other studies were conducted in the region to follow it up. In the Karamoja sub-region, Uganda most of the population are semi-nomadic pastoralists who keep dogs mostly for guarding property including livestock, herding, and hunting. These dogs roam freely in communities and if they are reservoirs for \u003cem\u003eLeishmania\u003c/em\u003e species, they could increase the risk of parasite transmission among wildlife, livestock, and humans [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite dogs being known as reservoirs in other regions, scant information exists on the possibility of a zoonotic component of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e in the Karamoja sub-region of Uganda. This study aimed to investigate whether the dog (\u003cem\u003eCanis familiaris, Linnaeus\u003c/em\u003e, 1758) is a potential reservoir for \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e in the Karamoja sub-region of Uganda.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eWe conducted a cross-sectional study that involved sampling domestic dogs in the Moroto and Amudat districts within the Karamoja sub-region of Uganda. The Karamoja sub-region is semi-arid, and is economically the poorest region in Uganda, with the population living on less than US\u003cspan\u003e$\u003c/span\u003e 1.3 and one or no meal per day, increasing vulnerability to leishmaniasis and other diseases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The Karamoja subregion communities are tribal, semi-nomadic pastoralists [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this current study, we carried out active dog screening in five villages, Rupa and Boma ground in Moroto district, and in Murut, Napodo, and Angataret villages in Amudat district (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample collection from dogs\u003c/h3\u003e\n\u003cp\u003eThrough the help of the Moroto and Amudat district veterinary offices and village health teams (VHTs), dog owners were sensitized and mobilized to bring their dogs to the respective sites on scheduled days. Following the recommended veterinary procedures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and with the help of a rope and Baskerville muzzle tied around the dog\u0026rsquo;s mouth, under the legs of the owner, 139 dogs were sampled. A 5 ml blood sample was collected from the cephalic vein of each dog into a plain vacutainer for ELISA screening. Next, the prescapular lymph node area was quickly shaved and disinfected. The lymph node cytological sample cells were obtained by puncture, and then suspended with serum in an EDTA tube [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The aliquots for ELISA were spun immediately after every field collection at 3500 rpm for 15 minutes and kept in 1.8 ml cryotubes, then stored at -196℃ in liquid nitrogen until laboratory diagnosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003eLeishmania\u003c/b\u003e \u003cb\u003eantibodies.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLeishmaniasis antibodies in the collected dog sera were examined using an indirect ELISA, INgezim Leishmania (Gold Standard Diagnostics, Spain) as described previously by Ledesma et al. (2017) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Briefly, positive, serum-free negative control, and dog sera samples were added accordingly. Microtiter immunoassay plates (Greiner, Frickenhausen, Germany) were coated with total soluble \u003cem\u003eL\u003c/em\u003e. \u003cem\u003einfantum\u003c/em\u003e promastigote antigens in 100 \u0026micro;l of coating buffer for 18 hours at 4\u0026deg;C in the dark. Free binding sites were blocked with a 2% casein solution for 1 h at 37\u0026deg;C. After three washes with 1XPBS-Tween (0.05%), plates were incubated with 100 \u0026micro;l of canine sera for 1 h at 37\u0026deg;C. Plates were washed three times with 1XPBS and incubated with 1:1000-fold diluted anti-canine IgG antibody (Sigma, USA) horseradish peroxidase-conjugate. The optical density (OD) was measured at 492 nm in an ELISA microplate spectrophotometer (SpectraMax Plus, USA).\u003c/p\u003e\n\u003ch3\u003eDNA extraction from dog lymph node samples\u003c/h3\u003e\n\u003cp\u003eDog lymph node DNA was extracted using the PureLink Spin Column Kit (Thermo Fisher Scientific, USA) as described by the manufacturer. This involved adding 200\u0026micro;l lymph node samples to a sterile 2 ml microcentrifuge tube followed by 20 \u0026micro;l of Proteinase K and 20 \u0026micro;l of Rnase A. 200 \u0026micro;l PureLink\u0026reg; Genomic Lysis/Binding Buffer was also added, and vortexed briefly for homogenous mixing. The sample mixture was incubated at 55\u0026deg;C for 30 minutes with brief vertexing at 10-minute intervals to encourage complete lysis of cells. Next, 200 \u0026micro;l 96% ethanol and 200 \u0026micro;l of PureLinkR Genomic Lysis/Binding were added to the lysate. The lysate (about 640 \u0026micro;l) was vortexed briefly and loaded on the PurelinkR spin column. The spin column was centrifuged at 10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 1 minute, and the collection tube and flow-through content were discarded. The spin column was transferred into a clean collecting tube and washed twice with appropriate wash buffers. Lastly, DNA elution was performed using 100 \u0026micro;l