CN114805554B - Refined egg yolk antibody injection for resisting streptococcus suis and haemophilus parasuis and preparation method thereof - Google Patents

Abstract

The invention discloses a colorless, transparent, high-potency double-linked quadrivalent refined egg yolk antibody injection against Streptococcus suis and Haemophilus parasuis which is comparable to human immune proteins. The yolk antibody contains Haemophilus parasuis serotype 4, serotype 5, serotype 13 and Streptococcus suis type 2 antigens; the strain preservation number of the Haemophilus parasuis serotype 4 is CVCC NO: 3953; The preservation number of the strain of Haemophilus parasuis serotype 5 is CVCC NO: 3956; the preservation number of the strain of Haemophilus parasuis serotype 13 is CVCC NO: 3958; the preservation number of the strain of Streptococcus parasuis type 2 is For CCTCC NO: M2021457. The purity of the egg yolk antibody of the invention can reach more than 90%, the antibody titer can reach 8log2 at the highest, and the antibody level can be maintained at more than 7log2 after being stored at room temperature for one year.

Description

Refined egg yolk antibody injection against Streptococcus suis and Haemophilus parasuis and its preparation method

technical field

The invention belongs to the field of preparation and application of egg yolk antibody, in particular to a refined egg yolk antibody injection against Streptococcus suis and Haemophilus parasuis and a preparation method thereof.

Background technique

Streptococcus suis (SS) and Haemophilus parasuis (Haemophilus parasuis, HPS) are two important pathogenic bacteria that cause upper respiratory tract infectious diseases in pigs. Low power and other problems seriously affect the healthy breeding of pigs.

Streptococcus suis (SS) is Gram-positive, aerobic or facultative anaerobic bacteria, which can not only invade the blood circulation to cause pneumonia, arthritis and lymphadenitis, but also cause meningitis and maternal infection through the blood-brain barrier and placental barrier. Pig miscarriage. At the same time, Streptococcus suis can infect people through wounds and respiratory tracts, causing human joint suppurative inflammation, meningoencephalitis and endocarditis, etc., which can cause death in severe cases. It has been listed as a second-class animal epidemic in the country. There are 33 serotypes of Streptococcus suis, of which type 2 (SS2) is the most widespread and is a zoonotic disease. Haemophilus parasuis (HPS) is a short rod-shaped Gram-negative bacterium that can cause Graves' disease in pigs, which is characterized by high fever, joint swelling, respiratory disorder and central nervous system needles. Piglets under the age of 10 days are very susceptible to infection from carrier sows, and piglets in the weaning period often develop the disease due to the decline in their maternal antibody levels. Currently identified Haemophilus parasuis includes 15 serotypes, among which types 4 and 5 are the most common. There are differences in the virulence of different serotypes, among which strains 4, 5 and 13 are medium and high virulence strains, which often cause symptoms such as myocarditis, pneumonia, pleurisy, meningitis and arthritis in pigs, and pose a huge threat to the healthy breeding of pigs.

Currently, the main methods for the prevention and treatment of Streptococcus suis and Haemophilus parasuis infections are immunization and antibiotic therapy. In terms of prevention, inactivated vaccines are mainly used in China.

Patent CN101721696A discloses a trivalent oil emulsion inactivated vaccine of Haemophilus parasuis serotypes 4, 5 and 13. It uses the three main popular serotypes and isolates of Haemophilus parasuis in my country as antigens. After 24 hours of culture and propagation in TSB broth, it is inactivated with 0.3% formaldehyde for 11 to 13 hours. The antigens of the three serotypes are as follows: It is prepared by mixing in a ratio of 1:1:1, adding an oil phase equal to the total volume of the antigen, and then emulsifying. The adjuvant of the vaccine is white oil, and the side effects of this adjuvant vaccine are relatively large, which will cause inflammation or swelling at the injection site; in addition, according to research data, the use of mineral oil adjuvants will stimulate and improve the quality of pigs such as pig rings to a certain extent. incidence of disease. Moreover, in the preparation process of the vaccine, the obtained antigen has no inactivation effect judgment and related inspection after inactivation, and it is impossible to know whether the antigen inactivation is complete, and the obtained antigen cannot be used as an antigen for vaccine production.

