WO2016119078A1 - 副猪嗜血杆菌lc株与lz-20100109株的联合应用 - Google Patents

副猪嗜血杆菌lc株与lz-20100109株的联合应用 Download PDF

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WO2016119078A1
WO2016119078A1 PCT/CN2015/000058 CN2015000058W WO2016119078A1 WO 2016119078 A1 WO2016119078 A1 WO 2016119078A1 CN 2015000058 W CN2015000058 W CN 2015000058W WO 2016119078 A1 WO2016119078 A1 WO 2016119078A1
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strain
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haemophilus parasuis
vaccine
protection
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PCT/CN2015/000058
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吴家强
张玉玉
于江
王可
杜以军
孙文博
李俊
王金宝
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山东省农业科学院畜牧兽医研究所
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Priority to PCT/CN2015/000058 priority Critical patent/WO2016119078A1/zh
Priority to US14/917,011 priority patent/US20180353592A1/en
Publication of WO2016119078A1 publication Critical patent/WO2016119078A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/116—Polyvalent bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0208—Specific bacteria not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the invention relates to the field of vaccines for Haemophilus parasuis in veterinary biological products, in particular to the combined application of Haemophilus parasuis L strain and LZ-20100109 strain in preparing bivalent inactivated vaccine.
  • Haemophilus parasuis is a NAD (Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide, coenzyme I)-dependent, non-moving Gram-negative bacillus, belonging to Pasteurella Pastteurellacea Haemophilus.
  • HPS often causes multiple serositis, arthritis, and meningitis in pigs, and the disease caused by the bacteria is also called porcine Gram's disease.
  • porcine Gram's disease According to the survey, HPS can affect young pigs from 2 weeks to 4 months old, mainly in the pre-weaning and pre-weaning stages, usually seen in pigs from 5 weeks to 8 weeks old, with an incidence rate of 40%. Up to 50%.
  • HPS can also infect sows, causing miscarriage, stillbirth or weak babies.
  • the scale and intensification of pig farms have been continuously improved, the density of pig breeding has become larger and larger, and the infection of Haemophilus parasuis has increased significantly, which has become the incidence and death of pigs in pig farms. An important reason.
  • some immunosuppressive diseases such as porcine reproductive and respiratory syndrome, porcine circovirus disease, etc. are often mixed with HPS or secondary infections, causing huge economic losses to the global pig industry, and also to Haemophilus parasuis. The diagnosis and comprehensive prevention of diseases bring more difficulties.
  • Haemophilus parasuis has become an important threat to the pig industry in China. Drug abuse has become more and more common in bacterial resistance. Vaccination with vaccine is an effective way to prevent and control Haemophilus parasuis. Due to the large number of HPS serotypes, the virulence and pathogenicity of different serotype strains are different, the immune cross protection between each serotype is very low, and the strains isolated in different places have strong local characteristics, so single strain The ability of vaccines to combat Haemophilus parasuis is very limited. In addition, domestic and foreign commercial H. parasuis vaccines cannot provide complete and effective immune protection against the disease. Exploring more cross-protective and immunogenic HPS and using it to develop efficient bivalent or multivalent H. parasuis vaccines is an urgent need for prevention and control of the disease.
  • Chinese Patent No. CN102399724A discloses a LC strain of Haemophilus parasuis, which has strong pathogenicity to pigs and has good immunogenicity, and is prepared by using the same.
  • the inactivated aluminum gel vaccine is safe and reliable, not only provides homologous attack protection, but also provides certain cross protection for heterologous attack such as serum type 4, type 5, type 10, type 12, type 14 and type 15 HPS. After immunization, it can produce strong immunity, and the morbidity and mortality of inoculated pigs are significantly reduced.
  • a Chinese invention patent with the publication number CN102851249A discloses a strain of Haemophilus parasuis LZ-20100109 and Application, it has strong pathogenicity to pigs and has good immunogenicity.
  • the inactivated aluminum gelatin vaccine prepared by it is safe and reliable, and can provide not only homologous attack protection, but also serum type 1 and type 2.
  • Heterologous attack such as type 4, type 12, type 14 and type 15 HPS provides certain cross-protection, which can produce strong immunity after immunization, and the morbidity and mortality of inoculated pigs are significantly reduced.
  • the vaccines disclosed in the above two patents do not have a good cross-protection rate for all pathogenic serotypes, such as serum type 13, which has a lower protection rate. Therefore, the search for a vaccine with good cross-protection rate for all pathogenic serotypes of Haemophilus parasuis is very necessary and has good application prospects.
