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.

Borrelia lonestari, Borrelia miyamotoi, and Borrelia theileri are members of HTBRF, or of Matestriate-tansmitted borreliae in Margos's classification. Borrelia lonestari was first identified in 1996 [7] and was initially considered pathogenic to humans in the southern United States [12,13]. However, subsequent research did not support the pathogenicity of B. lonestari in humans [14]. Amblyomma ticks and white-tailed deer are vectors and suspected reservoirs, respectively, for B. lonestari in North America [15][16][17]. Borrelia miyamotoi was confirmed in 2011 as a human pathogen [18]. Ixodes ticks are reported vectors of Borrelia miyamotoi in Asia, Europe, North America, and Russia [19], and deer may act as natural reservoirs for it [20]. Borrelia theileri is a causative agent of bovine borreliosis, identified in South Africa more than 100 years ago by Arnold Theiler, who first found the agent transmitted by Rhipicephalus sp. to cattle [21,22]. To date, Borrelia theileri has been reported in cattle, goats, sheep, and horses in Africa, North and South America, and Australia [23][24][25]. Rhipicephalus ticks, such as R. microplus, R. annulatus, R. evertsi, and R. decoloratus, are well-known vectors of Borrelia theileri. In addition, Borrelia theileri has recently been detected in head lice (Pediculus humanus) in the Republic of Congo [26].
Vertebrate hosts, including wild animals, act as reservoirs for Borrelia spirochetes and are crucial in the circulation and maintenance of them. For instance, Kumagai et al. (2018) discovered Borrelia spp. of the hard tick-borne relapsing fever borreliae in 25.9% of wild deer tested in Japan and suggested that wild deer could act as reservoir hosts for Borellia spp. [27]. Similarly, other studies have also reported the detection of Borrelia lonestari and antibodies against Borrelia burgdorferi in wild deer populations in America [16,28]. Furthermore, several serological studies have revealed that many species of African antelopes kept in zoos have antibodies against Borrelia burgdorferi [29][30][31]. However, only a few studies have been conducted to investigate Borrelia spirochetes in wild animals in Africa. For example, Borrelia infection was demonstrated in 9.2% of small mammals tested in West Africa [32], and Candidatus Borrelia fainii was detected in 27% of bats examined in Zambia [33]. In addition, to date, there has been no investigation of Borrelia in large wild animals in Africa.
We aimed to investigate the presence of Borrelia spirochetes in large and medium-sized wild animals and cattle in Zambia through molecular and phylogenetic analyses.

Materials and Methods
DNA samples extracted from the whole blood of wild animals and domestic cattle from two previous studies [34,35] were used to detect Borrelia in this study. A total of 140 DNA samples from wild animals, including 97 impalas (Aepyceros melampus), 37 hartebeests (Alcelaphus buselaphus), four lions (Panthera leo), and two wild dogs (Lycaon pictus), were previously collected in the greater Kafue ecosystem for investigating piroplasm diversity [34]. In addition, we used 488 cattle DNA samples with packed cell volume (PCV) value data from a previous African trypanosome investigation [35]. The cattle breed was mainly a cross between local breeds (Tonga and Baila) and exotic breeds (mostly Boran and Brahman). Thus, a total of 628 DNA samples from wild and domestic animals were screened for Borrelia spp.
Specific semiquantitative real-time PCR was used for the initial screening of Borrelia infection, using the THUNDERBIRD ® Probe qPCR Mix (TOYOBO, Osaka, Japan) and primers (Bor16S3F, 5 -AGCCTTTAAAGCTTCGCTTGTAG-3 ; Bor16S3R, 5 -GCCTCCCGTAGGAGT CTGG-3 ; Bor16S3P, 5 -6FAM-CCGGCCTGAGAGGGTGAACGG-TAMRA-3 ), which were designed to amplify a 148-bp fragment of the 16S ribosomal RNA gene (16S rDNA) of Borrelia. The specificity of the real-time PCR system for detection of Borrelia spp. was previously tested on DNA samples from 347 bacterial species [36]. All real-time PCRs were performed using a LightCycler 96 (Roche Diagnostics GmbH, Mannheim, Germany). The DNA of Candidatus Borrelia fainii strain Qtaro isolated from the patient in our previous study [33] and UltraPureTM distilled water (Invitrogen, Waltham, MA) were used as positive and negative controls, respectively, for each test. Samples with a cycle threshold level of log-based fluorescence <36 (~10-20 copies of spacer) were labelled positive, as described previously [36].
All positive samples from the real-time PCR were used for subsequent characterization based on three genes: flagellin (flaB), hypoxanthine-guanine phosphoribosyltransferase (hpt), and 16S rDNA using conventional PCR with the primers listed in Table 1. Briefly, PCRs were conducted using Ex-Taq HS (Takara, Shiga, Japan) with the following conditions: 1 min denaturation step at 98 • C followed by 35 cycles of 94 • C for 30 s, an appropriate annealing temperature (Table 1) for 30 s, and 72 • C for 30 s (1 min 30 s for 16S rDNA), and a final extension step at 72 • C for 5 min. For the negative and positive controls, UltraPureTM distilled water and DNA from Ca. Borrelia fainii strain Qtaro were added, respectively, instead of template DNA. The resulting PCR products were electrophoresed on a 1.2% agarose gel stained with Gel-Red (Biotium, Hayward, CA, USA) and visualized with a UV trans-illuminator. Sanger sequencing was performed using BigDye Terminator version 3.1 chemistry (Applied Biosystems, Foster City, CA, USA). Sequencing products were run on an ABI Prism 3500 Genetic Analyzer, according to the manufacturer's instructions. The sequence data were assembled using ATGC software version 6.0.4 (GENETYX, Tokyo, Japan). The DDBJ/EMBL/GenBank accession numbers for the sequences obtained were as follows: flab, LC656216-LC656235; 16S rDNA, LC656236-LC656247; and hpt, LC656248-LC656262 (Supplemental Table S1). The phylogenetic relationships for each gene were analyzed using the neighbor-joining and maximum likelihood methods with 1000 bootstraps implemented in MEGA X [40].
Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). For comparison of Borrelia spp. infection state in cattle and PCV value data of the samples, statistical significance (p < 0.05) was assessed using the Mann-Whitney test.

