CN110812473A - Triple inactivated vaccine for haemophilus parasuis disease, streptococcus suis disease and pasteurella multocida disease and preparation method thereof - Google Patents
Triple inactivated vaccine for haemophilus parasuis disease, streptococcus suis disease and pasteurella multocida disease and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological products for livestock, and particularly relates to a haemophilus parasuis, streptococcus suis and pasteurella multocida triple inactivated vaccine and a preparation method thereof. The triple inactivated vaccine for haemophilus parasuis, streptococcus suis and pasteurella multocida provided by the invention comprises an antigen concentrate and a nano aluminum adjuvant, wherein the antigen concentrate comprises inactivated haemophilus parasuis serotype 4H 23 strain, haemophilus parasuis serotype 5H 24 strain, streptococcus suis serotype 2S 23 strain, pasteurella multocida capsular type A P13 strain and pasteurella multocida capsular type B P11 strain. The triple inactivated vaccine can effectively prevent streptococcus suis, haemophilus parasuis and pasteurella multocida of pigs, achieves the aims of preventing more diseases with one injection and reducing stress, and reduces epidemic prevention cost.
Description
Technical Field
The invention belongs to the field of biological products for livestock, and particularly relates to a triple inactivated vaccine for haemophilus parasuis, streptococcus suis and pasteurella multocida and a preparation method thereof.
Background
Haemophilus Parasuis (HPS) is a opportunistic pathogen of the upper respiratory tract of pigs, invading the body under specific conditions causing a systemic infectious disease characterized by cellulolytic multiple serositis, arthritis, meningitis and high mortality. At present, haemophilus parasuis is one of the main bacterial diseases prevalent in China, the haemophilus parasuis can affect piglets and fattening pigs at the age of 2-25 weeks, the diseases are mainly developed after weaning and in the stage of conservation, the diseases are mostly found in the pigs at the age of 5-8 weeks, the disease incidence is 10% -90%, the death rate is 50-90% in severe cases, the death rate of infection of adult pigs is about 20%, the death rate of sows can be as high as 30% -40%, and huge economic losses are brought to the pig industry in China. KRG can be divided into at least 15 serotypes based on its serotyping, wherein the serotypes 4 and 5 are the main ones. The haemophilus parasuis lack immune cross protection among all serotypes, the haemophilus parasuis inactivated vaccine only has resistance protection effect on strains of the same serotype, and once the serotype of an epidemic strain is not consistent with the serotype of the vaccine, the vaccine has no protection effect on swinery.
Streptococcus Suis (SS) is a pathogenic bacterium causing streptococcosis in pigs, and pigs of different ages, breeds and sexes are susceptible to the disease under natural conditions and can cause clinical symptoms such as septicemia, meningitis, arthritis and the like. The streptococcus suis can be divided into 33 serotypes according to bacterial capsular polysaccharide, and through epidemiological analysis, the types 2, 7 and 9 are important serotypes causing swine infection, wherein the streptococcus suis type 2 has the strongest toxicity and is popular.
Swine pasteurellosis is an infectious disease of the respiratory system, focal infection and hemorrhagic septicemia of pigs caused by Pasteurella multocida (Pm), and is normally colonized in the oral cavity, nasopharynx and upper respiratory tract of healthy animals. According to the capsular polysaccharide of the bacteria, the swine pasteurella multocida can be classified into A, B, D, E, F type, swine pasteurellosis is also called swine plague, the swine pasteurellosis is mainly caused by A, B type infection, the pasteurella multocida infection can cause acute or sporadic infectious diseases, and the pathogenic bacteria can be infected with various pathogens in a synergistic manner, so that the morbidity and mortality of respiratory diseases of swine herds are increased, and serious economic loss is caused.
The market is mainly single vaccine and bivalent vaccine for the prevention and control of the streptococcus suis, the haemophilus parasuis and the pasteurella multocida, inactivated vaccines for the coinfection of the streptococcus suis, the haemophilus parasuis and the pasteurella multocida are still blank in China, commercial vaccines cannot meet clinical requirements, and the abuse of antibiotics causes the generation of drug-resistant strains, so that multivalent vaccines for the coinfection are more popular.
Most vaccines for animals are inactivated by formaldehyde, the inactivation of the formaldehyde is easily influenced by temperature, pH and concentration, and the inactivation of excessive formaldehyde can not only damage the antigenicity of the vaccine, but also cause a large amount of residues, and has influence on the edible safety of animal organisms and human beings. The aluminum adjuvant is one of the accepted vaccine adjuvants, and the application of the conventional aluminum adjuvant is limited due to the defects of high immune stress and the like, so that research and development of an inactivated vaccine which is simple in preparation method, can effectively prevent swine streptococcicosis, haemophilus parasuis and swine pasteurella multocida diseases, and is small in side reaction, safe and efficient are imperative.
Disclosure of Invention
The invention mainly aims to provide a safe and efficient triple vaccine capable of preventing co-infection of haemophilus parasuis, streptococcus suis and pasteurella multocida.
In order to achieve the purpose, the invention adopts the following technical scheme:
a triple inactivated vaccine for haemophilus parasuis, streptococcus suis and pasteurella multocida is characterized by comprising antigen concentrate and a nano aluminum adjuvant; the antigen concentrate comprises inactivated H23 type-4 Haemophilus parasuis serum, H24 type-5 type Haemophilus parasuis serum, S23 type-2 streptococcus suis serum, P13 type Pasteurella multocida capsule A and P11 type Pasteurella multocida capsule B antigen concentrates.
