Investigation on the status of Johne's disease based on agar gel immunodiffusion, ziehl-neelsen staining and nested PCR approach in two cattle farm

Abstract

Background and Methods: Paratuberculosis is a chronic disease of ruminant, caused by Mycobacterium avium subsp. paratuberculosis (Map), clinically infected animals produce high level of antibodies in blood and shed detectable amount of Map organisms in feces. Several serological and molecular tests are utilized for detection of antibodies or DNA of the organism in clinical samples. Present study indicates the status of paratuberculosis in two distinct cattle farms with different organizational set-ups viz. organized and unorganized. We used agar gel immunodiffusion (AGID) assay for the detection of antibodies in blood. Ziehl-Neelsen (ZN) staining of fecal smears was done to observe acid-fast bacilli and Nested PCR targeted to IS900 and f57 sequences, was performed to confirm the pathogen. Results: Sera samples of cattle, from organized farm, did not show any visible precipitating band with AGID assay. However, fecal smears of few cattle (3.57%) were positive for acid-fast bacilli. When confirmed with nested PCR, only one fecal sample (0.71%) was found positive for Map. In case of unorganized farm, a large number of cattle (38.75%) showed precipitating antibodies with AGID assay and the percentage of fecal smears that showed acid-fast bacilli was 26.62%. Nevertheless, fecal samples containing Map DNA was confirmed in 14.37% of fecal sample by nested PCR. Conclusions: An organized farm, with better hygiene and management practices, showed lesser occurrence of paratuberculosis in cattle in comparison to unorganized farm. Not all AGID assays positive cattle might be an efficient shedder of Map and mare detection of acid-fast bacilli in fecal smears did not always indicate the presence of Map organism. Cattle infected with JD were mostly in the age group of six years and above.