And with the pathogenic strains and mixed infection, it is difficult for existing vaccines to achieve the expected immune effect, and the antibody protection time produced by the vaccine is short, and multiple immunizations are required to obtain strong immunity, which also greatly stresses pigs. It is also treated with sensitive antibiotics, but with the extensive use of antibiotics, drug resistance is becoming more and more serious, the treatment effect is not good, and there are antibiotic residue problems in meat products.

Contents of the invention

In order to solve the technical problems existing in the prior art, the present invention provides a refined egg yolk antibody injection against Streptococcus suis and Haemophilus parasuis and its preparation method. Comparable to immune protein, it has better safety.

Yolk antibody does not have the problems of drug residue and drug resistance. It is a safe, efficient, and pollution-free biological product that can replace antibiotics for the daily prevention and treatment of Streptococcus suis and Haemophilus parasuis.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

The first aspect of the present invention is to provide an egg yolk antibody against Streptococcus parasuis and Haemophilus parasuis, the egg yolk antibody containing Haemophilus parasuis serotype 4, serotype 5, serotype 13 and Streptococcus parasuis 2 Type antigen;

The strain preservation number of the Haemophilus parasuis serotype 4 is CVCC NO: 3953;

The strain preservation number of the Haemophilus parasuis serotype 5 is CVCC NO: 3956;

The strain preservation number of the Haemophilus parasuis serotype 13 is CVCC NO: 3958;

The preservation number of the Streptococcus suis type 2 strain is CCTCC NO: M2021457.

The term "antigen", also known as immunogen, refers to any substance that stimulates an immune response, is a molecule that can be bound by an antibody, and can also induce a humoral and/or cellular immune response to B- and/or T-cells , also has one or more epitopes (B- and T-epitopes). The antigens include killed and inactivated bacteria, or cultured cell preparations and supernatants thereof. Wherein, the killed and inactivated bacteria refer to infectious organisms or pathogens that are no longer capable of replicating or growing. treatment), sonication, irradiation, heat, or any other commonly used method sufficient to prevent the replication or growth of an organism to achieve inactivation while maintaining its immunogenicity.

Further, the double quadrivalent inactivated vaccine contains Haemophilus parasuis serotype 4, serotype 5, serotype 13, and Streptococcus suis type 2 antigen dilutions before inactivation. The number of colonies is not less than 4×10 ⁹ CFU/mL.

further,

In the yolk antibody, the inactivated antigen dilutions of Haemophilus parasuis serotype 4, serotype 5, serotype 13, and Streptococcus suis type 2 were mixed in equal volumes.

Further, the total content of the inactivated antigen dilutions of Haemophilus parasuis serotype 4, serotype 5, serotype 13 and Streptococcus suis 2 is 50% (V/V) of the total amount of the vaccine.

Further, the double quadrivalent inactivated vaccine also includes an adjuvant; the content of the adjuvant is 50% (V/V) of the total amount of the vaccine.

The "total amount of vaccine" in the present invention refers to the total amount of antigen and adjuvant.

The term "adjuvant" refers to a compound that, when administered in conjunction with an antigen, enhances a subject's immune response to that antigen.

Further, the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant; further is Freund's complete adjuvant.

The second aspect of the present invention is to provide a method for preparing egg yolk antibody as described above, comprising:

(1) preparing Haemophilus parasuis serotype 4, serotype 5, serotype 13 and Streptococcus suis type 2 antigens into a double quadrivalent inactivated vaccine;

(2) Immune laying hens with double quadrivalent inactivated vaccine and separate and extract yolk antibody in immunized eggs.

The antibody prepared by the present invention with Haemophilus parasuis serum type 4, 5, and 13 strains can produce good immune protection effects on the three serotype strains. Live vaccines do not protect well against type 13 strains, and cannot even produce immune protection.

Further, the immunization method is leg and/or chest intramuscular injection 4 times, and each immunization injection is separated by 2 weeks.

Further, when the antibody titer reached 1:128, the immunized eggs were collected, and the egg yolk antibody was separated and extracted.

The third aspect of the present invention is to provide a use of the above-mentioned yolk antibody in the preparation of products for detecting, preventing and/or treating related diseases caused by Haemophilus parasuis and Streptococcus suis infection;

The product is selected from preparations, medicines or feed additives.

