CN103194412B - Serotype 5 haemophilus parasuis (HPs) vaccine strain - Google Patents

Abstract

The invention relates to the field of Haemophilus parasuis vaccines in veterinary biological products, and discloses a serotype 5 Haemophilus parasuis vaccine strain, which is classified and named as Haemophilus parasuis (Haemophilus parasuis), and the strain number is XX0306. It has been preserved in China Center for Type Culture Collection, preservation number: CCTCC NO:M 2013095, preservation date: March 21, 2013. The serotype 5 Haemophilus parasuis XX0306 strain of the present invention has strong pathogenicity to pigs, and has good immunogenicity, and the inactivated vaccine prepared by using it is safe and reliable, and has a strong antiviral effect on pigs challenged with homologous strains. Very good protective effect, the morbidity and mortality of the pig herd were significantly reduced after immunization. Regardless of whether it is a single vaccine or a combined vaccine, its immune effect is equal to or better than that of existing commercial vaccines on the market, and can effectively prevent the prevalence of Haemophilus parasuis.

Description

A vaccine strain of Haemophilus parasuis serotype 5

technical field

The invention relates to the field of Haemophilus parasuis disease vaccine in veterinary biological products, in particular to a serotype 5 Haemophilus parasuis strain. the

Background technique

Haemophilus parasuis (Haemophilus parasuis, HPs) is a Gram-negative coccus bacillus in the genus Haemophilus of Pasteurellaceae. Porcine Glasser's disease characterized by polyserositis, arthritis and meningitis. It mainly harms piglets aged 2 to 8 weeks, the incidence rate is generally 10% to 15%, and the mortality rate can reach more than 50%. the

Haemophilus parasuis has strict nutritional requirements, and its growth strictly requires factor V (NAD). At present, tryptone soy agar (TSA) medium and tryptone soy (TSB) medium are considered to be the most suitable culture medium for culturing Haemophilus parasuis base. On the TSA plate medium supplemented with NAD, cultured for 24 hours, gray-white colonies with the size of pinpoints can be seen, which are raised and round, with a smooth, moist surface and neat edges. It is difficult for HPs to grow on chocolate agar plates. It has been reported that HPs cannot be subcultured on chocolate agar plates and is not suitable for in vitro subcultures. However, chocolate agar plates can be used to isolate the bacteria and supply 5% to 10% for initial isolation and culture. Carbon dioxide (CO ₂ ) promotes growth. In liquid culture, serum and factor V (NAD) need to be added to broth or TSB medium, but increasing the concentration of factor V will not increase the bacterial concentration. Since Staphylococcus will release its own metabolite Factor V into the medium during the growth process, HPs and Staphylococcus aureus are co-inoculated on the blood agar plate for culture, and HPs grow well on both sides of Staphylococcus aureus. Staphylococcus colonies gradually become smaller as the distance increases. This phenomenon is called satellite phenomenon. HPs have extremely weak resistance to the external environment, and are easily killed in vitro.

There are great differences in antigenicity and virulence among HPs strains, and serological typing is one of the important methods to distinguish different strains. At present, the serotyping method (KRG) based on the thermostable antigen immunodiffusion test proposed by Kielstein et al. in 1992 has been accepted worldwide. This method divides HPs into 15 serotypes. The serotype of the isolate could not be determined. According to the seroepidemiological surveys in Japan, Germany, the United States and Spain, serotypes 4, 5 and 13 are the most prevalent. HPs is prevalent in my country. At present, there are reports of Haemophilus parasuis and the isolation of HPs in 12 provinces and cities including Hebei, Henan, Hubei, Hunan, Anhui, Shanghai, Guangdong, and Jiangxi. the

