Molecular genotyping of Babesia caballi.

Babesia caballi is an intra-erythrocytic parasite causing equine piroplasmosis. Three B. caballi genotypes (A, B, and C) have been identified based on the 18 S rRNA and rhoptry-associated protein (rap-1) gene sequences. These variant parasite genotypes compromise the diagnostic utility of the WOAH-recommended serological assays in declaring horses free of equine piroplasmosis. Although a gene encoding a spherical body protein 4 (sbp4) has recently been identified as a potential antigen for the serological detection of B. caballi, the ability of this antigen to detect the different geographical strains has not been determined. The molecular distinction between variant B. caballi genotypes is limited and therefore we developed molecular typing assays for the rapid detection and quantification of distinct parasite genotypes. Field samples were screened for the presence of B. caballi using an established multiplex equine piroplasmosis qPCR assay. In this study, B. caballi genotype A was not detected in any field samples screened. However, phylogenetic analysis of the amplified sbp4 and 18 S rRNA genes confirmed the phylogenetic groupings of the South African isolates into either B. caballi genotypes B or C. A multiple sequence alignment of the sbp4 gene sequences obtained in this study together with the published sbp4 sequences representing B. caballi genotype A, were used to identify conserved regions within the gene to design three primer pairs and three genotype-specific TaqMan minor-groove binder (MGB™) probes. The qPCR assays were shown to be specific and efficient in the detection and differentiation between B. caballi genotypes A, B, and C and could be used as a diagnostic assay to prevent the unintentional spread of variant B. caballi genotypes globally.

Distribution and Prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African Ticks: A Systematic Review and Meta-Analysis.

In Africa, ticks continue to be a major hindrance to the improvement of the livestock industry due to tick-borne pathogens that include Anaplasma, Ehrlichia, Rickettsia and Coxiella species. A systemic review and meta-analysis were conducted here and highlighted the distribution and prevalence of these tick-borne pathogens in African ticks. Relevant publications were searched in five electronic databases and selected using inclusion/exclusion criteria, resulting in 138 and 78 papers included in the qualitative and quantitative analysis, respectively. Most of the studies focused on Rickettsia africae (38 studies), followed by Ehrlichia ruminantium (27 studies), Coxiella burnetii (20 studies) and Anaplasma marginale (17 studies). A meta-analysis of proportions was performed using the random-effects model. The highest prevalence was obtained for Rickettsia spp. (18.39%; 95% CI: 14.23-22.85%), R. africae (13.47%; 95% CI: 2.76-28.69%), R. conorii (11.28%; 95% CI: 1.77-25.89%), A. marginale (12.75%; 95% CI: 4.06-24.35%), E. ruminantium (6.37%; 95% CI: 3.97-9.16%) and E. canis (4.3%; 95% CI: 0.04-12.66%). The prevalence of C. burnetii was low (0%; 95% CI: 0-0.25%), with higher prevalence for Coxiella spp. (27.02%; 95% CI: 10.83-46.03%) and Coxiella-like endosymbionts (70.47%; 95% CI: 27-99.82%). The effect of the tick genera, tick species, country and other variables were identified and highlighted the epidemiology of Rhipicephalus ticks in the heartwater; affinity of each Rickettsia species for different tick genera; dominant distribution of A. marginale, R. africae and Coxiella-like endosymbionts in ticks and a low distribution of C. burnetii in African hard ticks.

Development of Cathepsin L-like Real-Time PCR Assays for the Detection of African Animal Trypanosomosis (AAT) in South Africa.

