Preparation method and application of porcine circovirus para-triplet vaccine

Abstract

The invention relates to the field of preparation of animal husbandry vaccines, in particular to a preparation method and application of a porcine circovirus parasuis triple vaccine, and the method comprises the following steps: preparing and harvesting a first bacterial solution containing inactivated haemophilus parasuis; preparing and harvesting a second bacterial liquid containing the inactivated porcine circovirus; mixing the first bacterial liquid and the second bacterial liquid to obtain a mixed bacterial liquid; adding a TS6 adjuvant, an SP-oil adjuvant, Tween-80 and aluminum stearate into the mixed bacterial liquid; the method comprises the following steps: carrying out multiplication culture on mycoplasma hyopneumoniae, inducing in a culture system, collecting thalli, carrying out wall breaking, and collecting to obtain a culture solution containing a mycoplasma hyopneumoniae culture; adding the obtained culture solution of the mycoplasma hyopneumoniae culture into the mixed bacterium solution, then adding the mixed antigen stock solution of interleukin, aluminum stearate and squalane, stirring and mixing uniformly, and emulsifying in a high-speed shearing machine for 30 minutes; and quantitatively sub-packaging and sealing at 4 DEG C for storage for later use to obtain the triple inactivated vaccine.

Description

Technical Field

[0001] The invention relates to the field of animal husbandry vaccine preparation, and in particular to a preparation method and application of a porcine roundworm triple vaccine. Background Art

[0002] Porcine circovirus, Mycoplasma hyopneumoniae, and Haemophilus parasuis are all common pathogens in pig farming, which can easily cause diseases when the body's immunity is reduced.

[0003] Porcine circovirus (PCV) belongs to the genus Circovirus of the family Circoviridae. It is a small, non-enveloped, covalently closed, circular, single-stranded DNA virus with an average virus particle diameter of 17nm, making it the smallest animal virus found in veterinary medicine. Currently, PCV can be divided into three genotypes, namely PCV-1, PCV-2 and PCV3. PCV-1 is a non-pathogenic PCV, while PCV-2 and PCV-3 are both pathogenic to pigs and can cause PMWS (multisystemic wasting syndrome in weaned piglets), also known as PMWS-related PCV.

[0004] Mycoplasma hyopneumonia (Mhp) is a pathogen of chronic respiratory infectious diseases, which can cause Mycoplasmal pneumoniae of swine (MPS), also known as "swine pancreatic disease" or "porcine enzootic pneumonia (PEP)". The disease has a high infection rate and high infectivity, causing huge economic losses to the world's pig industry. Mycoplasma hyopneumonia often co-infects with pathogens such as porcine circovirus (PCV), increasing the severity and potential persistence of the disease, aggravating respiratory diseases and leading to an increase in the mortality rate of sick pigs. Studies have shown that the specific adhesion of Mhp to the cilia of pig respiratory epithelial cells is the key to causing mycoplasma pneumonia in swine.

[0005] Haemophilus parasuis (HPS) can cause polyserositis, arthritis and meningitis in pigs, which can affect suckling piglets from 2 weeks old to fattening pigs of 4 months old. The disease mainly occurs after weaning and in the nursery stage, and is more common in pigs aged 5 to 8 weeks. The incidence rate is generally 10% to 15%, and the mortality rate is as high as 50% in severe cases. It is easy to be mixed or secondary infected with Streptococcus suis, porcine reproductive and respiratory syndrome, mycoplasma pneumonia and other diseases. According to many literature reports, Haemophilus parasuis disease is easily misdiagnosed. Therefore, in clinical practice, controlling Haemophilus parasuis disease not only controls the disease itself but also other diseases, thereby improving the overall health level of the pig herd.

[0006] At present, the vaccines used in the market are only two-in-one vaccines that prevent Mycoplasma hyopneumoniae and porcine circovirus separately, or inactivated vaccines for Haemophilus parasuis. However, this is relatively limited in terms of disease prevention. In addition, the abuse of antibiotics has led to the emergence of drug-resistant strains, making drug treatment ineffective and wasteful; the residues of antibiotics also make it difficult for meat animals to pass when exported. Therefore, the use of vaccines to prevent infection with porcine circovirus, Mycoplasma hyopneumoniae and Haemophilus parasuis is currently the most economical and effective method. Summary of the invention

[0007] Based on this, the present invention proposes a preparation method and application of a porcine circovirus and mycoplasma hyopneumoniae triple vaccine to reduce the incidence of porcine circovirus, mycoplasma hyopneumoniae, and Haemophilus parasuis in pig farms and improve the survival efficiency of piglets.