The term "prevention and/or treatment" in relation to a bacterial infection refers to inhibiting the replication of the bacteria, inhibiting the spread of the bacteria or preventing the bacteria from colonizing their host, as well as alleviating the symptoms of the disease or condition caused by the bacterial infection. The treatment is considered to be therapeutically effective if there is a reduction in bacterial load, a reduction in symptoms of infection and/or an increase in food intake and/or growth.

Beneficial effect:

The present invention simultaneously realizes the treatment and prevention of Haemophilus parasuis serotypes 4, 5, 13 and 2 Streptococcus suis at the same time for the first time. The egg yolk antibody of the present invention can be used as feed addition or therapeutic biological preparation, while the existing inactivated vaccines only For immunization.

The refined egg yolk antibody injection of the present invention is colorless and transparent, has high potency, comparable to human immune protein, the purity of the antibody can reach more than 90%, and the antibody titer can reach up to 8log2, and the antibody level can be maintained at room temperature for one year. More than 7log2, it has better safety, and can achieve the effect of multiple preventions in one injection in clinical practice, reduce costs, and can meet the requirements of different users.

Description of drawings

Figure 1 is the purification and SDS-PAGE detection diagram of anti-Streptococcus suis IgY, the left side is the purified IgY; M: protein marker; 1 and 2 represent the purified anti-Streptococcus suis and Haemophilus parasuis IgY protein bands respectively.

Detailed ways

The present invention will be described in detail below in conjunction with examples, and these examples are for illustrative purposes only, and do not limit the scope of application of the present invention.

Unless otherwise specified, the experimental methods described in the present invention are conventional methods; the biological materials mentioned can be obtained from commercial channels.

Example 1

1. Method

1.1 Test animals

BALB/c mice, 15-20 g, were purchased from Sichuan Dashuo Experimental Animal Center; 160-day-old Roman commercial layer hens were purchased from Sichuan Zhengda.

1.2 Strain preservation

SMU-2020-ZQ2, a virulent strain of Streptococcus suis type 2, was isolated from pigs that died of sepsis in a pig farm in Mianyang City, Sichuan Province. The median lethal dose (LD ₅₀ ) for BALB/c mice was 2.1×10 ⁷ CFU, The strain is preserved in China Center for Type Culture Collection, the address is Wuhan University, No. 299, Bayi Road, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M2021457. Latin name: Streptococcus sp. SMU-2020-ZQ2.

The highly virulent isolates of Haemophilus parasuis type 4, type 5 and type 13 were all from dead pigs suffering from respiratory symptoms in a pig farm in Leshan City, Sichuan Province. Purchased from the Strain Preservation Management Center, the address is: No. 8 Zhongguancun South Street, Haidian District, Beijing. The preservation numbers are CVCC NO: 3953, CVCC NO: 3956, and CVCC NO: 3958. The preservation times are July 25, 2008, On July 28, 2008 and July 28, 2008, the LD ₅₀ for BALB/c mice was 4.6×10 ⁸ CFU, 3.3×10 ⁸ CFU and 5.6×10 ⁸ CFU, respectively.

1.3 Preparation of purified immune antigen

1.3.1 Preparation of Streptococcus suis type 2 immune antigen

Streptococcus suis type 2 (SMU-2020-ZQ2 strain) was inoculated on tryptone soy agar medium (TSA) containing 5% calf serum, placed in a constant temperature incubator at 37°C for 24 hours, and a single colony was picked ( CFU) inoculated into Tryptone Soy Broth (TSB) containing 5% calf serum and cultured for 18-24 hours, then diluted to 1×10 ⁹ CFU/mL bacterial solution with sterile PBS (0.01mol, pH7.2), Add formaldehyde to a final concentration of 0.3%, inactivate at 37°C for 72 hours, during which shake 5-6 times; centrifuge at 6000rpm to separate the precipitated bacteria, resuspend the bacteria with sterile PBS (0.01mol, pH7.2), and centrifuge again Wash twice, and dilute the bacterial solution with a bacterial concentration of 4×10 ⁹ CFU/mL with PBS. Take 100 μL of the bacterial solution and spread it on a TSA culture plate with 5% bovine serum, place it in a constant temperature incubator at 37°C for 72 hours, observe the bacterial colonies, and set up a negative control and a positive control of non-inactivated bacteria at the same time. If typical SS2 colonies appeared in the positive control, but no colonies appeared in the culture medium inoculated with the inactivated bacteria solution and the medium of the negative control group, it indicated that the SMU-2020-ZQ2 bacteria solution was completely inactivated.