African animal trypanosomosis (AAT), is an infectious parasitic disease of wildlife and livestock caused by multiple species and strains of Trypanosoma. In South Africa, it is restricted to northern KwaZulu-Natal (NKZN) and caused by Trypanosoma congolense and Trypanosoma vivax. A cross-sectional study was done to determine AAT prevalence in 384 goat samples and identify trypanosome species circulating in 60 cattle at dip tanks that are on the interface with the Hluhluwe-uMfolozi game reserve in NKZN. Both cattle and goat samples were analyzed using the buffy coat technique (BCT) and a polymerase chain reaction (PCR) assay targeting the internal transcribed spacer 1 (ITS) region. Cattle samples were further analyzed using an ITS quantitative real-time PCR (qPCR) assays designed for the detection of T. congolense, T. vivax, and T. brucei. None of the goat samples tested positive for Trypanosoma infections. The ITS qPCR assay detected Trypanosoma DNA in 30% of the cattle samples, while only 8.3% were positive with the ITS PCR and 11.7% were positive using BCT. Quantitative real-time PCR assays were designed to amplify a 98 bp, 137 bp, and 116 bp fragment of the cathepsin L-like (CATL) gene from T. brucei, T. theileri, and T. congolense, respectively. Each assay was shown to be efficient (>94%) and specific (109 to 102/101 copies/reaction) in the detection of Trypanosoma species. The CATL qPCR assays detected T. congolense and T. theileri infections in 33.3% of the cattle samples. The CATL qPCR assays also detected T. congolense infections in goats (23.1%) that were neither detected by BCT nor the ITS PCR. The CATL qPCR assays provide an additional, sensitive, and specific tool for Trypanosoma diagnostics. The presence of trypanosomes in goats suggests they might be potential reservoirs of infections to other livestock.

The Piroplasmida Babesia, Cytauxzoon, and Theileria in farm and companion animals: species compilation, molecular phylogeny, and evolutionary insights.

The order Piroplasmida, including the genera Babesia, Cytauxzoon, and Theileria is often referred to as piroplasmids and comprises of dixenous hemoprotozoans transmitted by ticks to a mammalian or avian host. Although piroplasmid infections are usually asymptomatic in wild animals, in domestic animals, they cause serious or life-threatening consequences resulting in fatalities. Piroplasmids are particularly notorious for the enormous economic loss they cause worldwide in livestock production, the restrictions they pose on horse trade, and the negative health impact they have on dogs and cats. Furthermore, an increasing number of reported human babesiosis cases are of growing concern. Considerable international research and epidemiological studies are done to identify existing parasite species, reveal their phylogenetic relationships, and develop improved or new drugs and vaccines to mitigate their impact. In this review, we present a compilation of all piroplasmid species, isolates, and species complexes that infect domestic mammals and which have been well defined by molecular phylogenetic markers. Altogether, 57 taxonomic piroplasmid entities were compiled, comprising of 43 piroplasmid species, 12 well-defined isolates awaiting formal species description, and two species complexes that possibly mask additional species. The extrapolation of the finding of at least 57 piroplasmid species in only six domestic mammalian groups (cattle, sheep, goat, horse, dog, and cat) allows us to predict that a substantially higher number of piroplasmid parasites than vertebrate host species exist. Accordingly, the infection of a vertebrate host species by multiple piroplasmid species from the same and/or different phylogenetic lineages is commonly observed. Molecular phylogeny using 18S rRNA genes of piroplasmids infecting domestic mammals results in the formation of six clades, which emerge due to an anthropocentric research scope, but not due to a possibly assumed biological priority position. Scrutinizing the topology of inferred trees reveals stunning insights into some evolutionary patterns exhibited by this intriguing group of parasites. Contrary to expectations, diversification of parasite species appears to be dominated by host-parasite cospeciation (Fahrenholz's rule), and, except for piroplasmids that segregate into Clade VI, host switching is rarely observed. When only domestic mammalian hosts are taken into account, Babesia sensu lato (s.l.) parasites of Clades I and II infect only dogs and cats, respectively, Cytauxzoon spp. placed into Clade III only infect cats, Theileria placed into Clade IV exclusively infect horses, wheras Theileria sensu stricto (s.s.) of Clade V infects only cattle and small ruminants. In contrast, Babesia s.s. parasites of Clade VI infect all farm and companion animal species. We outline how the unique ability of transovarial transmission of Babesia s.s. piroplasmids of Clade VI facilitates species diversification by host switching to other host vertebrate species. Finally, a deterioration of sequence fidelity in databases is observed which will likely lead to an increased risk of artifactual research in this area. Possible measures to reverse and/or avoid this threat are discussed.

