Histophilic somniphilus, hemagglutination antigen, hemagglutination inhibition antibody detection method, kit and application of histophilic somniphilus

Abstract

The invention discloses a histophilic somniphilus, a hemagglutination antigen, a hemagglutination inhibition antibody detection method, a kit and application of the histophilic somniphilus and the hemagglutination antigen. The method comprises the following steps: screening a histophilic sleep bacteria Yunnan isolate ZY25055 with high hemagglutination activity, culturing, and carrying out beta-propiolactone inactivation to prepare the histophilic sleep bacteria hemagglutination antigen. 1% hydroformylated sheep red blood cells are prepared by collecting sheep blood. Immunizing goat posterior jugular vein blood sampling with beta-propiolactone inactivated histophilic sleep bacteria liquid for three times, and centrifugally separating serum to prepare histophilic sleep bacteria standard positive serum. The method and the kit for detecting the histophilic sleeping bacteria hemagglutination inhibition antibody are established by utilizing the prepared histophilic sleeping bacteria hemagglutination antigen, 1% hydroformylated sheep red blood cells and histophilic sleeping bacteria standard positive serum, the method has no cross reaction with positive serum of common pathogens of cattle and sheep, and the method has the advantages of strong specificity, simplicity and convenience in operation, no need of complex equipment and the like, and can be used for detecting the histophilic sleeping bacteria hemagglutination inhibition antibody. The kit can be used for detecting the histophilic somniphilus antibody in bovine serum, sheep serum and goat serum.

Description

Technical Field

[0001] This invention relates to the field of veterinary microbiology detection technology, specifically to a Yunnan isolate ZY25055 of *Histophilus somni* (China Center for Type Culture Collection, accession number: CCTCC No: M 20251686) with high hemagglutination activity, hemagglutination antigen, and a detection method, kit, and application of a hemagglutination inhibition antibody against *Histophilus somni* established using this strain. Background Technology

[0002] Danish scholars Angen et al. [1] In 2003, the previously misnamed Histophilus ovis was corrected. [2] Haemophilus agni [3] Haemophilus somnus [4] A reclassification revealed that these three bacterial species belong to the same species, which was named *Histophilus somni*. To date, *Histophilus somni* is the only member of the genus *Histophilus*. *Histophilus somni* primarily infects cattle, causing meningitis, pneumonia, abortion, mastitis, and epididymitis. [5-9] Reports of Histophilia sleeper infection in sheep are rare, but it can cause meningitis, pneumonia, abortion, and mastitis in sheep. [10-13] Reports of Histophilia sleeper infection in goats are rare. [14-15] There are currently no reports of Histoplasma dorsifluum infection in other animal species. Currently, there are reports of Histoplasma dorsifluum infection in cattle or sheep in a few countries, including the United States, Argentina, Brazil, Canada, Mexico, Ethiopia, and Hungary. [14, 16-18] There have also been reports of Histophilia hirsuta infection in cattle, goats, and sheep in my country. [19-22] Histophilia slumberii infection causes significant economic losses to the global cattle, sheep, and goat farming industries.

[23] Therefore, establishing a rapid detection method for Histophilia hygrophila is of great significance for the prevention and control of its infection.

[0003] Regarding antibody detection, current domestic and international methods for detecting Histoplasma hirsutum antibodies mainly include agglutination tests (including plate agglutination test, microagglutination test, and tube agglutination test), complement fixation tests, and enzyme-linked immunosorbent assays (ELISA). [24-27] There is currently no antibody detection method for the hemagglutination inhibition test. However, the existing Histoplasma dormancy agglutination assay uses Histoplasma dormancy bacterial suspension as the antigen. The Histoplasma dormancy antigen-Histophilia dormancy antibody complex produced by the agglutination reaction consists of small, light grayish-white particles, which are difficult to observe and interpret with the naked eye. This results in a significant technical deficiency of low sensitivity. Furthermore, this antibody detection method also suffers from significant technical deficiency of poor specificity.

[28] This limitation restricts its application in detecting Histoplasma hygrophila antibodies. Existing complement fixation tests for Histoplasma hygrophila antibodies are extremely complex, with overly stringent requirements for reagent preparation and quantification. Furthermore, this method is susceptible to infection by other pathogens, leading to the presence of antigen-antibody complexes in the blood (or serum), which in turn results in a large number of false positives. [29-30] The numerous limitations of the aforementioned complement fixation test for detecting Histoplasma dormancy antibodies significantly restrict its application in detecting these antibodies. Furthermore, existing ELISA methods for detecting Histoplasma dormancy antibodies have not been validated for specificity using positive sera from common bovine and ovine pathogens such as Brucella, Pasteurella, Peutz-Russian peste des petits ruminants virus, ovine poxvirus, Clostridium perfringens, Mycoplasma caprineis, Escherichia coli, Salmonella, Staphylococcus, and Streptococcus. [24-27] This leads to a serious technical deficiency in the existing ELISA method for detecting Histoplasma dormans antibodies, resulting in poor specificity and limiting its application in this area. More importantly, existing agglutination, complement fixation, and enzyme-linked immunosorbent assay (ELISA) methods for detecting Histoplasma dormans antibodies are primarily used for bovine serum antibody detection, rarely for sheep antibody detection, and not for goat antibody detection. This significantly limits the application of these three existing methods for detecting Histoplasma dormans antibodies in bovine, sheep, and goat serum antibodies.

[0004] The surface of *Histomosomalemia dormans* contains filamentous hemagglutinin (FHA). [28, 31-33] It can agglutinate animal red blood cells, while most common bacteria and viruses in cattle and sheep (including Brucella abortus, Pasteurella multocida, Clostridium perfringens, Hemolytic Mansonia, Corynebacterium pseudotuberculosis, Escherichia coli, Salmonella enterica, Staphylococcus aureus, Streptococcus multifiliis, Cryptobacterium pyogenes, Mycoplasma bovis, Mycoplasma capsulatum, peste des petits ruminants virus, sheep stomatitis virus, and foot-and-mouth disease virus, etc.) do not have filamentous hemagglutinin protein on their surface and cannot agglutinate animal red blood cells.

[0005] References

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[26] Stephens LR, Little PB, Wilkie BN, et al. Humoral immunity inexperimental thromboembolic meningoencephalitis in cattle caused by Haemophilus somnus[J]. Am J Vet Res, 1981, 42(3): 468-473.

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[27] Pan Y, Fisher T, Olk C, et al. Detection of antibodies to the biofilm exopolysaccharide of Histophilus somni following infection in cattleby enzyme-linked immunosorbent assay[J]. Clin Vaccine Immunol, 2014, 21(10):1463-1467.

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[29] Zhu Liangquan, Wang Fang, Jiang Hui, et al. Comparative study on the diagnostic methods of complement fixation test for brucellosis in animals [J]. Chinese Journal of Veterinary Medicine, 2016, 36(2): 357-361.

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[30] Li Xiaojun, Deng Haiyan, Huang Jieying, et al. Research progress on laboratory diagnostic techniques for avian chlamydia diseases [J]. China Animal Quarantine, 2022, 39(12): 104-111.

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[31] Zekarias B, O'Toole D, Lehmann J, et al. Histophilus somni IbpAFic cytotoxin is conserved in disease strains and most carrier strains from cattle, sheep and bison[J]. Vet Microbiol, 2011, 149(1-2): 177-185.

