|Bradner, Laura -|
|Robbe-Austerman, Suelee -|
|Beitz, D -|
Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: February 4, 2012
Publication Date: February 19, 2012
Citation: Bradner, L., Robbe-Austerman, S., Beitz, D., Stabel, J.R. 2012. Optimization of methods for the detection of Mycobacterium avium subsp. paratuberculosis in milk and colostrum of naturally infected dairy cows. In: Proceedings of the 11th International Colloquium on Paratuberculosis, February 5-10, 2012, Sydney, Australia. p. 71-73. 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 primarily shed into the feces but it has also been isolated from the milk and colostrum of cows. Because of this, there exists concern about transfer of the organism from dam to calf and about the prevalence of MAP in the milk supply. The prevalence of MAP in milk is not well defined because of the complexity of the milk matrix. The objective of this study is to optimize the decontamination of whole raw milk for the isolation of viable MAP and compare recovery rates in liquid and solid culture mediums. The efficacy of two liquid culture mediums, TREK-ESP and BD Bactec 12B were compared for recovery thresholds, speed of recovery, incidence of contamination and reproducibility of results. Milk collected from a non-infected cow was spiked with MAP (102 to 108 cfu/ml). Variables investigated included concentration of hexadecylpyridinium chloride (HPC) as the primary decontaminant, temperature of decontamination, centrifugation speed, and time of incubation. It was found that neither length of exposure to HPC or concentration of HPC had significant effects on the recovery of MAP from milk. Because of known lethal effects of HPC on MAP, the most efficient decontamination parameters with the highest recovery rates of MAP were 0.75% HPC exposed for 5 hr at room temperature. Since increased temperature (>25°C) enhanced growth of microbial contaminants, room temperature was the optimal choice. In comparing the two liquid culture mediums, Bactec 12B was superior in recovery thresholds and speed of recovery of viable MAP. TREK-ESP culture demonstrated an increased incidence in false positive and false negative results that were not observed in Bactec 12B medium. Future decontamination studies will evaluate the efficacy of N-acetyl-L-cysteine (NALC)-sodium hydroxide. Optimized methods will be used to assess the frequency and level of MAP shed into milk during a complete lactation period of naturally infected dams.