1.3.2 Preparation of immune antigens of Haemophilus parasuis type 4, type 5 and type 13

Haemophilus parasuis type 4 (3953 strains), type 5 (3956 strains) and type 13 (3958 strains) were inoculated in TSA containing 5% calf serum and 1% nicotinamide adenine dinucleotide (NAD) Incubate at 37°C for 24h to 36h. Pick a single pinpoint-sized, smooth and transparent dewdrop-shaped colony, inoculate it again in TSB liquid medium containing 5% calf serum and 1% NAD, and cultivate it for 24-36 hours. Dilute the bacterial solution to 1×10 ⁹ CFU/mL, add formaldehyde to a final concentration of 0.3%, inactivate at 37°C for 72 hours, shake and shake 5 to 6 times during the period; mol, pH7.2) to resuspend the bacterial cells, centrifuge and wash twice, and dilute the bacterial solution with a bacterial concentration of 4×10 ⁹ CFU/mL with PBS. Take 100 μL of the bacterial solution and spread it on a TSA culture plate containing 5% calf serum and 1% NAD, place it in a constant temperature incubator at 37°C for 72 hours, observe the bacterial colonies, and set up negative controls and non-inactivated colonies at the same time positive control. If the positive control has typical pinpoint-sized, smooth and transparent dewdrop-shaped colonies, but no colonies appear in the medium inoculated with the inactivated bacteria solution and the medium of the negative control group, it indicates that the bacteria solution is completely inactivated.

1.3.3 Bacterin configuration

Mix the three serotype strains (3953, 3956 and 3958) of Streptococcus suis (SMU-2020-ZQ2 strain) and Haemophilus parasuis (strains 3953, 3958) that have been completely inactivated through testing, and mix them with complete Freund's adjuvant Equal volumes are mixed to make it a slightly viscous milky white suspension, which is the double quadrivalent inactivated vaccine of Streptococcus suis and Haemophilus parasuis respectively.

1.3.4 Safety inspection

The experimental steps of inactivated vaccine safety testing are as follows:

1) Sterility test: Take 100 μL of the inactivated vaccine and spread it on a TSA culture plate with 5% bovine serum, culture it at 37° C. for 24 hours, and observe whether there is any bacterial growth.

2) Safety test: Warm the inactivated vaccine at room temperature, and inoculate 6-8 week-old SPF BALB/c female mice by subcutaneous injection at multiple points on the neck and back respectively. They were raised in the animal room of the Veterinary Medicine Laboratory of Southwest University for Nationalities, and their food intake, mental state, and abnormalities such as redness and swelling at the injection site were observed.

The results of safety inspection: the inactivated vaccine was inoculated into BALB/c mice, full of energy, normal appetite, well absorbed at the injection site, without any adverse abnormalities such as redness and swelling, indicating that the Streptococcus suis and Haemophilus parasuis prepared in this study The dual quadrivalent inactivated vaccine has good safety and can be used in follow-up trials.

1.4 Flock immunization and egg collection

Select healthy high-yielding Roman commercial laying hens without major infectious diseases, especially those without Salmonella pullorum, and inject them with the double quadrivalent killed Streptococcus suis and Haemophilus parasuis prepared in this study through leg and/or chest muscles respectively. Live vaccine 1mL, immunized 4 times in a row, each interval of 2 weeks.

After the fourth immunization, the serum was collected to determine the titer. When the antibody agglutination titer reaches 1:2 ⁷ or more, it is considered qualified for immunity. Collect eggs every day, mark them well, and store them at 4°C for later use.

1.5 Determination of Titer of Quadrivalent Egg Yolk Antibody to Streptococcus suis and Haemophilus parasuis

After the fourth immunization, 3 eggs were randomly collected from the immunized group and the non-immunized group every 7 days, sterilized in 0.1% bromogeramine aqueous solution at 42°C for 15 minutes, taken out and dried in the air, and the egg yolk was separated with an egg white and yolk separator; Take 1 mL of the mixed egg yolk, add 9 mL of deionized water and mix again, and adjust the pH value of the egg yolk solution to 5.0-5.2; put it in a refrigerator at 4 °C for 4 hours, take the supernatant solution, centrifuged at 6000 rpm/min at 4°C for 25 min, and the supernatant was taken and stored for later use.

