sign in sign up
<<
Home , African wild dog

Veterinary Parasitology 291 (2021) 109381

Contents lists available at ScienceDirect

Veterinary Parasitology

journal homepage: www.elsevier.com/locate/vetpar

A shared pathogen: Babesia rossi in domestic , black-backed ( mesomelas) and African wild dogs ( pictus) in

Ntji Shabangu a, Barend L. Penzhorn a,b,c, Marinda C. Oosthuizen a,b, Ilse Vorster a, O. Louis van Schalkwyk d, Robert F. Harrison-White e, P. Tshepo Matjila a,* a Vectors and Vector-borne Diseases Programme, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa b Centre for Veterinary Wildlife Studies, Faculty of Veterinary Science, University of Pretoria, South Africa c National Zoological Garden, South African National Biodiversity Institute, P.O. Box 754, Pretoria, 0001, South Africa d Office of the State Veterinarian, Skukuza, , 1350, South Africa e Wildlife Damage – Research and Management, P.O. Box 783540, Sandton, 2146, South Africa

ARTICLE INFO ABSTRACT

Keywords: In sub-Saharan Africa, babesiosis in domestic dogs is caused primarily by Babesia rossi. Black-backed jackals African wild (Canis mesomelas), which are subclinical carriers of B. rossi, were a likely reservoir host from which Babesia rossi passed to domestic dogs. The role of other indigenous canids, e.g. African wild dogs (Lycaon pictus), as reservoirs Black-backed of B. rossi has not been elucidated. The question also arises whether genetic differences have arisen between Canis mesomelas B. rossi infecting domestic dogs and “ancestral” B. rossi in jackals. In a previous study we found that nearly one- Lycaon pictus Reservoir host third (27 of 91) of jackals were infected with B. rossi; this was confirmedby 18S rDNA sequence analysis. In this South Africa study, the near full-length B. rossi 18S rRNA gene was successfully amplified from 6 domestic dogs and 3 black- backed jackals. The obtained recombinant sequences were identical (100 %) to previously described B. rossi sequences of black-backed jackals in South Africa, and 99 % similar to B. rossi from dogs in South Africa and the . Although blood specimens from 5 (10 %) of 52 free-ranging African wild dogs (from Kruger National Park, South Africa, reacted with the B. rossi probe on RLB hybridisation, the presence of B. rossi could not be confirmed by amplification and sequencing, nor by multiplex, real-time PCR. Although African wild dogs they can be infected with B. rossi without showing clinical signs, our findings suggest that they are apparently not important reservoir hosts of B. rossi.

1. Introduction while adult ticks prefer carnivores, both canid and felid (Horak et al., 2018). Adult female H. elliptica, which ingest B. rossi when engorging on In sub-Saharan Africa, babesiosis in domestic dogs is caused pri­ an infected canid host, pass the infection transovarially; B. rossi is marily by Babesia rossi (Matjila et al., 2008a; Penzhorn, 2020). transmitted only by adult ticks of the subsequent generation (Lounsbury, Black-backed jackals (Canis mesomelas), which occur in two discrete 1904; Lewis et al., 1996). In areas where its off-host ecological re­ geographic ranges in southwestern and northeastern Africa (Kingdon quirements are met, H. elliptica is the most prevalent tick in black-backed and Hoffmann, 2013), harbour B. rossi without showing any clinical jackal populations (Penzhorn et al., 2020). These jackals are therefore signs (Neitz and Steyn, 1947; van Heerden, 1980). In a previous study likely reservoirs from which domestic dogs initially became infected we found that nearly one-third (27 of 91) of a free-ranging black-backed with B. rossi (Penzhorn, 2011; Penzhorn et al., 2017) jackal population was subclinically infected with B. rossi (Penzhorn Dogs are not native to sub-Saharan Africa, but have been present for et al., 2017). many centuries (Mitchell, 2015). It is postulated that through natural Haemaphysalis elliptica, the only confirmed vector of B. rossi, occurs selection, African dog types such as Africanis have developed the ability commonly in the more mesic parts of southern Africa (Theiler and to cope with B. rossi infection, while exotic dog breeds are highly sus­ Robinson, 1953). Immature stages feed primarily on murid , ceptible (Penzhorn, 2011). Once established, can be

