Preparation method and application of bovine hemophilus somnus positive serum

Abstract

The present application relates to the technical field of veterinary biological products, more specifically, it relates to a preparation method and application of bovine haemophilus somnus positive serum. The preparation medium, the preparation of bacteria liquid bovine haemophilus somnus H.S / NMG / 2023, the preparation of concentrated bacteria liquid bacteria, the preparation of immune antigen, immunization, and serum collection are prepared. The high-titer bovine haemophilus somnus specific positive serum is successfully prepared, which fills the blank of the positive serum in the current market, effectively solves the problem of lack of standard positive serum in bovine haemophilus somnus related detection and vaccine quality control, and the prepared positive serum can be widely used in bovine haemophilus somnus vaccine specificity test, differential test, foreign virus test and diagnostic detection and other key links, which significantly improves the accuracy and reliability of vaccine quality control.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, and more specifically, to a method for preparing and applying Haemophilus suis positive serum. Background Technology

[0002] Bovine Haemophilus somnus infection is a multisystemic infectious disease caused by Haemophilus somnus. Its clinical manifestations are complex and diverse, affecting multiple systems including the nervous, respiratory, digestive, and reproductive systems; therefore, it is often referred to as Haemophilus somnus syndrome. This pathogen can infect alone or in combination with other pathogens such as Mycoplasma bovis, Pasteurella multocida, hemolytic Mansonia solani, and bovine viral diarrhea virus, leading to bovine respiratory disease syndrome. Typical lesions include catarrhal pneumonia, purulent laryngitis, thromboembolic meningoencephalitis, arthritis, fibrinous pericarditis, and sudden death due to necrosis of the left ventricular papillary muscles caused by septic cardiovascular disease. Furthermore, Haemophilus somnus has been identified as a significant pathogen causing bovine reproductive disorders, leading to infertility, endometritis, and abortion at various stages of pregnancy.

[0003] Currently, immunization is considered the most effective strategy for controlling this disease. Studies abroad have reported that administering two doses of vaccine at regular intervals can significantly reduce morbidity and mortality. However, research on Haemophilus influenzae in my country is still insufficient, with few related reports. Significant gaps remain in vaccine development, diagnostic reagents, and serum products. Particularly noteworthy is the lack of commercially available Haemophilus influenzae positive serum, and no publicly reported methods for preparing its specific positive serum have been found. Therefore, developing effective vaccines, diagnostic tools, and serum products is of significant practical importance for strengthening the prevention and control of this disease. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a method for preparing Haemophilus bovis positive serum, the specific steps of which are as follows: S1. Preparation of culture media: Prepare solid culture medium Columbia blood agar basal medium and liquid culture medium Brain heart extract liquid medium, respectively; S2. Preparation of bacterial culture: The frozen bovine hemophagocytic HS / NMG / 2023 was streaked onto Columbia blood agar basal medium and cultured in a CO2 incubator; Single colonies that have grown were inoculated onto brain heart extract liquid culture medium, and cultured in a CO2 environment. The fermentation broth was obtained by shaking the culture on a shaker. S3. Preparation of concentrated bacterial culture: The bacterial culture was concentrated by centrifugation, and then the bacterial precipitate was collected and washed three times with phosphate buffer. The precipitate was then resuspended in sterile PBS to prepare concentrated bacterial culture. S4. Inactivation of bacterial solution: Add formaldehyde solution to bacterial solution for inactivation, and shake well during the process to prepare inactivated antigen bacterial solution; S5. Preparation of immunoantigen: Place the inactivated antigen bacterial solution in crushed ice, then place it in an ultrasonic cell disruptor for ultrasonic disruption, then freeze and thaw once, then ultrasonically disrupt once more, centrifuge the disrupted bacterial solution, and collect the supernatant to obtain the immunoantigen. S6. Immunization: Immunize healthy rabbits with immune antigens three times; S7. Serum collection: 14 days after the last immunization, rabbit blood was collected aseptically, and serum was separated. The serum was filtered twice under pressure through sterile filter membranes. The serum antibody titer was detected by agar diffusion test. Physical properties, sterility and specificity were tested. After passing the tests, the serum was aliquoted and stored to obtain positive serum for Haemophilus nigricans.

