Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome

Multiplex nested polymerase chain reactions (PCRs) were developed for the simultaneous detection and differentiation of genomic material of porcine circovirus 1 (PCV1), porcine circovirus 2 (PCV2), and porcine parvovirus (PPV) in formalin-fixed, paraffin-embedded tissues. Multiplex conventional and nested PCR and in situ hybridization were compared for their ability to detect the 3 viruses in such tissues. Xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for reliable and consistent PCR analyses. The DNA from PCV1, PCV2, and PPV was detected by both multiplex nested PCR and in situ hybridization in lymph-node tissue from 12 pigs experimentally co-infected with the 3 viruses, as well as in formalin-fixed, paraffin-embedded lymph-node tissue from 30 pigs with naturally occurring postweaning multisystemic wasting syndrome; the agreement rates for the 2 methods were 100% in both groups of pigs. Thus, multiplex nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for simultaneous detection of these 3 porcine viruses.     

Characterization of interstitial nephritis in pigs with naturally occurring postweaning multisystemic wasting syndrome

Kidney samples with interstitial nephritis from 26 pigs affected by postweaning multisystemic wasting syndrome (PMWS) were selected. A histologic evaluation was carried out to describe the type of inflammation and its relationship with viral load, as assessed by in situ hybridization (ISH). Of 26 cases, 10 revealed a tubulointerstitial, lymphoplasmacytic nephritis, 11 an interstitial granulomatous nephritis, and 5 both types of inflammation (mixed type). In 4 cases of granulomatous inflammation, the pattern was not classically nodular, and a population of macrophages and lymphocytes was present (interstitial lymphohistiocytic nephritis). ISH confirmed the presence of porcine circovirus type 2 (PCV2) nucleic acid in all cases. The epithelium of the renal tubules was the most constantly ISH-positive structure. In tubulointerstitial nephritis, the higher the number of positive inflammatory cells, the more severe the inflammation. The ISH reaction was more heterogeneous and unpredictable in granulomatous nephritis, with some epithelioid and giant cells positive by ISH. To quantify macrophages distributed in the three patterns of nephritis, immunohistochemical methods using anti-major histocompatibility complex II (anti-MHC-II) and anti-lysozyme antibodies were undertaken, and semiquantitative evaluation was carried out. MHC-II was mainly expressed by lymphocytes in tubulointerstitial nephritis, but did not always stain macrophages in cases of granulomatous (including lymphohistiocytic) nephritis; the anti-lysozyme antibody revealed macrophages when present in tissues. The amount of PCV2 nucleic acid was not apparently associated with the pattern of inflammation (tubulointerstitial or granulomatous). PCV2 load seems to reflect the severity of the lymphoplasmacytic inflammation but not that of granulomatous and lympho histiocytic types.     

Reproduction of postweaning multisystemic wasting syndrome in pigs by prenatal porcine circovirus 2 infection and postnatal porcine parvovirus infection or immunostimulation

Postweaning multisystemic wasting syndrome (PMWS) was reproduced in prenatally porcine circovirus 2 (PCV2)-infected pigs by either postnatal infection with porcine parvovirus (PPV) or by immunostimulation. Twenty-four randomly selected piglets from 3 sows, which had been experimentally infected during gestation with PCV2, were randomly divided into 3 groups; group 1 (prenatal PCV2 infection, with postnatal PPV infection), group 2 (prenatal PCV2 infection, with postnatal keyhole limpet hemocyanin, emulsified in incomplete Freund's adjuvant [KLH/ICFA] injection), and group 3 (prenatal PCV2 infection only). Twenty-four randomly selected piglets from 3 uninfected sows were randomly divided into 3 groups; group 4 (no prenatal infection, with postnatal PCV2 and PPV infection), group 5 (no prenatal infection, with postnatal PCV2 infection), and group 6 (negative control pigs). Body weight in negative control pigs (group 6) was increased significantly compared with pigs in groups 1, 2, and 4 at 49, 52, 56, 59, and 63 days of age. The granulomatous inflammatory reaction and lymphoid depletion that are typical lesions in pigs with PMWS were observed in the lymph node of piglets in groups 1, 2, and 4 at 63 days of age. Pigs in group 3 had significantly fewer PCV2-positive cells than those from groups 1, 2, 4, or 5. When the prenatally PCV2-infected pigs were infected with PPV or injected with immunostimulant in the postnatal period, they developed PMWS. Thus, factors that potentiate the progression of prenatal PCV2 infection to PMWS are postnatal infection with PPV or immune stimulation.     

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.     

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.     