  • the present invention provides Haemophilus parasuis LC strain and LZ-20100109.
  • the present invention is achieved by the following measures:
  • Haemophilus parasuis LC strain serum type 1 strain, with the accession number CGMCC No. 5257.
  • the combined application of the Haemophilus parasuis LC strain and the LZ-20100109 strain in the preparation of a bivalent inactivated vaccine against Haemophilus parasuis The H. parasuis LC strain and the LZ-20100109 strain were taken, activated and cultured in a suitable medium, and the culture was harvested, inactivated with a formaldehyde solution, and the hydrogenated alumina gel was made into Haemophilus parasuis. Inactivated vaccine.
  • the bivalent inactivated vaccine of Haemophilus parasuis is an aluminum gel vaccine.
  • the total antigen content of the Haemophilus parasuis LC strain and the LZ-20100109 strain in the aluminum gel vaccine is ⁇ 2.5 ⁇ 10 9 CFU/mL.
  • the ratio of the antigen content of the Haemophilus parasuis strain and the LZ-20100109 strain in the aluminum gel vaccine is (2:3) to (3:2).
  • the ratio of the antigen content of the Haemophilus parasuis LC strain and the LZ-20100109 strain in the aluminum gel vaccine is 2:3, 1:1 or 3:2.
  • the bivalent inactivated vaccine prepared by the combined application of Haemophilus parasuis LC strain and LZ-20100109 strain is safe and reliable, can not only provide homologous challenge protection for serum type 1 and type 5, but also serum Heterologous attack such as type 2, type 4, type 10, type 12, type 13, type 14 and type 15 HPS provides better cross protection, and the combined application of the two has obvious synergistic effect.
  • the bivalent inactivated vaccine of Haemophilus parasuis (type 1 LC strain + 5 type LZ-20100109 strain) can produce strong immunity after immunization of pigs, and the morbidity and mortality of inoculated pigs are significantly reduced. The effect is achieved or better than the market
  • the existing commercial vaccines have the advantage of competing with similar products at home and abroad, can effectively prevent the spread and spread of Haemophilus parasuis and reduce the economic losses caused by this disease, and have broad application prospects.
  • TSA medium Preparation of TSA medium: Weigh 4g TSA powder into 100mL distilled water, mix well, sterilize at 121 °C for 15min, cool to 50 °C ⁇ 60 °C, add final concentration of 0.001% NAD and 5% newborn calf serum, mix evenly After pouring the plate, spare.
  • TSB medium 3 g of TSB powder was weighed into 100 mL of distilled water and sterilized at 121 ° C for 15 min. Add 0.001% NAD and 5% newborn calf serum to the final concentration before use.
  • the HPS LC strain with the bacterial preservation number CGMCC No. 5257 and the HPS LZ-20100109 strain with the bacterial preservation number CGMCC No. 5802 were inoculated, and then inoculated with a suitable medium, and the culture was harvested and inactivated with a formaldehyde solution.
  • the hydrogenated alumina gel is made into a bivalent inactivated vaccine against Haemophilus parasuis.
  • HPS LC strain with the preservation number CGMCC No. 5257 and the HPS LZ-20100109 strain with the bacterial preservation number CGMCC No. 5802 were streaked into TSA medium and cultured at 37 °C. 24 ⁇ 36h, the activated LC strain and the LZ-20100109 strain single colony were densely streaked into TSA medium, cultured at 37 °C for 16-20 h, washed with PBS, collected the bacterial solution, and added with a final concentration of 20% glycerol. After passing the pure inspection, it is stored as a first-class seed below -20 °C. Store below -20 °C, should not exceed 1 month.
  • the secondary seed solution was inoculated into the TSB medium according to 1% of the medium amount, and an appropriate amount of defoaming agent was added to gradually increase the aeration amount.
  • the LC strain was cultured at 37 ° C for 12 hours to obtain the bacterial liquid
  • LZ- The 20100109 strain was cultured at 37 ° C for 14 h to harvest the bacterial liquid.
  • the sample is taken for pure inspection, and the inspection according to the current "Chinese Veterinary Pharmacopoeia" appendix should be pure.
  • TSA medium the live bacteria count according to the current "Chinese Veterinary Pharmacopoeia" appendix, as a reference for seedlings.
  • a final concentration of 0.3% formaldehyde solution was added to the bacterial solution by volume ratio, and inactivated at 37 ° C for 24 hours.
  • the live bacterial liquid was taken out and inoculated separately in TSA medium for inactivation test, and all should be grown aseptically.