[0005] Preferably, the solid culture medium in S1 is Columbia blood agar basal medium containing 3%-10% defibrinated sheep blood, and the liquid culture medium is liquid culture medium containing 0.5%-1% yeast extract brain heart extract.

[0006] Preferably, the *Haemophilus bovis* HS / NMG / 2023 in S2 was deposited on August 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC). The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. The classification name is *Haemophilus bovis*, the accession number is CGMCC No. 31702, the CO2 content is 5%, and the culture temperature is 37℃.

[0007] Preferably, in step S3, the centrifugation speed is 3500 r / min, the centrifugation temperature is 4℃, the centrifugation time is 20 min, the phosphate buffer is 0.01 M, the pH is 7.2, and the concentration of the concentrated bacterial solution is above 10 billion cfu / ml.

[0008] Preferably, in step S4, the bacterial solution is added to formaldehyde solution at 0.1% to 0.2% (v / v) of the bacterial solution volume and inactivated at 37°C for 8-10 hours.

[0009] Preferably, in step S5, the ultrasonic disruption is performed using a program of 200W ultrasonic for 5 seconds with a 3-second interval, for a total of 30 minutes. The centrifugation temperature is 4°C, the centrifugation speed is 12000 r / min, and the centrifugation time is 15 minutes.

[0010] Preferably, the healthy rabbits in S6 weigh between 2.25 kg and 3.0 kg and are used as experimental animals. Their serum is collected for testing to verify that they do not have antigens and antibodies such as bovine rotavirus, bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus, foot-and-mouth disease virus, and bovine Akabane virus.

[0011] Preferably, in S6, the first immunization is performed by injecting 2 mL into multiple muscle points in the leg, and the remaining two immunizations are performed every 14 days, with the immunization dose increasing by 1 mL each time.

[0012] Preferably, the serum in S7 is filtered under pressure through a 0.45μm sterile filter membrane and a 0.22μm sterile filter membrane, respectively, and stored at -80℃.

[0013] Another objective of this invention is to provide an application of Haemophilus suis positive serum, wherein the Haemophilus suis positive serum is used to prepare reagents or kits for the detection or identification of Haemophilus suis, wherein the Haemophilus suis positive serum is used for Haemophilus suis specificity tests, identification tests, exogenous virus tests or diagnoses, and wherein the Haemophilus suis positive serum is used to prepare Haemophilus suis positive serum standards.

[0014] The beneficial effects of this invention are as follows: A high-titer, specific positive serum for Haemophilus bovis has been successfully prepared, filling a gap in the current market for this type of positive serum. This effectively solves the problem of lacking standard positive serum in Haemophilus bovis-related detection and vaccine quality control. The prepared positive serum can be widely used in multiple key stages of Haemophilus bovis vaccine testing, including specificity testing, identification testing, exogenous virus testing, and diagnostic testing, significantly improving the accuracy and reliability of vaccine quality control. Simultaneously, it provides important support for the diagnostic technology of Haemophilus bovis infection and opens up a new technical path for the preparation of high-titer positive serum, possessing significant application value and promising prospects for widespread application.

[0015] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a negative serum of Haemophilus niger from an embodiment of the present invention; Figure 2 This is a positive serum of Haemophilus niger in an embodiment of the present invention. Detailed Implementation

[0017] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0018] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below. Example

[0019] Preparation of culture media: Culture media are divided into liquid culture media and solid culture media; Solid culture medium is prepared using conventional methods: 5% defibrinated sheep blood is added to Columbia blood agar basal medium. Columbia agar is used as the basal medium. Weigh 3.9g into 100mL of distilled water, autoclave at 121℃ for 15min, and when it cools to about 50℃, add 3-10mL of defibrinated sheep blood, gently shake to mix, and pour into a petri dish.

[0020] The liquid culture medium was prepared using standard methods: the base solution of the liquid culture medium was brain and heart extract liquid culture medium with 1% yeast extract added. Using brain and heart extract liquid culture medium as the base liquid culture medium, weigh 37.0g and 10g of yeast extract, heat and dissolve in 1000ml of purified water, autoclave at 121℃ for 15 minutes, and set aside.