Because veterinarians often use a large number of or even irrational antibacterial drugs clinically, bacteria in pig farms select drug-resistant bacteria under drug pressure. Therefore, it is becoming more and more difficult to use drugs to prevent and control Haemophilus parasuis disease, and with people's concerns about food safety, the application of antibiotics in the prevention and control of animal diseases will be less and will play a greater role is the corresponding vaccine. There are many HPs serotypes, and the virulence and pathogenicity of different serotype strains are quite different, and the immune cross-protection between each serotype is very low. Therefore, the existing commercial Haemophilus parasuis vaccine at home and abroad Neither can provide complete and effective protection against the disease. Therefore, the isolation and screening of general-purpose HPs strains with strong immunogenicity and the preparation of high-efficiency vaccines are the most important tasks in the prevention and control of Haemophilus parasuis disease. the

Contents of the invention

The technical problem to be solved by the present invention is to provide a serotype 5 Haemophilus parasuis vaccine strain (XX0306 strain), which has strong virulence and high immunogenicity. the

In order to solve the problems of the technologies described above, the technical scheme adopted in the present invention is as follows:

A vaccine strain of Haemophilus parasuis serotype 5, which is classified as Haemophilus parasuis (Haemophilus parasuis), the strain number is XX0306, and the strain number is XX0306 strain. It has been preserved in the China Type Culture Collection Center, address: China. Wuhan. Wuhan University, postcode 430072, deposit number: CCTCC NO:M 2013095, deposit date: March 21, 2013. The bacterial strain is a bacterial strain with strong virulence and high immunogenicity that the inventor collected and screened in Hengshui City, Hebei Province in April 2003. the

Application of the serotype 5 Haemophilus parasuis vaccine strain in the preparation of medicines for treating Haemophilus parasuis disease. the

Wherein, the drug is a vaccine, preferably a water-in-oil vaccine, or an oil-in-water vaccine, or a water-in-oil-in-water vaccine. the

Wherein, the vaccine strain of Haemophilus parasuis serotype 5 is combined with a pharmaceutically acceptable carrier or auxiliary material to form a drug, such as a monovalent vaccine, a multivalent vaccine or a combined vaccine and the like. the

Wherein, the serotype 5 Haemophilus parasuis vaccine strain contains more than 3.5×10 ⁹ CFU/mL of bacteria in the medicine (preferably the vaccine).

Beneficial effects: the serotype 5 Haemophilus parasuis XX0306 strain of the present invention has stable biological characteristics, strong pathogenicity to piglets, and good immunogenicity. The oil adjuvant inactivated vaccine prepared by using it is safe and reliable, can produce a higher level of antibody, has a long duration, and has a very good protective effect on pigs challenged by homologous strains. The incidence of pigs after immunization and The mortality rate is significantly reduced, and the immune effect is equal to or better than that of existing commercial vaccines on the market, which can effectively prevent the prevalence of Haemophilus parasuis. the

Detailed ways

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims. the

Example 1: Isolation, purification and identification of serotype 5 Haemophilus parasuis XX0306 strain. the

1. Culture medium for production

Preparation of TSA solid medium: Accurately weigh 40g of TSA powder, dissolve in 940mL of distilled water, shake well, and then sterilize by high-pressure steam at 121°C for 15min. 0.01% NAD of bacteria, mixed evenly and poured out for later use. the

Preparation of TSB liquid medium: Accurately weigh 30g of TSB powder, dissolve in 940mL of distilled water, shake well, and then sterilize by high-pressure steam at 121°C for 15 minutes. That's it. the

2. Isolation, purification and identification of Haemophilus parasuis

2.1 Separation and purification

In 2003, a large number of pigs suspected to be infected by Haemophilus parasuis appeared in a large-scale pig farm in Jiangsu, China. They were characterized by persistent fever, pleurisy, peritonitis, and arthritis. The lungs, pleural effusion, pericardial fluid, and The joint fluid was inoculated with TSA/NAD solid medium, cultured at 37°C and 5% CO ₂ for 24 to 48 hours, and the translucent raised small colonies were picked for smear, and Gram staining microscopic examination was performed, and Gram-negative small colonies were selected. For rod-shaped or pleomorphic microbacteria, perform subcloning culture on TSA/NAD solid medium twice. The blood agar solid medium and common agar solid medium were inoculated at the same time. As a result, the TSA/NAD solid medium grew well, and no colony was formed on the blood agar solid medium and common agar solid medium.

