Molecular characterization of Babesia caballi and Theileria equi, the aetiological agents of equine piroplasmosis, in South Africa

In an attempt to develop quantitative real-time PCR (qPCR) assays for the detection of equine piroplasms, sequence heterogeneity in the V4 hypervariable region of the 18S ribosomal RNA (rRNA) gene sequences within both Theileria equi and Babesia caballi from South Africa was discovered. A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was therefore carried out using horse and zebra samples from different geographical locations around South Africa. We evaluated the ability of a recently developed T. equi-specific qPCR assay in detecting all T. equi 18S rRNA variants identified in South Africa. We further present the first report on the development and application of a TaqMan minor groove binder (MGB™) qPCR assay, targeting the 18S rRNA gene, for the detection of B. caballi infections in equine blood samples. Despite the ability of the 18S rRNA T. equi- and B. caballi-specific qPCR assays to detect all known 18S rRNA gene sequence variants thus far identified in South Africa, the existence of as yet undetected variants in the field cannot be overlooked. Other qPCR assays targeting alternative genes could be developed which, used in conjunction with the 18S rRNA qPCR assays, may provide better confirmation of test results. A T. equi-specific qPCR assay targeting the equi merozoite antigen gene (ema-1) was recently developed for the detection of T. equi parasites in the midgut of Rhipicephalus (Boophilus) microplus nymphs. This assay was not able to detect T. equi in all South African samples that were confirmed positive by other molecular and serological assays. Sequence characterization of the ema-1 gene from South African isolates revealed the existence of variation in the regions where the qPCR primers and probes had been designed. Based on these observations, a conserved region of the ema-1 gene was selected and targeted in the development of an ema-1-specific TaqMan MGB™ qPCR assay, which was shown to have a higher sensitivity than the previously reported ema-1 qPCR assay. The rhoptry-associated protein (rap-1) gene from South African B. caballi isolates was also characterized following the failure of a B. caballi-specific competitive-inhibition enzyme-linked immunosorbent assay (cELISA) to detect B. caballi antibody in the sera of infected horses from South Africa. The genome walking PCR technique was used to amplify the complete rap-1 gene sequence from two South African B. caballi isolates. Significant heterogeneity in the rap-1 gene sequences and in the predicted amino acid sequences was found. Marked amino acid sequence differences in the carboxy-terminal region, and therefore the probable absence of the monoclonal antibody binding site, explains the failure of the cELISA to detect antibody to B. caballi in sera of infected horses in South Africa. This is the first comprehensive molecular study of the parasites that cause equine piroplasmosis in South Africa. Our results add further to the existing knowledge of piroplasmosis worldwide and will be invaluable in the development of further molecular or serological diagnostic assays.Thesis (PhD)--University of Pretoria, 2009.Veterinary Tropical Diseasesunrestricte

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 HluhluweuMfolozi 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 Belgian Directorate-General for Development Co-operation Framework Agreement.https://www.mdpi.com/journal/pathogensam2023Veterinary Tropical Disease

The significance of viral, bacterial and protozoan infections in zebra : a systematic review and meta-analysis of prevalence

TABLE S1 : PRISMA Checklists.TABLE S2 : Checklists based on Migliavaca et al. (2020).TABLE S3 : JBI critical appraisal checklists.TABLE S4 : Clinical signs, influencing factors and genotype/serotypes associated with microbial infections in zebra.Wild equids can harbour multi-host infectious agents that are able to affect other wildlife species, but also domestic animals and humans. The direct and indirect contact between wild and domestic equids is constantly increasing due to global movement of horses and equine products, the depletion of natural areas and climate and land-usage changes, which could result in burdensome epidemics. Nevertheless, currently there is a lack of adequate epidemiological data from zebra. Three electronic databases were searched from 10 to 20 March 2021 for publications reporting bacterial, viral and protozoan infections in zebra. Data for a total of 12 relevant variables were extracted from reviewed papers to undergo a qualitative analysis. Prevalence-reporting studies were subjected to meta-analysis for estimating the pooled prevalence and seroprevalence of microbials in wild zebra populations. We identified 30 pathogen species and the most represented were equine Herpesvirus 1 and 9, Bacillus anthracis, African horse sickness virus and Theileria equi. They were reported from all the three zebra species, both in captivity and wilderness. Pooled seroprevalences were estimated for the equine Orbiviruses AHSV (70%; 95% CI: 35–96%) and EEV (21%; 95% CI: 8–38%) and for the equine α-Herpesviruses EHV-1 (72%; 95% CI: 43–93%), EHV-4 (40%; 95% CI: 0–100%) and EHV-9 (58%; 95% CI: 9-98%), and pooled prevalences for the equine piroplasms T. equi (100%; 95% CI: 94–100%) and B. caballi (8%; 95% CI: 0–28%). Zebra is most probably a component of the reservoir from which AHSV, EHV-1 and T. equi can be directly or indirectly transmitted to horse populations, potentially causing disastrous epidemics. Zebra can also harbour zoonotic pathogens like B. anthracis, Brucella spp., A. phagocytophylum, CCHFV and T. brucei. Other agents like EHV-9, BPV-1 and BPV-2 have the potential to spread from zebra to other wild endangered animal species. We conclude that zebra is an important host of multiple and dangerous pathogens for both animals and humans. Comprehensive studies focused on the prevalence of infectious agents present in zebra populations and the associated risk factors are required.http://www.italian-journal-of-mammalogy.ithj2023Veterinary Tropical Disease