[0037]

[32] Shirbroun R M. Histophilus somni: Antigenic and genomic changes relevant to bovine respiratory disease[J]. Vet Clin North Am Food Anim Pract, 2020, 36(2): 279-295.

[0038]

[33] Angen Ø. Taxonomy of Histophilus somni[J]. Curr Top MicrobiolImmunol, 2016, 396: 1-14. Summary of the Invention

[0039] The technical problem to be solved by this invention is to overcome the lack of detection methods or kits for hemagglutination inhibition antibodies against Histoplasma dormatum in the existing technology, as well as the many major technical defects of existing detection methods for Histoplasma dormatum antibodies, such as poor specificity, complex operation, and limitation to bovine serum antibody detection only to sheep and goat serum antibody detection, and limitation to laboratory detection only to field detection in cattle, sheep, and goat farms. This invention provides a Yunnan isolate ZY25055 of Histoplasma dormatum with high hemagglutination activity (CCTCC accession number: M 20251686, accession date: July 23, 2025), and a detection method, kit, and application for Histoplasma dormatum hemagglutination inhibition antibodies established using this strain. This antibody detection method has many advantages, including high specificity, simple operation, speed, low cost, qualitative and relative quantitative detection of Histophilia hygrophila antibodies, antibody detection in bovine serum, sheep serum and goat serum, and application in laboratories as well as on-site detection in cattle farms, sheep farms and goat farms.

[0040] The first objective of this invention is to provide a Yunnan isolate ZY25055 of Histotrophic dormancy bacteria with high hemagglutination activity. Under the same bacterial concentration conditions, the hemagglutination activity of ZY25055 of Histotrophic dormancy bacteria in Yunnan Province is more than twice that of other Histotrophic dormancy bacteria isolates.

[0041] The second objective of this invention is to provide a method and kit for detecting *Histoplasma dormantis* hemagglutination inhibition antibodies using the Yunnan isolate ZY25055. The method includes the following steps: using bovine serum, sheep serum, and goat serum as the detection targets, a hemagglutination inhibition reaction is performed using the aforementioned method or kit. After the reaction, under the conditions that aldehyde-treated sheep erythrocytes in the negative control wells (without serum) are completely agglutinated and aldehyde-treated sheep erythrocytes in the blank control wells (without serum and hemagglutination antigen) are completely precipitated at the bottom center of the wells, the hemagglutination inhibition titer of the serum is determined by the maximum dilution factor that completely inhibits the agglutination of aldehyde-treated sheep erythrocytes. When the hemagglutination inhibition titer of the serum is less than or equal to 8, it is considered negative for *Histoplasma dormantis* antibodies; when the hemagglutination inhibition titer of the serum is greater than or equal to 16, it is considered positive for *Histoplasma dormantis* antibodies. A higher hemagglutination inhibition titer indicates a higher antibody concentration in the serum, thus achieving qualitative and quantitative detection of *Histoplasma dormantis* antibodies. The method or kit for detecting hemagglutination inhibition antibodies against Histophilia hygrophila described above can be applied to the detection of bovine serum antibodies, as well as sheep and goat serum antibodies; it can be applied to laboratory testing as well as on-site testing at cattle farms, sheep farms, and goat farms.

[0042] (1) The optimal conditions for the reaction between the hemagglutination antigen of Histophilia dormans and the antibody in bovine and ovine serum were: 30℃ for 30 min; the optimal conditions for hemagglutination inhibition after the addition of 1% aldehyde-treated sheep red blood cells were: 30℃ for 60 min.

[0043] (2) Qualitative test result judgment criteria: Under the condition that the aldehyde-treated sheep red blood cells in the negative control well (without serum) are completely agglutinated and the aldehyde-treated sheep red blood cells in the sodium phosphate buffer blank control well (without serum and hemagglutination antigen) are completely precipitated at the bottom center of the well, when the hemagglutination inhibition titer of the tested serum is less than or equal to 8, it is judged as negative for Histoplasma dormatum antibody; when the hemagglutination inhibition titer of the tested serum is greater than or equal to 16, it is judged as positive for Histoplasma dormatum antibody.

[0044] (3) Criteria for judging relative quantitative detection results: Under the conditions that the aldehyde-treated sheep red blood cells in the negative control well (without serum) are completely agglutinated and the aldehyde-treated sheep red blood cells in the sodium phosphate buffer blank control well (without serum and hemagglutination antigen) are completely precipitated at the center of the bottom of the well, when the hemagglutination inhibition titer of the tested serum is greater than or equal to 16, it is judged as positive for Histophilia hygrophila antibody, and the higher the hemagglutination inhibition titer, the higher the antibody concentration in the serum.

[0045] Mechanism and beneficial effects of the present invention:

[0046] This invention utilizes the unique characteristic of *Histoplasma dormantifolium* bacteria possessing filamentous hemagglutinin proteins on their surface, which can agglutinate animal erythrocytes. A Yunnan isolate of *Histoplasma dormantifolium*, ZY25055, exhibiting high hemagglutination activity, was screened. Hemagglutination antigens were prepared using bacterial suspensions of *Histoplasma dormantifolium* ZY25055 inactivated with β-propiolactone. β-propiolactone, a nucleic acid inactivating agent, acts directly on the pathogen's nucleic acid without affecting the outer membrane proteins, thus preserving the pathogen's immunogenicity and the hemagglutination activity of the filamentous hemagglutinin proteins. *Histoplasma dormantifolium* standard positive serum was prepared by immunizing goats with *Histoplasma dormantifolium* ZY25055. Sheep erythrocytes were prepared from sheep blood and aldehyde-treated with glutaraldehyde to prepare 1% aldehyde-treated sheep erythrocytes. Using prepared Histoplasma dormantii hemagglutination antigen, Histoplasma dormantii standard positive serum, and 1% aldehyde-treated sheep erythrocytes, a detection method and kit for detecting Histoplasma dormantii hemagglutination inhibition antibodies were established for the first time both domestically and internationally. This detection method has advantages such as high specificity, simple operation, and no need for complex equipment (only a common incubator is required). It can be applied to detect Histoplasma dormantii antibodies in bovine serum, sheep serum, and goat serum. After the reaction, the hemagglutination inhibition reaction results are directly observed with the naked eye, allowing for qualitative and relative quantitative detection of Histoplasma dormantii antibodies in the tested serum. The result interpretation criteria are... The standard is as follows: Under the conditions that aldehyde-treated sheep erythrocytes are completely agglutinated in the negative control well (without serum) and aldehyde-treated sheep erythrocytes are completely precipitated at the center of the bottom of the sodium phosphate buffer blank control well (without serum and hemagglutination antigen), the hemagglutination inhibition titer of the test serum is determined by the maximum dilution factor that completely inhibits the agglutination of aldehyde-treated sheep erythrocytes. When the hemagglutination inhibition titer of the test serum is less than or equal to 8, it is judged as negative for Histoplasma dormatum antibody; when the hemagglutination inhibition titer of the test serum is greater than or equal to 16, it is judged as positive for Histoplasma dormatum antibody. The higher the hemagglutination inhibition titer, the higher the antibody concentration in the serum.