猪断奶后多系统衰竭综合征诊断方法的系列研究

本研究对猪断奶后多系统衰竭综合征(PMWS)的诊断方法临床症状和病理变化、病毒的分离与鉴定、聚合酶链式反应(PCR)、间接免疫荧光试验(IFA)、间接酶联免疫吸附试验(IELISA)、免疫组织化学试验(IHC)等进行了系列研究,这些方法适用于猪断奶后多系统衰竭综合征的诊断.     

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.     

Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus

One-day-old gnotobiotic piglets were inoculated intranasally with in vitro passaged porcine circovirus 1 (PCV-1), PCV-2, and porcine parvovirus (PPV) alone or in combination (PCV-1/PCV-2, PCV-1/PPV, and PCV-2/PPV). Piglets were evaluated for 1) the development of porcine postweaning multisystemic wasting syndrome (PMWS), 2) distribution of viral antigens by immunochemistry, and 3) viremia and the presence of viral DNA in nasal and ocular secretions and feces. All single agent-infected piglets and piglets infected with PCV-1/PCV-2 or PCV-1/PPV were clinically asymptomatic. They were transiently viremic and seroconverted to homologous virus(es). At termination of the study on postinfection day (PID) 35, microscopic lesions were restricted to focal inflammatory cell infiltrates in livers and myocardia. One piglet given PCV-1/PPV was PPV viremic for 2 weeks after infection and had lymphangiectasia of the spiral and descending colon associated with granulomatous inflammation. All four PCV-2/PPV-inoculated piglets developed PMWS, characterized by sudden onset of depression and anorexia, icterus, and submucosal edema. One piglet became moribund on PID 27, and the remaining three piglets were euthanatized between PID 27 and PID 30 because of severe disease. Lymph nodes were small and the livers were mottled. Disseminated angiocentric granulomatous inflammation was present in all tissues examined except the brain. Multiple lightly basophilic intracytoplasmic inclusion bodies were identified in macrophages and histiocytes. PCV-2 antigen was widely distributed within macrophages; PPV antigen was sparse. Hepatocellular necrosis and bile retention were prominent. PCV-2 DNA was identified in ocular, fecal, and nasal secretions. Terminal sera contained antibodies to PPV (4/4) and PCV-2 (3/ 4). Production of PMWS in gnotobiotic swine appears to require PCV-2 and additional infectious agents such as PPV for full disease expression in gnotobiotic piglets.     

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.     

Reproduction of postweaning multisystemic wasting syndrome in pigs experimentally inoculated with a Swedish porcine circovirus 2 isolate.

In recent years, porcine circovirus type 2 (PCV2)-associated postweaning multisystemic wasting syndrome (PMWS) has been reported worldwide. However, to date, PMWS has not been reported in Sweden despite the demonstration of serum antibodies to a PCV2-like virus in Swedish pigs. This communication reports the experimental reproduction of clinical PMWS after inoculation of colostrum-deprived (CD) pigs, derived from a Northern Ireland herd, with an isolate of PCV2 virus recovered from a clinically normal Swedish pig that was necropsied in 1993. The clinical disease and histological lesions observed in CD pigs inoculated with this virus were indistinguishable from those observed in previous studies on CD pigs inoculated with a PCV2 virus isolate recovered from pigs with PMWS. These results highlight the disease potential of PCV2 isolated from regions apparently free of PMWS and suggest that the status of the host and its environment is an important factor in the development of clinical PMWS.     

Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome.

Postweaning multisystemic wasting syndrome (PMWS), an apparently new disease, has been recognized in swine herds in western Canada. Young pigs with this disease have progressive weight loss, tachypnea, dyspnea, and jaundice, accompanied by interstitial pneumonia, lymphadenopathy, hepatitis, and nephritis. We examined more than 400 pigs from more than 70 herds in Alberta, Saskatchewan, and Manitoba with cases of PMWS. A small virus was isolated from a range of tissues from 8 of 8 affected pigs examined. The agent was identified as a circovirus-like virus using electron microscopy, immunohistochemical staining with porcine and rabbit immune serum, and in situ hybridization. Immunohistochemical examination of tissues from more than 100 affected pigs has revealed widespread viral antigen, often contained in circovirus-like inclusion bodies, in lesions from numerous organs. Although Koch's postulates remain to be fulfilled, these results demonstrate a high degree of association between the presence of the circovirus-like virus and PMWS in affected swine.     

