#14-286

Complete

Date Full Report Received

08/02/2017

Date Abstract Report Received

08/02/2017

Oral fluids (OF) obtained from pigs using cotton ropes are becoming a popular sample type for use in the diagnosis of diseases in pigs. The genetic material, antigens and antibodies to viruses that infect pigs can be measured in OF from pigs. Test methods exploiting this sample type are available for diseases frequently seen in North American pigs. Similar test methods need to be evaluated for diseases that are foreign or absent, but yet pose a threat to the North American pig industry. Examples include diseases caused by foot and mouth disease virus (FMDV), African swine fever virus (ASFV), swine vesicular disease virus (SVDV) and classical swine fever virus (CSFV). This project was designed to partially address this need.
Specific objectives were to create a repository of OF, swab and serum samples from FMDV, CSFV, ASFV and SVDV-infected pigs; use these samples to develop and/or validate existing test methods for detecting the genome of individual viruses (singleplex PCR) and multiple viruses simultaneous (multiplex assay) for FMDV, CSFV, ASFV and SVDV in OF; and develop and/or validate existing test methods for the detection of antibodies to FMDV, CSFV, ASFV and SVDV in OF and serum specimens collected during the convalescent phase of infection.
For FMDV, groups of pigs were either directly inoculated intradermally in the heel bulb of one hind limb with cell culture supernatants containing FMDV or were inoculated by contact with the directly inoculated pigs. For SVDV, each pig was inoculated intradermally in the heel bulb of one hind limb with cell culture supernatants containing SVDV (4 groups of 4pigs /group). CSFV and ASFV inoculations were performed by administering virus to each animal through the nares and the mouth. Oral fluids were collected from each group of pigs using cotton ropes. Whole blood, serum and swabs of the mouth and nares were also collected from individual animals.

FMDV genome was detected in OF as early as one day after the animals were either injected with virus or exposed to infected animals and 21 days later FMDV could still be detected in OF when tested by a quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Virus in these OF collected at 1-5 days post infection (DPI) grew in cell cultures, meaning infectious virus could be recovered from these samples. Slightly more virus was detected in OF compared to oral and nasal swabs. FMDV antigen was detected in OF by both a rapid penside test and an enzyme –linked immunosorbent assay (ELISA). Antibodies to FMDV were also detected in OF. The immunoglobulin (Ig) most commonly secreted at the mucosal surface is IgA. IgA was thus the most reliably detected antibody in OF in response to FMDV infection starting at 14 DPI and peaking at 21 – 28 DPI.

SVDV genome was also detected in OF as early as one day after the animals were inoculated with virus and was still detectable at 21 DPI when tested by qRT-PCR. Similar to FMDV, SVDV in the OF collected at 1-5 DPI grew in cell cultures, meaning infectious virus could be recovered from these samples. Similarly, slightly more virus was detected in OF compared to oral and nasal swabs. OF was also a better sample type for SVDV detection when compared to serum. With a modified competitive ELISA based on commercially available monoclonal antibodies, antibodies to SVDV were detected in OF starting at 6 DPI. Antibodies of the IgM and IgA isotype were also detected in OF with IgM response starting at 6 DPI, reaching a maximum at 7 or 14 DPI and dropping at 21 DPI. The IgA response started at 7 DPI and peaked at 14 DPI.
CSFV genome was detected in OF at 10 to 14 days after the animals were inoculated with virus. One group remained negative for virus genome in OF throughout the experiment. Virus was detected in sera earlier than in OF, starting at day 6 – 7 after inoculation of the pigs. Using a commercially available IDEXX HerdCheck CSFV Ab ELISA and a partially validated modification of the manufacturer’s protocol, antibodies to CSFV were detected in OF starting at 14 – 21 DPI.
ASFV Malta ‘78 genome was detected in OF starting at 6 DPI to 21 DPI. Detection of virus in oral and nasal swabs mirrored detection in OF. However, whole blood was the best sample type for ASFV detection, becoming positive at 4 DPI and containing higher levels of virus genome in most pigs.
A fully integrated and automated assay for simultaneous detection and differentiation of FMDV, SVDV, CSFV and ASFV was developed. The fully integrated/automated assay was optimized, validated and used to successfully process and detect cell culture amplified viruses, as well as FMDV, SVDV, CSFV and ASFV in OF.
All these results demonstrate that OF can be used for the detection of genome and/or live virus of FMDV, SVDV, CSFV and ASFV. Additionally, FMDV antigen can be detected in OF. Furthermore, antibodies to these viruses can be detected in OF by a variety of serological assays, including competitive and isotype-specific (IgA and IgM) ELISAs. Likewise, detection of IgA in OF has potential use for detecting antibody response following vaccination. All these results point to the high potential for the use of OF for FMDV, SVDV, CSFV or ASFV surveillance employing both established and partially validated assays.

Principal investigator: Charles Nfon
Email: Charles.nfon@inspection.gc.ca
National Centre for Foreign Animal Disease, CFIA
1015 Arlington Street
Winnipeg, MB, Canada R3E 3M4
Phone: 1 204 789 2023; Fax: 1 204 789 2038
Co-investigators: Oliver Lung*, John Pasick*, Aruna Ambagala*, Jeffrey Zimmerman+ and Luis G. Giménez-Lirola+
Institutions: *National Centres for Animal Disease (NCAD) and +Iowa State University, Ames, Iowa