Genetic diversity in Babesia bovis from southern Africa and estimation of B. bovis infection levels in cattle using an optimised quantitative PCR assay

DATA AVAILABILITY : Data will be made available on request.Please read abstract in the article.https://www.elsevier.com/locate/ttbdisam2024Veterinary Tropical DiseasesSDG-03:Good heatlh and well-bein

Comparison of three nucleic acid-based tests for detecting Anaplasma marginale and Anaplasma centrale in cattle

Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1β gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1β sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp.The National Research Foundation (NRF) of South Africa (grant number 81840 awarded to Dr Nicola Collins) and Technology Innovation Agency (TIA), Tshwane Animal Health Cluster (grant TAHC12-00037 awarded to Professor Marinda Oosthuizen).http://www.ojvr.org/am2017GeneticsVeterinary Tropical Disease

Diagnosis of tick-borne diseases in cattle in Bushbuckridge Mpumalanga South Africa and identification of Theileria parva carriers

The Mnisi community is in the north-eastern corner of the Bushbuckridge Municipal Area, Mpumalanga Province, South Africa. This community is located at the livestock/wildlife interface sharing borders with several game reserves, and livestock are likely to be exposed to diseases with a wildlife reservoir, such as Corridor disease. Known tick vectors of important diseases such as Corridor disease, redwater, heartwater and anaplasmosis are present in the area. Although the farmers frequently dip their cattle in acaricide-filled dip tanks to control the tick burden, tick-borne diseases (TBDs) are still a major problem. This study was undertaken to determine if the symptoms of cattle in poor health in the Mnisi community could be attributed to TBDs. Corridor disease has previously been identified in cattle in the Mnisi community. Recent experimental studies have shown that T. parva DNA can be detected in infected cattle that survive the disease in the field. An additional aim of the study was therefore to identify T. parva carrier cattle in the area, and to search for evidence of selection of cattle-adapted T. parva parasites in carrier cattle. The study was conducted from July 2012 to June 2013. During the study period, samples from clinically sick cattle suspected of TBDs were collected to determine the cause of their symptoms. Blood smears from the clinically sick cattle were analysed using light microscopy while some cases were subjected to histopathology and T. parva-specific quantitative real-time polymerase chain reaction (qPCR). DNA extracted from blood samples and in some cases tissue samples collected from clinically sick cattle (n=137) was tested for the presence of haemoparasite DNA using the reverse line blot (RLB) hybridization assay. To identify T. parva carrier cattle, records from Hluvukani Animal Clinic and Bushbuckridge State Veterinary office were scrutinized to identify herds that may have been exposed to T. parva infection. Blood samples (n=670) were collected from herds that had recorded Corridor disease cases in the past three years, as well as herds that may have shared grazing with buffalo from the Kruger National Park and surrounding private game reserves. The indirect fluorescent antibody test (IFAT) was used to check for T. parva antibodies. Seropositive herds were revisited, as well as herds that had confirmed Corridor disease cases during the study period, and blood samples were collected (n=432). DNA extracted from these samples was screened for the presence of T. parva DNA using the T. parva-specific qPCR. In an attempt to find evidence of selection of cattle-adapted T. parva, the p67, p104 and PIM parasite genes were amplified from qPCR positive samples, and the amplicons were cloned and sequenced. Out of the 137 clinical disease cases examined from the study area, 24 cases of TBDs were diagnosed, of which 19 were Theileria related. The RLB hybridization assay confirmed the presence of tick-borne haemoparasites in the Mnisi community: 89 of the 137 clinical disease cases (65.0%) were found positive for one or more haemoparasite (Theileria, Babesia, Anaplasma and/or Ehrlichia species) while 48 (35.0%) were negative or below the detectable limit of the test. IFAT results indicated that there is a high seroprevalence of theileriosis (63.6%) in the Mnisi community area, but this may be due to cross reactions with other Theileria parasites known to be present (e.g. T. taurotragi). Fewer cattle (13.4%) were seropositive at the highest titre tested (160), and these are most likely to be associated with T. parva. In DNA extracted from blood samples from these seropositive herds, the T. parva-specific qPCR detected T. parva in eleven samples (2.6%). Eight of the eleven cattle were re-sampled six months later, but only one was still qPCR positive. All of the p104 and PIM sequences and two of the three p67 sequences were characteristic of buffalo-type T. parva alleles previously identified, implying that the T. parva infections in the cattle were transmitted directly from buffalo to cattle, and providing no evidence of selection of cattle-type alleles in the carrier animals. The study revealed that TBDs are a problem in the Mnisi community and surrounding area. Most important of the TBDs identified was Corridor disease, a notifiable disease in South Africa, which was the cause of most deaths among the cattle that were sampled. There was no evidence for the selection of cattle-derived T. parva alleles in any of the samples from T. parva carrier cattle, but a p67 sequence obtained from a clinical case was closely related to previously-identified alleles from cattle-derived isolates. Theileria parva DNA could only be detected in carrier cattle for a limited time post-exposure, suggesting that the infection will be cleared in infected animals before larvae or nymphs are available to pick up infections the following season. However, one bovine was still qPCR positive six months post-exposure, albeit with a very high Cp value (indicating a very low parasitaemia). The selection of T. parva parasites in cattle from the diverse T. parva population in African buffalo, therefore, remains a concern in the Mnisi community area, and at other livestock/wildlife interfaces in South Africa, but the risk is probably very low.Dissertation (MSc)--University of Pretoria, 2015.tm2016Veterinary Tropical DiseasesMS