Rapid Detection of Equine Piroplasms Using Multiplex PCR and First Genetic Characterization of Theileria haneyi in Egypt.

Equine Piroplasmosis (EP) is an infectious disease caused by the hemoprotozoan parasites Theileria equi, Babesia caballi, and the recently identified species T. haneyi. Hereby, we used a multiplex PCR (mPCR) targeting the 18S rRNA gene of T. equi and B. caballi for the simultaneous detection of EP in Egyptian equids and examined the presence of T. haneyi infections in Egypt. Blood samples from 155 equids (79 horses and 76 donkeys) collected from different governorates of Egypt were examined by mPCR and PCR targeting T. hayeni. The mPCR method revealed a prevalence of T. equi of 20.3% in horses and of 13.1% in donkeys and a prevalence of B. caballi of 1.2% in horses. B. caballi was not detected in donkeys in the current study. The mPCR method also detected coinfections with both species (2.5% and 1.3% in horses and donkeys, respectively). Additionally, we report the presence of T. haneyi in Egypt for the first time in 53.1% of the horse and 38.1% of the donkey tested samples. Coinfection with T. haneyi and T. equi was found in 13.5% of the samples, while infection with the three EP species was found in 1.9% of the samples.

First report of cystic echinococcosis in rhinos: A fertile infection of Echinococcus equinus in a Southern white rhinoceros (Ceratotherium simum simum) of Kruger National Park, South Africa.

Despite being a parasitic disease known since ancient times, some epidemiological aspects of cystic echinococcosis (CE) remain unclear. Many studies describe its prevalence and genotyping in populations of domestic animals and livestock, but data regarding wildlife are often scarce and incomplete. The available literature suggests that CE has never been reported in African rhinos. Considering the fragile conservation status of these species due to continued poaching, this study tries to clarify some neglected epidemiological aspects. In February 2020, an adult female of the Southern white rhinoceros, Ceratotherium simum simum (Burchell, 1817), was killed by poachers. The subsequent necropsy performed by the state veterinary team revealed the presence of seven cysts within the pulmonary tissue (four cysts in the right medio-caudal lobe and three cysts in the left medio-caudal lobe) with a diameter of between 1.5 and 2.3 cm. Given the state of decomposition of the carcass, only two of these were suitable for microscopic examination. Specimens were examined under 10x and 40x microscopic magnification for the confirmation of fertility of the cysts, based on the presence of numerous protoscoleces in different stages of maturation. A histopathological examination was also performed to describe the relationship between parasite and host tissue reaction. Cyst samples were subjected to PCR. The primers successfully amplified the expected fragments of the cox-1 and the nad-1 gene from the isolated genomic DNA, revealing high sequence identity with published sequences of Echinococcus equinus Williams & Sweatman, 1963 isolate G4 and E. equinus isolate SLG5-G4.

Molecular genotyping and epidemiology of equine piroplasmids in South Africa.

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.

Translocation a potential corridor for equine piroplasms in Cape mountain zebra (Equus zebra zebra).

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.

Multiplex hydrolysis-probe assay for the simultaneous detection of Theileria equi and Babesia caballi infections in equids.

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 heamoprotozoan 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 × 10-4 % parasitized erythrocytes (PE) for T. equi and 2.8 × 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.

Genetic diversity of piroplasms in plains zebra (Equus quagga burchellii) and Cape mountain zebra (Equus zebra zebra) in South Africa.