[0008] According to one aspect of the present invention, a method for preparing a triple vaccine of porcine roundworm and its application is provided, comprising:

[0009] preparing and harvesting a first bacterial liquid containing inactivated Haemophilus parasuis;

[0010] preparing and harvesting a second bacterial liquid containing inactivated porcine circovirus;

[0011] The inactivated porcine circovirus in the second bacterial solution is obtained by inactivating the porcine circovirus by using diethyleneimine and formaldehyde in combination;

[0012] Mixing the first bacterial liquid and the second bacterial liquid to obtain a mixed bacterial liquid;

[0013] Adding TS6 adjuvant and SP-oil adjuvant, Tween-80 and aluminum stearate to the mixed bacterial solution;

[0014] The Mycoplasma hyopneumoniae is taken for proliferation culture, and then induced in the culture system, and then the bacteria are collected, broken, and the culture fluid containing the Mycoplasma hyopneumoniae culture is collected;

[0015] The inclusion bodies were resuspended in urea lysis buffer, the supernatant was collected, and then Ni 2+ Purify the protein by column, renature the protein, and then filter and sterilize to obtain the culture fluid of Mycoplasma hyopneumoniae culture;

[0016] The obtained culture solution of Mycoplasma hyopneumoniae culture is added to the mixed bacterial solution, and then interleukin, aluminum stearate and squalane mixed antigen stock solution are added and stirred and mixed, and the mixture is placed in a high-speed shearing machine, stirred at 300 rpm for 10 minutes, and then emulsified at 3000 rpm for 30 minutes;

[0017] The mixture is quantitatively divided and sealed and stored at 4°C for later use to obtain the triple inactivated vaccine.

[0018] Furthermore, the first bacterial liquid containing inactivated Haemophilus parasuis is obtained by fermenting with culture medium and a fermenter, and then concentrating and inactivating the culture medium.

[0019] Furthermore, the culturing process of the Haemophilus parasuis in the first bacterial liquid includes:

[0020] Inoculate 1%-2% of the total culture medium with a strain of Haemophilus parasuis, add 1%-2% of lysed whole blood, mix well, and ferment in a fermenter at 37°C, set the dissolved oxygen content at 10%-15%, adjust the pH with sodium hydroxide, set the pH to 7.5-7.6, and ferment for 10-11 hours.

[0021] Furthermore, the inactivation step of the Haemophilus parasuis in the first bacterial liquid comprises: inactivating the first bacterial liquid after clarifying and concentrating the first bacterial liquid using a hollow fiber filter column, adding 0.3%-0.4% formaldehyde solution to the first bacterial liquid after concentration, inactivating the first bacterial liquid at 40° C. for 10-24 hours under stirring, so that the concentration of the inactivated Haemophilus parasuis in the first bacterial liquid is ≥10 7.0 CFU / ml.

[0022] Furthermore, the culturing process of the porcine circovirus in the second bacterial liquid includes:

[0023] Inoculate the inactivated porcine circovirus strain at 1%-2% of the total volume of the culture medium, add 0.1% Tween-80 and 0.5% glucose, mix well, and ferment in a fermenter at 37°C. Set the dissolved oxygen content to 10%-15%, adjust the pH with sodium hydroxide, and set the pH to 7.5-7.6. Ferment for 10-11 hours. After the porcine circovirus is multiplied and cultured, the concentration reaches 1.125×10 10 CFU / ml.

[0024] Furthermore, the concentration of diethyleneimine in the second bacterial liquid is 3-5 mmol / L, and the concentration of formaldehyde in the second bacterial liquid is 0.2-0.5 ml / L.

[0025] The second bacterial liquid is terminated and inactivated using a sodium thiosulfate solution.

[0026] Furthermore, the content of Tween-80 is 1-10% of the mass of the mixed bacterial solution;

[0027] The content of aluminum stearate is 1-3% of the mass of the mixed bacterial liquid;

[0028] The content of the TS6 adjuvant and the SP-oil adjuvant is 10 V / V%.

