Peer reviewed Case study Three cases of porcine respiratory disease complex associated with porcine circovirus type 2 infection

Perry A. Harms, DVM, MS; Patrick G. Halbur, DVM, PhD; Steven D. Sorden, DVM, PhD, Dipl ACVP

Summary cal cases are described in which there were pneumonia cases. Porcine circovirus type 2 Pneumonia associated with porcine concurrent infections with PCV2 and was diagnosed in 22% of cases, SIV in circovirus type 2 (PCV2) is being diag- PRRSV, PCV2 and SIV, and PCV2 and M 17%, and M hyopneumoniae in 14%. nosed in an increasing number of respira- hyopneumoniae. Prolonged and unusually Porcine circovirus type 2 was commonly tory disease cases in growing swine. Mul- severe clinical disease, unique histologic found in combination with other patho- tiple viral infections are commonly lesions of lymphoid depletion and airway gens. Of the 694 cases that were diagnosed diagnosed in cases of porcine respiratory fibrosis, and abundant PCV2 antigen with PCV2-associated pneumonia, both disease complex (PRDC) at the Iowa State within these lesions suggest that PCV2 is PCV2 and PRRSV were demonstrated in University Veterinary Diagnostic Labora- playing a pathogenic role in PRDC. 388 (56%), which is greater than the 42% tory. In the year 2000, porcine reproduc- Keywords: swine, porcine circovirus type prevalence of PRRSV in the total pool of tive and respiratory syndrome virus 2, porcine reproductive and respiratory pneumonia cases, suggesting an interaction (PRRSV) was present in 42% of these syndrome virus, swine influenza virus, por- between these two viruses. Concurrent in- cases, PCV2 was present in 22%, swine cine respiratory disease complex fection with PCV2 and M hyopneumoniae influenza virus (SIV) was present in 19%, was confirmed in 134 of the 694 cases and Mycoplasma hyopneumoniae was Received: May 14, 2001 (19%), and concurrent infection with present in 22%. In this report, three clini- Accepted: August 7, 2001 PCV2 and SIV was demonstrated in 83 cases (12%).

