| Pseudorabies(Pseudorabies,PR)caused by Pseudorabies virus is an acute infectious disease.It can cause a variety of diseases of domestic and wild animals with fever,strange itch(except pigs)and encephalomyelitis as the main symptoms.Pigs are the main host and source of infection of the virus.The sick piglets mainly showed neurologic symptoms and the mortality of suckling piglets could reach 100%.Pregnant sows infected with PRV can cause abortion,stillbirth and mummified fetus,which is a serious threat to China’s pig industry as an important infectious disease.The main measure to prevent and control pseudorabies is vaccination in China.At present,the most widely used strain of pseudorabies vaccine in China is pseudorabies virus Bartha-k61 and it has a gE gene natural deletion phenotype.Its virulence is greatly reduced while maintaining good immunogenicity.So it is recognized as a pretty vaccine strain in the world.In this study,St passage PRV cultures(Bartha-K61 strain)were mixed with formula ①,②,③ and conventional 5%sucrose gelatin,respectively,the freeze-dried method A and B were used to prepare the porcine PR heat-resistant and the porcine PR conventional protective agent live vaccines.The vaccines were randomly selected for physical characteristics,sterility,mycoplasmas,identification test,safety,potency,residual moisture,vacuum degree and aging resistance test,in order to screen out the appropriate heat-resistant protective agent formula and the matching method of freeze-drying.The results showed that the lyophilized vaccines were yellow spongy loose masses,easily separated from the bottle wall,dissolved rapidly after adding diluent,no bacteria,mold and mycoplasma grew,and only porcine PRV(Bartha-K61 strain)was contained in the vaccines,there were no local or systemic adverse reactions caused by the injection,the virus titer was not less than 106.25 TCID50/dose,the residual moisture content was less than 4.0%,the vacuum degree was purple glow;The aging resistance test showed that the lyophilized vaccine by formula ③and method A was superior to other combinations.Three batches of porcine PR heat-resistant vaccine were prepared by the above process and the efficacy was evaluated with porcine PR live vaccine(strain Bartha-K61),The first group was three batches of porcine PR heat-resistant vaccine,the second group was the porcine PR live vaccine.Respectively used virus titer and piglet test(25 healthy piglets of 20～25 days old were randomly divided into 5 groups,5 in each group;each of 5 piglets in 4 groups were respectively immunized with 1 dose for each group,the other group was blank control group,and 1.0 mL PRV GD1 virulent strain was attacked 10 days post-vaccination.The antibody(Ab)levels of different days after piglets immunization were detected by IDEXX Kit(25 healthy piglets aged 20～25 days were randomly divided into 5 groups,5 in each group;each of 5 piglets in 4 groups were immunized with 1 dose for each group,the other group was blank Control group.).The aging resistance test(the virus titer of two groups of vaccines was measured after 7,10,13 days storage at 37℃ and 18 and 24 months storage at 2～8℃).The results showed that the virus titer of the two groups of vaccines was no less than 106.6 TCID50/dose;the piglets in the immunized group were resistant to virulent virus attack,and the piglets in the control group were 5/5 died;the piglets in the immunized group were able to detect high titer Ab quickly,and the Ab was positive for at least 365 days;the virus titer of the live vaccine with PR heat-resistant protective agent could still reach 106.15 after being stored at 37℃ for 13 days and 2～8℃ for 24 months.The virus titer was significantly higher than porcine PR live vaccine.The former had a greater advantage in heat resistance.In this study,the protective efficacy of PR heat-resistant protective live vaccine agent on the mutant virulent strain was evaluated.Twenty piglets aged 20～25 days and twenty pregnant sows of 86～90 days old were respective randomly divided into two groups,10 in the immune group and 10 in the control group.After 10 days of immunization,1.0 mL of PRV ZLT and 1.0 mL of PRV HN variant virulent strain(virus titer 106.0 TCID50/mL)were attacked respectively,observed and measured the temperature for 14 days.The results showed that all the piglets in the immune group were resistant to the virulent strain attack,and all the piglets in the control group died;all the pregnant sows in the immune group were resistant to the virulent strain attack,without adverse reactions,and all the sows in the control group were sick.In conclusion,the suitable formulation of heat-resistant protectant ③ and freezedrying method A were selected in this study.There was no significant difference between the prepared PR heat-resistant protective live vaccine and the conventional live vaccine,but the heat-resistant performance was better.The process is stable and controllable.The vaccine can be stored at 2～8℃ and valid for 24 months,which reduces the storage conditions and the risk of losing efficacy in long-distance transportation of the vaccine.Moreover,it has a good protective effect on the epidemic strains and is of great significance in the practical application of the vaccine. |
