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Equine theileriosis, a tick-transmitted disease caused by the hemoprotozoan parasites Theileria equi and Theileria haneyi, affects equids throughout tropical and subtropical regions of the world. It is a significant regulatory concern in non-endemic countries, where testing for equine theileriosis is required prior to horse import to prevent parasite entry. Within endemic areas, infection causes significant morbidity and mortality, leading to economic losses. No vaccine for equine theileriosis is available, and current drug treatment protocols are inconsistent and associated with significant side effects. Recent work has revealed substantial genetic variability among equine theileriosis organisms, and analysis of ribosomal DNA from affected animals around the world indicates that the organisms can be grouped into five distinct clades. As these diverse parasites are capable of infecting a wide range of both tick and mammalian hosts, movement of different equine Theileria species between endemic countries, and eventually into non-endemic countries, is a significant concern. Furthermore, the substantial genetic variability of these organisms will likely render currently utilized importation diagnostic tests unable to detect all equine Theileria spp. To this end, more complete characterization of these diverse parasites is critical to the continued global control of equine theileriosis. This review discusses current knowledge of equine Theileria spp. in this context, and highlights new opportunities and challenges for workers in this field.

Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and β- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

Theileria equi (T. equi) is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and β- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

The persistence of African swine fever virus (ASFV) in endemic areas, with small-scale but regular outbreaks in domestic pigs, is not well understood. ASFV has not been detected using conventional diagnosis in these pigs or adjacent populations of resistant African wild pigs, that could act as potential carriers during the outbreaks. However, such data are crucial for the design of evidence-based control strategies. We conducted cross-sectional (1107 pigs) and longitudinal (100 pigs) monitoring of ASFV prevalence in local pigs in Kenya and Uganda. The horizontal survey revealed no evidence of ASFV in the serum or blood using either conventional or real-time PCR. One pig consistently tested positive using ELISA, but negative using PCR assays on blood. Interestingly, the isotype of the antibodies from this animal were strongly IgA biased relative to control domestic pigs and warthogs, suggesting a role for mucosal immunity. The tissues from this pig were positive by PCR following post-mortem. Internal organ tissues of 44 healthy pigs (28 sentinel pigs and 16 pigs from slaughter slabs) were tested with four different PCR assays; 15.9 % were positive for ASFV suggesting that healthy pigs carrying ASFV exist in the swine population in the study area. P72 and p54 genotyping of ASFV revealed very limited diversity: all were classified in genotype IX at both loci, as were virtually all viruses causing recent ASF outbreaks in the region. Our study suggests that carrier pigs may play a role in ASF disease outbreaks, although the triggers for outbreaks remain unclear and require further investigation. This study significantly increases scientific knowledge of the epidemiology of ASF in the field in Africa, which will contribute to the design of effective surveillance and control strategies.

This study was supported by the Ministry of Economy and Competitiveness (MINECO) from the Spanish Government (grant number AGL2016-78160-C2-1-R). The authors are also grateful to the Centres de Recerca de Catalunya (CERCA) Programme and Global Alliance for Research on African swine fever (GARA). The authors thank Frederic Paboeuf and Audrey Fougeroux for providing SPF and domestic pig samples. Most of the microorganisms living in a symbiotic relationship in different animal body sites (microbiota) reside in the gastrointestinal tract (GIT). Several studies have shown that the microbiota is involved in host susceptibilities to pathogens. The fecal microbiota of domestic and wild suids was analyzed. Bacterial communities were determined from feces obtained from domestic pigs (Sus scrofa) raised under different conditions: specific-pathogen-free (SPF) pigs and domestic pigs from the same bred, and indigenous domestic pigs from a backyard farm in Kenya. Secondly, the fecal microbiota composition of the African swine fever (ASF) resistant warthogs (Phacochoerus africanus) from Africa and a European zoo was determined. African swine fever (ASF) is a devastating disease for domestic pigs. African animals showed the highest microbial diversity while the SPF pigs the lowest. Analysis of the core microbiota from warthogs (resistant to ASF) and pigs (susceptible to ASF) showed 45 shared OTUs, while 6 OTUs were exclusively present in resistant animals. These six OTUs were members of the Moraxellaceae family, Pseudomonadales order and Paludibacter, Anaeroplasma, Petrimonas, and Moraxella genera. Further characterization of these microbial communities should be performed to determine the potential involvement in ASF resistance.

Control of Theileria equi, the primary cause of equine theileriosis, is largely reliant on acaracide use and chemosterilization with imidocarb dipropionate (ID). However, it is currently unknown if ID is effective against Theileria haneyi, the recently identified second causative agent of equine theileriosis, or if the drug maintains effectiveness against T. equi in the presence of T. haneyi co-infection. The purpose of this study was to address these questions using ID treatment of the following three groups of horses: (1) five T. haneyi infected horses, (2) three T. haneyi-T. equi infected horses, and (3) three T. equi-T. haneyi infected horses. Clearance was first evaluated using nPCR for each Theileria sp. on peripheral blood samples. ID failed to clear T. haneyi in all three groups of horses, and failed to clear T. equi in two of three horses in group two. For definitive confirmation of infection status, horses in groups two and three underwent splenectomy post-treatment. The T. equi-nPCR-positive horses in group two developed severe clinical signs and were euthanized. Remaining horses exhibited moderate signs consistent with T. haneyi. Our results demonstrate that ID therapy lacks efficacy against T. haneyi, and T. haneyi-T. equi co-infection may interfere with ID clearance of T. equi.

