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ARS Home » Pacific West Area » Pullman, Washington » Animal Disease Research Unit » Research » Publications at this Location » Publication #91656
Title: DIAGNOSIS OF MALIGNANT CATARRHAL FEVER BY PCR USING FORMALIN-FIXED EMBEDDED TISSUES

Author
item CRAWFORD, T. - WASHINGTON STATE UNIV.
item Li, Hong
item O'TOOLE, D. - UNIV. OF WYOMING

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/22/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: A previously-described PCR assay, which detects viral genetic material, for diagnosis of sheep-associated malignant catarrhal fever (SA-MCF) was adapted for use on formalin-fixed, paraffin wax-embedded tissues. Variables affecting its use were examined. Cases in cattle, deer and bison diagnosed as MCF by clinical signs or histological lesions were obtained from archived tissue banks at two veterinary diagnostic laboratories. Tissues from healthy animals and from cases of other common bovine viral diseases were examined as controls. A total of 86 tissue 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 SA-MCF, and did not yield false positive signals from healthy animals or from cases of other bovine viral diseases such as infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues could be used for the PCR examination, including spleen, lymph node, gut, brain, lung and kidney. Purified DNA provided a better result by PCR than did unpurified DNA samples. 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 samples. Prolonged fixation of tissues in formalin was deleterious to the target DNA, and progressively diminished PCR signals. Detection of genomic DNA of SA-MCF viral DNA by PCR worked successfully on archived tissues that were 15 years old.

Technical Abstract: A previously described PCR assay to detect specific ovine herpesvirus 2 (OHV-2) DNA for diagnosis of sheep-associated malignant catarrhal fever (SA MCF) was adapted for use on formalin fixed, paraffin wax- embedded tissues. Variables affecting its use were examined. Cases in cattle, deer and bison diagnosed as MCF by clinical signs or histological lesions were obtained from archived tissues at two veterinary diagnostic laboratories. 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 SA 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, gut, 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 cm-square blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and progressively diminished PCR signals after fixation exceeded 45 days. Detection of genomic DNA of OHV-2 by PCR worked successfully on archived tissues that were 15 years old.