Preferably, the volume ratio of the antigen concentrates of the haemophilus parasuis serotype 4H 23 strain, the haemophilus parasuis serotype 5H 24 strain, the streptococcus suis serotype 2S 23 strain, the pasteurella multocida capsular type A P13 strain and the pasteurella multocida capsular type B P11 strain is 1:1:1:1: 1.
Preferably, the content of the antigen concentrates of the haemophilus parasuis serotype 4H 23 strain, the haemophilus parasuis serotype 5H 24 strain, the streptococcus suis serotype 2S 23 strain, the pasteurella multocida capsular type A P13 strain and the pasteurella multocida capsular type B P11 strain is 5 x 107CFU/mL。
Preferably, the volume ratio of the nano aluminum adjuvant is 10%.
Preferably, the triple inactivated vaccine further comprises 1% (w/v) of thimerosal and a veterinarily acceptable vaccine diluent.
Preferably, the vaccine diluent is 0.9% NaCl solution or PBS solution.
The invention also aims to provide a preparation method of the triple inactivated vaccine for haemophilus parasuis, streptococcus suis and pasteurella multocida, which comprises the following steps:
s1, respectively proliferating H23 strain of haemophilus parasuis serotype 4, H24 strain of serotype 5, S23 strain of streptococcus suis serotype 2, P13 strain of pasteurella multocida capsule A and P11 strain of pasteurella multocida capsule B to obtain culture solutions;
s2, adding an inactivating agent accounting for 0.1-0.15% of the total volume of the culture solution into the culture solution obtained in the step S1, inactivating the mixture at 4 ℃ for 72 hours while stirring the mixture for several times, and performing water bath at 37 ℃ for 2 hours to obtain inactivated culture solutions of H23 serotype 4, H24 serotype 5, S23 serotype 2 of streptococcus suis, Pasteurella multocida capsular type A P13 and Pasteurella multocida capsular type B P11;
s3, concentrating the culture solutions of the haemophilus parasuis inactivated in the step S2 and containing H23 strain of serotype 4, H24 strain of serotype 5, S23 strain of streptococcus suis serotype 2, P13 strain of pasteurella multocida capsular type A and P11 strain of pasteurella multocida capsular type B, and adding 0.9% NaCl solution or PBS solution to resuspend the strains to obtain antigen concentrates of H23 strain of haemophilus parasuis serotype 4, H24 strain of haemophilus parasuis serotype 5, S23 strain of streptococcus suis serotype 2, P13 strain of pasteurella multocida type A and P11 strain of pasteurella multocida capsular type B;
s4, adding the antigen concentrates of the H23 type haemophilus parasuis serum 4 strain, the H24 type haemophilus parasuis serum 5 strain, the S23 type streptococcus suis serum 2 strain, the Pasteurella multocida capsular type A P13 strain and the Pasteurella multocida capsular type B P11 strain in the step S3 into an emulsifying machine, adding the nano aluminum adjuvant, the diluent and the thimerosal according to the proportion of the formula, stirring at a low speed, and mixing to obtain the inactivated vaccine of haemophilus parasuis, Streptococcus suis and Pasteurella multocida.
Preferably, the inactivator in step S2 is β -propiolactone.
Preferably, in the step S3, the bacteria of haemophilus parasuis serotype 4H 23, haemophilus parasuis serotype 5H 24, streptococcus suis serotype 2S 23, pasteurella multocida capsular type a P13 and pasteurella multocida capsular type B P11 are concentrated to have a bacterial content corresponding to a viable count of 5 × 10 before inactivation9FU/mL。
Preferably, the culture of haemophilus parasuis, streptococcus suis and pasteurella multocida is subjected to sterility, safety and efficacy tests, respectively, after step S4.
The embodiment 2-3 shows that after the triple inactivated vaccine is inactivated by β -propiolactone, the vaccine adopting nano aluminum as an adjuvant is injected into the weaned pigs, the local part of the vaccine injection has no adverse reaction, each organ has no abnormal lesion, and the injection part of the weaned pigs adopting formaldehyde inactivation has 2 cases of erythema, subcutaneous nodules and contact allergy, which shows that the β -propiolactone inactivated vaccine has good safety, and the vaccine adopting nano aluminum as the adjuvant has good absorption and no adverse reaction.
Through the embodiment 4, the conventional aluminum adjuvant vaccine 1 and the nano aluminum adjuvant vaccine 2 are respectively injected into the muscle of the weaned pig for 2mL respectively, secondary immunization is carried out, and each virulent strain is used for counteracting the toxin 14 days after the second immunization; the observation for 14 days after the virus attack shows that the conventional aluminum adjuvant vaccine and the nano aluminum adjuvant vaccine both have the protection effect on the virus attack of haemophilus parasuis (type 4 and type 5), streptococcus suis type 2 and pasteurella multocida (type A and type B), the protection rate of the conventional aluminum adjuvant vaccine and the nano aluminum adjuvant vaccine is basically over 80 percent, and the protection rate of the conventional aluminum adjuvant vaccine and the nano aluminum adjuvant vaccine is basically 100 percent, so that the triple inactivated vaccine of haemophilus parasuis, streptococcus suis and pasteurella multocida can generate better protection effect on corresponding pathogens, and the action effect of the nano aluminum vaccine is stronger than that of the conventional aluminum gel vaccine.