of DNA Elution Buffer, and the extracted DNA samples were kept at -20℃ until Polymerase Chain Reaction (PCR) analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003eLeishmania\u003c/b\u003e \u003cb\u003espp. in dog lymph node DNA by PCR\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe extracted DNA was subjected to PCR primers targeting the rRNA internal transcribed Spacer 2 region of \u003cem\u003eLeishmania\u003c/em\u003e spp. as described by De Almeida et al. (2011) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, the PCR was run in a reaction mixture of 50 \u0026micro;l, that included 1 \u0026micro;l of template DNA, 0.2 mM of each deoxynucleoside triphosphate, 0.2 \u0026micro;M each primer, 2.5 U of polymerase, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e. PCR conditions were 95\u0026deg;C for 5 min, followed by 40 cycles of 95\u0026deg;C for 30 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 1 min, and a final extension of 72\u0026deg;C for 7 min. The PCR included a negative control (DNA/RNA free water) and six positive controls (DNA samples of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003einfantum\u003c/em\u003e). Electrophoresis in 1.2% agarose gel at 100V was run to assess the PCR amplification.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGeneral characteristics of the sampled dogs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA total of 139 dogs (88 males, and 51 females) were sampled, 115 (83%) from Moroto, and 24 (17%) from Amudat district. All the sampled dogs were above 6 months old. They were categorized following the country and international veterinary age protocol [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], namely, juvenile (6\u0026ndash;12 months), young adult (aged 1 year or 12\u0026ndash;24 months), mature adult (2\u0026ndash;6 years), senior adults (7\u0026ndash;11 years), and geriatric (above 11 years). Demographic data, signalment (species, age, sex, breed, GPS, owner, and season), and dog owners\u0026rsquo; information were captured [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and other necessary data related to dog leishmaniasis, including clinical features (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Quality control was taken to exclude dogs that had not lived at least one transmission season in the VL endemic area, and those from which the required sample volume could not be collected during the study.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverall dog demographic and other clinical parameters during the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber examined (n\u0026thinsp;=\u0026thinsp;139)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (months or years)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJuvenile (6\u0026ndash;12 months)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24 (17.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYoung adult (aged 1 year or 12\u0026ndash;24 months)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41(29.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMature adult (2\u0026ndash;6 years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52 (37.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSenior adult (7\u0026ndash;11 years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 (15.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBreed\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAfrican shepherd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e132 (95%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnidentified breed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClinical parameters\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFever\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 (11%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphadenopathy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCachectic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 (50%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNasal or ocular lesion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDermatitis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43 (31%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpistaxis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (10%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLameness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCutaneous ulceration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (12%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of Leishmania antibodies by ELISA\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAll the ELISA test results from the 139 examined dog sera were negative for circulating \u003cem\u003eLeishmania\u003c/em\u003e spp. antibodies with an OD of \u0026lt;\u0026thinsp;0.25. As expected from the test kit, the positive control showed strong positivity/ validity with an OD of \u0026gt;\u0026thinsp;0.8.