Bontebok (Damaliscus pygargus pygargus) and blesbok (D. p. phillipsi) are classified as separate sub-species. The blesbok has a widespread distribution throughout South Africa and is listed as least concern by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Bontebok on the other hand is endemic within the Cape Floristic Region of the Western Cape in South Africa and has been listed as near-threatened species on the IUCN Red List of Threatened Species. Bontebok populations experienced a severe bottleneck and were brought back from the brink of extinction in the 1830s. Currently, the subspecies is threatened by hybridisation with blesbok resulting in fertile offspring. To date, molecular investigations using neutral markers have determined that genetic diversity in pure South African bontebok was significantly lower than in pure blesbok. Here, we investigated genetic diversity in bontebok, blesbok and hybrid individuals using microsatellites and an adaptive marker (toll-like receptor two (TLR2)). The study of single nucleotide polymorphisms (SNPs) revealed five mutations in TLR2 in different individuals and subspecies of D. pygargus. This included three non-synonymous and two synonymous mutations. The three amino acid substitution mutations were predicted to have no effect on protein function. Two of the five mutations, one of which resulted in an amino acid substitution, were not present in bontebok. The other three mutations were present to varying frequencies in the three groups. We confirm low adaptive and neutral diversity in bontebok. These mutations provide insights into the genetic diversity and relationships among the two sub-species of D. pygargus and may have implications for their conservation and management.

The Diederik Cuckoo Chrysococcyx caprius is an African species widely distributed south of the Sahara, which migrates seasonally between breeding and nonbreeding sites. It is currently unknown whether the species consists of a single panmictic population or if it is genetically structured. To investigate this, we analysed sequence variation in three mitochondrial and two nuclear gene regions in combination with morphological measurements in specimens from four localities. Phylogenetic relationships were estimated using maximum-likelihood methods and included samples of Klaas’s Cuckoo Chrysococcyx klaas, Red-chested Cuckoo Cuculus solitarius, and African Cuckoo Cuculus gularis. Haplotype networks and analysis of molecular variance were used to characterise the spatial distribution of genetic diversity. A principal component analysis was performed to investigate morphological variation among localities. Molecular analysis identified two divergent mitochondrial lineages, which were found to occur in sympatry in one South African locality (Limpopo Province). The magnitude of divergence between versus within these lineages was low (0.4–1%) yet significant (FST: 0.84–0.88). Lack of apparent phylogeographic structure provides support for the absence of physical barriers to gene flow in this species. The divergent mitochondrial lineages did not differ in morphological measurements. The emergence and persistence of shallow mitochondrial divergence among sympatric lineages in the Diederik Cuckoo could be linked to maternal divergence in host selection of these brood parasites — a hypothesis requiring additional data to be tested.

Several studies have shown that cytotoxic T lymphocytes (CTL) require CD4 + Th1 epitopes to generate strong immune responses to intracellular pathogens. However, not much is known about Ehrlichia ruminantizim epitopes, particularly those that can be considered potential candidates for inclusion in a multi-epitope vaccine. In order to identify CD4(+) Th1 epitopes that induce IFN gamma, a number of proteins previously identified as immunogenic were first screened to determine if they induce cellular immunity in tick infected immune sheep PBMC. Significant IFN-gamma production and other Th1 cytokines were evident for 10 recombinant proteins in all sheep tested. Secondly, peptides (n = 246) derived from the top 10 E. ruminantium vaccine candidate proteins were assayed using enzyme linked immunospot (ELISPOT) assay, quantitative real-time PCR and flow cytometry. Of the 246 peptides, 23 peptides, Erum0660 (p0660-42), Enun1150 (p1150-18, p1150-19), Erum2540 (p2540-6, p2540-16, p2540-19, p2540-20, p2540-21), Erum5420 (p5420-13, p5420-14), Erum7140 (p7140-6, p7140-7, p7140-12, p7140-13, p7140-20), Erum7320 (p7320-8, p7320-9, p7320.21), Erum7350 (p7350-9), Erum7360 (p7360-8), Erum7620 (p7620-2, p7620-12) and Erum8010 (p8010-8) were identified that stimulate the best and different cell mediated immune responses. Amino acid sequences of these peptides except for p7140-12, p7140-13, p7140-20, and p7350-9 were conserved between 13 different local strains. These peptides could efficiently induce memory CD4(+) T cells to rapidly proliferate and significantly increase IFN-gamma production in immune sheep PBMC. The upregulation of pro-inflammatory cytokines, which include, IL-1 alpha, IL-2, IL-12p40, TNF-alpha, IFN-gamma, inducible nitric oxide synthase (iNOS) and granulocyte-macrophage colony stimulating factor (GM-CSF) was also detected. Our results show that these peptides could serve as promising candidates for a multi-epitope vaccine against E. ruminantium.