[0029] Further, the content of aluminum stearate is 2-6% of the mass of the Mycoplasma hyopneumoniae culture material;

[0030] The interleukin is 2-6% of the mass of the culture material of Mycoplasma hyopneumoniae;

[0031] The squalane is 4-8% of the mass of the Mycoplasma hyopneumoniae culture material.

[0032] According to a second aspect of the present invention, a porcine roundworm triple vaccine prepared by a preparation method is provided.

[0033] According to the third aspect of the present invention, a porcine circovirus-mycoplasma pneumonia triple vaccine is provided for the prevention of porcine circovirus, Mycoplasma hyopneumoniae and Haemophilus parasuis in breeding pigs and piglets in pig farms, as well as the prevention of multisystemic wasting syndrome, porcine mycoplasma pneumonia, polyserositis, arthritis and meningitis, as well as sequential infections from multiple sources of infection in weaned piglets on pig farms.

[0034] It can be seen from the above technical scheme that the preparation method and application of the porcine roundworm triple vaccine provided by the present invention have the following beneficial effects:

[0035] The vaccine prepared by the preparation method of the present invention is mainly used for the prevention of porcine circovirus, Mycoplasma hyopneumoniae and Haemophilus parasuis in breeding pigs and piglets in pig farms, reducing the incidence of multisystemic wasting syndrome, porcine mycoplasma pneumonia, polyserositis, arthritis and meningitis in weaned piglets, as well as the incidence of successive infections from multiple infection sources, and improving the survival efficiency of piglets.

[0036] The advantages of the present invention are that diseases caused by porcine circovirus, Mycoplasma hyopneumoniae, and Haemophilus parasuis can be prevented at the same time, and a synergistic immune effect can be induced through the combination of multiple immunogens to produce a specific immune effect, thereby reducing the frequency of injections and changing the situation where at least two injections are required to prevent the above diseases. The immunization cost is reduced and the stress response of animals is reduced. The triple inactivated vaccine has a high potency content, and immunization is convenient and quick, thus avoiding the adverse reactions of multiple vaccinations and reducing breeding costs.

[0037] The present invention can use different types of porcine circovirus or porcine Haemophilus parasuis for mixed culture, and the immune protection effect for the same serotype can reach more than 80%. Compared with the current vaccines, a single injection can be used to immunize more serotypes, achieving the purpose of "one injection to prevent multiple diseases". It is easy to use, reduces pig stress, and does not have the hidden danger of spreading the virus. It is safe and reliable.

[0038] The present invention adopts a variety of adjuvants, including oily adjuvants and aqueous adjuvants, to reduce the side effects of redness, swelling and fever at the immunization site after immunization, will not affect the spirit and feeding conditions of the pigs after immunization, and effectively improves the immune effect and production performance of the immunized pigs. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0040] At present, the only vaccines used in the market are the two-in-one vaccine that prevents Mycoplasma hyopneumoniae and porcine circovirus separately, or the inactivated vaccine for Haemophilus parasuis. However, this is relatively limited in terms of disease prevention. In addition, the abuse of antibiotics has led to the emergence of drug-resistant strains, making drug treatment ineffective and wasteful; the residues of antibiotics also make it difficult for meat animals to pass when exported. Therefore, the use of vaccines to prevent infection with porcine circovirus, Mycoplasma hyopneumoniae and Haemophilus parasuis is currently the most economical and effective method.

[0041] Therefore, it is urgent to invent a new triple inactivated vaccine that can simultaneously prevent diseases caused by porcine circovirus, Mycoplasma hyopneumoniae, and Haemophilus parasuis to solve the above problems.

[0042] In view of some existing problems, the present invention provides a preparation method and application of a porcine roundworm triple vaccine, comprising:

[0043] preparing and harvesting a first bacterial liquid containing inactivated Haemophilus parasuis;

[0044] preparing and harvesting a second bacterial liquid containing inactivated porcine circovirus;

[0045] The inactivated porcine circovirus in the second bacterial solution is obtained by inactivating the porcine circovirus by using diethyleneimine and formaldehyde in combination;

[0046] mixing the first bacterial liquid with the second bacterial liquid to obtain a mixed bacterial liquid;

[0047] Add TS6 adjuvant and SP-oil adjuvant, Tween-80 and aluminum stearate to the mixed bacterial solution;