[0047] The detection method or kit for hemagglutination inhibition antibodies against histophilia established in this invention has the following advantages:

[0048] (1) High specificity: Because the surface of *Histoplasma dormans* has filamentous hemagglutinin protein, which can agglutinate animal red blood cells, specific antibodies against filamentous hemagglutinin protein can undergo antigen-antibody neutralization reaction with the filamentous hemagglutinin protein on the surface of *Histoplasma dormans*, resulting in a positive hemagglutination inhibition reaction. However, most common bacteria and viruses in cattle and sheep (including *Brucella abortus*, *Pasteurella multocida*, *Clostridium perfringens*, *Mannioblastus*, *Corynebacterium pseudotuberculosis*, *Escherichia coli*, *Salmonella enterica*, *Staphylococcus aureus*, *Streptococcus multifiliis*, *Cryptospirosis*, *Mycoplasma bovis*, *Mycoplasma capsulatum*, peste des petits ruminants virus, sheep pox virus, and foot-and-mouth disease virus, etc.) do not have filamentous hemagglutinin protein on their surface. Theoretically, the serum antibodies of these common bacteria and viruses in cattle and sheep do not contain filamentous hemagglutinin protein antibodies and cannot react with the filamentous hemagglutinin protein on the surface of *Histoplasma dormans*. The protein undergoes an antigen-antibody neutralization reaction, resulting in a negative hemagglutination inhibition reaction result (i.e., no cross-reaction). Therefore, theoretically, the hemagglutination inhibition antibody detection method or kit established in this invention has strong specificity. In fact, the specificity test in this invention further confirms that the hemagglutination inhibition antibody detection method or kit for *Histopspermia suis* has no cross-reaction with positive sera of *Brucella abortus*, *Pasteurella multocida*, *Clostridium perfringens*, *Mannioblastus*, *Corynebacterium pseudotuberculosis*, *Escherichia coli*, *Salmonella enterica*, *Staphylococcus aureus*, *Streptococcus multifiliis*, *Cryptospirosis*, *Mycoplasma bovis*, *Mycoplasma capsulatum*, peste des petits ruminants virus, sheep stomatitis virus, and foot-and-mouth disease virus. It has strong specificity and overcomes the huge technical defects of poor specificity in existing agglutination tests, complement fixation tests, and enzyme-linked immunosorbent assays for detecting *Histopspermia suis* antibodies.

[0049] (2) Simple operation and easy result interpretation: The detection method or kit for the hemagglutination inhibition antibody of Histoplasmosis suis established in this invention only requires a common constant temperature incubator (an incubator is not required when the room temperature is 25-32℃), and does not require complex equipment. The operation is simple, overcoming the huge technical defects of existing complement fixation test and enzyme-linked immunosorbent assay for the detection of Histoplasmosis suis antibodies, which are complicated to operate and require expensive equipment. In addition, in the result interpretation of the detection method for the hemagglutination inhibition antibody of Histoplasmosis suis established in this invention, the red blood cells are brightly colored, and it is easy to directly observe the red blood cell agglutination or precipitation at the bottom center of the well with the naked eye to determine the detection result, overcoming the huge technical defect of existing agglutination Histoplasmosis antibody detection methods where the result interpretation is difficult.

[0050] (3) Applied to qualitative and relative quantitative detection of Histoplasma sleeper antibodies: The Histoplasma sleeper hemagglutination inhibition antibody detection method or kit established in this invention can be used for both qualitative detection and relative quantitative detection of Histoplasma sleeper antibodies, overcoming the technical defect that the existing enzyme-linked immunosorbent assay (ELISA) Histoplasma sleeper antibody detection method cannot be applied to relative quantitative detection of antibodies.

[0051] (4) Wide range of detection targets: The detection method or kit for the hemagglutination inhibition antibody of Histophilia hymophilia established in this invention can be applied to bovine serum antibody detection as well as sheep and goat serum antibody detection, overcoming the huge technical defect that the existing agglutination test, complement fixation test and enzyme-linked immunosorbent assay for Histophilia hymophilia antibody detection methods can only be applied to bovine serum antibody detection.

[0052] (5) Wide range of applications: The detection method or kit for the hemagglutination inhibition antibody of Histophilia hymophilia established in this invention can be applied to laboratory testing as well as on-site testing in cattle farms and sheep farms. This overcomes the technical defects of existing complement fixation test and enzyme-linked immunosorbent assay (ELISA) for the detection of Histophilia hymophilia antibodies, which cannot be applied to on-site testing in cattle farms, sheep farms and goat farms.

[0053] In summary, the detection method or kit for *Histophilus dormatus* hemagglutination inhibition antibodies established by this invention is highly specific, easy to operate, time-saving, and requires no complex equipment. It can be applied to the detection of antibodies in bovine serum, sheep serum, and goat serum; it can be applied to laboratory testing as well as on-site testing in cattle farms, sheep farms, and goat farms, providing a reliable and rapid method for the detection of antibodies against *Histophilus dormatus* infection in cattle, sheep, and goats. Attached Figure Description

[0054] Figure 1 This is a schematic diagram showing the specific location of each well in a 96-well V-shaped micro-reaction plate.

[0055] Figure 2 The results show the effects of different inactivating agents on the hemagglutination titer of Histophilia hygrophila hemagglutination antigen.

[0056] Figure 3 The results show the screening results for Histophilia nocturia strains with high hemagglutination activity.

[0057] Figure 4 Results of optimized reaction conditions for the detection method of hemagglutination inhibition antibody for Histophilia hygrophila.

[0058] Figure 5 The results were used to determine the cutoff value of hemagglutination inhibition titer for positive serum in the detection method of histophilia hemagglutination inhibition antibody.

[0059] Figure 6 This is a specific test result for the detection method of hemagglutination inhibition antibody against Histophilia septicemia.

[0060] Figure 7 The result was validated for 4 units of Histotrophoblastic hemagglutination antigen.

[0061] Figure 8Qualitative detection results of Histophilia hygrophila antibodies in serum samples from cattle, sheep, and goats.

[0062] Figure 9 The results show the relative quantitative detection of Histophilia hygrophila antibodies in serum samples from cattle, sheep, and goats. Detailed Implementation

[0063] The following embodiments are further illustrations of the present invention, but not limitations thereof. Unless otherwise specified, the reagents, kits, consumables, and instruments used in the present invention are conventional reagents, kits, consumables, and instruments in this technical field.

[0064] Example 1: Isolation and identification of ZY25055, a type of hibernating histophilic bacterium derived from goats in Yunnan Province

[0065] In January 2025, a respiratory disease occurred in goats at a goat farm in Kunming, Yunnan Province. Lung samples were collected from dead goats and inoculated onto Columbia agar medium. After culturing at 37°C with 5% CO2 for 24 hours, a large number of translucent, pinhead-sized colonies grew. Single colonies were picked and inoculated onto Columbia agar medium and cultured at 37°C with 5% CO2 for 24 hours to obtain isolate ZY25055. The isolate is a carbon dioxide-loving bacterium that requires 5%–10% CO2 to grow.

[0066] 1. Gram staining identification

[0067] Colonies were picked up using an inoculation loop and transferred to a glass slide to prepare a bacterial smear. The smear was then stained with a bacterial Gram staining kit, and the bacterial morphology was observed under a microscope. The results showed that isolate ZY25055 was a Gram-negative micrococcus. The colony and bacterial morphology characteristics were similar to those of the histophila dormantivia type strain ATCC 43625. T Consistent.