espiratory disease is a major health in multiple organs, such as intestine, kid- Case One: Concurrent infection problem in growing swine in the ney, lymphoid tissue, liver, and lung. Inter- with PCV2 and PRRSV United States. Cases of respiratory stitial pneumonia is evident histologically A group of 2000 pigs averaging 34 kg ex- R 3, 5–7 1–4, 8 disease involving multiple pathogens are in many cases. Several reports perienced respiratory disease 2 to 3 weeks often referred to as porcine respiratory dis- describe cases of PMWS associated with after placement in a finishing barn. The ease complex (PRDC). Porcine reproduc- PCV2; however, the literature lacks clinical primary clinical signs, observed in 10 to tive and respiratory syndrome virus reports describing the involvement of 15% of the pigs, were inactivity, anorexia, (PRRSV) and swine influenza virus (SIV) PCV2 in PRDC. The following case re- and dyspnea. Though anorexia was ob- are the common viral agents associated ports exemplify the association of PCV2 served in some pigs, wasting was not a pre- with PRDC. Mycoplasma hyopneumoniae, with PRDC in the accessions of a US diag- senting sign. At necropsy, lungs of affected Pasteurella multocida, Streptocccus suis, and nostic laboratory with a heavy swine case pigs failed to collapse and were diffusely other bacterial agents may also be associ- load. mottled, red to purple, and rubbery. No ated. Porcine circovirus type 2 may be other gross lesions were present. Fresh and identified in cases of pneumonia. However, Case reports formalin-fixed tissues (lung, tonsil, spleen, it has most often been associated with In the year 2000, swine pneumonia was and ileum) from two pigs representative of postweaning multisystemic wasting syn- diagnosed in 3163 cases presented to the the group (healthy except for respiratory 1–4 drome (PMWS), which is characterized Iowa State University Veterinary Diagnos- signs) were submitted to the ISU-VDL by wasting, dyspnea, and occasionally jaun- tic Laboratory (ISU-VDL)(Table 1). Diag- early in the course of the outbreak. dice or pallor. This syndrome commonly nosis was based on gross and microscopic occurs in pigs between 8 and 16 weeks of lesions and demonstration of an agent by Histopathology revealed severe, diffuse, age. The defining histologic features of isolation, antigen detection, or identifica- lymphohistiocytic interstitial pneumonia PMWS include depletion of lymphocytes tion of nucleic acid by polymerase chain with marked type 2 pneumocyte hyperpla- in lymphoid tissues, occasional intracyto- reaction (PCR) or in situ hybridization in sia and hypertrophy, necrotic cellular de- plasmic inclusion bodies, and lympho- the tissues. The most common etiologic bris within alveolar spaces, moderate multi- histiocytic to granulomatous inflammation diagnosis was PRRSV, in 42% of the swine focal suppurative bronchopneumonia, and mild peribronchiolar fibrosis. Mild lym- PAH, PGH, SDS: Veterinary Diagnostic and Production Animal Medicine, College of Veterinary phocyte depletion was observed in Peyer’s Medicine, Iowa State University, Ames, IA 50011. patches, but not in tonsil or spleen. Nu- This article is available online at http://www.aasv.org/shap.html. merous cells positive for PCV2 antigen were detected in the alveolar septa and Harms PA, Halbur PG, Sorden SD. Three cases of porcine respiratory disease complex associated with porcine circovirus type 2 infection. J Swine Health Prod. 2002;10(1):27–30. around bronchioles by immunohistochem- Journal of Swine Health and Production — Volume 10, Number 1 27 airways (Figure 2). Swine influenza virus Table 1: Pathogens identified in 3163 swine pneumonia cases at the Iowa State University Veterinary Diagnostic Laboratory in the year 2000. H1N1 and Pasteurella multocida type A were isolated from the lung tissue. Immu- nohistochemistry did not detect PRRSV No. of cases Pathogen (% of total) antigen, and direct IFA on frozen lung sec- tions did not detect M hyopneumoniae anti- P4orcine reproductive and respiratory syndrome virus 1)32 (42% gen. The diagnosis was bronchiolitis and Pasteurella multocida 7)15 (23% bronchopneumonia due to SIV, PCV2, P4orcine circovirus type 2 6)9 (22% and P multocida infections. S9wine influenza virus 5)5 (17% The outbreak lasted for approximately 2 Mycoplasma hyopneumoniae 4)52 (14% weeks, with the group experiencing mortal- Streptococcus s8pp 3)9 (13% ity near 10% despite aggressive antibiotic Haemophilus parasuis 2)41 (8% therapy. Five percent of the pigs failed to Salmonella s5erovars 2)2 (7% reach market weight and were sold at a re- Actinobacillus pleuropneumoniae 1)73 (5% duced value.

Case Three: Concurrent infection istry (IHC) using antibodies derived Lung was the only formalin-fixed tissue with PCV2 and M hyopneumoniae against PCV2, and alveolar macrophages examined. Histopathology revealed multi- A group of 300 pigs weighing 77 to 82 kg were positive for PRRSV antigen by IHC.9 focal bronchial and bronchiolar epithelial experienced 10% morbidity and 2% mor- Small numbers of non-pathogenic bacteria necrosis, squamous metaplasia, and hyper- tality, with clinical signs of gauntness and were isolated from the lungs, spleen, and plasia. Moderate to marked multifocal peri- dyspnea, over a 2- to 3-week period. Two small intestine. Mycoplasma hyopneumoniae bronchial and peribronchiolar fibrosis and pigs were selected to represent the more was not detected by direct immuno- macrophages were present and often ex- recently affected pigs in the group. Nec- fluoresence assay (IFA), nor was SIV de- tended into the airway lamina propria. In ropsy revealed dark purple cranioventral tected by IHC in the lung tissues. On the addition, there was multifocal suppurative consolidation of 20 to 30% of the lungs, basis of these findings, the diagnosis was bronchopneumonia. Swine influenza virus and no other lesions. severe interstitial pneumonia due to con- antigen was identified by IHC in the nuclei Histopathology revealed fibrinosuppurative current PCV2 and PRRSV infections. of numerous bronchial and bronchiolar pleuritis, multiple neutrophil-rich thrombi, epithelial cells and occasional cells within Although the pigs were treated with antibi- mild to moderate peribronchiolar lym- the lung parenchyma (Figure 1). Immuno- otics in the water and feed, new pigs con- phoid hyperplasia, and mild fibrosis of the histochemistry also demonstrated large tinued to become affected for a 3- to 4- peribronchiolar alveolar septa. Lymphoid amounts of PCV2 antigen both within week period, with a total of 5% of the pigs tissues were not examined histologically. bronchial and bronchiolar epithelial cells removed and placed in hospital pens. Many Immunohistochemistry on formalin-fixed pigs continued to lose body condition de- and in mononuclear cells surrounding the spite individual treatments and were Figure 1: euthanized for humane reasons. Total mor- Section of lung from a pig with pneumonia associated with swine influenza virus (SIV) and porcine circovirus type 2, showing SIV antigen tality for the group was 9%, with an addi- (arrows) primarily within the bronchiolar epithelium (Case 2). tional 8% marketed as cull pigs at a