Real-Time and Conventional PCRs
Five out of 97 (5.1%) impala samples tested positive for the real-time PCR, while none of the samples from other wild animal species tested positive (Table 2). Subsequent PCRs for the characterization of detected Borrelia spp. were conducted using five positive impalas, and PCRs targeting flaB, 16S rDNA, and hpt genes successfully provided amplicons from four, two, and four impalas, respectively. On the other hand, 20 out of 488 (4.1%) cattle samples tested positive for the real-time PCR (Table 2). Additional PCRs targeting flaB, 16S rDNA, and hpt genes successfully produced amplicons from 16, 10, and 11 cattle, respectively.

Sequence Analysis
The flaB sequences from the four impalas had two variants with one nucleotide difference in 276-bp.

Phylogenetic Analysis
Phylogenetic trees were constructed to obtain information on the genetic association of our detected Borrelia spp. with other Borrelia species in the database. Based on the phylogenetic inference of the flaB gene, our detected Borrelia spp. from impalas and cattle were located within the clade of Borrelia theileri (Figure 1).  Similarly, the detected Borrelia spp. from impalas and cattle were positioned within the clade of Borrelia theileri in the phylogenetic trees based on the almost full-length 16S rDNA and the partial sequence of hpt ( Figure 2).

Discussion
In this study, we investigated the presence of Borrelia spirochetes in four different wild animal species and domestic cattle in the Kafue ecosystem in Zambia using molecular methods and successfully identified Borrelia theileri in impalas and cattle. To the best of our knowledge, this study provides the first evidence of Borrelia spirochetes in impalas in Africa and the first report of Borrelia theileri in cattle in Zambia.
Borrelia theileri is the causative agent of bovine borreliosis, first reported in cattle in South Africa in 1903 [21]. Zambia is a landlocked country located in south central Africa and shares its borders with Zimbabwe, Namibia, Botswana, Mozambique, Malawi, Tanzania, Democratic Republic of Congo, and Angola. Among these surrounding countries, only Botswana has reported cases of bovine borreliosis [41]. However, the previous report did not have any molecular information on the detected Borrelia theileri. The present study provides molecular evidence for a new geographical record of Borrelia theileri in Zambia. Furthermore, considering that cross-border trade is common in cattle and that wildlife migration occurs between Zambia and its neighbors, Borrelia theileri might have spread to the surrounding countries through infected animals.
A previous study in North Cameroon, using PCR, found that 17.9% (225/1260) of cattle were infected with Borrelia spp. [42]. However, out of 225 positive cattle, only 42 were confirmed to be infected with Borrelia theileri by sequence analysis. Thus, the infection rate of Borrelia theileri was 3.3% (42/1260) in the cattle population in North Cameroon. The present study also showed a similar infection rate (4.1%) in cattle to a previous study in North Cameroon. In addition, the present study revealed that 5.1% of the impalas in the Kafue ecosystem were infected with Borrelia theileri. However, there have been no

