1. Total Cases of PMWS and PCV2- Associated Disease at ISU-VDL 1993 1994 1995 1996 1997 1998 1999 14 15 18 37 130 449* * First full year using PCV IHC as routine diagnostic tool. 11

2. Cases of pneumonia submitted to the ISU-VDL from 1993-1999 PRRS Mhyo Pmult SIV APP PCV2Dz.120 218 360 117 262 —1993367 357 376 175 235 —1994708 417 563 291 220 01995714 624 638 384 298 11996869 731 653 447 271 20199710X 4X 2X 6X Flat 290XTrend859 894 715 350 230 1111998 1168 867 815 694 178 2901999

3. Postweaning Multisystemic Wasting Syndrome (PMWS) n primarily in 10-20 week old pigs in the U.S. n pneumonia n wasting n anemia, paleness, +/- icteric n +/- diarrhea n 2-50% morbidity (usually < 10%) n mortality of affected pigs may approach 100%

5. Age of Onset of PMWS -5 0 0 5 5 10 15 20 25 30 35 1234567891011121314151617181920 Age in Weeks Cases

6. Gross Lesions of PMWS n Lungs diffusely red-tan, noncollapsed, rubbery, with variable amounts of cranioventral consolidationdiffusely red-tan, noncollapsed, rubbery, with variable amounts of cranioventral consolidation n Lymph nodes diffusely enlarged and tandiffusely enlarged and tan n Other less common lesions Icterus, gastric ulcers, skin lesions (PDNS), enlarged kidneys, swollen or atrophied liverIcterus, gastric ulcers, skin lesions (PDNS), enlarged kidneys, swollen or atrophied liver n Gross lesions are rarely diagnostic - very similar to PRRS n Lungs diffusely red-tan, noncollapsed, rubbery, with variable amounts of cranioventral consolidationdiffusely red-tan, noncollapsed, rubbery, with variable amounts of cranioventral consolidation n Lymph nodes diffusely enlarged and tandiffusely enlarged and tan n Other less common lesions Icterus, gastric ulcers, skin lesions (PDNS), enlarged kidneys, swollen or atrophied liverIcterus, gastric ulcers, skin lesions (PDNS), enlarged kidneys, swollen or atrophied liver n Gross lesions are rarely diagnostic - very similar to PRRS

7. Microscopic Lesions of PMWS n Lung Lesions Bronchial and bronchiolar epithelial attenuation, hyperplasia, erosion, luminal obliteration & epithelial sequestrationBronchial and bronchiolar epithelial attenuation, hyperplasia, erosion, luminal obliteration & epithelial sequestration Lymphohistiocytic, proliferative interstitial pneumoniaLymphohistiocytic, proliferative interstitial pneumonia Peribronchiolar and septal fibroplasiaPeribronchiolar and septal fibroplasia n Lymphoid Lesions: Lymphoid depletion and replacement of follicles with macrophages, intracytoplasmic inclusions n Liver: Hepatocellular necrosis and lymphoplasmacytic inflammation n Kidney: Lymphoplasmacytic interstitial nephritis n Lung Lesions Bronchial and bronchiolar epithelial attenuation, hyperplasia, erosion, luminal obliteration & epithelial sequestrationBronchial and bronchiolar epithelial attenuation, hyperplasia, erosion, luminal obliteration & epithelial sequestration Lymphohistiocytic, proliferative interstitial pneumoniaLymphohistiocytic, proliferative interstitial pneumonia Peribronchiolar and septal fibroplasiaPeribronchiolar and septal fibroplasia n Lymphoid Lesions: Lymphoid depletion and replacement of follicles with macrophages, intracytoplasmic inclusions n Liver: Hepatocellular necrosis and lymphoplasmacytic inflammation n Kidney: Lymphoplasmacytic interstitial nephritis

8. Differential Diagnosis of Necrotizing Bronchitis and Bronchiolitis in Swine n n SIV and PRCV epithelial necrosis epithelial attenuation epithelial hyperplasia mononuclear cuffing n n SIV and PRCV epithelial necrosis epithelial attenuation epithelial hyperplasia mononuclear cuffing n n PCV2 epithelial necrosis epithelial attenuation epithelial hyperplasia mononuclear cuffing peribronchiolar fibrosis luminal obliteration epithelial sequestration n n PCV2 epithelial necrosis epithelial attenuation epithelial hyperplasia mononuclear cuffing peribronchiolar fibrosis luminal obliteration epithelial sequestration