significantly reduced value. ➜ Case Two: Concurrent infection ➜ with PCV2 and SIV This farm received feeder pigs weighing 23 kg from a single source every 3 weeks. One group of 700 pigs, housed in outside lots and weighing 54 to 64 kg, experienced an acute onset of respiratory disease with clini- cal signs (coughing, dyspnea, and lethargy) in 40% of the pigs. Field necropsies re- vealed tan-to-red, mottled lungs that failed to collapse, and cranioventral consolidation affecting 20 to 30% of the lung. No other gross lesions were present. On day 3 of the outbreak, necropsies were performed on two pigs selected to represent the group, and fresh and formalin-fixed lung tissues were submitted to the ISU-VDL for fur- ther investigation.

28 Journal of Swine Health and Production — January and February, 2002 absence of characteristic lesions suggests Figure 2: Section of lung from a pig with pneumonia associated with swine influenza virus and porcine cirovirus type 2 (PCV2), showing large numbers of that neither virus was contributing to the PCV2-antigen-positive cells (arrows) within the bronchiolar epithelium and disease in these pigs. within alveolar septa and spaces throughout the lung (Case 2). Section from the same pig as in Figure 1. Lymphoid depletion was mild and incon- sistent in Case One, and in Cases Two and Three, lymphoid tissues were not exam- ined, so the full extent of PCV2 infection

is not known. Some pigs on which necrop- ➜ sies were performed may have had undetec- ted lymphoid lesions characteristic of PMWS and might have progressed to de-