  • the inactivated LC strain and the LZ-20100109 strain bacterial solution were mixed in an appropriate ratio, and 2 parts of the mixed bacterial solution was added to sterilized 20% aluminum hydroxide gel to prepare a seedling.
  • thiomersal was added at 0.005% of the total amount of the vaccine, and the mixture was thoroughly stirred and mixed, and then allowed to stand at 2 to 8 ° C for 1 to 2 days, and the supernatant was discarded and concentrated to about one third of the total amount.
  • the total antigen content of the vaccine reached 2.5 ⁇ 10 9 CFU/ml.
  • Quantitative dispensing capping and sealing. Stir at any time during dispensing to make it evenly mixed.
  • the preparation of the bivalent inactivated vaccine is the same as that in the first embodiment, the HPS LC strain and the LZ-20100109 inactivated bacterial solution are mixed in an appropriate ratio, so that the ratio of the antigen contents of the two strains in the vaccine is 2:3, 1:1, respectively. 3:2, but the total antigen content was 2.5 x 10 9 CFU/ml.
  • These three antigen content ratio vaccines are called bivalent inactivated vaccines I, II, and III, respectively.
  • the preparation of the monovalent inactivated vaccine is the same as that of the first embodiment. The only difference is that the bacteria content of the HPS LC strain and the LZ-20100109 inactivated bacterial solution are adjusted to about 2.5 ⁇ 10 9 CFU/ml, and then respectively 2 parts of the inactivated bacterial solution and 1 part of sterilized 20% aluminum hydroxide gel were mixed to prepare seedlings.
  • the antigen content of the finished product monovalent inactivated vaccine was 2.5 ⁇ 10 9 CFU/ml.
  • Test method for each serotype 30 healthy and susceptible piglets from 3 weeks to 4 weeks old were randomly selected and randomly divided into 6 groups and 5 groups/group.
  • One group of cervical muscles injected bivalent inactivated vaccine I2.0ml/head, one group injected bivalent inactivated vaccine II2.0ml/head, one group injected bivalent inactivated vaccine III2.0ml/head, one group injected unit price Inactivated vaccine (type 1 LC strain) 2.0 ml/head, one group injected with monovalent inactivated vaccine (type 5 LZ-20100109 strain) 2.0 ml/head, and the other group received PBS 2.0 ml/head, interval 3 weeks later The immunization was boosted once in the same way at the same dose.
  • the piglets After the attack, the piglets showed a rise in body temperature above 0.5 °C for 2 days, depression, rough hair, difficulty breathing, severe joint swelling, lameness, difficulty in standing, clinical symptoms such as neurological symptoms or death, necropsy It can be found that the pericardial effusion, severe pericardial thickening, fibrinous exudate in the heart, lung surface or abdominal cavity, joint fluid in the joint cavity of the hind limb, and even jelly-like exudate can determine the incidence of piglets. .
  • the protective effect of the bivalent inactivated vaccine and the monovalent inactivated vaccine of Haemophilus parasuis on serum type 1 is shown in Table 1.
  • the control group had 100% (5/5) of the onset of the disease; the geometric mean titer of the type 1 antibody of the pigs in the immunization group of the bivalent inactivated vaccine was ⁇ 1:32, and the protection rate was above 80% (4/5); (1 type LC strain)
  • the average geometric titer of type 1 antibody in the immunized group was ⁇ 1:32, the protection rate was above 80% (4/5), and the monovalent seedling (type 5 LZ-20100109 strain) immunized group piglet type 1
  • the geometric mean titer of the antibody was only 1:16 and the protection rate was 60% (3/5).
  • the protective effect of the bivalent inactivated vaccine and the monovalent inactivated vaccine of Haemophilus parasuis on serum type 5 is shown in Table 2.
  • the control group had 100% (5/5) of the onset of the disease; the geometric mean titer of the type 5 antibody in the immunization group of the bivalent inactivated vaccine was ⁇ 1:32, and the protection rate was above 80% (4/5); (type 5 LZ-20100109 strain) immune group pig type 1 antibody geometric mean titer ⁇ 1:32, protection rate of 80% (4/5) or more; and monovalent seedling (type 1 LC strain) immunization group piglet type 1 The geometric mean titer of the antibody was only 1:24 and the protection rate was 60% (3/5).
  • HPS isolates of HPS, 2, 4, 10, 12, 13, 14 and 15 were selected to prepare the challenge bacteria solution, and the cross-protection effect of the bivalent inactivated vaccine against Haemophilus parasuis on heterologous serotypes was evaluated. And compared with the cross-protection of monovalent inactivated vaccines.