[0068] 2. Biochemical identification

[0069] Isolator ZY25055 was inoculated onto Columbia agar plates and incubated at 37°C for 24 h under 5% CO2 conditions. Colonies were eluted with sterile physiological saline, and the bacterial concentration was adjusted to 0.5 McFarland units with sterile physiological saline. Biochemical identification of the isolate was performed using API ID32 E biochemical identification strips. The results showed that isolate ZY25055 was positive for oxidase and ornithine decarboxylase; negative for catalase, urease, and arginine dihydrolase; it fermented glucose to produce acid, but did not ferment D-sucrose, D-maltose, D-trehalose, L-arabinose, L-rhamnose, D-mannitol, inositol, D-sorbitol, or calendula alcohol to produce acid. Most of the biochemical characteristics of isolate ZY25055 were similar to those of the dormant histophilic bacteria type strain ATCC 43625. T Consistent.

[0070] 3. Identification by nucleotide sequence analysis of 16S rRNA gene

[0071] Genomic DNA was extracted from isolate ZY25055 using a bacterial genomic DNA extraction kit and used as a PCR template. The 16S rRNA gene nucleotide sequence was amplified by PCR using universal primers 27F / 1492R (annealing temperature 57℃). The PCR amplification product was sequenced using primers 27F / 1492R, and the 16S rRNA gene nucleotide sequence of isolate ZY25055 was obtained as shown in SEQ ID NO.1.

[0072] SEQ ID NO.1:

[0073]

[0074] The 16S rRNA gene nucleotide sequence of isolate ZY25055 was submitted to the GenBank database and obtained the gene accession number PX396483. BLAST alignment of the 16S rRNA gene nucleotide sequence of isolate ZY25055 in the NCBI database showed that isolate ZY25055 is similar to the Histoplasma hirsutum type strain ATCC 43625. T The nucleotide sequence of the 16S rRNA gene (GenBank accession number NR_112238.1) showed 99.6% similarity, and it was further identified as Histomophilus hibernationis.

[0075] Based on the above colony and cell morphological characteristics, biochemical characteristics, and 16S rRNA gene nucleotide sequence similarity identification results, the isolated bacterium ZY25055 was finally identified as a hibernating histophilic bacterium.

[0076] The isolated and identified histophilic bacteria ZY25055 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251686, located at Wuhan University, Wuhan, China, on July 23, 2025.

[0077] Example 2: Preparation of 1% aldehyde-treated sheep erythrocytes

[0078] Blood was collected from Merino sheep. Glass beads were added and the mixture was gently shaken to remove fibrin. The blood was then added to 10 volumes of 0.05 mol / L pH 7.2 sodium phosphate buffer (Na₂HPO₄ / NaH₂PO₄ buffer) and mixed. The mixture was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. Next, 10 volumes of 0.05 mol / L pH 7.2 sodium phosphate buffer were added to the erythrocyte sedimentation volume and mixed. The mixture was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. This process was repeated three times to wash the erythrocytes. After discarding the supernatant, 5 volumes of 0.05 mol / L pH 7.2 sodium phosphate buffer at 4°C were added to the erythrocyte sedimentation volume and mixed. The mixture was gently shaken, and 5 volumes of 2% glutaraldehyde solution at 4°C were added dropwise. The mixture was then gently shaken at 4°C for 30 min to induce aldehyde reaction. The mixture was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The erythrocyte sedimentation volume was then washed with 10 volumes of 0.05 mol / L pH 7.2 sodium phosphate buffer. Resuspend the erythrocytes in sodium phosphate buffer (pH 7.2) at 1000 rpm, centrifuge and discard the supernatant. Repeat the above steps to wash the aldehyde-modified sheep erythrocytes three times. After discarding the supernatant, resuspend the erythrocyte pellet in 0.05 mol / L sodium phosphate buffer (pH 7.2) at 99 times the volume of the erythrocyte pellet. Then add sodium thimerosal solution to a final concentration of 0.005% to obtain 1% aldehyde-modified sheep erythrocytes. Store at 4°C for later use.

[0079] Example 3: Optimization of the inactivation method for hibernating histophilia

[0080] 1. Preparation of histophilic bacteria suspension

[0081] The Yunnan isolate ZY25055 of *Histotrophozoites hirsuta* (China Center for Type Culture Collection No.: M 20251686), obtained from Example 1, was inoculated onto Columbia agar medium and cultured at 37°C with 5% CO2 for 24 h. Colonies were washed away with sterile physiological saline and mixed thoroughly to obtain a bacterial suspension. The bacterial concentration was determined to be 4 × 10⁻⁶ using the plate count method. 9 CFU / mL (Colony Forming Units, CFU), adjust the bacterial concentration to 2 × 10⁻⁶ CFU using sterile physiological saline. 9 CFU / mL refers to the uninactivated histophilic hemagglutination antigen.

[0082] 2. Preparation of β-propiolactone-inactivated histiocytic hemagglutination antigen

[0083] In 1 mL of the above 2×10 9Add 2 μL of β-propiolactone (final β-propiolactone concentration in the bacterial culture is 0.2%) to CFU / mL *Histophilus dormatus* bacterial culture, mix well, inactivate at 25℃ for 5 h, centrifuge at 10000 rpm and discard the supernatant, add 10 mL of sterile physiological saline to resuspend the precipitate, centrifuge at 10000 rpm and discard the supernatant, add 1 mL of sterile physiological saline to resuspend the precipitate and mix well to obtain β-propiolactone-inactivated *Histophilus dormatus* hemagglutination antigen. Spread 10 μL of the inactivated bacterial culture onto Columbia agar medium and incubate at 37℃ with 5% CO2 for 24 h. No aseptic growth indicates complete inactivation. Add sodium thimerosal to the β-propiolactone-inactivated *Histophilus dormatus* hemagglutination antigen to a final concentration of 0.005% and store at 4℃ for later use.

[0084] 3. Preparation of formaldehyde-inactivated histiocytic hemagglutination antigen

[0085] In 1 mL of the above 2×10 9 Add 10 μL of 40% formaldehyde solution (final formaldehyde concentration in the bacterial culture is 0.4%) to a CFU / mL Histoplasma hirsutum bacterial culture, mix well, and inactivate at 25°C for 5 h. Centrifuge at 10,000 rpm and discard the supernatant. Add 10 mL of sterile physiological saline, centrifuge at 10,000 rpm and discard the supernatant. Resuspend the precipitate in 1 mL of sterile physiological saline and mix well to obtain formaldehyde-inactivated Histoplasma hirsutum hemagglutination antigen. Spread 10 μL of the inactivated bacterial culture onto Columbia agar medium and incubate at 37°C with 5% CO2 for 24 h. No aseptic growth indicates complete inactivation. Add sodium thimerosal to the formaldehyde-inactivated Histoplasma hirsutum hemagglutination antigen to a final concentration of 0.005% and store at 4°C for later use.

[0086] 3. Screening of the optimal inactivating agent

[0087] To evaluate the effects of β-propiolactone and formaldehyde, two inactivating agents, on the hemagglutination titer of *Histophilia hirsuta*, a hemagglutination test was performed on a disposable 96-well V-shaped microplate to determine the hemagglutination titer. To facilitate the description of the specific location of each well in the 96-well V-shaped microplate, the 8 rows were numbered A through H, and the 12 columns were numbered 1 through 12. A schematic diagram showing the specific location of each well in the numbered 96-well V-shaped microplate is shown below. Figure 1 Well A4 represents the well in the 4th column of the 1st row; wells A1 to A8 represent the wells in the 1st to 8th columns of the 1st row; and wells A1 to B12 represent the wells in the 1st to 12th columns of the 1st and 2nd rows. The specific steps of the blood coagulation test are as follows:

[0088] ① Take a 96-well V-shaped microplate and use a pipette to add 25 μL of 0.05 mol / L sodium phosphate buffer (pH=7.2) to each of wells A1 to C12;

[0089] ② Add the above-mentioned uninactivated, β-propiolactone-inactivated, and formaldehyde-inactivated Histoplasma hirsutum hemagglutination antigens to wells A1 to C1, respectively. Mix by pipetting and aspirating 5 times with an eight-channel pipette, and then add 25 μL of each antigen to wells A2 to C2. Mix by pipetting and aspirating 5 times with each antigen, and then add 25 μL of each antigen to wells A3 to C3. And so on. Dilute the uninactivated, β-propiolactone-inactivated, and formaldehyde-inactivated Histoplasma hirsutum hemagglutination antigens 2-fold to wells A11 to C11, respectively. Mix by pipetting and aspirating 5 times with each of wells A11 to C11, and then discard 25 μL of each antigen.