The genetic diversity of African pigs, whether domestic or wild has not been widely studied and there is very limited published information available. Available data suggests that African domestic pigs originate from different domestication centers as opposed to international commercial breeds. We evaluated two domestic pig populations in Western Kenya, in order to characterize the genetic diversity, breed composition and admixture of the pigs in an area known to be endemic for African swine fever (ASF). One of the reasons for characterizing these specific populations is the fact that a proportion of indigenous pigs have tested ASF virus (ASFv) positive but do not present with clinical symptoms of disease indicating some form of tolerance to infection. Pigs were genotyped using either the porcine SNP60 or SNP80 chip. Village pigs were sourced from Busia and Homabay counties in Kenya. Because bush pigs (Potamochoerus larvatus) and warthogs (Phacochoerus spp.) are known to be tolerant to ASFv infection (exhibiting no clinical symptoms despite infection), they were included in the study to assess whether domestic pigs have similar genomic signatures. Additionally, samples representing European wild boar and international commercial breeds were included as references, given their potential contribution to the genetic make-up of the target domestic populations. The data indicate that village pigs in Busia are a non-homogenous admixed population with significant introgression of genes from international commercial breeds. Pigs from Homabay by contrast, represent a homogenous population with a "local indigenous' composition that is distinct from the international breeds, and clusters more closely with the European wild boar than African wild pigs. Interestingly, village pigs from Busia that tested negative by PCR for ASFv genotype IX, had significantly higher local ancestry (>54%) compared to those testing positive, which contained more commercial breed gene introgression. This may have implication for breed selection and utilization in ASF endemic areas. A genome wide scan detected several regions under preferential selection with signatures for pigs from Busia and Homabay being very distinct. Additionally, there was no similarity in specific genes under selection between the wild pigs and domestic pigs despite having some broad areas under similar selection signatures. These results provide a basis to explore possible genetic determinants underlying tolerance to infection by ASFv genotypes and suggests multiple pathways for genetically mediated ASFv tolerance given the diversity of selection signatures observed among the populations studied.

The central variable region (CVR) within the B602L gene of the African swine fever virus (ASFV) is highly polymorphic within the 23 ASFV genotypes defined by sequencing of the C-terminal end of the p72 locus. Sequencing the p54 gene further discriminates ASFV genotypes that are conserved at the p72 locus. Variation in the thymidine kinase locus is a novel additional tool for ASFV genotyping whose application for this purpose is described for the first time herein. We evaluated genetic variation at these four polymorphic loci in 39 ASFV isolates obtained from outbreaks in Kenya and a region of Eastern Uganda between 2011 and 2013. Analysis of the p72 and p54 loci revealed high genetic conservation among these isolates; all clustered within p72 genotype IX and were similar to isolates associated with earlier outbreaks in East Africa. The thymidine kinase gene of the Kenyan isolates in this study were distinct relative to Southern African isolates and synonymous substitutions were observed among viruses from central Kenya. Analysis of the CVR within the B602L gene revealed two previously unknown polymorphisms that were restricted to Western Kenya and Eastern Uganda. A novel variant was revealed within CVR subgroup XXIV and a novel CVR subgroup XXIVa that contains tetrameric repeat F which has previously only been associated with p72 genotype I, was also identified for the first time in East Africa. Phylogeographic analysis of isolates based on CVR polymorphisms revealed rapid evolution and dissemination of variants present within ASFV genotype IX in East Africa. Electronic supplementary material The online version of this article (10.1007/s11262-017-1521-4) contains supplementary material, which is available to authorized users.

Equid herpesviruses types 1 (EHV-1) and 9 (EHV-9) are unusual among herpesviruses in that they lack strong host specificity, and the full extent of their host range remains unclear. The virus establishes latency for long periods and can be reactivated and shed, resulting in clinical disease in susceptible species. A sensitive and specific peptide-based enzyme-linked immunosorbent assay was developed to study the seroprevalence of both viruses in a broad range of species among both wild and captive populations. We used this assay to study the seroprevalences of EHV-1 and EHV-9 in a natural population of the highly endangered Grévy's zebra ( Equus grevyi) in Kenya, sampled during a 4-yr period (2012-15). The results were compared with those obtained from captive Grévy's zebras from a previous study. The wild population had a significantly higher seroprevalence of EHV-9 compared with the captive population, suggesting that captivity might reduce exposure to this serotype. In contrast, the seroprevalences of EHV-1 between captive and wild groups was not significantly different. The seroprevalence of EHV-9 was not significantly higher than EHV-1 in zebras within the wild Kenyan population.