Porcine circovirus type 2-associated cerebellar vasculitis in postweaning multisystemic wasting syndrome (PMWS)-affected pigs

Porcine circovirus type 2 (PCV2) is associated with several syndromes in growing pigs, including postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome. In the present study, a previously undescribed neurovascular disorder associated with a PCV2 infection is described. Sixteen pigs showed clinical signs of wasting and neurologic deficits. Acute hemorrhages and edema of cerebellar meninges and parenchyma due to a necrotizing vasculitis resulted in degeneration and necrosis of the gray and white matter. Few to numerous PCV2 DNA and antigen-bearing endothelial cells were detected in affected areas of the brain using in situ hybridization and immunohistochemistry. Conventional histochemical stains, as well as the detection of caspase 3 activity and DNA strand breaks by the terminal transferase dUTP nick end labeling assay, showed numerous apoptotic endothelial cells in the vascular lesions observed. Sequencing of various brain-derived PCV2-specific amplicons revealed a strong identity between different isolates and an 89 to 100% identity to previous isolates. The phylogenetic tree showed that there was no clustering of isolates correlating to clinical signs or geographic distribution. This previously undescribed PCV2-associated neurologic disease has features of both postweaning multisystemic wasting syndrome and, to a lesser extent, porcine dermatitis and nephropathy syndrome. The available evidence suggests that direct virus-induced apoptosis of endothelial cells plays a role in the pathogenesis of this unusual PCV2-associated cerebellar vasculitis.     

Haptoglobin and pig-major acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS)

The objective of this study was to determine the serum concentration levels of selected acute phase proteins (APP), haptoglobin (HPT) and pig-major acute phase protein (pig-MAP), in postweaning multisystemic wasting syndrome (PMWS) affected pigs and PCV2-subclinically infected pigs. In a first study, a group of 15 eight-week-old conventional pigs from a PMWS affected farm were bled and a complete necropsy, histopathology and in situ hybridisation to detect PCV2 were performed. Based on the results, pigs were classified as suffering from PMWS (n = 10) or healthy animals (n = 5). In a second study, a group of 45 pigs from another PMWS affected farm were selected and bled at 3, 7, 12 and 28 weeks of age. The assessment of PCV2 infection status in these pigs was retrospectively done by PCV2 PCR in serum samples. Selected APP were measured in the serum of all studied pigs by means of radial immunodiffusion. Mean HPT and pig-MAP levels were significantly increased (p = 0.004 and p = 0.0006 respectively) in PMWS-affected pigs when compared to levels found in healthy pigs (2.52 +/- 0.88 mg/mL vs. 1.06 +/- 0.73 mg/mL for HPT and 3.81 +/- 1.53 mg/mL vs. 0.76 +/- 0.34 mg/mL for pig-MAP). In the second study, no significant difference in mean HPT and pig-MAP values were observed between PCV2 PCR positive and negative pigs of any age. However, both APP increased significantly with age in PCV2 PCR negative pigs. Altogether, the present results suggest that APP levels are significantly increased in pigs that develop PMWS, but not in animals with a PCV2 subclinical infection.     

Changes in peripheral blood leukocyte populations in pigs with natural postweaning multisystemic wasting syndrome (PMWS)

The objective of the present study was to analyze, by flow cytometry, changes in PBMC subsets in pigs having postweaning multisystemic wasting syndrome (PMWS), a new condition associated to porcine circovirus type 2 (PCV2) infection. Thirteen acutely PMWS affected pigs were selected from a farm seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) and to Aujeszky's disease virus (ADV); 11 clinically healthy pigs were selected from a high health farm with no history of PMWS and free of the major swine pathogens, and used as a control group. All pigs were necropsied, and tissue samples were fixed in formalin; blood with EDTA anticoagulant was used to perform the flow cytometric analysis. PBMC were incubated with mAb against porcine CD3, CD4, CD8, CD25, CD45, IgM, SWC3, and SLA-Class II. Flow cytometric analysis showed substantial changes in leukocyte subsets in the peripheral blood of PMWS-affected pigs, which were characterized by an increase of monocytes, a reduction of T (mainly CD4(+)) and B-lymphocytes, and the presence of low-density immature granulocytes. Altogether, these changes would suggest an inability of acutely PMWS-affected pigs to mount an effective immune response.     

