Current status and molecular epidemiology of rabies virus from different hosts and regions in Malawi.

Although rabies is endemic in Malawi, there have been no studies in which rabies virus was systematically investigated and characterized in multiple animal hosts in that country. In order to provide molecular epidemiological data on rabies virus in Malawi, 683 suspected rabies case reports from 2008 to 2021 were examined, and 46 (dog = 40, cow = 5, and cat = 1) viable rabies-positive brain samples archived at the Central Veterinary Laboratory (CVL), Lilongwe, Malawi, were analyzed genetically. The results showed an increase in the submission of brain samples from 2008 to 2010, with the highest number of submissions observed in 2020. Of the 683 case reports analyzed for the period under review, 38.1% (260/683) (CI: 34.44 - 41.84) were confirmed by direct fluorescent antibody test. Among the confirmed cases, 65.4% (170/260) (CI: 59.23 - 71.09) were canine rabies. Further, phylogenetic analysis revealed that sequences from different animal hosts clustered together within the Africa 1b lineage, suggesting that the strains circulating in livestock are similar to those in domestic dogs. This finding supports the hypothesis that canine rabies is spilling over to livestock and emphasizes the need for further studies to provide data for effective control of rabies in Malawi.

Prevalence of trypanosomes and selected symbionts in tsetse species of eastern Zambia.

Insect symbionts have attracted attention for their potential use as anti-parasitic gene products in arthropod disease vectors. While tsetse species of the Luangwa valley have been extensively studied, less is known about the prevalence of symbionts and their interactions with the trypanosome parasite. Polymerase chain reaction was used to investigate the presence of Wolbachia and Sodalis bacteria, in tsetse flies infected with trypanosomes (Trypanosoma vivax, Trypanosoma congolense and Trypanosoma brucei). Out of 278 captured tsetse flies in eastern Zambia, 95.3% (n = 265, 95% CI = 92.8­97.8) carried endosymbionts: Wolbachia (79.1%, 95% CI 73.9­83.8) and Sodalis (86.3%, 95% CI 81.7­90.1). Overall, trypanosome prevalence was 25.5% (n = 71, 95% CI = 20.4­30.7), 10.8% (n = 30, 95% CI 7.1­14.4) for T. brucei, 1.4% (n = 4, 95% CI = 0.4­3.6) for both T. congolense and T. vivax, and 0.7% (n = 2, 95% CI 0.1­2.6) for T. b. rhodesiense. Out of 240 tsetse flies that were infected with Sodalis, trypanosome infection was reported in 40 tsetse flies (16.7%, 95% CI = 12.0­21.4) while 37 (16.8%, 95% CI 11.9­21.8) of the 220 Wolbachia infected tsetse flies were infected with trypanosomes. There was 1.3 times likelihood of T. brucei infection to be present when Wolbachia was present and 1.7 likelihood of T. brucei infection when Sodalis was present. Overall findings suggest absence of correlation between the presence of tsetse endosymbionts and tsetse with trypanosome infection. Lastly, the presence of pathogenic trypanosomes in tsetse species examined provided insights into the risk communities face, and the importance of African trypanosomiasis in the area.

Molecular Detection and Characterization of Rickettsia asembonensis in Human Blood, Zambia.

Rickettsia asembonensis is a flea-related Rickettsia with unknown pathogenicity to humans. We detected R. asembonensis DNA in 2 of 1,153 human blood samples in Zambia. Our findings suggest the possibility of R. asembonensis infection in humans despite its unknown pathogenicity.

In Vitro and In Vivo Trypanocidal Efficacy of Synthesized Nitrofurantoin Analogs.