* Corresponding author. E-mail address: [email protected] (P.T. Matjila). https://doi.org/10.1016/j.vetpar.2021.109381 Received 29 July 2020; Received in revised form 2 February 2021; Accepted 3 February 2021 Available online 14 February 2021 0304-4017/© 2021 Elsevier B.V. All rights reserved. N. Shabangu et al. Veterinary Parasitology 291 (2021) 109381 maintained in domestic dog populations in the absence of jackals clinical signs of babesiosis and B. rossi recovered form black-backed (Morters et al., 2020). The questions arises, therefore, whether genetic jackals; and (b) the prevalence of B. rossi in free-ranging African wild differences have arisen between B. rossi infecting domestic dogs and dogs, through molecular detection. “ancestral” B. rossi in jackals. In a previous study conducted in our laboratory, blood specimens 2. Materials and methods from 33 (30.8 %) of 107 black-backed jackals reacted with the B. rossi probe on Reverse Line Blot (RLB) assay (Penzhorn et al., 2017). To 2.1. Sample collection confirm the RLB results, two of the positive specimens were analysed further. After amplification and cloning of near full-length 18S rRNA Blood was collected into Ethylenediaminetetra-acetic acid (EDTA) gene from these two samples, six recombinants yielded sequences vacutainer tubes from the cephalic vein, refrigerated and transported to identical to that of B. rossi (L19079) in GenBank and differing by two the Department of Veterinary Tropical Diseases. As part of a previous base pairs from B. rossi (DQ111760). There were 1,502 positions in the study, blood samples (n = 75) were collected over a six- period final dataset. The original specimens were still available. More of the (2000–2006) from dogs presented at the Outpatients Clinic of the positive specimens could therefore be analysed further, to give a clearer Onderstepoort Veterinary Academic Hospital (OVAH) (Matjila et al., indication of possible genetic variation. 2008a) (Fig. 1). Blood samples from free-ranging (n = 91) and captive (n For comparison, blood specimens from 75 dogs presented to the = 6) black-backed jackals had been collected at Mogales Gate Biodi­ Outpatients Clinic of the Onderstepoort Veterinary Academic Hospital versity Centre, northwestern Gauteng, in 2012 and 2014 and at S.A. (OVAH) and tentatively diagnosed as suffering from babesiosis were Lombard Nature Reserve near Bloemhof, North West Province, South available. These specimens had been used in a previous study (Matjila Africa (Penzhorn et al., 2017) (Fig. 1). African wild dog blood samples (n et al., 2008a). = 52) were collected from a free-ranging population, primarily in the Occurrence and prevalence of B. rossi infection in other indigenous southern half of the KNP (Fig. 1). Additional blood samples from captive canid species has not been well documented. Some captive African wild African wild dogs (n = 6) were obtained from the biobank at the Na­ dogs (Lycaon pictus) have been shown to be infected (Matjila et al., tional Zoological Garden, South African National Biodiversity Institute, 2008b), but reports from free-ranging wild dogs are meagre (Peirce Pretoria, South Africa (Fig. 1). et al., 1995; van Heerden et al., 1995). Mortality due to babesiosis has been recorded in a zoo-born pup (Colly and Nesbit, 1992). On the other 2.2. DNA extraction hand, three 6-month-old African wild dogs infected intravenously with heparinised blood from a terminally sick domestic dog developed par­ Genomic DNA was extracted from 200 μL of EDTA anticoagulated ◦ asitaemias but showed no clinical signs of infection (van Heerden, whole blood (stored at 20 C) using the QIAamp®DNA mini kit (Qia­ 1980). Blood collected from individual African wild dogs on Day 29 after gen, Southern Cross Biotechnology, Hilden, Germany), following man­ infection and injected intravenously into three different domestic dogs ufacturer’s instructions. resulted in severe clinical signs, suggesting that African wild dogs could be reservoirs of infection (van Heerden, 1980). A general health survey 2.3. Reverse line blot hybridisation of the African wild dog population in the southern half of the Kruger National Park (KNP) where both black-backed jackals and H. elliptica The Reverse Line Blot (RLB) hybridization assay, as previously occur, offered the opportunity of examining a large set of blood speci­ described by Gubbels et al. (1999), was performed on all samples. The mens for the presence of haemoprotozoa. oligonucleotide probes and their sequences used for detecting pathogen The objectives of this study were to determine (a) whether there DNA with PCR-RLB hybridization are listed in Table 1. Babesia bovis and were genetic differences between B. rossi in domestic dogs showing Anaplasma centrale known positives were used as positive control to

Fig. 1. Map of the north-eastern part of South Africa, indicating the origins of the various blood specimens included in the study: (1) S.A. Lombard Nature Reserve, near Bloemhof, North West Province; (2) Mogale’s Gate Biodiversity Centre, Gauteng; Pretoria, Gauteng (Onderstepoort Veterinary Academic Hospital and National Zoological Garden; (4) Kruger National Park, Mpumalanga and Limpopo Provinces.

2 N. Shabangu et al. Veterinary Parasitology 291 (2021) 109381

Table 1 Table 1 (continued ) List of oligonucleotide probes and their sequences used for detecting pathogen ′ ′ Oligonucleotide probes Sequences (5 -3 ) Reference DNA with PCR-RLB hybridization. R = A / G, W = A / T are the symbols used to indicate degenerate positions. Babesia vogeli AGC GTG TTC GAG TTT Matjila et al. (2004) GCC ′ ′ Oligonucleotide probes Sequences (5 -3 ) Reference

