Date Full Report Received

02/13/2015

Date Abstract Report Received

02/13/2015

Investigation

Institution:
Primary Investigator:
Co-Investigators: Eric A. Nelson, Jane Christopher-Hennings, S. Lawson, A.. Singrey

Since April 2013 with the discovery of porcine epidemic diarrhea virus (PEDV) in the United States, the need for the development of diagnostic tests has been immediate. Real-time polymerase chain reaction (PCR) tests were developed within weeks of first detection to diagnose PEDV quickly and accurately. Next, the need for a test that determined the level and duration of neutralizing or “potentially protective” antibody was evident. Therefore, the objective of this study was to use the fluorescent focus neutralization (FFN) test to determine the levels of neutralizing antibody in sows in two different farms with two different feedback protocols. The first hypothesis was that we would be able to detect neutralizing antibody titers in sow serum within 3-4 weeks after an effective feedback protocol. Our second hypothesis was that the neutralizing antibody levels in serum and in milk at the time of farrowing would correlate to each other, indicating that the serum neutralizing antibodies may be transferred to milk. The third hypothesis was that there would still be detectable neutralizing antibody in the sow serum at 6 months at both sites with effective feedback protocols.

The research was conducted by collecting at least thirty serum samples from sows from two different commercial herds that conducted whole herd feedback protocols with PEDV after initial natural exposure of the virus to each herd. The serum samples were checked for the presence of neutralizing antibodies on the day of the feedback protocol (0 weeks), 3 weeks, ~6 weeks, and then ~6 months (week 24) after initial feedback. Piglet serum samples from corresponding sows were collected along with colostrum and milk samples from the sows. FFN testing was conducted on all samples for the detection of neutralizing antibodies. Inter and intra-comparative pairwise analysis was completed between the two sites and between the different collection dates to look for statistical significance.

The data has concluded that our FFN assay detected neutralizing antibody in sows at both sites by the third week after the initiation of the feedback protocol. The majority of the sow serum collected when the feedback protocol was initiated had titers of <1:20 ( interpretation=”negative),” indicating the whole herd had just been introduced to PEDV and had not developed neutralizing antibody towards the virus previously. After feedback exposure, site A sow serum titers ranged from 1:20 to 1:1280; whereas, site B sow serum ranged from <1:20 to 1:320. Statistical significance between site A and B was found during week 6 ( p-value=”0.008),” with site B having a lower geometric mean titer when compared to site A. This may indicate that differences in feedback protocols may play a role in the level of the PEDV neutralizing antibody titer in sow serum. Site A reported a more aggressive feedback regimen as compared to Site B.

Piglet loss was devastating during initial outbreaks with both sites reporting 100% loss of piglets for 2-3 weeks. Approximately 6 weeks after initial infection, piglet serum was collected from 12-18 day old piglets, and neutralizing antibody was measured by FFN testing. Detectable neutralizing antibodies were measured in 90% (27/30) of site A piglets tested as compared to only 52% (15/29) of site B piglets tested. Clinical signs at site A were reported as insignificant during this time period, but clinical signs at site B were reported as significant, requiring additional PCR and sequence testing. PCR testing indicated that shedding of the PEDV virus at site B was still continuing, and S1 PEDV sequencing confirmed that the shedding was still the same strain of PEDV that was originally introduced to the herd during the initial feedback process. Management practices at site B were re-evaluated and the herd cleaned up by week 24. Both sites A and B had successful duration of protection out to week 24 and successfully introduced sentinel pigs into their system without re-infection of PEDV. At week 24, 100% of sows tested from both site A and B had measurable neutralizing antibody in serum. Confidentiality reasons prevented production data from being shared for site production comparison to correlate to the testing data collected in this study.

Colostrum, milk, and serum were also collected from corresponding sows in site B. The data demonstrated that neutralizing antibody titers were higher in colostrum than serum, as expected. The levels of neutralizing antibodies were similar between serum and milk at both sites A and B. This limited data comparing the level of neutralizing antibody in serum and milk might suggest that measuring the level of neutralizing antibody in serum could be an indicator of the level of neutralizing antibody in the milk that piglets receive from the sow. Collection of serum versus milk might be a more convenient sample to collect and is certainly a better sample to handle for high-throughput laboratory testing.

In conclusion, the objective of this grant was to evaluate a testing method to measure duration of PEDV immunity in sows. At the time of the initiation of this project, the industry did not have any test options for measuring neutralizing “potentially protective” antibodies because PED virus neutralization testing had not been validated for production testing. This study has not only helped in the fine tuning of the development of the testing procedure, but it has established and validated FFN testing in a “real world” setting. The data collected in this study indicate that there might be value to the industry in FFN testing when needed for answering production issues when sites are having recurring infections of PEDV.

In this study, the feedback protocols were different between site A and B (eg. more frequent and several more feedbacks were given for site A vs. site B) which may have resulted in fewer clinical signs and higher neutralizing antibody titers against PEDV for site A at 6 weeks post exposure. However, other factors could have contributed to this difference such as the homogeneity of the feedback for consistent exposure of more sows, genetics, or other unknown factors.

Future studies need to be completed on sites that are having recurring issues with PEDV to determine the level and presence of neutralizing antibody at these sites and correlate the values to sites that do not have re-occurring issues. This data may be helpful in identification of better production protocols at sites with re-occurring infections of PEDV. Another benefit to the industry since the development and validation of the PEDV FFN test is that we can now evaluate the use of the FFN testing as a tool to measure vaccine response. This study has also shown that neutralizing antibodies can be detected in sow serum for at least 24 weeks. Both sites in this study had 100% detection of neutralizing antibody titers at 6 months and were able to successfully incorporate sentinel pigs into their systems without recurrence of PEDV, indicating duration of immunity for this period of time. Further studies with larger data sets are needed to fully understand and answer the question regarding the duration of immunity and how to best interpret results of serological assays, but this pilot study has provided invaluable insight on FFN testing for PEDV in serum, milk, and colostrum.

Contact Information: Travis Clement
Veterinary & Biomedical Sciences Department
Animal Disease Research & Diagnostic Laboratory
Box 2175, North Campus Drive
South Dakota State University
Brookings, SD 57007-1396
travis.clement@sdstate.edu