African trypanosomes cause diseases in humans and livestock. Human African trypanosomiasis is caused by Trypanosoma brucei rhodesiense and T. b. gambiense. Animal trypanosomoses have major effects on livestock production and the economy in developing countries, with disease management depending mainly on chemotherapy. Moreover, only few drugs are available and these have adverse effects on patients, are costly, show poor accessibility, and parasites develop drug resistance to them. Therefore, novel trypanocidal drugs are urgently needed. Here, the effects of synthesized nitrofurantoin analogs were evaluated against six species/strains of animal and human trypanosomes, and the treatment efficacy of the selected compounds was assessed in vivo. Analogs 11 and 12, containing 11- and 12-carbon aliphatic chains, respectively, showed the highest trypanocidal activity (IC50 < 0.34 µM) and the lowest cytotoxicity (IC50 > 246.02 µM) in vitro. Structure-activity relationship analysis suggested that the trypanocidal activity and cytotoxicity were related to the number of carbons in the aliphatic chain and electronegativity. In vivo experiments, involving oral treatment with nitrofurantoin, showed partial efficacy, whereas the selected analogs showed no treatment efficacy. These results indicate that nitrofurantoin analogs with high hydrophilicity are required for in vivo assessment to determine if they are promising leads for developing trypanocidal drugs.

Genetic diversity of rabies virus in different host species and geographic regions of Zambia and Zimbabwe.

Rabies is endemic in Zambia and Zimbabwe. The previously investigated strains of rabies virus in central Zambia belong to the Africa 1b lineage, with similar circulating virus strains found in the various tested hosts and regions. However, prior work assessed only limited regions and host species. Thus, this study aimed to more comprehensively determine the genetic diversity of rabies virus across regions of Zambia and Zimbabwe. RNA (n = 76) was extracted from positive direct fluorescent antibody test brain tissues from dog, cow, goat, cat, pig, human, and jackal collected from Zambia and Zimbabwe. The amplicons of the nucleoprotein and glycoprotein genes were obtained from all examined samples by nested RT-PCR and subsequently sequenced. A phylogenetic analysis of the N gene confirmed that all the endemic strains of rabies virus in Zambia and Zimbabwe belong to the Africa 1b lineage. The obtained viral gene sequences were phylogenetically divided into two clusters. Cluster II comprised only Zambian strains. In contrast, cluster I comprised both Zambia and Zimbabwe strains, with strains from Zimbabwe forming a distinct lineage from Zambian strains, implying viral genetic divergence due to geographical barriers. However, no evidence of clustering based on host or region was observed, implying the circulation of similar virus strains occurs in different hosts and regions of Zambia and Zimbabwe. The clustering of rabies virus strains from jackals with those from domestic animals provides evidence of similar virus strains circulating in both wildlife and domestic animals, and that the jackal might be one of the potential reservoirs of rabies virus infection. In this study, no strains circulating in Zimbabwe were detected in Zambia.

Characterization of tabanid flies (Diptera: Tabanidae) in South Africa and Zambia and detection of protozoan parasites they are harbouring.

Tabanids are haematophagous flies feeding on livestock and wildlife. In the absence of information on the relationship of tabanid flies and protozoan parasites in South Africa and Zambia, the current study was aimed at characterizing tabanid flies collected in these two countries as well as detecting protozoan parasites they are harbouring. A total of 527 tabanid flies were collected whereby 70·2% were from South Africa and 29·8% were from Zambia. Morphological analysis revealed a total of five different genera collected from the sampled areas namely: Ancala, Atylotus, Haematopota, Philoliche and Tabanus. DNA extracted from South African Tabanus par and Tabanus taeniola tested positive for the presence of Trypanosoma congolense (Savannah) and Trypanosoma theileri whilst one member from T. par was positive for Trypanosoma brucei species. DNA extracted from Zambian tabanid flies tested positive for the presence of Besnoitia species at 1·27% (2/157), Babesia bigemina 5·73% (9/157), Theileria parva 30·11% (30/157) and 9·82% (14/157) for Trypanosoma evansi. This study is the first to report on relationship of Babesia and Theileria parasites with tabanid flies. Further investigations are required to determine the role of tabanids in transmission of the detected protozoan parasites in livestock and wildlife in South Africa and Zambia.

Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa.

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.

Determination of the prevalence of trypanosome species in cattle from Monduli district, northern Tanzania, by loop mediated isothermal amplification.