Ehrlichia / Anaplasma - GGG GGA AAG ATT TAT Bekker et al. (2002) specific CGC TA validate the detections of Theileria, Babesia and Ehrlichia and Anaplasma Anaplasma bovis GTA GCT TGC TAT GRG Bekker et al. (2002) from the samples. AAC A Amplification of the V4 variable region (̴ 450–520 bp) of the 18S Anaplasma centrale TCG AAC GGA CCA TAC GC RLB Manual, Isogen rRNA gene was done using Theileria and Babesia genus-specific primers Anaplasma marginale GAC CGT ATA CGC AGC Bekker et al. (2002) ′ ′ TTG RLB-F (biotin 5 -GAC ACA GGG TAG TGA CAA G-3 ) and RLB-R (biotin- ′ ′ Anaplasma sp. Omatjenne CGG ATT TTT ATC ATA Bekker et al. (2002) 5 -CTA AGA ATT TCA CCT CTG ACA GT-3 ) (Gubbels et al., 1999). GCT TGC Subsequently, the V1 variable region (̴̴ 460–520 bp) of the parasite 16S Anaplasma phagocytophilum TTG CTA TAA AGA ATA Bekker et al. (2002) rRNA gene was amplified using the Ehrlichia and Anaplasma ATT AGT GG ′ genus-specific primers Ehr-F (biotin 5 -GGA ATT CAG AGT TGG ATC Ehrlichia canis TCT GGC TAT AGG AAA Bekker et al. (2002) ′ ′ TTG TTA MTG GYT CAG-3 ) and Ehr-R (biotin 5 -CGG GAT CCC GAG TTT GCC ′ Ehrlichia chaffeensis ACC TTT TGG TTA TAA RLB Manual, Isogen GGG ACT TYT TCT-3 ) (Bekker et al., 2002; Nijhof et al., 2005). A 25 μL ATA ATT GTT PCR reaction mixture was prepared, comprised of finalconcentration of Ehrlichia ruminantium AGT ATC TGT TAG TGG RLB Manual, Isogen 1X Platinum® Quantitative PCR SuperMix-UDG (LTC Tech SA, Johan­ CAG μ μ Theileria / Babesia genus- TTA TGG TTA ATA GGA Gubbels et al. nesburg, South Africa), 10 M of each primer, 5 L of genomic DNA and 7 specific RCR GTT G (1999) μL of ultra-pure water, with Babesia bovis and Anaplasma centrale posi­ Theileria genus-specific 1 ATT AGA GTG CTC AAA Nijhof tive control and ultra-pure water as a negative control. Amplification GCA GGC (Unpublished) was performed using Gene Amp®PCR system 9700 (Applied Biosystems, Theileria annae (=Babesia CCG AAC GTA ATT TTA Matjila et al. ) TTG ATT TG (2008a) South Africa), under stringent conditions according to the methods Theileria annulata CCT CTG GGG TCT GTG CA Georges et al. described by Nijhof et al. (2005). The PCR products were hybridized to (2001) Theileria, Babesia and Ehrlichia, Anaplasma genera- and species-specific Theileria buffeli GGC TTA TTT CGG WTT Gubbels et al. oligonucleotide probes, including B. rossi, B. canis, B. vogeli and GAT TTT (1999) Babesia gibsoni. Theileria bicornis GCG TTG TGG CTT TTT TCT Nijhof et al. (2005) G Theileria sp. (buffalo) CAG ACG GAG TTT ACT Oura et al. (2004) 2.4. 18S rRNA amplification, cloning, sequencing and phylogenetic TTG T analysis Theileria equi TTC GTT GAC TGC GYT Butler et al. (2008) TGG Theileria sp. () CTG TGT TTC TTT CCT Nijhof et al. (2005) Twelve samples that showed simultaneous probe signal with the TTG Theileria / Babesia group, genus specific 1, and Babesia rossi were Theileria mutans CTT GCG TCT CCG AAT Gubbels et al. selected for sequencing. The near full length 18 S rRNA gene (̴ 1600 bp) GTT (1999) of six domestic dog, four black-backed jackal and two African wild dog Theileria ovis TTG CTT TTG CTC CTT TAC Altay et al. (2007) ′ samples was amplifiedwith NBab_1 F (5 -AAG CCA TGC ATG TCT AAG GAG ′ ′ Theileria parva GGA CGG AGT TCG CTT TG Nijhof et al. (2005) TAT AAG CTT TT-3 ) and TB_Rev (5 -AAT AAT TCA CCG GAT CAC TCG- ′ Theileria sp. () GCT GCA TTG CCT TTT CTC Nijhof et al. (2005) 3 ) (Matjila et al., 2008c; Oosthuizen et al., 2008). Phusion Flash High C Fidelity PCR Master Mix (Thermo-Scientific™, South Africa) was used to Theileria separata GGT CGT GGT TTT CCT Schnittger et al. perform the PCR. Five separate reactions were prepared for each sample. CGT (2004) Theileria taurotragi TCT TGG CAC GTG GCT Gubbels et al. The reactions were then pooled and purified using the High Pure PCR TTT (1999) Product Purification Kit (Roche Diagnostics, Mannheim, Germany) Theileria velifera CCT ATT CTC CTT TAC GAG Gubbels et al. following the manufacturer’s instructions. T (1999) Cloning was subsequently performed with the CloneJET PCR Cloning Babesia genus-specific 1 ATT AGA GTG TTT CAA Nijhof ™ GCA GAC (Unpublished) Kit (Thermo-Scientific , South Africa,). The purifiedPCR fragment was Babesia genus-specific 2 ACT AGA GTG TTT CAA Nijhof ligated into the pJET 1.2 / blunt cloning vector and transformed into ACA GGC (Unpublished) competent E. coli JM109 cells (JM109 High EfficiencyCompetent Cells, Babesia bicornis TTG GTA AAT CGC CTT Nijhof et al. (2005) Promega, Madison, WI, USA). Colonies were picked and grown in GGT C imMedia Amp Liquid broth (LTC Tech SA, , South Africa). Babesia bovis CAG GTT TCG CCT GTA Gubbels et al. TAA TTG AG (1999) Isolation of recombinant plasmids was performed using High Pure Babesia caballi GTG TTT ATC GCA GAC Butler et al. (2008) Plasmid Isolation Kit (Roche Diagnostics, Mannheim, Germany), and the TTT TGT resulting plasmid DNA was sent for sequencing using vector primers ′ ′ Babesia canis TGC GTT GAC CGT TTG AC Matjila et al. (2004) pJET1.2 forward (5 -CGA CTC ACT ATA GGG AGA GCG GC3 and Babesia gibsoni CGT TTT TTC TTT GTT GG Nijhof et al. (2005) ′ ′ Babesia TTA TGC GTT TTC CGA Bosman et al. (2007) pJET1.2 reverse (5 -AAG AAC ATC GAT TTT CCA TGG CAG-3 ) at Inqaba CTG GC Biotechnical Industries, Pretoria, South Africa. Babesia leo ATC TTG TTG CCT TGC Bosman et al. (2007) The resultant sequences were assembled and edited using CLC ge­ AGC T nomics work bench version 7.5.1 (CLC Bio, Boston, MA, USA) and Babesia major TCC GAC TTT GGT TGG Nijhof et al. (2005) further identified by comparison with the GenBank database by ho­ TGT Babesia microti GRC TTG GCA TCW TCT Nijhof et al. (2005) mology searches made at the National Center for Biotechnology Infor­ GGA mation (NCBI) through BLASTn (Altschul et al., 1990). A multiple Babesia rossi CGG TTT GTT GCC TTT Matjila et al. (2004) sequence alignment was performed using Multiple Alignment with Fast GTG Fourier Transform (MAFFT) [version 7] program (Katoh and Standley, Babesia sp. (sable) GCT GCA TTG CCT TTT CTC Oosthuizen et al. 2013). The alignment was manually truncated to size of the smallest C (2008) sequence, using Bio-Edit version 7.2.5 (Hall, 1999). Phylogenetic trees were constructed with Neighbor joining and Maximum likelihood

