C. Rosell

Experimental Inoculation of Conventional Pigs with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus 2

ABSTRACT Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n = 5), PRRSV inoculated (n = 5), PCV2 inoculated (n = 7), and PRRSV and PCV2 inoculated (n = 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.

Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (Pcv) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of Pcv (which recognises both Pcv-i and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twentyeight of the 30 Spanish pigs and all three American pigs had Pcv in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyers patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that Pcv is present in tissues of almost all pigs affected by PDNS, and Pcv has to be considered as a possible agent involved in the pathogenesis of the syndrome.

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), Aujeszkys 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-i 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-i genome in its serum. Immunohistochemical, serological and PCR studies revealed that PRRSV and ADV were also present on the farm during the outbreak.

Serum antibodies to porcine circovirus type 1 and type 2 in pigs with and without PMWS

PORCINE circovirus (PCV) is a small, single-stranded, circular, covalently-closed DNA virus, which was first detected as a contaminant in the pig kidney cell line PK-15 (ATCC CCL-33) (Tischer and others 1974). PK-15-derived PCV is considered apathogenic (Tischer and others 1986, Allan and others 1995), and serological studies for the detection of PCV antibodies in pig sera from Germany, Canada, New Zealand, Great Britain, Northern Ireland and the USA have shown a widespread presence of PCV antibodies in fattening and adult pigs, ranging from 25 to 98 per cent of the investigated sera. Therefore, it has been suggested that PCV infection is ubiquitous throughout the world (Allan 1996). Since 1991 in Canada (Clark 1997) and during the past two years in several other countries, including the USA, France, Spain, the UK, Denmark, Italy and Germany, a new disease called postweaning multisystemic wasting syndrome (PMWS) has been identified. PMWS affects nursery and fattening pigs, and i s histologically characterised by lymphocyte depletion with histiocytic infiltrates within lymphoid organs, and bronchointerstitial pneumonia (Clark 1997, Segalés and others 1997). An unequivocal aetiological relationship between PCV and PMWS has not been demonstrated; however, pigs affected with PMWS are regularly infected with a novel strain of PCV. This new strain of PCV has a nucleotide sequence similarity of 75 per cent when compared with PCV derived from PK-15 cells (Hamel and others 1998, Meehan and others 1998); therefore, the nomenclature PCV type 1 (PCV-1) and PCV type 2 (PCV-2) has been suggested for the PK-15and PMWS-derived PCV, respectively (Allan and others 1999). Antigenic differences between PCV-1 and PCV-2 do exist, as some monoclonal antibodies raised against PCV-1 are not able to recognise PCV-2, and monoclonal antibodies raised against PCV-2 cannot recognise PCV-1 in infected cell monolayers by immunofluorescence (Allan and others 1999). In addition, hyperimmune rabbit serum raised by immunisation with PCV-2 showed a high specific titre against PCV-2 but only a low titre against PCV-1 in cell monolayers (Allan and others 1998). The aim of this study was to compare serum antibody titres against PCV-1 and PCV-2 in necropsied pigs, with and without PMWS, using an immunoperoxidase monolayer assay (IPMA) technique. A total of 90 oneto five-month-old pigs from 37 different Spanish farms were submitted to the laboratory of the Veterinary School of Barcelona for pathological examination between May 1997 (when PMWS was recognised in Spain) and July 1998. Pigs were bled at the jugular vein and a 10 ml blood sample was collected in a Vacutainer (Venoject; Terumo Europe). The sample was allowed to clot, centrifuged and then the serum was frozen at –80°C until required for testing. The pigs were necropsied and tissue samples (including the lungs, lymphoid organs, livers and kidneys) were fixed in 10 per cent buffered formalin, embedded in paraffin wax and routinely processed for histopathology. Pigs were classified as PCV-2Short Communications

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 Aujeszkys 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.

