Mogućnost upotrebe rt-pcr tehnike u utvrđivanju prisustva virusa goveđe virusne dijareje u spermi priplodnih bikova

Autor: Milovan Jovicin, Tamaš Petrović, Sava Lazić, Bosiljka Djuricic
Jazyk: srbština
Rok vydání: 2005
Předmět:
Zdroj: Veterinarski Glasnik, Vol 59, Iss 3-4, Pp 371-381 (2005)
Veterinarski Glasnik
ISSN: 2406-0771
0350-2457
Popis: 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. Virus goveđe virusne dijareje (BVD-a) je značajan zdravstvenoekonomski patogen kod goveda koji može da se izlučuje i širi i putem sperme trajno i akutno inficiranih bikova. Nativna sperma šest bikova, za koju je izolacijom virusa i RT-PCR metodom utvrđeno da je negativna na BVD virus, eksperimentalno je inokulisana desetostrukim razređenjima necitopatogenim 22146 sojem BVD virusa titra 105,5. Na ovaj način dobijena su razređenja BVD virusa od 10-1 do 10-6 (5 x 104 TCID/50 do 0,5 TCID/50 BVD virusa) u 0,1 ml nativne sperme. Iz ovako inokulisane sperme obavljena je reizolacija virusa na kulturi ćelija FTB u mikrotitar ploči sa 96 bazenčića, u kojoj je svaki uzorak inficirane sperme postavljen u tri primerka, a svaki od njih je titriran od razređenja 1:2 do 1:256. Prisustvo BVD virusa je dokazano tehnikom fluorescentnih antitela u drugoj slepoj pasaži na kulturi ćelija FTB. Za kulturu ćelija je utvrđen izrazito toksičan efekat nativne sperme do razređenja 1:64. Virus BVD-a je reizolovan iz sperme u sva tri primerka uzoraka sperme sa 5 x 104, 5 x 103 i 5 x 102 TCID/50, a nije reizolovan iz sperme sa 50, sa 5 i sa 0,5 TCID/50 BVD virusa u 0,1 ml nativne sperme. Istovremeno izvedeno je dokazivanje prisustva BVD virusnog genoma RT-PCR metodom u istim uzorcima virusom inokulisane nativne sperme bikova. Pozitivan rezultat je utvrđen u nativnoj spermi sa 5 x 104, 5 x 103, 5 x 102 i 50 TCID/50 BVD virusa u 0,1 ml nativne sperme. Eksperiment je ukazao da RT-PCR metoda ima prednosti u odnosu na izolaciju BVD virusa iz uzoraka nativne sperme bikova. To su: brzina ispitivanja (jedan do dva dana) i veća osetljivost (deset puta u odnosu na izolaciju virusa). Izolacija virusa traje najmanje deset dana, a njena manja osetljivost je, pre svega, rezultat citotoksičnog efekta nativne sperme bikova na kulturu ćelija.
Databáze: OpenAIRE
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