3 N. Shabangu et al. Veterinary Parasitology 291 (2021) 109381 methods as implemented in the Molecular Evolutionary Genetics Anal­ gene sequences were compared by determining the number of base ysis version 7 (MEGA 7) software package (Kumar et al., 2016). The differences per near full-length 18S rRNA gene sequence. The observed evolutionary history inferred using Maximum likelihood was based on sequence similarities were subsequently confirmed by phylogenetic the Tamura-Nei parameter model (TN93 + G + I for trees generated analyses. Neighbor-joining and maximum likelihood analyses confirmed from B. rossi sequences (Tamura and Nei, 1993), and the the relationship between the obtained sequences and related Babesia and Hasegawa-Kishino-Yano model (Hasegawa et al., 1985) was used for Hepatozoon sequences previously deposited in Genbank. There was no construction of trees from the H. canis sequences. The bootstrap analysis significant difference in the topology of the or in the was done for the percentage of replicate trees in which the associated bootstrap values found. Representative trees obtained by the Maximum taxa clustered together in 1000 replicates (Felsenstein, 1985). The ge­ Likelihood method are shown in Figs. 2 and 3. netic distances between the sequences were estimated by determining the number of base differences between the sequences using MEGA 7 4. Discussion (Kumar et al., 2016). The 18S rRNA gene sequences of the sequences identified in this Our findingsconfirmed that B. rossi from domestic dogs at OVAH and study were submitted to GenBank (MT683382-MT683385, MT762140- black-backed jackals were similar, as had been reported previously MT762141; MT774531). (Penzhorn et al., 2017). The area served by the OVAH comprises, urban, peri-urban and rural areas. Black-backed jackals occur in the area. Since 2.5. Multiplex, real-time PCR assay adult female H. elliptica ticks engorging on an infected host transmit B. rossi transovarially, the transfer of B. rossi from jackal to dog, and vice Specimens that reacted with the B. rossi probes on RLB were sub­ versa, can therefore not be ruled out. For B. rossi to adapt to dogs, strict jected to multiplex, real-time PCR assay for B. rossi and Babesia vogeli dog-to-dog transmission only should occur over an extended period. (Troskie et al., 2019). In a study in , virtually no differences were found in B. rossi infecting domestic dogs: sequences obtained from 450 bp amplicons 3. Results from 39 dogs displayed 99.74 % homology with partial sequences of 18S rRNA gene of B. rossi in GenBank (Takeet et al., 2017). Black-backed No DNA was detected in the captive African wild dog specimens (n = jackals are confined to southwestern and northeastern Africa, but 6) from the National Zoological Garden biobank. The RLB hybridization Nigeria falls within the wide distribution of the side-striped jackal (Canis assay revealed positive haemoparasite detection in domestic dogs, adustus) (Kingdon and Hoffmann, 2013), from which B. rossi was black-backed jackals and free-ranging African wild dogs (Table 2). described (Nuttall, 1910). The status of the side-striped jackal as The near full-length 18S rRNA gene (~ 1 600 bp) was successfully possible reservoir host of B. rossi has not been elucidated. Since amplified from six domestic dog, four free-ranging black-backed jackal H. elliptica is restricted to southern and East Africa, at least one other and two free-ranging African wild dog samples. Two of the fiveAfrican vector must be involved in the transmission of B. rossi in West Africa. wild dogs specimens selected for showing B. rossi signals on RLB were The single record of H. elliptica from Nigeria (Adamu et al., 2014) is successfully sequenced. Attempts at generating near full-length parasite regarded as a misidentification. 18S rRNA sequences for the three other specimens were unsuccessful, Dogs presented to the OVAH are mostly exotic breeds, which are despite an attempt at using internal primers to generate smaller fully susceptible to B. rossi infection due to lack of previous exposure fragments. (Mellanby et al., 2011; Liu et al., 2018). A recent study of B. rossi in BLASTn homology searches revealed that the obtained recombinant free-roaming dogs in Zenzele township, Gauteng, South Africa, pre­ sequences from six domestic dogs (n = 11) and three black-backed sented an interesting picture of convalescent carrier state or self-induced jackals (n = 8) were identical (100 %) to previously described B. rossi recovery in domestic dogs due to repeated exposure to B. rossi (Morters sequences of black-backed jackals in South African (accession numbers et al., 2020). Although resistance to or tolerance of B. rossi infections has KY463430-KY463434) (Penzhorn et al., 2017), and further revealed been postulated in Africanis-type dogs (Penzhorn, 2011), the impor­ sequence similarity (99 %) to B. canis (presumably B. rossi) from a South tance of acquired resistance to B. rossi infection in endemic areas cannot African dog (L19079) (Allsopp et al., 1994) as well as B. rossi from be overlooked (Morters et al., 2020). Pathogenic B. rossi is ascribed to eastern Sudan (DQ111760) (Oyamada et al., 2005). Multiplex, real-time BREMA1 gene (Babesia rossi erythrocytes membrane antigen gene 1) PCR assays of the five African wild dog specimens that reacted with B. (Matjila et al., 2009), the presence of specific B. rossi genotypes viz, rossi probes on RLB were negative for B. rossi and Babesia vogeli. genotype 19 is associated with virulence whilst genotype 29 is associ­ Partial recombinant sequences (n = 2) from one black-backed jackal ated with a virulence in domestic dogs (Peloakgosi-Shikwambani, positive for B. rossi on RLB, as well as four recombinant sequences ob­ 2018). The absence of the BREMA1 gene was associated with less tained from two African wild dogs revealed sequence identity (100 %) to pathogenic B. rossi in dogs (Adamu et al., 2014). It is unknown if this Hepatozoon sp. of pale (Vulpes pallida) from gene is present in subclinical reservoir hosts such as black-backed (KJ499493, KJ499502-KJ499507) (Maia et al., 2014) and 99 % jackals. There is a need to investigate B. rossi genotypes in susceptible sequence similarity to Hepatozoon sp. described from black-backed and resistant domestic dogs as well as reservoir hosts to elucidate on the jackals in South Africa (MG919973-MG919987) (Penzhorn et al., 2018). gene expression patterns that render susceptibility and resistance to Estimated evolutionary divergence of the observed B. rossi and hosts. An understanding of pathogenic pathways of B. rossi in susceptible H. canis gene sequences and published Babesia and Hepatozoon 18S rRNA and resistant hosts would enhance an understanding of co- of

Table 2 Occurrence of haemoparasites in African wild dogs from the Kruger National Park and domestic dogs from the Onderstepoort Veterinary Academic Hospital, as determined by Reverse Line Blot hybridization assay.

