1. Iowa State University College of Veterinary Medicine Diagnostic sampling strategies and classification of breeding herds for PRRS virus Pilot training school in PRRS diagnostics, 2012 Zagreb, Croatia Dr. Derald Holtkamp September 24, 2012

2. Iowa State University College of Veterinary Medicine Objective Provide a herd classification system for describing the PRRSV status of herds, based upon a set of definitions that reflect the biology and ecology of PRRS virus Why? –Required for implementation of regional and national efforts towards PRRSV control and elimination –Necessary to facilitate communication between veterinarians, swine producers, genetic companies, and other industry participants

3. Iowa State University College of Veterinary Medicine Background A definitions committee was formed to develop a herd classification system for describing the PRRS virus status of herds in July of 2009 The committee was formed jointly by –American Association of Swine Veterinarians (AASV) –United States Department of Agriculture (USDA) PRRS-CAP

4. Iowa State University College of Veterinary Medicine Background Committee was chaired by Dr. Bob Morrison and composed of researchers and veterinarians from private practice, industry, and representatives from the American Association of Swine Veterinarians (AASV) and National Pork Board (NPB) –Drs. Dyneah Augsburger, Lisa Becton, Steve Henry, Derald Holtkamp, Dale Polson, Max Rodibaugh, Bob Rowland, Harry Snelson, Barb Straw, Montse Torremorell, Paul Yeske, Jeff Zimmerman (in alphabetical order) Input was also received from the AASV PRRS Task Force

5. Iowa State University College of Veterinary Medicine Background The herd classification system proposed by the committee was approved by the AASV Board of Directors on March 9, 2010 –A recommendation from the AASV PRRS Task force was made to the Board to approve the herd classification system A manuscript describing the herd classification system and related terminology has been published in the Journal of Swine Health and Production (JSHAP) as a peer reviewed commentary Holtkamp D.J., Polson D.D., Torremorell M. and committee members Morrison B. (chair), Classen D.M., Becton L., Henry S., Rodibaugh M.T., Rowland R.R., Snelson H., Straw B., Yeske P., Zimmerman J. 2011. Terminology for classifying swine herds by PRRS virus status. J. Swine Health Prod. 19:1 44-56.

6. Iowa State University College of Veterinary Medicine Basis for classification of a herd The herd classification system is based upon the shedding and exposure status of the herd –Shedding status is negative, uncertain or positive –Exposure (current or past infection) status is negative or positive

7. Iowa State University College of Veterinary Medicine Diagnostic evidence is based upon periodic herd tests Rely upon PCR and ELISA For the purpose of establishing a negative or uncertain shedding status and negative exposure status, no distinction is made between diagnostic results that are positive due to vaccine usage versus those due to wild-type virus exposure

8. Iowa State University College of Veterinary Medicine What is an “uncertain” shedding status An uncertain shedding status is used when diagnostic data is available to suggest shedding may have ceased in a herd but without sufficient confidence to support a negative shedding status –Lack of power in the sampling and testing strategy! An uncertain shedding status is considered transitory in the progress towards eliminating PRRS virus from an infected herd

9. Iowa State University College of Veterinary Medicine Classification system for breeding herds Herd categoryShedding status Exposure status Positive Unstable (I) Positive Positive Stable (II-A) Uncertain Positive Positive Stable (undergoing elimination) (II-B) Uncertain – undergoing elimination Positive Provisional Negative (III) NegativePositive Negative (IV) Negative Roman numerals were assigned to each category to emphasize that the status of herds must follow a specific order as they progress from Positive Unstable to Negative

10. Iowa State University College of Veterinary Medicine Classification of breeding herds – I Positive shedding and exposure status Herd categoryCriteria Supporting Evidence Required I “Positive Unstable” default Herds that do not meet the criteria for any of the other categories (II through IV) are category I by default. None required.