  • the cross-protection effect of the bivalent inactivated vaccine of H. parasuis and the monovalent inactivated vaccine on the heterologous serotype is shown in Table 3. It can be seen from Table 3 that the bivalent inactivated vaccine against Haemophilus parasuis has a significant synergistic effect on the cross-protection of serum type 13 HPS, and its serotypes 4, 10, 12, 14 and 15 Type HPS has a strong cross-protection effect. Therefore, the bivalent inactivated vaccine of Haemophilus parasuis (type 1 LC strain + 5 type LZ-20100109 strain) is a vaccine with broad market application prospects and is worth promoting.
  • the cross-protection rate of the bivalent inactivated vaccine compared to the monovalent vaccine, the serotype 4, 10, 12, 14 and 15 with high cross-protection rate of the monovalent vaccine is also the cross-protection rate of the bivalent vaccine.
  • Higher, and serotypes with low cross-protection rate of monovalent vaccines, such as type 2 and type 13, bivalent vaccines also have a high cross-protection rate, especially for serum type 13 and bivalent vaccines relative to monovalent vaccines. Better, enhanced cross protection.

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Abstract

保藏编号CGMCC No.5257的副猪嗜血杆菌LC株与保藏编号CGMCC No.5802的LZ-20100109株在制备二价灭活疫苗中的联合应用。副猪嗜血杆菌LC株与LZ-20100109株联合应用制备的二价灭活疫苗安全可靠，不仅能为血清1型和5型提供同源攻毒保护，还可为血清2型、4型、10型、12型、13型、14型和15型HPS等异源攻毒提供一定的交叉保护，二者联合应用具有明显的增效作用。免疫猪体后能够产生较强免疫力，接种猪群的发病率和死亡率均明显减少，能够有效预防副猪嗜血杆菌病的流行和传播，降低经济损失，有着广阔的应用前景。

Description

副猪嗜血杆菌LC株与LZ-20100109株的联合应用 技术领域
本发明涉及兽医生物制品中副猪嗜血杆菌病疫苗领域，特别涉及副猪嗜血杆菌LC株与LZ-20100109株在制备二价灭活疫苗中的联合应用。
背景技术
副猪嗜血杆菌(Haemophilus parasuis，HPS)是一种NAD(Nicotinamide adenine dinucleotide，烟酰胺腺嘌呤二核苷酸即辅酶I)依赖型、不运动的革兰氏阴性细小杆菌，属于巴斯德菌科(Pastteurellacea)嗜血杆菌属(Haemophilus)。HPS常引起猪的多发性浆膜炎、关节炎以及脑膜炎，由该菌引起的病又称为猪革拉斯氏病。据调查，HPS可以影响从2周龄到4月龄的青年猪，主要在断奶前后和保育阶段发病，通常见于5周龄～8周龄的猪，发病率可达40％，严重时死亡率可达50％。HPS还可感染生产母猪，引起流产、产死胎或弱仔等。随着养猪业的不断发展，猪场规模化、集约化程度的不断提高，猪群饲养密度越来越大，副猪嗜血杆菌感染有明显增加趋势，已成为猪场保育猪发病和死亡的一个重要原因。另外，一些免疫抑制性疾病如猪繁殖与呼吸综合征、猪圆环病毒病等经常与HPS混合感染或继发感染，对全球的养猪业造成巨大的经济损失，也给副猪嗜血杆菌病的诊断和综合防治带来更多的难度。