velop PMWS. The variability in growth

rate subsequent to the respiratory disease in ➜ the pigs in these cases may have been due ➜ to chronic effects of respiratory disease or development of PMWS in some animals. Regardless of their PMWS status, these cases are significant because of the contri- bution of PCV2 to the respiratory disease observed. Many PRDC outbreaks may be complicated by PMWS. These two diseases occur in pigs of similar ages; therefore, consideration of PCV2 as a possible patho- gen in PRDC should be independent of the diagnosis of PMWS. lung tissue demonstrated large amounts of period, experiencing severe respiratory dis- Infection with PCV2 was clearly demon- PCV2 antigen within the alveolar septa ease with a prolonged recovery period, and strated in these cases, but because most and macrophages surrounding bronchioles. a large percentage of the affected pigs died pigs seroconvert to PCV2 prior to slaugh- Mycoplasma hyopneumoniae antigen was or did not recover well (17% deaths and ter,11,12 it might be argued that PCV2 in- detected by direct IFA on frozen lung tis- culls in this case). Interstitial pneumonia fection was incidental. Disease in these pigs sue sections. Large numbers of P multocida with peribronchiolar fibrosis and identi- occurred during the early- to mid-finishing type A were isolated from the lungs. Nei- fication of both PCV2 and PRRSV in the period, which coincides with the time ther PRRSV nor SIV was detected by IHC tissues are common findings in similar when seroconversion to PCV2 commonly or virus isolation. The diagnosis was cases. occurs.12 However, we believe that PCV2 PCV2-associated bronchointerstitial pneu- played a significant role in the disease pro- Case Two (concurrent infection with monia with mycoplasmal pneumonia and cess in these herds for three reasons. First, PCV2 and SIV) represents acute outbreaks pneumonic pasteurellosis. The pleuritis and clinical disease was longer, more severe, and of respiratory disease. This case differs from pulmonary arterial thrombosis suggested a resulted in greater numbers of low-weight Case One in the sudden onset of disease concurrent or earlier bacterial septicemia. pigs than would be expected with PRRSV and is also in contrast to the chronic nature or SIV alone. Second, fibrous tissue prolif- This group of pigs continued to cough de- of a “wasting“ disease as previously de- eration around airways has been associated spite treatment with chlortetracycline (22 scribed for PMWS. Case Two had an acute with PCV2 infection,13 but has not been mg per kg) in the feed for an extended pe- clinical onset typical of a swine influenza induced by experimental PRRSV or SIV riod. The group took longer to close out outbreak, but microscopic lesions, such as inoculation. Third, the unique clinical than normal. Mortality was less than 5%, peribronchiolar fibrosis and infiltrates of signs and microscopic lesions observed but many of the deaths occurred in older macrophages, were more chronic, recovery were associated with numerous intrale- pigs at heavier weights, when investments was prolonged, and mortality was much sional PCV2-antigen-positive cells. in feed, time, and facilities are greater. higher than is commonly associated with swine influenza.10 In the year 2000, IHC for PCV2 was per- Discussion formed on 2030 swine cases submitted to Case Three (concurrent infection with These cases represent PRDC in pigs in- the ISU-VDL at the request of the submit- PCV2 and M hyopneumoniae) is included fected concurrently with a recognized ting veterinarians or due to suspicious because the clinical features are consistent swine respiratory pathogen and PCV2. clinical signs or lesions, but was positive in Case One (concurrent infection with with those commonly seen in PRDC, but only 694 cases (34%). The pathogenesis of PCV2 and PRRSV) is representative both no evidence of PRRSV or SIV, the most PCV2-associated disease is complex, and clinically and diagnostically of a large num- frequently identified viruses, was demon- the role of PCV2 as a primary pathogen in ber of cases of swine respiratory disease strated. Failure to detect PRRSV or SIV by PMWS or as a secondary respiratory submitted to the ISU-VDL. These pigs IHC or virus isolation does not preclude pathogen is unclear. The classification of were affected in the early- to mid-finishing the presence of either virus. However, the Journal of Swine Health and Production — Volume 10, Number 1 29 pathogens as primary or secondary often Implications 9. Sorden SD, Harms PA, Nawagitgul P, Cavanaugh D, Paul PS. Development of a polyclonal-antibody- oversimplifies the interactions that occur in • Disease associated with PCV2 based immunohistochemical method for the detec- PRDC. Studies using a M hyopneumoniae infection is most often found in tion of type 2 porcine circovirus in formalin-fixed, and PRRSV coinfection model show that conjunction with one or more other paraffin-embedded tissue. J Vet Diagn Invest. these pathogens can act synergistically to pathogens. 1999;11:528–530. 10. Easterday BC, Reeth KV. Swine influenza. In: produce more severe respiratory disease • Infection with PCV2 is commonly Straw BE, D’Allaire S, Mengeling WL, Taylor DJ, than either pathogen alone.14 There is ex- demonstrated in outbreaks of PRDC eds. Diseases of Swine. 8th ed. Ames, IA: Iowa State perimental evidence that synergism occurs and may be contributing to the University Press;1999:277–290. between PCV2 and parvovirus15 and be- severity of the lesions and the severity 11. Tischer I, Bode L, Peters D, Pociuli S, Germann 16, 17 B. Distribution of antibodies to porcine circovirus tween PCV2 and PRRSV, producing and duration of clinical disease in in swine populations of different breeding farms. lesions and clinical signs of PMWS in both these cases. Arch Virol. 