Genus-specific probes Host n Babesia rossi Babesia vogeli Theileria sp. (sable) T/Ba Babesia 1 Babesia 2 E/Ab

African wild dog 52 5 (10 %) 0 0 4 (8%) 44 (85 %) 19 (31 %) 3 (6%) Domestic dog 75 66 (86 %) 1 (1%) 10 (13 %) 69 (92 %) 67 (89 %) 0 0

a Theileria/Babesia. b Ehrlichia/Anaplasma.

4 N. Shabangu et al. Veterinary Parasitology 291 (2021) 109381

Fig. 2. Maximum likelihood tree showing the evolutionary relationships of the represented Babesia 18S rDNA sequences obtained from a domestic dog (D1C3) (GenBank MT683383) and a black-backed jackal (J11C5) (GenBankMT683385) with published sequences. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993). A discrete Gamma distribution was used to model evolutionary rate dif­ ferences among sites (5 categories (+G, parameter = 0.4223)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 36.86 % sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. There were a total of 1526 positions in the finaldataset. Sarcocystis muris, Neospora caninum and Toxoplasma gondii were used as outgroup.

Fig. 3. Maximum likelihood tree showing the evolutionary relationships of the represented Hepatozoon 18S rDNA sequence obtained from an African wild dog (RE52C1) (GenBank MT762141) with published sequences. The evolutionary history was inferred by using the Maximum Likelihood method based on the Hasegawa- Kishino-Yano model (Hasegawa et al., 1985). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.2713)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 44.25 % sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 18 nucleotide sequences. There were a total of 583 positions in the final dataset. Dactylosoma ranarum and Hemolivia mariae were used as outgroup.

5 N. Shabangu et al. Veterinary Parasitology 291 (2021) 109381 host-parasite relationships. Hoffmann, 2013). Their role, if any, in the epidemiology of canine RLB indicated the presence of Theileria sp. (sable) in 13 % of the babesiosis is unknown. Both species inhabit the more arid central domestic dogs. This piroplasm was incriminated in causing clinical signs and western parts of Southern Africa, where ecological conditions are in dogs (Matjila et al., 2008c). Although also reported from dogs in not optimal for off-host survival of H. elliptica. It seems unlikely, there­ Nigeria (Adamu et al., 2014), from cattle (Yusufmia et al., 2010), Afri­ fore, that these foxes serve as reservoir hosts for B. rossi. can buffalo (Syncerus caffer) (Eygelaar et al., 2015) and various ante­ lopes, recent findings suggest that Theileria sp. (sable) is restricted to 5. Conclusions tragelaphine (Pienaar et al., 2020). Yet in Croatia, Theileria capreoli, which infects various cervid species, was found in 14 % of 108 Our study confirmed that B. rossi from domestic dogs and black- grey (Canis lupus), the ancestors of domestic dogs (Beck et al., backed jackals were similar, as had been reported previously. We 2017). The identity of the Theileria spp. affecting dogs in South Africa could not confirmthe presence of B. rossi DNA in African wild dogs from should be clearly established. KNP. Although they can be infected with B. rossi without showing RLB identifiedB. rossi in ca. 10 % of 52 African wild dogs in the KNP, clinical signs, our findings and those reported in the literature from but the identity of the Babesia could not be confirmed by cloning and various other free-ranging African wild dog populations indicate that sequencing, nor by multiplex, real-time PCR. Piroplasms presumed to be prevalence of B. rossi infection is rare. This suggests that African wild Babesia canis sensu lato were seen on blood smears of 2 (6.9 %) of 29 dogs are apparently not important reservoir hosts of B. rossi. African wild dogs in KNP, while 6 (40 %) of 15 African wild dogs were serologically positive (van Heerden et al., 1995). All 46 African wild Ethics declarations dogs examined in KNP were infested with ticks, including H. elliptica, the only known vector of B. rossi (van Heerden et al., 1995). Piroplasms This study was approved by the Ethics Committee of the resembling B. canis s.l. were seen on blood smears from 1 (6.3 %) of 16 University of Pretoria (ref. V031-17), the Animal Use and Care Com­ African wild dogs in National Park, (Peirce et al., mittee of South African National Parks (ref. 013/16), and the Depart­ 1995), but no piroplasms were seen on blood smears from 12 African ment of Agriculture, Forestry and Fisheries (ref. 12/11/1/1/6) in terms wild dogs from KwaZulu-Natal, South Africa (Flacke et al., 2010). In a of Section 20 of the Animal Diseases Act (Act No. 35 of 1984). general survey, no Babesia DNA was detected in blood specimens from 154 African wild dogs from , , and South Africa CRediT authorship contribution statement (Hluhluwe-iMfolozi Park, Madikwe, Pilanesberg and Venetia) (Prager et al., 2012), nor was Babesia DNA was detected in 11 free-ranging Af­ Ntji