[0048] The Mycoplasma hyopneumoniae is taken for proliferation culture, and then induced in the culture system, and then the bacteria are collected, broken, and the culture fluid containing the Mycoplasma hyopneumoniae culture is collected;

[0049] The inclusion bodies were resuspended in urea lysis buffer, the supernatant was collected, and then Ni 2+ Purify the protein by column, renature the protein, and then filter and sterilize to obtain the culture fluid of Mycoplasma hyopneumoniae culture;

[0050] The obtained culture solution of Mycoplasma hyopneumoniae culture was added to the mixed bacterial solution, and then interleukin, aluminum stearate and squalane mixed antigen stock solution were added and stirred and mixed, and the mixture was placed in a high-speed shearing machine and stirred at 300 rpm for 10 minutes, and then emulsified at 3000 rpm for 30 minutes;

[0051] The mixture is quantitatively divided and sealed and stored at 4°C for later use to obtain the triple inactivated vaccine.

[0052] The vaccine prepared by the preparation method of the present invention is mainly used for the prevention of porcine circovirus, Mycoplasma hyopneumoniae and Haemophilus parasuis in breeding pigs and piglets in pig farms, reducing the incidence of multisystemic wasting syndrome, porcine mycoplasma pneumonia, polyserositis, arthritis and meningitis in weaned piglets, as well as the incidence of successive infections from multiple infection sources, and improving the survival efficiency of piglets.

[0053] The advantages of the present invention are that diseases caused by porcine circovirus, Mycoplasma hyopneumoniae, and Haemophilus parasuis can be prevented at the same time, and a synergistic immune effect can be induced through the combination of multiple immunogens to produce a specific immune effect, thereby reducing the frequency of injections and changing the situation where at least two injections are required to prevent the above diseases. The immunization cost is reduced and the stress response of animals is reduced. The triple inactivated vaccine has a high potency content, and immunization is convenient and quick, thus avoiding the adverse reactions of multiple vaccinations and reducing breeding costs.

[0054] The present invention can use different types of porcine circovirus or porcine Haemophilus parasuis for mixed culture, and the immune protection effect for the same serotype can reach more than 80%. Compared with the current vaccines, a single injection can be used to immunize more serotypes, achieving the purpose of "one injection to prevent multiple diseases". It is easy to use, reduces pig stress, and does not have the hidden danger of spreading the virus. It is safe and reliable.

[0055] Furthermore, the first bacterial liquid containing inactivated Haemophilus parasuis is fermented in a culture medium and a fermenter, and then concentrated and inactivated to obtain the first bacterial liquid.

[0056] Furthermore, the culturing process of Haemophilus parasuis in the first bacterial liquid includes:

[0057] Inoculate 1%-2% of the total culture medium with a strain of Haemophilus parasuis, add 1%-2% of lysed whole blood, mix well, and ferment in a fermenter at 37°C, set the dissolved oxygen content at 10%-15%, adjust the pH with sodium hydroxide, set the pH to 7.5-7.6, and ferment for 10-11 hours.

[0058] Furthermore, the inactivation step of Haemophilus parasuis in the first bacterial solution includes: inactivating the first bacterial solution after clarifying and concentrating the first bacterial solution using a hollow fiber filter column, adding 0.3%-0.4% formaldehyde solution to the first bacterial solution after concentration, inactivating the solution at 40° C. for 10-24 hours under stirring, and making the concentration of the inactivated Haemophilus parasuis in the first bacterial solution ≥

[0059] 10 7. 0CFU / ml.

[0060] Furthermore, the culture process of porcine circovirus in the second bacterial liquid includes:

[0061] Inoculate the inactivated porcine circovirus strain at 1%-2% of the total volume of the culture medium, add 0.1% Tween-80 and 0.5% glucose, mix well, and ferment in a fermenter at 37°C. Set the dissolved oxygen content to 10%-15%, adjust the pH with sodium hydroxide, and set the pH to 7.5-7.6. Ferment for 10-11 hours. After the porcine circovirus is multiplied and cultured, the concentration reaches 1.125×10 10 CFU / ml.

[0062] Furthermore, the concentration of diethyleneimine in the second bacterial solution is 3-5 mmol / L, and the concentration of formaldehyde in the second bacterial solution is 0.2-0.5 ml / L.

