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Association of porcine circovirus 2 with porcine respiratory disease complex
The Veterinary Journal The Veterinary Journal 166 (2003) 251–256 www.elsevier.com/locate/tvjl
Association of porcine circovirus 2 with porcine respiratory disease complex J. Kim, H.-K. Chung, C. Chae
*
Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Kyounggi-Do, Republic of Korea Accepted 24 September 2002
Abstract A retrospective study was performed on natural cases of porcine respiratory disease complex (PRDC) to determine the association and prevalence of PRDC with porcine circovirus 2 (PCV2) and other co-existing pathogens in Korea. Histologically, alveolar septa were markedly thickened by infiltrates of mononuclear cells. Moderate to marked multifocal peribronchial and peribronchiolar fibrosis were present and often extended into the airway lamina propria. Among the 105 pigs with PRDC, 85 were positive for PCV2, 66 were positive for porcine reproductive and respiratory syndrome virus (PRRSV), 60 were positive for porcine parvovirus (PPV), and 14 were positive for swine influenza virus (SIV). There were 80 co-infections and 25 single infections. A coinfection of PCV2 with another additional bacterial pathogen is frequently diagnosed in PRDC. The combination of PCV2 and Pasteurella multocida (38 cases) was most prevalent followed by PCV2 and Mycoplasma hyopneumoniae (33 cases). The consistent presence of PCV2, but lower prevalence of other viral and bacterial pathogens in all pigs examined with PRDC, has led us to speculate that PCV2 plays an important role in PRDC. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Pneumonia; Porcine circovirus; Porcine respiratory disease complex; Prevalence
1. Introduction Porcine respiratory disease complex (PRDC) is a serious health problem in growing and finishing pigs typically around 16–22 weeks of age. PRDC is characterized by slow growth, decreased feed efficiency, lethargy, anorexia, fever, cough, and dyspnoea (Halbur, 1998; Thacker, 2001). Pneumonia in pigs with PRDC is due to a combination of both viral and bacterial agents, such as porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, and Pasteurella multocida (Halbur, 1998; Thacker, 2001). Although the aetiology of PRDC involves multiple pathogens and varies from farm-to-farm, M. hyopneumoniae and PRRSV are two of the most common
*
Corresponding author. Tel.: +82-31-290-2736; fax: +82-31-2944588. E-mail address: [email protected] (C. Chae). 1090-0233/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1090-0233(02)00257-5
pathogens isolated from pigs exhibiting PRDC (Dee, 1996; Thacker et al., 1999). Postweaning multisystemic wasting syndrome (PMWS) is clinically characterized by poor body condition, dyspnoea, pallor of the skin, and sometimes icterus (Allan and Ellis, 2000; Kim et al., 2002). Porcine circovirus 2 (PCV2) is identified as an aetiological agent for PMWS (Allan and Ellis, 2000; Choi and Chae, 1999; Choi et al., 2000; Kim et al., 2001). In addition, co-infection with PCV2 and porcine parvovirus (PPV) induces more severe lesions and clinical disease (Allan et al., 1999; Ellis et al., 1999; Kennedy et al., 2000; Krakowka et al., 2000). PCV2/PPV co-infection has been demonstrated in a significant number of field cases of PMWS in Korea and Western Canada (Ellis et al., 2000; Kim et al., 2002). The extent of the involvement of PCV2 in swine disease other than PMWS is currently poorly understood. Recently, PCV2 has been identified in association with lesions in pigs with PRDC (Harms et al., 2002). However, the prevalence of PCV2 and co-infection with other viral and bacterial pathogens in pigs with PRDC has not been
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determined but could be of significant importance in epidemiological survey programs. This paper describes the prevalence of PCV2, microscopic lesions, and other coexisting microorganisms in pigs with PRDC in Korea.
2. Materials and methods
used to compare mean positive cells per unit area for PCV2, PRRSV, and PPV in same lung tissue samples from pigs with PRDC, and to compare mean PCV2positive cells per unit area in pigs infected with PCV2 only and co-infected with PCV2 and other viral pathogens. Statistical significance was assumed at P < 0:05. Statistical analysis was performed InStat package (GraphPad Softwear).
2.1. Animals A total of 105 pigs with PRDC were selected from 105 different herds on the basis of age and clinical history (Dee, 1996; Halbur, 1998; Thacker, 2001). All pigs were epidemiologically unrelated. A common clinical herd history was sought with animals approximately 18– 20 weeks of age experiencing severe outbreaks of respiratory disease, resulting in elevated mortality and an increased number of cull pigs. Morbidity ranged from 30% to 70% with an average mortality of 4% to 6%. 2.2. Diagnosis of bacterial pathogens At necropsy, samples of lung were cultured for bacteria as previously described (Cho and Chae, 2001; Choi et al., 2001). In situ hybridization was used for the diagnosis of M. hyopneumoniae in formalin-fixed, paraffin wax-embedded tissues (Kwon and Chae, 1999). 2.3. Diagnosis of viral pathogens Tissues from each pig were collected in 10% neutral buffered formalin, and after one or two daysÕ fixation were dehydrated through graded alcohols and xylene and then embedded in paraffin wax. Four serial sections (4 lm) were prepared from each lung tissues for PCV2, PRRSV, SIV, and PPV. In situ hybridization was used for the diagnosis of viral pathogens (PCV2, PRRSV, SIV, and PPV) (Cheon and Chae, 2000; Choi and Chae, 2000; Kim and Chae, 2001; Jung et al., 2002).