2.2 Biochemical identification

Inoculate the TSA/NAD solid medium with the pure culture liquid, and culture it at 37°C and 5% CO ₂ for 24-48 hours. Observing the colony shape, it can be seen that the size of the needle tip is round, the edges are neat, flat, and the small colonies are off-white translucent bulges. Gram-stained smear of a single colony was Gram-negative and pleomorphic, and it was a single small coccus or elongated filamentous bacilli.

Put the pure culture liquid on the defiberized sheep blood agar solid medium and make a cross streak with Staphylococcus aureus, and cultivate it under the condition of 37°C and 5% CO ₂ for 24-48 hours, and the colonies are small and transparent, and the more The growth near the staphylococcus is better, showing a typical satellite growth phenomenon, and there is no hemolysis around the colony of Haemophilus parasuis on the blood agar plate.

Inoculate the pure culture bacteria solution on TSA/NAD solid medium, and then paste circular filter paper pieces impregnated with X, V, and X+V factors respectively. Cultivate at 37°C and 5% CO ₂ for 24-48 hours. As a result, only colonies grow around the discs containing V and X+V factors, indicating that the strain only depends on V factor, not X factor.

Inoculate the micro-biochemical reaction tubes with the pure culture bacteria solution, supplement with NAD at a final concentration of 0.01%, and place them at 37°C for static culture, and determine the results of biochemical characteristics after 48 hours. The results showed that the isolated strain fermented glucose, sucrose, and fructose, but did not ferment maltose, galactose, xylose, and fructose. The indole test, oxidase test, and urease test were negative, and the nitrate reduction test and contact enzyme test were positive. Biochemical characterization of Haemophilus suis. According to the origin of the strain, it was named XX0306 strain. the

2.3 Determination and analysis of OmpA gene sequence

2.3.1 Primer Design Two primers were designed according to the gene sequence of Haemophilus parasuis OmpA in GenBank. The primer sequences are: P1:5'-gctccacaagctaacactttc-3', P2:5'-catagaaacttcttttgaacc-3', provided by Shanghai Sangon Biology Synthesized by Engineering Co., Ltd., the size of the amplified fragment of the primer is 1038bp. the

2.3.2 Preparation of cell DNA template Take 1.5mL of XX0306 strain culture, centrifuge at 10000r/min for 5min, discard the supernatant, resuspend the precipitate in 200μL of double distilled water, bathe in water at 100℃ for 10min, then centrifuge at 12000r/min for 5min to collect the supernatant , which is the PCR template, and stored at -20°C for future use. the

2.3.3 PCR amplification and sequencing Use the extracted bacterial DNA as a PCR template. The PCR amplification system is: 10×PCR Buffer 5.0 μL, MgCl ₂ (25mM) 2.5 μL, dNTP (2.5mM) 4.0 μL, primer P1 (50 μM) 0.5 μL, primer P2 (50 μM) 0.5 μL, template DNA 3.0 μL, Add 0.5 μL of Taq enzyme (5U/μL) to 50 μL with double distilled water. The reaction conditions were: 95°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 1 min, 30 cycles, and 72°C final extension for 10 min. Take 10 μL of the PCR amplification product, use 1% agarose gel to identify by electrophoresis at 120V, and use the standard strain as a positive control. The result shows a specific band with the same size as the standard strain of 1038bp, indicating that the isolated XX0306 strain is Haemophilus parasuis.