Examination for a viral co-factor in postweaning multisystemic wasting syndrome (PMWS)

In order to test the hypothesis that a putative co-factor for the development of postweaning multisystemic wasting syndrome (PMWS) in pigs could be of viral origin, we performed extensive virological examinations on organ material from pigs diagnosed with PMWS originating from within a Danish PMWS-transmission study. Virus isolation attempts were carried out on a large panel of different cell types including primary pig kidney cells and lung macrophages, primary rabbit kidney cells and seven established cell lines (MARC-145, ST117, PK15, BHK21, HeLa, Vero, and MDCK). Although these represent cells with susceptibility to a wide range of known viruses, the results did not provide evidence for a specific virus other than PCV2 contributing to the development of PMWS. Furthermore, in order to test whether specific genotypes of PCV2 may trigger the switch from PCV2 infection to clinical disease, we compared complete DNA genome sequences of PCV2 derived from PMWS-positive as well as PMWS-negative pigs. On the basis of the DNA sequences, the PCV2 isolates were divided into two groups. Group 1 consisting of one isolate originating from a herd unaffected by PMWS, with group 2 consisting of nine isolates originating from four PMWS-affected herds, four PMWS-positive pigs plus one unaffected herd. The PCV2 genomes from the two groups showed 95.5% identity. Alignment analyses of the sequences encoding the replicase and capsid protein from group 1 and group 2 PCV2 isolates showed two amino acid differences encoded in the replicase protein, while 19 amino acid differences were predicted among the capsid protein sequences. The PCV2 DNA sequence analysis supports recent observations from studies in USA as well as Europe, which suggest that strain variations may influence the clinical outcome of PCV2 infection.     

Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review

Postweaning multisystemic wasting syndrome, caused by porcine circovirus type 2 (PCV2), is a recently described clinical condition which affects nursery and growing pigs. PMWS was initially recognized in Canada in 1991 and nowadays is considered to be worldwide distributed. Clinically, PMWS most representative symptoms include wasting, unthriftness, paleness of the skin, respiratory distress, diarrhea and sometimes icterus. PCV2 infection occurs in both PMWS affected and non-affected farms, and viral seroconversion shows a typical pattern, with declining of colostral antibodies during the lactating and nursery periods, with the lowest levels at the end of the nursery period, and active seroconversion of almost all pigs during the grower period. Although antibodies to PCV2 have been detected as early as 1969, no explanation for the emergence of this disease in the 90s has been established. Macroscopic lesions associated with PMWS are quite unspecific, but histopathological lesions in lymphoid tissues (lymphocyte depletion with histiocytic infiltration) are almost unique for this disease. These lesions together with other clinical and laboratorial findings suggest that severely affected pigs may be immunosuppressed. The criteria used for the diagnosis of PMWS include the existence of compatible clinical signs, presence of characteristic microscopic lesions and detection of PCV2 within these lesions. Because of the lack of appropriate treatment or vaccination against PCV2, zootechnical changes have been proposed in affected farms to reduce the so-called "infection pressure" due to PCV2 as well as to any other pathogen.     

Pathological investigations of pigs with porcine multisystemic wasting syndrome (PMWS)

Extract

Pathological changes and immunohistochemical (IHC) examination for porcine circovirus-2 (PCV-2) in nine weaner pigs from a property with PMWS are reported. The pigs were in moderate to poor body condition, and several had diarrhoea and noisy respiration. The outstanding pathological changes related to microscopic lesions in lymphoid tissues, including lymph nodes, palatine tonsils, Peyer's patches, peribronchial lymphoid aggregates and spleen. While there was variability of expression of change between and within cases, the main features of lesions could be summarised as depletion of lymphoid cells, infiltration of histiocytes (often syncytial), and the presence of botryoid intra-cytoplasmic inclusion bodies in macrophages. The changes in lymphoid tissue were typical of those described in PMWS. Also characteristic of this syndrome were histiocyte-dominated cellular inflammatory infiltrates in kidney, liver and lung. There was strong correlation between IHC positivity for PCV-2 and the lesions in lymphoid, pulmonary and renal tissues. Concurrent diseases that are probably promoted by immune suppression may complicate the diagnosis of PMWS. In the present cases, salmonellosis, attaching and effacing Escherichia coli infections, and secondary bacterial bronchopneumonia were identified.     

