Abstract
The bovine viral diarrhea (BVD) virus is a significant health-economic pathogen in cattle which can be excreted and spread also through sperm of persistently or acutely infected bulls. Native sperm of 6 bulls, found to be negative to the BVD virus by isolating the virus and using the RT-PCR method, was experimentally infected with a tenfold dilution of the non-cytopathogen 22146 strain of the BVD virus with a titer of 105,5. This way, dilutions of the BVD virus from 10-1 to 10-6 (5 x 104 TCID/50 do 0,5 TCID/50 in 0.1 ml native sperm were obtained. From sperm infected in this way, the virus was reisolated on FTB cell culture in a microtiter plate with 96 pools in which each sample of the infected sperm was set up in three samples, and each of them was titrated to a dilution of 1:2 to 1:256. The presence of the BVD virus was proven using the technique of fluorescent antibodies in a second blind passage on FTB culture cells. For cell culture an extremely toxic effect of native sperm to a dilution of 1:64 was established. The BVD virus was reisolated from sperm in all three sperm samples with 5 x 104, 5 x 103 i 5 x 102 TCID/50, and it was not reisolated from sperm with 50, with 5, and with 0.5 TCID/50 BVD virus in 0.1 ml native sperm. At the same time, the presence of the BVD viral genome was proved using the RT-PCR method in the same samples of artificially infected native sperm of bulls. A positive re suit was established in native sperm with 5 x 104, 5 x 103, 5 x 102 and 50 TCID/50 BVD virus n 0.1 ml native sperm. The experiment proved that the RT-PCR method has advantages over the isolation of the BVD virus from samples of native sperm of bulls. These are: shortterm investigations (1 to 2 days) and greater sensitivity (10 times bigger than the isolation of the virus). The isolation of the virus takes at least 10 days, and its greater sensitivity is primarily a result of the cyrotoxic effect of native sperm of bulls on cell culture.
Highlights
Eksperiment je ukazao da reverzne transkripcije - polimeraza lan~ane reakcije (RT-PCR) metoda ima prednosti u odnosu na izolaciju BVD virusa iz uzoraka nativne sperme bikova
Petrovi} i sar: Mogu}nost upotrebe RT-PCR tehnike u utvr|ivanju prisustva virusa gove|e virusne dijareje u spermi priplodnih bikova odnosu na izolaciju virusa)
Petrovi} i sar: Mogu}nost upotrebe RT-PCR tehnike u utvr|ivanju prisustva virusa gove|e virusne dijareje u spermi priplodnih bikova moe da izoluje iz sperme bikova posle pojavljivanja specifi~nih antitela u krvi, iako je on prisutan u spermi Š8, 9, 12, 131
Summary
Virus gove|e virusne dijareje (BVD-a) je zna~ajan zdravstvenoekonomski patogen kod goveda koji moe da se izlu~uje i {iri i putem sperme trajno i akutno inficiranih bikova. Petrovi} i sar: Mogu}nost upotrebe RT-PCR tehnike u utvr|ivanju prisustva virusa gove|e virusne dijareje u spermi priplodnih bikova odnosu na izolaciju virusa). Petrovi} i sar: Mogu}nost upotrebe RT-PCR tehnike u utvr|ivanju prisustva virusa gove|e virusne dijareje u spermi priplodnih bikova moe da izoluje iz sperme bikova posle pojavljivanja specifi~nih antitela u krvi, iako je on prisutan u spermi Š8, 9, 12, 131. Kao materijal u ispitivanju osetljivosti metoda za utvr|ivanje prisustva virusa BVD u spermi kori{}eni su uzorci nativne sperme {est bikova i necitopatogeni (ncp) soj virusa BVD 22146, titra 105.5, na sekundarnoj kulturi }elija fetalnog tele}eg bubrega (FTB). Petrovi} i sar: Mogu}nost upotrebe RT-PCR tehnike u utvr|ivanju prisustva virusa gove|e virusne dijareje u spermi priplodnih bikova u 0,1 ml inficiranih uzoraka sperme. Pozicija u genomu i predvi|ena veli~ina umnoene sekvence genoma odgovaraju referentnom BVDV soju NADL Š161
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