Lori Hassard

Characterization of novel circovirus DNAs associated with wasting syndromes in pigs

Porcine circovirus (PCV) was initially recognized as a contaminant of continuous pig kidney cell lines and was not thought to be pathogenic. Antibodies reactive to the cell culture isolate of PCV (PCV PK-15) are prevalent in the swine population worldwide. Recently, PCV PK-15-like antigen and nucleic acid were demonstrated in lesions associated with wasting syndromes in pigs in North America and Europe. Monoclonal antibodies raised to circoviruses isolated from pigs with wasting syndromes highlighted differences between these circoviruses and the PCV PK-15 cell culture isolate. This has led to speculation that a new pathogenic PCV may have emerged in the swine populations of several countries. We report the cloning and characterization of novel circovirus DNAs purified from virus isolates made from tissues of North American and European pigs with wasting syndromes. These North American and European circoviruses form a closely related group at the nucleotide sequence level (> 96% intra-group nucleotide sequence identity) but exhibit < 80% nucleotide sequence identity with the PCV PK-15 cell culture isolate. This report provides evidence for a new type of possibly pathogenic PCV. We propose that these new circoviruses should be referred to as PCV2 as opposed to the original PK-15 cell culture isolate, which should be referred to as PCV1.

Reproduction of Lesions of Postweaning Multisystemic Wasting Syndrome in Gnotobiotic Piglets

Neonatal gnotobiotic piglets were inoculated with tissue homogenates and low- and high-passage cell culture material to determine if the lesions of the newly described porcine postweaning multi-systemic wasting syndrome (PMWS) could be reproduced. For this, 17 3-day-old gnotobiotic piglets were inoculated intranasally with pelleted chloroform-treated, filtered extracts from cell cultures, filter-sterilized homogenates of lymphoid tissue from PMWS-affected piglets, or control materials. Piglets were maintained in germ-free isolators for up to 5 weeks after infection prior to euthanasia and collection of samples for analysis. All piglets inoculated with the viral inocula developed lesions typical of PMWS, including generalized lymphadenopathy, hepatitis, nephritis, interstitial pneumonia, myocarditis, and gastritis. Porcine circovirus (PCV), as well as porcine parvovirus (PPV), was detected in tissues by virus reisolation, polymerase chain reaction analysis, or immunohistochemistry. All infected piglets developed moderate to high titers of antibody to PCV and moderate titers to PPV. No lesions, virus, or virus-specific antibodies were detected in sham-inoculated or uninoculated control piglets. These studies demonstrate that the lesions of PMWS can be experimentally reproduced in gnotobiotic piglets using filterable viral agents derived from pigs with PMWS and provide an experimental basis for further investigation into the pathogenesis and control of this emerging infectious disease in swine.

Coinfection by Porcine Circoviruses and Porcine Parvovirus in Pigs with Naturally Acquired Postweaning Multisystemic Wasting Syndrome

Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Recently, the disease has been reproduced with inocula containing a newly described porcine circovirus (PCV), designated PCV 2, and porcine parvovirus (PPV). In order to determine if these viruses interact in naturally acquired PMWS, affected tissues from field cases were examined by immunohistochemistry (IHC) and polymerase chain reaction (PCR) for PCV 2 and PPV, as well as by PCR for the other recognized porcine circovirus, PCV 1. Porcine circovirus 2 was detected by PCR or IHC in affected fixed or frozen tissues from 69 of 69 cases of PMWS collected over 3 years from 25 farms. Porcine parvovirus was detected in 12 of the same cases, and PCV 1 was detected in 9 of 69; however, an apparent decrease was found in the sensitivity of the PCRs used to detect the latter 2 viruses when fixed tissue from the same cases were compared with the use of frozen tissues. Porcine circovirus 2 was not detected by PCR in affected tissues from 16 age-matched pigs that had Streptococcus suis-associated disease. Electron microscopic examination of plasma pooled from 15 pigs with PMWS revealed the presence of PCV and PPV, whereas these viruses were not observed in pooled plasma from 5 age-matched clinically normal pigs. These results confirm and extend previous findings documenting a consistent association of PCV 2 with PMWS. As well, infection by PPV or PCV 1 or both may be an important cofactor in the pathogenesis of some, but apparently not all, cases of PMWS.

Pathology of Newcastle disease in double-crested cormorants from Saskatchewan, with comparison of diagnostic methods.

Newcastle disease (ND) in juvenile double-crested cormorants (Phalacrocorax auritus) occurred several times since 1975, but there are relatively few studies on its pathology and diagnosis. In order to describe the distribution of Newcastle disease virus (NDV) and associated lesions in cormorants with ND and to compare diagnostic methods, 25 cormorants with nervous signs from a ND epizootic in Saskatchewan in 1995 (NDE cormorants) were compared with 18 negative control cormorants. Tissues of these birds were examined by necropsy, histology, virus isolation, immunohistochemistry, serology, and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. The NDE cormorants had a characteristic non-suppurative encephalomyelitis, with a significantly higher prevalence of neuronal necrosis, gliosis, perivascular infiltration with mononuclear cells, and endothelial hypertrophy than control cormorants. These lesions were found more frequently in the cerebellum and brain stem than in other parts of the central nervous system. Immunohistochemically, NDV antigen was limited to neurons, glial and endothelial cells in the central nervous system, and to tubular epithelial cells in the kidney. Newcastle disease virus was isolated with the highest prevalence (4/5) and the highest concentration (104.8 ELD50/g) from the kidney. The virus isolates often did not agglutinate erythrocytes in the standard hemagglutination test; the presence of NDV was confirmed by use of an indirect immunoperoxidase assay. By RT-PCR, NDV was detected in kidney and jejunum of a NDE cormorant. There was no significant difference between sensitivity of histology, virus isolation, and serology for detecting ND in NDE cormorants.

Development and application of a microneutralization ELISA for the detection of antibodies to bovine respiratory syncytial viruses

A microneutralization enzyme-linked immunosorbent assay (ELISA) was developed to detect specific antibodies to bovine respiratory syncytial viruses (BRSVs) in cattle sera using a monoclonal antibody to the fusion protein of the virus. Serum from 20 naturally exposed, 24 experimentally infected, and 15 immunized cattle were evaluated using 3 different BRSV isolates. Antibody titers determined with the micro-neutralization ELISA were compared with those derived from a classical virus neutralization assay, an indirect ELISA, and a fusion inhibition assay. These studies demonstrated a high degree of correlation (usually 0.90) among the assays. Furthermore, the results showed that immunization of cattle with one isolate (subgroup) of BRSV induced antibody responses that cross-reacted with at least 2 disparate isolates. These results document the utility of the microneutralization ELISA in assessing functionally important antibody responses to BRSVs in cattle.