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

ABSTRACT: 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.

SUBMITTER: Mtshali MS

PROVIDER: S-EPMC3751629 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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

Mtshali Moses Sibusiso MS Mtshali Phillip Senzo PS

BMC veterinary research 20130808

<h4>Background</h4>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 pro ...[more]

PMID: 23927555

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Project description:BackgroundBabesia bigemina and B. bovis are two economically important hemoparasites affecting both cattle and buffaloes involved in dairy and beef production. In Colombia, although some parasitological and serological studies suggest an endemicity of these pathogens in areas under 1000 m, little is known about its molecular prevalence in different host. The objective of this study was to estimate the prevalence and molecular traits of these parasites in cattle and buffaloes from two Colombian regions.MethodsBetween 2014 and 2016, a three-point longitudinal survey was designed in farms from Caribbean and Orinoquia regions to evaluate the molecular prevalence of B. bigemina and B. bovis using a nested PCR (n-PCR) targeting hypothetical protein (hyp) and rhoptry-associated protein (RAP-1) genes, respectively. A total of 1432 cattle, 152 buffalo and 1439 Rhipicephalus microplus samples were analyzed. Moreover, phylogenetic relationship of isolates was analyzed using the 18S rRNA gene.ResultsA molecular prevalence of 31.6% (24.2% for B. bigemina and 14.4% for B. bovis), 23.6% (6.5% for B. bigemina and 17.7% for B. bovis) and 4.3% (3.5% for B. bigemina and 1.0% for B. bovis) was observed in cattle, buffaloes and Rhipicephalus microplus, respectively. Higher values of infection were observed during the wet season and late wet season; nevertheless, other variables such as age, production type, sex, breed and babesiosis control were also significantly associated with infection. Prevalence analysis showed that B. bovis infection was higher in cattle that coexist with buffaloes, when compared to those which did not. For each species, phylogenetic analyses revealed a high genetic diversity of isolates without clusters related to the isolation source.ConclusionsTo our knowledge, this is the first longitudinal survey that evaluates through molecular methods, the infection of B. bigemina and B. bovis in two important livestock regions from Colombia. This study reveals that the prevalence of infection by Babesia spp., in cattle and buffaloes are modulated by seasonal variations, host factors and vector traits. Our results provide new insights on the epidemiological aspects of infection of Babesia spp., in cattle and buffaloes, which must be taken into consideration when babesiosis control programs are implemented in the study area.

Project description:BackgroundBabesiosis is an important haemoparasitic disease, caused by the infection and subsequent intra-erythrocytic multiplication of protozoa of the genus Babesia that impacts the livestock industry and animal health. The distribution, epidemiology and genetic characterization of B. bigemina, B. bovis, and B. ovata in cattle in China as well as the prevalence of these protozoan agents were assessed.MethodsA total of 646 blood specimens from cattle, dairy cattle and yaks from 14 provinces were collected and tested for the presence of the three Babesia species via a specific nested PCR assay based on the rap-1 and ama-1 genes. The PCR results were confirmed by DNA sequencing. Gene sequences and the genetic characterization were determined for selected positive samples from each sampling area.ResultsOf a total of 646 samples, 134 (20.7 %), 60 (9.3 %) and 10 (1.5 %) were positive for B. bovis, B. bigemina and B. ovata infections, respectively. Mixed infections were found in 7 of 14 provinces; 43 (6.7 %) samples were infected with B. bovis and B. bigemina. Three samples (0.5 %) exhibited a co-infection with B. bovis and B. ovata, and 6 (0.9 %) were infected with all three parasites. The rap-1a gene of B. bovis indicated a high degree of sequence heterogeneity compared with other published rap-1a sequences worldwide and was 85-100 % identical to B. bovis rap-1a sequences in Chinese isolates. B. bigemina rap-1c and B. ovata ama-1 genes were nearly identical, with 97.8-99.3 % and 97.8-99.6 % sequence identity, respectively, in GenBank.ConclusionsPositive rates of B. bovis and B. bigemina infection are somewhat high in China. The B. bovis infection in yaks was first reported. The significant sequence heterogeneity in different variants of the rap-1a gene from Chinese B. bovis isolates might be a great threat to the cattle industry if RAP-1a protein is used as immunological antigen against Babesia infections in China. The data obtained in this study can be used to plan effective control strategies against babesiosis in China.