9. Porcine Circovirus: Diagnosis n Clinical signs and gross lesions very similar to PRRS n unique microscopic lesions and inclusion bodies n immunohistochemistry (IHC), in situ hybridization (ISH) to confirm association of virus with lesion n virus isolation and serology available in some labs offered at ISU VDLoffered at ISU VDL ubiquitous virus so value and interpretation of serologic information is difficultubiquitous virus so value and interpretation of serologic information is difficult attempt virus isolation in recurrent cases to have virus available for potential autogenous vaccine productionattempt virus isolation in recurrent cases to have virus available for potential autogenous vaccine production

10. PMWS Case Observations n PMWS concurrent infections confirm PRRSV in 60% of the cases,confirm PRRSV in 60% of the cases, –suspect PRRSV is involved in 80% of the cases other common concurrent infectionsother common concurrent infections –SIV, HPS/S. suis, M. hyo. n Mortality much higher in concurrent infection cases acute SIV + circovirus + M. hyo. pneumonia casesacute SIV + circovirus + M. hyo. pneumonia cases –2-10% mortality in finishers chronic PRDC due to PRRSV + M hyo + PMWSchronic PRDC due to PRRSV + M hyo + PMWS –10-15% mortality in finishers

11. PMWS Case Observations n Duration of disease in a system varies often a single batch problem in smaller, single-site herds that use internal gilt replacementsoften a single batch problem in smaller, single-site herds that use internal gilt replacements increasing number of cases where PMWS has become endemic in multiple building finishing sitesincreasing number of cases where PMWS has become endemic in multiple building finishing sites

12. Porcine Circovirus: Pathogenesis n PMWS strains (Type 2) differ from PK/15 cell culture strains (Type 1) genetically types 1 and 2 are quite differentgenetically types 1 and 2 are quite different –multiplex PCR can differentiate PCV1 is nonpathogenic in growing pigs (Allan et al.)PCV1 is nonpathogenic in growing pigs (Allan et al.) evidence that “shaker pig disease” may be caused by PCV1 or PCV2evidence that “shaker pig disease” may be caused by PCV1 or PCV2 n High seroprevalence of PCV2 but limited cases tested 27 high health herds with no clinical evidence of PMWStested 27 high health herds with no clinical evidence of PMWS –100% or herds seropositive for PCV2 –high percent of animals within a herd positive –passive antibody decay by 10-20 weeks

13. Experimental Reproduction of PMWS n Ellis et al., 1999 reproduced PMWS lesions in CDCD pigs inoculated with tissue filtrates; both PCV2 and PPV detectedreproduced PMWS lesions in CDCD pigs inoculated with tissue filtrates; both PCV2 and PPV detected n Allan et al. 1999; and Kennedy et al. 2000 inoculated colostrum-deprived pigs with PCV2 alone, PPV alone, and PCV2+PPV; reproduced severe disease and lesions typical of PMWS in dually-inoculated group whereas only mild lesions and subclinical disease was reproduced in the PCV2 only groupinoculated colostrum-deprived pigs with PCV2 alone, PPV alone, and PCV2+PPV; reproduced severe disease and lesions typical of PMWS in dually-inoculated group whereas only mild lesions and subclinical disease was reproduced in the PCV2 only group n Krakowka et al. 2000 reproduced disease and PMWS lesions in gnotobiotic pigs co-infected with PCV2 and PPVreproduced disease and PMWS lesions in gnotobiotic pigs co-infected with PCV2 and PPV

14. Experimental PCV2/PRRSV Co-Infection n Field cases suggested interaction n Objective: Reproduce disease seen in field casesReproduce disease seen in field cases Develop model to study the interactionDevelop model to study the interaction Develop model for evaluating intervention strategiesDevelop model for evaluating intervention strategies n Field cases suggested interaction n Objective: Reproduce disease seen in field casesReproduce disease seen in field cases Develop model to study the interactionDevelop model to study the interaction Develop model for evaluating intervention strategiesDevelop model for evaluating intervention strategies

15. Experimental Design n n 4 groups of 3 week old CD/CD pigs Control 9 pigs PCV2 19 pigs PRRSV 13 pigs PRRSV/PCV2 17 pigs n n Intranasal inoculation of PRRSV and PCV2 n n Scheduled necropsies at 7,10,14,21,35,49 days post-inoculation n n 4 groups of 3 week old CD/CD pigs Control 9 pigs PCV2 19 pigs PRRSV 13 pigs PRRSV/PCV2 17 pigs n n Intranasal inoculation of PRRSV and PCV2 n n Scheduled necropsies at 7,10,14,21,35,49 days post-inoculation

16. Clinical Respiratory Scores 0 1 2 3 4 5 6 71011-1415-21 Days Control PCV PRRSV PCV/PRRSV

17. Mean Gross Lung Scores 0 10 20 30 40 50 60 70 80 90 100 7-1011-1415-21 Days % Lung Affected Control PCV PRRSV PCV/PRRSV