3. Results 3.1. Microscopic lesions Alveolar septa were markedly thickened by infiltrates of macrophages and fewer lymphocytes and plasma cells. Many alveolar septa were entirely lined by hypertrophied type 2 pneumocytes, and alveolar spaces contained abundant necrotic debris (Fig. 1). Moderate to marked multifocal peribronchial and peribronchiolar fibrosis were present and often extended into the airway lamina propria (Fig. 2). 3.2. Prevalence of viral pathogens In situ hybridization results for the 105 pigs with PRDC are presented in Table 1. Of the 105 pigs, 85 were positive for PCV2, 66 were positive for PRRSV, 60 were positive for PPV, and 14 were positive for SIV. Eighty were co-infected and 25 were singly infected (Table 1). The morphology of host cells was preserved despite the relatively high temperature required during parts of the incubation procedure. The signal intensity varied within and between histological structures in any one section and between pigs. Positive cells for PCV2 and
2.4. Morphometric analysis One section from each formalin-fixed lung tissue sample was taken from 105 pigs with PRDC for morphometric analysis (Kim et al., 2003). To obtain quantitative data, the morphometric analysis of the in situ hybridization slides was performed with NIH Image J Program (National Institutes of Health). For each condition, three fields were randomly selected and the number of positive cells per unit area (0.25 mm2 ) counted. The mean number of positive cells was expressed per unit area. 2.5. Statistical analysis All data are given as means standard deviation (SD). The unpaired t test using WelshÕs correction was
Fig. 1. Pigs with porcine respiratory disease complex (PRDC). Alveolar septa are markedly thickened by infiltrates of macrophages and alveolar spaces contain abundant necrotic debris. Haematoxylin & eosin.
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Fig. 2. Pigs with PRDC. Moderate to marked multifocal peribronchial and peribronchiolar fibrosis are present and often extended into the airway lamina propria. Haematoxylin and eosin.
Fig. 3. Pigs with PRDC. PCV2 DNA is detected in macrophages. In situ hybridization; nitroblue tetrazolium/5-bromocresyl-3-indolylphosphate, methyl green counterstain.
Table 1 Prevalence of viral pathogens in 105 pigs with porcine respiratory disease complex
tissue sections treated with DNase I for PCV2 and PPV, or RNase A for PRRSV and SIV prior to in situ hybridization (data not shown). Sections from positive control pigs for each virus had strong hybridization signal. Negative control pigs gave consistently negative results for these viruses tested.
PCV2
PRRS
SIV
PPV
Number of pigs
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
17 14 0 8 1 36 2 7 4 1 2 1 2 0 4 6
PCV2, porcine circovirus 2; PRRSV, porcine reproductive and respiratory syndrome virus; SIV, swine influenza virus; PPV, porcine parvovirus.
PPV typically exhibited a dark brown to black reaction products, mainly in the nucleus but occasionally in the cytoplasm, without any background staining. In contrast, PRRSV and SIV nucleic acid was located almost exclusively within the cytoplasm of cells. Distinct positive labelling was scattered throughout the alveolar septa and spaces of the lung. The positive cells for PCV2 (Fig. 3), PRRSV, SIV, and PPV generally had large oval nuclei and aboundant cytoplasm resembled macrophages. Less intense hybridization signals for PRRSV were also detected very occasionally in cells that resembled type II pneumocytes. Bronchial and bronchiolar epithelial cells had positive hybridization signals for SIV. No hybridization signal was consistently seen in
3.3. Prevalence of bacterial pathogens Among the 105 pigs, 56 were co-infected with both virus and bacteria. A co-infection of PCV2 with another additional bacterial pathogen is frequently diagnosed in PRDC. The combination of PCV2 and P. multocida (38 cases) was shown to be the most prevalent followed by PCV2 and M. hyopneumoniae (33 cases) (Table 2). 3.4. Morphometric analysis Significantly more PCV2-positive cells were detected in the lung (P < 0:05Þ than PRRSV and PPV in the same tissues from pigs with PRDC (Table 3). Statistical analysis of mean PCV2-positive cells per unit area in pigs coinfected with PCV2 and PPV, and pigs co-infected with PCV2, PRRSV, and PPV showed significant differences in these pigs infected with PCV2 only and pigs co-infected with PCV2 and PRRSV. There was no significant difference in the mean PCV2-positive cells per unit area between pigs co-infected with PCV2 and PPV, and pigs co-infected with PCV2, PRRSV, and PPV (Table 4).