[0021] Preparation of bacterial culture 1) Primary seed propagation: First, retrieve the bovine Haemophilus hygroscopicus HS / NMG / 2023 bacteria frozen at -80℃. After thawing, use an inoculation loop to take a small amount of bacterial solution and streak it onto a Columbia blood agar plate. Incubate at 37℃ with 5% CO2 for 16–20 hours. Then, pick a single colony and inoculate it into 10 ml of brain heart extract yeast extract liquid medium. Incubate at 37℃ with 5% CO2 for 16–20 hours. This will serve as the primary seed culture, stored at 2–8℃ for large-scale culture. The culture period should not exceed 7 days.

[0022] 2) Secondary seed propagation: Take one primary seed, inoculate 4 ml of bacterial culture into 200 mL of brain heart extract yeast extract liquid culture medium, and incubate at 37℃, 5% CO2 environment with shaking at 120 r / min for 8-12 h. After passing the purity test, store at 2-8℃ as secondary seed. 3) Preparation of bacterial culture: Haemophilus silicosis was cultured in shake flasks. 2% (v / v) of the secondary seed culture was added to the liquid culture medium containing brain and heart extract and yeast extract. The volume of the liquid culture medium was controlled at a ratio of 1:3 to the container volume. After thorough mixing, the culture was incubated at 37℃ and 5% CO2 with shaking at 120 rpm for 8–12 hours. After the bacterial culture was completed, samples were taken for purity testing according to the appendix of the 2015 edition of the Chinese Veterinary Pharmacopoeia. The samples should be pure. Simultaneously, viable cell counts were performed on the samples.

[0023] Preparation of concentrated bacterial culture: The fermentation broth was concentrated by centrifugation. The centrifugation conditions were: 3500 r / min, 4℃, centrifugation for 20 min. The bacterial precipitate was then collected and washed three times with phosphate buffer (0.01M, pH 7.2). The precipitate was then resuspended in sterile PBS to prepare a concentrated bacterial culture with a concentration of more than 10 billion cfu / ml.

[0024] Inactivation of bacterial solution: Add formaldehyde solution at 0.1% to 0.2% (v / v) of the concentrated bacterial solution and inactivate at 37°C for 8-10 hours, shaking several times during the process. Prepare inactivated antigen bacterial solution. The inactivation test shall be carried out in accordance with the appendix of the 2015 edition of the Chinese Veterinary Pharmacopoeia. There should be no sterile growth.

[0025] Preparation of immunoantigen: Place the Haemophilus bovis suspension in crushed ice, then place it in an ultrasonic cell disruptor and sonicate for 30 minutes at 200W for 5 seconds with a 3-second interval. Then freeze-thaw once, and sonicate again. Centrifuge the disrupted suspension at 4℃ and 12000r / min for 15 minutes, and collect the supernatant to obtain the immunoantigen.

[0026] Immunization: Screening for serum negative for Haemophilus bovis (before immunization) Figure 1 As shown, rabbits weighing 2.25-3.0 kg were used as experimental animals. Serum samples were collected and tested to verify that none of them had antigens and antibodies against bovine rotavirus, bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus, foot-and-mouth disease virus, or bovine Akabane virus. Basic immunization: 2 mL of prepared Haemophilus influenzae type b (HABB) immunogen was injected into the leg muscles of each rabbit at multiple sites. Booster immunization: After basic immunization, Haemophilus influenzae type b (HABB) immunogen was injected into the leg and back muscles of each rabbit at multiple sites every 14 days for 3 times, with the immunization dose increasing by 1 mL each time.

[0027] Serum collection: 14 days after the last immunization, rabbit blood was collected aseptically, serum was separated, and the serum was filtered under pressure through a 0.45μm sterile filter membrane and then through a 0.22μm sterile filter membrane. The serum antibody titer was detected by agar diffusion test, and physical properties, sterility and specificity were tested. After passing the tests, the serum was aliquoted and stored at -80℃.