[0090] ③ Wells A12 to C12 are used as negative control wells without hemagglutination antigen;

[0091] ④ Add 25 μL of 1% aldehyde-treated sheep red blood cells to each of wells A1 to C12 in a suspended position (without the pipette tip touching the liquid in the well);

[0092] ⑤ Place the 96-well V-shaped micro-reaction plate on a microplate shaker and shake for 1 min. After standing at 30℃ for 60 min, observe the results. Under the condition that the aldehyde-treated sheep red blood cells in wells A12 to C12 (negative control wells) are completely precipitated at the center of the bottom of the well, the hemagglutination titer of the hemagglutination antigen is determined by the maximum dilution factor that can completely agglutinate the aldehyde-treated sheep red blood cells.

[0093] ⑥ According to the above criteria, the hemagglutination titers of non-inactivated, β-propiolactone-inactivated, and formaldehyde-inactivated Histoplasma hirsutum hemagglutination antigens were 512, 512, and 32, respectively. Figure 2 The results indicate that formaldehyde inactivation significantly reduces the hemagglutination titer of *Histophilus dormatus* (from 512 to 32), while β-propiolactone inactivation does not affect the hemagglutination titer (which remains at 512 before and after inactivation). These results suggest that β-propiolactone is the optimal inactivating agent for *Histophilus dormatus*, and the optimal inactivation method is at 2 × 10⁻⁶ ppm. 9 Adding β-propiolactone to a final concentration of 0.2% to the CFU / mL bacterial culture and inactivating it at 25℃ for 5 h under these inactivation conditions can completely inactivate Histomophilus dormatus without affecting its hemagglutination titer.

[0094] Example 4: Screening of highly hemagglutinating histophilic bacteria strains

[0095] 1. Strains

[0096] Sixteen strains of *Histotrophozoa hygrophila* were isolated from cattle, sheep, and goats in Yunnan Province and are deposited in the Key Laboratory of Tropical and Subtropical Animal Viral Diseases, Yunnan Academy of Animal Science and Veterinary Medicine. The source of the strains and their GenBank accession numbers for 16S rRNA genes are shown in Table 1.

[0097] Table 1. Sources of *Histomophilus dormatoides* strains and screening results of strains with high hemagglutination activity

[0098]

[0099] 2. Preparation of β-propiolactone-inactivated histiocytic hemagglutination antigen

[0100] The optimal method for inactivating Histophilia nigricans as described in Example 3 (at 2 × 10) 9 Hemagglutination antigens for 16 strains of Histomophilus dormatus were prepared by adding β-propiolactone to a final concentration of 0.2% in CFU / mL bacterial culture and inactivating the bacteria at 25℃ for 5 h.

[0101] 3. Screening of highly hemagglutinating histophilic bacteria strains

[0102] According to the hemagglutination titer determination method for the hemagglutination antigen of *Histophilus dormatus* as described in Example 3, the hemagglutination titers of the above 16 *Histophilus dormatus* strains were determined. The order of adding the hemagglutination antigen samples of the 16 *Histophilus dormatus* strains was as follows: hemagglutination antigen samples of strains YN240861, YN240862, YN240863, YN240907, YN240908, YN240909, YN2409010 and YN24101 were added to wells A1 to H1 of the 96-well V-shaped microplate No. 1, respectively; hemagglutination antigen samples of strains YN24102, YN24126, YN24127, ZY25052, ZY25053, ZY25054, ZY25055 and ZY25067 were added to wells A1 to H1 of the 96-well V-shaped microplate No. 2, respectively. The test results showed that the hemagglutination titers of the 16 Histophilus dormans isolates ranged from 64 to 512, with strain ZY25055 exhibiting the highest hemagglutination titer (512), which was more than twice that of the other Histophilus dormans isolates (Table 1). Figure 3 This indicates that the *Histophilus dormatus* isolate ZY25055 (China Center for Type Culture Collection strain accession number: CCTCC No: M 20251686) is most suitable for preparing *Histophilus dormatus* hemagglutination antigen.

[0103] Example 5: Preparation of high hemagglutination titer Histophilia hygrophila hemagglutination antigen

[0104] The Yunnan isolate ZY25055 of *Histophilus hirsutus* (China Center for Type Culture Collection No.: M 20251686), which showed high hemagglutination titer (high hemagglutination activity) as screened in Example 4, was inoculated into Columbia agar medium and cultured at 37°C with 5% CO2 for 24 h. Colonies were washed away with sterile physiological saline and mixed thoroughly to obtain a bacterial suspension. The bacterial concentration was determined to be 4 × 10⁻⁶ using the plate count method. 9 CFU / mL, adjust the bacterial concentration to 2×10⁻⁶ CFU / mL with sterile physiological saline. 9CFU / mL yielded 50 mL of uninactivated Histoplasma hygrophila hemagglutination antigen. Then, following the optimal inactivation method for Histoplasma hygrophila described in Example 3, the concentration was increased to 2 × 10⁻⁶ CFU / mL. 9 CFU / mL bacterial culture was inactivated at 25°C for 5 h by adding β-propiolactone to a final concentration of 0.2%. The supernatant was discarded after centrifugation at 10,000 rpm, and the precipitate was resuspended in 50 mL of sterile physiological saline. The mixture was then centrifuged again at 10,000 rpm, the supernatant was discarded, and the precipitate was resuspended in 50 mL of sterile physiological saline and mixed well. Finally, sodium thimerosal was added to a final concentration of 0.005%, yielding 50 mL of β-propiolactone-inactivated Histomonia solani hemagglutination antigen (bacterial concentration 2 × 10⁻⁶). 9 CFU / mL (containing sodium thimerosal to a final concentration of 0.005%) is the *Histoplasma sleepans* hemagglutination antigen used in the detection method or kit for *Histoplasma sleepans* hemagglutination inhibition antibody described in this invention. The hemagglutination titer of this *Histoplasma sleepans* hemagglutination antigen was determined to be 512 using the hemagglutination test method described in Example 3. It was then diluted 1:128 (where 128 = 512 / 4) in 0.05 mol / L sodium phosphate buffer at pH 7.2 to obtain 4 units of *Histoplasma sleepans* hemagglutination antigen.

