Diagnosis of malignant catarrhal fever by PCR using formalin-fixed, paraffin-embedded tissues.
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A previously described polymerase chain reaction (PCR) assay (amplification of a 238-bp fragment of ovine herpesvirus 2 [OHV-2] genomic DNA) for diagnosis of sheep-associated malignant catarrhal fever (MCF) was adapted for use on formalin-fixed, paraffin-embedded tissues. Variables affecting its use were examined. Archived tissues from cattle, white-tailed deer (Odocoileus virginianus), and bison (Bison bison) diagnosed with MCF by clinical signs or histologic lesions were obtained from 2 veterinary diagnostic laboratories. Tissues from healthy animals and from animals diagnosed with other common bovine viral diseases were examined as controls. A total of 86 blocks from 37 suspect MCF cases were examined. Forty-one blocks from healthy animals and animals with unrelated viral diseases were examined as controls. The assay was specific for sheep-associated MCF and did not yield false-positive signals from healthy animals or from cases of infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues were suitable substrates, including spleen, lymph node, intestine, brain, lung, and kidney. Extracted DNA provided a more suitable target than did unextracted tissue lysate. The highest levels of viral DNA were present in lymphoid organs and intestine, but the data indicate that in acute clinical cases, most organs contain sufficient viral DNA to serve as a suitable diagnostic specimen. Fixation of 0.5-cm3 blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and PCR signals progressively diminished after fixation for >45 days. Detection of genomic DNA of OHV-2 by PCR was successful for archived tissues that were 15 years old. (+info)
Quantitation of sheep-associated malignant catarrhal fever viral DNA by competitive polymerase chain reaction.
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A single-step, competitive polymerase chain reaction technique was developed to quantitate sheep-associated malignant catarrhal fever (SA-MCF) viral DNA. The assay employed coamplification of a fixed quantity of target DNA with graded amounts of a competitor, generated by truncation of the target sequence lying between the 2 primer binding sites. The assay yielded a linear response (r = 0.98) for DNA measurement within the range of 30-300,000 copies. Amplification efficiency analysis by coamplification of target and competitor in equal copy numbers for various numbers of cycles showed that the relative abundance of the coamplified products remained constant with increasing cycle numbers up to 40. Reproducibility was assessed by repetitively assaying a set of blind-coded samples from a variety of animals and tissues. Results indicated that the assay is reliable and reproducible for quantitation of SA-MCF viral DNA in samples from asymptomatically infected sheep and from animals with clinical SA-MCF. (+info)
Malignant catarrhal fever (MCF) was diagnosed by clinical signs and lesions in five out of six white-tailed deer (Odocoileus virginianus) in a North American zoo. The clinical signs and histopathological lesions in these deer were typical of MCF. Antibody to an epitope conserved among the MCF viruses was detected in the sera collected from the deer. PCR failed to amplify viral sequences from DNA extracted from peripheral blood leukocytes (PBL) and/or spleens of the deer with primers specific for ovine herpesvirus 2 (OHV-2) or specific for alcelaphine herpesvirus 1 (AHV-1). By using degenerate primers targeting a conserved region of a herpesviral DNA polymerase gene, a DNA fragment was amplified from the PBL or spleens of all six deer and sequenced. Alignment of the sequences demonstrated that the virus in the deer belongs to the Gammaherpesvirinae subfamily, exhibiting 82% identity to OHV-2, 71% to AHV-1, and 60% to a newly identified bovine lymphotropic herpesvirus. This virus, which causes classical MCF in white-tailed deer, is a newly recognized agent belonging to the MCF group of gammaherpesviruses. It is the third reported pathogenic MCF virus, genetically distinct but closely related to OHV-2 and AHV-1. The reservoir for the virus has not been identified. (+info)
Epizootic malignant catarrhal fever in three bison herds: differences from cattle and association with ovine herpesvirus-2.
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Three bison herds in Colorado experienced high mortality from malignant catarrhal fever (MCF). In comparison with cattle, the bison had a more rapidly progressive disease, fewer clinical signs, and milder inflammatory histologic lesions. There was consistent association with ovine herpesvirus-2 (OHV-2). Contact with sheep was not consistent. Of 17 animals in herd A, 15 died of acute MCF; 1 was slaughtered while healthy; and 1 developed clinical signs of MCF, was treated with corticosteroids and antibiotics, and died of fungal abomasitis and rhinitis after 5 months. In herds B and C, approximately 300 of 900 and 18 of 20 died of MCF following brief clinical disease. The nearest sheep were 1 mile away from herd A, but direct contact with sheep could be documented in herds B and C. Complete gross and histologic examinations were conducted on 34 animals, including all animals in herd A, and MCF was diagnosed in 31. In addition, field necropsies were performed on all dead animals in herd B and most in herd C and MCF was diagnosed on the basis of the gross lesions in most animals. Clinical signs of each animal in herd A were recorded. Illness was brief, usually 8-48 hours. Clinical signs were subtle; separation from the herd was often observed. In all 3 herds, hemorrhagic cystitis and multifocal ulceration of the alimentary tract were consistently found at necropsy. Mild lymphocytic vasculitis was present in multiple organs. Ovine herpesvirus-2 was found by polymerase chain reaction (PCR) in 71 of 105 formalin-fixed tissue specimens from 29 of 31 animals with MCF. In herd A, blood samples from 13 animals were collected at 5 time points and tested by PCR for the presence of OHV-2 viral sequences in peripheral blood leukocytes. Nine bison with a positive PCR test and 4 with negative results prior to clinical illness died of MCF. (+info)
Recognition of another member of the malignant catarrhal fever virus group: an endemic gammaherpesvirus in domestic goats.