18. Summary of Findings: PCV2- PRRSV Co-infection n PCV2 Group Minimal respiratory diseaseMinimal respiratory disease Mortality of 40% between 10-30 DPIMortality of 40% between 10-30 DPI Mild necrotizing bronchiolitis, minimal pneumoniaMild necrotizing bronchiolitis, minimal pneumonia Lymphoid depletion, necrotizing hepatitisLymphoid depletion, necrotizing hepatitis n PRRSV group Moderate respiratory diseaseModerate respiratory disease Mortality = 0%Mortality = 0% Interstitial pneumonia, no airway lesionsInterstitial pneumonia, no airway lesions n PCV2 Group Minimal respiratory diseaseMinimal respiratory disease Mortality of 40% between 10-30 DPIMortality of 40% between 10-30 DPI Mild necrotizing bronchiolitis, minimal pneumoniaMild necrotizing bronchiolitis, minimal pneumonia Lymphoid depletion, necrotizing hepatitisLymphoid depletion, necrotizing hepatitis n PRRSV group Moderate respiratory diseaseModerate respiratory disease Mortality = 0%Mortality = 0% Interstitial pneumonia, no airway lesionsInterstitial pneumonia, no airway lesions

19. Summary of Findings: PCV2-PRRSV Co-infection n PCV2/PRRSV group Severe respiratory diseaseSevere respiratory disease Mortality >90% between 10-21 DPIMortality >90% between 10-21 DPI Necrotizing and ulcerative bronchiolitisNecrotizing and ulcerative bronchiolitis Severe interstitial pneumoniaSevere interstitial pneumonia Lymphoid depletion, necrotizing hepatitisLymphoid depletion, necrotizing hepatitis n PCV2/PRRSV group Severe respiratory diseaseSevere respiratory disease Mortality >90% between 10-21 DPIMortality >90% between 10-21 DPI Necrotizing and ulcerative bronchiolitisNecrotizing and ulcerative bronchiolitis Severe interstitial pneumoniaSevere interstitial pneumonia Lymphoid depletion, necrotizing hepatitisLymphoid depletion, necrotizing hepatitis

20. Experimental Evidence for PRRSV- Induced Potentiation of PMWS n Harms et al, 2000 Reproduction of severe disease and lesions typical of PMWS in CDCD pigs co-infected with PCV2 and PRRSVReproduction of severe disease and lesions typical of PMWS in CDCD pigs co-infected with PCV2 and PRRSV Less severe disease, mortality, and lesions in PCV2 only groupLess severe disease, mortality, and lesions in PCV2 only group No evidence of PPV infectionNo evidence of PPV infection n Allan et al., 2000 Experimental infection of CD piglets with PCV2 and PRRSV potentiates PCV2 replicationExperimental infection of CD piglets with PCV2 and PRRSV potentiates PCV2 replication

21. Activation of the Immune System May Lead to Increased Susceptibility to PCV2-induced PMWS? n Allan et al., 2000 Vet Rec; Krakowka et al., Vet Path, IN PRESS. Activation of macrophages in lymphoid tissues of colostrum fed pigs may lead to potentiation of PCV2 replication and development of clinical disease and lesions typical of PMWS –7/7 colostrum fed pigs inoculated with PCV2 and then immunized with keyhole limpet haemocyanin (in Freund’s incomplete adjuvant) developed clinical disease and lesions typical of PMWS whereas subclinical disease and minimal lesions were reproduced in PCV2-inoculated pigs without immunization –3/14 pigs vaccinated with M. hyo (2 dose) and APP (1 dose) developed PMWS whereas 0/14 unvaccinated pigs developed PMWS

22. Control of PMWS n Confirm PMWS/circovirus by necropsy, histopath, IHC, ISH n Identify farm, site, or system specific concurrent infections eliminate PRRSV from the mix if possibleeliminate PRRSV from the mix if possible –work to avoid simultaneous coinfection –focus on breeding herd stabilization and pig flow eliminate/control SIV with vaccinationeliminate/control SIV with vaccination further explore role of PPV and use of PPV vaccinefurther explore role of PPV and use of PPV vaccine minimize effects of M. hyo. with vaccination and medicationminimize effects of M. hyo. with vaccination and medication treat bacterial coinfectionstreat bacterial coinfections anti-inflammatories (aspirin, ketoprofen) usefulanti-inflammatories (aspirin, ketoprofen) useful

23. PMWS Summary and Conclusions n PMWS has become a major disease in the U.S. n Field and experimental evidence supports a major role for PCV2 in PMWS n Field evidence also supports a role for PCV2 in porcine respiratory disease complex