[0105] Example 6: Preparation of Standard Positive Serum for Histophilia hygrophila

[0106] 1. Preparation of histophilic bacteria immunogen

[0107] The Yunnan isolate ZY25055 of *Histophila dormantivia* (China Center for Type Culture Collection, accession number: CCTCC No: M 20251686) was inoculated into Columbia agar medium and cultured at 37°C with 5% CO2 for 24 h. Colonies were washed away with sterile physiological saline and mixed to obtain 20 mL of bacterial suspension. The bacterial concentration was determined to be 4 × 10⁻⁶ using the plate count method. 9 CFU / mL, add 40 μL β-propiolactone (final β-propiolactone concentration in bacterial culture is 0.2%), inactivate at 25℃ for 5 h, centrifuge at 10000 rpm, discard the supernatant, add 20 mL sterile physiological saline to resuspend the precipitate, which is the Histoplasma hirsutum immunogenic antigen, with a concentration of 4 × 10⁻⁶. 9 CFU / mL.

[0108] 2. Preparation of Standard Positive Serum for Histophilia hygrophila

[0109] The above-mentioned Histophilia hygrophila immunogen (concentration of 4×10) was used to treat the above-mentioned Histophilia hygrophila immunogen. 9A 12-month-old Tougamburg dairy goat was vaccinated with 0.5 mL of serum subcutaneously and 0.5 mL intramuscularly via the neck. This was repeated every 10 days for a total of three vaccinations. Ten days after the third vaccination, 100 mL of blood was collected from the jugular vein and placed in a centrifuge tube. The tube was tilted slightly and allowed to stand until the serum was completely separated. The tube was then centrifuged at 10,000 rpm for 10 minutes, and 25 mL of the supernatant was collected. 25 μL of 5% sodium thimerosal solution was added and mixed thoroughly (the final concentration of sodium thimerosal in the serum was 0.005%) to obtain the standard positive serum for Histoplasma hirsutum.

[0110] Example 7: Establishment of a method or kit for detecting hemagglutination inhibition antibodies against histophilia hygrophila

[0111] 1. Optimization of reaction conditions for the detection method of hemagglutination inhibition antibody against histophilia in sleep

[0112] To optimize the optimal temperature and time for the reaction between *Histoplasma dormatum* hemagglutination antigen and bovine / ovine serum antibodies in the hemagglutination inhibition assay, as well as the optimal temperature and time for the hemagglutination inhibition reaction after the introduction of 1% aldehyde-treated sheep erythrocytes, the hemagglutination inhibition assay was performed, with the following steps:

[0113] ① Add 25 μL of 0.05 mol / L sodium phosphate buffer (pH=7.2) to each of wells A1 to A12 of the 96-well V-shaped microplate No. 1;

[0114] ② Add the standard positive serum of Histophilia dormantii to well A1, mix by pipetting 5 times, and then add 25 μL to well A2. Mix by pipetting 5 times and then add 25 μL to well A3, and so on. Dilute the standard positive serum 2-fold to well A10. Mix by pipetting 5 times in well A10 and then discard 25 μL. Well A11 is the undiluted serum well as the negative control. Add 25 μL of 0.05 mol / L sodium phosphate buffer (pH=7.2) to well A12 as the blank control.

[0115] ③ Add 25 μL of the 4 units of Histotrophoblastic hemagglutination antigen described in Example 5 to each of wells A1 to A11 (without the pipette tip touching the liquid in the well), place on a microplate shaker and shake for 1 min, then let stand at 25°C (room temperature) for 30 min to react;

[0116] ④ Add 1% aldehyde-treated sheep red blood cells as described in Example 2 to each well (without the pipette tip touching the liquid in the well), suspended in the air, and shaken on a microplate shaker for 1 min. Incubate at 25°C (room temperature) for 60 min. Observe the hemagglutination inhibition titer directly with the naked eye. The results show that, under the reaction conditions at 25°C, the hemagglutination inhibition titer of the standard positive serum is 256 (…).Figure 4 Reaction plate #1).

[0117] ⑤ Repeat steps ① to ④ above (changing 25℃ (room temperature) in steps ③ and ④ to 30℃), and determine the hemagglutination inhibition titer of standard positive serum under 30℃ reaction conditions on a 96-well V-shaped microplate (No. 2). The results show that the hemagglutination inhibition titer of standard positive serum under 30℃ reaction conditions is 512 ( Figure 4 Reaction plate #2).

[0118] In summary, the hemagglutination inhibition titer (512) of the Histotrophozoites standard positive serum under reaction conditions at 30℃ is twice that of the hemagglutination inhibition titer (256) under reaction conditions at 25℃ (room temperature). Therefore, the optimal reaction conditions for the Histotrophozoites hemagglutination inhibition antibody detection method established in this invention are: the optimal conditions for the reaction between Histotrophozoites hemagglutination antigen and bovine / sheep serum antibody are 30℃ for 30 min; the optimal conditions for the hemagglutination inhibition reaction after adding 1% aldehyde-treated sheep erythrocytes are 30℃ for 60 min.

[0119] 2. Establishment of a method or kit for detecting hemagglutination inhibition antibodies against histophiliae during sleep.

[0120] The method or kit for detecting Histoplasmosis hemagglutination inhibition antibodies is obtained by combining 1% aldehyde-treated sheep erythrocytes prepared in Example 2, Histoplasmosis hemagglutination antigen with a hemagglutination titer of 512 prepared in Example 5, Histoplasmosis standard positive serum prepared in Example 6, sodium phosphate buffer with a molar concentration of 0.05 mol / L and a pH of 7.2, and a disposable 96-well V-shaped microplate. The basic operating steps of the hemagglutination test and hemagglutination inhibition test used in this method or kit are modified from conventional operating steps. Specific modifications include:

[0121] (1) The buffer used was 0.05 mol / L sodium phosphate buffer (Na2HPO4 / NaH2PO4 buffer) with pH=7.2, instead of the commonly used PBS buffer (phosphate buffered saline) and saline.

[0122] (2) In the hemagglutination inhibition test, the optimal conditions for the reaction between the hemagglutination antigen of Histophilia dormans and the antibody in bovine and ovine serum were 30℃ for 30 min; the optimal conditions for the hemagglutination inhibition reaction after adding 1% aldehyde-treated sheep red blood cells were 30℃ for 60 min.

[0123] 3. Establishment of the cutoff value (judgment criterion) for hemagglutination inhibition titer of positive serum in qualitative and relative quantitative detection.

[0124] The hemagglutination inhibition titer of 150 healthy bovine and ovine serum samples (including 50 dairy bovine serum, 50 sheep serum, and 50 goat serum) uninfected with *Histophilus dormatus* was determined under optimal reaction conditions using the established method or reagents for detecting hemagglutination inhibition antibodies. The method was as follows: Seven bovine and ovine serum samples were added to wells A1–G1 of each 96-well V-shaped microplate, and serially diluted 2-fold to wells A10–G10. Negative control wells (column 11) (without serum), blank control wells (column 12) (without serum and hemagglutination antigen), and positive controls (wells H1–H10) were also established. The results showed that aldehyde-coated sheep erythrocytes in the negative control wells were completely agglutinated, and in the blank control wells, aldehyde-coated sheep erythrocytes were completely precipitated at the center of the bottom of the well. The standard positive serum hemagglutination inhibition titer was 512 (a standard positive serum hemagglutination inhibition titer of 256, 512, or 1024 indicates a valid standard positive serum control). Figure 5 All controls were valid, and the hemagglutination inhibition titers of Histophilia hygroscopicus antibodies in all 150 bovine and ovine serum samples were less than or equal to 8, with an average hemagglutination inhibition titer of 4. Some serum hemagglutination inhibition antibody test results are shown below. Figure 5 Therefore, the threshold value for hemagglutination inhibition titer of negative serum is set at 8, and the threshold value for hemagglutination inhibition titer of positive serum is set at 16. That is, when the hemagglutination inhibition titer of the tested serum is less than or equal to 8, it is judged as negative for Histoplasma dormatum antibody; when the hemagglutination inhibition titer of the tested serum is greater than or equal to 16, it is judged as positive for Histoplasma dormatum antibody. Furthermore, the higher the hemagglutination inhibition titer, the higher the antibody concentration in the serum.