[0063] The second bacterial liquid was inactivated by using sodium thiosulfate solution.

[0064] Furthermore, the first bacterial liquid and the second bacterial liquid can also be expanded and cultured in a primary bioreactor and a secondary bioreactor.

[0065] Furthermore, the culture method of Mycoplasma hyopneumoniae adopts commonly used culture technology.

[0066] Furthermore, the content of Tween-80 is 1-10% of the mass of the mixed bacterial solution;

[0067] The content of aluminum stearate is 1-3% of the mass of the mixed bacterial liquid;

[0068] The content of TS6 adjuvant and SP-oil adjuvant was 10 V / V%.

[0069] Further, the content of aluminum stearate is 2-6% of the mass of the Mycoplasma hyopneumoniae culture material;

[0070] Interleukin is 2-6% of the mass of the culture of Mycoplasma hyopneumoniae;

[0071] Squalane is 4-8% by mass of the culture medium of Mycoplasma hyopneumoniae.

[0072] Furthermore, porcine circovirus, Mycoplasma hyopneumoniae, and Haemophilus parasuis can be concentrated using hollow fiber membranes after cultivation.

[0073] The present invention adopts a variety of adjuvants, including oily adjuvants and aqueous adjuvants, wherein interleukin is a cytokine adjuvant, aluminum stearate is an aqueous adjuvant, and the others are all oily adjuvants, and squalane can also effectively alleviate the discomfort of the immunization site. The side effects of redness, swelling and fever at the immunization site after immunization can be reduced, and the spirit and feeding of the pigs after immunization will not be affected, and the immune effect and production performance of the immunized pigs are effectively improved. Aluminum stearate can also play the role of a suspending agent, enhancing the stability of the vaccine.

[0074] Furthermore, a porcine roundworm triple vaccine prepared by a preparation method is provided.

[0075] The advantages of the present invention are that diseases caused by porcine circovirus, Mycoplasma hyopneumoniae, and Haemophilus parasuis can be prevented at the same time, and a synergistic immune effect can be induced through the combination of multiple immunogens to produce a specific immune effect, thereby reducing the frequency of injections and changing the situation where at least two injections are required to prevent the above diseases. The immunization cost is reduced and the stress response of animals is reduced. The triple inactivated vaccine has a high potency content, and immunization is convenient and quick, thus avoiding the adverse reactions of multiple vaccinations and reducing breeding costs.

[0076] The present invention can use different types of porcine circovirus or porcine Haemophilus parasuis for mixed culture, and the immune protection effect for the same serotype can reach more than 80%. Compared with the current vaccines, a single injection can be used to immunize more serotypes, achieving the purpose of "one injection to prevent multiple diseases". It is easy to use, reduces pig stress, and does not have the hidden danger of spreading the virus. It is safe and reliable.

[0077] Furthermore, a porcine circovirus-mycoplasma pneumonia triple vaccine is provided for the prevention of porcine circovirus, Mycoplasma hyopneumoniae, and Haemophilus parasuis in breeding pigs and piglets in pig farms, as well as the prevention of multisystemic wasting syndrome, porcine mycoplasma pneumonia, polyserositis, arthritis and meningitis in weaned piglets, as well as sequential infections from multiple sources of infection.

[0078] The advantages of the present invention are that diseases caused by porcine circovirus, Mycoplasma hyopneumoniae, and Haemophilus parasuis can be prevented at the same time, and a synergistic immune effect can be induced through the combination of multiple immunogens to produce a specific immune effect, thereby reducing the frequency of injections and changing the situation where at least two injections are required to prevent the above diseases. The immunization cost is reduced and the stress response of animals is reduced. The triple inactivated vaccine has a high potency content, and immunization is convenient and quick, thus avoiding the adverse reactions of multiple vaccinations and reducing breeding costs.

[0079] The present invention can use different types of porcine circovirus or porcine Haemophilus parasuis for mixed culture, and the immune protection effect for the same serotype can reach more than 80%. Compared with the current vaccines, a single injection can be used to immunize more serotypes, achieving the purpose of "one injection to prevent multiple diseases". It is easy to use, reduces pig stress, and does not have the hidden danger of spreading the virus. It is safe and reliable.