11. Iowa State University College of Veterinary Medicine Classification of breeding herds – II Uncertain shedding and positive exposure status Herd category Criteria Supporting Evidence Required II-A “Positive Stable” Starts after a 90 day period of sustained lack of viremia in weaning age pigs and no clinical signs of PRRS in the breeding herd. Starts after a 90 day period of sustained lack of viremia in weaning age pigs and no clinical signs of PRRS in the breeding herd. has not Herd has not initiated an elimination program. Test serum from weaning age pigs by PCR. No positive results over a 90 day period (4 consecutive negative herd tests sampling every 30 days or more frequently) and no clinical signs consistent with PRRS observed in breeding herd. II-B “Positive Stable” (undergoing elimination) Same as II-A except;has Same as II-A except; Herd has initiated an elimination program and intends to become Negative. Same as II-A Same as II-A

12. Iowa State University College of Veterinary Medicine Definition of elimination & when it begins Definition Initiation of elimination 1) cease entry of PRRS virus positive animals and 2) discontinue intentional exposure to live virus. Elimination by any method requires 1) cease entry of PRRS virus positive animals and 2) discontinue intentional exposure to live virus. Elimination begins when Elimination begins when last seropositive breeding replacements are introduced or when the last intentional exposure to any live PRRS virus occurs in the herd, whichever is later. depopulation / repopulationherd rollovers For the breeding herd all PRRS virus elimination procedures can be placed into two categories: depopulation / repopulation and herd rollovers

13. Iowa State University College of Veterinary Medicine Classification of breeding herds – III Negative shedding and positive exposure status Herd categoryCriteria Supporting Evidence Required Provisional Negative (III) Only for herds that have completed a herd rollover Only for herds that have completed a herd rollover Starts 60 days after “negative breeding replacements” are first introduced during a herd rollover with diagnostic evidence that they remain uninfected Starts 60 days after “negative breeding replacements” are first introduced during a herd rollover with diagnostic evidence that they remain uninfected. If growing pigs are present at the same premises, a confirmation of negative exposure status in that sub- population is also required. Test serum from negative breeding replacements by ELISA. No positive results, after ruling out false positives, at least 60 days after the initial introduction of negative breeding replacements. Test serum from growing pigs by ELISA. No positive results, after ruling out false positives

14. Iowa State University College of Veterinary Medicine Classification of breeding herds - IV Negative shedding and exposure status For herds established Negative by “herd rollover” Herd categoryCriteria Supporting Evidence Required Negative (IV) All breeding animals in the herd are seronegative by ELISA Test serum from adult breeding animals by ELISA. No positive results, after ruling out false positives, subsequent to completion of rollover -confirmed by breeding animal inventory lists from production records.

15. Iowa State University College of Veterinary Medicine Recommended protocol to assess PRRS virus status of Category II-A or II-B breeding herds “rule out”Trying to “rule out” that virus is circulating in the herd In other words we want to say with some confidence that PRRS virus is not being shed and transmitted to other pigs in the herd Need to choose the right test –Need a sensitive test Need to look in the right place Need to carefully choose sample size and frequency –Prevalence is likely low as we are transitioning from Category I to II so we need sufficient sample size to find the small percentage of animals that may still be shedding virus

16. Iowa State University College of Veterinary Medicine Recommended protocol to assess PRRS virus status of Category II-A or II-B breeding herds Requirements Test(s) performedPCR Animals testedWeaning age pigs Specimen(s) collectedSerum (blood, notch/swab, tail/swab) Sampling or whole-herd testing (every animal in population)Sampling If sampling; Targeted subpopulation (if any) sampledLight weight males from gilt litters may increase sensitivity (Optional) Systematic sampling procedureOne pig per litter, random Minimum number of samples per herd test30 samples - Determined by target prevalence to be detected of 10% and 95% confidence level for any population size Pooling strategies (if any)Pools of 5 Procedures to rule out false positivesNone Minimum number of periodic herd testsMinimum of 4 to account for variation in prevalence and increase confidence of finding positives if present Frequency of herd tests (minimum frequency)Every 30 days or more frequently to confirm status Frequency to reconfirm status after initial tests depends upon reason for classifying herd Decision rules that classify the herdNone positive over a 90 day period (4 consecutive negative herd tests if sampling every 30 days); No clinical signs in breeding herd Target prevalence to be detected10% Confidence level (for any population size)> 95% This is the test of choice