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of Leishmania DNA\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAll the dog lymph node sample aspirates were analyzed by PCR targeting the rRNA internal transcribed spacer 2 region, and all were negative for \u003cem\u003eLeishmania\u003c/em\u003e spp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows a representative 1.2% agarose gel obtained from PCR amplification of an rRNA internal transcribed Spacer 2 region of \u003cem\u003eLeishmania\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe Karamoja sub-region in Uganda has reported over 100 VL new cases annually since 2013 and this is likely to worsen with the current increase in human population, changes in land use and climate if no interventions are taken. The zoonotic reservoir(s) for VL in the Karamoja sub-region of Uganda have not been elucidated. To our knowledge, this is the first attempt to identify VL nonhuman reservoirs within this region. We investigated the possibility that dogs are potential reservoirs for VL within the Karamoja sub-region of Uganda.\u003c/p\u003e \u003cp\u003eBased on our findings, we did not detect any circulating \u003cem\u003eLeishmania\u003c/em\u003e spp. antibodies in dog sera or \u003cem\u003eLeishmania\u003c/em\u003e spp. parasites from DNA extracted from lymph node aspirates. Previous related studies elsewhere including in Machakos District, Kenya [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], Sudan [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the Middle East (such as Yemen, United Arab Emirates, Syria, Saudi Arabia), and North African countries including Morocco, Algeria, Tunisia, Libya, Egypt [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], Suriname in South America [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and other countries in the Old World [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] have reported dogs \u003cem\u003eCanis familiaris\u003c/em\u003e as the main nonhuman reservoir for VL parasites. In Machakos District, Kenya, two (0.7%) of the 288 examined dogs were VL positive with \u003cem\u003eLeishmania\u003c/em\u003e amastigotes, enlarged and darkened livers and spleens [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In Sudan, three (6%) of the 51 examined dogs had \u003cem\u003eLeishmania\u003c/em\u003e zymodemes known to be present in human VL and post-kala-azar dermal leishmaniasis cases [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Studies by Zijlstra and El-Hassan (2001) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] in Sudan stated that Sudanese rodents and some carnivores serve as reservoirs of human visceral disease. Hoogstraal and Heyneman (1969) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] detected \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e responsible for VL in rodents and a few wild mammals: grass rats (\u003cem\u003eArvicanthis niloticus\u003c/em\u003e), spiny mouse (\u003cem\u003eAcomys albigena\u003c/em\u003e), serval \u003cem\u003e(Felis serval\u003c/em\u003e) and genet (\u003cem\u003eGenetta genetta\u003c/em\u003e) in Sudan, while Sixl et al. (1987) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] also detected \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e in jackal (\u003cem\u003eCanis\u003c/em\u003e sp.) in Sudan.\u003c/p\u003e \u003cp\u003eThe currently recognized high VL human cases in the Karamoja sub-region could be associated with socio-economic factors like pastoralism, the social-cultural lifestyle of living in “manyatta” houses poorly built using clay, and with holes that allow sand flies to pass in and/or through and breed. We observed several termite hills/mounds around domestic settings, kraals, and grazing grounds during the present study. The termite mounds serve as important breeding and resting sites for the sand flies in the study area. Also, the pastoralists sit and sleep on the raised termite mounds while grazing and guarding livestock, this increases the chance of human-sand fly interaction/ bites. On another related risk aspect, the Karamojong environmental settings possess lots of acacia-balanites woodland and black cotton soils, these are known to be good habitats for sand flies including \u003cem\u003ePhlebotomus orientalis\u003c/em\u003e, the \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e vector and the causative agent for VL [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e "},{"header":"Limitations","content":"\u003cp\u003eOur study has limitations that might have affected the results. The first one relates to the sample size of the dogs (n = 139), we might likely have missed positive dogs as we sampled around 5% of dogs per village. Secondly, dogs were sampled from mass vaccination campaigns instead of considering dogs from or near homesteads reporting VL cases. Thirdly, we did not sample other reservoir hosts like livestock, and wild animals like rodents known to be reservoirs for \u003cem\u003eLeishmania\u003c/em\u003e parasites, nor did we monitor the status and feeding trends of the phlebotomine sand fly vectors. Future studies could benefit from sampling more dogs within VL hotspot villages and from homesteads reporting a significant number of human cases and examining the status of \u003cem\u003eLeishmania\u003c/em\u003e parasites in sand flies, and their feeding trends.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe current study has demonstrated that dogs might not be reservoirs for \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e in the Karamoja sub-region of Uganda. Other domestic animals (like cattle, goats, rabbits, camels, and sheep), and wild rodents could be the potential reservoirs for this disease. Studies investigating the presence of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e in domestic animals and wild rodents are required to address this gap.