[0080] The technical solution of the present invention is described in detail below through preferred embodiments. It should be noted that the specific embodiments below are only used for illustration and are not used to limit the present invention.

[0081] Example 1: Preparation of samples.

[0082] Take Haemophilus parasuis and inoculate the strain at 1%-2% of the total amount of the culture medium, add lysed blood cells at 1%-2%, mix well, ferment in a fermenter at 37°C, set the dissolved oxygen content at 10%-15%, adjust the pH with sodium hydroxide, set the pH to 7.5-7.6, ferment for 10-11 hours, perform proliferation culture, and harvest a first bacterial liquid containing Haemophilus parasuis, wherein the concentration of the first bacterial liquid is ≥10 7. 0CFU / ml;

[0083] The first bacterial solution is clarified and concentrated by a hollow fiber filter column, and then inactivated by adding a 0.3% to 0.4% formaldehyde solution to the first bacterial solution after the concentration treatment, and inactivating the solution at 40° C. for 10 to 24 hours under stirring;

[0084] After washing, a first bacterial liquid containing inactivated Haemophilus parasuis is separated and obtained;

[0085] Inoculate the strain at 1%-2% of the total volume of the culture medium, add 0.1% Tween-80 and 0.5% glucose, mix well, and ferment in a fermenter at 37°C. Set the dissolved oxygen content to 10%-15%, adjust the pH with sodium hydroxide, and set the pH to 7.5-7.6. Ferment for 10-11 hours. After the porcine circovirus is multiplied and cultured, the concentration reaches 1.125×10 10 CFU / ml.

[0086] The inactivated porcine circovirus in the second bacterial solution is inactivated by combining diethyleneimine and formaldehyde, wherein the concentration of diethyleneimine in the second bacterial solution is 3-5mmol / L, and the concentration of formaldehyde in the second bacterial solution is 0.2-0.5ml / L, and sodium thiosulfate solution is used for termination inactivation.

[0087] TS6 adjuvant and SP-oil adjuvant were added to the concentrate, the adjuvant content was 10 V / V%, as well as Tween-80 and part of aluminum stearate.

[0088] The Mycoplasma hyopneumoniae is taken for proliferation culture, and then induced in the culture system, and then the bacteria are collected, the cell wall is broken, and the culture fluid containing the Mycoplasma hyopneumoniae culture is collected.

[0089] The inclusion bodies were resuspended in urea lysis buffer, the supernatant was collected, and then Ni 2+ Purify the protein by column, renature the protein, and then filter and sterilize to obtain the culture fluid of Mycoplasma hyopneumoniae culture;

[0090] The culture fluid of the Mycoplasma hyopneumoniae culture was added to the mixed solution, and then the interleukin, aluminum stearate and squalane mixed antigen stock solution were added and stirred and mixed, and then placed in a high-speed shearing machine, stirred at 300 rpm for 10 minutes, and then emulsified at 3000 rpm for 30 minutes. The mixture was quantitatively divided and sealed and stored at 4°C for later use, and the triple inactivated vaccine was obtained.

[0091] Sample 1:

[0092] The triple inactivated vaccine was prepared by the above method, except that 50 parts of a first bacterial liquid containing inactivated Haemophilus parasuis; 60 parts of a culture medium of Mycoplasma hyopneumoniae; 40 parts of a second bacterial liquid containing inactivated porcine circovirus; 10 parts of Tween-80; 6 parts of aluminum stearate; 3 parts of interleukin; and 4 parts of squalane were used as sample 1.

[0093] Sample 2:

[0094] The above method is used to prepare a bivalent inactivated vaccine, except that the method uses 30 parts of a first bacterial liquid containing inactivated Haemophilus parasuis, 40 parts of a culture medium of Mycoplasma hyopneumoniae, 30 parts of a second bacterial liquid containing inactivated porcine circovirus, 1 part of Tween-80, 2 parts of aluminum stearate, 1 part of interleukin, and 2 parts of squalane as sample 2.

[0095] Sample 3:

[0096] The above method was used to prepare a bivalent inactivated vaccine, except that 40 parts of a first bacterial liquid containing inactivated Haemophilus parasuis; 50 parts of a culture medium of Mycoplasma hyopneumoniae; 35 parts of a second bacterial liquid containing inactivated porcine circovirus; 5 parts of Tween-80; 4 parts of aluminum stearate; 2 parts of interleukin; and 3 parts of squalane were used as sample 3.

