Date Full Report Received

05/12/2015

Date Abstract Report Received

05/12/2015

Investigation

Institution:
Primary Investigator:
Co-Investigators: Megan Niederwerder

Porcine epidemic diarrhea virus (PEDV) has recently emerged in the US.  The purpose of this investigation was to determine tissue localization, shedding pattern, virus carriage, antibody response, and aerosol transmission of PEDV following oral/nasal inoculation of 4-week-old feeder pigs.
Experimental Animals:  Thirty-three PEDV naive 3-week-old feeder pigs obtained from a high health commercial source were allowed to acclimate for one week prior to inoculation.   The study was conducted under BSL2 containment at the Biosecurity Research Institute at Kansas State University.  Twenty-three Group A pigs were inoculated with the PEDV challenge material.  Five Group B pigs were not inoculated, but were comingled with inoculated Group A animals approximately 6 hours post inoculation (PI). Five aerosol transmission Group C pigs were not inoculated, but were housed in a separate pen in the same common animal room as Groups A and B.
The challenge material used in this study was a pool of gut-derived intestinal contents that has been used as “feedback” inocula for controlled exposure of a sow herd in a commercial swine production unit.  This material tested negative for PRRS and PCV and produced a PEDV nucleic acid “CT titer” of 22. Challenged animals (Group A) were inoculated at 4 weeks of age via the oral and intranasal routes with 5 ml of inocula per route. Following inoculation, the animals were observed daily for clinical symptoms.  Nasal and fecal swabs as well as serum samples were collected prior to challenge and at days 0-7, 9, 14, 21, 28, 35, and 42 post inoculation (PI).  Pen oral fluid samples were also collected at the same time points for Groups A/B and the aerosol control Group C.

PEDV shedding was monitored by performing real-time PCR on fecal and nasal swab samples and oral fluids.  Serum samples were collected in order to monitor viremia and antibody response. Fresh and formalized tissues were collected from randomly selected Group A pigs at days 0, 2,4,7,9,14,21,28, 35, and 42 PI in order to monitor tissue tropism of the virus and histopathology.

Experimental data indicate the following:
Mild clinical signs appeared 2 days post inoculation and resolved by 8 days post inoculation.
Fecal and nasal swabs were PCR positive in the inoculated group at 48 hours post inoculation.
Peak fecal shedding occurred 5 to 6 days post challenge and was significantly higher than that in the nasal swabs.
Most group A and B animals were negative by fecal and/or nasal swab testing at 21 days post inoculation; however, some animals shed virus as long as 35 days PI  The inoculated piglets were qRT-PCR positive in fecal and nasal swabs to 21 DPI.
Productive transmission did not appear to occur in the aerosol control group in spite of the fact that PEDV nucleic acid could be detected in the nares of some of those animals and oral fluids.
Room environmental samples were collected at 14 days post inoculation and the data demonstrate that viral nucleic acid was abundant on the walls, pens, and food bins on both the inoculated and aerosol control areas in the challenge room.  Due to the possibility of a false positive PCR reaction, questionable samples were retested and the reaction products were sequenced to determine if the product was PEDV specific. All questionable reactions demonstrated the presence of PEDV viral nucleic acid.  PEDV viremia was clearly detected in 3 of the 5 contact controls and 9 of the 22 inoculated animals. No detectable viremia was detected in any of the aerosol control animals. The raw data suggest that there seems to be a correlation with viremia and extended duration of shedding either fecal or nasal.  Serological data (IFA) show that pre-inoculation samples were negative and that there was significant seroconversion in all of the inoculated and contact control animals. There is no evidence of seroconversion in the aerosol control group in spite of the clear demonstration of PEDV nucleic acid in nasal and oral fluid samples. 

Atrophic enteritis was observed in the jejunum and ileum of affected piglets from 2 to 8 DPI, and corresponded to positive antigen detection by IHC. Mesenteric lymph node and small intestine were the primary sites of antigen detection by IHC and tissue qRT-PCR, and most inoculated group A piglets in were qRT-PCR positive in the intestinal tissue samples out to the end of the study. Tissue blocks were sent to Dr. Madson at ISU for PEDV preliminary immunohistochemistry (IHC) evaluation.  IHC results were subsequently confirmed and results expanded at KSVDL.  The only samples that tested positive for the presence of viral antigen were tissues from the GI tract.  Turbinates, trachea, lung, bronchial lymph nodes, spleen, and other visceral tissues were all negative for PEDV as evaluated by IHC.

A complete set of serum samples has been provided to 5 laboratories (~1,200 samples) for assay development/standardization.  In addition, 3 complete sets of oral fluid samples and tissues have been provided to other laboratories.  These samples have been used for assay development and standardization across the different diagnostic laboratories.

The experimental results demonstrate that aerosol transmission did not occur in this study.  These results seem to be in conflict with reports from the field that implicate aerosol transmission, but lack confirmation via bioassay.  Factors like disinfectant and ultraviolet inactivation of PEDV, sensitivity of the indicator animal (nursing pigs vs. weaned pigs) and infectious dose as a function of route of exposure need to be investigated in order to gain insight into modes of transmission of PEDV. 

The tissue PCR positivity for PED nucleic acid at day 43 post inoculation was an unexpected finding which provides insight into virus carriage and potential transmission of the virus long after the clinical disease has abated.  In view of these findings, additional animal co-mingle studies will need to be conducted to determine the actual duration of horizontal transmission between infected and naïve pigs.