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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Publications at this Location » Publication #288918


Location: Infectious Bacterial Diseases Research

Title: Optimization of hexadecylpyridinium chloride decontamination for culture of Mycobacterium avium subsp. paratuberculosis from milk

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 Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2013
Publication Date: 5/1/2013
Publication URL:
Citation: Bradner, L., Robbe-Austerman, S., Beitz, D.C., Stabel, J.R. 2013. Optimization of hexadecylpyridinium chloride decontamination for culture of Mycobacterium avium subsp. paratuberculosis from milk. Journal of Clinical Microbiology. 51(5):1575-1577.

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: Cows in advanced stages of Johne’s disease shed Mycobacterium avium subsp. paratuberculosis (MAP) into both their milk and feces, allowing for transmission of the bacteria between animals. The objective of this study was to formulate an optimized protocol for the isolation of MAP from milk and colostrum including parameters such as chemical decontamination with hexadecylpyridinium chloride (HPC) alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and efficacy of solid, Herrold’s egg yolk medium (HEY), and liquid, BACTEC 12B and para-JEM, media. For each experiment, raw milk samples from a known Johne’s disease-free cow were inoculated with 10**2-10**8 CFU/ml of live MAP. Results indicated that increasing time of exposure to HPC from 5 to 48 hours, as well as increasing concentration of HPC from 0.75 to 1.50% decreased the recovery of viable MAP for all culture media. Additional treatment of milk samples with antibiotics following HPC decontamination decreased the recovery of viable MAP more than did treatment with HPC alone. The BACTEC 12B medium proved to be the superior medium for isolating MAP from milk by having a lower rate of contamination and threshold of detection than did either the para-JEM or HEY media. The optimal decontamination protocol determined was 0.75% HPC exposed for 5 hours at room temperature. This study demonstrates the efficiency of the recovery of MAP from milk is highly dependent upon the culturing protocol, and such protocols should be optimized to ensure that low concentrations of MAP in milk can be detected.