[0125] The criteria for qualitative testing are as follows: when the serum hemagglutination inhibition titer is less than or equal to 8, it is considered negative for Histoplasma sleeper antibody; when the serum hemagglutination inhibition titer is greater than or equal to 16, it is considered positive for Histoplasma sleeper antibody.

[0126] The criteria for relative quantitative detection are as follows: when the hemagglutination inhibition titer of the tested serum is greater than or equal to 16, it is determined to be positive for Histoplasma sleeper antibody, and the higher the hemagglutination inhibition titer, the higher the antibody concentration in the serum.

[0127] Example 8: Performance evaluation of the detection method or kit for hemagglutination inhibition antibody against histophilia suis

[0128] 1. Specificity evaluation

[0129] (1) Positive sera of common bacteria and viruses in cattle and sheep

[0130] The sources of positive sera for 15 common bovine and ovine bacteria and viruses are shown in Table 2.

[0131] Table 2. Sources of bacterial and viral positive sera and specificity test results of the Histophilia suis antibody detection method.

[0132]

[0133] In Table 2, the strains used to prepare positive sera—including hemolytic Mansonia solani ASV17113, Corynebacterium pseudotuberculosis ASV220610, Escherichia coli ASV210723, Salmonella enterica ASV201158, Staphylococcus aureus ASV201020, Streptococcus multifiliis ASV210759, and Cryptobacterium pyogenes YN170843—were isolated and preserved by the Yunnan Provincial Key Laboratory of Tropical and Subtropical Animal Viral Diseases, Yunnan Academy of Animal Science and Veterinary Medicine. The vaccines used to prepare positive sera—Brucella abortus, Pasteurella multocida, Clostridium perfringens, Mycoplasma bovis, Mycoplasma caprineis, peste des petits ruminants virus, sheep stomatitis virus, and foot-and-mouth disease virus—were all commercially available vaccines.

[0134] Positive sera for Brucella abortus, Pasteurella multocida, Clostridium perfringens, Mycoplasma bovis, Mycoplasma caprineis, peste des petits ruminants virus, sheep stomatitis virus, and foot-and-mouth disease virus were prepared by immunizing cattle and sheep with commercially available vaccines. Positive sera for Mansonia hemolyticus, Corynebacterium pseudotuberculosis, Escherichia coli, Salmonella enterica, Staphylococcus aureus, Streptococcus multifiliis, and Cryptococcus pyogenes were prepared by using strains isolated and preserved by the Yunnan Academy of Animal Science and Veterinary Medicine according to the positive serum preparation method described in Example 6.

[0135] (2) Specificity evaluation of the detection method for hemagglutination inhibition antibody against histophilia suis

[0136] According to the optimized method for detecting hemagglutination inhibition antibodies against histophilia in Example 7, the positive sera of the above 15 common bovine and ovine bacteria and viruses were measured. The order of adding the positive sera of the 15 common bovine and ovine bacteria and viruses was as follows: Brucella abortus A19 strain, Pasteurella multocida 45-2 strain, and Clostridium perfringens C60 strain were added to wells A1 to H1 of the No. 1 96-well V-shaped microplate, respectively. Positive serum samples of strains ASV17113 (hemolytic Manslaughter ASV), strain ASV220610 (Corynebacterium pseudotuberculosis ASV), strain ASV210723 (Escherichia coli ASV), strain ASV201158 (Salmonella enterica ASV), and strain ASV201020 (Staphylococcus aureus) were added to wells A1-H1 of a 96-well V-shaped microplate. Positive serum samples of strains ASV210759 (Streptococcus multifiliis), strain YN170843 (Cryptobacter pyogenes), strain HB150 (Mycoplasma bovis), strain M87-1 (Mycoplasma caprineis), strain Clone9 (Pelteorum purulent ruminant virus), strain HCE (oral pox virus), and strain O (foot-and-mouth disease virus) and strain OJMS (foot-and-mouth disease virus) were added, as well as standard positive serum of Histoplasmosis dormancy (positive control). The test results showed that aldehyde-treated sheep erythrocytes in the negative control wells (A11-H11 wells) were completely agglutinated, and in the blank control wells (A12-H12 wells), aldehyde-treated sheep erythrocytes were completely precipitated at the center of the bottom of the well. The hemagglutination inhibition titer of the standard positive serum control was 512 (a hemagglutination inhibition titer of 256, 512, or 1024 indicates that the standard positive serum control is valid). Figure 6 The control group was found to be effective. The hemagglutination inhibition titers of *Histoplasma dormatum* antibody in the positive sera of 15 common bovine and ovine bacteria and viruses were all less than or equal to 8, and were therefore determined to be negative for *Histoplasma dormatum* antibody (Table 2). Figure 6 The results indicate that the detection method or kit for hemagglutination inhibition antibody against sleep-loving histophilia established in this invention has no cross-reactivity with positive sera of Brucella abortus, Pasteurella multocida, Clostridium perfringens, Hemolytic Mansonia, Corynebacterium pseudotuberculosis, Escherichia coli, Salmonella enterica, Staphylococcus aureus, Streptococcus multifiliis, Cryptobacterium pyogenes, Mycoplasma bovis, Mycoplasma caprineis, peste des petits ruminants virus, sheep stomatitis virus, and foot-and-mouth disease virus, and has strong specificity.

[0137] 2. Stability Evaluation

[0138] The 1% aldehyde-treated sheep erythrocytes described in Example 2 (containing a final concentration of 0.005% sodium thimerosal preservative) do not affect the hemagglutination titer of *Histoplasma dormica* hemagglutination antigen and the hemagglutination inhibition titer of *Histoplasma dormica* antibody when stored at 4°C for 6 months, avoiding the drawback of needing to prepare fresh erythrocytes immediately. The *Histoplasma dormica* hemagglutination antigen described in Example 5 (containing a final concentration of 0.005% sodium thimerosal preservative) does not change its hemagglutination titer when stored at 4°C for 6 months. The *Histoplasma dormica* standard positive serum described in Example 6 (containing a final concentration of 0.005% sodium thimerosal preservative) does not change its hemagglutination inhibition titer when stored at 4°C for 6 months. Therefore, the *Histoplasma dormica* hemagglutination antibody detection kit described in Example 7 has a shelf life of more than 6 months.

[0139] Example 9: Application of a method or kit for detecting hemagglutination inhibition antibodies against *Histopterus hirsutus* in the detection of antibodies in bovine, ovine, and goat serum samples.

[0140] 1. Validation of 4 units of histophilic hemagglutination antigen

[0141] Before each detection of Histoplasma dormatum antibodies in bovine and ovine serum, the hemagglutination titer of the 4 units of Histoplasma dormatum hemagglutination antigen described in Example 5 or the 4 units of Histoplasma dormatum hemagglutination antigen newly prepared using the Histoplasma dormatum hemagglutination antigen labeled with a titer of 512 in the kit described in Example 7 needs to be verified by a hemagglutination test. The specific steps are as follows:

[0142] ① Take a 96-well V-shaped microplate and add 25 μL of 0.05 mol / L pH=7.2 sodium phosphate buffer to each of wells A1 to A5 using a pipette;

[0143] ② Add 4 units of the above-mentioned Histophilus dormancy hemagglutination antigen to well A1, mix by pipetting 5 times, and then add 25 μL to well A2. Mix by pipetting 5 times, and then add 25 μL to well A3. And so on, dilute the Histophilus dormancy hemagglutination antigen 2-fold to well A4. Mix by pipetting 5 times in well A4, and then discard 25 μL.