近年来，副猪嗜血杆菌病已经成为我国养猪业的重要威胁，药物滥用导致细菌的耐药性越来越普遍，采用疫苗免疫是预防和控制副猪嗜血杆菌病的有效途径。由于HPS血清型众多，不同血清型菌株的毒力与致病力差异较大，各个血清型之间免疫交叉保护力很低，且不同地方分离的菌株具有较强的地方性特征，因此单一菌株疫苗抵抗副猪嗜血杆菌病的能力非常有限。另外，现有国内外的副猪嗜血杆菌商品化疫苗不能对该病提供完全有效的免疫保护。发掘更多的交叉保护性好且免疫原性强的HPS并利用其研制高效的二价或多价副猪嗜血杆菌疫苗是该病防控的迫切需要。
公开号为CN102399724A的中国专利，公开了一种一种副猪嗜血杆菌LC株，副猪嗜血杆菌LC株对猪有较强的致病性，且具有良好的免疫原性，应用其制备的灭活铝胶疫苗安全可靠，不仅能提供同源攻毒保护，还可为血清4型、5型、10型、12型、14型和15型HPS等异源攻毒提供一定的交叉保护，免疫后能够产生较强免疫力，接种猪群的发病率和死亡率均明显减少。
公开号为CN102851249A的中国发明专利，公开了一种副猪嗜血杆菌LZ-20100109株及其 应用，对猪有较强的致病性，且具有良好的免疫原性，应用其制备的灭活铝胶疫苗安全可靠，不仅能提供同源攻毒保护，还可为血清1型、2型、4型、12型、14型和15型HPS等异源攻毒提供一定的交叉保护，免疫后能够产生较强免疫力，接种猪群的发病率和死亡率均明显减少。
但上述两个专利公开的疫苗，并不是对所有致病血清型都具有好的交叉保护率，比如说血清13型，两种疫苗的保护率较低。因此，寻求一种对所有副猪嗜血杆菌致病血清型都具有好的交叉保护率的疫苗，是非常必须且具有良好应用前景的。
发明内容
为了解决以上现有国内外的副猪嗜血杆菌商品化疫苗各个血清型之间免疫交叉保护力低、免疫原性不强的问题，本发明提供了副猪嗜血杆菌LC株与LZ-20100109株在制备各个血清型之间免疫交叉保护力强的二价灭活疫苗中的联合应用。
本发明是通过以下措施实现的：
副猪嗜血杆菌(Haemophilusparasuis，)LC株，血清1型菌株，其保藏编号为CGMCC No.5257。
副猪嗜血杆菌(Haemophilus parasuis)LZ-20100109株，血清5型菌株，其保藏编号为CGMCC No.5802。
所述的副猪嗜血杆菌LC株与LZ-20100109株在制备副猪嗜血杆菌病二价灭活疫苗中的联合应用。取所述的副猪嗜血杆菌LC株与LZ-20100109株，活化后分别接种适宜培养基培养，收获培养物，用甲醛溶液灭活，加氢氧化铝胶制成副猪嗜血杆菌病二价灭活疫苗。
优选所述副猪嗜血杆菌病二价灭活疫苗为铝胶疫苗。
所述铝胶疫苗中副猪嗜血杆菌LC株与LZ-20100109株的抗原总含量≥2.5×109CFU/mL。
所述铝胶疫苗中副猪嗜血杆菌LC株与LZ-20100109株的抗原含量之比为(2∶3)～(3∶2)。
优选所述铝胶疫苗中副猪嗜血杆菌LC株与LZ-20100109株的抗原含量之比为2∶3、1∶1或3∶2。
本发明的有益效果：副猪嗜血杆菌LC株与LZ-20100109株联合应用制备的二价灭活疫苗安全可靠，不仅能为血清1型和5型提供同源攻毒保护，还可为血清2型、4型、10型、12型、13型、14型和15型HPS等异源攻毒提供较好的交叉保护，二者联合应用具有明显的增效作用。副猪嗜血杆菌病二价灭活疫苗(1型LC株+5型LZ-20100109株)免疫猪体后能够产生较强免疫力，接种猪群的发病率和死亡率均明显减少，其免疫效果达到或优于市场上 现有的商品化疫苗，具备了与国内外同类产品竞争的优势，能够有效预防副猪嗜血杆菌病的流行和传播，降低本病造成的经济损失，有着广阔的应用前景。
具体实施方式
为了更好的理解本发明，下面结合具体实施例来进一步说明。
副猪嗜血杆菌LC株，血清1型，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为CGMCC No.5257。
副猪嗜血杆菌LZ-20100109株，血清5型，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为CGMCC No.5802。
实施例1
生产用培养基
