| Pseudorabies virus (PrV) was the agent of acute infectious disease in many domestic and wild animals. Swine was the natural host and reservoir of PrV. Pseudorabies was of most important economical to pig industry. Rapid, accurate diagnosis and immunization with safe, effective vaccine were main measurements to prevent the disease. In order to provide the pig industry with good quality vaccine and a on-site diagnostic method, the research containing 3 related experiments, was conducted. The research level occupied advancing position in China. 1. The study on the Ea TK7gG7LacZ+ attenuated live vaccineThe different propagation titers on BHK-21 cell and their left virulence to Balb/C mice showed that Ea field virus was of highest virulence, Bartha strains the second, the TK gene-negative mutants possessed the lowest virulence. The LD50 of Ea field virus, TK7gG7LacZ+ mutant and Bartha strain was 102.5TCID50, 104.0TCID50 and 1060TCID50, respectively. By injecting the vaccine virus titers ranging from 1050 to 1070TCID50 to pregnant sow and natal piglets, no adverse reactions, such as abortion, stillbirth, week piglets in pregnant and fever response, growth retardation in piglets were observed, showing the safety of the TK7gG7LacZ+ mutant.The immunization dosage was determined by measuring the neutralization antibody levels and sero-conversion rate in the vaccinated sero-negative pigs. No statistics differentiation of neutralization antibody titers existed between 1050~1070TCID5o and 1030~1040TCID50, but antibody level of 1050TCID50 was much higher than that of 1040TCID50, the sero-conversion of the two dosage ranges were 100 and 80 percentage. Taking consideration of antibody lever and sero-conversion rate, 1050TCID50 TK7gG7LacZ+ mutant was chosen as the immunization dosage. The culture liter of TK7gG7LacZ+ mutant on chick embryo blast fiber cell was 1063TCID50/lmL, as much 13 times as the determined immunization dosage. The live vaccine was prepared according the standard method. The safety test showed that the vaccine was safe to the Balb/C mice and pregnant sows, 15-day-old pigs. Furthermore, the fact that no variation of the body temperature appeared in the immunized pigs and sentinel pigs were still sero-negative revealed that the mutant could not be transmissible between pigs. The duration of immunization efficacy to the sero-negative pigs could last 5-6 months. The immune effect could be reduced by maternally derived antibody (MDA). To overcome the interference of MDA, the two immunizations at interval of four weeks, instead of higher dosage of one immunization, seems more effective. The sows accepted immunizations two times duringgestation or one vaccination at about two months prior to farrow could provide more MDA up to 2 months after birth, similar to the MDA titer from sows vaccinated two tunes with oil emulsion inactivated vaccine. Comparison among the Ea TK'/gG" vaccine, Begonia, NIA-783 and Bartha strain indicated that TK-/gG- vaccine could provide Balb/C mice protection against fatal infection of Ea field virus, while the rest of vaccines provided only 60-80% protection. The antibody levels of the TK-/gG- mutant, Begonia and NIA-783 were similar, while that of Bartha was the lowest. The vaccine could be kept under -20 and 4-8 for 12 months and 6 month, respectively, without changes of the vaccine property.In short, the vaccine based on the TK-/gG- mutant was studied in laboratory conditions. The field trial will be next step.2. The development of latex agglutination test (LAT) to detect antibody against swine pseudorabies virusPseudorabies virus (Ea strain), isolated from the farms in Hubei province, was cultivated in BHK-21 cell. The viral supernatant was precipitated by ammonium sulfate, concentrated with PEG. The procedure that the concentrated virus liquid was mingled with equal volume of latex formerly treated by trypsin could produce antigen for LAT use. Later, the antigen mixed with PrV positive serum, the immunized pigs as well as clinically infected pigs sera and the... |
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