In silico and phylogenetic analyses of partial BbRAP-1, BbCP2, BbSBP-4 and BbßTUB gene sequences of Babesia bovis isolates from cattle in South Africa.

BACKGROUND: Bovine babesiosis is one of the most economically important tick-borne diseases threatening the livestock industry globally including South Africa. This disease is induced by members of Babesia bovis species. Antigenic variations among geographical strains of B. bovis, and these heterogeneities are cited as the mechanism by which parasites evade from host immune system and they hamper the successful development of a single vaccine that could confer absolute protection. Given the economic importance of livestock industry in South Africa, the extent of genetic diversity among field isolates of B. bovis merits extensive investigation. In this study, we genetically characterized partial genes of B. bovis and studied the phylogenetic relationship among B. bovis isolates of South African origin. The genes, which were PCR-amplified from bovine samples collected from different locations across South Africa, coded for rhoptry-associated protein 1 (BbRAP-1), cysteine peptidase 2 (BbCP2), spherical body protein 4 (BbSBP-4) and ß-tubulin (BbßTUB). Phylogenies were inferred from newly determined sequences using the neighbour-joining approach. RESULTS: Nested PCR assays with gene-specific primers indicated that, of the 54 bovine samples tested, 59.3% (32/54; 95% CI = 46.0-71.3%), 27.8% (15/54; 95% CI = 17.6-40.9%), 37.0% (20/54; 95% CI = 25.4-50.4%) and 29.6% (16/54; 95% CI = 19.1-42.8%) possessed BbRAP-1, BbCP2, BbSBP-4 and BbßTUB fragments, respectively. Sequencing of PCR-generated fragments revealed that nucleotide sequences of each of the four genes were highly conserved among the B. bovis isolates examined. Phylogenetic analyses of BbCP2, BbSBP-4 and BbßTUB sequences indicated a close phylogenetic relatedness among South African-derived sequences and those of global B. bovis strains. CONCLUSION: The data reported in this study indicated that there is a high conservation among the genes of B. bovis isolates from cattle in South Africa. These findings give an indication that immunologically important proteins encoded by these genes could potentially be considered for exploitation as viable candidates for inclusion in recombinant subunit vaccines.

Investigation of false positives associated with loop-mediated isothermal amplification assays for detection of Toxoplasma gondii in archived tissue samples of captive felids.

Toxoplasma gondii is a ubiquitous protozoan parasite that infects humans and many different animals, including felids. Many molecular and serologic tests have been developed for detection of T. gondii in a wide range of hosts. Loop-mediated isothermal amplification (LAMP) is a field-friendly technique that lacks the practical drawbacks of other molecular and serologic tests, and LAMP assays have been successfully developed for detection of T. gondii in fresh tissue samples. In the current study, both a previously published and a de-novo designed LAMP assay were compared to a quantitative real-time (q)PCR assay, for the detection of T. gondii in archived formalin-fixed, paraffin-embedded (FFPE) tissue samples from captive wildlife. The LAMP assays produced conflicting results, generating both false positives and false negatives. Furthermore, the LAMP assays were unable to positively identify samples with low levels of parasites as determined by qPCR and histopathology. Therefore, these LAMP assays may not be the most suitable assays for detection of T. gondii in archived FFPE and frozen tissue samples.

Application of crude and recombinant ELISAs and immunochromatographic test for serodiagnosis of animal trypanosomosis in the Umkhanyakude district of KwaZulu-Natal province, South Africa.

A total of 231 serum samples were collected from sheep (n=9), goats (n=99) and cattle (n=123) in northeastern KwaZulu-Natal, South Africa. Trypanosome infection was detected using Trypanosoma brucei brucei crude antigen (TbbCA) and T. congolense crude antigen (TcoCA) ELISA assays. Recombinant antigen (T. evansi GM6 which consisted of 4 repeat domains, TeGM6-4r) ELISA and immunochromatographic test (ICT) were also used. Crude antigen ELISA, TeGM6-4r-ELISA and ICT detected 27.3%, 29% and 19.9% of trypanosome seropositive samples, respectively. Trypanosome infection prevalence in cattle and goats was 35.8-46.3% and 0-9.1%, respectively. Out of 9 sheep serum samples, 2-4 sera (22.2-44.4%) were positive. The detection performance of crude and recombinant antigen ELISAs was relatively similar (K=0.6-0.7); both are recommended for reference diagnosis and large scale epidemiological surveys. There is potential application for ICT in on-site diagnosis, but its sensitivity should be improved.

