Development of species-specific polymerase chain reaction (PCR), real-time PCR and loop mediated isothermal amplification (LAMP) assays for detection of anaplasma marginale strains in South Africa
Khumalo, Zamantungwa Thobeka Happiness
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Anaplasma marginale is a virulent intra-erythrocytic pathogen that causes bovine anaplasmosis, its closely related species, Anaplasma centrale causes mild sickness. The pathogen is transmitted biologically by tick vectors and mechanically through blood contaminated fomites. It has a worldwide distribution extending from tropical to subtropical regions in correlation with the vector distribution. Bovine anaplasmosis is often characterised by progressive anaemia, jaundice, decreased milk production, abortion and a sudden death. The commonly used method for the diagnosis of A. marginale of infected cattle in South Africa is microscopic examination of Giemsa stained blood smears anddetection of antibodies from serum using cELISA. However the diagnostic methods have limitations in cases of low parasitemia and in carrier cattle (microscopy) and they fail to differentiate closely related Anaplasma spp due to antigenic similarity (serology), the detection limitations of the diagnostic methods influenced the aim of this study which is to develop molecular species -specific assays for the detection of A. marginale strains in South Africa, specifically Including conventional polymerase chains reaction, real-time polymerase chain reaction and loop-mediated isothermal amplification assay. Chapter one of this study discusses bovine anaplasmosis and its causative agent A. marginale, the diversity of the strains transmission, distribution, clinical signs, treatment and economic importance of the disease. The first objective of this study was to develop a species specific conventional PCR for detection of A. marginale in cattle in South African regions based on msp1b gene. The conventional PCR primers were designed through visual inspection and were named F3 and 83 primers. In the specificity test, the primers were specific whereby the amplified only A. marginale DNA and did not amplify control DNA's: A. centrale, Babesia bovis, B. bigemina and Ehrlihia rumunantium. The sensitivity of the conventional PCR primers was examined using a 10 ng/ul DNA and the detection limit of the assay was 0.01 ng/ul, The assay was validated on field samples to confirm the infection of the cattle with A. marginale, out of 144 samples, (60%) infection rate was obtained with the newly developed onventional PCR, the homogeneity of the sequences were confirmed with the GenBank, the maximum similarity varied from 94 - 100%. The second objective of this study was to develop a species-specific real-time PCR for detection of A. marginale in cattle in South African regions based on msp1b gene. The real-time PCR primers and probes were designed using Genescript program, one set of primer (Prf 2, PrR2, and Pr82) was chosen to carry out the study as it showed high sensitivity with the detection limit of 0.001 ng/ul .The specific and sensitive TaqMan based real-time PCR was successfully developed for the of A. marginale infections in South Africa. Validation of the assay on field samples showed that the rate of infection was 74% in different sampled provinces of South Africa. The third objective of this study was to develop loop-mediated isothermal amplification for the detection of A. marginale in South African regions based on msp1b gene. The LAMP primers were designed using primer Explorer version 4, the LAMP primers were named LAF3, LA-83, LA-FIP, LA-BIP,LA-LF and LA-LB. The LAMP assay showed positive results with specific amplification, but as far as the validation of the assay false positive results were obtained, troubleshooting involved the addition of additives, changing of primer purification and manufacturers, however the results were not consistent, false positive results were obtained, speculations were that it could be possible contamination of the laboratory resulting in the amplification of control DNA and distilled water. The first three objectives of this study were achieved. The newly developed assays were further compared for specificity, sensitivity and detection performance on field derived samples. The developed assays are specific and sensitive; they form a good tool of diagnosis of bovine anaplasmosis, with each assay having its own unique characteristic over the other, they are sensitive giving a correct determination of the infection status, aiding in compiling of epidemiological information. These assays will aid in understanding the major constraint to develop control measures due to the genetic diversity of A. marginale, and will also help in constructing of phylogenetic tree between strains from South Africa and other countries.