The effect of the mode of sampling on BHV-1 detection in infected cattle by dot-blot hybridization.

The BHV-1 genome in nasal swabs and washings was detected by dot-blot hybridization using the 32P-pUR-1 probe (1.8 kb EcoRI-HindIII random fragment of BHV-1 DNA ligated into the pUC-9 plasmid) as early as on day 1 after the experimental infection of cattle. In dependence on the sampling method, differences were observed in the maximum of hybridization signals. During nasal swab analyses maximum amounts of BHV-1 differed in the individual samples (day 1-3). Hybridization signals obtained at the analysis of BHV-1 DNA nasal washings did not vary but showed a continuous maximum on day 2 after infection. Nasal washings proved to be more advantageous for detection of the BHV-1 genome by the hybridization technique.

Detection of bovine herpesvirus 1 with various types of DNA probes.

The method of dot-blot hybridization on nitrocellulose filters by various types of DNA probes (ds recombinant plasmids, ss recombinant M13 phages and a 42bp synthetic oligonucleotide) was used for BHV-1 detection. The highest sensitivity was achieved with the 32P-pUR1 probe (1.8 kb random EcoRI-HindIII fragment inserted into pUC9) which detected the BHV-1 genome in 5 x 10(3) infected MDBK cells. Using the pUR1 probe, no cross hybridization was observed with other herpesviruses: BHV-2, 3, 4, and Aujeszky's disease virus. The 32P-pUR1 probe detected BHV-1 in nasal swabs of calves as early as on day 1 after experimental infection. The maximum intensity of BHV-1 detection occurred on day 1-3. The 32P-pUR1 probe also detected BHV-1 in field samples of nasal swabs from cows and calves.

[Detection of bovine herpesvirus-1 using the dot-blot hybridization method]. / Detekcia bovinného herpesvírusu-1 metódou dot-blot hybridizácie.

With the development of molecular biology in the 1980s methods of microorganism detection start to innovate. One of the main advantages of the molecular-genetic methods, namely hybridization of nucleic acids and PCR methods, is the detection of genome of microorganism without the need for cellular cultivation. To detect BHV-1 (etiological agens IBR-IPV) the dot-blot hybridization method on nitrocellulose filters was used together with different types of DNA probes (two-fiber recombinant plasmids, one-fiber recombinant phages M 13 and 40 bp synthetic oligonucleotide). Genome DNA BHV-1 was isolated from samples (virions, infested cells, nasal smears and secretions by phenol extraction). The highest sensitivity of detection was achieved with 32P-pUR-1 probe (1.8kb random EcoRI-Hind III fragment ligated into plasmid pUC 9) which detected genome BHV-1 in 5 x 10(3) infested MDBK cells. This probe did not respond with herpetic viruses BHV-2, BHV-3, BHV-4 and the virus of Aujeszky's disease. The quality of pUR-1 probe was further tested for IBR diagnostics in animals experimentally infested with the virus BHV-1 (intranasal infection). BHV-1 could be detected in nasal smears and secretions in experimentally infested calves as early as on the first day following infection, while the agens amount reached its peak on the days 2-3 and on the days 6-7 the occurrence of virus fell markedly. When digoxigenin-pUR-1, i.e. non-radioactively marked probe, the virus presence was confirmed only on the days 2-3, in the time of the highest occurrence of infection agens. To detect the virus through the dot-blot hybridization nasal secretions were confirmed as better compared with nasal smears. The technology of virus isolation on cell cultures confirmed also the occurrence of agens as soon as on the first day from infection, with maximum on the days 2-5, but much more reliably it detected the virus on the days observed from the day 3 and their peak was obtained on the day 6 from infection. Experiments, comparing classical methods of IBR diagnostics (detection of specific antibodies, the method of isolation on cellular cultures) with the dot-blot hybridization using the samples obtained from farms with natural occurrence of IBM, are under progress.

[Isolation and control of the functional quality of mRNA of bovine leukocyte interferon]. / Izolácia a kontrola funkcnej kvality mRNA bovinného leukocytárneho interferónu.

For the construction of the cDNA library total cellular RNA was separated from bovine leucocytes induced with NDV (inductor of interferons) for five hours. Guanidinisothiocyanate and ultracentrifugation in the gradient of CsCl were used for the separation of the total RNA (Chirgwin et al., 1979). Bands 18S and 28S were detected in the samples of RNA by electrophoresis in an MOPS buffer (Fig. 1). mRNA was separated from the mixture of RNA via affinity chromatography on oligo(dT)-cellulose (Aviv and Leder, 1972). The function quality of mRNA BoIFN-alpha was verified by microinjection into the oocytes of Xenopus laevis using a microinjector of our own construction (Fig. 2). The microinjector was calibrated by injection of a radioactive 51Cr solution (Fig. 3). It was found out on the basis of CPE inhibition measurements that the injected oocytes synthesized 320-640 U/ml BoIFN-alpha (16-32 U/50 microliters; Tab. I).