Preparation And Assessment Of Feline Calicivirus Inactivated Vaccine

Feline calicivirus?FCV?,which belongs to Caliciviridae,Vesivirus virus genus,is an important pathogen that causes oral and upper respiratory tract disease of cats.The main clinlcal symptoms of FCV infection include dental ulcer,Chronic stomatitis,rhinitis,conjunctivitis and pneumonia.It can also cause clinical symptoms such as ulcerative dermatitis,acute arthritis,enteritis,abortion and limping,etc.Despite of a low mortality rate,the incidence of the disease is high with the death rate of more than 30%in some virulent strains infections.FCV is distributed worldwide,highly contagious among the cats and mainly infects kittens within 6⁸4 days.FCV can proliferate in oral and upper respiratory tract,then cause inflammation of the upper respiratory tract after infection.The clinical symptoms will decrease in 14¹8 days,but it can still be expelled through saliva,ocular and nasal secretions,which make it a dangerous source of infection.Since FCV infection is often accompanied with Feline herpesvirus 1 and Feline panleucopenia virus,it has posed much difficulty to prevention and treatment.At present,vaccine injection is still the major approach of prevention.However,there is no clinically licensed vaccine researched and developed independently and we still has to rely on imported commercial vaccines,which can no longer meet the demand of the domestic market.Therefore,it is necessary to carry out the study on feline infectious rhino-conjunctivitis vaccine.5 FCV strains of had been isolated in our previous study,and both the pathogenicity and immunogenicity were compared.In this study,FCV CH-JL2 with strong pathogenicity and good immunogenicity was selected in preparation for inactivated vaccine and evaluation of immune effect.Firstly,animal infection test of FCV CH-JL2 was performed.Each non-immunized healthy felis catus,6¹1 weeks old,was inoculated 0.5ml dose via oronasal with viral titers of 10^8.97 TCID₅₀/mL,10^7.97 TCID₅₀/mL and 10^6.97 TCID₅₀/mL,respectively.The pathogenicity was estimated by monitoring clinical symptoms and detecting body temperature,detoxification situation,histopathological change and virus distribution in tissues in the following 14 days.The results showed that all titers of FCV CH-JL2 could invade felis catus with morbidities of 100%,50%and 25%respectively.The kittens began to discharge virus and exhibited typical clinical symptoms like fervescence,nasal secretions increase and oral ulcer 5 days after challenged with a viral titer of 10⁸.97.97 TCID₅₀/mL.Nevertheless,no FCV in lungs,hearts,splenic organ,weasand and whole body organizations was detected.The results indicate that CH-JL2 may not belong to FCV VSD and can only cause upper respiratory infection rather than general infection.Secondly,the screening of virus inactivation conditions and vaccine preparation were studied.The inactivation conditions are as follows:viral fluid was inactivated by adding 0.2%volume fraction of formaldehyde for 48 h in a constant temperature rocking bed at 22??120 r/min?;Then the inactivated FCV CH-JL2 viral fluid was mixed with aluminum hydroxide adjuvant at the ratio of 9:1 to prepare the inactivated vaccine.The inactivated vaccine was qualified through phy-chemical test,aseptic test and germ-free test without any pollution.Moreover,there should be no adverse reaction to the injected kittens.Next,the test kittens were challenged with 1mL(10^6.5TCID₅₀/mL)inactivated vaccine subcutaneously and intramuscularly.The best immunization way was determined by criteria of clinical observation and antibody content monitoring.The results showed that there was no significant difference in the antibody level produced by the two immune methods.But the subcutaneous injection was more convenient,easier to control in actual operation and had a lower probability of scleroma.Thirdly,each cat was injected with 1mL(10⁸TCID50/mL?10⁷TCID₅₀/mL?10⁶TCID₅₀/mL)inactivated vaccine subcutaneously accompanied with a second immunization 21 days later.The serum antibody in peripheral blood was detected at a 7 day interval each.Animal infection test was established at 21 day after second immunization.The infection state and the best immune dose were evaluated according to the results of clinical symptoms and RT-PCR detection.Our results showed that the vaccine titer of 10⁸TCID₅₀/mL could provide better and more stable protection with a protection rate of 100%?5/5?.Last,the inoculation safety tests including single dose,repeated single dose and super dose of 170601batch inactivated vaccine were performed,then tested animal temperature was measured in fixed-point along with clinical observation for 14 days.The results revealed that the kittens in each group presented normal temperatures,good mental state and appetite,and no adverse reactions were observed in the whole body after vaccination.It proved that the inoculations of single dose,repeated single dose and super dose of inactivated vaccine were safe for cats.The inactivated FCV CH-JL2 vaccine was successfully prepared in this study.It possesses good quality,safety and strong immunogenicity via physical and chemical test,exotic viruse detection,germ-free test,effectiveness and safety evaluation,which will provide statistical reference for further research and development of FCV vaccines.