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The goals of this research are to evaluate a three-step procedure to identify the presence of live Mycobacterium avium subspecies paratuberculosis (MAP) comprised of, i) affinity-based bacterial extraction, ii) broth enrichment and iii) mRNA-based identification, for the potential to improve sensitivity and reduce detection time of the contamination or infection. Current procedures utilize extensive sample processing and decontamination to separate the relatively few MAP from large numbers of microbial contaminants. Although the sensitivity of testing by culture on agar is accepted as poor, and takes 6-18 weeks for results, it is widely applied as the best method available to detect live MAP. At present there is no recommended standard laboratory protocol for live MAP tracking in milk or other sample types. Given this lack of standards for sample preparation, decontamination, and culture there is considerable variability in assay sensitivity, contamination rates and time-to-detection among laboratories when processing identical sample types.
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Using various sample types, the study shall determine assay reliability, specificity, detection range, and cost. The study will examine the procedures for their application in clinical and food laboratories. Specifically, the study will focus on the following 3 components of the assay:<ol type="a">
<li> Evaluate monoclonal antibody-based immunomagnetic capture for rapid and specific isolation of MAP cells.
<li> Evaluate the potential of broth enrichment to increase the sensitivity of the assay.
<li> Optimize an RNA-based protocol to detect MAP-specific transcripts, hence viable MAP, directly in the captured MAP or after periods of broth enrichment. </ol>
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MAP is the cause of Johne's disease (JD), and affects approximately 37% of Ontario dairy herds. Feces, milk, blood and lymph nodes from infected animals can be contaminated with MAP bacteria. The infectious dose of MAP is not known. Some MAP bacteria escape killing during pasteurization and present an exposure risk to susceptible humans. Currently, it is not known what factors affect MAP persistence in the environment, it's survival in milk, or in meat. The potential for contamination of milk and meat within the Ontario dairy industry are not known.
Expected Impact of Project Outcomes on Food Safety in Ontario:
Through collaboration with the University of Guelph Laboratory Services Division the study will develop an assay for application in diagnostic service to test both specimens for disease and food products for contamination. The impetus for the development of a widely available, reliable, sensitive and rapid assay is that it will enable the following:<ol type="a">
<li> <b>Disease management:</b> Reliable data are needed on the biological and on-farm environmental sources of live MAP. Informed strategies can then be implemented to reduce transmission within and among herds. There are no effective treatments or vaccines. Identifying and removing sub-clinically infected animals and other on-farm sources of infection will best achieve control of JD. Wild ruminant and non-ruminant species infected with MAP are a concern as they can act as reservoirs for the disease within a farm and as vectors carrying the disease between farms and so interfere with efforts to control the disease in livestock.
<li> <b>Human Food Safety:</b> Pasteurization studied over a wide range of temperatures and time combinations either report effective killing of MAP or tailing with live bacteria. It is apparent that live MAP is present in retail milk. Blood, lymph node tissue, feces and milk of animals with subclinical disease can contain MAP organisms. Concerns regarding the zoonotic potential of MAP indicate that the contamination of all products designated for human consumption be accurately assessed, then reduced or eliminated and monitored. </ol><P> For more information, please visit the <a href="http://www.omafra.gov.on.ca/english/research/foodsafety/index.html" target="_blank">Ontario Ministry of Agriculture, Food & Rural Affairs (OMAFRA) Food Safety Research Program</a>.