[0028] The prepared positive serum for Haemophilus suicidal bacteria was subjected to physical properties, sterility, and potency tests. Physical property test: The prepared positive serum of Haemophilus suicidal bacteria should be a colorless or pale yellow clear liquid.

[0029] Sterility test: The prepared serum of Haemophilus nigricans positive was tested according to the appendix of the current Chinese Pharmacopoeia and showed no sterile growth.

[0030] Potency test: Take the prepared positive serum of Haemophilus bovis, such as... Figure 2 The serum samples (3 samples, numbered 1-1, 1-2, and 1-3) were diluted to 1:2, 1:4, 1:8, 1:16, and 1:32, respectively, and tested using an agar diffusion test. The results are shown in Table 1. Table 1 shows that when the *Haemophilus bovis* positive serum samples 1-1, 1-2, and 1-3 were diluted to 1:32, specific antigen-antibody precipitation lines could still be detected, indicating that the prepared *Haemophilus bovis* positive serum had a high titer.

[0031] Table 1. Results of the agar agar assay for Haemophilus influenzae in bovine stupor

[0032] (—) indicates that, Figure 1 The line showing no antigen-antibody precipitation is indicated by (+). Figure 2 The image shows visible lines of antigen-antibody precipitation.

[0033] Comparative Example 1 The preparation method of Haemophilus bovis immunogen in Comparative Example 1 differs from that in the Example only in that the 10 billion CFU / ml immunogen in the Example is replaced with 5 billion CFU / ml immunogen.

[0034] Preparation of culture media: Culture media are divided into liquid culture media and solid culture media; Solid culture medium is prepared using conventional methods: 5% defibrinated sheep blood is added to Columbia blood agar basal medium. Columbia agar is used as the basal medium. Weigh 3.9g into 100mL of distilled water, autoclave at 121℃ for 15min, and when it cools to about 50℃, add 3-10mL of defibrinated sheep blood, gently shake to mix, and pour into a petri dish.

[0035] The liquid culture medium was prepared using standard methods: the base solution of the liquid culture medium was brain and heart extract liquid culture medium with 1% yeast extract added. Using brain and heart extract liquid culture medium as the base liquid culture medium, weigh 37.0g and 10g of yeast extract, heat and dissolve in 1000ml of purified water, autoclave at 121℃ for 15 minutes, and set aside.

[0036] Preparation of bacterial culture 1) Primary seed propagation: First, retrieve the bovine Haemophilus hygroscopicus HS / NMG / 2023 bacteria frozen at -80℃. After thawing, use an inoculation loop to take a small amount of bacterial solution and streak it onto a Columbia blood agar plate. Incubate at 37℃ with 5% CO2 for 16–20 hours. Then, pick a single colony and inoculate it into 10 ml of brain heart extract yeast extract liquid medium. Incubate at 37℃ with 5% CO2 for 16–20 hours. This will serve as the primary seed culture, stored at 2–8℃ for large-scale culture. The culture period should not exceed 7 days.

[0037] 2) Secondary seed propagation: Take one primary seed, inoculate 4 ml of bacterial culture into 200 mL of brain heart extract yeast extract liquid culture medium, and incubate at 37℃, 5% CO2 environment with shaking at 120 r / min for 8-12 h. After passing the purity test, store at 2-8℃ as secondary seed. 3) Preparation of bacterial culture: Haemophilus silicosis was cultured in shake flasks. 2% (v / v) of the secondary seed culture was added to the liquid culture medium containing brain and heart extract and yeast extract. The volume of the liquid culture medium was controlled at a ratio of 1:3 to the container volume. After thorough mixing, the culture was incubated at 37℃ and 5% CO2 with shaking at 120 rpm for 8–12 hours. After the bacterial culture was completed, samples were taken for purity testing according to the appendix of the 2015 edition of the Chinese Veterinary Pharmacopoeia. The samples should be pure. Simultaneously, viable cell counts were performed on the samples.