4. Discussion The results of this survey indicate that PCV2 is widely prevalent in pigs with PRDC and should be considered a major respiratory pathogen. Most pigs with PRDC had
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Table 2 Prevalence of bacterial pathogens in 85 porcine circovirus 2 (PCV2)-infected pigs with porcine respiratory disease complex PCV2
P. multocida
M. hyopneumoniae
A. pleuropneumoniae
H. parasuis
Number of pigs
+ + + + + + + + + + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
12 17 0 2 0 1 0 0 10 0 0 0 3 0 3 37
PCV2, porcine circovirus 2.
more than one pathogen associated with respiratory disease. These observations underscore the importance of conducting a complete diagnostic investigation, even if one respiratory pathogen has been identified. Moreover, laboratory diagnosis is required because our study has shown that there is considerable overlap in the clinical manifestations of respiratory disease induced by Table 3 Comparison of postive cells of porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV) in pigs with porcine respiratory disease complex Viruses
Number of pigs
Numbers of positive cells (0.25 mm2 )
PCV2 PRRSV PPV
85 66 61
142:71 141:36a;b 45:38 92:97 24:33 11:32
a b
Mean standard deviation. Significant difference from other groups (P < 0:05).
Table 4 Comparison of positive cells of porcine circovirus 2 (PCV2) in pigs infected with PCV2 only and pigs co-infected with other viral pathogens Virus infection
Number of pigs
Numbers of positive cells (0.25 mm2 )
PCV2 single infection PCV2 + PRRSV co-infection PCV2 + PPV co-infection PCV2 + PRRSV + PPV co-infection
17 15
128:31 32:37a ;b 118:40 39:12
10
187:60 54:37
43
212:09 65:57
PCV2, porcine circovirus 2; PRRSV, porcine reproductive and respiratory syndrome virus; SIV, swine influenza virus; PPV, porcine parvovirus. a Mean standard deviation. b Significant difference from other groups (P < 0:05).
the different pathogens. Compared with other viral pathogens, the consistent presence of PCV2 in lung lesions examined, indicated that PCV2 may play an important role in developing PRDC. Detection of hybridization signals provided a molecular evidence that these cells were infected with PCV2. These results indicate that the major subcellular site of infection was the nucleus of macrophages-like cells in lung tissues. However, in the previous studies (Choi and Chae, 1999; Choi et al., 2000; Kim et al., 2002), PCV2 DNA has been found mainly in the cytoplasm of macrophages-like cells in lymphoid tissues. It is not possible to explain the difference in subcellular distribution of PCV2, which could be due to different tissues or to different strains of PCV2. Further studies on different subcellular sites of PCV2 infection between lung and lymphoid tissues are needed. PCV2-associated PRDC should be differentiated from PMWS clinically and histopathologically because of considerable diagnostic overlap between the two conditions. PMWS is characterized by cachexia, dyspnoea, and occasionally jaundice or pallor in young pigs, typically between 8 and 16 weeks of age (Allan and Ellis, 2000; Harding and Clark, 1997; Kim et al., 2002), while PRDC is characterized by slow growth, decreased feed efficiency, lethargy, anorexia, fever, cough, and dyspnoea in growing and finishing pigs typically around 16–22 weeks of age (Halbur, 1998; Thacker, 2001). Histologically, PCV2-associated PRDC is characterized by widespread granulomatous inflammation, multinucleated giant cells, and variable numbers of intracytoplasmic basophilic viral inclusion bodies within infiltrating histiocytes and macrophages (Allan et al., 1999; Choi and Chae, 1999; Choi et al., 2000; Ellis et al., 1999; Kennedy et al., 2000; Kim et al., 2002; Krakowka et al., 2000). In contrast, the most striking and consistent lesions of PCV2-associated PRDC in our study was bronchointerstitial pneumonia
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without evidence of characteristic lesions in non-respiratory tissues in this study. The presence of prolonged and unusually severe clinical respiratory disease, bronchointerstitial pneumonia with peribronchial and peribronchiolar fibrosis, and abundant PCV2 nucleic acid and/or antigen associated with the pulomonary lesions is suggestive of PCV2-associated PRDC. The interaction or synergism of PCV2 with other respiratory pathogens may be important. In our study, over 55% of cases diagnosed as PRDC had evidence of concurrent infections of both PCV2 and PRRSV. Pigs coinfected with PCV2 and PRRSV had a bronchointerstitial pneumonia. This bronchointerstitial pneumonia is compatible with the typical lesions seen in field cases of PRDC at our diagnostic laboratory. The findings suggest that infection of pigs with PCV2 or co-infection of pigs with both PCV2 and