The recovered and identified positive PCR amplification products were sequenced by Shanghai Sangon Bioengineering Co., Ltd. The sequence result of the OmpA gene of Haemophilus parasuis XX0306 strain is shown in SEQ ID No.1. The sequence was compared with the sequence published in GenBank by BLAST, and the homology with the corresponding sequence of Haemophilus parasuis existing at home and abroad Up to 95% or more. the

2.4 Serological identification of Haemophilus parasuis XX0306 strain

According to Kleletein-Rapp-Gabriedson (KRG) agar diffusion serotyping method, the serotype of Haemophilus parasuis XX0306 strain was identified. Inoculate Haemophilus parasuis XX0306 strain in TSB medium at a ratio of 5%, culture it at 37°C and 5% CO ₂ for 18-24 hours, then concentrate the bacterial solution to a bacterial content of 1×10 ¹⁰ CFU/ mL, the bacterial solution was centrifuged at 5000r/min for 10min, and the supernatant was used for typing antigens in the agar diffusion test. Prepare a 1% agar plate and punch holes (6 holes around the middle hole), with a hole diameter of 5.0mm and a hole distance of 3.0mm. . Add 6 μl of Haemophilus parasuis standard strain rabbit anti-hyperimmune serum to the 6 surrounding wells (the upper well is marked as 1, add type 1 hyperimmune serum, and the sera of other serotypes are in order of ascending order of serotypes) Add samples in the clockwise direction), add 6 μl of heat-treated typing antigen of the strain to be tested in the middle hole, incubate at 37°C in a wet box, observe and record the results at 24h, 48h, and 72h respectively, and set a standard strain control at the same time. The agar diffusion test showed that the high-pressure extracted antigen of Haemophilus parasuis XX0306 strain had a precipitation line only with the positive serum of the standard strain of Haemophilus parasuis type 5, and had no precipitation line with the serotypes of other standard strains, indicating that the antigen of the H. Haemobacterium XX0306 strain was serotype 5.

2.5 Animal challenge test

Serum type 5 Haemophilus parasuis XX0306 strain was cultured in TSB/NAD liquid medium, counted live bacteria, and after concentration, the infectious titer reached 1×10 ⁹ CFU/ml, and injected intraperitoneally at 50 to 60 days old to be healthy and easy Infect 5 pigs, 5ml/head. Another control group was set up, with 5 heads, and TSB/NAD medium was injected intraperitoneally, 5ml/head. After challenge, they were reared as usual, and there was no antibiotic in the feed. Observe for 14 days, measure body temperature every day, and observe clinical symptoms. Autopsy was performed 14 days after the attack, and the number of cases was calculated based on clinical symptoms and autopsy lesions. At the same time, the tissue disease materials of sick pigs were taken for bacterial isolation and identification.

The results of the challenge showed that after 14 days of continuous observation, the five experimental pigs in the challenge group of Haemophilus parasuis XX0306 serotype 5 all developed disease successively; the control pigs were normal. The clinical symptoms were as follows: one day after the experimental pigs were infected with the isolated strain of Haemophilus parasuis, their body temperature increased, and then they gradually showed loss of appetite, rough coat, red ears and whole body skin, increased nasal fluid, dyspnea, and joint swelling in the later stage , lying on the ground. Necropsy revealed that the diseased pigs and dead pigs had pleural and peritoneal effusions, varying degrees of pleural and peritoneal adhesions, joint swelling, increased mucus in the joint cavity, pulmonary hemorrhage, and lymph node swelling. The results are shown in Table 1. the

Bacterial isolation was carried out using the tissue disease materials challenged by the isolated strains. Results Bacteria similar to the standard strain of Haemophilus parasuis were isolated from 4 diseased pigs in the XX0306 strain challenge group. At the same time, all isolated bacteria were identified by PCR, and a specific band appeared at 1038bp, which proved that the isolated pathogen was Haemophilus parasuis. the

Table 1 The virulence test results of Haemophilus parasuis XX0306 strain

"-" means not immunized or not applicable.

