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. OBJECTIVES: The overall goal of this proposal is to develop a quantitative real-time PCR (qPCR) assay for use in bulk tank milk for the detection of Mycobacterium bovis (M. bovis). Research in Phase I will demonstrate that M. bovis can be extracted from bulk milk samples and detected by a quantitative real-time PCR assay (qPCR) with sufficient sensitivity and specificity for TB surveillance. The technical objectives have been divided into three specific tasks. 1. Design and optimize qPCR assay to detect M. bovis: The objective is to design a qPCR test that will be able to detect M. bovis with a detection limit of one copy of genomic DNA per PCR reaction. Primer sets for M. bovis PCR detection protocols will be assessed using dilutions of M. bovis genomic DNA. Successful completion of this objective will require working with NVSL/APHIS laboratory to acquire genomic DNA from well characterized strains of M. bovis. 2. Establish qPCR assay sensitivity and specificity for diagnosing M. bovis in milk samples: The goal of this task is to determine analytical sensitivity and specificity of the M. bovis qPCR assay in a milk sample matrix. Raw milk samples will be spiked with known numbers of M. bovis bacillus Calmette-Guerin (BCG) organism. The design will include optimization protocols to isolate and detect bacterial DNA from milk samples to obtain a detection limit of less than 3 organisms in one milliliter of milk. 3. Validate M. bovis qPCR assay for bulk milk samples: The third task will focus on testing field milk samples from M. bovis positive (collaboration with research group in Mexico) and negative herds to validate the bulk milk qPCR test. The following set of experiments will be conducted: 1) milk samples will be collected from TB-positive animals and tested using a dilution series with milk from uninfected animals (accredited TB-free herds), 2) bulk milk samples will be collected from farms with different levels of TB prevalence and tested for the presence of M. bovis, and 3) bulk milk samples from farms across Michigan to study prevalence of M.bovis in bulk tank milk. These technical objectives will answer the following questions. 1) Do the reagents designed to recognize M. bovis DNA work efficiently in the qPCR format 2) Can M. bovis be successfully recovered from milk samples and DNA isolated for a diagnostic assay 3) Does the real-time PCR for M. bovis detect the presence of the organism in the bulk milk supply from herds with varying numbers of TB-infected animals Overall, phase I research will be able to address the question; can bulk milk screening for M. bovis be used effectively in a TB surveillance program for dairy herds and milk products
