#14-160

Complete

Date Full Report Received

06/27/2016

Date Abstract Report Received

06/27/2016

Investigation

Institution:
Primary Investigator:

Porcine epidemic diarrhea (PED), caused by a coronavirus (PEDV), is a newly emerged enteric disease of swine, which was confirmed in the United States for the first time in April 2013. Since then, PED has caused unprecedented challenges to the US swine industry due to an extremely high mortality (up to 100%) in pre-weaning pigs and significant productivity loss in growing and breeding pigs. While still unanswered how PEDV was initially introduced to US swine, imported feed ingredient(s) has been suspected as a source for introduction to the US. Feedstuff has also been identified as a contributing factor to spreading of PEDV within the US. While positive PCR finding of PEDV RNA in feed or feedstuffs raised the serious question about the cleanness of environment at both collection and manufacturing sites and manufacturing quality control processes of raw materials to supply and storage of final products, infectivity of such PCR-positive materials in pigs has been extremely difficult to prove through feeding trials. Knowing that PCR-based tests detects the presence of targeted genetic material regardless of live or dead, a different and reliable way to assess the infectivity of these materials was necessary.

PEDV has been known to be difficult to isolate or propagate in cell culture (a 5-6% success rate). Furthermore, complete feed, feed ingredients or environmental samples are not matrices which work well in a cell culture system due to cytotoxicity. All these facts reduce the value of cell culture-based testing to assess if a material in question contains an infectious virus. Alternatively, swine bioassay is an excellent tool to access infectivity as pigs are the natural hosts. However, swine bioassay is resource-driven (i.e., expensive) and a biologically variable system which can be confounded by many factors, not to mention its longer turnaround. The following proposed study was for a proof-of-concept to address the need of diagnostic procedures for determining potential live virus contamination of feed or feedstuffs. Since small intestine is the ultimate place for enteric viral pathogens to replicate, leading to the disease due to functional disruption of villi and other parts, we explored an ex-vivo bioassay for PEDV in feed or feedstuffs using “Tied Small Intestinal Segment (TSIS)” by maintaining the intact structure of intestine outside of the pig under laboratory conditions. As neonates are born with intestinal tissues with fully functional enterocytes which are the most susceptible cells to PEDV, the specific objective of the study is to determine if TSIS from neonatal pigs will be a cost-effective diagnostic tool for rapid and reliable determination of infectivity of PEDV.

A tied small intestinal segment (TSIS) was a cross-sectional piece of small intestine (6-10 cm in length) tied-off and liquid material (i.e., inoculum) injected into the lumen. The TSIS was kept moist by immersing  in sterile cell culture media within a sterile petri dish and maintained at 37°C in a humidified CO2 incubator to mimic the body condition. Hence TSIS is a miniaturized natural pig gut with functional enterocytes along with villous structure. Intestinal segments from CDCD or snatch-farrowed piglets obtained from PEDV and TGEV negative farms were used of the TSIS. To supply sufficient number of TSIS for the study, piglets of the same litter were used as intestine donor.  Three separate experiments were conducted to address the following qusetions:

1. How long TSIS can be maintained in vitro without detrimental loss or alteration of villous structure?
2. Is TSIS permissive to coronaviruses and rotaviruses and all intestinal segments are equally susceptible to these viruses?
3. How sensitive is TSIS method for viral infectivity assay?

Tied small intestinal segments could be maintained in vitro up to 72 hours without significant loss of enterocytes and villous structure under optimized conditions regardless of sample matrices (i.e., media or feed extracts) tested. Virus infection and growth in TSIS was evident to a degree by PCR or IHC staining when inoculated with 2ml cell-culture derived PEDV or TGEV containing as low as 102 PFU/ml at 72 hours post inoculation (PI) but not at 48 hours PI. Such an evidence of virus growth was not observed in TSIS inoculated with swine influenza virus at a rate of up to 106 TCID50/ml within 72 hours. Jejunum and ileum were permissive to both coronaviruses regardless of source of intestine (i.e., CDCD piglets or snatch-farrowed piglets). When feed or feed ingredients, which were positive for PEDV nucleic acid by PCR at Iowa State University Veterinary Diagnostic Laboratory, were tested in TSIS, no infectivity was detected.  Some of the samples which were tested by swine bioassays also did not show the presence of infectious PEDV.

The study outcome suggests that TSIS can be an ex-vivo bioassay tool to measure the presence of an infectious enteric viral pathogen in feed or non-traditional sample matrices.  While this new method would be better than cell-culture based assessment, further optimization and refining is necessary to significantly enhanced the sensitivity of TSIS-based testing for infectivity.  Some examples are: a) the ability to maintain TSIS integrity for longer than 72 hours and b) availability of assays for tissues to demonstrate virus infection/replication in enterocytes which are more sensitive than IHC.

Contact info:
Kyoung-Jin Yoon, Professor
College of Veterinary Medicine, Iowa State University, Ames, IA 50011
Email: kyoon@iastate.edu
Phone: 515-294-1083