2.6 Determination of immunogenicity

TSB/NAD liquid medium was used to culture Haemophilus parasuis XX0306 strain of serum type 5. After counting viable bacteria, the viable culture was inactivated with a final concentration of 0.3% formaldehyde solution for 12 hours, and the number of bacteria was concentrated to 4×10 ⁹ CFU/ ml, after passing the inactivation test, prepare the vaccine by emulsifying according to the water-oil phase ratio of 1:3. A total of 25 healthy susceptible pigs around 2 weeks old were selected and randomly divided into 5 groups with 5 pigs in each group. Groups 1 to 3 were immunized with 0.5ml, 1ml, and 2ml of XX0306 strain vaccine respectively, the fourth group was the challenge control group, and the fifth group was the healthy control group. Three weeks after the first immunization, the vaccine immunization groups of each group were given a booster immunization with the same dose of vaccine. 42 days after the first immunization, except for 5 pigs in the healthy control group, all the pigs in the immunization group and the challenge control group were transferred to the challenge animal room. Groups 1 to 3 and Group 4 were each injected intraperitoneally with the concentrated live bacterial culture of XX0306 strain (the number of bacteria was (1×10 ⁹ CFU/ml), 5ml/head4; group 5 was injected with TSB intraperitoneally. /NAD culture medium, 5ml/head.Observed 14 days after challenged poison, necropsy.Based on clinical symptom and necropsy pathological changes calculation sickness number and challenged virus protection rate.Immune challenged virus protection test result shows, challenged poisonous control group 5 pigs All of them became ill (2 of them died), and typical clinical symptoms and obvious pathological changes of Haemophilus parasuis appeared. None of the experimental pigs in the healthy control group developed the disease. 2 of the 5 pigs in the XX0306 strain 0.5mL immunization group Clinical symptoms and pathological changes appeared, only 1 of the 5 pigs in the 1mL immunization group had necropsy lesions, and the protection rates of the challenge reached 3/5 and 4/5 respectively; while none of the experimental pigs in the 2mL immunization group developed disease, and the challenge The protection rate reached 5/5. See Table 2 for the details of immune attack protection.

Table 2 The results of the immunogenicity assay of Haemophilus parasuis XX0306 strain

"-" means not applicable. the

It can be seen from the results in Table 2 that the minimum immune protective dose of Haemophilus parasuis XX0306 strain is 1 mL containing 1×10 ⁹ CFU/mL of viable bacteria.

Example 2: Application of serotype 5 Haemophilus parasuis XX0306 strain in Haemophilus parasuis disease vaccine. the

1 strain

The strain used for seedling production is Haemophilus parasuis XX0306 strain serotype 5, and its preservation number is CCTCC M 2013095. The morphology of XX0306 strain's bacteria and colonies conformed to the standard strain of Haemophilus parasuis, and its biochemical and cultural characteristics were stable. It had strong toxicity to piglets and good immunogenicity. the

2 Preparation of strains for production

2.1 Primary production seed culture After dissolving the freeze-dried material of the basic seeds of Bacillus parasuis type 5, inoculate it on TSA/NAD solid medium, cultivate it at 37°C for 24 hours, select more than 5 typical colonies for each plant, and inoculate them on In TSB/NAD liquid medium, shake at 180r/min at 37°C for 18-24 hours. Then the culture is harvested, and after passing the pure inspection, it is used as the primary production seed and stored at 2-8°C for no more than 2 days. the

2.2 Propagation of secondary production seeds Take primary production seed cultures, inoculate TSB/NAD liquid medium at a volume ratio of 1:100, and culture at 37°C for 18-24 hours with shaking at 180r/min. After the medium has passed the pure inspection, it is used as secondary production seeds and stored at 2-8°C for no more than 2 days. the

2.3 Preparation of seedling-making bacteria liquid Take the secondary production seed culture liquid, inoculate it in a culture tank filled with TSB/NAD liquid medium at a volume ratio of 1:100, culture it with shaking at 37°C for 24 hours, harvest and take samples, according to the current " The appendix of the Veterinary Pharmacopoeia of the People's Republic of China conducts pure inspection and viable count. There was no growth of miscellaneous bacteria in each batch of vaccine preparation solution, and the number of viable bacteria reached 4.5×10 ⁹ CFU/ml.

