Paratuberculosis (Johnes disease) is a costly infectious intestinal disease of cattle caused by Mycobacterium paratuberculosis (MAP). The disease affects ruminants worldwide, causing weight loss and decreased milk production in affected animals. The United States Animal Health Association (USAHA) approved a resolution (#12) in October, 2006 stating "The United States Animal Health Association (USAHA) recommends that the United States Department of Agriculture (USDA), Agricultural Research Services (ARS) and the research community have a greater focus on development of quantitative based tests for detecting Mycobacterium avium paratuberculosis (MAP) in bulk tank milk." Purpose: The overall goal of this project is to develop a sensitive diagnostic test, using real-time PCR, to determine the presence and magnitude of MAP infection in dairy herds using pipeline in-line milk filters as a surrogate composite test sample. Towards this goal, Tetracore has developed and produced the only quantitative real-time PCR Johnes Disease test available in the North American marketplace. This assay has been demonstrated to be significantly more sensitive than both ELISA and IS900 PCR testing, detecting the presence of less than 30 organisms/gram of fecal material in several hours rather than the weeks required for MAP culture of samples. This assay will be applied to the detection of MAP organisms in milk filters, and protocols will be developed specific for this test sample. OBJECTIVES: Paratuberculosis (Johnes disease) is a costly infectious intestinal disease of cattle caused by Mycobacterium paratuberculosis (MAP). The disease affects ruminants worldwide, causing weight loss and decreased milk production in affected animals. Economic losses to the dairy industry attributed to Johnes disease are in the millions of dollars. A study conducted by the National Animal Health Monitoring System (NAHMS) in 1996 showed that at least 22% of US dairy herds are infected, although current estimates exceed 60%. Infected herds must be identified to target these herds for control of the disease and prevent movement of animals from infected to uninfected herds. Numerous studies have also shown that MAP organisms can be found in the milk of infected animals. On dairy farms, milk from all animals is collected and mixed in a bulk tank twice daily. Thus, the bulk tank milk represents a pooled sample from all lactating cows in the herd. If this sample can be tested in a highly sensitive manner, it would only be necessary to test one sample to identify a herd as infected. Similarly, as milk moves through the milking system pipeline to the bulk tank, it passes through a filter which traps gross debris, and potentially, MAP organisms. The overall goal of this project is to develop a sensitive diagnostic test, using real-time PCR, to determine the presence and magnitude of MAP infection in dairy herds using pipeline in-line milk filters as a surrogate composite test sample. APPROACH: Pipeline in-line milk filters from dairy herds with known levels of MAP infection will serve as surrogate composite test samples for the detection of MAP. The USDA-approved Tetracore Johnes test will be used for both detection and quantification of MAP from the milk filters. During the Phase I program, the team will demonstrate that MAP can be efficiently extracted from bulk tank milk filters and detected by real-time PCR. This will be performed by first spiking the MAP organisms in raw milk onto clean milk filters and developing an efficient extraction protocol for their isolation. As manure contamination of milk likely contributes the majority of MAP organisms, we will also spike raw milk with fecal samples with known concentrations of MAP organisms. The Tetracore real-time PCR test will be used to detect the presence of these organisms and approximate the efficiency of the extraction protocol. The optimized extraction protocol will then be tested on field-derived bulk milk filters obtained from dairy farms with demonstrated Johnes disease
