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Location: Infectious Bacterial Diseases Research

Title: Short communication: Application of an N-acetyl-L-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subsp. paratuberculosis from milk of naturally infected cows

item Bradner, L - Iowa State University
item Robbe-austerman, S - Animal And Plant Health Inspection Service (APHIS)
item Beitz, D - Iowa State University
item Stabel, Judith

Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/2/2014
Publication Date: 6/1/2014
Publication URL:
Citation: Bradner, L., Robbe-Austerman, S., Beitz, D.C., Stabel, J.R. 2014. Short communication: Application of an N-acetyl-L-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subsp. paratuberculosis from milk of naturally infected cows. Journal of Dairy Science. 97(6):3694-3699.

Interpretive Summary: Johne's disease is a chronic, debilitating intestinal disorder in cattle characterized by diarrhea, reduced feed intake, weight loss and death. Cattle usually become infected as young calves by ingesting feces containing the causative bacteria. However, symptoms of disease do not usually present themselves until the animals reach 3 to 5 years of age or even older. During this time the animal is infected and may be shedding the organism in its feces without showing any clinical signs of disease. In addition to reduced production by these animals through reduced milk production, they also present a potential infective threat to the rest of the herd. Shedding of this bacterium into the milk of infected dams is one mode of transmission to young calves. However, there is very little data to determine how much shedding occurs. This is due to the difficulty in culturing the bacterium from milk and colostrum. The present study evaluates the best method for decontamination and culturing of milk for optimal recovery of the bacterium. These results are critical for diagnostic laboratories so that proper methods can be employed to assess exposure of calves on-farm.

Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne’s disease and, therefore, is a route of transmission for infection in youngstock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of MAP from individual bovine milk samples from known infected herds. Decontamination of milk samples (n = 17) with either 0.75% hexadecylpyridinium chloride for 5 hours or N-acetyl-L-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15 minutes prior to culture in BACTEC 12B, para-JEM, and Herrold’s egg yolk (HEY) media was compared. Results demonstrated that treatment with NALC-NaOH resulted in a lower percentage (6%) of contaminated samples than did treatment with HPC (47%), regardless of culture medium. The decontamination protocol (NALC-1.5% NaOH) was then applied to milk samples (n = 144) collected from cows at 7 US dairies. Recovery of viable MAP from the milk samples was low, regardless of culture medium, with recovery from 2 samples cultured in BACTEC 12B medium, 1 sample cultured in para-JEM medium, and no viable MAP recovered on HEY medium. However, 32 cows were fecal culture positive and 13 milk samples were positive by direct PCR, suggesting that a number of cows were actively shedding MAP at the time of milk collection. Contamination rates were similar across media, with 39.6, 34.7, and 41.7% of samples contaminated after culture in BACTEC 12B, para-JEM, and HEY media, respectively. Herd-to-herd variation had a major impact on sample contamination, with the percentage of contaminated samples ranging from 4% to 83%. It was concluded that decontamination of milk with NALC-1.5% NaOH prior to culture in BACTEC 12B medium was the most efficacious method for the recovery of viable MAP from milk, though the ability to suppress the growth of contaminating microorganisms varied greatly between herds.