Immunosuppression in postweaning multisystemic wasting syndrome affected pigs

The present review concentrates on the clinical, pathological and immunological aspects of pigs suffering from PMWS which strongly suggest that PCV2 may be, in particular conditions, a cause of secondary immunodeficiency in pigs. From a clinical point of view, the lack of antibiotic therapy response against the disease, the existence of a litter effect and the concurrence of other disease syndromes and well-known secondary pathogens, such as Pneumocystis carinii, Chlamydia spp. and Aspergillus spp., may account as features of immunosuppression in PMWS. Furthermore, pathologic, immunohistologic and flow cytometric studies also suggest that pigs with PMWS may be immunosuppressed. Lymphocyte depletion of follicular and interfollicular areas together with macrophage infiltration of lymphoid tissues is a unique lesion, which is the basic feature of PMWS affected pigs. These findings are highly correlated with the decrease of circulating B- and T-cells and the diminution of these cell types in lymphoid organs, and with the increase of macrophage/monocytes lineage cells both in peripheral blood and lymphoid tissues in both naturally and experimentally PMWS affected pigs. The altered populations of cells participating in the immune system response both in blood and tissues suggests, at least in those severely PMWS affected pigs, a transient inability of diseased pigs to mount an effective immune response. From these points of view, strong suspicions on the immunosuppressive status of PMWS affected pigs do exist; however, future studies are needed to characterise the exact role of PCV2 on the immune system of pigs affected with PMWS.     

Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs

The aim of the study was to determine the presence of swine hepatitis E virus (HEV) RNA and antibodies in postweaning multisystemic wasting syndrome-affected (n=114) and non-affected (n=46) pigs and the possible association with hepatitis lesions. Forty-four pigs were RT-PCR positive (28.2%); 25 of them were PMWS cases, while 19 were non-PMWS pigs. In both groups, HEV RT-PCR results were associated with hepatitis (OR=5.61 for PMWS-affected pigs and OR=5.17 for non-PMWS affected pigs; p=0.01). No interaction was detected in a logistic regression between PMWS occurrence and HEV infection for the development of hepatitis lesions. Seropositivity to HEV was more likely to occur in pigs with hepatitis (51.9%) compared to pigs without hepatitis (36.1%; p=0.03). Significant differences in optical densities were notices comparing the lesional stage of pigs (p=0.009). While pigs with slight or moderate hepatitis were seropositive, pigs with more severe lesions were seronegative to HEV. These results indicate that swine HEV infection can be a significant contributor to the development of moderate hepatitis in pigs regardless of the PMWS status.     

Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS)

Postweaning multisystemic wasting syndrome (PMWS) affects nursery and growing pigs, and is characterized by wasting, failure to thrive, pale skin, respiratory distress, diarrhoea and sometimes jaundice. Macroscopic findings are aspecific, but lymphocyte depletion in lymphoid tissues is one of the histological hallmarks [Vet. Q. 24 (2002) 109]. Spontaneous cases of PMWS were studied to evaluate proliferative activity and apoptosis as mechanisms involved in the pathogenesis of cell depletion in lymph nodes. The presence of Porcine Circovirus type 2 (PCV2) genome in the processed material was confirmed by in situ hybridization (ISH). The lymph node pattern of depletion was graded as initial, intermediate or final stage according to histological criteria in 10 superficial inguinal nodes from piglets with PMWS which died spontaneously or were slaughtered by euthanasia. The apoptotic and proliferative fraction were investigated by monoclonal antibody MIB1 immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labeling) methods, respectively, and compared to three normal cases. One-way analysis of variance (ANOVA) comparison between the MIB1 index (number of positive cells per 100 nuclei) in controls and PMWS cases revealed a decrease of proliferation in both lymphoid and medulla-like tissues in the initial group (respectively, P=0.0017 and 0.024) but not in the intermediate (respectively, P=0.25 and 0.88) or final (respectively, P=0.47 and 0.81) cohorts. The apoptotic index (number of apoptosis/apoptotic bodies in 100 cells) revealed a statistically significant decrease only in the initial group (one-way ANOVA P=0.05). The proliferation/apoptosis ratio (MIB1/APO ratio) assessed to determine cell turnover disclosed a significant decrease of cell turnover from initial to final PMWS cases (Spearman's rank test: P=0.027). Decreased cell proliferation and not increased apoptosis seems to be the most important variable leading to cell depletion in PMWS lymphoid tissues.     