Project description: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 ?-tubulin (Bb?TUB). Phylogenies were inferred from newly determined sequences using the neighbour-joining approach.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 Bb?TUB 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 Bb?TUB sequences indicated a close phylogenetic relatedness among South African-derived sequences and those of global B. bovis strains.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.

Project description:Tick-borne Babesia bigemina is responsible for acute and potentially lethal hemolytic disease in cattle. The development of genetic manipulation tools necessary to the better understanding of parasite biology is currently limited by the lack of a complete parasite genome and experimental tools such as transfection. Effective promoters, required to regulate expression of transgenes, such as the elongation factor-1 alpha (ef-1?), have been identified in other apicomplexans such as Babesia bovis and Plasmodium falciparum.The B. bigemina ef-1a locus was defined by searching a partial genome library of B. bigemina (Sanger Institute). Presence of an intron in the 5' untranslated region was determined by 5' Rapid Amplification of cDNA Ends (RACE) analysis. Promoter activity was determined by measurement of luciferase expression at several time points after electroporation, efficiency of transfections and normalization of data was determined by quantitative PCR and by the percentage of parasitized erythrocytes.The ef-1? locus contains two identical head to head ef-1? genes separated by a 1.425 kb intergenic (IG) region. Significant sequence divergence in the regions upstream of the inverted repeats on each side of the B. bigemina IG region suggest independent regulation mechanisms for controlling expression of each of the two ef-1? genes. Plasmid constructs containing the 5' and 3' halves of the IG regions controlling the expression of the luciferase gene containing a 3' region of a B. bigemina rap-1a gene, were generated for the testing of luciferase activity in transiently transfected parasites. Both halves of the ef-1? IG region tested showed the ability to promote high level production of luciferase. Moreover, both B. bigemina ef-1? promoters are also active in transiently transfected B. bovis and conversely, a B. bovis ef-1? promoter is active in transiently transfected B. bigemina.Collectively these data demonstrate the existence of two distinct promoters with homologous and heterologous promoter function in B. bigemina and B. bovis which is described for the first time in Babesia species. This study is of significance for development of interspecies stable transfection systems for B. bigemina and for B. bovis.

Project description:BackgroundAmong protozoan parasites in the genus Babesia, Babesia bigemina is endemic and widespread in the East African region while the status of the more pathogenic Babesia bovis remains unclear despite the presence of the tick vector, Rhipicephalus microplus, which transmits both species. Recent studies have confirmed the occurrence of R. microplus in coastal Kenya, and although B. bovis DNA has previously been detected in cattle blood in Kenya, no surveillance has been done to establish its prevalence. This study therefore investigated the occurrence of B. bovis in cattle in Kwale County, Kenya, where R. microplus is present in large numbers.MethodsA species-specific multiplex TaqMan real-time PCR assay targeting two Babesia bovis genes, 18S ribosomal RNA and mitochondrially-encoded cytochrome b and B. bigemina cytochrome b gene was used to screen 506 cattle blood DNA samples collected from Kwale County for presence of Babesia parasite DNA. A sub-set of 29 B. bovis real-time PCR-positive samples were further amplified using a B. bovis-specific spherical body protein-4 (SBP-4) nested PCR and the resulting products sequenced to confirm the presence of B. bovis.ResultsA total of 131 animals (25.8%) were found to have bovine babesiosis based on real-time PCR. Twenty-four SBP4 nucleotide sequences obtained matched to B. bovis with a similarity of 97-100%. Of 131 infected animals, 87 (17.2%) were positive for B. bovis while 70 (13.8%) had B. bigemina and 26 (5.1%) were observed to be co-infected with both Babesia species. A total of 61 animals (12.1%) were found to be infected with B. bovis parasites only, while 44 animals (8.7%) had B. bigemina only. Babesia bovis and B. bigemina infections were detected in the three Kwale sub-counties.ConclusionThese findings reveal high prevalence of pathogenic B. bovis in a Kenyan area cutting across a busy transboundary livestock trade route with neighbouring Tanzania. The Babesia multiplex real-time PCR assay used in this study is specific and can detect and differentiate the two Babesia species and should be used for routine B. bovis surveillance to monitor the spread and establishment of the pathogen in other African countries where B. bigemina is endemic. Moreover, these findings highlight the threat of fatal babesiosis caused by B. bovis, whose endemic status is yet to be established. GRAPHICAL ABTRACT.

Dataset Information

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

Publications

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

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