The invention also carries out efficacy test on the triple inactivated vaccine, the single inactivated vaccine or the bivalent inactivated vaccine respectively through the embodiment 5, and the results show that when the triple inactivated vaccine, the single inactivated vaccine and the bivalent inactivated vaccine are used for attacking viruses by haemophilus parasuis type 4, streptococcus suis type 2, and pasteurella multocida types A and B, the immune protection rates of the triple inactivated vaccine to the weaned piglets are not obviously different, but when the haemophilus parasuis type 5 and the pasteurella multocida type B are used for attacking the viruses, the immune protection rates of the triple inactivated vaccine to the weaned piglets are superior to those of the single inactivated vaccine and the bivalent inactivated vaccine; in the toxin attacking parts of the haemophilus parasuis type 5 and the swine pasteurella multocida type B strains, the immune protection rates of the triple inactivated vaccine to the weaned pigs are 10/10, and the immune protection rates of the bivalent inactivated vaccine to the weaned pigs are 9/10, so that the triple inactivated vaccine provided by the invention is superior to the bivalent inactivated vaccine and the single inactivated vaccine in the immune efficiency of the haemophilus parasuis type 5 and the swine pasteurella multocida type B strains. Compared with the inoculation dose, the triple inactivated vaccine is injected with 2mL by one needle, and the single inactivated vaccine or the double inactivated vaccine is injected with 4mL by at least 2 needles, so the triple inactivated vaccine provided by the invention reduces the immunization cost, has high titer and is convenient and quick to immunize.
Compared with the prior art, the invention has the following beneficial effects:
(1) the strains of haemophilus parasuis, streptococcus suis and pasteurella suicidentalis provided by the invention are rejuvenated by sensitive pigs, have strong immunogenicity, wide antigen spectrum and stable genetic performance, and have more pertinence when being used as strains.
(2) The haemophilus parasuis, streptococcus suis and pasteurella multocida triple inactivated vaccine provided by the invention adopts nano-aluminum as an adjuvant, has strong adsorption capacity, improves targeted delivery of antigens, greatly reduces adverse reactions of the antigens, improves the utilization rate of the antigens to the maximum extent, improves safety and achieves one-injection multiple prevention.
(3) The vaccine has high titer and convenient and quick immunization.
Detailed Description
The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.
Strain: haemophilus parasuis serotype 4H 23 strain (GDMCC NO: 60722, deposited at: Guangdong provincial collection of microorganisms), serotype 5H 24 strain (GDMCC NO: 60723, deposited at: Guangdong provincial collection of microorganisms), Streptococcus suis serotype 2S 23 strain (GDMCC NO: 60724, deposited at: Guangdong provincial collection of microorganisms), porcine pasteurella multocida capsule type A P13 strain (GDMCC NO: 60720, deposited at: Guangdong provincial collection of microorganisms), porcine pasteurella multocida capsule type B P11 strain (GDMCC NO: 60721, deposited at: Guangdong provincial collection of microorganisms)
EXAMPLE 1 obtaining of antigen solution of Strain
1. Isolation identification and virulence test of H23 strain and H24 strain of haemophilus parasuis
1.1 isolation and identification of Haemophilus parasuis H23 and H24 strains
Animals: a fresh sick pig with the clinical manifestations of asthma, pleuritis, peritonitis, pericarditis, arthritis or encephalitis and the like is collected from the south China in 2016-2018 and is a piglet in the nursery period of 1-2 weeks after weaning.
The method comprises the following steps: aseptically collecting lung, heart blood and joint fluid of a sick pig, respectively inoculating the sick pig to a TSA culture medium (containing 5% bovine serum, 0.002% NAD and 100 mu g/mL lincomycin), culturing the sick pig in an incubator at 37 ℃ for 24-48 hours, observing the bacterial colony morphology, selecting a suspicious colony for pure culture, carrying out 16S rRNA identification after carrying out amplification culture on the suspicious colony, and carrying out the following primer sequences:
an upstream primer: 5'-GGCTTCGTCACCCTCTGT-3', respectively;
a downstream primer: 5'-GTGATGAGGAGGGGTGGTGT-3', respectively;
the 16S rRNA PCR product is sent to Shanghai biological engineering Co., Ltd for sequencing.
As a result: suspicious colonies grow on colonies with the needle point sizes, the needle points are round, smooth and moist, colorless and transparent, the edges are regular, the colonies which are closer to the staphylococcus aureus are larger, the colonies which are farther away are smaller, and an obvious satellite phenomenon appears, the suspicious colonies are preliminarily proved to be haemophilus parasuis, the preliminarily proved haemophilus parasuis is subjected to sequencing identification, the sequencing result is subjected to Blast comparison with a known sequence in a GenBank, the comparison result shows that a gene fragment obtained by PCR is 822bp, the similarity with the known haemophilus parasuis is 99-100%, and the separated strain is determined to be haemophilus parasuis.
1.2 serotype identification of Haemophilus parasuis H23 and H24 strains
An agglutination test is carried out by using the haemophilus parasuis standard positive serum, and the result shows that 85 strains are haemophilus parasuis H23 strains which are serogroup 4; 110 strains are haemophilus parasuis H24 strains are serotype 5, and serotype typing identification is carried out on the strains by a PCR method, and the primer sequences are as follows:
type 4 upstream primer: 5'-GGTTAAGAGGTAGAGCTAAGAATAGAGG-3', respectively;
type 4 downstream primer: 5'-CTTTCCACAACAGCTCTAGAAACC-3', respectively;
type 5 upstream primer: 5'-TAGTTGGTATATTATTTTCT-3', respectively;
type 5 downstream primer: 5'-AGAATGCATCTGTACCACTAAG-3', respectively;
the product is sent to Shanghai biological engineering Limited company for sequencing, and Blast comparison is carried out on the product and a known sequence in GenBank, and the result shows that the gene fragment obtained by the PCR of the H23 strain is 320bp, the similarity of the gene fragment and the wcIP gene of the known haemophilus parasuis serotype 4 is 100 percent, and the separated H23 strain is determined to be the haemophilus parasuis serotype 4; the gene fragment obtained by PCR of the H24 strain is 560bp, the similarity with the funk gene of the known haemophilus parasuis serotype 5 is 100%, and the separated haemophilus parasuis H24 strain is determined to be haemophilus parasuis serotype 5.