An exploratory study on risk factors for postweaning multisystemic wasting syndrome (PMWS) in Spain

An exploratory case-control study was carried out in Spain in 2002/2003, involving 62 pig farms of different production systems to assess risk factors that, in association with PCV2 infection, induce postweaning multisystemic wasting syndrome (PMWS) expression. To achieve this objective two groups of farms selected according to their PMWS status were compared: "cases" (farms with clinical PMWS, n = 32) and "controls" (farms without clinical PMWS, n = 30). A filled-in questionnaire and 45 blood samples (15 sows, and two groups of 15 pigs of 12 and 20 weeks of age, respectively) were obtained from each farm. Additionally, two to three diseased pigs were necropsied and relevant tissues to diagnose PMWS collected when PMWS was clinically suspected ("case" farms). A statistical analysis to compare "case" versus "control" farms was performed with the variables obtained from the questionnaire (191 variables) and the serologic test results (20 variables). Data were analysed using conditional logistic regression with a nested n:m matched design taking into account the farm size. Three variables were found significant in the final model: two related to vaccination scheme and one to PCV2 seroprevalence in growing pigs. Vaccination of gilts against PRRSV increased the odds of PMWS expression and vaccination of sows against atrophic rhinitis was related to decreased odds of the disease; however, the possibility that those two factors could be spurious effects (due to the small sample size) or confounding variables cannot be ruled out. On the other hand, a higher prevalence of antibodies to PCV2 at 12 weeks of age was observed in pigs from "case" farms than in pigs from "control" farms. This result suggests that an earlier infection with PCV2 might be a risk factor for PMWS expression.     

Association of lymphopenia with porcine circovirus type 2 induced postweaning multisystemic wasting syndrome (PMWS)

The composition of peripheral blood leukocyte populations was studied following experimental PCV2-infection in 3-week-old piglets. Four of 10 PCV2-infected piglets developed clinical and pathological symptoms consistent with postweaning multisystemic wasting syndrome (PMWS) between 14 and 21 days post-inoculation (p.i.), and were characterised as PMWS-affected. Only these four PMWS-affected piglets, but neither the non-symptomatic infected nor control animals, developed a clear leukopenia. Kinetic analysis demonstrated a clear loss of both CD21(+) B and CD3(+) T lymphocytes in the PMWS-affected piglets. By CD3/CD4/CD8 triple labelling, the influence of PCV2 infection on all T cell sub-populations was discernible. A loss of CD3(+)CD4(+)CD8(+) memory/activated Th lymphocytes was particularly notable. However, all T lymphocyte sub-populations-CD3(+)CD4(+)CD8(+) memory Th, CD3(+)CD4(+)CD8(-) nai;ve Th, CD3(+)CD4(-)CD8(+) Tc and CD3(+)CD4(-)CD8(-) gammadelta TCR(+) lymphocytes-were susceptible to PCV2 infection-induced lymphopenia. CD3(-)CD4(-)CD8(+) NK cells were also depleted in the PMWS-affected animals, but granulocytes and monocytes were less affected. In conclusion, PCV2 infection induces primarily a lymphopenia, but only in animals which subsequently develop PMWS. The lymphopenia can be identified early p.i., particularly with the B lymphocytes. Memory/activated Th lymphocytes might be affected more than the other T cell sub-populations, but as time progressed a collapse of both T and B cell populations was clear.     