The antibody titer was determined by plate agglutination test. Add 50 μL of sterilized PBS (0.01 mol, pH 7.2) to the center of each grid of the glass plate, then pipette 50 μL of the treated egg yolk antibody sample into the first grid, mix well with a pipette tip, and pipette Add 50 μL of the mixture to the second compartment, mix thoroughly, and sequentially perform doubling dilutions to the eighth compartment (that is, dilute 1024 times), aspirate 50 μL and discard. The 9th, 10th and 11th grids were used as antigen control and positive and negative serum controls respectively. Then add 50 μL of antigen for detection to each droplet, and stir well with a toothpick. After 2 to 5 minutes, observe the agglutination reaction of each grid on the glass plate. The negative serum control grid is slightly uniformly turbid (the antigen is diluted by 2 times), and it is judged as negative, marked as "-", and the positive control grid has flocculent blocks. If it is judged as positive, it is marked as "+". The more clots that appear and the more transparent the liquid, the stronger the positive reaction, which is marked as "+", "++", "+++", and "#" from weak to strong. The highest dilution of the serum with agglutination phenomenon above "++" is the titer of the serum (the yolk antibody has been diluted by 2log2 during the extraction process).

The yolk antibody of immunized chickens began to detect specific antibodies 7 days after the second immunization, and the antibody titers reached 8log2 and 9log2 respectively 7 days after the fourth immunization for Streptococcus suis type 2, Haemophilus parasuis types 4, 5 and 13 , 8log2 and 8log2, the antibody level can be maintained above 7log2 one year after the 4 immunizations.

1.6 Extraction and purification of IgY antibody

Collect qualified eggs after immunization, sterilize them in 0.1% bromogeramine aqueous solution at 42°C for 15 minutes, take them out and dry them in the air, separate the egg yolks with an egg white and yolk separator, and weigh them. Add purified water to dilute the homogenate at a ratio of 1:6~9. Use 1moL/L hydrochloric acid solution to adjust the pH to 4.8-6.0, and stand at 2-8°C for 1-24h. Centrifuge at 4000-20000r/min, remove the precipitate, and take the supernatant. Add 10%-35% ammonium sulfate to the supernatant for salting out, mix thoroughly, and precipitate overnight. The prepared precipitate was centrifuged at 4000-20000 r/min for desalting, and then the precipitate was taken to obtain the crude extract of egg yolk antibody, and the ammonium sulfate supernatant was removed. The prepared precipitate was redissolved with 1/150-1/300 volume of weakly acidic buffer, adjusted to pH 4.8-6.0, fully dissolved overnight at 2-8°C, and the supernatant was taken. The obtained supernatant was subjected to 54-60°C for 8-20 hours to inactivate the microorganisms in the antibody. After inactivation, the supernatant was ultrafiltered with an ultrafiltration membrane with a relative molecular weight cut-off of 50-100 kD. The ultrafiltrate was sterilized and filtered to obtain the IgY stock solution. The obtained IgY stock solution is added with a stabilizer according to the detected antibody titer to make an egg yolk antibody preparation.

In the process of preliminary preparation of egg yolk antibody, the present invention found that the purity of the crude egg yolk antibody was not high, and the protection efficiency shown in animal experiments was not increased. Therefore, IgY can be further purified by ultrafiltration after extraction with ammonium sulfate , reducing impurity proteins, the purified IgY has higher purity and fewer impurity bands, as shown by SDS-PAGE non-reducing electrophoresis (Fig. The purity of the IgY antibody analyzed by gel imaging software was above 90%.

SDS-PAGE denaturation electrophoresis showed that the extracted anti-Streptococcus suis and Haemophilus parasuis bivalent tetravalent yolk antibody IgY was distinguished into two main bands, 65kDa was the IgY heavy chain, and 25kDa was the IgY light chain. It was determined that the agglutination titers of the purified and refined IgY products against Streptococcus suis type 2, Haemophilus parasuis types 4, 5 and 13 all reached above 8log2.

