| Haemophilus parasuis(HPs)is a member of Haemophilus genus,Pasteurellaceae family,mainly colonizes in the upper respiratory tract of pigs.HPs is a symbiotic bacterium and normally presents in healthy pigs.When pigs are exposed to stress environment,poor breeding and management conditions,or infected with immunosuppressive viruses,which will cause immune suppression,resulting in secondary infection of HPs.Current pig farms that outbreak HPs mainly use antibiotics to control the disease,which poses a risk of bacterial resistance.Therefore,development of an effective HPs vaccine will be of great significance to prevent and control this disease.However,HPs has a variety of serotypes,and the cross-reactive protection between different serotypes is relatively poor,which adds difficulty for the vaccine development.Isolation and identification of candidate vaccine strains with the prevalent serotypes is the key step for the development of effective vaccines.Jiangsu province is a large breeding province in China,and the prevalence of HPs is also relatively complicated.The main purpose of this study was to select effective vaccine candidate strains in Jiangsu province based on the epidemic data of HPs and to prepare inactivated vaccine,and evaluate the immune efficacy of the protype inactivated vaccine.This study provides epidemiological data for the prevention and control of HPs infection in Jiangsu province,and a feasible idea for the control of the disease.The experimental results are as follows:1 Epidemiological investigation of HPs in Jiangsu provinceFrom 2013 to 2015,13 large-scale pig farms and 97 backyard pig farms were suspected to HPs infection in Yancheng,Taizhou,Lianyungang,Xuzhou,Suqian,Yangzhou,Suzhou,Nantong,Huaian of Jiangsu province.A total of 317 samples of diseased pigs were collected from these pig farms,and the clinical symptoms of the pigs were observed and recorded.The clinical symptoms were mainly as follows:emaciation,disordered fur,depression,swollen joints,ameness,trembling(pain),ataxia,palpebral swelling,cough,hoarseness,dyspnea and fever.Autopsy examination showed the typical features of HPs infection:arthritis,joint with yellow fluid,a fibrous exudation on heart,pleural visible fibrin exudation,peritonitis,pericardium accumulate yellow fluid,heart and lung surface with yellow fibrin,dilated vessels in the brain hyperemia,pig cadaver tended to be not stiff.The positive rate of HPs was 11.54%-27.55%among tested samples,and the average infection rate was 20.50%,suggesting different degrees of HPs infection in different regions of Jiangsu province,and also in different pig farms.At the same time,HPs infected pigs with all stages of life:the positive rate of weaned pigs was 16.05%,23.56%for nursery pigs,10.53%for finishing pigs,and 11.11%for other pigs.The most serious infections were observed in weaned pigs and nursery pigs,there was significant difference in HPs infection rates among different age groups.Regular viruses were detected in HPs positive pigs.The result revealed that HPs infected pigs was often co-infected with porcine circovirus(PCV2)and porcine pseudorabies virus(PRV)in all tested areas of Jiangsu province,mixed infection rate as high as 30.77%.In particular,the co-infection with porcine circovirus(PCV2)was serious and the infection rate was 26.15%,which provided data for the prevention and control of HPs in Jiangsu province 2 Isolation and identification of HPsIn order to understand the serotypes and characteristics of HPs in Jiangsu province,the HPs positive samples collected from regional large scale pig farms and backyard pig farms in Jiangsu province,and the samples of diseased pigs submitted to Yangzhou university animal hospital were also collected to isolate HPs.In addition,nasal swabs from healthy pigs were also collected from a slaughter house in Yangzhou for HPs isolation.HPs was inoculated on the chocolate medium for culture,and the 16sRNA of HPs was detected by PCR.The positive strains were subcloned and preserved.Serotypes of all isolates were determined by slide agglutination test,the results showed that the HPs in Jiangsu province were mainly serotype 4,serotype 5 and serotype 12,accounting for 14.29%,12.98%and 36.36%,respectively.Among them,18 strains were non-serotypeable,accounting for 23.38%of the total isolates,and the serotyped strains accounted for 76.62%of all strains.The results showed that most prevalent HPs serotypes were 4,5 and 12,respectively,which provided epidemiological data for prevention and control of HPs and vaccine immunity in Jiangsu province.In some pig farms with