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A novel gammaherpesvirus in goats that is herein tentatively designated as caprine herpesvirus-2 was identified based on the sequence of a fragment from the herpesvirus DNA polymerase gene. Sequence alignment analysis revealed that the virus sequence isolated from goats was 67% identical to the homologous sequence from alcelaphine herpesvirus-1, 71% identical to ovine herpesvirus-2 and 73% identical to a recently recognized herpesvirus causing malignant catarrhal fever in white-tailed deer. Combined serological and PCR-survey data demonstrated that this virus is endemic in goats and its transmission pattern may be similar to that of ovine herpesvirus-2 in sheep. (+info)
Constitutive activation of Lck and Fyn tyrosine kinases in large granular lymphocytes infected with the gamma-herpesvirus agents of malignant catarrhal fever.
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Large granular lymphocytes (LGL) with a T or natural killer (NK) lymphoblast morphology and indiscriminate (non-major histocompatibility complex-linked) cytotoxicity for a variety of target cells can be derived in culture from the tissues of animals infected with either alcelaphine herpesvirus-1 (AlHV-1) or ovine herpesvirus-2 (OvHV-2). In this study, LGL survival in the absence of exogenous interleukin-2 was inhibited by the protein kinase inhibitor genestein, but not the p70 s6 kinase inhibitor rapamycin. Constitutive activation of the src kinases Lck and Fyn was demonstrated in a bovine LGL line infected with OvHV-2 and in two rabbit LGL lines infected with AlHV-1. The p44 erk1 and p42 erk2 mitogen-activated protein kinases (MAPK) were also constitutively activated in the LGLs but not control T cells. Lck and Fyn kinase activity in the LGLs did not increase after mitogen (concanavalin A or concanavalin A plus phorbol ester) stimulation of the cells, in contrast to control T cells. Control T cells, but not the LGLs, proliferated after mitogen stimulation. An analysis of tyrosine phosphorylated proteins in the cells indicated that the LGLs exhibited some similarities and differences to activated control T cells. The results demonstrate that the activated phenotype of the LGLs, associated with malignant catarrhal fever virus infection and in the absence of exogenous interleukin-2, involves constitutively activated Lck and Fyn kinases. These are normally crucial for the initial activation of T cells via several cell-surface receptors (e.g. the T-cell receptor and CD2). The inability of the LGLs to proliferate in response to mitogen may be due to an inability of Lck and Fyn to become further activated after mitogen stimulation. (+info)
A simpler, more sensitive competitive inhibition enzyme-linked immunosorbent assay for detection of antibody to malignant catarrhal fever viruses.
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An earlier competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) was developed for detection of specific antibody against malignant catarrhal fever (MCF) viruses (MCFV) in ruminants. In this study, the indirect CI-ELISA was improved by conjugating the monoclonal antibody 15-A directly with horseradish peroxidase and by developing a method of producing precoated, dried antigen plates. This new test is referred to as a direct CI-ELISA. The reformatted test yielded a significantly improved sensitivity, and the time required was reduced to about one-sixth of the previous time. Of 37 MCF cases in cattle that were confirmed by histopathology and polymerase chain reaction (PCR) assay, 37 (100%) were positive by the new test, whereas the indirect CI-ELISA detected only 23 (62%). The direct CI-ELISA detected antibody to MCFV in 100% of 48 sheep that had been defined as infected with ovine herpesvirus 2 (OvHV-2) by PCR, whereas the indirect CI-ELISA detected only 41 (85%). Comparison of antibody titers measured by the 2 assays for sera collected from OvHV-2-infected sheep and from cattle, bison, and deer with clinical sheep-associated MCF revealed that the direct CI-ELISA offered a 4-fold increase in analytical sensitivity over the indirect format. The number of seropositive animals detected by the direct CI-ELISA among apparently normal cattle and bison was 2-3 times greater than the number detected by the indirect CI-ELISA, indicating that a significant percentage of normal cattle and bison are subclincally infected with MCFV. (+info)
Ovine herpesvirus-2 glycoprotein B sequences from tissues of ruminant malignant catarrhal fever cases and healthy sheep are highly conserved.
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Ovine herpesvirus-2 (OHV-2) infection has been associated with malignant catarrhal fever (MCF) in susceptible ruminants. In order to further investigate whether OHV-2 is an aetiological agent for sheep-associated (SA) MCF in cattle and bison, the entire sequences of OHV-2 glycoprotein B (gB) from different sources of viral DNA were compared. Target DNA was derived from tissues of bovine and bison cases of SA-MCF, from a lymphoblastoid cell line established from another bovine case of SA-MCF, and from a healthy sheep. The divergence between deduced amino acid sequences of OHV-2 gB ranged from 0.5 to 1.2%. The high degree of similarity between gB sequences from a healthy sheep and clinical cases of SA-MCF in cattle and bison suggests that OHV-2 is an ovine virus that is occasionally transmitted to other ruminant species, in which it can cause severe disease. (+info)