Comparison of Polymerase Chain Reaction and Virus Isolation for Detection of Epizootic Hemorrhagic Disease Virus in Clinical Samples from Naturally Infected Deer

Abstract

Abstract. We compared our recently reported reverse transcriptase polymerase chain reaction (PCR)-based assay for detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples with different virus isolation (VI) procedures. Thirty-six blood samples and 1 spleen sample from deer were assessed by the EHDV PCR assay and VI in baby hamster kidney (BHK)-21 cells and embryonated chicken eggs (ECE). The EHDV PCR assay detected EHDV RNA from 6 blood samples obtained from deer during 1988-1989 outbreaks of epizootic hemorrhagic disease and from the spleen and blood samples of a deer with clinical hemorrhagic disease in 1992. The 6 blood samples from the 1988-1989 outbreaks and the spleen sample from the 1992 case were VI positive on BHK-21 cell culture. The blood from the same deer with the PCR-and VI-positive spleen was VI negative in BHK-21 cells and ECE. All EHDV isolates were identified as EHDV serotype 2 by a plaque inhibition test. The results of this study indicate that the sensitivity of the previously described EHDV PCR assay is comparable to or greater than that of the VI method in BHK-21 cell culture or ECE. The EHDV PCR assays could provide a superior diagnostic alternative to the current cumbersome and time-consuming VI procedures.Epizootic hemorrhagic disease virus (EHDV) is an finitive diagnosis of EHDV infection presently requires arthropod-borne, double-stranded RNA virus of the isolation of the virus. Various laboratories have difgenus Orbivirus, family Reoviridae, 4,6 that causes fatal ferent methods for isolation of EHDV, including inhemorrhagic disease in white-tailed deer (Odocoileus travenous (i.v.) inoculation of embryonated chicken virginianus). 5,8,9,11 Other domestic and wild ruminants eggs (ECE) or isolation on cell lines. The VI technique may also be infected with the virus. 13,15 The possibility is laborious, expensive, and time consuming, and a of clinical disease in domestic and wild ruminants has final result requires 2-4 weeks. led to restrictions on the international movement of livestock and their germplasm if the animals are EHDV positive by serology or virus isolation (VI). 22Diagnosis of EHDV infection by traditional methods includes evaluation of clinical signs, pathologic changes, serology, and VI. Because clinical signs and pathologic changes caused by EHDV are indistinguishable from those produced by bluetongue virus (BLU), an orbivirus related to EHDV, confirmation of EHDV infection under field conditions by these methods is unreliable.14,19 Serology may provide a good indication of EHDV infection if there is a 4-fold increase in the antibody titer in samples taken 2 weeks apart. However, the difficulty in obtaining paired serum samples from field-infected animals makes a definitive diagnosis of EHDV infection by serology impractical. De-

Evaluation of Epizootic Haemorrhagic Disease Virus Infection in Sentinel Calves from the San Joaquin Valley of California

“…Despite the extended nature of the study, only a few EHDV isolates were recovered. A lack of success in isolating EHDV from the blood samples of sentinel calves has been reported previously (Shope et al, 1960;Pearson et al, 1992;Aradaib et al, 1994Aradaib et al, , 1995. This could be attributed to viraemia being missed because of infrequent sampling.…”

“…The sentinel calves were fed a ration of concentrates and hay with free access to water. Processing of the blood samples for virus isolation (VI) was as described previously (Aradaib et al, 1995). The multiplex EHDV RT-PCR for simultaneous detection of EHDV serogroup and identi¢cation of EHDV-1 and EHDV-2 has been described previously (Aradaib et al, 1998).…”

Development of polymerase chain reaction for specific identification of epizootic hemorrhagic disease virus serotype 1

“…In previous studies we have described a simple, rapid, sensitive and specific EHDV PCR-assay for detection of EHDV-serogroup in cell culture and in clinical samples from naturally and experimentally infected animals [2,3]. Comparison of PCR technology to conventional virus isolation and serology for detection of EHDVinfection was also studied [5]. Recently, we developed a PCR-based assay for specific identification ofEHDV serotype 2 in celt culture and a variety of clinical specimens [6].…”

“…The rapidity, sensitivity and specificity of the PCR assay would greatly facilitate detection of EHDV infection during an outbreak among susceptible ruminants. It is worth mentioning that the first report of the successful use of PCR for detection of EHDV serogroup [2][3][4][5], the development of a PCR-based assay for specific identification of EHDV-2 [6], and specific identification of EHDV-1, described in this study, should provide the basis for future diagnostic techniques.…”

Short Communication: A Simple and Rapid Method for Detection of African Horse Sickness Virus Serogroup in Cell Cultures Using RT-PCR

“…Serology is useful in epidemiological studies to identify previous infection with AHSV. The agar gel immunodi¡usion (AGID) test, currently used as standard serological test, is complicated by cross-reactions between related orbiviruses (Aradaib et al, 1995). The problems associated with the use of the AGID test have been solved by using monoclonal antibodies (MAbs) in competitive ELISA (cELISA) technique (Laviada et al, 1992).…”