severe HPs infections,drug sensitivity tests were conducted based on isolates collected from these farms,the results showed that the isolated strains were highly sensitive to ceftriaxone,cefotaxime,enoxacin,pioneer mycin V,compound neomycin,poly-mycin B,azithromycin and other drugs.The sensitive drugs were applied to the HPs infection farms for treatment,and an ideal therapeutic effect was obtained.At the same time,the drug sensitivity test results showed that isolates from pig farms using antibiotics with a long-term experienced serious antibiotic resistance.3 Preparation of a protype trivalent inactivated vaccine for HPsAccording to the epidemiological investigation results,the prevalent serotypes of HPs in Jiangsu province are serotype 4,serotype 5 and serotype 12,respectively.Isolates with serotypes 4,5 and 12 were selected and used as candidate vaccine strains to prepare the protype trivalent inactivated vaccine.HPs was cultured in vitro and then inactivated at 37?for 24 h using formaldehyde at a final concentration of 0.3%.Three inactivated vaccine strains were mixed to prepare trivalent inactivated vaccine,and the final concentration of each strain was 2.0 ×109 CFU/mL respectively.After the preparation of the trivalent vaccine,the inactivation confirmation and the quality test of the trivalent vaccine(including property test,endotoxin test and formaldehyde residue test)were conducted to determine the inactivation effect and safety of the vaccine.Finally,the inactivated HPs was emulsified with the 206 adjuvant at a volume ratio of one to one,the protype inactivated trivalent vaccine was qualified.4 The immune efficacy of protype trivalent inactivated vaccine against HPs challengeA protype trivalent inactivated vaccine was developed using the prevalent serotypes 4,5 and 12 strains that widely popular in Jiangsu province,the immune efficacy was evaluated in susceptible pigs.The experimental pigs were divided into 10 groups with 5 pigs in each group:pigs in the three groups immunized with HPs trivalent inactivated vaccine(15 piglets);pigs in other three groups immunized with HPs commercial inactivated vaccine(15 piglets);challenge control group(15 piglets,three group);healthy control group(5 piglets).The experimental pigs were primarily immunized by subcutaneous injection into the neck of pigs,boosted 30 days post-prime vaccination at the same way,animals in both vaccination-challenge group and challenge-only group were challenged by serotypes 4,5 and 12 strains respectively at 14 days post-boost vaccination,all experimental animals were monitored till 14 days post-challenge.Animal experiments results showed that the average antibody titer in serum of piglets after boost immunization was more than 1:16,and the immune protection rate of type 4 strain was comparable to that of commercial vaccine immunization group,both were 20%.However,the efficacy of the trivalent inactivated vaccine group was higher than that of the commercial vaccine against either serotype 5 or serotype 12 strain challenge,reaching 60%,while the protection rate of the commercial vaccine against serotype 5 strain challenge was 40%,and the protection rate of the commercial vaccine against serotype 12 strain challenge was only 20%,the difference is very significant.Therefore,the trivalent inactivated vaccines of HPs preliminarily developed in this experiment have an overall protective effect better than that of commercial vaccines,and have better immune protection against HPs serotypes 4,5 and 12,which will play a very important role in preventing and controlling HPs infection in the pig industry of Jiangsu province.In conclusion,this study conducted epidemiological investigation on HPs in large-scale pig farms and backyard pig farms in most districts of Jiangsu,Subsequently,positive samples were identified by serological method,and the prevalence of HPs in Jiangsu province was analyzed.The results showed that HPs was widespread in Jiangsu province,and the main prevalent strains of HPs were serotype 4,serotype 5 and serotype 12,respectively.A protype trivalent inactivated vaccine was successfully prepared with the prevalent serotype 4,serotype 5 and serotype 12 strains.The immune efficacy experiment of trivalent inactivated vaccine results showed that the efficacy of trivalent inactivated vaccine is significantly higher than that of the tested commercial vaccine.This study provided basic data for the epidemiology of HPs infection in Jiangsu province,and provided a protype HPs vaccine for HPs infection in this area. |
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