Optimization and Validation of A Real-Time RT-PCR Assay For Rapid Detection of FMDv for Use in Bulk Tank Milk Samples

Principal Investigator(s): ,

Purpose

Foot and Mouth Disease (FMD) is a highly contagious viral disease of cloven-hoofed animals including pigs and cattle. While the disease itself is typically not lethal to animals and does not cause disease in humans, it can spread rapidly through susceptible livestock and wildlife populations with devastating effects on livestock industries and a country’s economy. Rapid detection and diagnosis of suspect cases and a rapid response, typically involving slaughter of infected animals and possibly vaccination of animals in close proximity to an outbreak, is essential for effective control and eradication of the disease. The economic impacts of FMD on countries endemic for the disease are both local and national due to reduced animal productivity and strict trade restrictions with other countries. Countries, such as the U.S., that are recognized by the World Organization for Animal Health as being free from FMD, enjoy the benefits of export trade and high yield livestock product production. The impact of an epidemic of this disease in a FMD free country is particularly devastating as it results in sudden economic and social turmoil including culling of large numbers of livestock, loss of consumer confidence, freezing of export markets, food shortages, and even losses in product movement and trade within the country due to the adoption of restrictions needed to limit the spread of the outbreak.

The objectives of this study were to define, optimize, and standardize RNA extraction and polymerase chain reaction (PCR)-mediated detection of Foot and Mouth Disease virus (FMDv) from bulk tank milk and obtain data suitable for development of a national FMD surveillance plan to assist the dairy industry in the event of an outbreak of FMD. In this regard the purpose of the test would be for efficient screening of individual cow’s milk or bulk tank milk for the presence of FMDv contamination or for confirmatory testing for FMD in a dairy cow when milk is provided as a diagnostic sample. This study, spearheaded by the U.S. Department of Agriculture Animal and Plant Health Inspection Service (USDA APHIS) and Department of Homeland Security Office of Science and Technology, represented a consortium of key collaborators including the National Veterinary Services Laboratories’ Foreign Animal Disease Diagnostic Laboratory (NVSL-FADDL); the Institute for Infectious Animal Diseases (IIAD); the Pirbright Institute in the United Kingdom; U.S. dairy producers; the National Animal Health Laboratory Network (NAHLN); the Centers for Epidemiology and Animal Health (CEAH); the Center of Veterinary Biologics (CVB); and on-going international collaborators in Bangladesh and Africa. For development of a screening tool for FMD surveillance in the dairy industry, the project focused on the USDA real-time Reverse-transcriptase PCR (rRT-PCR) previously validated using other sample types and adapted to high-throughput diagnosis, and on selection and optimization of a companion high-throughput RNA extraction method.

Outcomes and Impacts

In this study, IIAD, members of the NAHLN, Wisconsin Veterinary Diagnostic Laboratory, and NVSL-FADDL optimized and standardized methods for FMDv RNA extraction from bulk tank milk samples followed by rRT-PCR analysis, as validated and currently employed by USDA APHIS, FADDL and NAHLN. International studies in experimentally infected dairy cows and in international bulk tank milk samples from endemic countries or countries experiencing outbreaks of FMD were used to help validate and obtain modeling data for the design of a U.S. FMD surveillance plan utilizing bulk tank milk as a sample and rRT-PCR as a detection method. A large negative cohort study was conducted in the NAHLN in collaboration with State Animal Health Officials and the dairy industry.

For the dairy industry there is a recognized need for rapid diagnostics and surveillance to enable early detection and support the permitting and subsequent movement of raw milk (and other activities) from uninfected premises to a processing plant. Raw milk and even some pasteurized milk, depending on the method of pasteurization, from FMD infected cattle represents not only a significant source of disease spread during an outbreak of FMD but also a potentially valuable sample source for FMDv detection or surveillance during and in recovery from a disease outbreak. The outcomes of this study provided data on the projected sensitivity, practicality and specificity of the testing procedure. Data obtained during this study and included in the dossier will be instrumental in providing scientific support for determining how this sample type could be utilized in a post-outbreak FMD surveillance plan and how it might be utilized to support the Secure Milk Supply business continuity efforts.

A high-throughput nucleic acid extraction method and a rapid diagnostic assay such as bulk tank milk FMD rRT-PCR are valuable tools to support situational awareness and rapid decision making in the response to an outbreak of FMD with potential to contribute to early detection or added confidence of disease freedom during or in recovery from an FMD outbreak.  Such tools will be essential to supporting the Secure Milk Supply plan and maintaining continuity of business for the dairy industry in the face of an FMD outbreak.

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