Diagnostic Assay for Mycobacterium bovis in Bulk Tank Milk

Bovine tuberculosis is a well-known zoonotic disease which affects cattle world-wide. Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis (bTB), is a slow-growing bacteria, for which cattle are a host and significant reservoir. Consumption of raw milk and milk products has been associated with tuberculosis in humans. M. bovis accounts for up to 10% of human tuberculosis (TB) cases in developing countries and is increasing in cattle in the U.S. and U.K. Consequently, many developed nations have embarked on campaigns to eradicate M. bovis from cattle populations, or at least to control the spread of infection. The success of these eradication and control programs has been mixed and largely hindered by the presence of numerous wildlife reservoirs such as white-tailed deer, badger, and opossum. Raw milk from herds infected with M. bovis is a significant threat to national food safety and upon diagnosis, the herds are either depopulated or quarantined, which leads to major economic losses. Current ante-mortem tests for detecting the presence of bTB involve the use of costly and invasive techniques such as tuberculin skin testing and/or whole blood testing for gamma interferon production. The cost and disruption associated with these testing procedures are the major obstacles to the implementation of routine, industry-wide surveillance programs. The overall goal of this project is to develop a simple diagnostic test, using real-time PCR, to detect M. bovis organism in bulk milk. Using bulk milk samples as a test matrix will allow easier implementation of a national bTB surveillance program for the protection of the U.S. dairy industry. Specifically, research in Phase I will design and develop reagents for a real-time PCR assay to identify M. bovis DNA isolated from milk samples. The majority of the work will be devoted to resolving the potential challenges of isolating mycobacteria and its DNA when employing milk as test matrix. The newly designed assay will be used to determine shedding levels of M. bovis in milk from naturally-infected cows. Based on measured shedding levels and the analytical sensitivity of the new assay, collection and processing procedures will be developed for bulk tank testing. Finally, bulk milk samples from infected and bTB-free dairy herds will be tested to determine the diagnostic sensitivity and specificity of the assay for M. bovis. In Phase II, the milk-based, real-time PCR assay for M. bovis will be further validated in large scale field trails, and the test performance evaluated and presented to the United States Animal Health Association for recognition as an "official" test for herd-level screening for bTB. Early detection by an ongoing surveillance program can lead to the implementation of intervention strategies that will prevent severe economic losses resulting from spread of M. bovis infection. Such a test will directly benefit dairy producers and will be a very attractive tool for milk marketing and processing cooperatives to ensure safety of the nation's milk supply.