SR9081 - Mycobacterium Avium Subspecies Paratuberculosis in the Ontario Dairy Industry: Strategies for Detection of the Bacterium in Dairy Products

Researcher:

Drs. Lucy Mutharia, Dept. of Microbiology and Joseph Odumeru, Laboratory Services Division, University of Guelph

Objectives:

1. Optimize sample decontamination protocols for capture and culture of MAP cells.
   
2. Develop protocols for direct extraction of MAP DNA from samples.
   
3. Define the minimum incubation periods for detection of viable MAP.
   
4. Generate baseline information on the prevalence of MAP in dairy products, to assist dairy Farmers of Ontario in the management of MAP transmission in herds.

Expected Benefits:

1. To provide tools that can be applied to the long-term management of MAP infections and to food safety issues of MAP contamination.
   
2. This project is of benefit to management of disease, to farm production and to increase consumer confidence in the dairy industry.

Summary of Research Results:

Mycobacterium avium subspecies paratuberculosis or MAP is the cause of Johne's disease (also called paratuberculosis) a common and chronic disease of the intestines that affects ruminant animals (such as cattle, sheep, goats and deer) and some non-ruminant animals. The disease is found in animals all over the world including cattle in all regions of Canada. Cattle with the late stage or clinical disease show wasting, have chronic diarrhea, are in poor health and eventually die. Since there are no vaccines to protect against infection, it is important to control the disease spread from herd-to-herd, and within a herd. Because MAP bacteria are shed in feces and in milk from the infected animals, the disease can be easily spread to the young animals though fecal contaminated foods, water or objects. To control disease spread, infected animals should be removed from the herd before they begin to shed the bacterium. It is not easy to identify such animals or to detect the MAP bacterium in feces or milk. After infection the animal, for a number of years, will appear healthy while it may be shedding the bacterium and potentially spreading the disease. Identifying the bacterium in cow feces or in milk is also complicated. MAP is extremely slow growing (takes 6-16 weeks before it can be identified by growth on lab media), and cultures can be overgrown by contaminants. Feces and milk from infected but healthy animals may only contain a few cells of this bacterium relative to other contaminants. To detect MAP bacterium, samples are treated with chemical reagents to selectively kill the contaminants while hopefully retaining live most of the MAP in the sample. Finally, there are not very reliable tests to identify infected animals by blood-based test or by skin tests (as done for bovine tuberculosis).

Our research goals are to develop assays for rapid and reliable detection of MAP bacterium from cow feces and milk. Ideally, the assays will detect samples contaminated with only a few cells of MAP. Another research focus is to develop assays that differentiate contamination of foods or water with live and dead MAP cells. Only the live bacterium poses a risk to animals.

Our approaches included: (a) using specific antibody reagents to rapidly and selectively capture and concentrate MAP bacterium from the samples; (b) determining the optimal conditions and reagents that selectively kill contaminants while maintaining live most of the MAP bacteria, (c) modifying culture media and conditions to support the growth of this organisms, (d) detection in a sample of MAP-specific nucleic acids as a quick indicator of the target bacterium.

We have met some of the goals of this research. We developed an assay that combines enrichment of the bacterium from the sample, with selective killing of contaminants (retaining maximum live MAP) and rapid detection of MAP by its nucleic acids. Using this assay we can reliably detect, by culture, 10 MAP bacteria per gram of feces or per milliliter of milk. Our next goal is to optimize the conditions of an assay that differentiates live from dead MAP based on detection of specific nucleic acids and bacteriophages and without the need to culture the bacterium. Our methods will have application in the areas of disease control and food safety. The tests could be applied to cow feces or to cow milk to identify infected animals and to foods to identify contamination.

Using our methods, we tested over 144 matched cow milk and fecal samples. We showed that if only the milk or feces are tested, up to 30% of the infected cattle would not be identified. For example, at the time of sample collection, a cow may be shedding MAP bacterium in the feces or milk or in both.

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