Theileria parva are intracellular protozoal parasites responsible for three disease syndromes in cattle, namely East Coast fever (ECF), Corridor disease (CD) and Zimbabwean theileriosis. The increase in reports of CD outbreaks in recent years has raised questions about the probability of adaptation of buffalo-derived T. parva strains in cattle herds adjacent to game reserves. A cross-sectional study was conducted from March 2016 to December 2018 to investigate the extent of occurrence of T. parva infections in cattle in the CD-controlled area of KwaZulu-Natal Province. Blood samples were collected from 1137 cattle from 14 herds and analysed by quantitative real-time PCR (qPCR) and indirect fluorescent antibody test (IFAT) to determine the prevalence of T. parva. A total of 484 samples from 4 of the 14 herds were further tested on qPCR for the presence of T. taurotragi infections. The data were analysed using descriptive statistics and a chi-square test was used to assess association between variables. The overall prevalence of T. parva was 1.3% (95%CI:1-2%) and 19.9% (95%CI:17-22%) on qPCR and IFAT, respectively. The qPCR positive samples were detected in March and May while IFAT positive samples were detected in all seasons sampled, with higher numbers during summer months. The Pearson Chi-squared test showed that T. parva prevalence rates based on both qPCR and IFAT were positively associated with herds with previous history of CD outbreaks (chi(2) = 8.594, p = 0.003; chi(2) = 69.513, p < 0.001, respectively). The overall prevalence of T. taurotragi was 39.4% (95% CI: 35-44%) with the herd-level prevalence ranging between 35.0% and 43.4%. Possible cross-reaction of T. parva IFAT to T. taurotragi was detected on few samples, however, there was no significant association between T. taurotragi infections and IFAT positivity (chi(2) = 0.829, p = 0.363). Results from this study demonstrated the extent of occurrence of subclinical carriers and the level of exposure to T. parva infections in cattle populations at a livestock/game interface area of KwaZulu-Natal Province. The molecular and seroprevalence rates were low when compared with other areas where cattle-adapted T. parva infections are endemic. The adaptation of buffalo-derived T. parva in cattle population resulting in cattle-cattle transmissions seem to be unlikely under the current epidemiological state.

Theileria parva is a protozoan parasite transmitted by the brown-eared ticks, Rhipicephalus appendiculatus and Rhipicephalus zambeziensis. Buffaloes are the parasite’s ancestral host, with cattle being the most recent host. The parasite has two transmission modes namely, cattle–cattle and buffalo–cattle transmission. Cattle–cattle T. parva transmission causes East Coast fever (ECF) and January disease syndromes. Buffalo to cattle transmission causes Corridor disease. Knowledge on the genetic diversity of South African T. parva populations will assist in determining its origin, evolution and identify any cattle–cattle transmitted strains. To achieve this, genomic DNA of blood and in vitro culture material infected with South African isolates (8160, 8301, 8200, 9620, 9656, 9679, Johnston, KNP2, HL3, KNP102, 9574, and 9581) were extracted and paired-end whole genome sequencing using Illumina HiSeq 2500 was performed. East and southern African sample data (Chitongo Z2, Katete B2, Kiambu Z464/C12, Mandali Z22H10, Entebbe, Nyakizu, Katumba, Buffalo LAWR, and Buffalo Z5E5) was also added for comparative purposes. Data was analyzed using BWA and SAMtools variant calling with the T. parva Muguga genome sequence used as a reference. Buffalo-derived strains had higher genetic diversity, with twice the number of variants compared to cattle-derived strains, confirming that buffaloes are ancestral reservoir hosts of T. parva. Host specific SNPs, however, could not be identified among the selected 74 gene sequences. Phylogenetically, strains tended to cluster by host with South African buffalo-derived strains clustering with buffalo-derived strains. Among the buffalo-derived strains, South African strains were genetically divergent from other buffalo-derived strains indicating possible geographic sub-structuring. Geographic sub- structuring was also observed within South Africa strains. The knowledge generated from this study indicates that to date, ECF is not circulating in buffalo from South Africa. It also shows that T. parva has historically been present in buffalo from South Africa before the introduction of ECF and was not introduced into buffalo during the ECF epidemic.

Heartwater is an economically important tick-borne disease of ruminants in Africa. The current commercial vaccine uses live Ehrlichia ruminantium from blood of infected sheep, requires antibiotic treatment during infection, needs to be administered intravenously and does not protect against all South African isolates. An attenuated tissue culture vaccine not requiring antibiotic treatment and effective against different field strains in small groups of goats and sheep was reported previously. The objective of the present study was to test safety and efficacy of this vaccine administered by intramuscular (i.m.) inoculation in larger groups of sheep, Angora goats and cattle. Animals were vaccinated via intravenous (i.v.) and i.m. routes and received E. ruminantium homologous challenge by feeding of infected ticks or by i.v. inoculation of infected blood. For vaccine titration in sheep and goats, the optimum safe and efficacious dose was determined using 2 ml equivalent of 102–105 culture-derived live elementary bodies (EBs). Similarly, the vaccine was titrated in cattle using 5 ml containing 105–107 EBs. Seventy percent of i.v. vaccinated and 9.7% of i.m. vaccinated Angora goats receiving 105 EBs, developed severe reactions to vaccination and were treated. These treated animals and the remaining 90.3% of i.m.- vaccinated goats showed 100% protection against i.v. or tick challenge. Sheep and Angora goats vaccinated i.m. with 104 EBs had no vaccination reactions and were fully protected against i.v. or tick challenge. Similarly, vaccinated cattle (dose 106 EBs) did not react to vaccine inoculation and were fully protected against i.v. or tick homologous challenge. Control non-vaccinated animals reacted severely to challenge and required oxytetracycline treatment. This successfully demonstrated that Angora goats, sheep and cattle can be safely vaccinated with the attenuated E. ruminantium Welgevonden vaccine via the i.m. route, with no clinical reactions to vaccination and 100% protection against virulent i.v. and homologous tick challenge.

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.

Quantitative real-time PCR assays previously developed for the detection of Theileria equi and Babesia caballi, were combined in a single multiplex TaqMan qPCR platform for the simultaneous detection of both heamo-protozoan parasites in equids. The multiplex equine piroplasmosis (M-EP) qPCR assay was shown to be efficient and specific. The detection limit was determined to be 1.4 x 10(-4) % parasitized erythrocytes (PE) for T. equi and 2.8 x 10(-4) % PE for B. caballi. The effect of differential DNA concentrations on the outcome of the M-EP qPCR for each target species was also investigated. The data demonstrated that the assay could reliably detect both targets, over a range of at least 1000-fold difference in target concentrations, without loss of sensitivity. The assay was subsequently evaluated on 243 field samples collected from areas where limited tick control strategies were implemented. The IFAT detected circulating T. equi and B. caballi antibodies in 100% and 92% of the samples, respectively. The M-EP qPCR assay detected T. equi parasite DNA in 98% of the samples, while B. caballi could only be detected in 6% of the samples tested, confirming that B. caballi infections generally occur at extremely low parasitaemias that rarely exceed 1%. The developed M-EP qPCR assay therefore serves as a reliable tool for the rapid diagnosis and epidemiological survey of equine piroplasmosis.