Phylogenetic exploration and haemato-biochemical significance of Anaplasma marginale infection in water buffalo

Buffalo blood samples (8,352) were screened microscopically during 2017-2020 for Anaplasma species and 104 samples were found positive. Later on, same samples were confirmed positive using genomic DNA PCR technique. For efficiency of PCR technique, 335 blood samples were randomly chosen. Microscopy revealed Anaplasma species in erythrocytes of 1.19% (4 samples) of the total blood samples (n=335). Initially PCR assay was standardized targeting 16S rRNA for detection of genus Anaplasma with expected amplicon size of 781 bp. Thereafter, msp1 beta gene was targeted for species level identification of Anaplasma (A. marginate) with expected amplicon size of 407 bp. A total of 14 samples out of 335 samples (4.17%) were positive by PCR. Randomly, two confirmed positive samples were sent for bidirectional sequencing and the sequencing results were submitted in GenBank (MW269530, MW269531), to further confirm the Anaplasma marginale infection in buffalo. The study showed higher specificity and sensitivity of PCR test in comparison to blood smear examination. Erythron of infected animals showed significantly low level of mean Hb, PCV, RBC counts, MCH, MCHC and high level of mean MCV, indicating regenerative hypochromic macrocytic anaemia. Infected animals had a significantly low mean level of total protein, albumin and globulin and significantly high level of ALT, AST, total bilirubin, direct and indirect bilirubin. The current study provides indication of the association between A. matginale infection and probable liver damage or hepatic fatty degeneration. Nevertheless, advanced studies are warranted to explain the precise pathophysiological mechanism of this association.