Joaquim Segalés

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.

PCV-2 genotype definition and nomenclature

SIR, — To date, at least three different phylogenetic groups of porcine circo-virus type 2 (pcv-2) have been recognised ([Gagnon and others 2007][1], [Dupont and others 2008][2], [Grau-Roma and others 2008][3], [Timmusk and others 2008][4]). However, the mandate of the International Committee on

Porcine circovirus type 2 (PCV2) infections: clinical signs, pathology and laboratory diagnosis.

Clinical signs and pathological features are still the corner-stones to suspect and diagnose overt disease associated with PCV2 infection. The clinico-pathological scope of this viral infection has been expanded over time. From the initial description of postweaning multisystemic wasting syndrome, some enteric, respiratory and reproductive disorders have been subsequently linked with PCV2. Porcine dermatitis and nephropathy syndrome, an immunocomplex disease, has also been associated with infection by this virus. All together, these conditions have been grouped under the name of porcine circovirus diseases (PCVD) or porcine circovirus associated diseases (PCVAD). The precise mechanisms by which a PCV2 infected pig develops a PCV2 subclinical infection or a clinical PCVD/PCVAD are still to be fully elucidated, but inferences based upon clinical, gross and histologic findings from field cases of disease have been useful to suggest the pathogenesis of this viral infection. The objective of the present review is to update the current knowledge on the clinical and pathological scope of PCV2 infections, as well as on their diagnosis. Moreover, a proposal on a unified PCVD/PCVAD terminology and clearly defined diagnostic criteria for these conditions are also given.

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.

Porcine circovirus type 2 (PCV2) vaccination of conventional pigs prevents viremia against PCV2 isolates of different genotypes and geographic origins.

The efficacy of recently developed porcine circovirus type 2 (PCV2) vaccines has not been tested yet against PCV2 isolates of the two proposed genotypes. In the present work, the efficacy of a subunit vaccine containing PCV2 capsid protein was evaluated by using a challenge model with four different PCV2 isolates of different genotype and geographic origin. The vaccine prevented the development of viremia in all cases as well as significantly decreased nasal and faecal shedding of the virus. Also, the vaccine elicited PCV2-specific neutralizing antibodies to PCV2 even in the presence of maternally derived immunity.

Characterisation of PCV-2 isolates from Spain, Germany and France

The new isolated circovirus variant PCV-2 is discussed to be the etiological agent of a new emerging swine disease with a variable morbidity and high lethality, postweaning multisystemic wasting syndrome (PMWS). PMWS has been diagnosed in North America and West Europe. Clinical signs include dyspnea, loss of weight, lymph node enlargement and lymphocyte depletion in lymphoid tissues. This report describes the characterisation of PCV-2 isolates from animals affected with PMWS from Germany, Spain and France. We could demonstrate the presence of circovirus by electron microscope, in situ hybridisation and PCR. PCR revealed incidence of PCV-2 in many tissues of one infected animal with the exception of heart and nervous system. The phylogenetic analysis of all PCV-2 isolates yet published in the database, showed relationship of isolates from Spain, Germany and France, with three sequences from Canada determined recently and two isolates from Taiwan, while other North American sequences display a separate cluster. PCR screening of randomly collected organ samples from German pigs not affected with PMWS, revealed a rate of infection with PCV-1 of 5% and with PCV-2 of 26.8%, while blood samples showed a lower incidence.

A review of viral diseases of the European wild boar: effects of population dynamics and reservoir rôle.

Abstract There has been a worldwide increase in the number and geographical spread of wild boar populations in recent decades leading to an increase in both the circulation of disease agents and greater contact with domestic animals and humans. Diseases affect the population dynamics of wildlife but the effects of most viral diseases on the European wild boar are largely unknown. Many viral diseases present in domestic pig populations are also present in wild boars where they can provide a disease reservoir, as is clearly the case with classical swine fever, but little is known about other viral diseases such as porcine circovirus diseases or hepatitis E. This review considers the current scientific knowledge of the effects of viral diseases on wild boar populations and their rôle as potential disease reservoirs. The focus is on those viral diseases of domestic swine and wild boars that are included as notifiable by the Office International des Epizooties (OIE).

First report of post-weaning multisystemic wasting syndrome in pigs in Spain

Surgery or cryosurgery is one of the most common treatments for squamous cell carcinoma (Kwa and others 1992, Withrow and Madewell 1996). However, in the flock studied the location of the tumour on the eyelid rendered this type of treatment unsuitable. Bovine squamous cell carcinoma are responsive to immunotherapy, which results in the complete or partial regression of the tumours in the majority of cases (Spradbrow and others 1977). However, the application of immunotherapy to the condition in sheep has so far proved negative, and has resulted in the enhancement of tumour growth and -an increased risk of metastasis (Jun 1978, Jun and others 1979, Swan and others 1983). Chemotherapy with cyclophosphamide (200 mg weekly for 10 weeks) induced the partial or complete regression of the early lesions in a flock of sheep (Jun and Johnson 1979); the treatment was subsequently recommended for all the sheep in the flock with early lesions, but owing to the cost, the owner decided not to apply it. The clinical and pathological characteristics of the outbreak of squamous cell carcinoma described here suggest that the breed of sheep, its lack of skin pigmentation, the farms southern latitude, and factors related to the type of farming played a decisive role in the development of squamous cell carcinoma in these sheep.

Pathogenesis of postweaning multisystemic wasting syndrome caused by Porcine circovirus 2: An immune riddle.

Summary.Postweaning multisystemic wasting syndrome (PMWS) is a disease of pigs first recognised in North America in 1997 and subsequently reported worldwide that is caused by Porcine circovirus 2 (PCV2), a member of the family Circoviridae. The most consistent feature of PMWS is a generalized depletion of lymphocytes. Secondary infections with opportunistic organisms are common. There is evidence that the destruction of thymic lymphocytes has a central role in the pathogenesis of PMWS. Pigs with PMWS have altered cytokine responses to mitogens and recall antigens. It remains unknown what cells are primarily infected and are permissive for the replication of PCV2. Macrophages and dendritic cells commonly contain virus in their cytoplasm but may not be the primary source of the large amounts of virus found in tissues of diseased pigs. There is evidence that PCV2, like mammalian parvoviruses, requires cells in the S phase of the cell cycle for replication. It has been difficult to reproduce PMWS experimentally although some protocols have been developed which involve antigenic stimulation with other agents that presumably increase the number of permissive cells entering S phase of the cell cycle. In addition to reviewing the literature attempts are made to identify key unresolved areas that should be the focus of future research.

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.