Preparation Of Feline Herpes Virus Type Ⅰ Inactivated Vaccine And Evaluation Of Immune Effect In Pet Cats

Feline herpesvirus type 1（FHV-1）is the pathogen which causes Feline infectious rchinotracheitis（FIR）in cats.It is a double-stranded DNA virus of the Herpesviridae,αherpesvirus subfamily.The virus is widespread worldwide and has a high degree of species specificity.After FHV-1 infects the animal,it often lurks in the trigeminal ganglion of their body.It is very easy to form a recessive infection and detoxify for life.It recurs periodically,posing a serious threat to the health of cats,cheetahs,tigers and other wild cats.As the number of pet cats in our country is increasing day by day,once there are poisonous cats in small environments such as feedlots,it will subsequently cause continuous infection of susceptible kittens,which will lead to uninterrupted epidemics of the disease in the area.At present,there is no commercial vaccine for feline herpes virus in our country,and there is no vaccine that has passed the national examination and approval for import.Therefore,this experiment prepares vaccines with independently isolated and identified strains,laying the foundation for the development of vaccines with independent intellectual property rights.In this study,the FHV-1 CH-B strain isolated and identified in our laboratory in the early stage was used as the seed strain,and the FHV-1 CH-B strain virus liquid with the virus titer of107.84 TCID50/mL was obtained after the expansion of the culture.Different concentrations of formaldehyde,diethylene imine（BEI）andβ-propiolactone（BPL）were selected to inactivate the FHV-1 CH-B strain virus liquid,and the inactivating agents were screened.The final inactivation conditions were determined as follows:After FHV-1 CH-B strain virus liquid was mixed with 0.2%formaldehyde in volume fraction,it was inactivated in a constant temperature shaker at 37°C with 120r/min for 48 h.The FHV-1 CH-B strain virus solution inactivated by formaldehyde was mixed with AL（OH）₃ adjuvant and CRM adjuvant respectively,and injected into mice.The serum antibodies（Ig G）and white blood cells in the mice were detected.The content of interleukin 1β（IL-1β）,interleukin 6（IL-6）,interferon gamma（IFN-γ）,tumor necrosis factor（TNF-α）,and the titer of neutralizing antibodies were used to screen adjuvants,The AL（OH）₃ adjuvant was determined to be the adjuvant prepared by the FHV-1 CH-B strain virus liquid inactivated vaccine.After FHV-1 CH-B strain virus liquid was inactivated by formaldehyde,it was mixed with AL（OH）₃ adjuvant in a ratio of 9:1 to prepare an inactivated vaccine.The inactivated vaccine passed the laboratory test and was free of pollution.There were no adverse reactions in the injected mice and cats.The FHV-1 inactivated vaccines with virus content of 10^7.84TCID₅₀/mL,10^6.84TCID₅₀/mL,10^5.84TCID₅₀/mL were injected subcutaneously,and the each of the experimental cats was immunized at a dose of 1mL,and the peripheral blood of cat was collected every 7 days for separation.In the serum,the indirect ELISA method was used to determine the serum antibody（Ig G）content,interleukin 4（IL-4）,IFN-γ,neutralizing antibody and other indicators,and the second immunization was performed on the 21st day.On the 42nd day,the FHV-1 infection kitty model established in the laboratory was used to conduct an animal challenge test.According to the body temperature,weight,clinical symptoms,detoxification amount,and tissue lesions of the experimental cat,the minimum immunization dose was screened.The results showed that the FHV-1 inactivated vaccine with a virus titer of10^7.84TCID₅₀/mL had a protection rate of 100%（5/5）for the experimental cat.The safety test of the inactivated vaccine was carried out.Through 14-day clinical observation,there was no abnormal change in the mental state,feeding situation,and main biochemical indicators of the experimental cats in each group.Meanwhile,there was no adverse reactions were seen,which proved the safety of the homemade inactivated vaccine was good.In this study,an inactivated FHV-1 CH-B strain vaccine was successfully prepared.Laboratory tests,potency and safety evaluations proved that the FHV-1 CH-B strain inactivated vaccine had a certain preventive effect on FHV-1 infection in kittens.These results provide effective reference data for the further research of FHV-1 vaccine.