PRRSV could account for the clinical conditions and lesions currently being attributed to PRDC. Evidence from the diagnostic submission strongly supports an important role for PCV2 in PRDC and enhanced PRDC with co-infection of PCV2 and PRRSV. The interactions between different pathogens play an important role in developing PRDC. For example, pigs infected with M. hyopneumoniae have an increased duration of PRRSV-induced pneumonia (Thacker et al., 1999). In contrast to the potentiation observed with dual infection with M. hyopneumoniae and PRRSV, pigs infected with both SIV and M. hyopneumoniae lacked the significant increase in severity and duration of SIVinduced pneumonia (Thacker et al., 2001). There is experimental evidence that synergism occurs between PCV2 and PPV (Allan et al., 1999; Ellis et al., 1999; Kennedy et al., 2000; Krakowka et al., 2000), and between PCV2 and PRRSV (Allan et al., 2000; Harms et al., 2001). Experimental co-infection with PCV2 and PRRSV induces more severe lesions and clinical signs (Harms et al., 2001). Further study is needed to determine the interaction between PCV2 and other pathogens, especially M. hyopneumoniae. The course of the respiratory disease may be more severe, prolonged, and refractory to therapy than PRDC uncomplicated by concurrent PCV2 infection in Korean herds. Therefore, identifying the involvement of PCV2 in an outbreak of PRDC enables the veterinarian to develop a more accurate prognosis for PRDC. Although it may be unclear whether PCV2 is a primary pathogen, synergistic pathogen, secondary, or opportunistic pathogen, demonstration of lung lesions associated with PCV2 DNA certainly suggest that PCV2 has an important role in PRDC.
Acknowledgements The research was supported by Ministry of Agriculture, Forestry and Fisheries-Special Grants Research
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Program (MAFF-SGRP), and Brain Korea 21 Project, Republic of Korea.
References Allan, G.M., Ellis, J.A., 2000. Porcine circoviruses: a review. J. Vet. Diagn. Invest. 12, 3–14. Allan, G.M., Kennedy, S., McNeilly, F., Foster, J.C., Ellis, J.A., Krakowka, S.J., Meehan, B.M., Adair, B.M., 1999. Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. J. Comp. Pathol. 121, 1–11. Allan, G.M., McNeilly, F., Ellis, J., Krakowka, S., Meehan, B., Mcnair, I., Walker, I., Kennedy, S., 2000. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiate PCV2 replication. Arch. Virol. 145, 2421–2429. Cheon, D.-S., Chae, C., 2000. Comparison of virus isolation, reverse transcription-polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine reproductive and respiratory syndrome virus from naturally aborted fetuses and stillborn piglets. J. Vet. Diagn. Invest. 12, 582–587. Cho, W.-S., Chae, C., 2001. Genotypic prevalence of apxIV in Actinobacillus pleuropneumoniae field isolates. J. Vet. Diagn. Invest. 13, 175–177. Choi, C., Chae, C., 1999. In-situ hybridization for the detection of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. J. Comp. Pathol. 121, 265–270. Choi, C., Chae, C., 2000. Distribution of porcine parvovirus in porcine circovirus-2-infected pigs with postweaning multisystemic wasting syndrome as shown by in-situ hybridization. J. Comp. Pathol. 123, 302–305. Choi, C., Chae, C., Clark, E.G., 2000. Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction. J. Vet. Diagn. Invest. 12, 151–153. Choi, C., Kim, B., Cho, W.-S., Kwon, D., Cheon, D.-S., Chae, C., 2001. Capsular serotype, toxA gene, and antimicrobial susceptibility profiles of Pasteurella multocida isolated from pigs with pneumonia in Korea. Vet. Rec. 149, 210–212. Dee, S., 1996. The porcine respiratory disease complex: are subpopulations important? J. Swine Health Prod. 4, 147–149. Ellis, J.A., Bratanich, A., Clark, E.G., Allan, G., Meehan, B., Haines, D.M., Harding, J., West, K.H., Krakowka, S., Konoby, C., Hassard, L., Martin, K., McNeilly, F., 2000. Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome. J. Vet. Diagn. Invest. 12, 21–27. Ellis, J., Krakowka, S., Lairmore, M., Haines, D., Bratanich, A., Clark, E., Allan, G., Konoby, C., Hassard, L., Meehan, B., Martin, K., Harding, J., Kennedy, S., McNeilly, F., 1999. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J. Vet. Diagn. Invest. 11, 3–14. Halbur, P.G., 1998. Porcine respiratory disease. Proc. Int. Pig Vet. Soc. Congr. 15, 1–10. Harding, J.C.S., Clark, E.G., 1997. Recognizing and diagnosing postweaning multisystemic wasting syndrome (Pmws). J. Swine Health Prod. 5, 201–203. Harms, P.A., Halbur, P.G., Sorden, S.D., 2002. Three cases of porcine respiratory disease complex associated with porcine circovirus type 2 infection. J. Swine Health Prod. 10, 27–30. Harms, P.A., Sorden, S.D., Halbur, P.G., Bolin, S.R., Lager, K.M., Morozov, I., Paul, P.S., 2001. Experimental reproduction of severe disease in Cd/Cd pigs concurrently infected with type 2 porcine