1.7 Yolk IgY antibody hemolysis test

Aseptically collect a few milliliters of rabbit blood, stir the blood with a glass rod, remove fibrinogen, and make it into defibrillated blood. Add 10 times the volume of 0.9% sodium chloride solution, shake well, centrifuge at 1000-1500r/min for 15 minutes, remove the supernatant, and then wash the precipitated red blood cells with 0.9% sodium chloride solution 2 to 3 times according to the above method.

The resulting erythrocytes were made into a 2% suspension with 0.9% sodium chloride solution. The prepared egg yolk antibody was diluted 1:3 with 0.9% sodium chloride solution as the test solution, with sterile normal saline as the negative control and the uninactivated Streptococcus suis culture as the positive control, respectively adding EP tube. Then add 2% erythrocyte suspension and 0.9% sodium chloride solution in turn, mix well, immediately place in a 37°C ± 0.5°C incubator for incubation, observe once every 12 hours, continuously observe for 6 days, and record and analyze result.

The results showed that the negative control tube had no hemolysis and aggregation, and the positive control tube had hemolysis; at the same time, the solution in the test substance tube did not undergo hemolysis and aggregation within 144 hours, indicating that the egg yolk IgY antibody prepared in this study had no hemolysis. Can be injected.

1.8 In vitro antibacterial test of Streptococcus suis and Haemophilus parasuis quadrivalent purified yolk antibody injection

1.8.1 Evaluation of antibacterial effect of Streptococcus suis and Haemophilus parasuis quadrivalent purified yolk antibody injection on Streptococcus suis type 2 in vitro

Streptococcus suis SMU-2020-ZQ2 was inoculated into TSB containing 5% bovine serum and cultured until the bacterial concentration reached 10 ⁷ CFU/mL, after 200 μL of diluted bacterial suspension and 200 μL egg yolk IgY antibody or PBS (control group).

After incubating at 37°C for 1h and 2h, respectively, after 10-fold dilution with PBS, take 0.1mL and apply it to TSA medium containing 5% serum, and incubate at 37°C for 24h, and count the number of bacterial colonies in each well. Each dilution factor was repeated three times, each sample was repeated three times, and each time 3 parallels were performed. Bacteria reduction percentage: [CFU(bacteria+PBS)-CFU(bacteria+antibody)]/CFU(bacteria+PBS)×100.

1.8.2 Evaluation of antibacterial effect of Streptococcus suis and Haemophilus parasuis quadrivalent purified egg yolk antibody injection on three kinds of Haemophilus parasuis in vitro

Three different serotypes of Haemophilus parasuis were respectively inoculated into TSB containing 5% bovine serum and 1% NAD and cultured until the bacterial concentration reached 10 ⁷ CFU/mL.

After 200 μL of diluted bacterial suspension and 200 μL of egg yolk IgY antibody or PBS (control group), incubate at 37°C for 1 hour and 2 hours, respectively, after 10-fold dilution with PBS, take 0.1 mL and spread it on TSA containing 5% serum medium, and incubated at 37°C for 24 h, counting the number of bacterial colonies in each well. Each dilution factor was repeated three times, each sample was repeated three times, and each time 3 parallels were performed. Calculate the percent reduction in bacteria.

The results of the antibacterial test are shown in Table 1.

Table 1 Bacteria content (cfu/ml) in culture medium with culture time (h)

Compared with the PBS group, the IgY finished products could significantly inhibit the growth of Streptococcus suis and Haemophilus parasuis in vitro.

1.9 Passive protection test on mice by dual quadrivalent refined egg yolk antibody injection against Streptococcus suis and Haemophilus parasuis

Eighty BALB/c mice were randomly divided into 8 groups, with 10 mice in each group. Among them, 7 groups were instilled with 0.5 mL of yolk IgY antibody once a day for 3 consecutive days. Group 1 was treated with 0.5 mL of normal saline as the control group. Each group used 5LD50 of Streptococcus suis type II, Haemophilus parasuis types 4, 5 and 13 to challenge mice by intramuscular injection, and judged the protection rate of egg yolk IgY antibody to mice.

Table 2 shows the results of the passive protection test of egg yolk IgY antibody on mice.