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVisceral leishmaniasis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003erRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRibosomal ribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003e This study experimental plan was reviewed and approved by the Institutional Review Board (or Ethics Committee) of the Vector Control Division Research and Ethics Committee, and the Ministry of Health Uganda under protocol number (VCDREC167).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to participate\u003c/strong\u003e \u003cp\u003eIn all cases, dog owners were informed about the study's purpose and provided consent to sample their dogs.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to publish\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis investigation received financial support funding from the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), with grant number P22-00833.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eCDK, AM, IA, TAO, EA, MM, JMF, and JRO conceived and designed this study. EM, BM, MPB, JMF, and JRO carried out field data collection and dog sampling. CDK, SCT, GPF, JRO, EA, and JRO conducted laboratory ELISA, DNA extraction, and PCR tests. CDK, AM, IA, TAO, MM, JMF, and JRO conducted statistical analyses. CDK, AM, IA, TAO, EA, MM, JMF, and JRO wrote the first draft of the manuscript. All authors have read and approved the manuscript to be submitted for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe would also like to thank the District Veterinary Officers of Moroto and Amudat districts, the Village Health Teams, and the dog owners for their voluntary assistance during the sample collection exercise.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data supporting this study's findings are available from the corresponding author, upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJones CM, Welburn SC (2021) Leishmaniasis Beyond East Africa. 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J Nanjing Med Univ 23(2):79\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1007-4376(09)60032-0\u003c/span\u003e\u003cspan address=\"10.1016/S1007-4376(09)60032-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"ba7877dd-95ef-41d2-a38b-44473a231c17","identifier":"10.13039/100004423","name":"World Health Organization","awardNumber":"P22-00833","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Makerere University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dog, Leishmania donovani, Karamoja sub-region, Uganda","lastPublishedDoi":"10.21203/rs.3.rs-6424553/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6424553/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo date, the reservoir host for visceral leishmaniasis, a neglected tropical disease caused by \u003cem\u003eLeishmania donovani\u003c/em\u003e, is unknown, although studies pointing to dogs, domestic animals, and rodents are emerging. We aimed to investigate whether the dog (\u003cem\u003eCanis familiaris\u003c/em\u003e, Linnaeus, 1758) is a potential reservoir for \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovan\u003c/em\u003ei in the Karamoja sub-region of Uganda. Blood and lymph node aspirates were collected from dogs (n\u0026thinsp;=\u0026thinsp;139) in disease endemic villages of Amudat and Moroto districts. An indirect enzyme-linked immunosorbent assay was used to detect anti-\u003cem\u003eLeishmania\u003c/em\u003e IgG antibodies in serum. DNA extracted from lymph node aspirates was subjected to a polymerase chain reaction (PCR) targeting the rRNA internal transcribed spacer region of \u003cem\u003eLeishmania\u003c/em\u003e species. All sera from 139 dogs did not demonstrate any evidence of circulating antibodies against \u003cem\u003eLeishmania\u003c/em\u003e, as optical density (OD) values were below 0.25. Similarly, all the dog lymph node DNAs (n\u0026thinsp;=\u0026thinsp;139) were negative for \u003cem\u003eLeishmania\u003c/em\u003e parasites. Although our results indicate that dogs may not be reservoirs for \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e, studies utilizing larger sample sizes are recommended. Furthermore, the presence of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edonovani\u003c/em\u003e in sand flies and other suspected reservoirs, such as domestic animals and wild rodents, needs to be investigated.\u003c/p\u003e","manuscriptTitle":"Detection of Leishmania donovani status in dogs (Canis familiaris, Linnaeus, 1758) in the Karamoja sub-region of Uganda.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-14 08:53:52","doi":"10.21203/rs.3.rs-6424553/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f17f0378-fbb0-407f-9a92-0a6f267ace71","owner":[],"postedDate":"April 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47006226,"name":"Animal Science"}],"tags":[],"updatedAt":"2025-04-14T08:53:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-14 08:53:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6424553","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6424553","identity":"rs-6424553","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}