VALIDATION OF NON-NESTED AND REAL-TIME PCR FOR DIAGNOSIS OF SHEEP-ASSOCIATED MALIGNANT CATARRHAL FEVER IN CLINICAL SAMPLES

Authors: Traul, Donald; Taus, Naomi; OAKS, J - WSU; OTOOLE, D - UNIV OF WYOMING; RURANGIRWA, F - WSU; Li, Hong

Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: 7/15/2006
Publication Date: 10/15/2006
Citation: Traul, D., Taus, N.S., Oaks, J.L., Otoole, D., Rurangirwa, F.R., Li, H. 2006. Validation of non-nested and real-time pcr for diagnosis of sheep-associated malignant catarrhal fever in clinical samples . Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. 131.

Interpretive Summary: 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. 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. 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.

Technical Abstract: 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. 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. 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.