[0097] Sample 4:

[0098] The above method was used to prepare a bivalent inactivated vaccine, except that 42 parts of a first bacterial liquid containing inactivated Haemophilus parasuis; 46 parts of a culture medium of Mycoplasma hyopneumoniae; 33 parts of a second bacterial liquid containing inactivated porcine circovirus; 4 parts of Tween-80; 4 parts of aluminum stearate; 2 parts of interleukin; and 4 parts of squalane were used as sample 4.

[0099] Control group 1:

[0100] A commercially available bivalent vaccine for porcine circovirus and Mycoplasma hyopneumoniae.

[0101] Control group 2:

[0102] Commercially available Haemophilus parasuis vaccines.

[0103] Embodiment 1:

[0104] 35 healthy weaned piglets aged 28-35 days were divided into 7 groups, each with 5 piglets, each of which was intramuscularly injected with 2 ml of the vaccine prepared in Example 1, containing 1 dosage, and the second immunization was performed 21 days after the first immunization; the remaining 5 piglets were used as a blank control group without immunization. After immunization, the body temperature of the animals was measured and the clinical manifestations were observed.

[0105] 14 days after the second vaccination, porcine circovirus type 2 was challenged intravenously, and Haemophilus parasuis was challenged intraperitoneally. 2.0×106 CFU porcine circovirus type 2 and 7.0×109 CFU Haemophilus parasuis 4-MD03220 strain were used respectively. After the challenge, the clinical symptoms and mortality were observed, and the protection rate of each batch of vaccine was calculated.

[0106] 1. Body temperature after vaccination

[0107] The vaccinated pigs had no obvious temperature response, normal appetite and spirit, no other visible clinical reactions, and no inflammatory reactions such as swelling at the injection site (see Table 1).

[0108] Table 1: Average body temperature changes of piglets (28-35 days old) after vaccination

[0109]

[0110] As shown in Table 1, the observation results after the vaccine was administered to healthy weaned piglets aged 28 to 35 days with 2 ml of the vaccine showed that, except for the temperature in experimental group 2, which increased and returned to normal levels after 1 day, the rest of the vaccinated pigs did not show an increase in body temperature, and the average temperature increase did not exceed 1°C. In addition, all piglets showed normal appetite and spirit after vaccination, with no other visible clinical reactions.

[0111] 2. Vaccine immune efficacy determination:

[0112] Fourteen days after the second vaccination, the piglets were given 2.0×106 CFU porcine circovirus type 2 and 7.0×109 CFU of Haemophilus parasuis type 4 (MD03220), respectively. The results are shown in Table 2.

[0113] Table 2: Animal experiment results (%)

[0114]

[0115]

[0116] The vaccine is mainly used for preventing pig breeders and piglets from porcine circovirus, mycoplasma hyopneumoniae and Haemophilus parasuis in pig farms, reducing the incidence of multisystemic wasting syndrome, mycoplasma pneumonia, polyserositis, arthritis and meningitis in weaned piglets, and multiple infection sources, and improving the survival efficiency of piglets. The advantages of the present invention are that it can prevent diseases caused by porcine circovirus, mycoplasma hyopneumoniae and Haemophilus parasuis at the same time, and different types of porcine circovirus, mycoplasma hyopneumoniae and Haemophilus parasuis can be mixed and cultured, and the immune protection effect for the same serotype reaches more than 80%. Compared with the current vaccine, more serotypes can be immunized with a single injection, achieving the purpose of "one injection to prevent multiple diseases", and it is easy to use, reduces the stress of pigs, and there is no hidden danger of spreading the virus, which is safe and reliable.