17. Iowa State University College of Veterinary Medicine Recommended protocol to assess PRRS virus status of Category II-A or II-B breeding herds Requirements Test(s) performedPCR Animals testedWeaning age pigs Specimen(s) collectedSerum (blood, notch/swab, tail/swab) Sampling or whole-herd testing (every animal in population)Sampling If sampling; Targeted subpopulation (if any) sampledLight weight males from gilt litters may increase sensitivity (Optional) Systematic sampling procedureOne pig per litter, random Minimum number of samples per herd test30 samples - Determined by target prevalence to be detected of 10% and 95% confidence level for any population size Pooling strategies (if any)Pools of 5 Procedures to rule out false positivesNone Minimum number of periodic herd testsMinimum of 4 to account for variation in prevalence and increase confidence of finding positives if present Frequency of herd tests (minimum frequency)Every 30 days or more frequently to confirm status Frequency to reconfirm status after initial tests depends upon reason for classifying herd Decision rules that classify the herdNone positive over a 90 day period (4 consecutive negative herd tests if sampling every 30 days); No clinical signs in breeding herd Target prevalence to be detected10% Confidence level (for any population size)> 95% We don’t have unlimited money available for diagnostics

18. Iowa State University College of Veterinary Medicine Recommended protocol to assess PRRS virus status of Category II-A or II-B breeding herds Requirements Test(s) performedPCR Animals testedWeaning age pigs Specimen(s) collectedSerum (blood, notch/swab, tail/swab) Sampling or whole-herd testing (every animal in population)Sampling If sampling; Targeted subpopulation (if any) sampledLight weight males from gilt litters may increase sensitivity (Optional) Systematic sampling procedureOne pig per litter, random Minimum number of samples per herd test30 samples - Determined by target prevalence to be detected of 10% and 95% confidence level for any population size Pooling strategies (if any)Pools of 5 Procedures to rule out false positivesNone Minimum number of periodic herd testsMinimum of 4 to account for variation in prevalence and increase confidence of finding positives if present Frequency of herd tests (minimum frequency)Every 30 days or more frequently to confirm status Frequency to reconfirm status after initial tests depends upon reason for classifying herd Decision rules that classify the herdNone positive over a 90 day period (4 consecutive negative herd tests if sampling every 30 days); No clinical signs in breeding herd Target prevalence to be detected10% Confidence level (for any population size)> 95% This is where we expect to find virus

19. Iowa State University College of Veterinary Medicine Recommended protocol to assess PRRS virus status of Category II-A or II-B breeding herds Requirements Test(s) performedPCR Animals testedWeaning age pigs Specimen(s) collectedSerum (blood, notch/swab, tail/swab) Sampling or whole-herd testing (every animal in population)Sampling If sampling; Targeted subpopulation (if any) sampledLight weight males from gilt litters may increase sensitivity (Optional) Systematic sampling procedureOne pig per litter, random Minimum number of samples per herd test30 samples - Determined by target prevalence to be detected of 10% and 95% confidence level for any population size Pooling strategies (if any)Pools of 5 Procedures to rule out false positivesNone Minimum number of periodic herd testsMinimum of 4 to account for variation in prevalence and increase confidence of finding positives if present Frequency of herd tests (minimum frequency)Every 30 days or more frequently to confirm status Frequency to reconfirm status after initial tests depends upon reason for classifying herd Decision rules that classify the herdNone positive over a 90 day period (4 consecutive negative herd tests if sampling every 30 days); No clinical signs in breeding herd Target prevalence to be detected10% Confidence level (for any population size)> 95% How many and how frequently to sample

