Detection of Foot-and-Mouth Disease Virus in Swine Meat Juice.

Foot-and-mouth disease virus (FMDV) is a highly contagious agent that impacts livestock industries worldwide, leading to significant financial loss. Its impact can be avoided or minimized if the virus is detected early. FMDV detection relies on vesicular fluid, epithelial tags, swabs, serum, and other sample types from live animals. These samples might not always be available, necessitating the use of alternative sample types. Meat juice (MJ), collected after freeze-thaw cycles of skeletal muscle, is a potential sample type for FMDV detection, especially when meat is illegally imported. We have performed experiments to evaluate the suitability of MJ for FMDV detection. MJ was collected from pigs that were experimentally infected with FMDV. Ribonucleic acid (RNA) was extracted from MJ, sera, oral swabs, and lymph nodes from the same animals and tested for FMDV by real-time reverse transcription polymerase chain reaction (rRT-PCR). MJ was also tested for FMDV antigen by Lateral Flow Immunoassay (LFI). FMDV RNA was detected in MJ by rRT-PCR starting at one day post infection (DPI) and as late as 21 DPI. In contrast, FMDV RNA was detected in sera at 1-7 DPI. Antigen was also detected in MJ at 1-9 DPI by LFI. Live virus was not isolated directly from MJ, but was recovered from the viral genome by transfection into susceptible cells. The data show that MJ is a good sample type for FMDV detection.

Field evaluation of two commercial RT-rtPCR assays for porcine reproductive and respiratory syndrome virus detection using sera from ill and healthy pigs, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious respiratory virus causing severe morbidity in pigs worldwide. Control strategies for PRRSV often rely on detecting PRRSV, culling or isolating sick pigs, disinfecting pig barns, vaccination, and monitoring for virus spread. Given the high economic impact of PRRSV on pig farms, there is a great need for rapid and reliable PRRSV detection assays. We compared the performance of 2 commercial reverse-transcription real-time PCR (RT-rtPCR) assays, the VetMAX PRRSV NA and EU reagents (ABI assay) and the PRRSV general RT-rtPCR kit (Anheal assay), for the molecular detection of PRRSV in sera collected from pigs in China. Between June and September 2015, sera were collected from 219 healthy and 104 suspected PRRSV-infected pigs on 4 farms in China. Employing blinding, the 2 assays were run by 2 laboratories (Guangzhou Animal Health Inspection Institute [GAHII] and Sun Yat-sen University [SYSU] laboratories) and compared. Although both assays detected PRRSV with 100% specificity at both laboratories, the sensitivity (95% vs. 78% at GAHII; 94% vs. 72% at SYSU Laboratory) and the reproducibility (kappa value 0.933 vs. 0.931) were slightly better for the ABI assay compared to the Anheal assay.

Detection of foot-and-mouth disease virus: comparative diagnostic sensitivity of two independent real-time reverse transcription-polymerase chain reaction assays.

Rapid and accurate diagnosis is central to the effective control of foot-and-mouth disease (FMD). It is now recognized that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role in the routine detection of FMD virus (FMDV) in clinical samples. The aim of this study was to compare the ability of 2 independent real-time RT-PCR (rRT-PCR) assays targeting the 5' untranslated region (5'UTR) and RNA polymerase (3D) to detect FMDV in clinical samples. There was concordance between the results generated by the 2 assays for 88.1% (347 of 394) of RNA samples extracted from suspensions of epithelial tissue obtained from suspect FMD cases. The comparison between the 2 tests highlighted 19 FMDV isolates (13 for the 5'UTR and 6 for the 3D assay), which failed to produce a signal in 1 assay but gave a positive signal in the other. The sequence of the genomic targets of selected isolates highlighted nucleotide substitutions in the primer or probe regions, thereby providing an explanation for negative results generated in the rRT-PCR assays. These data illustrate the importance of the continuous monitoring of circulating FMDV field strains to ensure the design of the rRT-PCR assay remains fit for purpose and suggest that the use of multiple diagnostic targets could further enhance the sensitivity of molecular methods for the detection of FMDV.

Use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus.

OBJECTIVE: To evaluate a portable real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay designed to detect all 7 viral serotypes of foot-and-mouth disease virus (FMDV). DESIGN: Laboratory and animal studies. STUDY POPULATION: Viruses grown in tissue culture and animals experimentally infected with FMDV. PROCEDURE: 1 steer, pig, and sheep were infected with serotype O FMDV. Twenty-four hours later, animals were placed in separate rooms that contained 4 FMDV-free, healthy animals of the same species. Oral and nasal swab specimens, oropharyngeal specimens obtained with a probang, and blood samples were obtained at frequent intervals, and animals were observed for fever and clinical signs of foot-and-mouth disease (FMD). Samples from animals and tissue cultures were assayed for infectious virus and viral RNA. RESULTS: The assay detected viral RNA representing all 7 FMDV serotypes grown in tissue culture but did not amplify a panel of selected viruses that included those that cause vesicular diseases similar to FMD; thus, the assay had a specificity of 100%, depending on the panel selected. The assay also met or exceeded sensitivity of viral culture on samples from experimentally infected animals. In many instances, the assay detected viral RNA in the mouth and nose 24 to 96 hours before the onset of clinical disease. CONCLUSIONS AND CLINICAL RELEVANCE: The assay reagents are produced in a vitrified form, which permits storage and transportation at ambient temperatures. The test can be performed in 2 hours or less on a portable instrument, thus providing a rapid, portable, sensitive, and specific method for detection of FMDV.