Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

Abstract

The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900-PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

Detection of Mycobacterium paratuberculosis in raw cow’s milk using polymerase chain reaction (PCR) technique

“…In addition, the zoonotic potential of M. paratuberculosis via consumption of contaminated milk and milk products is of public health importance due to frequent isolation of M. paratuberculosis from Crohn's disease patients (22)(23)(24)(25). The PCR used in our study offer sensitive and rapid method for detection and confirmation of the presence of M. paratuberculosis in clinical samples (6,7,11). Unlike, culture method that requires special media and experienced personnel, PCR might be conducted in few hours in the equipped laboratory, while culture method might need several months to consider negative results (6,7,25,26).…”

“…In this study, M. paratuberculosis was detected in (6%) 3/50 of tested cow's milk, which indicates direct shedding of the pathogen in the raw milk. Since paratuberculosis is a chronic disease and clinical signs may only appear after years of infection, the secretion of M. paratuberculosis in milk is considered as main risk factor for transmission and spreading of the disease to calves (6,11,20,21). In addition, the zoonotic potential of M. paratuberculosis via consumption of contaminated milk and milk products is of public health importance due to frequent isolation of M. paratuberculosis from Crohn's disease patients (22)(23)(24)(25).…”

“…Unlike, culture method that requires special media and experienced personnel, PCR might be conducted in few hours in the equipped laboratory, while culture method might need several months to consider negative results (6,7,25,26). The PCR technique allowed the detection of fewer M. paratuberculosis cells in 2 mL of milk, making this procedure is rapid, sensitive and cost-effective for the diagnosis of clinical and subclinical paratuberculosis (11,27). In a study targeting cattle population in south of Iraq 29/81 (35.8%) were positive for M. paratuberculosis using PCR from buffy coat of seropositive cows detected by ELISA, and this highlighted the importance of PCR as a confirmatory tool (28).…”

“…However, this method is cumbersome and timeconsuming, and cultured samples may need up to 3 months to consider negative (7). Therefore, the diagnosis is mainly relied on serological (8-10) and molecular methods (6,7,11,12). Paratuberculosis has been confirmed previously in several studies in Iraq.…”

BIO-INCIDENCE OF Mycobacterium avium SUBSPECIES paratuberculosis IN THE PASTEURIZED LIQUID MILK, FLAVOURED MILK AND MILK POWDER COMMERCIALLY SOLD BY LEADING MARKET BRANDS IN INDIA

“…Sample positive in any one of the six tests was considered as positive. The reasons for such high bio-incidence of MAP in milk samples was due to high secretion of MAP by domestic livestock in milk, colostrum and feces, in-ability of pasteurization temperature to inactivate MAP, transmission of MAP from one generation to next generation by semen (Khol et al, 2010), during pregnancy (Whittington & Windsor, 2009) and through milk (Gilardoni et al, 2016;Grant et al, 2017) and colostrums (Pithua et al, 2011), absence of 'indigenous diagnostic kits, high cost of imported kits, poor sensitivity of imported kits, lack of priority for control, absence of control strategies, vaccines etc. (Bastida & Juste, 2011).…”