Seventy EDTA blood samples collected from plains zebra (Equus quagga burchellii) and Cape mountain zebra (Equus zebra zebra) were screened for the presence of piroplasm parasite DNA using quantitative T. equi-specific and B. caballi-specific TaqMan real-time PCR (qPCR) tests. T. equi parasite DNA was detected in 60 samples, 19 of which were also positive for B. caballi. Approximately 1480bp of the piroplasm 18S rRNA gene was amplified and sequenced from 17 samples, while the V4 hypervariable region of the 18S rRNA gene was amplified, cloned and sequenced from 31 samples. BLASTN analysis revealed that all of the sequences obtained were most similar to T. equi genotypes and not B. caballi genotypes. Although Babesia parasites were present in some of these samples, as indicated by qPCR, the parasitaemia may have been too low to allow detection by cloning of PCR products from a mixed infection. Sequence analyses of both the full-length and the V4 hypervariable region of the T. equi 18S rRNA gene revealed the existence of 13 new T. equi sequences from zebra, confirming the existence of sequence heterogeneity in the rRNA genes of the parasites that cause equine piroplasmosis, and further suggesting that there may be additional, as yet unidentified, T. equi and B. caballi 18S rRNA sequences present in the horse and zebra populations in South Africa. The occurrence of previously unrecognized sequence variation could pose a potential problem in the implementation of diagnostic tests targeting the 18S rRNA gene.

Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGB assay for the detection of T. equi.

Although a quantitative real-time PCR assay (qPCR) assay for the detection of Theileria equi has been developed and evaluated, it is possible that additional, as yet undetected 18S rRNA gene sequence variants may exist. A qPCR assay targeting a different gene, used in conjunction with the T. equi 18S rRNA qPCR assay, could assist in the detection of all T. equi genotypes in field samples. A T. equi ema-1-specific qPCR (Ueti et al., 2003) was tested on 107 South African field samples, 90 of which tested positive for T. equi antibody using the immuno-fluorescent antibody test (IFAT). The qPCR assay performed poorly, as T. equi was detected in only 67 of the 90 IFAT-positive field samples at quantification cycle (C(q)) values ranging from 27 to 39.95. Furthermore, a high C(q) value of 36.18 was obtained from DNA extracted from a South African in vitro-cultured T. equi WL isolate [1.38% parasitized erythrocytes (PE)] when a low C(q) value (indicative of a high T. equi DNA concentration) was expected. Approximately 600 bp of the ema-1 gene from 38 South African samples were sequenced and BLASTN analysis confirmed all sequences to be merozoite surface protein genes, with an identity of 87.1-100% to previously published T. equi ema-1 gene sequences. Alignment of the sequences revealed extensive sequence variations in the target regions of the primers and probes (Ueti et al., 2003), explaining the poor performance of the qPCR assay. Based on these observations, we developed a new TaqMan minor-groove binder (MGB) probe-based qPCR assay, targeting a more conserved region of the ema-1 gene. This assay was shown to be efficient and specific, and the detection limit, defined as the concentration at which 95% of T. equi-positive samples are detected, was determined to be 1.4 x 10(-4)% PE. The two ema-1 assays were compared by testing 41 South African field samples in parallel. The results suggested that the new assay was more sensitive than the original assay, as T. equi was detected in more samples and at lower C(q) values when the new assay was used. Phylogenetic analyses of the 18S rRNA gene sequences and ema-1 amino acid sequences from the same samples showed inconsistencies between the clades, indicating that the T. equi 18S rRNA genetic groups previously identified in South Africa may not represent distinct T. equi lineages. It is possible that the different T. equi ema-1 genotypes could be related to antigenic variability and pathogenicity and may be associated with clinical differences in equine piroplasmosis cases, but this remains to be elucidated.

Sequence heterogeneity in the gene encoding the rhoptry-associated protein-1 (RAP-1) of Babesia caballi isolates from South Africa.