[0117] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a porcine roundworm triple vaccine and its application, characterized in that: include: preparing and harvesting a first bacterial liquid containing inactivated Haemophilus parasuis; preparing and harvesting a second bacterial liquid containing inactivated porcine circovirus; The inactivated porcine circovirus in the second bacterial solution is obtained by inactivating the porcine circovirus by using diethyleneimine and formaldehyde in combination; Mixing the first bacterial liquid and the second bacterial liquid to obtain a mixed bacterial liquid; Adding TS6 adjuvant and SP-oil adjuvant, Tween-80 and aluminum stearate to the mixed bacterial solution; The Mycoplasma hyopneumoniae is taken for proliferation culture, and then induced in the culture system, and then the bacteria are collected, broken, and the culture fluid containing the Mycoplasma hyopneumoniae culture is collected; The inclusion bodies were resuspended in urea lysis buffer, the supernatant was collected, and then Ni 2+ Purify the protein by column, renature the protein, and then filter and sterilize to obtain the culture fluid of Mycoplasma hyopneumoniae culture; The obtained culture solution of Mycoplasma hyopneumoniae culture is added to the mixed bacterial solution, and then interleukin, aluminum stearate and squalane mixed antigen stock solution are added and stirred and mixed, and the mixture is placed in a high-speed shearing machine, stirred at 300 rpm for 10 minutes, and then emulsified at 3000 rpm for 30 minutes; The mixture is quantitatively divided and sealed and stored at 4°C for later use to obtain the triple inactivated vaccine.

2. The triple vaccine according to claim 1, characterized in that The first bacterial liquid containing inactivated Haemophilus parasuis is obtained by fermenting with culture liquid and a fermentation tank, and then concentrating and inactivating the culture liquid.

3. The triple vaccine according to claim 2, characterized in that: The culturing process of the Haemophilus parasuis in the first bacterial liquid comprises: Inoculate 1%-2% of the total culture medium with a strain of Haemophilus parasuis, add 1%-2% of lysed whole blood, mix well, and ferment in a fermenter at 37°C, set the dissolved oxygen content at 10%-15%, adjust the pH with sodium hydroxide, set the pH to 7.5-7.6, and ferment for 10-11 hours.

4. The triple vaccine according to claim 2, characterized in that The inactivation step of the Haemophilus parasuis in the first bacterial liquid comprises: inactivating the first bacterial liquid after clarifying and concentrating the first bacterial liquid using a hollow fiber filter column, adding 0.3%-0.4% formaldehyde solution to the first bacterial liquid after concentration, inactivating the first bacterial liquid at 40° C. for 10-24 hours under stirring, so that the concentration of the inactivated Haemophilus parasuis in the first bacterial liquid is ≥10 7.0 CFU / ml.

5. The triple vaccine according to claim 1, characterized in that: The culturing process of the porcine circovirus in the second bacterial liquid includes: Inoculate the inactivated porcine circovirus strain at 1%-2% of the total volume of the culture medium, add 0.1% Tween-80 and 0.5% glucose, mix well, and ferment in a fermenter at 37°C. Set the dissolved oxygen content to 10%-15%, adjust the pH with sodium hydroxide, and set the pH to 7.5-7.

6. Ferment for 10-11 hours. After the porcine circovirus is multiplied and cultured, the concentration reaches 1.125×10 10 CFU / ml.

6. The triple vaccine according to claim 1, characterized in that The concentration of diethyleneimine in the second bacterial solution is 3-5 mmol / L, and the concentration of formaldehyde in the second bacterial solution is 0.2-0.5 ml / L. The second bacterial liquid is terminated and inactivated using a sodium thiosulfate solution.

7. The triple vaccine according to claim 1, characterized in that The content of Tween-80 is 1-10% of the mass of the mixed bacterial liquid; The content of aluminum stearate is 1-3% of the mass of the mixed bacterial liquid; The content of the TS6 adjuvant and the SP-oil adjuvant is 10 V / V%.

8. The triple vaccine according to claim 1, characterized in that The content of aluminum stearate is 2-6% of the mass of the Mycoplasma hyopneumoniae culture material; The interleukin is 2-6% of the mass of the culture material of Mycoplasma hyopneumoniae; The squalane is 4-8% of the mass of the Mycoplasma hyopneumoniae culture material.

9. A porcine roundworm triple vaccine prepared by the preparation method described in claims 1-8.

10. A triple vaccine of porcine circovirus, mycoplasma hyopneumoniae and Haemophilus parasuis obtained according to claim 9 for the prevention of porcine circovirus, mycoplasma hyopneumoniae and Haemophilus parasuis in breeding pigs and piglets in pig farms, as well as for the prevention of multisystemic wasting syndrome, porcine mycoplasma pneumonia, polyserositis, arthritis and meningitis in weaned piglets, and successive infections from multiple sources of infection.

Images