| Classical swine fever (CSF) caused by the CSF virus (CSFV) is a highly fatal and contagious virus disease affecting pigs, among the highest notifiable diseases to the World Organization for Animal Health (OIE). Hog Cholera Lapinized Virus (HCLV) strain is considered as the safest and most potent one for the preparation of the attenuated CSF vaccine, the only one for the attenuated CSF vaccine in China. CSFV infected only pigs. The inoculation of HCLV could induce rabbit high body temperature, but no CPE in the cell cultures. The rabbit high body temperature reaction (RHBTR) test is used to test the titration of virus and the main method for detection of efficiency of the vaccine products. However, there are some problems when RHBTR test is employed to do the efficiency test, such as differences between individuals and about 3-9 d. In addition, the direct immunofluorescence antibody method is not adapted to monitor the contamination of Bovine Viral Diarrhea Virus (BVDV) in the CSFV vaccine. It was reported that the immunogenicity of the CSFV vaccine would decrease after contaminated with BVDV, resulting in the failure of immunization protection. BVDV in the CSFV vaccine could cause the pig illness and death. Therefore, it is necessary to establish novel techniques to substitute the rabbit test to do the efficiency test.(?)s reported that FQ-PCR and McAb-FITCID50 were used to determine the titre of HCLV and test the efficiency of HCLV vaccine. The FQ-PCR specific to diagnosed BVDV contaminated in the HCLV vaccine was also studied in the paper.1. HCLV FQ-PCR and BVDV FQ-PCR A pair of specific TaqMan-MGB fluorescent probes and a pair of primers are designed referring to some available complete sequences of CSFV, BVDV and BDV, constructed the positive standard substance, the FQ-RCR kit is established to quantity the nucleic acid of HCLV and BVDV. The results showed that the sensitivity of HCLV FQ-PCR is 4.35 nucleic acid copy of template, and the relation coefficient between CT value and template content is 0.9998, amplification efficiency is 101.14%,40 quality control samples have been stored six months, the coefficient of variation in CT value is 1.1-4.0%; when used BVDV FQ-PCR method, the relation coefficient between CT value and template content is 0.9978, amplification efficiency is 95.68%,20 quality control samples have been stored six months, the coefficient of variation in CT value is 1.1-2.4%. When detected the CSFV, BVDV, BDV, PRRSV, FMDV and other pathogens, both the HCLV FQ-PCR and BVDV FQ-PCR kits only can test HCLV and BVDV, indicated that the HCLV FQ-PCR and BVDV FQ-PCR kits established in this experiment have a high specificity, sensitivity and stability.2. McAb-FITCID50 method was established. The monoclonal antibody (McAb) against CSFV produced by the WH303 hybridoma cell (from VLA) was purified and labeled by FITC. The cell culture infected by the HCLV, stained with the McAb-FITC, showed the specific fluorescence by the microscopes. According to the data, the TCID50 titre of HCLV in the vaccine samples was determined. Negative results were obtained and showed no cross-reaction from the samples infected with BVDV, BDV, PRRSV, FMDV and other pathogens detected by the CSFV McAb-FITCID50 method.3. Application of the HCLV FQ-PCR and McAb-FITCID50 kits. Total of 34 samples from 17 batches of the 4 manufactures diluted by 107 times were detected, using RHBTR test, HCLV FQ-PCR, and McAb-FITCID50. Data showed that 11 samples were negative, two rabbits showed no fever reaction, detected by RHBTR test, with the CT value of HCLV stock liquid 21.15-27.30 by FQ-PCR, corresponding viral content of 8.80×102copy/μL-6.52×104copy/μL, and the TCID50 number 10-0.6/0.1mL-10-2.5/0.1mL by McAb-FITCID50. Twelve samples were suspicious, one rabbit typical fever and one no, with the CT value is 17.47-23.70, the viral content is 1.10×104copy/μL-8.55×105copy/μL, and the TCID50 number is 10-2.4/0.1mL-10-3.76/0.1mL. The CT value and TCID50 of the eleven positive samples, two rabbits showed typical fever, were 17.10-20.81 with the viral content of 8.27×104copy/μL-1.11×106copy/μL, and 10-2.88/0.1mL-10-4.54/0.1mL, respectively.4.The comamination of BVDV test of CSFV vaccine samples by BVDV FQ-PCR. A total of 58 semi-finished vaccine products and 36 finished vaccine products from different bathes of several manufactures were detected by BVDV FQ-PCR. The positive rate was 20.7% and 22.2%, respectively. This finding suggests that the contamination of BVDV in the CSFV vaccine be serious.Conclusion:The HCLV-FQ-PCR and McAb-FITCID50 kits established in this study could be used to substitute the RHBTR test to do the efficiency test of semi-finished CSFV vaccine, monitoring vaccine production. The BVDV-FQ-PCR could be employed to monitor the BVDV contamination of semi-finished and finished HCLV vaccine. Data obtained here showed that these novel three techniques could be used to control the quality of CSFV vaccine with high sensitivity, stability, and specificity. |
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