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circovirus and porcine reproductive and respiratory syndrome virus. Vet. Pathol. 38, 528–539. Jung, T., Choi, C., Chae, C., 2002. Localization of swine influenza virus in naturally infected pigs. Vet. Pathol. 39, 10–16. Kennedy, S., Moffett, D., McNeilly, F., Meehan, B., Ellis, J., Krakowka, S., Allan, G.M., 2000. Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus. J. Comp. Pathol. 122, 9–24. Kim, J., Chae, C., 2001. Differentiation of porcine circovirus 1 and 2 in formalin-fixed, paraffin-embedded tissues from pigs with postweaning multisystemic wasting syndrome by in-situ hybridisation. Res. Vet. Sci. 70, 265–269. Kim, J., Choi, C., Han, D.U., Chae, C., 2001. Simultaneous detection of porcine circovirus 2 and porcine parvovirus in pigs with postweaning multisystemic wasting syndrome by multiplex PCR. Vet. Rec. 149, 304–305. Kim, J., Chung, H.-K., Jung, T., Cho, W.-S., Choi, C., Chae, C., 2002. Postweaning multisystemic wasting syndrome of pigs in Korea: prevalence, microscopic lesions and coexisting microorganisms. J. Vet. Med. Sci. 64, 57–62.
Kim, J., Choi, C., Chae, C., 2003. Pathogenesis of postweaning multisystemic wasting syndrome reproduced by co-infection with Korean isolates of porcine circovirus-2 and porcine parvovirus. J. Comp. Pathol., in press. Krakowka, S., Ellis, J.A., Meehan, B., Kennedy, S., McNeilly, F., Allan, G., 2000. Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus. Vet. Pathol. 37, 254–263. Kwon, D., Chae, C., 1999. Detection and localization of Mycoplasma hyopneumoniae Dna in lungs from naturally infected pigs by in situ hybridization using a digoxigenin-labeled probe. Vet. Pathol. 36, 308–313. Thacker, E.L., 2001. Porcine respiratory disease complex – what is it and why does it remain a problem? Pig J. 48, 66–70. Thacker, E.L., Halbur, P.G., Ross, R.F., Thanawongnuwech, R., Thacker, B.J., 1999. Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia. J. Clin. Microbiol. 37, 620–627. Thacker, E.L., Thacker, B.J., Janke, B.H., 2001. Interaction between Mycoplasma hyopneumoniae and swine influenza virus. J. Clin. Microbiol. 39, 2525–2530.
Association of porcine circovirus 2 with porcine respiratory disease complex J. Kim, H.-K. Chung, C. Chae
*
Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Kyounggi-Do, Republic of Korea Accepted 24 September 2002
Abstract A retrospective study was performed on natural cases of porcine respiratory disease complex (PRDC) to determine the association and prevalence of PRDC with porcine circovirus 2 (PCV2) and other co-existing pathogens in Korea. Histologically, alveolar septa were markedly thickened by infiltrates of mononuclear cells. Moderate to marked multifocal peribronchial and peribronchiolar fibrosis were present and often extended into the airway lamina propria. Among the 105 pigs with PRDC, 85 were positive for PCV2, 66 were positive for porcine reproductive and respiratory syndrome virus (PRRSV), 60 were positive for porcine parvovirus (PPV), and 14 were positive for swine influenza virus (SIV). There were 80 co-infections and 25 single infections. A coinfection of PCV2 with another additional bacterial pathogen is frequently diagnosed in PRDC. The combination of PCV2 and Pasteurella multocida (38 cases) was most prevalent followed by PCV2 and Mycoplasma hyopneumoniae (33 cases). The consistent presence of PCV2, but lower prevalence of other viral and bacterial pathogens in all pigs examined with PRDC, has led us to speculate that PCV2 plays an important role in PRDC. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Pneumonia; Porcine circovirus; Porcine respiratory disease complex; Prevalence
1. Introduction Porcine respiratory disease complex (PRDC) is a serious health problem in growing and finishing pigs typically around 16–22 weeks of age. PRDC is characterized by slow growth, decreased feed efficiency, lethargy, anorexia, fever, cough, and dyspnoea (Halbur, 1998; Thacker, 2001). Pneumonia in pigs with PRDC is due to a combination of both viral and bacterial agents, such as porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, and Pasteurella multocida (Halbur, 1998; Thacker, 2001). Although the aetiology of PRDC involves multiple pathogens and varies from farm-to-farm, M. hyopneumoniae and PRRSV are two of the most common
*
Corresponding author. Tel.: +82-31-290-2736; fax: +82-31-2944588. E-mail address: [email protected] (C. Chae). 1090-0233/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1090-0233(02)00257-5