3 Inactivation of bacteria solution

Take 300mL of Haemophilus parasuis culture and add it into 500mL bottles, divide into 3 bottles. Add 10% formaldehyde solution dropwise to each bottle, mix well while adding, so that the final concentration of formaldehyde in the three bottles of culture is 0.1%, 0.2%, 0.3% and 0.4%, and then transfer to another bottle and put it at 37 ° C to extinguish Live, shake once every 2h. Start timing when the liquid temperature in the container reaches 37°C, and take samples at 6h, 9h, 12h, 15h, 18h, 21h, and 24h after the start of inactivation, and conduct pure and inactivation tests. When sampling at each time point of the four formaldehyde concentrations, all bacteria grew. Formaldehyde solutions with a final concentration of 0.3% and 0.4% inactivated Haemophilus parasuis XX0306 strains had no viable bacteria detected at 18h and 12h respectively, as shown in Table 3. Therefore, the inactivation method of the bacterial solution is to inactivate the formaldehyde solution with a final concentration of 0.3% for 18 hours, and shake it every 2 hours. The inactivated bacterial liquid is qualified after being tested for aseptic growth, and the inactivated bacterial liquid is used as the antigen for seedling production. the

Table 3 The results of different concentrations of formaldehyde inactivating the culture medium of Haemophilus parasuis XX0306 strain

"+" positive means the growth of Haemophilus parasuis; "-" negative means no growth of Haemophilus parasuis. the

4 Preparation of the vaccine

4.1 Preparation of Haemophilus parasuis oil-adjuvanted inactivated vaccine

4.1.1 Preparation of oil phase Take 94 parts of white oil for injection and 806 parts of Siben-806 parts, stir well, sterilize at 121°C for 30 minutes, cool to room temperature for later use. the

4.1.2 Preparation of water phase Take 96 parts of Haemophilus parasuis strain XX0306 bacterial liquid that passed the inactivation test, and 4 parts of Tween-80, stir and mix well for later use. the

4.1.3 Seedling preparation and packaging Take 2.5 parts of the oil phase and add it to a sterile beaker, start the homogenizer, 10000r/min, after homogenizing for 30-60s, slowly add 1 part of the water phase, continue emulsification, 12000-13000r/min min, 5-8 minutes, add thimerosal solution with a final concentration of 0.005% before the end of emulsification to make a water-in-oil emulsion. Take 10ml of the vaccine and centrifuge at 3000r/min for 15min, the lower layer of the test tube will separate out the aqueous phase less than 0.5ml, and the emulsification is qualified. Quantitatively dispense the emulsified vaccine, seal it with a cover, label it, and store it at 2-8°C. the

4.2 Preparation of Haemophilus parasuis water-adjuvanted inactivated vaccine

4.2.1 Preparation of adjuvant Take Seppic ISA 201 VG adjuvant, heat to 31°C, and set aside. the

4.2.2 Antigen preparation Take the liquid of Haemophilus parasuis strain XX0306 that has passed the inactivation test, heat it to 31°C, and set it aside. the

4.2.3 Seedling production and sub-packaging are based on the mass ratio of ISA 201 VG adjuvant and antigen at 1:1 (for example, 10 g of Haemophilus parasuis strain XX0306, and 10 g of ISA 201 VG adjuvant). Each phase was heated to 31°C prior to mixing. Add the aqueous antigen medium to ISA 201 VG, keep the temperature at 30°C, and mix with low shear force to obtain a stable preparation. After the qualified emulsified vaccine is mixed evenly, it is quantitatively dispensed, sealed with a cover, labeled, and stored at 2-8°C. the