Bovine African trypanosomosis (BAT) remains one of the major vector-borne diseases with serious impediment to cattle production and economic advancement in sub-Saharan Africa. The present study evaluated the performance of the trypanosome-species-specific loop-mediated isothermal amplification (LAMP), using parasite DNA obtained from 295 indigenous Tanzanian short horn Zebu (TSHZ) and Boran crosses in Monduli district within northern Tanzania, against routine microscopy on Giemsa-stained blood films. Compared to parasitological data in which the prevalence of BAT was estimated at 2.4% (95% CI 0.7-4.1%), LAMP increased the prevalence to 27.8% (95% CI 22.3-32.5%), of which 11.9% (95% CI 8.2-15.6%) were monolytic infections with Trypanosoma vivax, while 13.6% (95% CI 9.7-17.5%) were coinfections of either T. vivax and Trypanosoma brucei subspecies or T. vivax and Trypanosoma congolense, respectively. Among the T. brucei subspecies detected, 0.7% (95% CI 0-1.7%) were human-infective Trypanosoma brucei rhodesiense. Our study is in concordance with previous reports and suggests that LAMP is a potential tool for routine diagnosis of trypanosomes in domestic animals in BAT endemic regions. According to LAMP, T. vivax seems to be the predominant trypanosome species circulating among the indigenous Monduli cattle. Importantly, the detection of T. b. rhodesiense in cattle in such wildlife-domestic-animal-human-interface areas poses a risk of contracting human African trypanosomiasis (HAT) by local communities and tourists. Continuous trypanosome surveillances in domestic animals, humans, and tsetse flies using sensitive and specific tests such as LAMP are recommended.

Dogs' health and demographics in wildlife-populated and tsetse-infested villages of Mambwe district, eastern Zambia.

Good dog-keeping practices and access to veterinary care are essential for the well-being of dogs. As the main causes of morbidity and mortality in the rural canine population in Zambia are poorly understood, we followed a cohort of 162 indigenous dogs for six months in wildlife-populated and tsetse-infested villages of Mambwe district, eastern Zambia to gain deeper insights. Dogs lacked basic home and veterinary care, they were often starved and burdened with ticks, and some passed live adult worms in their stool. The frequent exposure of dogs to tsetse bites and consumption of fresh raw game meat and bones puts them at greater risk of acquiring African trypanosomiasis. Nearly 20 % of dogs were lost to follow-up, with the main causes being poor health (58.1 %), predation by wild carnivores (29 %), and owner culling or euthanasia (12.9 %). We observed that indigenous dogs' general well-being and survival were largely influenced by their environment, infectious diseases, injuries sustained during interaction with conspecifics and wildlife, and community attitudes and practices associated with dog ownership.

Molecular detection and characterization of Anaplasma spp. in cattle and sable antelope from Lusaka and North-Western provinces of Zambia.

Rickettsiales of the genus Anaplasma are globally distributed tick-borne pathogens of animals and humans with complex epidemiological cycles. Anaplasmosis is an important livestock disease in Zambia but its epidemiological information is inadequate. This study aimed to detect and characterize the species of Anaplasma present in domestic and wild ruminants in Zambia with a focus on the infection risk posed by the translocation of sable antelope (Hippotragus niger) from North-Western Province to Lusaka Province. Archived DNA samples (n = 100) extracted from whole blood (sable n = 47, cattle n = 53) were screened for Anaplasmataceae using 16S rRNA partial gene amplification followed by species confirmation using phylogenetic analysis. Out of the 100 samples, Anaplasma species were detected in 7% (4/57) of the cattle and 24% (10/43) of the sable antelope samples. Of the 14 positive samples, five were determined to be A. marginale (four from cattle and one from sable), seven were A. ovis (sable) and two were A. platys (sable). Phylogenetic analysis of the 16S rRNA partial gene sequences revealed genetic proximity between A. ovis and A. marginale, regardless of host. The detection of Anaplasma in wildlife in Zambia shows the risk of transmission of Anaplasma species associated with wildlife translocation.

Sequence Diversity of Tp1 and Tp2 Antigens and Population Genetic Analysis of Theileria parva in Unvaccinated Cattle in Zambia's Chongwe and Chisamba Districts.