pathogens isolated from pigs exhibiting PRDC (Dee, 1996; Thacker et al., 1999). Postweaning multisystemic wasting syndrome (PMWS) is clinically characterized by poor body condition, dyspnoea, pallor of the skin, and sometimes icterus (Allan and Ellis, 2000; Kim et al., 2002). Porcine circovirus 2 (PCV2) is identified as an aetiological agent for PMWS (Allan and Ellis, 2000; Choi and Chae, 1999; Choi et al., 2000; Kim et al., 2001). In addition, co-infection with PCV2 and porcine parvovirus (PPV) induces more severe lesions and clinical disease (Allan et al., 1999; Ellis et al., 1999; Kennedy et al., 2000; Krakowka et al., 2000). PCV2/PPV co-infection has been demonstrated in a significant number of field cases of PMWS in Korea and Western Canada (Ellis et al., 2000; Kim et al., 2002). The extent of the involvement of PCV2 in swine disease other than PMWS is currently poorly understood. Recently, PCV2 has been identified in association with lesions in pigs with PRDC (Harms et al., 2002). However, the prevalence of PCV2 and co-infection with other viral and bacterial pathogens in pigs with PRDC has not been
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J. Kim et al. / The Veterinary Journal 166 (2003) 251–256
determined but could be of significant importance in epidemiological survey programs. This paper describes the prevalence of PCV2, microscopic lesions, and other coexisting microorganisms in pigs with PRDC in Korea.
2. Materials and methods
used to compare mean positive cells per unit area for PCV2, PRRSV, and PPV in same lung tissue samples from pigs with PRDC, and to compare mean PCV2positive cells per unit area in pigs infected with PCV2 only and co-infected with PCV2 and other viral pathogens. Statistical significance was assumed at P < 0:05. Statistical analysis was performed InStat package (GraphPad Softwear).
2.1. Animals A total of 105 pigs with PRDC were selected from 105 different herds on the basis of age and clinical history (Dee, 1996; Halbur, 1998; Thacker, 2001). All pigs were epidemiologically unrelated. A common clinical herd history was sought with animals approximately 18– 20 weeks of age experiencing severe outbreaks of respiratory disease, resulting in elevated mortality and an increased number of cull pigs. Morbidity ranged from 30% to 70% with an average mortality of 4% to 6%. 2.2. Diagnosis of bacterial pathogens At necropsy, samples of lung were cultured for bacteria as previously described (Cho and Chae, 2001; Choi et al., 2001). In situ hybridization was used for the diagnosis of M. hyopneumoniae in formalin-fixed, paraffin wax-embedded tissues (Kwon and Chae, 1999). 2.3. Diagnosis of viral pathogens Tissues from each pig were collected in 10% neutral buffered formalin, and after one or two daysÕ fixation were dehydrated through graded alcohols and xylene and then embedded in paraffin wax. Four serial sections (4 lm) were prepared from each lung tissues for PCV2, PRRSV, SIV, and PPV. In situ hybridization was used for the diagnosis of viral pathogens (PCV2, PRRSV, SIV, and PPV) (Cheon and Chae, 2000; Choi and Chae, 2000; Kim and Chae, 2001; Jung et al., 2002).
3. Results 3.1. Microscopic lesions Alveolar septa were markedly thickened by infiltrates of macrophages and fewer lymphocytes and plasma cells. Many alveolar septa were entirely lined by hypertrophied type 2 pneumocytes, and alveolar spaces contained abundant necrotic debris (Fig. 1). Moderate to marked multifocal peribronchial and peribronchiolar fibrosis were present and often extended into the airway lamina propria (Fig. 2). 3.2. Prevalence of viral pathogens In situ hybridization results for the 105 pigs with PRDC are presented in Table 1. Of the 105 pigs, 85 were positive for PCV2, 66 were positive for PRRSV, 60 were positive for PPV, and 14 were positive for SIV. Eighty were co-infected and 25 were singly infected (Table 1). The morphology of host cells was preserved despite the relatively high temperature required during parts of the incubation procedure. The signal intensity varied within and between histological structures in any one section and between pigs. Positive cells for PCV2 and
2.4. Morphometric analysis One section from each formalin-fixed lung tissue sample was taken from 105 pigs with PRDC for morphometric analysis (Kim et al., 2003). To obtain quantitative data, the morphometric analysis of the in situ hybridization slides was performed with NIH Image J Program (National Institutes of Health). For each condition, three fields were randomly selected and the number of positive cells per unit area (0.25 mm2 ) counted. The mean number of positive cells was expressed per unit area. 2.5. Statistical analysis All data are given as means standard deviation (SD). The unpaired t test using WelshÕs correction was
Fig. 1. Pigs with porcine respiratory disease complex (PRDC). Alveolar septa are markedly thickened by infiltrates of macrophages and alveolar spaces contain abundant necrotic debris. Haematoxylin & eosin.