Shabangu: Writing - review & editing, Investigation, Method­ rican wild dogs from two populations in (Williams et al., 2014). ology, Investigation. Barend L. Penzhorn: Writing - original draft, On RLB, a large number of specimens (44/52; 84.6 %) reacted Writing - review & editing. Marinda C. Oosthuizen: Formal analysis, positively with Babesia-specific probe 1. Partial recombinant sequences Methodology. Ilse Vorster: Formal analysis. O. Louis van Schalkwyk: from two of these specimens indicated 100 % sequence identity to Investigation. Robert F. Harrison-White: Investigation. P. Tshepo Hepatozoon sp. of pale foxes (Vulpes pallida) from North Africa (Maia Matjila: Writing - review & editing, Project administration, Funding et al., 2014). Detecting Hepatozoon spp. when using PCR primers acquisition. designed for Babesia has been reported (Silaghi et al., 2012). Infection with Hepatozoon spp. appears to be frequent in KNP African wild dogs. Declaration of Competing Interest Hepatozoon sp., presumed to be H. canis, were reported from 26 (89.7 %) of 29 blood smears from KNP African wild dogs (van Heerden et al., The authors report no declarations of interest. 1995). High prevalences of Hepatozoon spp. in free-ranging African wild dogs have also been reported elsewhere: in Serengeti, Tanzania, 81.5 % Acknowledgments (13/16) individuals were infected (Peirce et al., 1995) and in Zambia, 55 % (6/11) individuals were infected (Williams et al., 2014). Hepatozoon This research was funded by an AgriSETAgrant to NS and an NRF spp. have also been reported from captive African wild dogs in South Scarce Skills scholarship to NS. The map was drawn by Ms Estelle Africa (Matjila et al., 2008b). Mayhew. Although 6% (3/52) of the African wild dog specimens reacted positively with the Ehrlichia/Anaplasma genus-specific probe on RLB, References none reacted positively with the Ehrlichia canis-specific probe. Experi­ mental infection demonstrated that African wild dogs are susceptible to Adamu, M., Troskie, M., Oshadu, D.O., Malatji, D.P., Penzhorn, B.L., Matjila, P.T., 2014. Occurrence of tick-transmitted pathogens in dogs in Jos, Plateau State, Nigeria. canine ehrlichiosis (van Heerden, 1979). In a previous survey in KNP, Paras. Vectors 7, 119. https://doi.org/10.1186/1756-3305-7-119. none of 29 African wild dogs were seropositive to E. canis (van Heerden Alexander, K.A., Conrad, P.A., Gardner, I.A., Parish, C., Appel, M., Levy, M.G., Lerche, N., et al., 1995). Although ehrlichiosis has been confirmedin domestic dogs Kat, P., 1993. Serologic survey for selected microbial pathogens in African wild dogs (Lycaon pictus) and sympatric domestic dogs (Canis familiaris) in Maasai-Mara, belonging to KNP staff (Neitz and Thomas, 1938), the number of do­ Kenya. J. Zoo Wildl. Med. 24, 140–144. mestic dogs in the KNP is low. Furthermore, Rhipicephalus sanguineus Allsopp, M., Cavalier-Smith, T., De Waal, D.T., Allsopp, B., 1994. Phylogeny and sensu lato, the only known vector of E. canis, has not been reported from evolution of the piroplasms. Parasitology 108, 147–152. African wild dogs in KNP (van Heerden et al., 1995). In the Maasai-Mara Altay, K., Dumanli, N., Aktas, M., 2007. Molecular identification, genetic diversity and distribution of Theileria and Babesia species infecting small ruminants. Vet. Parasitol. Game Reserve, Kenya, all 16 African wild dogs tested were seronegative 147, 161–165. for E. canis, while 23 (16 %) of 148 domestic dogs in the same area were Altschul, S., Gish, W., Miller, W., Myers, E., Lipman, D., 1990. 3.1 Sequence searches- – seropositive (Alexander et al., 1993). In northeastern KwaZulu-Natal, challenges. Mol. Biol. 215, 403 410. Beck, A., Huber, D., Polkinghorne, A., Kurilj, A.G., Benko, V., Mrljak, V., Relji´c, S., South Africa, 20 (83 %) of 24 African wild dogs were seropositive to Kusak, J., Reil, I., Beck, R., 2017. The prevalence and impact of Babesia canis and E. canis (Flacke et al., 2010). In the Laikipia area, northern Kenya, 70 (80 Theileria sp. in free-ranging grey (Canis lupus) populations in Croatia. Par. Vect. %) of 88 African wild dogs and 56 (80 %) of 65 domestic dogs from the 10, 168. Bekker, C.P., De Vos, S., Taoufik, A., Sparagano, O.A., Jongejan, F., 2002. Simultaneous same general area were seropositive to E. canis (Woodroffe et al., 2012). detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia This strongly suggested that domestic dogs serve as reservoir of infection ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization. Vet. for African wild dogs. Microbiol. 89, 223–238. Bosman, A.-M., Venter, E.H., Penzhorn, B.L., 2007. Occurrence of Babesia felis and Two other indigenous canids occur in southern Africa: Babesia leo in various wild felid species and domestic in Southern Africa, based (Vulpes chaama) and bat-eared fox (Otocyon megalotis) (Kingdon and on reverse line blot analysis. Vet. Parasitol. 144, 33–38.