[0038] Preparation of concentrated bacterial culture: The fermentation broth was concentrated by centrifugation. The centrifugation conditions were: 3500 r / min, 4℃, centrifugation for 20 min. The bacterial precipitate was then collected and washed three times with phosphate buffer (0.01 M, pH 7.2). The precipitate was then resuspended in sterile PBS to prepare a concentrated bacterial culture with a concentration of more than 5 billion cfu / ml.

[0039] Inactivation of bacterial solution: Add formaldehyde solution at 0.1% to 0.2% (v / v) of the concentrated bacterial solution and inactivate at 37°C for 8-10 hours, shaking several times during the process. Prepare inactivated antigen bacterial solution. The inactivation test shall be carried out in accordance with the appendix of the 2015 edition of the Chinese Veterinary Pharmacopoeia. There should be no sterile growth.

[0040] Preparation of immunoantigen: Place the Haemophilus bovis suspension in crushed ice, then place it in an ultrasonic cell disruptor and sonicate for 30 minutes at 200W for 5 seconds with a 3-second interval. Then freeze-thaw once, and sonicate again. Centrifuge the disrupted suspension at 4℃ and 12000r / min for 15 minutes, and collect the supernatant to obtain the immunoantigen.

[0041] Immunization: Rabbits weighing 2.25-3.0 kg were selected as experimental animals and screened for negative serum against Haemophilus bovis (before immunization). Serum samples were collected to verify that they did not have antigens and antibodies against bovine rotavirus, bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus, foot-and-mouth disease virus, or bovine Akabane virus. Basic immunization: 2 mL of prepared Haemophilus bovis immunogen was injected into the leg muscles of each rabbit at multiple sites. Booster immunization: After basic immunization, the prepared Haemophilus bovis immunogen was injected into the leg and back muscles of each rabbit at multiple sites every 14 days for 3 immunizations, with the dose increasing by 1 mL each time.

[0042] Serum collection: 14 days after the last immunization, rabbit blood was collected aseptically, serum was separated, and the serum was filtered under pressure through a 0.45μm sterile filter membrane and then through a 0.22μm sterile filter membrane. The serum antibody titer was detected by agar diffusion test, and physical properties, sterility and specificity were tested. After passing the tests, the serum was aliquoted and stored at -80℃.

[0043] Potency test: The prepared positive serum of Haemophilus bovis (3 serum samples, numbered 1-1, 1-2, and 1-3) was diluted 1:2, 1:4, 1:8, 1:16, and 1:32, and tested using an agar diffusion test. The test results are shown in Table 2. Table 2. Results of the agar agar assay for Haemophilus influenzae in bovine stupor

[0044] Comparative Example 2 The preparation method of Haemophilus niger immunogen in Comparative Example 2 differs from that in the Example in that booster immunization 3 in the Example is replaced with booster immunization 1 time.

[0045] Preparation of culture media: Culture media are divided into liquid culture media and solid culture media; Solid culture medium is prepared using conventional methods: 5% defibrinated sheep blood is added to Columbia blood agar basal medium. Columbia agar is used as the basal medium. Weigh 3.9g into 100mL of distilled water, autoclave at 121℃ for 15min, and when it cools to about 50℃, add 3-10mL of defibrinated sheep blood, gently shake to mix, and pour into a petri dish.

[0046] The liquid culture medium was prepared using standard methods: the base solution of the liquid culture medium was brain and heart extract liquid culture medium with 1% yeast extract added. Using brain and heart extract liquid culture medium as the base liquid culture medium, weigh 37.0g and 10g of yeast extract, heat and dissolve in 1000ml of purified water, autoclave at 121℃ for 15 minutes, and set aside.

[0047] Preparation of bacterial culture 1) Primary seed propagation: First, retrieve the bovine Haemophilus hygroscopicus HS / NMG / 2023 bacteria frozen at -80℃. After thawing, use an inoculation loop to take a small amount of bacterial solution and streak it onto a Columbia blood agar plate. Incubate at 37℃ with 5% CO2 for 16–20 hours. Then, pick a single colony and inoculate it into 10 ml of brain heart extract yeast extract liquid medium. Incubate at 37℃ with 5% CO2 for 16–20 hours. This will serve as the primary seed culture, stored at 2–8℃ for large-scale culture. The culture period should not exceed 7 days.

