A PCR for differentiate between Anaplasma marginale and A. centrale

ABSTRACT

Background: Anaplasma marginale ssp. centrale (A. centrale) exhibits low pathogenicity and therefore is used as a live vaccine against bovine anaplasmosis in several countries. During production of the vaccine, accidental contamination with Anaplasma marginale (A. marginale) is a risk that can jeopardize the entire batch of vaccine. Due to limitation of microscopic examination to detect low levels of parasitaemia, the present study aims to standardize a polymerase chain reaction assay using primers for the msp4 gene of Anaplasma sp. for detecting and differentiating with greater sensitivity and specificity the species of A. centrale and A. marginale in blood samples from experimentally infected cattle.Materials, Methods & Results: The DNA extraction was performed from frozen blood. Erythrocytes infected with known A. centrale, A. marginale served as positive control and the erytrocytes infected with Babesia bovis and Babesia bigemina served as the negative control polymerase chain reaction. PCR was standardized from annealing temperature variations of the primers, magnesium chloride concentration, amounts concentration of primers and DNA concentration of rickettsiae. By PCR method, it was analyzed the DNA from blood samples of 13 cattle positive to A. marginale by microscopic examination from smear stained with Giemsa. The PCR assay was specific for A. centrale and A. marginale, presented 100% identity without presenting cross-reactivity with other bovine hemoparasites. The detection limits of the PCR were 0.25 pg and 0.125 pg of DNA for detection of A. centrale and A. marginale DNA respectively.Discussion: A. marginale is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia being considered the main agent of bovine anaplasmosis.[...]