Nested PCR detection and phylogenetic analysis of Babesia bovis and Babesia bigemina in cattle from Peri-urban localities in Gauteng Province, South Africa.

Babesia bovis and Babesia bigemina are tick-borne hemoparasites causing babesiosis in cattle worldwide. This study was aimed at providing information about the occurrence and geographical distribution of B. bovis and B. bigemina species in cattle from Gauteng province, South Africa. A total of 268 blood samples collected from apparently healthy animals in 14 different peri-urban localities were tested using previously established nested PCR assays for the detection of B. bovis and B. bigemina species-specific genes encoding rhoptry-associated protein 1 (RAP-1) and SpeI-AvaI restriction fragment, respectively. Nested PCR assays revealed that the overall prevalence was 35.5% (95% confidence interval [CI]=± 5.73) and 76.1% (95% CI=± 5.11) for B. bovis and B. bigemina, respectively. PCR results were corroborated by sequencing amplicons of randomly selected samples. The neighbor-joining trees were constructed to study the phylogenetic relationship between B. bovis and B. bigemina sequences of randomly selected isolates. Analysis of phylogram inferred with B. bovis RAP-1 sequences indicated a close relationship between our isolates and GenBank strains. On the other hand, a tree constructed with B. bigemina gp45 sequences revealed a high degree of polymorphism among the B. bigemina isolates investigated in this study. Taken together, the results presented in this work indicate the high incidence of Babesia parasites in cattle from previously uncharacterised peri-urban areas of the Gauteng province. These findings suggest that effective preventative and control measures are essential to curtail the spread of Babesia infections among cattle populations in Gauteng.

Molecular diagnosis and phylogenetic analysis of Babesia bigemina and Babesia bovis hemoparasites from cattle in South Africa.

BACKGROUND: Babesia parasites, mainly Babesia bovis and B. bigemina, are tick-borne hemoparasites inducing bovine babesiosis in cattle globally. The clinical signs of the disease include, among others, anemia, fever and hemoglobinuria. Babesiosis is known to occur in tropical and subtropical regions of the world. In this study, we aim to provide information about the occurrence and phylogenetic relationship of B. bigemina and B. bovis species in cattle from different locations in nine provinces of South Africa. A total of 430 blood samples were randomly collected from apparently healthy cattle. These samples were genetically tested for Babesia parasitic infections using nested PCR assays with species-specific primers. RESULTS: Nested PCR assays with Group I primer sets revealed that the overall prevalence of B. bigemina and B. bovis in all bovine samples tested was 64.7% (95% CI = 60.0-69.0) and 35.1% (95% CI = 30.6-39.8), respectively. Only 117/430 (27.2%) animals had a mixed infection. The highest prevalence of 87.5% (95% CI = 77.2-93.5) for B. bigemina was recorded in the Free State province collection sites (Ficksburg, Philippolis and Botshabelo), while North West collection sites had the highest number of animals infected with B. bovis (65.5%; 95% CI = 52.7-76.4). Phylograms were inferred based on B. bigemina-specific gp45 and B. bovis-specific rap-1 nucleotide sequences obtained with Group II nested PCR primers. Phylogenetic analysis of gp45 sequences revealed significant differences in the genotypes of B. bigemina isolates investigated, including those of strains published in GenBank. On the other hand, a phylogeny based on B. bovis rap-1 sequences indicated a similar trend of clustering among the sequences of B. bovis isolates investigated in this study. CONCLUSION: This study demonstrates the occurrence of Babesia parasites in cattle from different provinces of South Africa. It was also noted that the situation of Babesia parasitic infection in cattle from certain areas within the surveyed provinces had either reached endemic stability or was progressing towards stability.