Development And Evaluation Of A Bivalent Inactivated Vaccine Against Haemophilus Parasuis

Haemophilus parasuis(HPS)can cause Glazer’s disease in pigs,which can infecte the piglets age range from 2 weeks to 4 months,especially in weaned piglets at 3 months of age.The clinical symptoms are mainly joints swelling,lameness,convulsions,spasms and other symptoms.Pathological changes are mainly characterized by systemic serositis such as peritonitis and pleurisy.In the early stage of infection,there will be yellow inflammatory exudate in the abdominal cavity and chest cavity.A large amount of cellulosic protein exudate will appear in the late stage of infection,leading to organs such as abdominal cavity and chest cavity sticky.Since its first report in 1910,HPS has become a global epidemic,causing enormous harm to the global pig industry.HPS is divided into 15 serotypes and some strains cannot be classified.The most popular serotypes in China are type 4 and type 5.At present,the use of antibiotics is restricted in the clinic,and inactivated vaccines are mainly used to control Haemophilus parasuis,but the domestic commercial inactivated vaccines have poor efficacy and large side effects.This study developed a more efficient H.parasuis inactivated vaccines based on the popular serotypes 4 and 5 in China to make a certain contribution to the prevention of Haemophilus parasuis.1.Isolation and identification of HPS and detection of biological characteristics.This experiment used mPCR method to identify serological typing of clinical isolates and study the biological characteristics of HPS.The results are as follows:A total of 10 strains were isolated from the clinical,3 of which were serotype 4 strains,3 strains were serotype 5,and serotypes 1,6,7,and 13 strains each.According to the clinical infection situation,one strain of serotype 4 and serotype 5 with strong virulence was selected from the clinical isolates,named YC16A strain and SQ15C strain,and biological characteristics studied.The results showed that the biological characteristics of the two strains were similar,and the colony morphology was round,smooth and translucent.It was grown on TSA medium for 12 h and was needle-shaped.The diameter was about 1 mm at 24 h.After incubation for 8 hours in TSB liquid medium,the log phase was entered and cultured for 20 hours,and the plateau phase was started.The number of viable cells reached the highest at 12-16 h in TSB liquid medium,and then gradually decreased.2.Establishment of an animal infection model of HPS YC16A strain and SQ15C strain.In this experiment,an animal model was established by 21-day-old piglets infected with Haemophilus parasuis YC16A strain and SQ15C strain.The virulence of the strain and the SQ15C strain was determined from four aspects:LD50,clinical symptoms,anatomical pathological changes and pathological tissue sections.The results were as follows:LD50 of YC16A strain and SQ15C strain infected with 21-day-old piglets were 1.44×109 CFU(P ≤ 0.05)and 8.41 × 108 CFU(P ≤ 0.05),respectively.YC16A strain and SQ15C strain were infected with 21-day-old piglets,and their body temperature increased,joint swelling,and symptoms such as convulsions and convulsions appeared later.Anatomical findings of abdominal cavity,pleural effusion,arthritis and other symptoms,severe cases of fibrin protein exudation leading to adhesion between the abdominal cavity and chest organs.Pathological observation revealed serous exudation in the alveoli,and a large number of red blood cells,neutrophils and macrophages were found in the alveoli and spleen.3.Development of the bivalent inactivated vaccine against Haemophilus parasuis.In order to develop a effective bivalent inactivated Haemophilus parasuis serovars 4,5 vaccine,the HPS serovars 4 YC16A strain and the serovars 5 SQ15C strain isolated by our laboratory were killed by formaldehyde completely.Thoroughly mixed with 10%Montanide GEL 02 PR water-soluble adjuvant to prepare a bivalent inactivated vaccine.The 21-day-old piglets were inoculated with the bivalent inactivated vaccine,and the effect of the vaccine was evaluated against the antibody level after immunization,the immune protection rate,the clinical symptoms after the challenge with HPS,and the pathological changes of the necropsy.After 2 weeks of vaccination,the results showed that the selfmade vaccine produced higher antibody levels;the immune protection rate of the self-made vaccine against HPS serovars 4,5 strains was 80%,100%,respectively.While the immune protection rate of commercial vaccine was only 80%and 80%,the negative group mortality rate is 100%.After the challenge,the piglets in the self-made vaccine group had no obvious clinical symptoms,anatomical pathological changes and histopathological damage.In the commercial vaccine group,the piglets showed transient temperature rise,and the abdominal cavity and pleural effusion were found by necropsy.In the negative control group,the temperature of the piglets increased,and the latter showed symptoms such as convulsions and convulsions,and the necropsy revealed a large amount of fibrinous exudation symptoms in the abdominal cavity and thoracic cavity.In summary,the bivalent inactivated vaccine developed by our laboratory has a better immune effect and can effectively prevent the infection of HPS serovars 4,5 strains in clinical practice.In this study,an animal model of HPS infected piglets was established,and a bivalent inactivated vaccine of HPS YC16A strain and SQ15C strain was developed.The results showed that the vaccine produced a better immune effect through the immune challenge protection test.