East Coast Fever (ECF), caused by Theileria parva, is a major constraint to improved livestock keeping in east and central Africa, including Zambia. To understand the dynamics and determine the candidates for immunization in Zambia's Chongwe and Chisamba districts, a combination of Tp1 and Tp2 gene sequencing and microsatellite analysis using nine markers was conducted from which an abundance of Muguga, Kiambu, Serengeti and Katete epitopes in the field samples was obtained. Phylogenetic analysis showed six (Tp1) and three (Tp2) clusters with an absence of geographical origin clustering. The majority of haplotypes were related to Muguga, Kiambu, Serengeti and Katete, and only a few were related to Chitongo. Both antigens showed purifying selection with an absence of positive selection sites. Furthermore, low to moderate genetic differentiation was observed among and within the populations, and when vaccine stocks were compared with field samples, Chongwe samples showed more similarity to Katete and less to Chitongo, while Chisamba samples showed similarity to both Katete and Chitongo and not to Muguga, Kiambu or Serengeti. We conclude that the use of Katete stock for immunization trials in both Chongwe and Chisamba districts might produce desirable protection against ECF.

Healthcare Management of Human African Trypanosomiasis Cases in the Eastern, Muchinga and Lusaka Provinces of Zambia.

Human African trypanosomiasis (HAT) is a neglected tropical disease that has not received much attention in Zambia and most of the countries in which it occurs. In this study, we assessed the adequacy of the healthcare delivery system in diagnosis and management of rHAT cases, the environmental factors associated with transmission, the population at risk and the geographical location of rHAT cases. Structured questionnaires, focus group discussions and key informant interviews were conducted among the affected communities and health workers. The study identified 64 cases of rHAT, of which 26 were identified through active surveillance and 38 through passive surveillance. We identified a significant association between knowledge of the vector for rHAT and knowledge of rHAT transmission (p < 0.028). In all four districts, late or poor diagnosis occurred due to a lack of qualified laboratory technicians and diagnostic equipment. This study reveals that the current Zambian healthcare system is not able to adequately handle rHAT cases. Targeted policies to improve staff training in rHAT disease detection and management are needed to ensure that sustainable elimination of this public health problem is achieved in line with global targets.

Characterization of influenza A viruses isolated from wild waterfowl in Zambia.

Although the quest to clarify the role of wild birds in the spread of the highly pathogenic H5N1 avian influenza virus (AIV) has yielded considerable data on AIVs in wild birds worldwide, information regarding the ecology and epidemiology of AIVs in African wild birds is still very limited. During AIV surveillance in Zambia (2008-2009), 12 viruses of distinct subtypes (H3N8, H4N6, H6N2, H9N1 and H11N9) were isolated from wild waterfowl. Phylogenetic analyses demonstrated that all the isolates were of the Eurasian lineage. Whilst some genes were closely related to those of AIVs isolated from wild and domestic birds in South Africa, intimating possible AIV exchange between wild birds and poultry in southern Africa, some gene segments were closely related to those of AIVs isolated in Europe and Asia, thus confirming the inter-regional AIV gene flow among these continents. Analysis of the deduced amino acid sequences of internal proteins revealed that several isolates harboured particular residues predominantly observed in human influenza viruses. Interestingly, the isolates with human-associated residues exhibited higher levels of virus replication in the lungs of infected mice and caused more morbidity as measured by weight loss than an isolate lacking such residues. This study stresses the need for continued monitoring of AIVs in wild and domestic birds in southern Africa to gain a better understanding of the emergence of strains with the potential to infect mammals.

Prevalence of Trypanosoma sp. in cattle from Tanzania estimated by conventional PCR and loop-mediated isothermal amplification (LAMP).

This study compared the prevalence of trypanosome infections estimated by PFR-loop-mediated isothermal amplification (LAMP) with conventional polymerase chain reaction (PCR) tests. One hundred forty eight cattle blood samples were collected from Robanda village, Mara region, Tanzania in April 2008. In conventional PCR, four sets of primers, specific for the detection of Trypanosoma sp., Trypanosoma brucei rhodesiense, Trypanosoma vivax, and Trypanozoon, as well as a modified LAMP were used. Conventional PCR detected no infection or up to 8, 1, and 3 infections with Trypanosoma congolense savannah, Trypanozoon, and T. vivax, respectively, whereas LAMP detected additional 44 Trypanozoon positive cases. Our results clearly indicate that the prevalence of Trypanozoon spp. in cattle in Robanda village estimated by PFR-LAMP (30.4%) was significantly higher than the estimates by PCR assays (0.6-2%). As such, future studies should target epidemiological surveys of Trypanozoon and T. brucei rhodesiense infections in possible reservoir animals by LAMP to further elucidate the actual prevalence of these parasites.