1.3 virulence test of H23, H24 strains of Haemophilus parasuis
25 weaned piglets of 21 days old are divided into 5 groups randomly, wherein 1 group of control groups is injected with 1mL of sterile normal saline in the abdominal cavity, and the other 4 groups are injected with different doses of H23 and H24 strains, and the conditions such as morbidity and mortality of the weaned piglets are observed and recorded after 2 weeks of continuous observation after injection.
The results of virulence test of H23 and H24 strains of Haemophilus parasuis are shown in Table 1, and the results show that 2X 10 strains were intraperitoneally injected9CFU、1×109After the strain of the haemophilus parasuis H23 of CFU, 5 experimental weaned piglets all suffered from the disease, and were injected with the abdominal cavity with 1 x 109CFU、5×108After the strain of the haemophilus parasuis H24 of CFU, 5 test weaned piglets all suffer from the disease, and the test result shows that the infection amount of the haemophilus parasuis H23 is 1 multiplied by 109The infection amount of the strain of the CFU/Haemophilus parasuis H24 is 5 multiplied by 108CFU/only.
TABLE 1 result of toxin challenge by H23 and H24 strains of Haemophilus parasuis
2. Isolation and identification of S23 strain of streptococcus suis and virulence test
2.1 isolation and preliminary characterization of Streptococcus suis S23 Strain
Animals: the affected pigs with meningitis, septicemia, endocarditis, multiple serositis, bronchopneumonia and arthritis were collected from south China in 2016-2018.
The method comprises the following steps: aseptically collecting diseased tissues such as hearts, livers, lungs, spleens, lymph nodes and the like of sick pigs, respectively inoculating the diseased tissues to a sheep blood agar plate, culturing the diseased tissues in a biochemical incubator at 37 ℃ for 24-48 hours, observing the colony morphology, selecting a single suspicious colony which is small in colony, transparent and slightly sticky in grey, or has a hemolytic ring for gram staining microscopic examination, selecting a single suspected colony, streaking again, inoculating the single suspected colony to a blood plate for purification and bacteria preservation, and performing GDH gene sequencing identification after strain amplification culture, wherein the used primer sequences are as follows:
an upstream primer: 5'-GCAGCGTATTCTGTCAAAACG-3', respectively;
a downstream primer: 5'-CCATGGACAGATAAAGATGG-3', respectively;
as a result: and performing Blast comparison with a known sequence in GenBank, wherein the sequencing result shows that the gene fragment obtained by PCR of the streptococcus suis S23 strain is 689bp, the similarity with the known streptococcus suis GDH gene is 99-100%, and the separated strain is determined to be streptococcus suis.
2.2 serotype identification of Streptococcus suis S23 Strain
Serotype identification is carried out on more than 200 strains of streptococcus suis which are preliminarily verified, agglutination test is carried out by using standard positive serum of streptococcus suis, and the result shows that 62 strains are serotype 2; and serotype typing identification is carried out on the DNA fragment by a PCR method, and the primer sequences are as follows:
the upstream primer is as follows: 5'-TTCGTATTAACTTACTTGGCGT-3', respectively;
the downstream primer is: 5'-TAAATCCCCATATGCCAAATCC-3', respectively;
and (3) sequencing the PCR product by Shanghai Bioengineering Co., Ltd, performing Blast comparison with a known sequence in GenBank, and determining that the separated S23 strain is Streptococcus suis serotype 2, wherein the gene fragment obtained by the PCR of the S23 strain is 450bp and the similarity with the known Streptococcus suis serotype 2 gene is 99-100%.
2.3 virulence test of S23 Strain of Streptococcus suis
25 weaned piglets of 21 days old are divided into 5 groups randomly, wherein 1 group of the control groups is injected with 1mL of sterile physiological saline intravenously, the other 4 groups are injected with S23 strains with different doses, the injection is continuously observed for 2 weeks, and the morbidity, the mortality and other conditions are observed and recorded.
The virulence test results of S23 strain of Streptococcus suis are shown in Table 2, and the results show that 1X 10S 23 strain is injected intravenously9CFU、5×108After CFU of S.suis S23, all the 5 weaned piglets died, indicating that the minimum lethal dose of S23 is 5X 108 CFU/pig.
TABLE 2 Streptococcus suis S23 strain challenge results
3. Isolation and identification of porcine pasteurella multocida strain P13 and P11 and virulence test
3.1 isolation and preliminary characterization of porcine Pasteurella multocida strains P13 and P11
Animals: fresh sick pig disease material with clinical manifestations of respiratory system disease symptoms is collected from south China in 2013-2018.