Dynamics of porcine circovirus type 2 infection in a herd of pigs with postweaning multisystemic wasting syndrome

OBJECTIVE: To determine the pattern of infection for porcine circovirus type 2 (PCV2) in a herd of pigs with postweaning multisystemic wasting syndrome (PMWS). ANIMALS: 29 sows and 250 pigs. PROCEDURE: Blood samples were collected from all 3-, 7-, and 12-week old pigs and 59 pigs at 28 weeks of age. Pigs that died during the study were necropsied. Porcine parvovirus and PCV2 antibodies were assayed. A polymerase chain reaction (PCR) was used to detect PCV2 genome in serum of selected pigs. RESULTS: The PMWS started when pigs were 8 weeks old, with a prevalence of 30% in 8- to 10-week-old pigs. Eighty-three pigs died during the period between 3 and 12 weeks of age. Microscopic lesions consistent with PMWS were observed, and PCV2 nucleic acid was detected (50 of 68 pigs). Antibodies to PCV2 decreased from 3 to 7 weeks of age, increased at 12 weeks of age, and were maintained until 28 weeks of age. One sow had a positive result for PCR of serum. Nine, 37 and 8 pigs had PCV2 genome in serum obtained at 7, 12, and 28 weeks of age, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with PCV2 coincided with severe clinical signs; however, infected 28-week-old pigs did not have evidence of disease. Immunity declined over time in young pigs. A long duration of PCV2 viremia was apparent in a high percentage of infected pigs, which may affect transmission and persistence of the virus in a herd.     

Potentiation of porcine circovirus 2-induced postweaning multisystemic wasting syndrome by porcine parvovirus is associated with excessive production of tumor necrosis factor-alpha

This study investigated the potentiation of porcine circovirus 2 (PCV2)-induced postweaning multisystemic wasting syndrome by porcine parvovirus (PPV) and found it was associated with excessive production of tumor necrosis factor-alpha (TNF-alpha). Colostrum-deprived conventional pigs were inoculated intranasally with PCV2 or PPV alone or in combination (PCV2 and PPV). In vitro assay of TNF-alpha, obtained from alveolar macrophages coinfected with PCV2 and PPV, showed a significant increase in TNF-alpha compared to single infection of macrophages with either PCV2 or PPV alone (P < 0.05). All pigs inoculated with PCV2 and PPV developed severe postweaning wasting syndrome, whereas clinical signs (e.g., weight loss) were present but perhaps less severe in either PCV2- or PPV-inoculated pigs. Compared to the pigs inoculated with PCV2 or PPV alone, pigs inoculated dually with PCV2 and PPV showed significantly (P < 0.05) increased levels of TNF-alpha. Levels of TNF-alpha in the sera were reversely correlated with the body weight in pigs experimentally infected with dual inoculation of PCV2 and PPV (r(s) = -0.92, P < 0.001). These data suggest that a potentiation of PPV in PCV2-induced PMWS is associated with the excessive production of TNF-alpha.     

断奶仔猪多系统衰竭综合征

断奶仔猪多系统衰竭综合征(PMWS) 是由PCV2引起的多发于断奶仔猪的一种疾病, 常对养猪生产造成重大损失。本文就PMWS的临床特征、病原、病理变化、诊断标准、促发因素、控制等方面进行了综述。     

Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2

The objectives of this study were to investigate the interactions between Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2) and to establish a model for studying the pathogenesis of and testing intervention strategies for the control of PCV2-associated porcine respiratory disease complex (PRDC). Sixty-seven pigs were randomly assigned to four groups. Group 1 (n=17) pigs served as controls, group 2 (n=17) pigs were inoculated with M. hyopneumoniae, group 3 (n=17) pigs were dual infected with M. hyopneumoniae and PCV2, and group 4 (n=16) pigs were inoculated with PCV2. Pigs were inoculated intratracheally with M. hyopneumoniae at 4 weeks of age followed by intranasal inoculation with PCV2 at 6 weeks of age. Dual-infected pigs had moderate dyspnea, lethargy, and reduced weight gain. The overall severity of macroscopic lung lesions, PCV2-associated microscopic lesions in lung and lymphoid tissues, and the amount of PCV2-antigen associated with these lesions were significantly (P <0.05) higher in dual-infected pigs compared with all other groups. Four of 17 (23.5%) dual-infected pigs had decreased growth rate and severe lymphoid depletion and granulomatous lymphadenitis associated with high amounts of PCV2-antigen consistent with postweaning multisystemic wasting syndrome (PMWS). PCV2-antigen in lung tissue was most often associated with M. hyopneumoniae-induced peribronchial lymphoid hyperplasia, suggesting that this is an important site for PCV2 replication in the lung. This study indicates that M. hyopneumoniae potentiates the severity of PCV2-associated lung and lymphoid lesions, increases the amount and prolongs the presence of PCV2-antigen, and increases the incidence of PMWS in pigs.