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1.
Parasit Vectors ; 11(1): 5, 2018 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-29298712

RESUMO

BACKGROUND: Only a few studies have examined the presence of Anaplasma marginale and Anaplasma centrale in South Africa, and no studies have comprehensively examined these species across the whole country. To undertake this country-wide study we adapted a duplex quantitative real-time PCR (qPCR) assay for use in South Africa but found that one of the genes on which the assay was based was variable. Therefore, we sequenced a variety of field samples and tested the assay on the variants detected. We used the assay to screen 517 cattle samples sourced from all nine provinces of South Africa, and subsequently examined A. marginale positive samples for msp1α genotype to gauge strain diversity. RESULTS: Although the A. marginale msp1ß gene is variable, the qPCR functions at an acceptable efficiency. The A. centrale groEL gene was not variable within the qPCR assay region. Of the cattle samples screened using the assay, 57% and 17% were found to be positive for A. marginale and A. centrale, respectively. Approximately 15% of the cattle were co-infected. Msp1α genotyping revealed 36 novel repeat sequences. Together with data from previous studies, we analysed the Msp1a repeats from South Africa where a total of 99 repeats have been described that can be attributed to 190 msp1α genotypes. While 22% of these repeats are also found in other countries, only two South African genotypes are also found in other countries; otherwise, the genotypes are unique to South Africa. CONCLUSIONS: Anaplasma marginale was prevalent in the Western Cape, KwaZulu-Natal and Mpumalanga and absent in the Northern Cape. Anaplasma centrale was prevalent in the Western Cape and KwaZulu-Natal and absent in the Northern Cape and Eastern Cape. None of the cattle in the study were known to be vaccinated with A. centrale, so finding positive cattle indicates that this organism appears to be naturally circulating in cattle. A diverse population of A. marginale strains are found in South Africa, with some msp1α genotypes widely distributed across the country, and others appearing only once in one province. This diversity should be taken into account in future vaccine development studies.


Assuntos
Anaplasma centrale/classificação , Anaplasma marginale/classificação , Anaplasmose/microbiologia , Doenças dos Bovinos/microbiologia , Coinfecção/veterinária , Variação Genética , Genótipo , Anaplasma centrale/genética , Anaplasma centrale/isolamento & purificação , Anaplasma marginale/genética , Anaplasma marginale/isolamento & purificação , Anaplasmose/epidemiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Bovinos , Doenças dos Bovinos/epidemiologia , Chaperonina 60/genética , Coinfecção/epidemiologia , Coinfecção/microbiologia , Epidemiologia Molecular , Reação em Cadeia da Polimerase Multiplex , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , África do Sul/epidemiologia
2.
Onderstepoort J Vet Res ; 84(1): e1-e9, 2017 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-28155283

RESUMO

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 msp1ß gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1ß 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.


Assuntos
Anaplasma centrale , Anaplasma marginale , Anaplasmose/diagnóstico , Doenças dos Bovinos/diagnóstico , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Anaplasma centrale/genética , Anaplasma marginale/genética , Anaplasmose/microbiologia , Animais , Bovinos , Doenças dos Bovinos/microbiologia , DNA Bacteriano/genética , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade
3.
Ticks Tick Borne Dis ; 5(6): 624-31, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25024014

RESUMO

Bovine anaplasmosis caused by infection of cattle with Anaplasma marginale has been considered to be endemic in South Africa, an assumption based primarily on the distribution of the tick vectors of A. marginale and serological studies on the prevalence of anaplasmosis in Limpopo, Free State, and North West. However, molecular evidence of the distribution of anaplasmosis has only been reported in the Free State province. In order to establish effective control measures for anaplasmosis, epidemiological surveys are needed to define the prevalence and distribution of A. marginale in South Africa. In addition, a proposed control strategy for anaplasmosis is the development of an A. marginale major surface protein 1a (MSP1a)-based vaccine. Nevertheless, regional variations of this gene would need to be characterized prior to vaccine development for South Africa. The objectives of the present study were therefore to conduct a national survey of the prevalence of A. marginale in South Africa, followed by an evaluation of the diversity and evolution of msp1a in South African strains of A. marginale. To accomplish these objectives, species-specific PCR was used to test 250 blood samples from cattle collected from all South African provinces (including 26 districts and municipalities), except the Free State province where similar studies were reported previously. The prevalence of A. marginale ranged from 65% to 100%, except in Northern Cape province where A. marginale was not detected. A correlation was found between the prevalence and genetic diversity of A. marginale MSP1a. Additionally, the genetic diversity of the A. marginale MSP1a was found to evolve under negative and positive selection, and 23 new tandem repeats in South Africa were shown to have evolved from the extant tandem repeat 4. Despite the MSP1a genetic variability, some types of tandem repeats were found to be conserved among the A. marginale strains, and low-variable peptides in MSP1a tandem repeats were subsequently identified. The results of this research confirmed that anaplasmosis is endemic in South Africa. The results of the molecular characterization of the MSP1a can then be used as the basis for development of new and novel vaccines for anaplasmosis control in South Africa.


Assuntos
Anaplasma marginale/genética , Anaplasmose/epidemiologia , Doenças dos Bovinos/microbiologia , Variação Genética , Doenças Transmitidas por Carrapatos/microbiologia , Sequência de Aminoácidos , Anaplasma marginale/classificação , Anaplasma marginale/isolamento & purificação , Anaplasmose/microbiologia , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Evolução Molecular , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , África do Sul/epidemiologia , Doenças Transmitidas por Carrapatos/epidemiologia , Carrapatos/microbiologia
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