Edward Clark

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.

Myocarditis and Abortion Associated with Intrauterine Infection of Sows with Porcine Circovirus 2

Porcine circovirus 2 (PCV2) is a recently identified agent that has been associated with post-weaning multisystemic wasting syndrome (PMWS) in swine populations. In this report, the potential spectrum of disease associated with PCV2 is expanded by evidence of vertical transmission and associated reproductive failure. PCV2 was isolated from a litter of aborted piglets from a farm experiencing late-term abortions and stillbirths. Severe, diffuse myocarditis was present in 1 piglet associated with extensive immunohistochemical staining for PCV2 antigen. Variable amounts of PCV2 antigen were also present in liver, lung, and kidney of multiple fetuses. The presence of other agents that have been associated with fetal lesions and abortion in swine, including porcine parvovirus, porcine reproductive respiratory syndrome virus, encephalomyocarditis virus, and enterovirus, could not be established.

Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland

A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1).

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.

Bovine respiratory syncytial virus.

Since the first report of BRSV in the 1970s, the understanding of this agent and its respective disease has increased dramatically. Current evidence supports a major role for this virus in bovine respiratory disease. Advances in diagnostics have increased the ability to demonstrate this virus in field outbreaks of respiratory disease. The clinical signs and pathologic features have been well described, and vaccines are available to aid in prevention and control. Still, many questions remain to be answered with respect to BRSV. It appears there may be antigenic subgroups of BRSV, but the epidemiologic significance and relevance to immunization of this remains unknown. The question of differences in virulence among isolates of this virus has yet to be addressed. From an epidemiologic standpoint, the means by which BRSV perpetuates in the cattle population has yet to be elucidated. Although progress has been made in understanding the pathogenesis and immune response to BRSV, the mechanism of disease production and immune protection is incomplete. Lastly, efficacy testing of existing vaccines need to continue, as well as the development of new vaccines and new approaches to vaccination.The current knowledge is reviewed in regards to the importance of bovine respiratory syncytial virus in the bovine respiratory disease complex. The epidemiology, clinical disease, pathologic findings, pathogenesis, diagnosis, treatment, and prevention of this viral disease are discussed.

Diagnosis of persistent bovine viral diarrhea virus infection by immunohistochemical staining of formalin-fixed skin biopsy specimens.

The objective of this study was to evaluate the efficacy of immunohistochemical (IHC) staining for diagnosis of persistent bovine viral diarrhea virus (BVDV) infection using formalin-fixed, paraffin-embedded skin biopsy specimens. Skin from 41 of 42 calves shown to be persistently infected (PI) with BVDV by repeated virus isolation more than 3 weeks apart were immunohistochemically positive for BVDV antigen. Positive IHC staining was most pronounced in the keratinocytes and in hair follicle epithelium, hair matrix cells of the hair bulb, and the dermal papilla. All of the skin sections from 10 calves experimentally infected postnatally with BVDV (105 median tissue culture infective doses [TCID50]) and biopsied on days 0, 5, 7, and 9 postinfection were negative for viral antigen. Ten calves from a second group experimentally infected with a higher dose of BVDV (108 TCID50) were biopsied when viremic between 10 and 14 days postinfection and 4 calves exhibited positive IHC staining for BVDV; however, staining in these skin biopsies was confined to small foci in the nonfollicular epidermis and follicular ostia. This staining was distinct from that observed in skin obtained from PI cattle. Skin biopsy represents an effective method for identifying animals PI with BVDV.

Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction.

This report describes the first diagnosis of porcine circovirus (PCV) infection in weaned pigs with postweaning multisystemic wasting syndrome in Korea by immunohistochemistry and polymerase chain reaction. The most unique lesions were multifocal granulomatous inflammation affecting lymph nodes, liver, and spleen, characterized by infiltrates of epithelioid macrophages and multinucleated giant cells. Circoviral antigen was detected in formalin-fixed sections and was usually present in large, round, dendritic cells in the white pulp of spleen and remnants of follicles in lymph nodes. Lymphoid follicles in the tonsils also contained PCV antigen. A 530–bp DNA fragment of circovirus was successfully amplified from all tested lymph nodes, liver, and spleen.

Typhlocolitis Caused by Clostridium Difficile in Suckling Piglets

phe obsoleta quadrivittata) was treated with radiation therapy, which delayed local recurrence.11 Cobalt therapy was unsuccessful in the treatment of a lymphosarcoma in an Indian python (Python molurus) and an angiosarcoma in a spitting cobra (Naja nigrocollis).7,11 Chemotherapy has been used rarely in reptiles.8 Cytosine arabinoside has been unsuccessfully used in the treatment of a lymphosarcoma in a rhinoceros viper.9 In this kingsnake, chemotherapy was not selected because the tumor appeared localized and without metastases.

The effect of formalin-inactivated vaccine on respiratory disease associated with bovine respiratory syncytial virus infection in calves.

The effect of vaccination with a formalin-inactivated, alum-precipitated (FI), bovine respiratory syncytial virus (BRSV) vaccine on BRSV induced respiratory disease in calves was investigated. Six month old BRSV-naive calves were vaccinated with either a FI, a modified live virus (MLV), or virus antigen negative control vaccine (n = 4 per group). One month after the second vaccination, the calves were aerosol challenged with lung wash from a newborn calf infected with a field isolate of BRSV. Moderate to severe clinical disease occurred in all calves. Calves that received FI vaccine had a significantly earlier (day 2 vs. day 4-5) onset of pyrexia and dyspnea (P < 0.05). Pulmonary lesions, consisting of cranioventral atelectasis and dorsal emphysema, occurred in all groups. Two calves that received MLV, and three that received FI vaccine, had reduced pneumonic lung area relative to controls. Vaccination with the FI vaccine resulted in more rapid onset of clinical disease, but ultimately, reduced pulmonary pathology in most recipients.