Detection of Porcine Circovirus Types 1 and 2 in Serum and Tissue Samples of Pigs with and without Postweaning Multisystemic Wasting Syndrome

ABSTRACT Presence of porcine circovirus type 1 (PCV1) and PCV2 was studied in sera and superficial inguinal lymph nodes from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs by using in situ hybridization and PCR. PCV1 and PCV2 were found in less than 3% and more than 50% of the samples, respectively. The most sensitive technique and site was PCR in superficial inguinal lymph nodes, but in situ hybridization correlated better with presence of characteristic lesions.

Hepatitis and staging of hepatic damage in pigs naturally infected with porcine circovirus type 2.

A total of 100 liver samples from pigs with postweaning multisystemic wasting syndrome (PMWS) were studied. All livers were evaluated microscopically and were staged based on the severity and localization of lesions. Presence of porcine circovirus type 2 (PCV-2) was evaluated using an in situ hybridization technique. Eighty-eight of 100 livers (88%) had a variable degree of lymphohistiocytic hepatitis, with apoptotic bodies, disorganization of hepatic plates, and/or perilobular fibrosis. Twelve pigs did not have microscopic liver lesions. Four stages of hepatic damage were established based on intensity and distribution of the lesions. Viral nucleic acid was detected in 70 of 100 livers (70%). Target cells for PCV-2 infection included Kupffer cells, hepatocytes, and inflammatory infiltrates. According to distribution of PCV-2 nucleic acid, four basic labeling patterns were identified. This study shows that liver damage is a frequent microscopic finding in cases of PMWS and hepatocytes are a target cell for PCV-2 infection and replication. Therefore, PCV-2 should be considered a new hepatitis-inducing viral agent in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection status in pigs naturally affected with post-weaning multisystemic wasting syndrome (PMWS) in Spain

The purpose of the study reported here was to determine the prevalence of porcine reproductive and respiratory syndrome virus (PRRSV) in pigs affected with post-weaning multisystemic wasting syndrome (PMWS), a disease believed to be caused by porcine circovirus type 2 (PCV2). From May 1997 to February 2000, PMWS was diagnosed in 277 pigs (from 120 farms) submitted to the Veterinary Pathology Diagnostic Service, Veterinary School of Barcelona, Spain. In each case, the PMWS diagnosis was based on clinical history and the detection, by in situ hybridization, of nucleic acid of PCV2 in characteristic histologic lesions. Antigens for PRRSV were detected by immunohistochemistry in tissues of 66 (23.8%) of the same 277 pigs. Sera, which were available for 93 of the 277 pigs, were tested for PRRSV by a multiplex reverse transcription-polymerase chain reaction (RT-PCR). A total of 33 of these sera were RT-PCR positive, three for a North American strain(s) of PRRSV. In addition, 76 of the 93 sera were tested for antibodies to PCV2 (indirect immunoperoxidase) and PRRSV (enzyme-linked immunoassay). Antibodies for PCV2 and PRRSV were detected, respectively, in 56 (73.9%) and 43 (56.6%) of the 76 sera. Collectively, these results suggest that while infection with PRRSV may be common, it is not an essential component of PMWS.

Intestinal chiamydial infection concurrent with postweaning multisystemic wasting syndrome in pigs

L. Carrasco, DVM, PhD, M. J. Bautista, DVM, PhD, J. C. G6mezViramandos, DVM, PhD, E. Ruiz-Villamor, DVM, M. A. Sierra, DVM, PhD, Departmento Anatomia y Anatomia Patol6gica Comparadas, Facultad de Veterinaria, Edificio de Sanidad Animal, Campus Universitario de Rabanales, Universidad de C6rdoba, C6rdoba, Spain J. Segales, DVM, PhD, C. Rosell, DVM, UD Anatomia Patol6gica, Facultat de Veterinaria, Universitat Aut6noma de Barcelona, Barcelona, Spain Intestinal chiamydial infection concurrent with postweaning multisystemic wasting

AUJESZKY'S DISEASE VIRUS INFECTION CONCURRENT WITH POSTWEANING MULTISYSTEMIC WASTING SYNDROME IN PIGS

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