The method comprises the following steps: aseptically collecting blood, respiratory tract secretion, local edema fluid, thoracic cavity exudate, lesion lymph node, liver, spleen and the like of a dead pig to prepare a contact sheet or a smear, inoculating the contact sheet or the smear to a fresh blood plate, culturing for 24 hours at 37 ℃, observing the bacterial colony morphology, then selecting a dew-like round suspicious microcolony with a smooth and moist surface, selecting the suspicious microcolony of gram-negative bacilli with two-pole coloration under a gram-colored mirror, purifying and preserving the bacteria on the fresh blood plate, and identifying the specific Kmt gene of the porcine pasteurella multocida. The primer sequences used were as follows:
an upstream primer: 5'-ATCCGCTATTTACCCAGTGG-3', respectively;
a downstream primer: 5'-GCTGTAAACGAACTCGCCAC-3', respectively;
the gene product is sent to Shanghai biological engineering Limited company for sequencing, and Blast comparison is carried out on the gene product and a known sequence in GenBank, and the result shows that the gene fragment obtained by PCR is 457bp, the similarity with the known porcine pasteurella multocida Kmt gene is 99-100%, and the separated strain is determined to be the porcine pasteurella multocida.
3.2 serotype identification of porcine Pasteurella multocida strains P13, P11
Identifying the capsular group of the swine pasteurella multocida by using standard capsular group strain stereotyping serum of 100 strains of swine pasteurella multocida P13 and P11, wherein the result shows that 60 strains of swine pasteurella multocida P13 are of type A, and 5 strains of swine pasteurella multocida P11 are of type B; and serotype typing identification is carried out on the DNA fragment by a PCR method, and the primer sequences are as follows:
type A upstream primer: 5'-GATGCCAAAATCGCAGTCAG-3', respectively;
a type downstream primer: 5'-TGTTGCCATCATTGTCAGTG-3', respectively;
b type upstream primer: 5'-CATTTATCCAAGCTCCACC-3', respectively;
b type downstream primer: 5'-GCCCGAGAGTTTCAATCC-3', respectively;
the PCR product is sent to Shanghai biological engineering Limited company for sequencing, and Blast comparison is carried out on the PCR product and a known sequence in GenBank, the result shows that the gene fragment obtained by the PCR of the P13 strain is 1048bp, the similarity with the known porcine pasteurella multocida capsular type A gene is 99-100%, and the separated P13 strain is determined to be porcine pasteurella multocida capsular type A; the gene fragment obtained by PCR of the P11 strain is 758bp, the similarity with the known porcine pasteurella multocida capsular B type gene is 99-100%, and the separated P11 strain is determined to be porcine pasteurella multocida capsular B type.
3.3 virulence test of porcine Pasteurella multocida strains P13, P11
50 weaned piglets of 21 days old are taken and randomly divided into 10 groups, wherein 2 groups of the control groups are injected with 1mL sterile physiological saline subcutaneously, the other 8 groups are injected with different dosages of porcine pasteurella multocida P13 and P11 respectively, after injection, the continuous observation is carried out for 2 weeks, and the morbidity, mortality and other conditions of the piglets are observed and recorded.
The results of the virulence test of the porcine pasteurella multocida strains P13 and P11 are shown in Table 3, and the results show that subcutaneous injection is 1X 1010After the CFU porcine pasteurella multocida P13 strain, all the weaned piglets die in 5 tests and are injected with 5 multiplied by 10 subcutaneously8After the CFU porcine pasteurella multocida P11 strain, all the 5 weaned piglets die, which shows that the minimum lethal dose of the porcine pasteurella multocida P13 strain is 1 multiplied by 1010The minimum lethal dose of CFU/porcine Pasteurella multocida P11 strain is 5 × 108CFU/only.
TABLE 3 porcine Pasteurella multocida P13, P11 strains challenge results
Example 2 preparation of a triple inactivated vaccine against Haemophilus parasuis, Streptococcus suis and Pasteurella multocida
1. Seed preparation for production
First-order seed propagation: a freeze-dried Haemophilus parasuis serotype 4H 23 strain (GDMCC No: 60722, the preservation place: Guangdong province microbial strain preservation center) and a Haemophilus parasuis serotype 5H 24 strain (GDMCC No: 60723, the preservation place: Guangdong province microbial strain preservation center) are inoculated in TSA containing 5% of calf serum and 0.002% of NAD, a freeze-dried Streptococcus suis serotype 2S 23 strain (GDMCC No: 60724, the preservation place: Guangdong province microbial strain preservation center) is inoculated in TSA containing 5% of calf serum, a pig pasteurella multocida type A capsule P13 strain (GDMCC No: 60720, the preservation place: Guangdong province microbial strain preservation center) and a pig pasteurella multocida capsule B11 strain (GDMCC No: 60721, the preservation place: Guangdong province microbial strain preservation center) are inoculated in TSA containing 5% of calf serum, and a single cell culture box is arranged at 36-37 ℃ to select good growing colonies after 18-24 hours, inoculating into TSB nutrient medium, culturing at 36-37 deg.C for 14-18 hr in biochemical incubator to serve as first-stage seed, and storing at 2-8 deg.C for no more than 5 generations.
And (3) secondary seed propagation: and respectively taking the first-stage seeds of the haemophilus parasuis serum 4 type H23 strain, the haemophilus parasuis serum 5 type H24 strain, the streptococcus suis serum 2 type S23 strain, the pasteurella suis multocida capsular A type P13 strain and the pasteurella suis multocida capsular B type P11 strain to inoculate in the serum of a calf with 5 percent of TSB, wherein the haemophilus parasuis serum 4 type H23 strain and the haemophilus parasuis serum 5 type H24 strain are inoculated in a TSB culture medium containing 5 percent of calf serum and 0.002 percent of NAD, culturing for 12-16 hours at 36-37 ℃, sampling and taking the qualified second-stage seeds as pure inspection according to the appendix of the pharmacopoeia of the existing Chinese beast, and immediately storing the second-stage seeds at 2-8 ℃ for a service life not more than 4 days.