6 N. Shabangu et al. Veterinary Parasitology 291 (2021) 109381

Butler, C., Nijhof, A., Van Der Kolk, J., De Haseth, O., Taoufik, A., Jongejan, F., Oosthuizen, M.C., Zweygarth, E., Collins, N.E., Troskie, M., Penzhorn, B.L., 2008. Houwers, D., 2008. Repeated high dose imidocarb dipropionate treatment did not Identification of a novel Babesia sp. from a sable (Hippotragus Harris, eliminate Babesia caballi from naturally infected horses as determined by PCR- 1838). J. Clin. Microbiol. 46, 2247–2251. reverse line blot hybridization. Vet. Parasitol. 151, 320–322. Oura, C., Bishop, R., Wampande, E., Lubega, G., Tait, A., 2004. Application of a reverse Colly, L.P., Nesbit, J.W., 1992. Fatal acute babesiosis in a juvenile wild dog (Lycaon line blot assay to the study of haemoparasites in cattle in . Int. J. Parasitol. pictus). J. S. Afr. Vet. Assoc. 63, 36–38. 34, 603–613. Eygelaar, D., Jori, F., Mokopasetso, M., Sibeko, K.P., Collins, N.E., Vorster, I., Troskie, M., Oyamada, M., Davoust, B., Boni, M., Dereure, J., Bucheton, B., Hammad, A., Itamoto, K., Oosthuizen, M.C., 2015. Tick-borne haemoparasites in (Syncerus Okuda, M., Inokuma, H., 2005. Detection of Babesia canis rossi, B. canis vogeli, and caffer) from two wildlife areas in Northern Botswana. Par. Vect. 8, 26. Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and Felsenstein, J., 1985. Confidencelimits on phylogenies: an approach using the bootstrap. sequencing methodologies. Clin. Diagn. Lab. Immunol. 12, 1343–1346. Evolution 39, 783–791. Peirce, M., Laurenson, M., Gascoyne, S., 1995. Hepatozoonosis in and wild dogs Flacke, G., Spiering, P., Cooper, D., Gunther, M.S., Robertson, I., Palmer, C., Warren, K., in the Serengeti ecosystem. Afr. J. Ecol. 33, 273–275. 2010. A survey of internal parasites in free-ranging African wild dogs (Lycaon pictus) Peloakgosi-Shikwambani, K., 2018. Analysis of Babesia Rossi Transcriptome in Dogs from KwaZulu-Natal, South Africa. S. Afr. J. Wildl. Res. 40, 176–180. Diagnosed With Canine Babesiosis. PhD Dissertation. University of South Africa, Georges, K., Loria, G., Riili, S., Greco, A., Caracappa, S., Jongejan, F., Sparagano, O., Pretoria. 2001. Detection of haemoparasites in cattle by reverse line blot hybridisation with a Penzhorn, B.L., 2011. Why is Southern African canine babesiosis so virulent? An note on the distribution of ticks in Sicily. Vet. Parasitol. 99, 273–286. evolutionary perspective. Parasit. Vectors 4, 51. https://doi.org/10.1186/1756- Gubbels, J., De Vos, A., Van Der Weide, M., Viseras, J., Schouls, L., De Vries, E., 3305-4-51. Jongejan, F., 1999. Simultaneous detection of bovine theileria and Babesia species by Penzhorn, B.L., 2020. Don’t let sleeping dogs lie: unravelling the identity and reverse line blot hybridization. J. Clin. Microbiol. 37, 1782–1789. of Babesia canis, Babesia rossi and Babesia vogeli. Parasit. Vectors 13, 184. https://doi. Hall, T.A., 1999. BioEdit: A User-friendly Biological Sequence Alignment Editor and org/10.1186/s13071-020-04062-w. Analysis Program for Windows 95/98/NT. Nucleic Acids Symposium Series. Penzhorn, B.L., Vorster, I., Harrison-White, R.F., Oosthuizen, M.C., 2017. Black-backed Information Retrieval Ltd, London, pp. 95–98. jackals (Canis mesomelas) are natural hosts of Babesia rossi, the virulent causative Hasegawa, M., Kishino, H., Yano, T., 1985. Dating the human-ape split by a molecular agent of canine babesiosis in sub-Saharan Africa. Parasit. Vectors 10, 124. https:// clock of mitochondrial DNA. J. Mol. Evol. 22, 160–174. doi.org/10.1186/s13071-017-2057-0. Horak, I.G., Heyne, H., Williams, R., Gallivan, G.J., Spicket, A.M., Bezuidenhout, J.D., Penzhorn, B.L., Netherlands, E.C., Cook, C.A., Smit, N.J., Vorster, I., Harrison-White, R. Estrada-Pena,˜ A., 2018. The Ixodid Ticks (Acari: Ixodidae) of Southern Africa. F., Oosthuizen, M.C., 2018. Occurrence of Hepatozoon canis (Adeleorina: Springer Nature, Cham, Switzerland. Hepatozoidae) and Anaplasma spp. (Rickettsiales: Anaplasmataceae) in black-backed Katoh, K., Standley, D.M., 2013. MAFFT multiple sequence alignment software version 7: jackals (Canis mesomelas) in South Africa. Parasit. Vectors 11, 158. https://doi.org/ improvements in performance and usability. Mol. Biol. Evol. 30, 772–780. 10.1186/s13071-018-2714-y. Kingdon, J., Hoffmann, M., 2013. of Africa. 5. Carnivores, Pangolins, Equids Penzhorn, B.L., Harrison-White, R.F., Stoltsz, W.H., 2020. Completing the cycle: and . Bloomsbury, London, 560 pp.. Haemaphysalis elliptica, the vector of Babesia rossi, is the most prevalent tick infesting Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics black-backed jackals (Canis mesomelas), an indigenous reservoir host of B. rossi in analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. South Africa. Ticks Tick-borne Dis. 11, 101325. https://doi.org/10.1016/j.ttbdis/ Lewis, B.D., Penzhorn, B.L., De Waal, D.T., Lopez´ Rebollar, L.M., 1996. Isolation of a 2019.101325. South African vector-specific strain of Babesia canis. Vet. Parasitol. 63, 9–16. Pienaar, R., Josemans, A., Latif, A.A., Mans, B.J., 2020. The host-specificity of Theileria Liu, Y.H., Wang, L., Xu, T., Guo, X., Li, Y., Yin, T.T., Yang, H.C., Hu, Y., Adeola, A.C., sp. (sable) and Theileria sp. (sablelike) in African Bovidae and detection of novel Sanke, O.J., Otecko, N.O., 2018. Whole-genome sequencing of African dogs provides Theileria in antelope and . Parasitol. 147, 213–224. https://doi.org/10.1017/ insights into adaptations against tropical parasites. Mol. Biol. Evol. 35, 287–298. S003118201900132X. Lounsbury, C.H., 1904. Ticks and malignant jaundice of the dog. J. Comp. Pathol. Ther. Prager, K.C., Mazet, J.A.K., Munson, L., Cleaveland, S., Donnelly, C.A., Dubovi, E.J., 7, 113–129. Gunther, M.S., Lines, R., Mills, G., Davies-Mostert, H.T., McNutt, J.W., Maia, J.P., Alvares, F., Boratynski,´ Z., Brito, J.C., Leite, J.V., Harris, D.J., 2014. Molecular Rasmussen, G., Terio, K., Woodroffe, R., 2012. The effect of protected areas on assessment of Hepatozoon (Apicomplexa: Adeleorina) infections in wild canids and pathogen exposure in endangered African wild dog (Lycaon pictus) populations. Biol. rodents from North Africa, with implications for transmission dynamics across Conserv. 