Effect of wing length on the prevalence of trypanosomes in Glossina morsitans morsitans in eastern Zambia.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) transmit trypanosomiasis (sleeping sickness in humans and nagana in livestock). Several studies have indicated that age, sex, site of capture, starvation and microbiome symbionts, among others, are important factors that influence trypanosome infection in tsetse flies. However, reasons for a higher infection rate in females than in males still largely remain unknown. Considering that tsetse species and sexes of larger body size are the most mobile and the most available to stationary baits, it was hypothesized in this study that the higher trypanosome prevalence in female than in male tsetse flies was a consequence of females being larger than males. METHODS: Black screen fly rounds and Epsilon traps were used to collect tsetse flies in eastern Zambia. Measurement of wing vein length and examination for presence of trypanosomes in the flies were carried out by microscopy. Principal component method was carried out to assess the potential of wing vein length as a predictor variable. The multilevel binary logistic regression method was applied on whole data, one-method data and one-sex data sets to evaluate the hypothesis. RESULTS: Data derived from a total of 2195 Glossina morsitans morsitans were evaluated (1491 males and 704 females). The wing length variable contributed the highest variance percentage (39.2%) to the first principal component. The variable showed significant influence on prevalence of trypanosomes when the analysis was applied on the whole data set, with the log odds for the prevalence of trypanosomes significantly increasing by 0.1 (P = 0.032), per unit increase in wing length. Females had higher trypanosome prevalence rates than males, though not always significant. Furthermore, moving from females to males, wing length significantly reduced by 0.2 (P < 0.0001). CONCLUSIONS: We conclude that wing length is an important predictor variable for trypanosome prevalence in Glossina morsitans morsitans and could partially explain the higher prevalence of trypanosomes in females than in males. However, reasonably representative population data are required for analysis-a serious challenge with the current tsetse sampling methods. Thus, analysis combining data from mobile and stationary methods that include both sexes' data could be useful to verify this hypothesis.

Policy and Linkages in the Application of a One Health System for Reporting and Controlling African Trypanosomiasis and Other Zoonotic Diseases in Zambia.

The capacity to detect, control and manage emerging and re-emerging zoonotic diseases in Africa has been limited by a lack of utilisation of available reporting structures and policies to support programmes at national and local levels. This study explored the impact of the Zambian government policies on animal and human disease reporting and management and on One Health opportunities. An in-depth review and analysis of strengths, weaknesses, opportunities, and threats in the existing policies and reporting structures in the departments responsible for Veterinary Services, Health, and Wildlife, was conducted. According to our findings, sub-optimal implementation of existing policies related to the control of zoonotic diseases was impacting disease reporting, and reporting structures play an important role in effective and sustainable reporting of zoonotic diseases. Further, the study explored capacities and strategies in trypanosomiasis control as a case study that could prompt effective adoption of a One Health approach, and as such, the study suggests measures that could help to assess the performance of a One Health system in the control of African trypanosomiasis and other zoonotic diseases.

Challenges in the Diagnostic Performance of Parasitological and Molecular Tests in the Surveillance of African Trypanosomiasis in Eastern Zambia.

African animal trypanosomiasis (AAT) control programs rely on active case detection through the screening of animals reared in disease endemic areas. This study compared the application of the polymerase chain reaction (PCR) and microscopy in the detection of trypanosomes in cattle blood in Mambwe, a rural district in eastern Zambia. Blood samples were collected from 227 cattle and tested for infection with trypanosomes using microscopy and Ribosomal RNA Internal Transcribed Spacers (ITS)-PCR. Microscopy on the buffy coat detected 17 cases, whilst thin and thick smears detected 26 cases and 28 cases, respectively. In total, microscopy detected 40 cases. ITS-PCR-filter paper (FP) on blood spots stored on FP detected 47 cases, and ITS-PCR-FTA on blood spots stored on Whatman FTA Classic cards detected 83 cases. Using microscopy as the gold standard, ITS-PCR-FTA had a better specificity (SP) and sensitivity (SE) (SP = 72.2%; SE = 77.5%; kappa = 0.35) than ITS-PCR-FP (SP = 88%; SE = 60%; kappa = 0.45). The prevalence of Trypanosoma brucei s.l. was higher on ITS-PCR-FTA (19/227) than on ITS-PCR-FP (0/227). Our results illustrate the complexities around trypanosomiasis surveillance in rural Africa and provide evidence of the impact that field conditions and staff training can have on diagnostic results, which in turn impact the success of tsetse and trypanosomiasis control programs in the region.

