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Validation of Non-nested and Real-Time PCR for Diagnosis of
Sheep-Associated Malignant Catarrhal Fever
in Clinical Samples |
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D. L. Traul1,
N. S. Taus1, J. L. Oaks2, D. O’Toole3,
F. R. Rurangirwa2, and H. Li1 |
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Sheep-associated malignant catarrhal fever (SA-MCF), a
herpesviral disease caused by ovine herpesvirus 2 (OvHV-2), can pose
a significant diagnostic challenge to veterinary clinicians in that
its clinical presentation makes it difficult to differentiate from
several other common viral diseases. The classical signs of corneal
edema, fever, and generalized lymphadenopathy, are not always
evident, particularly in acute cases in highly susceptible species
such as deer and bison. Although the predominant microscopic
lesions of MCF, (lymphoproliferation, mucosal inflammation, and
vasculitis) are highly suggestive, they may either be absent or so
mild that the diagnostician has difficulty in differentiating them
from similar lesions that can accompany bovine virus
diarrhea/mucosal disease, infectious bovine rhinotracheitis and
epizootic hemorrhagic disease. In situ immunohistochemistry
or immunofluorescence has been unsuccessful, despite many attempts,
for demonstration of viral antigen in tissues.
The previous
cloning and sequencing of a fragment of the OvHV-2 genome resulted
in the development of a nested PCR assay specific for the virus,
which advanced the ability to detect OvHV-2 DNA in animals with
clinical MCF. However, the use of this PCR as a routine diagnostic
method for clinical MCF in veterinary diagnostic laboratories is
problematic because the nested amplification format has the
potential for contamination from the amplicons. Recent preliminary
data revealed that high levels of OvHV-2 DNA are present in the
blood and tissues of various animals, including cattle, bison, deer,
and other exotic ruminant species that developed clinical MCF.
Based on these data, we hypothesized that OvHV-2 DNA in the blood
and tissues of ruminants with clinical MCF could be detected by
non-nested PCR and real-time PCR. In this report, we validated
non-nested and real-time PCR for detection of OvHV-2 DNA in samples
from clinically affected animals. Two sets of tissue or blood
samples were collected: one set consisted of 97 samples from
naturally-affected animals with clinical or histopathologic, and
nested-PCR evidence of MCF, as well as animals with
experimentally-induced MCF; the second set consisted of 100 samples
from animals without clinical MCF (defined as no MCF clinical signs
or histological lesions, and OvHV-2 nested PCR-negative). Among 97
positive samples defined by nested PCR from clinically affected
animals, 95 (98%) were positive by non-nested PCR and 93 (96%) were
positive by real-time PCR, respectively. Neither non-nested PCR nor
real-time PCR resulted in positive signal from any of 100 negative
control samples. The data confirmed that both non-nested and
real-time PCR maintained the high specificity and had adequate
sensitivity for detection of OvHV-2 DNA in clinical samples from
animals with SA-MCF. |
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1Animal
Diseases Research Unit, USDA-Agricultural Research Service,
Washington State University, Pullman, WA 99164
2Washington
Animal Disease Diagnostic Laboratory and Department of Veterinary
Microbiology and Pathology, Washington State University, Pullman, WA
99164
3Wyoming
State Veterinary Laboratory, University of Wyoming, Laramie, WY
82070 |
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