The Investigation On Diagnosis And Immune Technology Of Viral Diarrhea

Bovine viral diarrhea virus (BVDV) and bovine rotavirus (BRV) are the leading etiologic agents of the severe diarrheal disease in young animals worldwide. BVDV is a major viral pathogen in cattle and causes considerable economic losses throughout the world. Surveys of herds indicate that 30-95% of the cattle have been infected with BVDV. Depending on the stage of gestation at the time of transplacental infection, the outcome may e embryonic death, abortion, stillgirth or the birth of immunotolerant calves persistently infected with BVDV. They are also at risk of developing fatal mucosal disease if superinfected with a cytopathic biotype of the virus. BRV is classified as a genus in the family Reovirridae, and could cause severe diarrheal illness which may lead to dehydration. According to the survey, the estimate was that 60-80% of the cows were infected by BRV, accounting for about 0-50% deaths from severe diarrhea.First, according to the published sequences of the BVDV strains, a pairs of specific primers was designed, and then the RT-PCR method was established for detecting the bovine viral diarrhea virus. Using the specific primers 196bp bands could be obtained from the BVDV by this method. Clinical samples were detected using the RT-PCR method, and 14 out of 23 samples were positive. The results showed that the RT-PCR was very specific and sensitive, and could detect 1pg virus` RNA. So it was a quick and exact method to detect the BVDV.Second, a duplex RT-PCR method was established in order to detect bovine rotavirus and bovine viral diarrhea virus in the same time. Two pairs of primers were designed according to the sequences of the BRV`s VP7 gene and the BVDV`s 5`-UTR gene. The PCR products were 342bp and 196bp respectively. The results showed that the RT-PCR was very sensitive and specific, which could detect 1pg virus` RNA, and could be applied for clinical detection and epidemiological investigation.Third, the bovine rotavirus CHLY was proliferated in the MA-104 cell to prepare the antigen. When the titer of the antigen solution reached 106TCID50/ml, the virus was inactivated by formaldehyde solution. The oil adjuvant and Al(OH)3 adjuvant were added into the antigen solution respectively, and then the mice and the rabbits were inoculated with the vaccine. Collected the blood serum and detected the IgG using the ELISA method. The results showed that serum anti-rotavirus antibody titers were significantly increased in the vaccinated mice after the second immunization compared to the control group(P<0.01), no significant difference was detected between the Al(OH)3 adjuvant group and oil adjuvant group(P>0.05); high rotavirus special serum IgG antibodies titers were detected in the young rabbits born to the vaccinated group, was significantly different from the control group(P<0.01). The results of these experiments indicated that the formaldehyde-inactivated Bovine rotavirus vaccine had good immunogenicity and could significantly improve the anti-rotavirus antibodies titer.Forth, cows were immunized infected bovine rotavirus vaccine, and then the effect of passive antibody from colostrums on immunity to bovine rotavirus infections in neonatal calves was investigated. The cows were immunized at 2 and 1months before calving, and the IgG, IgA and neutralizing rotavirus antibody in the colostrums or serum were determined. A virulent bovine rotavirus was given orally to the calves at 21days after birth, and the infection rates after challenge were compared between groups. The results showed that the colostrums and serum anti-rotavirus antibody titers in the vaccinated group were significantly increased compared to control group(P<0.01); all six calves born to control cows and one born to vaccinated cow were observed to develop diarrhea after challenge. The incubation and infection rates were significantly different between two groups(P<0.01).