| Swine influenza(SI)is a highly contagious respiratory disease caused by swine influenza virus(SIV)in pigs of different age.The clinical signs of swine influenza are fever,mental depression,loss of appetite or appetite elimination,dyspnea,abdominal breathing and paroxysmal cough.SIV could damages the epithelial cells of respiratory tract and the infected pigs will be prone to secondary infection with respiratory diseases,which increase the mortality.Many studies have shown that SIV plays the critical role in the interspecies transmission chain of"poultry-pig-human",which not only causes losses to the pig industry,but also affect public health.Swine influenza has become an epidemic disease worldwide.At present,the prevalent swine influenza virus in China is mainly H1N1 and H3N2 subtypes.In this study,nasal swabs from the swines were collected in farms with suspected swine influenza in Liaoning and Heilongjiang provinces during 2006 and 2007.Two SIV strains belong to H1N1 and H3N2 subtypes were isolated and identified,swine influenza bivalent inactivated vaccine were developed using the strains in order to provide basic data for swine influenza virus research and set the foundation for development of swine influenza vaccine in China.1.Isolation and identification of swine influenza virus H1N1 and H3N2 subtypesNasal swabs were collected in farms with suspected influenza in Liaoning and Heilongjiang provinces.Tendays-old SPF chicken embryos were inoculated with each nasal swab for virus isolation.Hemagglutination test,EID50 detection,HA and NA genes amplifying with RT-PCR,sequencing and sequence alignment,purity test(including bacteria and mycoplasma),exogenous virus detection and virulence test were conducted to identify the virus.Finally,swine influenza bivalent inactivated vaccine was developed using the strains and the immunogenicity of the vaccine was tested.Results showed that both the hemagglutination(HA)titer of two strains were more than 1:128.Both the titer of the strains reached 106.5EID50/mL.The genes H1(668 bp)and N1(615 bp)genes of strain LN were PCR-amplified and proved to belong to SIV H1N1 subtype.And H3(668 bp)and N2(246 bp)genes of strain HLJ were PCR-amplified and proved to belong to SIV H3N2 subtype.The purity test results showed that the two strains were sterile,without mycoplasma and exogenous virus contamination.The virulence test showed that the two strains SIV could cause 100%(5/5)of healthy susceptible pigs infected and the vaccine developed with the two strains could provide 100%protective rate against the two isolates challenge.The above results showed that two isolates meet the requirements of master seed virus for vaccine development.2.Optimization of production process of swine influenza bivalent inactivated vaccine(strain H1N1 LN+H3N2 HLJ)The antigen was developed in MDCK cell line culture process.Comparing the inactivation effect between two inactivators(formaldehyde and BEI),comparing the emulsifying process between domestic white oil and imported ISA206 adjuvant and evaluation of safety and protective effect of the swine influenza bivalent vaccine on health susceptible piglets were conducted.Results showed that BEI had little side effects on the HA of the viruses,no residue,and the inactivation test method was easy to operate and observe.It was proved that ISA206 adjuvant is the better adjuvant for inactivated vaccine of swine influenza,which is W/O/W type,low viscosity and easy to inject.Piglets were immunized with the vaccine mixed with ISA206 adjuvant and challenged with SIV-H1N1 LN strain and SIV-H3N2 HLJ strain by intratracheal.Results showed that the protective rates for both H1N1 LN and H3N2 HLJ were 100%(5/5).It indicated that the swine influenza bivalent inactivated vaccine has better protective efficacy.3.Safety evaluation of the swine influenza bivalent vaccineIn order to evaluate the safety of the swine influenza bivalent inactivated vaccine,the vaccine was injectioned into neck muscle,safety tests of one single dose,repeated injection with one single dose,overdose vaccination,and over dose inoculation of pigs of inapplicable age were carried out.The body temperature of all the vaccinated pigs was normal,the state of mind and appetite were good,and no adverse