TSA培养基配制：称取4g TSA粉末加入100mL蒸馏水中，充分混匀后，121℃灭菌15min，冷却至50℃～60℃时，加入终浓度0.001％NAD和5％新生牛血清，混合均匀后倾倒平板备用。
TSB培养基配制：称取3g TSB粉末加入100mL蒸馏水中，121℃灭菌15min。临用前加入终浓度0.001％NAD和5％新生牛血清，混合均匀后配制而成。
副猪嗜血杆菌LC株与LZ-20100109株在制备副猪嗜血杆菌病二价灭活疫苗中的联合应用。
取菌种保藏编号为CGMCC No.5257的HPS LC株与菌种保藏编号为CGMCC No.5802的HPS LZ-20100109株，活化后分别接种适宜培养基培养，收获培养物，用甲醛溶液灭活，加氢氧化铝胶制成副猪嗜血杆菌病二价灭活疫苗。
优选操作步骤如下：
1.1生产用种子制备
1.1.1一级种子繁殖取菌种保藏编号为CGMCC No.5257的HPS LC株与菌种保藏编号为CGMCC No.5802的HPS LZ-20100109株，分别划线接种于TSA培养基，37℃培养24～36h，取活化后的LC株与LZ-20100109株单菌落密集划线接种于TSA培养基，37℃培养16～20h后，用PBS冲洗，收集菌液，加入终浓度为20％的甘油，经纯粹检验合格后，-20℃以下保存作为一级种子。-20℃以下保存，应不超过1个月。
1.1.2二级种子繁殖取一级种子按总体积1％的量接种于TSB培养基中，37℃180r/min振荡培养，LC株培养12～14h，LZ-20100109株培养16～18h，经纯检(镜检无杂菌污染)合格后，作为二级种子。2～8℃保存，应不超过8h。
1.2制苗菌液的制备
用发酵罐通气培养，将二级种子液按照培养基量的1％接种于TSB培养基，加入适量消泡剂，以逐渐增大通气量的方法，LC株37℃培养12h收获菌液，LZ-20100109株37℃培养14h收获菌液。菌液培养完成后取样作纯粹检验，按现行《中国兽药典》附录进行检验，应纯粹。同时用TSA培养基，按现行《中国兽药典》附录进行活菌计数，作为配苗参考。
1.3菌液灭活
按体积比向菌液中加入终浓度0.3％甲醛溶液，37℃灭活24h。取灭活菌液，分别接种于TSA培养基作灭活检验，均应无菌生长。
1.4疫苗配制
将灭活的LC株和LZ-20100109株菌液以适当的比例混合，按混合菌液2份加灭菌的20％氢氧化铝胶1份进行配苗。同时，按疫苗总量的0.005％加入硫柳汞，充分搅拌混合后2～8℃静置1～2日，抽弃上清约浓缩成全量的三分之一。疫苗的抗原总含量达到2.5×109CFU/ml。
1.5分装
定量分装，加盖密封。分装时随时搅拌，使其混合均匀。
1.6成品检验
1.6.1物理性状测定本品静置后，上层为透明清亮液体，下层为灰白色沉淀，充分振摇后，呈均匀混悬液。
1.6.2无菌检验按现行《中国兽药典》附录进行检验，应无菌生长。
1.6.3甲醛、汞类防腐剂残留量测定分别按现行《中国兽药典》附录进行测定，应符合规定。
1.6.4安全检验选取3周龄～4周龄健康易感仔猪5头，各颈部肌肉注射疫苗4mL，观察2周，接种仔猪体温、精神和食欲均正常，全部健活，且注苗局部无疫苗引起的不良反应。
1.6.5效力检验取3周龄～4周龄健康易感仔猪20头，分为4组，5头/组。其中2组仔猪各颈部肌肉注射疫苗2.0ml，另外2组仔猪各颈部肌肉注射PBS 2.0ml作为对照，间隔21日每头猪以相同剂量相同途径重复注射1次。2周后采血，分离血清，采用微量凝集试验测定血清抗体效价。免疫组仔猪血清1型抗体和血清5型抗体的几何平均效价均≥1∶32，对照组仔猪血清1型抗体和血清5型抗体的效价均≤1∶4。采血后，取免疫猪5头和对照猪5头，各腹腔注射LC株菌液2.0ml(约4.5×109CFU)，连续观察14日，期间对濒死猪进行剖检，对照组应至少4头发病或死亡，免疫组应至少保护4头；取免疫猪5头和对照猪5头，各腹腔注射LZ-20100109株菌液2.0ml(约4.5×109CFU)，连续观察14日，期间对濒死猪进行剖检，对照猪应至少4头发病或死亡，免疫猪应至少保护4头。
实施例2
副猪嗜血杆菌病二价灭活疫苗与单价灭活疫苗的效力对比试验
2.1二价灭活疫苗的制备
二价灭活疫苗的制备同实施例1，HPS LC株、LZ-20100109株灭活菌液按照适当的比例混合，使疫苗中两株菌的抗原含量之比分别为2∶3、1∶1、3∶2，但抗原总含量为2.5×109CFU/ml。这三种抗原含量比例的疫苗分别称为二价灭活疫苗I、II、III。
2.2单价灭活疫苗的制备