Occurrence of blood-borne tick-transmitted parasites in common tsessebe (Damaliscus lunatus) antelope in Northern Cape Province, South Africa

Blood samples were collected from 71 tsessebes relocated from the deproclaimed Vaalbos National Park to Mokala National Park, South Africa. DNA was extracted from the samples and the reverse line blot (RLB) hybridization technique was used to detect and identify any haemoparasites present. Six samples hybridized to the Theileria/Babesia genus-specific probe, the Theileria genus-specific probe and the Theileria sp. (sable) probe, while 3/6 also hybridized to the Theileria separata probe. Full-length 18S rRNA genes of the Theileria spp. detected were amplified, cloned and sequenced. Two novel Theileria 18S rRNA gene sequences were identified which are phylogenetically very closely related to both Theileria sp. (sable) and T. separata. All animals appeared to be in good health. It seems likely, therefore, that these Theileria spp. do not cause disease under normal circumstances. Nevertheless, care should be taken when translocating wild animals, as introduction of novel piroplasm parasites into new areas could cause clinical disease and losses in naïve wildlife and domestic animals, and new parasite species could become established in areas in which they previously did not occur.National Research Foundation grant UID 44403 to B.L. Penzhorn.http://www.elsevier.com/locate/vetparab201

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.B.S.M.E, National research center, (NRC)http://www.mdpi.com/journal/pathogenspm2022Veterinary Tropical Disease

Molecular characterisation of Eucalyptus grandis PGIP

Coniothyrium zuluense is the causal agent of a serious Eucalyptus stem canker disease in South Africa (Wingfield et al., 1997). Eucalypts are the most important hardwood plantations in the world, and in South Africa these hardwoods occupy approximately 1.5 million hectares of plantation area, an area that is soon to be increased by an additional 600 000 hectares. As exotics, Eucalyptus plantations are constantly exposed to infection by fungal pathogens such as C. zuluense, which by secreting cell-¬wall degrading enzymes contribute to the degradation of plant cell walls and subsequent reduction and in the quality of timber produced. This ultimately affects the South African paper, pulp and timber industries. Selection of resistant clones through traditional breeding methods is the most common method currently employed in overcoming the problem of fungal infection. The genetic manipulation of Eucalyptus trees for enhanced resistance to fungal diseases is an alternative to the time-consuming and tedious approach of conventional breeding. The identification of several antifungal proteins, particularly polygalacturonase-inhibiting proteins (PGIPs) from various plant species including Eucalyptus, lead to the hypothesis that over-expression of these proteins could potentially reduce pathogen attack. However, prior to the expression of PGIPs in plants, isolation and molecular characterization of these genes are required. The aims of this study were therefore (l) to clone and characterize the complete Eucalyptus grandis pgip gene, (2) to transform Nicotiana tabacum (tobacco) plants with the E. grandis pgip gene and (3) to test for inhibition of C. zuluense PGs by PGIPs extracted from transgenic tobacco plants. This forms the first step towards the generation of E. grandis clones that are more disease tolerant. A review of the role of fungal endopolygalacturonases and polygalacturonase¬inhibitors in plant-pathogen interactions are presented in chapter I. Strategies employed to isolate and characterize pgip genes from a range of plant species are highlighted and the importance ofPGIPs in disease resistance is discussed. In chapter 2, the molecular cloning and characterization of the E. grandis pgip gene is discussed. The work presented in this chapter is a follow up on work previously conducted by Chimwamurombe (2001). Previously, a partial Eucalyptus pgip gene sequence was obtained with the use of degenerate oligonucleotide primers. In this study, the complete Eucalyptus pgip gene was obtained through the employment of genome walking strategies. Transformation of Nicotiana tabacum cv LA Burley plants with the Eucalyptus pgip gene and the molecular characterization of transgenic tobacco plants is discussed in chapter 3. The transformation and expression of foreign genes in tobacco plants is a well-established protocol, making tobacco the most appropriate candidate plant for assessing the functionality of the plant transformation construct. The production of endopolygalacturonases from virulent C. zuluense isolates and the subsequent PGIP assays conducted to determine levels of PG inhibition are included in this chapter. This thesis consists of three independent chapters representing studies on the molecular characterization of an E. grandis pgip gene and focusing on the potential for inhibition of PGs produced by C. zuluense by Eucalyptus PGIP extracted from transgenic tobacco plants. Repetition of certain aspects in the individual chapters has been unavoidable and the thesis is presented following a uniform style.Dissertation (MSc)--University of Pretoria, 2003.GeneticsUnrestricte

Putative clinical piroplasmosis in a Burchell's zebra (Equus quagga burchelli)

A 10-year-old tame zebra gelding was presented after suffering from lethargy, nervousness, reported anaemia and icterus as well as a decreased appetite. These symptoms were seen over some months, with changing severity. The animal was immobilised, treated, and blood specimens were submitted for haematology and biochemistry. This report describes molecular characterisation of Theileria equi recovered from this animal, as well as the clinical findings, treatment and historical relevance of piroplasmosis in zebra in southern Africa