20. Iowa State University College of Veterinary Medicine Recommended protocol to assess PRRS virus status of Category II-A or II-B breeding herds Requirements Test(s) performedPCR Animals testedWeaning age pigs Specimen(s) collectedSerum (blood, notch/swab, tail/swab) Sampling or whole-herd testing (every animal in population)Sampling If sampling; Targeted subpopulation (if any) sampledLight weight males from gilt litters may increase sensitivity (Optional) Systematic sampling procedureOne pig per litter, random Minimum number of samples per herd test30 samples - Determined by target prevalence to be detected of 10% and 95% confidence level for any population size Pooling strategies (if any)Pools of 5 Procedures to rule out false positivesNone Minimum number of periodic herd testsMinimum of 4 to account for variation in prevalence and increase confidence of finding positives if present Frequency of herd tests (minimum frequency)Every 30 days or more frequently to confirm status Frequency to reconfirm status after initial tests depends upon reason for classifying herd Decision rules that classify the herdNone positive over a 90 day period (4 consecutive negative herd tests if sampling every 30 days); No clinical signs in breeding herd Target prevalence to be detected10% Confidence level (for any population size)> 95% Will save money and time - OK?

21. Iowa State University College of Veterinary Medicine Recommended protocol to assess PRRS virus status of Category II-A or II-B breeding herds Requirements Test(s) performedPCR Animals testedWeaning age pigs Specimen(s) collectedSerum (blood, notch/swab, tail/swab) Sampling or whole-herd testing (every animal in population)Sampling If sampling; Targeted subpopulation (if any) sampledLight weight males from gilt litters may increase sensitivity (Optional) Systematic sampling procedureOne pig per litter, random Minimum number of samples per herd test30 samples - Determined by target prevalence to be detected of 10% and 95% confidence level for any population size Pooling strategies (if any)Pools of 5 Procedures to rule out false positivesNone Minimum number of periodic herd testsMinimum of 4 to account for variation in prevalence and increase confidence of finding positives if present Frequency of herd tests (minimum frequency)Every 30 days or more frequently to confirm status Frequency to reconfirm status after initial tests depends upon reason for classifying herd Decision rules that classify the herdNone positive over a 90 day period (4 consecutive negative herd tests if sampling every 30 days); No clinical signs in breeding herd Target prevalence to be detected10% Confidence level (for any population size)> 95% How do we use the individual animal test results to make a diagnosis for the herd (ie. classification)?

22. Iowa State University College of Veterinary Medicine Recommended protocol to assess PRRS virus status of Category III breeding herds Requirements Test(s) performedELISA Animals testedNegative breeding replacements (negative when entered – serve as sentinels) Specimen(s) collectedSerum Sampling or whole-herd testing (every animal in population)Sampling If sampling; Targeted subpopulation (if any) sampledNone Systematic sampling procedureRandom sample from multiple ages of breeding replacements Minimum number of samples per herd test60 samples - Determined by target prevalence to be detected of 5% and 95% confidence level for any population size Pooling strategies (if any)None Procedures to rule out false positivesFor positive and suspect samples, retest by ELISA and perform IFA and PCR; resample population if still positive Minimum number of periodic herd tests 1 Frequency of herd tests (minimum frequency)1 at least 60 days after initial introduction of negative breeding replacements Frequency to reconfirm status after initial test depends upon reason for classifying herd Decision rules that classify the herdNo positive after ruling out false positives Target prevalence to be detected5% Confidence level (for any population size)95%

23. Iowa State University College of Veterinary Medicine Recommended protocol to assess PRRS virus status of Category IV herds established by herd rollover Requirements Test(s) performedELISA Animals testedAdult breeding animals Specimen(s) collectedSerum Sampling or whole-herd testing (every animal in population)Sampling If sampling; Targeted subpopulation (if any) sampledNone Systematic sampling procedure (if any)Random sample from multiple parities and stages of gestation and boars Minimum number of samples per herd test60 samples - Determined by target prevalence to be detected of 5% and 95% confidence level for any population size Pooling strategies (if any)None Procedures to rule out false positivesFor positive and suspect samples, retest by ELISA and perform IFA and PCR; resample if still positive Minimum number of periodic herd tests1 Frequency of herd tests (minimum frequency)1 test after the herd has been category III for a minimum of 1 year Frequency to reassess status after initial test depends upon reason for classifying herd Decision rules that classify the herdNone positive after ruling out false positives Target prevalence to be detected5% Confidence level (for any population size)95%

24. Iowa State University College of Veterinary Medicine Acknowledgements American Association of Swine Veterinarians (AASV) PRRS-CAP, USDA NIFA Award 2008-55620-19132 National Pork Board (NPB)