Muco-cutaneous candidiasis in two pigs with postweaning multisystemic wasting syndrome

In two distinct commercial swine herds, poor weight gain and an increased number of animals showing wasting were observed among nursery and growing pigs. Cases of postweaning multisystemic wasting syndrome (PMWS) and infection with Haemophilus parasuis had been previously diagnosed in these herds. One growing wasted pig from each herd was necropsied and showed enlarged lymph nodes. Pseudomembranous material adhered to the dorsum of the tongue, soft and hard palate in case 1, and in case 2, fibrinous material was seen as whitish plaques on the oesophageal surface with hyperkeratosis of the non-glandular stomach. The main histological lesions in both cases were found in lymphoid tissues with a multifocal accentuated lymphohistiocytic infiltrate, areas of lymphoid depletion and intracytoplasmic inclusions in histiocytic cells in lymph nodes and Payer's patches. Focally, extensive ulceration was found in the stratified pavement epithelium of the tongue with necrosis and necrosuppurative infiltrate in case 1; in case 2, there was ulceration in the stomach with lymphohistiocytic infiltrate in the submucosa and ulceration in the mucosa of the oesophagus associated with yeast cells and pseudo-hyphae. Candida albicans was isolated from the oral cavity lesions. Immunohistochemistry of the lymph nodes was positive for porcine circovirus 2 (PCV2). The association between PMWS and mucocutaneous candidiasis reported here supports the potential immunosuppressive state of PMWS infected pigs.     

西班牙和丹麦受感染猪场的猪个体感染断奶后多系统衰竭综合征的风险因素

研究人员对来自西班牙(n=3)和丹麦(n=10)13家感染断奶后多系统衰竭综合征(PostweaningMuhisystemic Wasting Syndrome,PMWS)的猪场进行了2项前瞻性纵向研究.猪的血液样本从第1周开始纵向采集直到爆发PMWS为止.相同日龄的健康或衰竭性猪都进行安乐死、尸体解剖和组织病理学描述.通过淋巴病变、原位杂交或免疫组织化学方法检测这些组织中的PCV2,从而确诊PMWS.血清分析在纵向采集的血清样本中进行,以检测针对PCV2、猪繁殖与呼吸综合征病毒(Porcine Reproductive And Respiratory Syndrome Virus,PRRSV)、猪细小病毒(Porcine ParvoVirus,PPV)、猪流感病毒(Swine Influenza Virus,SIV)、细胞内罗森氏菌(Lawsonia intracellularis,law)、猪肺炎支原体(Mycoplasma hyopneumoniae)、猪伪狂犬病病毒(Aujeszky's Disease Virus,ADV)和沙门菌(Salmonella spp.)的抗体.Cox比例风险模型(Cox proportional hazards model)被用来研究血清转换和抗所研究病原体母体免疫的同步效应.     

Occurrence of swine salmonellosis in postweaning multisystemic wasting syndrome (PMWS) affected pigs concurrently infected with porcine reproduction and respiratory syndrome virus (PRRSV)

Fourteen diseased pigs from four farms in which there had been an outbreak of salmonellosis were investigated. Granulomatous inflammation with depletion of lymphocytes was observed in the swollen lymph nodes in these pigs. Antigens to porcine circovirus type 2 (PCV2) were immunolabeled in the lesions along with detection of viral DNA as PCV2 by polymerase chain reaction (PCR). In addition, antigens to porcine reproductive respiratory syndrome virus (PRRSV) were immunodetected in the lungs and Salmonella Choleraesuis was isolated from the affected pigs. The nine salmonellosis affected pigs, five (55.6%) with salmonellosis and PMWS concurrently infected with PRRSV were much higher than those infected with salmonellosis and PMWS (22.2%) or with salmonellosis and PPPRV (22.2%).     

Immunohistochemical MHC-II and interleukin 2-R (CD25) expression in lymph nodes of pigs with spontaneous postweaning multisystemic wasting syndrome (PMWS)

Immunohistochemical expression of immunocompetent cells bearing major histocompatibility complex (MHC-II) and interleukin 2-R (IL2-R) (CD25) molecules was performed on lymph nodes with spontaneous postweaning multisystemic wasting syndrome (PMWS). Control lymph nodes displayed intense diffuse immunoreactivity to MHC-II in both follicles and interfollicular areas. A marked reduction of follicular MHC-II immunoreactivity and inconsistent staining of histiocytes in interfollicular areas was observed in PMWS cases with a slight lymphoid depletion; in those cases with moderate to severe lymphoid depletion, there was a progressive decrease in MHC-II expression. In controls and in slightly depleted nodes, IL2-R was equally expressed in interfollicular tissue and in follicles, whereas in moderate and severe cases, it was detected in interfollicular remnants only. Immunohistochemical staining was scored semiquantitatively. The mean MHC-II score was significantly reduced in PMWS cases compared with controls (Spearman test), whereas there was no difference in the IL2-R score. The evident reduction of MHC-II immunoreactivity suggests an impairment in MHC-II linked antigen presenting cell expression.     