5 Inspection of finished vaccines

5.1 Appearance The appearance of Haemophilus parasuis oil adjuvant inactivated vaccine is milky white uniform emulsion. The appearance of Haemophilus parasuis water-adjuvanted inactivated vaccine is a milky white emulsion, and there is no obvious water precipitation at the bottom. the

5.2 The dosage form is water-in-oil type. Take a clean straw, suck up a small amount of vaccine and drop it in cold water. Except for the first drop that diffuses in the form of cloud, each subsequent drop does not spread, indicating that the dosage form is stable. Haemophilus parasuis water-adjuvanted inactivated vaccine is water-in-oil-in-water type; take a clean straw, suck a small amount of vaccine and drop it in cold water, part of the droplet will self-dilute and make the water appear milky white, which means it is water-in-water Water-in-oil type. the

5.3 Stability Take 10ml of the vaccine and add it to a centrifuge tube, centrifuge at 3000r/min for 15 minutes, the water phase precipitated at the bottom of the tube does not exceed the regulation of 0.5ml. the

5.4 Viscosity The appendix of the current "Chinese Veterinary Pharmacopoeia" shall be inspected and shall meet the regulations. the

5.5 Sterility test The test was carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and all the prepared vaccines grew aseptically. the

5.6 Thimerosal residues According to the current "Chinese Veterinary Pharmacopoeia" appendix, the residues of thimerosal in the vaccine are 0.006-0.007%, which is in line with the provisions of the General Rules for Veterinary Biological Products. the

5.7 Determination of formaldehyde content According to the current appendix of "Chinese Veterinary Pharmacopoeia", the formaldehyde content in the vaccine is 0.03-0.11%, which is in line with the provisions of the General Rules for Veterinary Biological Products. the

5.8 Safety inspection Inactivated Haemophilus parasuis vaccine prepared in the laboratory was injected into the neck muscle in a single dose (2mL/head), single dose repetition (2mL/head, 2 times) and large dose repetition (4mL/head) 2-week-old healthy susceptible piglets and 90-day-pregnant sows injected intramuscularly with a large dose (4mL/head) in the neck, 6 in each group. Observe the clinical manifestations of piglets and sows after immunization, and record the local and systemic reactions of piglets and sows, as well as the farrowing conditions of sows; measure rectal temperature before vaccination and within 14 days after vaccination, and high-dose vaccination is safe In the experimental group, 30, 60 and 90 days after immunization, 2 pigs were selected and killed, and local tissue biopsy was performed on the injection site. The results showed that the appetite of the experimental pigs was normal and the mental condition was good; 100% of the piglets were healthy and alive, and there was no stillbirth or abortion; 24-48 hours after the vaccine immunization, the body temperature of the piglets and sows did not rise above 1 ℃, but it was obvious. Transient and short-lived, it does not have adverse effects on pigs. Obvious inflammatory reaction was observed in the group immunized with the bivalent inactivated Haemophilus parasuis vaccine 30 days after the first immunization, but no obvious inflammatory reaction was observed in tissue sections 60 and 90 days after the first immunization. It shows that the prepared vaccine is safe and reliable. the

5.9 Efficacy test Select 5 2-week-old healthy susceptible piglets, intramuscularly inject 1 vaccine (2mL) into each neck, and boost immunization with the same dose once 21 days later. At the same time, pigs with the same conditions were set up as the challenge control group and 5 healthy control groups. They were challenged 42 days after the first immunization, observed for 14 days after the challenge, and autopsyed. protection rate. The results showed that all the test pigs in the two challenge control groups developed the disease, and the protection rate of the immune group was 100% (5/5). None of the pigs in the healthy control group developed the disease. All efficacy tests are qualified, indicating that the prepared vaccine has very good immune protection for piglets. the

Claims (1)

1. A vaccine strain of Haemophilus parasuis serotype 5, which is classified and named as Haemophilus parasuis ( Haemophilus parasuis ), the strain number is XX0306, and has been preserved in the China Center for Type Culture Collection, preservation number: CCTCC NO: M 2013095, date of deposit: March 21, 2013.