2. Culture medium for seedling preparation: commercially available TSB liquid media.
3. Preparation of bacterial solution for seedling production
Inoculating 1-2% of the culture amount of secondary seed solution of Haemophilus parasuis serotype 4, Haemophilus parasuis serotype 5, Streptococcus suis serotype 2, Pasteurella multocida capsular type A and Pasteurella multocida capsular type B, respectively, and culturing at 36-37 deg.C for 12-20 hr.
4. Pure inspection: after the culture of the bacterial liquid is finished, the existing appendix of Chinese animal pharmacopoeia is subjected to pure inspection and is required to be pure.
5. Counting viable bacteria: counting viable bacteria according to the appendix of the current Chinese veterinary pharmacopoeia, and taking the counting result as the number of cultured bacteria for reference during vaccine preparation.
6. And (2) inactivating, namely, respectively taking the secondary seed solutions of the haemophilus parasuis serotype 4, the haemophilus parasuis serotype 5, the streptococcus suis serotype 2, the pasteurella multocida capsule A and the pasteurella multocida capsule B obtained by culturing in the step 2, slowly adding β -propiolactone solution according to 0.1-0.15 percent of the total amount, inactivating for 72 hours at 4 ℃, stirring for a plurality of times during the inactivation, then carrying out water bath for 2 hours at 37 ℃, hydrolyzing an inactivating agent, or slowly adding formaldehyde solution according to 0.4 percent of the total amount of the bacterial solution, inactivating for 24 hours at 37 ℃, reversing and uniformly mixing the inactivated solution for a plurality of times during the inactivation, and carrying out inactivation inspection according to the supplement of the current Chinese veterinary pharmacopoeia and requiring aseptic growth.
7. Concentration: concentrating the inactivated five bacterial liquids, and re-suspending the bacteria with 0.9% NaCl solution or PBS to make the bacteria content equal to the number of viable bacteria before inactivation 5 × 109CFU/mL。
8. Seedling preparation: the inactivated and concentrated haemophilus parasuis serum 4 type H23 strain, haemophilus parasuis serum 5 type H24 strain, streptococcus suis serum 2 type S23 strain, pasteurella multocida capsular A type P13 strain and B type P11 strain are taken and mixed according to the volume ratio of 1:1:1:1:1, the inactivated and concentrated antigen, nano aluminum gel or conventional aluminum hydroxide gel, sterile 0.9% physiological saline and thimerosal are mixed in an emulsifying machine and are fully stirred and mixed, and the vaccine components are shown in the table 4.
TABLE 4 inactivated vaccine component
Note: "/" indicates no addition.
9. And (3) checking a semi-finished product: after inactivation and addition of an adjuvant, samples are taken according to the appendix of the pharmacopoeia of the people's republic of China for aseptic examination, and the cells are required to grow aseptically.
10. Subpackaging: and (4) after the aseptic inspection is qualified, performing batch subpackage, stirring uniformly during subpackage, performing subpackage, covering a bottle cap, and pressing an aluminum cap.
Example 3 sterility and safety testing of triple inactivated vaccines
Materials: vaccine 1 and vaccine 2. Wherein, the vaccine 2 is prepared by inactivating each bacterial liquid according to two inactivation modes, and the preparation method is the same as that of the example 2 according to the components of the vaccine 2.
The first inactivation mode adopts β -propiolactone solution as the inactivator to inactivate each bacteria liquid, and the specific mode is that β -propiolactone solution is slowly added according to 0.1-0.15% of the total bacteria liquid, and is inactivated for 72 hours at 4 ℃, and is stirred for a plurality of times, and then is put in water bath at 37 ℃ for 2 hours to hydrolyze the inactivator;
the second inactivation mode is to adopt formaldehyde solution as an inactivator to inactivate various bacteria liquid, and the specific mode is as follows: slowly adding formaldehyde solution 0.4% of the total amount of the bacteria solution, inactivating at 37 deg.C for 24 hr, and mixing the inactivated solution by inversion for several times.
The inactivation of the vaccine 1 adopts β -propiolactone solution as an inactivator to inactivate each bacterial liquid, and the specific mode is that β -propiolactone solution is slowly added according to 0.1-0.15 percent of the total amount of the bacterial liquid, the bacterial liquid is inactivated for 72 hours at 4 ℃, the mixture is stirred for a plurality of times during the inactivation, and then the inactivator is hydrolyzed in water bath at 37 ℃ for 2 hours;
1. sterility testing
The samples were taken according to the appendix of the current Chinese veterinary pharmacopoeia for sterility test, and the results showed that each inactivated vaccine should grow aseptically.
2. Target animal safety inspection
Injecting 10 SPF Kunming mice with the weight of 18-22g into each group, and injecting vaccine 2 with different inactivators with the volume of 0.5mL into the mice subcutaneously respectively for 10 days; meanwhile, 21-day-old weaned piglets were randomly grouped into 5 groups, 4mL of double vaccine dose was injected into each neck muscle, and the remaining 10 were control groups and observed for 14 days.
3. Results
The results of the sterility test performed by sampling according to the appendix of the current Chinese veterinary pharmacopoeia are shown in Table 5, and the results show that each inactivated vaccine grows aseptically, which indicates that each vaccine is qualified in sterility test.