[0048] 2) Secondary seed propagation: Take one primary seed, inoculate 4 ml of bacterial culture into 200 mL of brain heart extract yeast extract liquid culture medium, and incubate at 37℃, 5% CO2 environment with shaking at 120 r / min for 8-12 h. After passing the purity test, store at 2-8℃ as secondary seed. 3) Preparation of bacterial culture: Haemophilus silicosis was cultured in shake flasks. 2% (v / v) of the secondary seed culture was added to the liquid culture medium containing brain and heart extract and yeast extract. The volume of the liquid culture medium was controlled at a ratio of 1:3 to the container volume. After thorough mixing, the culture was incubated at 37℃ and 5% CO2 with shaking at 120 rpm for 8–12 hours. After the bacterial culture was completed, samples were taken for purity testing according to the appendix of the 2015 edition of the Chinese Veterinary Pharmacopoeia. The samples should be pure. Simultaneously, viable cell counts were performed on the samples.

[0049] Preparation of concentrated bacterial culture: The fermentation broth was concentrated by centrifugation. The centrifugation conditions were: 3500 r / min, 4℃, centrifugation for 20 min. The bacterial precipitate was then collected and washed three times with phosphate buffer (0.01M, pH 7.2). The precipitate was then resuspended in sterile PBS to prepare a concentrated bacterial culture with a concentration of more than 10 billion cfu / ml.

[0050] Inactivation of bacterial solution: Add formaldehyde solution at 0.1% to 0.2% (v / v) of the concentrated bacterial solution and inactivate at 37°C for 8-10 hours, shaking several times during the process. Prepare inactivated antigen bacterial solution. The inactivation test shall be carried out in accordance with the appendix of the 2015 edition of the Chinese Veterinary Pharmacopoeia. There should be no sterile growth.

[0051] Preparation of immunoantigen: Place the Haemophilus bovis suspension in crushed ice, then place it in an ultrasonic cell disruptor and sonicate for 30 minutes at 200W for 5 seconds with a 3-second interval. Then freeze-thaw once, and sonicate again. Centrifuge the disrupted suspension at 4℃ and 12000r / min for 15 minutes, and collect the supernatant to obtain the immunoantigen.

[0052] Immunization: Rabbits weighing 2.25-3.0 kg with negative serum for Haemophilus bovis (before immunization) were selected as experimental animals. Their serum was collected for testing to verify that they did not have antigens and antibodies against bovine rotavirus, bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus, foot-and-mouth disease virus, bovine Akabane virus, etc. Basic immunization: 2 mL of prepared Haemophilus bovis immunogen was injected into the leg muscles of each rabbit at multiple sites.

[0053] Serum collection: 14 days after immunization, rabbit blood was collected aseptically, serum was separated, and the serum was filtered under pressure through a 0.45μm sterile filter membrane and then through a 0.22μm sterile filter membrane. The serum antibody titer was detected by agar diffusion test, and physical properties, sterility and specificity were tested. After passing the tests, the serum was aliquoted and stored at -80℃.

[0054] Potency test: The prepared positive serum of Haemophilus niger (3 serum samples, numbered 1-1, 1-2, and 1-3) was diluted at 1:2, 1:4, 1:8, 1:16, and 1:32, and tested using the agar diffusion test. The test results are shown in Table 3.