单价灭活疫苗的制备同实施例1，唯一不同之处是需要先将HPS LC株、LZ-20100109株灭活菌液的含菌量均调整至约2.5×109CFU/ml，然后分别按灭活菌液2份加灭菌的20％氢氧化铝胶1份进行配苗。成品单价灭活疫苗中抗原含量为2.5×109CFU/ml。
2.3同源保护检验
每一血清型的检验方法：随机选取3周龄～4周龄健康易感仔猪30头，随机分成6组，5头/组。其中1组颈部肌肉注射二价灭活疫苗I2.0ml/头，1组注射二价灭活疫苗II2.0ml/头，1组注射二价灭活疫苗III2.0ml/头，1组注射单价灭活疫苗(1型LC株)2.0ml/头，1组注射单价灭活疫苗(5型LZ-20100109株)2.0ml/头，另外1组注射PBS 2.0ml/头，间隔3周后各组以相同剂量相同途径加强免疫1次。2周后采血，分离血清，采用微量凝集试验测定血清抗体效价。采血后，分别腹腔注射LC株或LZ-20100109株菌液(约4.5×109CFU)。攻毒后，连续观察2周，记录仔猪发病与死亡情况。攻毒后，仔猪出现体温升高0.5℃以上并持续2日、精神沉郁、被毛粗乱、呼吸困难，严重者关节肿大，跛行，站立困难，出现神经症状或死亡等临床症状，剖检发现心包积液、严重者心包膜增厚、心脏、肺脏表面或腹腔内有纤维素性渗出物、后肢关节腔内关节液增多甚至有胶冻样渗出物等病变，即可判定仔猪发病。
副猪嗜血杆菌病二价灭活疫苗与单价灭活疫苗对血清1型的保护作用如表1所示。对照组仔猪均100％(5/5)发病；二价灭活疫苗免疫组仔猪1型抗体的几何平均效价均≥1∶32，保护率均在80％(4/5)以上；单价苗(1型LC株)免疫组仔猪1型抗体的几何平均效价≥1∶32，保护率在80％(4/5)以上；而单价苗(5型LZ-20100109株)免疫组仔猪1型抗体的几何平均效价仅达1∶16，保护率60％(3/5)。
表1副猪嗜血杆菌病二价灭活疫苗与单价灭活疫苗对血清1型的保护效果
Figure PCTCN2015000058-appb-000001
Figure PCTCN2015000058-appb-000002
副猪嗜血杆菌病二价灭活疫苗与单价灭活疫苗对血清5型的保护作用如表2所示。对照组仔猪均100％(5/5)发病；二价灭活疫苗免疫组仔猪5型抗体的几何平均效价均≥1∶32，保护率均在80％(4/5)以上；单价苗(5型LZ-20100109株)免疫组仔猪1型抗体的几何平均效价≥1∶32，保护率在80％(4/5)以上；而单价苗(1型LC株)免疫组仔猪1型抗体的几何平均效价仅达1∶24，保护率60％(3/5)。
表2副猪嗜血杆菌病二价灭活疫苗与单价灭活疫苗对血清5型的保护效果
Figure PCTCN2015000058-appb-000003
这些试验结果表明在副猪嗜血杆菌病二价灭活疫苗中，1型LC株与5型LZ-20100109株之间存在增效现象。
2.4交叉保护检验
选取HPS血清2、4、10、12、13、14和15型7个HPS分离株分别制备攻毒菌液，评估副猪嗜血杆菌病二价灭活疫苗对异源血清型的交叉保护作用，并与单价灭活疫苗的交叉保护作用进行比较。
每株HPS分离株的交叉保护检验方法：
随机选取3周龄～4周龄健康易感仔猪90头，分为6组，每组15头，其中3组分别颈部肌肉注射二价灭活疫苗I、II、III 2mL/头，1组注射单价灭活疫苗(1型LC株)2mL/头，1组注射单价灭活疫苗(5型LZ-20100109株)2mL/头，另1组仔猪颈部肌肉注射PBS 2.0 ml/头作为对照组，间隔3周后以同样剂量加强免疫1次。2周后，腹腔注射HPS分离株菌液进行攻毒，每株HPS分离株的攻毒剂量见表3，连续观察2周，记录仔猪发病与死亡情况。
副猪嗜血杆菌病二价灭活疫苗与单价灭活疫苗对异源血清型的交叉保护作用如表3所示。由表3可以看出，副猪嗜血杆菌病二价灭活疫苗对血清13型HPS的交叉保护具有明显的增效作用，同时其对血清4型、10型、12型、14型和15型HPS都有较强的交叉保护作用。因此副猪嗜血杆菌病二价灭活疫苗(1型LC株+5型LZ-20100109株)是一种具有广阔市场应用前景的疫苗，值得推广。
表3副猪嗜血杆菌病二价灭活疫苗与单价灭活疫苗对异源血清型的交叉保护作用
Figure PCTCN2015000058-appb-000004
Figure PCTCN2015000058-appb-000005
从上表可以看出，二价灭活疫苗相比于单价疫苗，单价疫苗交叉保护率高的血清型4型、10型、12型、14型、15型，二价疫苗的交叉保护率也较高，而对单价疫苗交叉保护率低的血清型，如2型、13型，二价疫苗也具有较高的交叉保护率，特别是对血清13型，二价疫苗相对于单价疫苗，具有更好的、增强的交叉保护效果。