TABLE 5 sterility test results
The safety inspection results of target animals are shown in table 6, the results show that 0.5mL of vaccine is injected subcutaneously into the mice, the mice are all healthy and alive, the safety and reliability of the vaccine are shown, the results of different vaccines injected intramuscularly into the weaned pigs are shown in table 5, the weaned pigs are all healthy and alive, the body temperature and mental diet are normal, the local part of the suckling pig injected with the β -propiolactone inactivated vaccine has no adverse reaction, and after killing by dissection, each organ has no abnormal lesion, and after the formaldehyde inactivated vaccine is injected, 2 weaned pigs have erythema and subcutaneous nodules and contact allergy at the injection part, the safety of the β -propiolactone inactivated vaccine is shown to be good, so the vaccine for efficacy inspection is inactivated by β -propiolactone.
TABLE 6 target animal safety test for different inactivator vaccines
The results of injecting the vaccine with double adjuvant to the weaned piglets are shown in table 7, and the results show that the vaccine with nano aluminum as adjuvant has no adverse reaction on the local part, and after killing, each organ has no abnormal lesion, and 2 weaned piglets injected with the conventional aluminum hydroxide gel adjuvant vaccine have red rash and subcutaneous nodules, which indicates that the vaccine with nano aluminum as adjuvant has better absorption and no adverse reaction.
Table 7 target animal safety test for different adjuvants of vaccines
Example 4 potency test for triple inactivated vaccine
1 materials and methods
Materials: vaccine 1 and vaccine 2
The method comprises the following steps: healthy and susceptible 21-day-old weaned piglets of 75 are selected and randomly divided into 3 groups, 2 immunization groups and 1 control group, and each group has 25 piglets. The immunization group is characterized in that 2mL of vaccine 1 and vaccine 2 are respectively injected intramuscularly in an immunization group, 1 injection dose is contained, secondary immunization is carried out according to the same operation after 21 days, 14 days after secondary immunization, strain of haemophilus suis H23 strain (HPS-4), haemophilus parasuis H24 strain (HPS-5), streptococcus suis type 2S 23 strain (SS-2), pasteurella multocida P13 strain (Pm-A) and pasteurella multocida P11 strain (Pm-B) are respectively used for virus challenge, and statistical results are observed for 14 days after virus challenge. The immune group weaned piglets are protected by more than 4/5, and the control group weaned piglets are attacked by more than 4/5, so that the immunity is judged to be qualified.
2 results
The result of the virus attack of each strain is shown in Table 8, and the results show that the vaccine 1 and the vaccine 2 show that the immune group weaned piglets have protection above 4/5 after being attacked by the haemophilus parasuis H23 strain, the haemophilus parasuis H24 strain, the streptococcus suis type 2S 23 strain, the pasteurella multocida P13 strain and the pasteurella multocida P11 strain, the control group of weaned piglets with diseases above 4/5 or death shows that the vaccine 1 and the vaccine 2 are qualified in the tests of the efficacy of haemophilus parasuis type 4 and 5, streptococcus suis type 2, pasteurella multocida type A and B strains, wherein the vaccine 2 adopting the nano aluminum adjuvant achieves 5/5 protection (100 percent) on the efficacy test of the five strains, the vaccine 1 adopting the conventional aluminum hydroxide adjuvant achieves 4/5 protection (80%) on the efficacy test of the five strains, which shows that the vaccine adopting the nano aluminum adjuvant has stronger effect than the vaccine adopting the conventional aluminum hydroxide adjuvant.
TABLE 8 vaccine efficacy test results
Example 5 comparison of efficacy tests of triple inactivated vaccine against Haemophilus parasuis, Streptococcus suis, and Pasteurella multocida with Single inactivated vaccine and Dual inactivated vaccine
1. Materials: vaccine 2, vaccine 3, vaccine 4, vaccine 5, vaccine 6, vaccine 7 and vaccine 8
2. Method of producing a composite material
Selecting healthy and susceptible 21-day-old weaned piglets, carrying out intramuscular injection of 2mL of vaccine for the immunization group prime immunization respectively, containing 1 injection dose, carrying out secondary immunization according to the same operation after 21 days, and carrying out virus challenge by using various virulent strains of haemophilus parasuis type 4 and 5, streptococcus suis type 2, and pasteurella multocida types A and B14 days after secondary immunization; the statistics of 14 days after challenge were observed, and the experimental groups and challenge strains are shown in Table 9.
TABLE 9 Experimental groups and challenge strains
3. Results
The results of the efficacy test comparison of the haemophilus parasuis, streptococcus suis and pasteurella multocida triple inactivated vaccines and the single-combined inactivated vaccines and the double inactivated vaccines are shown in the table 10, and the results show that the triple inactivated vaccines, the single-combined inactivated vaccines and the double inactivated vaccines have no obvious difference in the immune protection rate of weaned pigs when the haemophilus parasuis type 4, streptococcus suis type 2, pasteurella multocida type A and B strains are subjected to strong virus challenge, but the triple inactivated vaccines are superior to the single-combined inactivated vaccines and the double inactivated vaccines in the immune protection rate of the weaned pigs when the haemophilus parasuis type 5 and pasteurella multocida B strains are subjected to strong virus challenge; in the toxin attacking parts of the haemophilus parasuis type 5 and the swine pasteurella multocida type B strains, the immune protection rates of the triple inactivated vaccines to the weaned pigs are 10/10, and the immune protection rates of the bivalent inactivated vaccines to the weaned pigs are 9/10, so that the triple inactivated vaccines are superior to the bivalent inactivated vaccines and the single inactivated vaccines in the immune efficiency of the toxin attacking of the haemophilus parasuis type 5 and the swine pasteurella multocida type B strains. Compared with the inoculation dose, the triple inactivated vaccine is injected with 2mL by one needle, and the single inactivated vaccine or the double inactivated vaccine is injected with 4mL by at least 2 needles, so the triple inactivated vaccine provided by the invention is more convenient to use, and saves time and labor.