Toll-like receptors (TLR) are a family of proteins that signal activation of the innate immune response through the recognition of a variety of pathogen molecular compounds. Here, we characterized the complete TLR9 gene in Cape mountain zebra (Equus zebra zebra) from three populations in South Africa and compared sequences to a variety of horse and donkey breeds. Overall, we identified six single nucleotide polymorpHisms (SNPs). A single SNP (G586S) was non-synonymous, whereas the remaining SNPs were synonymous. The G586S alteration was detected in Cape mountain zebra populations with varying frequency. In addition, adaptive diversity was found to be discordant with variation based on neutral markers. The mutation is unique to the Cape mountain zebra when compared to other equid species. The structure of TLR9 is relatively conserved and the resulting amino acid substitution was found to have minimal interaction with active sites in the protein. Future studies can explore the effects of this potentially functional mutation which will contribute to our understanding of genetic diversity within adaptive sites of the Cape mountain zebra genome.

Recently reported substantial genetic diversity within Theileria equi 18S rRNA gene sequences has led to the identification of five genotypes A, B, C, D, and E, complicating molecular and serological diagnosis. In addition, T. haneyi has lately been reported as a species closely related to the T. equi 18S rRNA genotype C (Knowles et al., 2018). Theileria spp. of this group have a monophyletic origin and are therefore referred to as Equus group to distinguish them from the remaining Theileria lineages (Jalovecka et al., 2019). In this study, we report on the development of genotype-specific quantitative real-time PCR assays capable of detecting and distinguishing between each parasite genotype. Alignment of complete 18S rRNA sequences available on GenBank allowed for the design of a single primer pair and five TaqMan minor groove binder (MGB™) probes specific for each genotype (A–E). The assays, evaluated as qPCR simplex and two qPCR multiplex formats (Multiplex EP–ABC and Multiplex EP–DE), were shown to be both efficient and specific in the detection of T. equi genotypes. The developed qPCR assays were used to study (i) the intra-specific diversity of parasite genotypes within horse and zebra, (ii) the inter-specific differences in parasite genotype diversity in horses as compared to zebra, and (iii) the geographic distribution of T. equi 18S rRNA genotypes in South Africa. In addition, (iv) the presence of T. haneyi in South Africa was evaluated. An assessment of 342 equine field samples comprising 149 field horses, 55 racehorses, and 138 wild zebra confirmed the previously reported presence of T. equi 18S rRNA genotypes A, B, C, and D, and absence of genotype E in South African equids. Theileria equi genotypes A, B, C, and D, were detected in zebra, whereas only genotypes A, C and D, could be identified in field horses, and only genotypes A and C in racehorses. Genotypes B and D were the dominant genotypes identified in zebra in South Africa, while horses were predominantly infected with T. equi genotypes A and C. The greater diversity of T. equi genotypes in zebra suggests that it is an ancestral host for this piroplasmid lineage. Importantly, evidence is presented that each identified T. equi genotype segregates independently in each of the three studied equid populations reinforcing the notion that they represent individual separate entities corresponding to species. Preliminary investigations of the relationship between T. equi genotype C infections and Theileria haneyi, suggest that in addition to the five currently known T. equi genotypes, South African equids are also infected with T. haneyi.