J. Kim et al. / The Veterinary Journal 166 (2003) 251–256
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Fig. 2. Pigs with PRDC. Moderate to marked multifocal peribronchial and peribronchiolar fibrosis are present and often extended into the airway lamina propria. Haematoxylin and eosin.
Fig. 3. Pigs with PRDC. PCV2 DNA is detected in macrophages. In situ hybridization; nitroblue tetrazolium/5-bromocresyl-3-indolylphosphate, methyl green counterstain.
Table 1 Prevalence of viral pathogens in 105 pigs with porcine respiratory disease complex
tissue sections treated with DNase I for PCV2 and PPV, or RNase A for PRRSV and SIV prior to in situ hybridization (data not shown). Sections from positive control pigs for each virus had strong hybridization signal. Negative control pigs gave consistently negative results for these viruses tested.
PCV2
PRRS
SIV
PPV
Number of pigs
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
17 14 0 8 1 36 2 7 4 1 2 1 2 0 4 6
PCV2, porcine circovirus 2; PRRSV, porcine reproductive and respiratory syndrome virus; SIV, swine influenza virus; PPV, porcine parvovirus.
PPV typically exhibited a dark brown to black reaction products, mainly in the nucleus but occasionally in the cytoplasm, without any background staining. In contrast, PRRSV and SIV nucleic acid was located almost exclusively within the cytoplasm of cells. Distinct positive labelling was scattered throughout the alveolar septa and spaces of the lung. The positive cells for PCV2 (Fig. 3), PRRSV, SIV, and PPV generally had large oval nuclei and aboundant cytoplasm resembled macrophages. Less intense hybridization signals for PRRSV were also detected very occasionally in cells that resembled type II pneumocytes. Bronchial and bronchiolar epithelial cells had positive hybridization signals for SIV. No hybridization signal was consistently seen in
3.3. Prevalence of bacterial pathogens Among the 105 pigs, 56 were co-infected with both virus and bacteria. A co-infection of PCV2 with another additional bacterial pathogen is frequently diagnosed in PRDC. The combination of PCV2 and P. multocida (38 cases) was shown to be the most prevalent followed by PCV2 and M. hyopneumoniae (33 cases) (Table 2). 3.4. Morphometric analysis Significantly more PCV2-positive cells were detected in the lung (P < 0:05Þ than PRRSV and PPV in the same tissues from pigs with PRDC (Table 3). Statistical analysis of mean PCV2-positive cells per unit area in pigs coinfected with PCV2 and PPV, and pigs co-infected with PCV2, PRRSV, and PPV showed significant differences in these pigs infected with PCV2 only and pigs co-infected with PCV2 and PRRSV. There was no significant difference in the mean PCV2-positive cells per unit area between pigs co-infected with PCV2 and PPV, and pigs co-infected with PCV2, PRRSV, and PPV (Table 4).
4. Discussion The results of this survey indicate that PCV2 is widely prevalent in pigs with PRDC and should be considered a major respiratory pathogen. Most pigs with PRDC had
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Table 2 Prevalence of bacterial pathogens in 85 porcine circovirus 2 (PCV2)-infected pigs with porcine respiratory disease complex PCV2
P. multocida
M. hyopneumoniae
A. pleuropneumoniae
H. parasuis
Number of pigs
+ + + + + + + + + + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
12 17 0 2 0 1 0 0 10 0 0 0 3 0 3 37
PCV2, porcine circovirus 2.
more than one pathogen associated with respiratory disease. These observations underscore the importance of conducting a complete diagnostic investigation, even if one respiratory pathogen has been identified. Moreover, laboratory diagnosis is required because our study has shown that there is considerable overlap in the clinical manifestations of respiratory disease induced by Table 3 Comparison of postive cells of porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV) in pigs with porcine respiratory disease complex Viruses
Number of pigs
Numbers of positive cells (0.25 mm2 )
PCV2 PRRSV PPV
85 66 61
142:71 141:36a;b 45:38 92:97 24:33 11:32
a b
Mean standard deviation. Significant difference from other groups (P < 0:05).