Molecular characterization and population genetics of Theileria parva in Burundi's unvaccinated cattle: Towards the introduction of East Coast fever vaccine.

Theileria parva (T. parva) is a protozoan parasite that causes East Coast fever (ECF). The disease is endemic in Burundi and is a major constraint to livestock development. In this study, the parasite prevalence in cattle in six regions namely; Northern, Southern, Eastern, Western, Central and North Eastern was estimated. Furthermore, the sequence diversity of p67, Tp1 and Tp2 genes was assessed coupled with the population genetic structure of T. parva using five satellite markers. The prevalence of ECF was 30% (332/1109) on microscopy, 60% (860/1431) on ELISA and 79% (158/200) on p104 gene PCR. Phylogenetic analysis of p67 gene revealed that only allele 1 was present in the field samples. Furthermore, phylogenetic analysis of Tp1 and Tp2 showed that the majority of samples clustered with Muguga, Kiambu and Serengeti and shared similar epitopes. On the other hand, genetic analysis revealed that field samples shared only two alleles with Muguga Cocktail. The populations from the different regions indicated low genetic differentiation (FST = 0.047) coupled with linkage disequilibrium and non-panmixia. A low to moderate genetic differentiation (FST = 0.065) was also observed between samples and Muguga cocktail. In conclusion, the data presented revealed the presence of a parasite population that shared similar epitopes with Muguga Cocktail and was moderately genetically differentiated from it. Thus, use of Muguga Cocktail vaccine in Burundi is likely to confer protection against T. parva in field challenge trials.

Genetic Diversity of African Trypanosomes in Tsetse Flies and Cattle From the Kafue Ecosystem.

We clarified the genetic diversity of Trypanosoma spp. within the Kafue ecosystem, using PCR targeting the internal transcribed spacer 1 and the cathepsin L-like cysteine protease (CatL) sequences. The overall prevalence of Trypanosoma spp. in cattle and tsetse flies was 12.65 and 26.85%, respectively. Cattle positive for Trypanosoma vivax had a significantly lower packed cell volume, suggesting that T. vivax is the dominant Trypanosoma spp. causing anemia in this area. Among the 12 operational taxonomic units (OTUs) of T. vivax CatL sequences detected, one was from a known T. vivax lineage, two OTUs were from known T. vivax-like lineages, and nine OTUs were considered novel T. vivax-like lineages. These findings support previous reports that indicated the extensive diversity of T. vivax-like lineages. The findings also indicate that combining CatL PCR with next generation sequencing is useful in assessing Trypanosoma spp. diversity, especially for T. vivax and T. vivax-like lineages. In addition, the 5.42% prevalence of Trypanosoma brucei rhodesiense found in cattle raises concern in the community and requires careful monitoring of human African trypanosomiasis.

Evidence of Borrelia theileri in Wild and Domestic Animals in the Kafue Ecosystem of Zambia.

Members of the genus Borrelia are arthropod-borne spirochetes that are human and animal pathogens. Vertebrate hosts, including wild animals, are pivotal to the circulation and maintenance of Borrelia spirochetes. However, information on Borrelia spirochetes in vertebrate hosts in Zambia is limited. Thus, we aimed to investigate the presence of Borrelia spirochetes in wild animals and cattle in Zambia. A total of 140 wild animals of four species and 488 cattle DNA samples from /near the Kafue National Park were collected for real-time PCR screening, followed by characterization using three different genes with positive samples. Five impalas and 20 cattle tested positive using real-time PCR, and sequence analysis revealed that the detected Borrelia were identified to be Borrelia theileri, a causative agent of bovine borreliosis. This is the first evidence of Borrelia theileri in African wildlife and cattle in Zambia. Our results suggest that clinical differentiation between bovine borreliosis and other bovine diseases endemic in Zambia is required for better treatment and control measures. As this study only included wild and domestic animals in the Kafue ecosystem, further investigations in other areas and with more wildlife and livestock species are needed to clarify a comprehensive epidemiological status of Borrelia theileri in Zambia.