reactions were found in the injection site and the whole body.The results indicated that the 3 batches of vaccines were safe for the pigs.4.Minimal immune dose testTen health susceptible pigs were inoculated with 0.25 ml,0.5 ml,1 ml and 2 ml vaccine,respectively,the vaccine was injectioned into neck muscle,piglets without immunization was as control group.Booster immunization after 14 days,at 14 days after booster immunization,5 pigs were randomly selected from each dose of immunization group and control group and challenged with SIV-H1N1 LN strain(106.5SEID50/mL).The other pigs were challenged with SIV-H3N2 HLJ strain(106.5EID50/mL),4 mL per each.Results showed that the morbidity rate of all control group are 100%,the protective rates of the vaccinated pigs with 0.25 ml dose was 0/5,the protective rates of the vaccinated pigs with 0.5 ml dose was 80%(4/5),both of the protective rates of the vaccinated pigs with 1 ml and 2 ml dose were 100%(5/5).In addition,the HI antibody titers of pigs inoculated with diverse doses of vaccine were different,and the protective rates were different.It indicated that there is a correlation between the HI antibody level and the protection rate,and the vaccinated pigs could be protected when HI antibody titer not less than 1:160.In order to ensure the immune efficacy of the vaccine,the routine immunization dose of the vaccine was determined as 2 ml per pig5.Efficacy test of 3 batches bivalent vaccineTen health susceptible piglets were vaccinated with 3 batches swine influenza bivalent vaccine(H1N1+H3N2),every pig is vaccinated with 2ml by injectioned into neck muscle,piglets without immunization was as control group.Booster immunization after 14 days,at 14 days after booster immunization,5 pigs were randomly selected and challenged with SIV-H1N1 LN strain(106.5EID50/mL).5 pigs were randomly selected and challenged with SIV-H3N2 HLJ strain(106.5EID50/mL),4 mL per each.Results showed that the protective rates of 3 batches of vaccine were 100%(5/5),80%(4/5),100%(5/5),respectively.It is demonstrated that the vaccine has better protective efficacy.6.Cross protection of vaccination against domestic H1N1 and H3N2 subtype epidemic strainsTen 4-week-old pigs were vaccinated with the vaccine,every pig is vaccinated with 2ml by injectioned into neck muscle,piglets without immunization was as control group.Booster immunization after 14 days,at day 14 after booster immunization,5 pigs were randomly selected from the vaccination group and the control group and challenged with the domestic epidemic strain SIV H1N1 SD731(106.5EID50/mL).The other 5 pigs from the vaccination group and the control group were challenged with the domestic epidemic strain SIV H3N2 JS(106.5EID50/mL),4 mL per each.Results showed that the protective rate of vaccinated pigs challenged with SIV H1N1 SD731 strain and H3N2 JS strain was 80%(4/5)and 100%(5/5),respectively.It indicated that the vaccine has a good cross protection effect on the challenge with SIV H1N1 and H3N2 prevalent in China.7.Immune duration testTwenty 4-week-old pigs were vaccinated with the vaccine,every pig is vaccinated with 2ml by injectioned into neck muscle,piglets without immunization was as control group.Booster immunization after 14 days,blood samples were collected every month to 6 months,and the titer of HI antibody was determined by hemagglutination inhibition test.Three months after vaccination,5 pigs were randomly selected from vaccinated group and control group and challenged with SIV-H1N1 LN strain(106.5EID50/mL),the other 5 pigs were randomly selected from immunized group and control group and challenged with SIV-H3N2 HLJ strain(106.5EID50/mL),4 mL per each.Results showed that the protective rate of all the immunized pigs were 100%(5/5),while the morbidity of control group was only 60%(3/5).The challenge test after 6 months did not performed,and the duration of vaccine immunization was determined according to the HI antibody level.It is found that HI antibody titers against H1N1 and H3N2 of 8/10 pigs were not less than 1:160 after 6 months.According to the correlation between HI antibody level and protection rate,the duration of vaccine immunization was determined as 6 months. |
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