Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.
The products of the BoLA-DRB3 locus are important molecules in the bovine immune response. Several techniques have been used to study and define this locus but they are generally time consuming and limited in their ability to detect novel alleles. In this study we used denaturing gradient gel electrophoresis (DGGE), and direct sequencing, for BoLA-DRB3-typing. First, modified locus-specific primers were used in polymerase chain reaction (PCR) to amplify a 240 bp fragment of exon 2 of BoLA-DRB3 from the genomic DNA of 22 cattle and one pair of twin calves. The reverse primer included a GC-rich clamp to improve the physical separation of the BoLA-DRB3 alleles by DGGE. The denaturing gradient needed to produce separation of alleles was determined using perpendicular DGGE, and this gradient was then applied to parallel denaturing gels. The optimal time for producing allele separation was determined using a time-series analysis. The bands representing individual BoLA-DRB3 alleles were excised from the gels, reamplified, and the nucleotide sequence determined using fluorescent-based automated cycle sequencing. The nucleotide sequences of the separated bands were then compared to published BoLA-DRB3 alleles. A gradient of 10-15% acrylamide combined with a 15-50% ureaformamide gradient was successfully used to separate BoLA-DRB3 alleles in all individuals examined. Nucleotide sequencing showed that the 24 animals possessed 13 BoLA-DRB3 alleles, all of which have been previously described. The BoLA-DRB3 genotypes included 20 heterozygotes and two homozygotes. Three BoLA-DRB3 alleles were seen in each of the twin calves, possibly due to leukochimerism. The technique is reliable and rapid, and avoids cloning alleles prior to nucleotide sequencing and therefore offers distinct advantages over previous techniques for BoLA-DRB3-typing.
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