[0055] Table 3. Results of Haemophilus dormancy agar agar assay

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing positive serum of Haemophilus influenzae type 2 (BCI), characterized in that: The specific steps of the preparation method are as follows: S1. Preparation of culture media: Prepare solid culture medium Columbia blood agar basal medium and liquid culture medium Brain heart extract liquid medium, respectively; S2. Preparation of bacterial culture: The frozen bovine hemophagocytic HS / NMG / 2023 was streaked onto Columbia blood agar basal medium and cultured in a CO2 incubator; Single colonies that have grown were inoculated onto brain heart extract liquid culture medium, and cultured in a CO2 environment. The fermentation broth was obtained by shaking the culture on a shaker. S3. Preparation of concentrated bacterial culture: The bacterial culture was concentrated by centrifugation, and then the bacterial precipitate was collected and washed three times with phosphate buffer. The precipitate was then resuspended in sterile PBS to prepare concentrated bacterial culture. S4. Inactivation of bacterial solution: Add formaldehyde solution to bacterial solution for inactivation, and shake well during the process to prepare inactivated antigen bacterial solution; S5. Preparation of immunoantigen: Place the inactivated antigen bacterial solution in crushed ice, then place it in an ultrasonic cell disruptor for ultrasonic disruption, then freeze and thaw once, then ultrasonically disrupt once more, centrifuge the disrupted bacterial solution, and collect the supernatant to obtain the immunoantigen. S6. Immunization: Immunize healthy rabbits with immune antigens three times; S7. Serum collection: 14 days after the last immunization, rabbit blood was collected aseptically, and serum was separated. The serum was filtered twice under pressure through sterile filter membranes. The serum antibody titer was detected by agar diffusion test. Physical properties, sterility and specificity were tested. After passing the tests, the serum was aliquoted and stored to obtain positive serum for Haemophilus nigricans.

2. The method for preparing Haemophilus influenzae positive serum according to claim 1, characterized in that: The solid culture medium in S1 is Columbia blood agar basal medium containing 3%-10% defibrinated sheep blood, and the liquid culture medium is liquid culture medium containing 0.5%-1% yeast extract brain and heart extract.

3. The method for preparing positive serum of Haemophilus influenzae type 2 (HAIB) according to claim 1, characterized in that: The *Haemophilus bovis* HS / NMG / 2023 strain S2 was deposited on August 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC). The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The classification name is *Haemophilus bovis*, the accession number is CGMCC No. 31702, the CO2 content is 5%, and the culture temperature is 37℃.

4. The method for preparing Haemophilus influenzae positive serum according to claim 1, characterized in that: In S3, the centrifugation speed is 3500 r / min, the centrifugation temperature is 4℃, the centrifugation time is 20 min, the phosphate buffer is 0.01M, the pH is 7.2, and the concentration of the concentrated bacterial solution is above 10 billion cfu / ml.

5. The method for preparing Haemophilus influenzae positive serum according to claim 1, characterized in that: In step S4, the bacterial solution is taken and added to formaldehyde solution at 0.1% to 0.2% (v / v) of the bacterial solution volume, and then inactivated at 37°C for 8-10 hours.

6. The method for preparing Haemophilus influenzae positive serum according to claim 1, characterized in that: In S5, the ultrasonic disruption is performed using a program of 200W ultrasound for 5 seconds, with a 3-second interval, for 30 minutes. The centrifugation temperature is 4℃, the centrifugation speed is 12000r / min, and the centrifugation time is 15 minutes.

7. The method for preparing Haemophilus influenzae positive serum according to claim 1, characterized in that: The healthy rabbits in S6 weighed between 2.25 kg and 3.0 kg and were used as experimental animals. Their serum was collected for testing, and it was verified that they did not have antigens and antibodies such as bovine rotavirus, bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus, foot-and-mouth disease virus, and bovine Akabane virus.

8. The method for preparing Haemophilus influenzae positive serum according to claim 1, characterized in that: The S6 immunization was performed three times. The first immunization involved injecting 2 mL into multiple muscle points in the leg, and the remaining two immunizations were performed every 14 days, with the immunization dose increasing by 1 mL each time.

9. The method for preparing a positive serum of Haemophilus influenzae type 2 (HAIB) according to claim 1, characterized in that: The serum in S7 was filtered under pressure through a 0.45μm sterile filter membrane and a 0.22μm sterile filter membrane, respectively, and stored at -80℃.

10. The application of the positive serum for Haemophilus influenzae type 2 (HAIB) according to claim 1, characterized in that: The positive serum of Haemophilus suis is used to prepare reagents or kits for the detection or identification of Haemophilus suis. The positive serum of Haemophilus suis is used for specific tests, identification tests, exogenous virus tests or diagnoses of Haemophilus suis. The positive serum of Haemophilus suis is used to prepare Haemophilus suis positive serum standards.

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