Characterizationofbacteriacausingbovinemastitisbyamplifiedribosomal DNA restriction analysis (ARDRA) method

Abstract

Mastitis is an important constraint that accounts for high economic loss in dairy farms across the world. The prevalence and distribution of mastitis in dairy cows in Bangladesh is well-documented while most of thesefarms are confronted with problems of clinical and subclinical mastitis. This research was conducted to assess the rapid detection of bovine subclinical mastitis by DNA based ARDRA method and to validate its efficiency to identify other associated organisms. Traditional biochemical tests such as California mastitis test (CMT) was 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 template, amplification of 16s ribosomal DNA by previously described universalprimers (27F and 1492R) was successfully achieved. Subsequent restriction digestion (with Hae-III) of PCR amplicons (n=51) revealed characteristic restriction pattern 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 profile also was investigated to develop hypothesis associated with drug resistance patterns. The study shows that, in absence of sequencing facilities, ARDRA can be a useful approach to efficiently characterize the mastitis-causing bacteria indicating diagnostic implications.