TABLE 10 Haemophilus parasuis disease, Streptococcus suis disease, Pasteurella multocida triple inactivated vaccine and single inactivated vaccine,
Potency test comparison of bivalent inactivated vaccine
According to the immune challenge test results, compared with single-combined inactivated vaccines and double-combined inactivated vaccines, the triple inactivated vaccine for haemophilus parasuis, streptococcus suis and pasteurella multocida provided by the invention can provide good immune protection for the challenge of haemophilus parasuis, streptococcus suis and pasteurella multocida, and the triple inactivated vaccine provided by the invention has a good protection effect.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A triple inactivated vaccine for haemophilus parasuis, streptococcus suis and pasteurella multocida is characterized by comprising antigen concentrate and a nano aluminum adjuvant;
the antigen concentrate comprises inactivated H23 type-4 Haemophilus parasuis serum, H24 type-5 type Haemophilus parasuis serum, S23 type-2 streptococcus suis serum, P13 type Pasteurella multocida capsule A and P11 type Pasteurella multocida capsule B antigen concentrates.
2. The triple inactivated vaccine according to claim 1, wherein the volume ratio of the antigen concentrates of the haemophilus parasuis serotype 4H 23 strain, the haemophilus parasuis serotype 5H 24 strain, the streptococcus suis serotype 2S 23 strain, the pasteurella multocida capsule type A P13 strain and the pasteurella multocida capsule type B P11 strain is 1:1:1: 1.
3. The triple inactivated vaccine according to claim 1, wherein the antigen concentrates of the H23 serotype 4 strain of Haemophilus parasuis, the H24 serotype 5 strain of Haemophilus parasuis, the S23 serotype 2 strain of Streptococcus suis, the P13 serotype A capsule of Pasteurella multocida, and the P11 serotype B capsule of Pasteurella multocida all have an antigen concentrate content of 5 x 107CFU/mL。
4. The triple inactivated vaccine according to claim 1, wherein the nano aluminum adjuvant is 10% by volume.
5. The triple inactivated vaccine according to claim 1, further comprising 1% (w/v) of thimerosal and a veterinarily acceptable vaccine diluent.
6. The method of claim 5, wherein the vaccine diluent is 0.9% NaCl solution or PBS solution.
7. The preparation method of the triple inactivated vaccine of claim 1 is characterized by comprising the following steps:
s1, respectively proliferating strains of haemophilus parasuis serotype 4H 23 strain, serotype 5H 24 strain, streptococcus suis serotype 2S 23 strain, pasteurella multocida capsular type A P13 strain and pasteurella multocida capsular type B P11 strain to respectively obtain culture solutions;
s2, adding an inactivating agent accounting for 0.1-0.15% of the total volume of the culture solution into the culture solution obtained in the step S1, inactivating the mixture at 4 ℃ for 72 hours while stirring the mixture for several times, and performing water bath at 37 ℃ for 2 hours to obtain inactivated culture solutions of H23 serotype 4, H24 serotype 5, S23 serotype 2 of streptococcus suis, Pasteurella multocida capsular type A P13 and Pasteurella multocida capsular type B P11;
s3, concentrating the culture solutions of the haemophilus parasuis inactivated in the step S2 and containing H23 strain of serotype 4, H24 strain of serotype 5, S23 strain of streptococcus suis serotype 2, P13 strain of pasteurella multocida capsular type A and P11 strain of pasteurella multocida capsular type B, and adding 0.9% NaCl solution or PBS solution to resuspend the bacteria to obtain antigen concentrates of H23 strain of haemophilus parasuis serotype 4, H24 strain of haemophilus parasuis serotype 5, S23 strain of streptococcus suis serotype 2, P13 strain of pasteurella multocida capsular type A and P11 strain of pasteurella multocida;
s4, adding the antigen concentrates of the H23 type haemophilus parasuis serum 4 strain, the H24 type haemophilus parasuis serum 5 strain, the S23 type streptococcus suis serum 2 strain, the Pasteurella multocida capsular type A P13 strain and the Pasteurella multocida capsular type B P11 strain in the step S3 into an emulsifying machine, adding the nano aluminum adjuvant, the diluent and the thimerosal according to the proportion of the formula, stirring at a low speed, and mixing to obtain the inactivated vaccine of haemophilus parasuis, Streptococcus suis and Pasteurella multocida.
8. The method for preparing the triple inactivated vaccine according to claim 7, wherein the inactivating agent in the step S2 is β -propiolactone.
9. The method for preparing the triple inactivated vaccine according to claim 7, wherein the bacteria of Haemophilus parasuis serotype 4H 23, Haemophilus parasuis serotype 5H 24, Streptococcus suis serotype 2S 23, Pasteurella multocida capsular type A P13 and Pasteurella multocida capsular type B P11 in the step S3 have a bacterial content after concentration corresponding to a viable count of 5 x 10 before inactivation9FU/mL。
10. The method for preparing the triple inactivated vaccine according to claim 7, wherein the cultivation of Haemophilus parasuis, Streptococcus suis and Pasteurella multocida is respectively subjected to sterility test, safety test and efficacy test after step S4.
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