Claims (5)

  1. 副猪嗜血杆菌LC株与LZ-20100109株在制备副猪嗜血杆菌病二价灭活疫苗中的联合应用，
    所述副猪嗜血杆菌LC株保藏编号为CGMCC No.5257，副猪嗜血杆菌LZ-20100109株保藏编号为CGMCC No.5802。
  2. 根据权利要求1所述的联合应用，其特征在于所述副猪嗜血杆菌病二价灭活疫苗为铝胶疫苗。
  3. 根据权利要求1或2所述的联合应用，其特征在于所述铝胶疫苗中副猪嗜血杆菌LC株与LZ-20100109株的抗原总含量≥2.5×109CFU/mL。
  4. 根据权利要求1-3这任一项所述的联合应用，其特征在于所述铝胶疫苗中副猪嗜血杆菌LC株与LZ-20100109株的抗原含量之比为(2∶3)～(3∶2)。
  5. 根据权利要求1-3这任一项所述的联合应用，其特征在于所述铝胶疫苗中副猪嗜血杆菌LC株与LZ-20100109株的抗原含量之比为2∶3、1∶1或3∶2。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106729686A (zh) * 2016-12-16 2017-05-31 青岛易邦生物工程有限公司 一种副猪嗜血杆菌四价灭活疫苗

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112831442B (zh) * 2021-02-07 2023-06-20 河南省农业科学院畜牧兽医研究所 一株具有交叉保护力的血清14型副猪嗜血杆菌及其应用
CN113018426B (zh) * 2021-02-07 2023-06-09 河南省农业科学院畜牧兽医研究所 用于制备副猪嗜血杆菌疫苗的组合菌株、八价灭活疫苗及其应用
CN113018425B (zh) * 2021-02-07 2023-09-19 河南省农业科学院畜牧兽医研究所 猪肺炎支原体与副猪嗜血杆菌二联五价灭活疫苗及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101721696A (zh) * 2009-12-18 2010-06-09 西南民族大学 猪副嗜血杆菌血清4型、5型、13型三价油乳剂灭活疫苗
CN102220272A (zh) * 2011-06-01 2011-10-19 武汉科前动物生物制品有限责任公司 一种高密度培养副猪嗜血杆菌用于疫苗制备的方法
CN102399724A (zh) * 2011-11-08 2012-04-04 山东省农业科学院畜牧兽医研究所 一种副猪嗜血杆菌lc株及其应用
CN102851249A (zh) * 2012-04-01 2013-01-02 山东省农业科学院畜牧兽医研究所 一种副猪嗜血杆菌lz-20100109株及其应用
CN102908615A (zh) * 2011-08-01 2013-02-06 普莱柯生物工程股份有限公司 新型副猪嗜血杆菌病三价灭活疫苗及制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101721696A (zh) * 2009-12-18 2010-06-09 西南民族大学 猪副嗜血杆菌血清4型、5型、13型三价油乳剂灭活疫苗
CN102220272A (zh) * 2011-06-01 2011-10-19 武汉科前动物生物制品有限责任公司 一种高密度培养副猪嗜血杆菌用于疫苗制备的方法
CN102908615A (zh) * 2011-08-01 2013-02-06 普莱柯生物工程股份有限公司 新型副猪嗜血杆菌病三价灭活疫苗及制备方法
CN102399724A (zh) * 2011-11-08 2012-04-04 山东省农业科学院畜牧兽医研究所 一种副猪嗜血杆菌lc株及其应用
CN102851249A (zh) * 2012-04-01 2013-01-02 山东省农业科学院畜牧兽医研究所 一种副猪嗜血杆菌lz-20100109株及其应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106729686A (zh) * 2016-12-16 2017-05-31 青岛易邦生物工程有限公司 一种副猪嗜血杆菌四价灭活疫苗

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