Immunohistochemical characterisation of PCV2 associate lesions in lymphoid and non-lymphoid tissues of pigs with natural postweaning multisystemic wasting syndrome (PMWS)

The lymphoid, renal, pulmonary, and hepatic lesions of naturally occurring postweaning multisystemic wasting syndrome (PMWS) affected pigs have been studied by means of immunohistology. Ten conventionally reared pigs showing acute clinical signs of PMWS were selected from a farm on which animal were seronegative to porcine reproductive and respiratory virus and to Aujeszky's disease virus. All pigs were positive in tests for porcine circovirus type 2 by ISH and IHC. Monoclonal and polyclonal antibodies to CD3, CD79alpha, CD45RA (3C3/9), lysozyme, SLA-II-DQ (BL2H5), and MAC387 were used to characterise cells in PMWS lesions. The most relevant changes were reduction or loss of B and T lymphocytes, increased numbers of macrophages, and partial loss and redistribution of antigen presenting cells throughout lymphoid tissues compared to uninfected controls. The characteristics of lymphoid lesions in the present study strongly suggest an immunosuppressive effect of PMWS in affected pigs.     

Brain lesions in pigs affected with postweaning multisystemic wasting syndrome.

Anti-porcine circovirus type 2 (anti-PCV2) immunostaining was associated with cerebellar lymphohistiocytic vasculitis combined with hemorrhages (50 pigs) or with lymphohistiocytic meningitis (23 pigs) in pigs naturally affected with postweaning multisystemic wasting syndrome (PMWS). The animals originated from 12 farms in Rio Grande do Sul, Brazil. In total, 456 unthrifty 3- to 5-month-old postweaning pigs confirmed as PMWS cases were necropsied. Although most findings mimicked those extensively reported in PMWS-affected pigs, there were distinctive brain lesions that included multiple hemorrhages in the cerebellar leptomeninges associated with lymphohistiocytic vasculitis and fibrinoid degeneration in vessels of the cerebellum and periventricular areas (69 pigs). These vascular lesions were also seen in conjunction with lymphohistiocytic meningitis (38 additional pigs). PCV2 antigen was immunohistochemically demonstrated in the cytoplasm and nuclei from intralesional perivascular macrophages and endothelial-like cells in brain tissues. Together these findings suggest that these lesions were caused by PCV2.     

Clinical and pathological observations on pigs with postweaning multisystemic wasting syndrome

The aim of this work was to characterise the lesions and agents present in clinically normal and clinically affected pigs on a farm during an outbreak of postweaning multisystemic wasting syndrome (PMWS), and to evaluate the diagnostic techniques for detecting porcine circovirus type 2 (PCV-2) and other microorganisms. Four pigs in the early stage and 11 pigs in the late stage of the disease, and eight clinically normal pigs were necropsied. Samples of lymphoid tissue and serum were also obtained from 12 slaughter pigs from the same farm. The tissues were examined histopathologically, and in situ hybridisation, serology and PCR were used to detect porcine circovirus type 1 (PCV-1) and/or PCV-2 in tissues and/or sera. The presence of porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) were also investigated. Characteristic microscopical lesions of PMWS were observed in the lymphoid tissues of the pigs in all three necropsied groups; the lesions were most common and severe in the pigs in the early stage of the disease, less so in the pigs in the late stage of the disease, and least in the clinically normal pigs. PCV-2 infection was detected in all the necropsied pigs by in situ hybridisation and PCR. Only three pigs had the PCV-1 genome in serum or lymph node tissue. In contrast, the slaughter pigs had no microscopical lesions and no PCV-2 nucleic acid in their serum or tissues, and only one of them had the pCV-1 genome in its serum. Immunohistochemical, serological and PCR studies revealed that PRRSV and ADV were also present on the farm during the outbreak.