150, 15–22. taxonomic groups. J. Wildl. Dis. 50, 837–848. Schnittger, L., Yin, H., Qi, B., Gubbels, M.J., Beyer, D., Niemann, S., Jongejan, F., Matjila, P.T., Penzhorn, B.L., Bekker, C., Nijhof, A., Jongejan, F., 2004. Confirmation of Ahmed, J.S., 2004. Simultaneous detection and differentiation of Theileria and occurrence of Babesia canis vogeli in domestic dogs in South Africa. Vet. Parasitol. Babesia parasites infecting small ruminants by reverse line blotting. Parasitol. Res. 122, 119–125. 92, 189–196. Matjila, P.T., Leisewitz, A.L., Jongejan, F., Penzhorn, B.L., 2008a. Molecular detection of Silaghi, C., Woll, D., Hamel, D., Pfister, K., Mahling, M., Pfeffer, M., 2012. Babesia spp. tick-borne protozoal and ehrlichial infections in domestic dogs in South Africa. Vet. and Anaplasma phagocytophilum in questing ticks, ticks parasitizing rodents and the Parasitol. 155, 52–157. parasitized rodents - Analyzing the host-pathogen-vector interface in a metropolitan Matjila, P.T., Leisewitz, A.L., Jongejan, F., Bertschinger, H.J., Penzhorn, B.L., 2008b. area. Parasit. Vectors 5, 191. https://doi.org/10.1186/1756-3305-5-191. Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon Takeet, M.I., Oyewusi, A.J., Abakpa, S.A.V., Daramola, O.O., Peters, S.O., 2017. Genetic pictus) in South Africa. Vet. Parasitol. 157, 123–127. diversity among Babesia rossi detected in naturally infected dogs in Abeokuta, Matjila, P.T., Leisewitz, A.L., Oosthuizen, M.C., Jongejan, F., Penzhorn, B.L., 2008c. Nigeria, based on 18S rRNA gene sequences. Acta Parasitol. 62, 192–198. Detection of a Theileria species in dogs in South Africa. Vet. Parasitol. 157, 34–40. Tamura, K., Nei, M., 1993. Estimation of the number of nucleotide substitutions in the Matjila, P.T., Carcy, B., Leisewitz, A.L., Schetters, T., Jongejan, F., Gorenflot, A., control region of mitochondrial DNA in humans and . Mol. Biol. Evol. Penzhorn, B.L., 2009. Preliminary evaluation of the BrEMA1 gene as a tool for 10, 512–526. associating Babesia rossi genotypes and clinical manifestation of canine babesiosis. Theiler, G., Robinson, B.N., 1953. Zoological Survey of the Union of South Africa. Tick J. Clin. Microbiol. 47, 3586–3592. Survey. Part VII – Distribution of Haemaphysalis leachi, the yellow dog tick. Mellanby, R.J., Handel, I.G., Clements, D.N., Bronsvoort, B.Mde C., Lengeling, A., Onderstepoort J. Vet. Res. 26, 83–91. http://hdl.handle.net/2263/58696. Schoeman, J.P., 2011. Breed and sex risk factors for canine babesiosis in South Troskie, M., de Villiers, L., Leisewitz, A., Oosthuizen, M.C., Quan, M., 2019. Development Africa. J. Vet. Int. Med. 25, 1186–1189. and validation of a multiplex, real-time PCR assay for Babesia rossi and Babesia vogeli. Mitchell, P., 2015. Did disease constrain the spread of domestic dogs (Canis familiaris) Ticks Tick-borne Dis. 10, 421–432. https://doi.org/10.1016/j.ttbdis.2018.12.004. into sub-Saharan Africa? Azania: Archaeol. Res. Afr. 50, 92–135 doi: 11080/ Van Heerden, J., 1979. The transmission of canine ehrlichiosis to the wild dog Lycaon 0067270X.2015.1006441. pictus (Temminck) and black-backed jackal Canis mesomelas Schreber. J. S. Afr. Vet. Morters, M.K., Archer, J., Ma, D., Matthee, O., Goddard, A., Leisewitz, A.L., Matjila, P.T., Assoc. 50, 245–248. Wood, J.L.N., Schoeman, J.P., 2020. Long-term follow-up of owned, free-roaming Van Heerden, J., 1980. The transmission of Babesia canis to the wild dog Lycaon pictus dogs in South Africa naturally exposed to Babesia rossi. Int. J. Parasitol. 50, 103–110. (Temminck) and black-backed jackal Canis mesomelas (Schreber). J. S. Afr. Vet. https://doi.org/10.1016/j.ijpara.2019.11.006. Assoc. 51, 119–120. Neitz, W.O., Steyn, H.P., 1947. The transmission of Babesia canis (Piana and Galli- Van Heerden, J., Mills, M.G.L., Van Vuuren, M.J., Kelly, P.J., Dreyer, M.J., 1995. An Valerio, 1895) to the black-backed jackal [Thos mesomelas (Schreber)], with a investigation into the health status and diseases of wild dogs (Lycaon pictus) in the discussion of the classification of the piroplasms of the . J. S. Afr. Vet. Med. Kruger National Park. J. S. Afr. Vet. Assoc. 66, 18–27. Assoc. 18, 1–12. Williams, B.M., Berentsen, A., Shock, B.C., Teixiera, M.R., Dunbar, M.R., Becker, M.S., Neitz, W.O., Thomas, A.D., 1938. Rickettsiosis in the dog. J. S. Afr. Vet. Med. Assoc. 9, Yabsley, M.J., 2014. Prevalence and diversity of Babesia, Hepatozoon, Ehrlichia, and 166–172. Bartonella in wild and domestic carnivores from Zambia, Africa. Parasitol. Res. 113, Nijhof, A., Pillay, V., Steyl, J., Prozesky, L., Stoltsz, W., Lawrence, J., Penzhorn, B.L., 911–918. Jongejan, F., 2005. Molecular characterization of Theileria species associated with Woodroffe, R., Prager, K.C., Munson, L., Conrad, P.A., Dubovi, E.J., Mazet, J.A.K., 2012. mortality in four species of African antelopes. J. Clin. Microbiol. 43, 5907–5911. Contact with domestic dogs increases pathogen exposure in endangered African wild Nuttall, G.H.F., 1910. On haematozoa occurring in wild in Africa. 1. Piroplasma dogs (Lycaon pictus). PLoS One 7, e30099. rossi n. sp. and Haemogegarinia canis adusti n. sp. found in the jackal. Parasitology 3, Yusufmia, S.B.A.S., Collins, N.E., Nkuna, R., Troskie, M., Van Den Bossche, P., 108–112. Penzhorn, B.L., 2010. Occurrence of Theileria parva and other haemoprotozoa in cattle at the edge of Hluhluwe-iMfolozi Park, KwaZulu-Natal, South Africa. J. S. Afr. Vet. Assoc. 81, 45–49.

7