Table 4 Comparison of positive cells of porcine circovirus 2 (PCV2) in pigs infected with PCV2 only and pigs co-infected with other viral pathogens Virus infection
Number of pigs
Numbers of positive cells (0.25 mm2 )
PCV2 single infection PCV2 + PRRSV co-infection PCV2 + PPV co-infection PCV2 + PRRSV + PPV co-infection
17 15
128:31 32:37a ;b 118:40 39:12
10
187:60 54:37
43
212:09 65:57
PCV2, porcine circovirus 2; PRRSV, porcine reproductive and respiratory syndrome virus; SIV, swine influenza virus; PPV, porcine parvovirus. a Mean standard deviation. b Significant difference from other groups (P < 0:05).
the different pathogens. Compared with other viral pathogens, the consistent presence of PCV2 in lung lesions examined, indicated that PCV2 may play an important role in developing PRDC. Detection of hybridization signals provided a molecular evidence that these cells were infected with PCV2. These results indicate that the major subcellular site of infection was the nucleus of macrophages-like cells in lung tissues. However, in the previous studies (Choi and Chae, 1999; Choi et al., 2000; Kim et al., 2002), PCV2 DNA has been found mainly in the cytoplasm of macrophages-like cells in lymphoid tissues. It is not possible to explain the difference in subcellular distribution of PCV2, which could be due to different tissues or to different strains of PCV2. Further studies on different subcellular sites of PCV2 infection between lung and lymphoid tissues are needed. PCV2-associated PRDC should be differentiated from PMWS clinically and histopathologically because of considerable diagnostic overlap between the two conditions. PMWS is characterized by cachexia, dyspnoea, and occasionally jaundice or pallor in young pigs, typically between 8 and 16 weeks of age (Allan and Ellis, 2000; Harding and Clark, 1997; Kim et al., 2002), while PRDC is characterized by slow growth, decreased feed efficiency, lethargy, anorexia, fever, cough, and dyspnoea in growing and finishing pigs typically around 16–22 weeks of age (Halbur, 1998; Thacker, 2001). Histologically, PCV2-associated PRDC is characterized by widespread granulomatous inflammation, multinucleated giant cells, and variable numbers of intracytoplasmic basophilic viral inclusion bodies within infiltrating histiocytes and macrophages (Allan et al., 1999; Choi and Chae, 1999; Choi et al., 2000; Ellis et al., 1999; Kennedy et al., 2000; Kim et al., 2002; Krakowka et al., 2000). In contrast, the most striking and consistent lesions of PCV2-associated PRDC in our study was bronchointerstitial pneumonia
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without evidence of characteristic lesions in non-respiratory tissues in this study. The presence of prolonged and unusually severe clinical respiratory disease, bronchointerstitial pneumonia with peribronchial and peribronchiolar fibrosis, and abundant PCV2 nucleic acid and/or antigen associated with the pulomonary lesions is suggestive of PCV2-associated PRDC. The interaction or synergism of PCV2 with other respiratory pathogens may be important. In our study, over 55% of cases diagnosed as PRDC had evidence of concurrent infections of both PCV2 and PRRSV. Pigs coinfected with PCV2 and PRRSV had a bronchointerstitial pneumonia. This bronchointerstitial pneumonia is compatible with the typical lesions seen in field cases of PRDC at our diagnostic laboratory. The findings suggest that infection of pigs with PCV2 or co-infection of pigs with both PCV2 and PRRSV could account for the clinical conditions and lesions currently being attributed to PRDC. Evidence from the diagnostic submission strongly supports an important role for PCV2 in PRDC and enhanced PRDC with co-infection of PCV2 and PRRSV. The interactions between different pathogens play an important role in developing PRDC. For example, pigs infected with M. hyopneumoniae have an increased duration of PRRSV-induced pneumonia (Thacker et al., 1999). In contrast to the potentiation observed with dual infection with M. hyopneumoniae and PRRSV, pigs infected with both SIV and M. hyopneumoniae lacked the significant increase in severity and duration of SIVinduced pneumonia (Thacker et al., 2001). There is experimental evidence that synergism occurs between PCV2 and PPV (Allan et al., 1999; Ellis et al., 1999; Kennedy et al., 2000; Krakowka et al., 2000), and between PCV2 and PRRSV (Allan et al., 2000; Harms et al., 2001). Experimental co-infection with PCV2 and PRRSV induces more severe lesions and clinical signs (Harms et al., 2001). Further study is needed to determine the interaction between PCV2 and other pathogens, especially M. hyopneumoniae. The course of the respiratory disease may be more severe, prolonged, and refractory to therapy than PRDC uncomplicated by concurrent PCV2 infection in Korean herds. Therefore, identifying the involvement of PCV2 in an outbreak of PRDC enables the veterinarian to develop a more accurate prognosis for PRDC. Although it may be unclear whether PCV2 is a primary pathogen, synergistic pathogen, secondary, or opportunistic pathogen, demonstration of lung lesions associated with PCV2 DNA certainly suggest that PCV2 has an important role in PRDC.
Acknowledgements The research was supported by Ministry of Agriculture, Forestry and Fisheries-Special Grants Research
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Program (MAFF-SGRP), and Brain Korea 21 Project, Republic of Korea.
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