Titration of Marek's disease cell-associated vaccine virus (CVI 988) of reconstituted vaccine and vaccine ampoules from Dutch hatcheries.

Thirty-one outbreaks of Marek's disease (MD) were reported in the Netherlands and retrospectively analyzed. The outbreaks occurred mostly in vaccinated commercial layer and a few breeder flocks of several breeds; however, the cause of the outbreaks could not be established. Therefore, in a prospective study, the occurrence of true vaccine failures was assessed onfive hatcheries. The plaque-forming units (PFU) of MD vaccine per chicken dose were determined through in vitro assays on vacine ampoules (2 to 5 per hatchery) and samples of reconstituted vaccine (approximately 22 per hatchery). All forty reconstituted vaccine samples of hatcheries 1 and 4 showed PFU doses <10(3). In hatchery 4, 14 samples showed extreme low PFU (< or = 10 PFU). In hatcheries 2, 3, and 5, the numbers of MD vaccine suspensions with a titer > or = 10(3) PFU, which is the standard required, were 1 (5%), 17 (77%), and 3 (14%), respectively. Some vaccine ampoules showed < 10(3) PFU per chicken dose. This study shows the usefulness to assess the PFU per chicken dose of reconstituted MD vaccine and vaccine ampoules to unravel true vaccine failures, which could result in disease outbreaks in the field.

Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk.

A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were constructed, added to milk samples, and co-amplified with viral DNA using the same primers for both templates. Specificity, sensitivity, and reproducibility of the two PCR assays were examined. In both PCR assays, all 31 BHV4 strains examined were scored positive, whereas 14 unrelated viruses scored negative. Sensitivity studies showed that two-ten copies of BHV4 DNA were detectable by the gB-PCR, while one-three copies could be detected by the TK-PCR. For the detection of BHV4 in milk samples, the gB-PCR amplification was found to be ten-times, and the TK-PCR was found to be 55-times more sensitive than virus isolation. BHV4 DNA was detected by gB-PCR and TK-PCR in 93 and 95%, respectively, of 61 milk samples collected from cows infected intramammarily with BHV4, while only 61% were positive by virus isolation. Four out of 48 cows with clinical mastitis were positive for BHV4-gB and BHV4-TK DNA, whereas no BHV4 DNA was detected in milk from control cows. Considerable agreement was seen between the results of the two PCR assays, and both methods were considered as rapid and reliable tests for the screening of BHV4 DNA in bovine milk. The less laborious gB-PCR might be the recommended test of choice for screening large amounts of milk samples for the presence of BHV4.

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains.

Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex.

Spontaneous BHV1 recombinants in which the gI/gE/US9 region is replaced by a duplication/inversion of the US1.5/US2 region.

In a bovine herpesvirus 1 (BHV1) vaccine strain, a spontaneous BHV1 mutant (Za) was found that arose from a recombination between two isomeric forms of the BHV1 genome. In this Za mutant one end of the U(S) region, containing part of the US1.5 gene, was found duplicated in an inverted orientation at the other end of the U(S) region. Concurrently, a 2.7 kb deletion was found in Za that encompasses both the US8 (gE) and US9 gene. Analysis of the in vitro growth properties of a genetically modified BHV1gE(-) mutant showed that at 11 hours post infection BHV1gE(-) viruses were secreted ten times more efficiently than wild type virus. Using this observation we developed a protocol to enrich for spontaneous gE deletion mutants in a BHV1 field isolate and found another mutant (Rof3) with similar properties as the Za mutant. Rof3 has a duplication/inversion of the US1.5 gene and part of the US2 gene and a simultaneous 3.5 kb deletion that encompasses the US7 (gI), US8 (gE) and US9 genes. The nucleotide sequences of the recombination points of both recombinants were determined and compared. No obvious sequence similarities were found, suggesting that non-homologous recombination events led to the observed recombinations. The implications for the use of BHV1 gE deletion mutants as marker or diva vaccines are discussed.