|Bradner, Laura -|
|Robbe-Austerman, Suelee -|
|Beitz, Donald -|
Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 19, 2013
Publication Date: July 1, 2013
Repository URL: http://handle.nal.usda.gov/10113/57200
Citation: Bradner, L., Robbe-Austerman, S., Beitz, D.C., Stabel, J.R. 2013. Chemical decontamination with n-acetyl-l-cysteine-sodium hydroxide improves recovery of viable Mycobacterium avium subsp. paratuberculosis organisms from cultured milk. Journal of Clinical Microbiology. 51(7):2139-2146. 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 a novel method for decontamination prior to culture 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 milk and feces of cows with advanced Johne’s disease, allowing transmission of MAP among animals. The objective of this study was to formulate an optimized protocol for the isolation of MAP from milk. Parameters investigated included chemical decontamination with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH), 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, culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 10**2-10**8 cfu/ml of live MAP. Results indicate increased length of exposure to NALC-NaOH from 5 to 30 min and increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of MAP. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable MAP more than treatment with NALC-NaOH alone. The BACTEC 12B medium was the superior medium of the three evaluated for isolation of MAP from milk, achieving the lowest threshold of detection. The optimal conditions of NALC-NaOH decontamination were determined to be 1.50% NaOH exposed for 15 min, followed by culture in BACTEC 12B medium. This study demonstrates that the severity of the detrimental effect of chemical decontamination on the recovery of viable MAP is highly dependent upon the chemical(s) used and the sample matrix. Therefore, it is important to optimize milk decontamination protocols to ensure low concentrations of MAP can be detected.