Development of a Mutiplex RT-qPCR Assay for Surveillance of Foreign Animal Diseases During Routine Testing of Oral Fluid Samples

Principal Investigator(s): ,


Foreign animal diseases (FAD) pose grave and catastrophic economic burden on the U.S. swine industry and other livestock industries through economic loss associated with disease as well as with trade barriers. Highly contagious FADs, such as African Swine Fever (ASF), Classical Swine Fever, (CSF) and Foot and Mouth Disease (FMD), are severe threats to the swine industry and have potential major economic and social consequences. The United States is considered to be ASF free, and CSF and FMD were eradicated from the U.S. in 1978 and 1929, respectively. However, in many parts of the world they are still endemic and thus pose serious threats globally through reintroductions. Developed countries with extensive swine production, including the United States, conduct disease surveillance to curtail the possible disease introduction and spread.

Oral fluids containing saliva and serum components has a similar representation of analytes found in blood, and can be used for disease diagnosis. Oral fluid-based diagnostic testing holds great potential for FAD surveillance. The inherent presence of CSF virus (CSFv) and FMD virus (FMDv) in the oral fluid and ASF virus (ASFv) in tonsils and pharynx indicates the likelihood of their successful detection. The oral fluid sampling cost effectiveness, ease of use, user and animal friendliness, coupled with optimized protocols and reagents for subsequent detection, make oral-fluid based FAD diagnostics well suited for FAD surveillance and outbreak testing.

The objective of this project is to develop an optimized oral fluid nucleic acid purification method and a multiplex reverse transcriptase quantitative PCR assay (mpRT-qPCR) that can be used to simultaneously detect several economically important swine endemic (PRRSV, PCV2, SIV, and M. hyopneumoniae) and foreign animal diseases (ASFv, CSFv, and FMDv) from the same sample, in the same assay. The mpRT-qPCR assay approach for swine disease pathogens detection was chosen due to its cost effectiveness, ease of use, and adaptability (ability to add, modify, and update assays).

Outcomes and Impacts

Oral fluid sampling holds a great potential for being the single source diagnostic sample for surveillance and monitoring of FAD and endemic swine diseases. Oral fluids are collected from a group of pigs and represent each individual pig sample contribution (if all pigs chew on the rope). Group sampling is cost effective, enables greater sampling number and group disease surveillance. Oral fluid sampling is simple, inexpensive, safe, and less labor and animal intensive. The method is user-friendly and easily trained and transferrable to farmers and animal care specialists. The ease of use and cost effectiveness enables multiple sampling to monitor endemic disease progression and intervention measures and enables rapid sampling during disease outbreaks.

The deliverables of this project will include optimized reagents and protocols for pathogen nucleic acid purification and detection using oral fluids. The optimized mpRT-qPCR reagents will also enable future assay additions/modifications, and maintenance; this capability ensures that novel and emerging pathogenic strains/isolates are detectable. This assay will enable an economic and rapid method for foreign animal disease surveillance in the background of endemic disease testing using oral fluids. By using a single protocol that is routinely used for diagnostics of endemic diseases with the capacity for simultaneous FAD detection, no additional personnel training, Standard Operating Procedures development, reagents or purchases are required.  Continuous and routine use of this protocol ensures highly trained personnel are immediately available during outbreak situations.

Rapid and accurate diagnosis is essential for effective control and eradication of a FAD in swine, but swine affected by an FAD may have clinical signs indistinguishable from other endemic diseases currently seen in the country. The profound public health and economic impact of swine diseases (FAD and endemic) warrants improvements in swine disease surveillance and diagnostics methods and tools. The ability to perform surveillance and monitoring of endemic and FADs rapidly, accurately, and economically using one single diagnostic sample is highly desirable. In addition, embedding the FADs into routine surveillance activities will allow the assays to be used regularly and provide a convenient sample that could be utilized without altering sample collection techniques during a disease outbreak.