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) developed for the detection of antibody specific for Babesia caballi was used to test sera collected from 1237 South African horses. None of these samples tested positive using the cELISA, although 63 samples tested positive for B. caballi antibody using the indirect fluorescent antibody test (IFAT). We therefore characterized the rap-1 gene that codes for the antigen (rhoptry-associated protein, RAP-1) used in the cELISA, from South African B. caballi isolates. Three sets of primers were designed to amplify the complete gene and flanking regions (approximately 1800 bp), but only one set of primers yielded PCR products, and we were only able to amplify a region at the 5' end of the gene (615 bp) from ten South African B. caballiin vitro-cultured isolates. Sequence data from seven of these were obtained. The sequences showed between 79% and 81% identity to B. caballirap-1 gene sequences that have been reported in the literature (accession numbers: AF092736 and AB017700). The GenomeWalker Universal kit (Clonetech) was used to amplify the regions flanking the 615bp B. caballirap-1 fragment from two South African isolates. Amplified products were cloned into the pGEM-T Easy vector and sequenced. The complete rap-1 gene sequence, comprising a single open reading frame of 1479 bp that encodes a protein consisting of 493 amino acids, was obtained from the two South African isolates. This sequence data was used to redesign the amplification primers and rap-1 homologues were obtained from a further eight isolates. BLASTP analysis indicated an amino acid identity of between 57.9% and 65.1% to the two RAP-1 protein sequences, AF092736 and AB017700, with most differences occurring at the carboxy-terminus. The amino acid sequence differences probably explain why it was not possible to detect B. caballi antibody in IFAT positive sera from South Africa using the cELISA. Redesigning the current cELISA using a conserved epitope of the RAP-1 antigen, or a more conserved protein as the target antigen, may overcome this problem.

Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa.

A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The B. caballi TaqMan MGB qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported Theileria equi-specific qPCR assay and showed that it was able to detect the 12 T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either T. equi or B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating T. equi and B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected T. equi parasite DNA in 73% of the samples, but none of the samples were positive for B. caballi, although 19 T. equi-positive samples also hybridized to the Babesia genus-specific probe. This could indicate a mixed T. equi and B. caballi infection in these samples, with either the B. caballi parasitaemia at a level below the detection limit of the B. caballi RLB probe, or the occurrence of a novel Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The B. caballi TaqMan MGB qPCR assay was able to detect B. caballi parasite DNA in 78% of the samples. The T. equi-specific qPCR assay could positively detect T. equi DNA in 80% of the samples. These results suggest that the qPCR assays are more sensitive than the RLB assay for the detection of T. equi and B. caballi infections in field samples.

Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa.

A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was conducted using samples collected from horses and zebra from different geographical locations in South Africa. A total of 488 samples were tested for the presence of Theileria equi and/or Babesia caballi using the reverse line blot hybridization assay. Ten percent of the samples hybridized to the Theileria/Babesia genus-specific probe and not to the B. caballi or T. equi species-specific probes, suggesting the presence of a novel species or genotype. The small subunit of rRNA gene (18S; approximately 1600bp) was amplified and sequenced from 33 of these 488 samples. Sequences were compared with published sequences from the public sequence databases. Twelve distinct T. equi and six B. caballi 18S rRNA sequences were identified. Alignments demonstrated extensive sequence variation in the V4 hypervariable region of the 18S rRNA gene within T. equi. Sequence variation was also found in B. caballi 18S rRNA genes, although there was less variation than observed for T. equi. Phylogenetic analysis based on 18S rRNA gene sequences revealed three T. equi clades and two B. caballi clades in South Africa. The extent of sequence heterogeneity detected within T. equi and B. caballi 18S rRNA genes was unexpected since concerted evolution is thought to maintain homogeneity within repeated gene families, including rRNA genes, in eukaryotes. The findings reported here show that careful examination of variants of the 18S rRNA gene of T. equi and B. caballi is required prior to the development of molecular diagnostic tests to detect these parasites in horses. Species-specific probes must be in designed in regions of the gene that are both conserved within and unique to each species.