PCR detection of the sheep-associated agent of malignant catarrhal fever

Abstract

From a genomic library previously constructed from a lymphoblastoid cell line (LCL) propagated from a bovine case of sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine herpesvirus-2 (OHV-2), several OHV-2 clones were identified and characterised by hybridisation using probes from the unique region of the Alcelaphine herpesvirus-1 (AVH-1) genome. Nucleotide sequence from one clone was generated and the predicted amino acid sequence was found to contain regions of homology with the 140 and 160 kDa tegument proteins of Epstein-Barr virus and herpesvirus saimiri respectively. Oligonucleotide primers were constructed and a polymerase chain reaction (PCR) test was developed for the detection of OHV-2 viral DNA. Amplified product was identified by restriction with RsaI and BmyI. The primers were highly specific for OHV-2 DNA with a limit of detection of 6.4 pg of genomic DNA derived from the parent LCL. This was estimated to correspond to one diploid bovine cell. The PCR was successfully applied to detect OHV-2 DNA in peripheral blood leucocytes (pbl) from clinical cases of SA-MCF and normal sheep.

Malignant Catarrhal Fever in Free-Ranging Cervids Associated With Ovhv-2 and CPHV-2 Dna

“…The amount of target DNA used in all PCRs was about 0.5 mg per reaction, which was reduced from the previous protocols. The OvHV-2-specific PCR was based on the primers initially developed by Baxter et al (1993), targeting a region of open reading frame 75, and the detailed procedure was carried out as described by Li et al (1995).…”

“…DNA from the samples was isolated with DNeasy Tissue kit (Qiagen Nordic, Oslo, Norway) according to the manufacturer's instructions. A single PCR, specific for OvHV-2, was performed using the outer primer set (556/755) of the protocol described by Baxter et al (1993). The PCR was performed with the use of HotStar Taq DNA polymerase (Qiagen), with a final concentration of Mg 2+ of 1.5 mM.…”

“…The MCF-associated viruses (MCFV) occur endemically in latently infected carrier species that spread infection to MCFV-susceptible species. Included in this group are the well-known alcelaphine herpesvirus 1 (AlHV-1) (Plowright et al, 1960) and ovine herpesvirus 2 (OvHV-2) (Baxter et al, 1993), endemic in wildebeest (Connochaetes spp.) and domestic sheep, respectively, and two further viruses identified in later years, caprine herpesvirus 2 (CpHV-2) endemic in domestic goats (Li et al, 2001a), and a virus of unknown origin causing MCF in white-tailed deer (Odocoileus virginianus) (MCFV-WTD) (Li et al, 2000;Kleiboeker et al, 2002).…”

Malignant Catarrhal Fever in Bison, Acute and Chronic Cases

“…The MCF gamma herpesviruses are thought to replicate in lymphocytes. 1,3 Presence of the viral sequences usually was correlated with lymphoid infiltration, lymphocytic vasculitis, or lymphocytic proliferation in the tissue. However, viral sequences were often detected in lung but were not correlated with histologic lesions.…”

Quantitation of Sheep-Associated Malignant Catarrhal Fever Viral DNA by Competitive Polymerase Chain Reaction

“…3,14 Recent improvements in methods for detection of the genomic DNA of the SA virus have provided for improved accuracy in diagnosis of clinical MCF and enabled studies that are beginning to better define the epidemiology of this agent. 1,6,7 Accurate quantitation of both extracellular and intracellular viral DNA is essential to delineate shedding patterns, organ-specific virus loads, and other aspects of pathogenesis and epidemiology. The polymerase chain reaction (PCR) technique is a sensitive method for detecting DNA, which has been applied to detection of OHV-2 DNA.…”