Discussion
In this study, we investigated the presence of Borrelia spirochetes in four different wild animal species and domestic cattle in the Kafue ecosystem in Zambia using molecular methods and successfully identified Borrelia theileri in impalas and cattle. To the best of our knowledge, this study provides the first evidence of Borrelia spirochetes in impalas in Africa and the first report of Borrelia theileri in cattle in Zambia.
Borrelia theileri is the causative agent of bovine borreliosis, first reported in cattle in South Africa in 1903 [21]. Zambia is a landlocked country located in south central Africa and shares its borders with Zimbabwe, Namibia, Botswana, Mozambique, Malawi, Tanzania, Democratic Republic of Congo, and Angola. Among these surrounding countries, only Botswana has reported cases of bovine borreliosis [41]. However, the previous report did not have any molecular information on the detected Borrelia theileri. The present study provides molecular evidence for a new geographical record of Borrelia theileri in Zambia. Furthermore, considering that cross-border trade is common in cattle and that wildlife migration occurs between Zambia and its neighbors, Borrelia theileri might have spread to the surrounding countries through infected animals.
A previous study in North Cameroon, using PCR, found that 17.9% (225/1260) of cattle were infected with Borrelia spp. [42]. However, out of 225 positive cattle, only 42 were confirmed to be infected with Borrelia theileri by sequence analysis. Thus, the infection rate of Borrelia theileri was 3.3% (42/1260) in the cattle population in North Cameroon. The present study also showed a similar infection rate (4.1%) in cattle to a previous study in North Cameroon. In addition, the present study revealed that 5.1% of the impalas in the Kafue ecosystem were infected with Borrelia theileri. However, there have been no studies on the infection rate of Borrelia theileri in large and middle-sized wildlife. Nevertheless, previous studies in Japan showed that 10.6% (25/235) and 25.9% (165/638) of wild sika deer were infected with a Borrelia sp., similar to Borrelia lonestari, which is a closely related species to Borrelia theileri [27,43].
Zambia is an endemic region of the East Coast Fever (ECF), a fatal disease in cattle caused by the blood parasite Theileria parva [44] and Redwater or bovine babesiosis caused by Babesia bigemina and Babesia bovis [45]. Another severe cattle disease, African animal trypanosomiasis, caused by Trypanosoma spp., is also endemic to Zambia [42]. Animals infected with these diseases have common clinical symptoms, such as fever, anemia, jaundice, hemoglobinuria, and swollen lymph nodes [44][45][46], and these diseases often result in death. Therefore, these diseases are of great veterinary and economic importance, as they have a considerable effect on livestock production [44]. In contrast, bovine borreliosis shows mild but similar symptoms, such as fever, lethargy, and anemia [42]. In this study, Borrelia infection was not related to anemia, statistically. Nevertheless, these symptoms can affect cattle performance and reduce their production. Furthermore, the livelihoods of an estimated 700 million rural African poor are supported or maintained by livestock ownership [47]. In addition to its intrinsic value, livestock provide a flow of food and income over time, helping to increase farm productivity; for most rural communities, livestock production is their only available livelihood [47]. Therefore, recognizing the existence of Borrelia theileri in Zambia was helpful in the clinical differentiation between bovine borreliosis and other bovine diseases for better treatment and control measures, which would ensure that farmers bring healthier and more valuable products to the market.
Human activities and associated land use changes have caused an increase in proximity between domestic and wild animals in many places, resulting in the mutual exchange of pathogens [20,48]. As is often the case near national parks in Zambia, domestic and wild animals often share a grazing place. In this study, we detected Borrelia theileri, which had sequences identical to those of cattle and impalas. Thus, the same strain of Borrelia theileri might be circulating between cattle and impalas in the Kafue ecosystem. Furthermore, Espinaze et al. (2018) revealed that large and middle-sized animals were highly connected by the tick species they shared, facilitating cross-infection with ticks and the transmission of tick-borne pathogens, including Borrelia theileri [49]. In particular, domestic animals can play an important role in accelerating the spread of ticks and tick-borne pathogens in Southern African animal communities [49], which means that these ticks and tick-borne pathogens could be introduced into the Kafue ecosystem. Therefore, although the pathogenicity of Borrelia theileri in wild animals is not known, limiting contact between cattle and wild animals might be a good conservation measure for reducing tick-borne diseases in the Kafue ecosystem. Furthermore, investigations of vector tick species for Borrelia theileri in the Kafue ecosystem are required for understanding their natural life cycle in the ecosystem.
In this study, we revealed the presence of Borrelia theileri, a causative agent of bovine borreliosis in wild impalas and domestic cattle in the Kafue ecosystem in Zambia. Our findings indicate the circulation of spirochetes between impalas and cattle. Furthermore, bovine borreliosis has veterinary and economic implications in livestock, even though the clinical presentation is not as severe as in other bovine diseases. As the study was conducted with samples collected only from the Kafue ecosystem, further investigations are required to clarify comprehensive Borrelia theileri status in the country by expanding the sampling area and including additional wild animal and livestock species, especially ungulates.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms9112405/s1, Table S1: Accession numbers of the DNA sequences obtained in this study.