1995;140:737–743. models as well as a respiratory component • Lesions associated with PCV2 12. Walker IW, Konoby CA, Jewhurst VA, McNair in the concurrent-PRRSV-infection model. infections can be found in acute I, McNeilly F, Meehan BM, Cottrell TS, Ellis JA, Allan GM. Development and application of a com- While the primary or secondary role of respiratory disease and are not limited petitive enzyme-linked immunosorbent assay for the PCV2 in PMWS or PRDC continues to be to a wasting syndrome. detection of serum antibodies to porcine circovirus debated, the demonstration of unique le- type 2. J Vet Diagn Invest. 2000;12:400–405. sions in the lungs associated with large Acknowledgements 14. Thacker EL, Halbur PG, Ross RF, Thanawongnuwech R, Thacker BJ. Mycoplasma amounts of viral antigen suggest that We gratefully acknowledge Dr Jeffery hyopneumoniae potentiation of porcine reproduc- PCV2 is an important pathogen in some Blythe, Dr Douglas Quam, and Dr Tho- tive and respiratory syndrome virus-induced pneu- cases of PRDC. mas Pollack for the original submissions monia. J Clin Microbiol. 1999;37:620–627. and follow-up on these cases. 15. Kennedy S, Moffett D, McNeilly F, Meehan B, Although we do not have a clear means to Ellis J, Krakowka S, Allan GM. Reproduction of solve the problem, a diagnosis of PMWS or lesions of postweaning multisystemic wasting syn- References — refereed drome by infection of conventional pigs with por- PCV2-associated disease is important for 2. Meehan BM, McNeilly F, Todd D, Kennedy S, cine circovirus type 2 alone or in combination with several reasons. Good production practices Jewhurst VA, Ellis JA, Hassard LE, Clark EG, porcine parvovirus. J Comp Pathol. 2000;122:9–24. such as attention to pig flow, environment, Haines DM, Allan GM. Characterization of novel 16. Allan GM, McNeilly F, Ellis J, Krakowka S, pig density, and nutrition play an impor- circovirus DNAs associated with wasting syndromes Meehan B, McNair I, Walker I, Kennedy S. Experi- in pigs. J Gen Virol. 1998;79:2171–2179. mental infection of colostrum deprived piglets with tant role in preventing many diseases and 3. Morozov I, Sirinarumitr T, Sorden SD, Halbur porcine circovirus 2 (PCV2) and porcine reproduc- are also recommended to moderate the PG, Morgan MK, Yoon KJ, Paul PS. Detection of a tive and respiratory syndrome virus (PRRSV) severity of disease associated with PCV2.8 novel strain of porcine circovirus in pigs with potentiates PCV2 replication. Arch Virol. Secondly, identifying the involvement of postweaning multisystemic wasting syndrome. 2000;145:2421–2429. J Clin Microbiol. 1998;36:2535–2541. 17. Harms PA, Sorden SD, Halbur PG, Bolin S, PCV2 in an outbreak of PRDC enables the 4. Nayar GP, Hamel A, Lin L. Detection and char- Lager K, Morosov I, Paul PS. Experimental repro- veterinarian to develop a more accurate acterization of porcine circovirus associated with duction of severe disease in CD/CD pigs concur- prognosis for the group of pigs. The course postweaning multisystemic wasting syndrome in rently infected with type 2 porcine circovirus and of the disease may be more severe, pro- pigs. Can Vet J. 1997;38:385–386. PRRSV. Vet Pathol. 2001;38:528–539. 5. Rosell C, Segales J, Plana-Duran J, Balasch M, 18. Royer RL, Nawagitgul P, Halbur PG, Paul PS. longed, and refractory to therapy than Rodriguez-Arrioja GM, Kennedy S, Allan GM, Susceptibility of porcine circovirus type 2 to com- PRDC uncomplicated by concurrent McNeilly F, Latimer KS, Domingo M. Pathological, mercial and laboratory disinfectants. J Swine Health PCV2 infection. Understanding the poten- immunohistochemical, and in-situ hybridization Prod. 2001;9:281–284. tial outcome, the veterinarian and producer studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs. References — non refereed can make informed decisions regarding the J Comp Pathol. 1999;120:59–78. 1. Harding JCS, Clark EG. Recognizing and diag- choice and effectiveness of antimicrobial 6. Allan GM, McNeilly F, Kennedy S, Daft B, nosing postweaning multisystemic wasting syn- therapy in controlling secondary bacterial Clarke EG, Ellis JA, Haines DM, Meehan BM, drome (PMWS). Swine Health Prod. 1997;5:201– infections in PRDC. Thirdly, identifying Adair BM. Isolation of porcine circovirus-like vi- 203. ruses from pigs with a wasting disease in the USA 13. Sorden SD, Thacker BJ, Harms PA, PCV2-associated disease in a group of pigs and Europe. J Vet Diagn Invest. 1998;10:3–10. Siranarumitr T, Morosov I, Paul PS. Retrospective allows swine veterinarians to monitor dis- 7. Kiupel M, Stevenson GW, Mittal SK, Clark EG, study of porcine circovirus infection in porcine tis- ease outcomes as they make management Haines DM. Circovirus-like viral associated disease sues submitted to the Iowa State University Veteri- in weaned pigs in Indiana. Vet Pathol. 1998;35:303– nary Diagnostic Laboratory using in situ hybridiza- changes, develop intervention strategies, or 307. tion. Proc Am Assoc Vet Lab Diagn. San Diego, CA. 18 make disinfectant choices for control of 8. Harding F, Clark E, Strokappe J, Willson P, Ellis October 1999;55. the PCV2-associated disease. Systematic J. Postweaning multisystemic wasting syndrome: evaluation of intervention strategies by Epidemiology and clinical presentation. Swine practitioners will help us all in developing Health Prod. 1998;6:249–254. effective control strategies.

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