The Cape mountain zebra (Equus zebra zebra) is a subspecies of mountain zebra endemic to South Africa. The Cape mountain zebra experienced near extinction in the early 1900's and their numbers have since recovered to more than 4,800 individuals. However, there are still threats to their long-term persistence. A previous study reported that Cape mountain zebra had low genetic diversity in three relict populations and that urgent conservation management actions were needed to mitigate the risk of further loss. As these suggestions went largely unheeded, we undertook the present study, fifteen years later to determine the impact of management on genetic diversity in three key populations. Our results show a substantial loss of heterozygosity across the Cape mountain zebra populations studied. The most severe losses occurred at De Hoop Nature Reserve where expected heterozygosity reduced by 22.85% from 0.385 to 0.297. This is alarming, as the De Hoop Nature Reserve was previously identified as the most genetically diverse population owing to its founders originating from two of the three remaining relict stocks. Furthermore, we observed a complete loss of multiple private alleles from all populations, and a related reduction in genetic structure across the subspecies. These losses could lead to inbreeding depression and reduce the evolutionary potential of the Cape mountain zebra. We recommend immediate implementation of evidence-based genetic management and monitoring to prevent further losses, which could jeopardise the long term survival of Cape mountain zebra, especially in the face of habitat and climate change and emerging diseases.

Translocation of animals in fragmented habitats is an important means of dispersal and gene flow, however, the movement of animals has led to the spread of various diseases globally and wildlife are often the reservoirs of these diseases. Currently, Cape mountain zebra are translocated within South Africa as a management method for augmentation of isolated and fragmented populations. The movement of pathogens due to translocations in local regions have gone largely unchecked, particularly where there may still be isolated regions that can be negatively affected. Equine piroplasmosis is a tick-borne disease caused by Theilaria equi and/or Babesia caballi reported to occur in equids (Bhoora et al., 2010; Zweygarth et al., 2002). Here, the presence of T. equi and B. caballi was detected in 137 clinically healthy Cape mountain zebra from three South African reserves, Mountain Zebra National Park (MZNP), De Hoop Nature Reserve (DHNR) and Karoo National Park (KNP) using the multiplex EP real-time PCR (qPCR) assay. We observed 100% prevalence for T. equi and identified only one animal from MZNP with B. caballi. These results affirm that precautions should be taken prior to founding new populations of Cape mountain zebra and that potential farms and properties adjacent to prospective reserves should be screened for the presence of the organisms in order to mitigate risks of infection to domestic animals.

Since CD8 T cells play an important role in resistance to infection with heartwater, effective vaccines against this disease will likely require identification of antigens that contain CD8+ T cell epitopes responsible for cytotoxic T lymphocyte (CTL) responses. With the use of the fluorescent antigen-transfected target cell (FATT)-CTL assay, IFN-γ ELISPOT and flow cytometry, peptides that induce CTL, proliferation of CD8+T cells and IFN-γ production were identified as possible target antigens for vaccine development. Of particular relevance was the finding that different peptides from different antigens were able to elicit varied cytotoxic activities by immune peripheral blood mononuclear cells (PBMC) from heartwater immune tick-infected sheep. Several peptides derived from Erum0660, Erum2330, Erum2540, Erum2580 and Erum5000 induced CTL in immune sheep PBMC. Peptide Erum2540-6 was the only peptide that induced significant CTL, CD8+CD45RO+ and CD8+IFN-γ+ by PBMC from all three sheep, and Erum2540 and p2540-20 induced the highest % CTL response in all three outbred sheep. These results suggest that these epitopes may be of major importance in heartwater recombinant vaccine development.