Table 2 Passive protection test results of egg yolk IgY antibody to BALB/c mice

The results showed that the mice with normal saline instillation showed obvious clinical symptoms after challenge: weight loss, dyspnea, messy coat, depression, loss of appetite, etc. They began to die 16 hours after challenge, and all died within 48 hours . The mice instilled with egg yolk IgY antibody have good protective effects on Streptococcus suis type II, Haemophilus parasuis type 4, 5 and 13 strains, and the protection efficiency is higher than 90%. On the 3rd day after being challenged with Streptococcus suis type II strain, 2 mice died, and the survival protection rate of all others was 90%; on the 2nd day after being challenged with Haemophilus parasuis type 4 strain, 1 mouse died, and the rest survived The protection rate was 90%; no mice died after inoculation with Haemophilus parasuis type 5 strain, and the protection rate was 100%; 3 days after inoculation with Haemophilus parasuis type 13 strain, one mouse died, and all other mice died. The survival protection rate was 90%.

In summary, the present invention provides a dual quadrivalent purified egg yolk antibody injection against Streptococcus suis type 2 and Haemophilus parasuis types 4, 5, and 13. After purification, the purity of IgY in the egg yolk antibody can reach Reach more than 90%, no hemolysis, and the agglutination titer to Streptococcus suis type 2, Haemophilus parasuis type 4, 5 and 13 all reach more than 8log2; the obtained IgY finished products can significantly inhibit pig chain in vitro The growth of H. parasuis type 2 and H. parasuis types 4, 5, and 13 has a protection rate of more than 90% for challenged mice, especially the protection rate of H. parasuis type 5 strain reaches 100%. It provides a safe, efficient, and pollution-free biological product for the clinical treatment of Streptococcus suis and Haemophilus parasuis, which can replace antibiotics for daily prevention and treatment of Streptococcus suis and Haemophilus parasuis.

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any skilled person familiar with the profession, Within the scope of not departing from the technical solution of the present invention, the technical content disclosed above can be used to make some changes or be modified into equivalent embodiments of equivalent changes, but all the content that does not depart from the technical solution of the present invention shall be based on the technical essence of the present invention Any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the technical solution of the present invention.

Claims (4)

1. a kind of preparation method of the refined yolk antibody injection of anti-Streptococcus suis and Haemophilus parasuis, is characterized in that, comprises: (1) Prepare the double quadrivalent inactivated vaccine by preparing Haemophilus parasuis serotype 4, serotype 5, serotype 13 and Streptococcus suis type 2 antigens; The strain preservation number of the Haemophilus parasuis serotype 4 is CVCC NO: 3953; The strain preservation number of the Haemophilus parasuis serotype 5 is CVCC NO: 3956; The strain preservation number of the Haemophilus parasuis serotype 13 is CVCC NO: 3958; The strain preservation number of Streptococcus suis type 2 is CCTCC NO: M2021457; (2) Immune laying hens with a double quadrivalent inactivated vaccine and separate and extract the yolk antibody in the immunized eggs; the immunization method is 4 times of leg and/or chest muscle injections, with an interval of 2 weeks between each immunization injection; When the antibody titer reached 1:128, the immunized eggs were collected 7 days after the fourth immunization, and the yolk antibody was isolated and extracted. The antibody titers of Streptococcus suis type 2, Haemophilus parasuis type 4, 5 and 13 were Reach 8log2, 9log2, 8log2 and 8log2; The two-unit quadrivalent inactivated vaccine contains Haemophilus parasuis serotype 4, serotype 5, serotype 13, and Streptococcus suis 2 antigen dilutions, and the number of colonies before inactivation is not less than 4×10 ⁹ CFU/ mL; In the yolk antibody, the inactivated antigen dilutions of Haemophilus parasuis serotype 4, serotype 5, serotype 13, and Streptococcus suis type 2 were mixed in equal volumes. 2. The preparation method of yolk antibody injection according to claim 1, characterized in that, the inactivated antigen diluent of said Haemophilus parasuis serotype 4, serotype 5, serotype 13, and Streptococcus suis type 2 The total content is 50% of the total vaccine (V/V). 3. the preparation method of yolk antibody injection according to claim 1, is characterized in that, described double quadrivalent inactivated vaccine also comprises adjuvant; Described adjuvant content is 50% (V/V ); The adjuvant is Freund's complete adjuvant. 4. The yolk antibody described in any one of claims 1 to 3 is used in the preparation, detection, prevention and/or treatment of Haemophilus parasuis serotype 4, serotype 5, serotype 13, and Streptococcus suis type 2 infection. Use in products for relevant diseases; The product is selected from preparations, medicines or feed additives.