| Summary: | 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 and
detection 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 B3 primers. In the specificity test, the primers were specific whereby the amplified only
A. marginale DNA and did not amplify control DNAâs: 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
conventional 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 PrB2) 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-B3, 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.
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