Characterization of bacteria causing bovine mastitis by amplified ribosomal DNA restriction analysis (ARDRA) method

Abstract

Mastitis is an important constraint that accounts for high economic losses in dairy farms worldwide. The prevalence and distribution of mastitis in dairy cows in Bangladesh are well-documented, with many farms facing problems of clinical and subclinical mastitis. This research aimed to assess the rapid detection of bovine subclinical mastitis by the DNA-based ARDRA method and to validate its efficiency in identifying other associated organisms. Traditional biochemical tests such as the California mastitis test (CMT) were used for initial screening, followed by direct DNA extraction from 146 CMT-positive milk samples (n=196) collected from 5 different dairy farms in Chittagong. Using the extracted DNA as a template, amplification of 16s ribosomal DNA by previously described universal primers (27F and 1492R) was successfully achieved. Subsequent restriction digestion (with Hae-III) of PCR amplicons (n=51) revealed characteristic restriction patterns indicating six different groups of organisms in 38 cases where complete digestion was found. Further sequence analysis from corresponding PCR products and bioinformatic analysis revealed the identity of the responsible pathogens. Plasmid profiles were also investigated to develop hypotheses associated with drug resistance patterns. The study shows that, in the absence of sequencing facilities, ARDRA can be a useful approach to efficiently characterize mastitis-causing bacteria, indicating diagnostic implications.