[0144] ③ No hemagglutination antigen was added to well A5 as a negative control well;

[0145] ④ Add 25 μL of 1% aldehyde-treated sheep red blood cells to each of wells A1 to A5 in a suspended position (without the pipette tip touching the liquid in the well);

[0146] ⑤ Place the 96-well V-shaped micro-reaction plate onto a micro-plate shaker and shake for 1 min. After standing at 30℃ for 60 min, observe the results.

[0147] ⑥ Validation criteria for 4 units of Histoplasmosis hemagglutination antigen: Under the condition that the aldehyde-treated sheep erythrocytes in the negative control well are completely precipitated at the center of the bottom of the well, the aldehyde-treated sheep erythrocytes in the 1 unit hemagglutination antigen well must achieve 100% agglutination, and the erythrocytes in the 0.5 unit hemagglutination antigen well must achieve 50% agglutination to meet the requirements. If the actual hemagglutination titer of 4 units of Histoplasmosis hemagglutination antigen is not 4, then 4 units of Histoplasmosis hemagglutination antigen need to be prepared again according to the actual hemagglutination titer. Validation results show that the aldehyde-treated sheep erythrocytes in the negative control well (well A5) are completely precipitated at the center of the bottom of the well, the aldehyde-treated sheep erythrocytes in the 1 unit antigen well (well A2) agglutinate 100%, and the aldehyde-treated sheep erythrocytes in the 0.5 unit hemagglutination antigen well (well A3) agglutinate 50%. Figure 7 The results meet the above-mentioned verification criteria for 4 units of Histoplasmosis dormatoid hemagglutination antigen, indicating that the 4 units of Histoplasmosis dormatoid hemagglutination antigen described in Example 5 or the 4 units of Histoplasmosis dormatoid hemagglutination antigen newly prepared using the Histoplasmosis dormatoid hemagglutination antigen with a titer of 512 in the kit described in Example 7 can be used for the detection of Histoplasmosis dormatoid antibodies in bovine and ovine serum.

[0148] 2. Qualitative detection of Histophilia dormans antibodies in bovine, sheep, and goat serum samples.

[0149] (1) Serum samples from cattle, sheep and goats

[0150] Three bovine serum samples (numbered N1, N2, and N3) were obtained from bovines with respiratory diseases at a dairy farm in Yunnan Province; two sheep serum samples (numbered MY1 and MY2) were obtained from sheep with respiratory diseases at a sheep farm in Yunnan Province; and two goat serum samples (numbered SY1 and SY2) were obtained from goats with respiratory diseases at a goat farm in Yunnan Province. The sources of the seven bovine, sheep, and goat serum samples are shown in Table 3.

[0151] Table 3. Sources of serum samples from cattle, sheep, and goats, and qualitative and relative quantitative detection results of antibodies against Histophilia dormans.

[0152]

[0153] The seven bovine, sheep, and goat serum samples in Table 3 are all preserved in the Key Laboratory of Tropical and Subtropical Animal Viral Diseases of Yunnan Academy of Animal Science and Veterinary Medicine.

[0154] (2) Qualitative detection of Histophilia dormans antibodies in serum samples from cattle, sheep and goats

[0155] Using the optimized method or kit for detecting Histoplasma dormantii hemagglutination inhibition antibodies described in Example 7, Histoplasma dormantii antibodies in the above seven bovine, sheep, and goat serum samples were detected. In the qualitative detection, the serum samples only needed to be serially diluted 2-fold to 32-fold, saving the amount of detection reagents and 96-well microplates. The specific steps are as follows:

[0156] ① Take a 96-well V-shaped microplate and add 25 μL of 0.05 mol / L pH=7.2 sodium phosphate buffer to each of wells A1 to H7 using a pipette;

[0157] ② Add N1 bovine serum, N2 bovine serum, N3 bovine serum, MY1 sheep serum, MY2 sheep serum, SY1 goat serum, SY2 goat serum, and the positive serum of Histophilia hygroscopicus to wells A1-H1, respectively. Mix by pipetting 5 times with an eight-channel pipette, and then add 25 μL to each well corresponding to wells A2-H2. Mix by pipetting 5 times with each well, and then add 25 μL to each well corresponding to wells A3-H3. Continue this process, diluting each of the seven bovine, sheep, and goat serums and the positive serum of Histophilia hygroscopicus standard by 2-fold to wells A5-H5. Mix by pipetting 5 times with each well in wells A5-H5, and then discard 25 μL from each well. Wells A6-H6 are used as negative controls without serum. Wells A7-H7 are used as blank controls with 25 μL of 0.05 mol / L pH=7.2 sodium phosphate buffer.

[0158] ③ Add 25 μL of the above-verified 4 units of Histophilic hemagglutination antigen in each of wells A1 to H6 (without the pipette tip touching the liquid in the well), place on a microplate shaker and shake for 1 min, then let stand at 30°C for 30 min.

[0159] ④ Add 1% aldehyde-modified sheep erythrocytes as described in Example 2 to each well (without the pipette tip touching the liquid in the well), suspended in the air, and shake on a microplate shaker for 1 min. Incubate at 30°C for 60 min. Visually observe the hemagglutination inhibition titer to determine the hemagglutination inhibition effect. The results showed that the aldehyde-modified sheep erythrocytes in the negative control wells (A6-H6) were completely agglutinated, the aldehyde-modified sheep erythrocytes in the blank control wells (A7-H7) were completely precipitated at the center of the bottom of the well, and the aldehyde-modified sheep erythrocytes in the standard positive serum control wells (H1-H5) were completely precipitated at the center of the bottom of the well. Figure 8 All controls were valid, and the hemagglutination inhibition titers of N3 dairy bovine serum, MY2 sheep serum, and SY1 goat serum were less than or equal to 8 (Table 3). Figure 8 The results indicated that the antibodies against Histoplasma sleeper were negative; the hemagglutination inhibition titers of N1 bovine serum, N2 bovine serum, MY1 sheep serum, and SY2 goat serum were greater than or equal to 16 (Table 3). Figure 8 The result indicated a positive result for Histophilia hygrophila antibody.

[0160] 3. Relative quantitative detection of Histophilia hygrophila antibodies in bovine, ovine, and goat serum samples.

[0161] Using the optimized method or kit for detecting *Histoplasma dormantis* hemagglutination inhibition antibodies described in Example 7, the *Histoplasma dormantis* antibodies in the above seven bovine, ovine, and goat serum samples were detected. For relative quantification, the tested serum samples needed to be serially diluted 2-fold to 1024-fold. The specific steps are as follows:

[0162] ① Take a 96-well V-shaped microplate and add 25 μL of 0.05 mol / L pH=7.2 sodium phosphate buffer to each of wells A1 to H12 using a pipette;

[0163] ② Add N1 bovine serum, N2 bovine serum, N3 bovine serum, MY1 sheep serum, MY2 sheep serum, SY1 goat serum, SY2 goat serum, and the positive serum of Histophilia hygroscopicus to wells A1-H1, respectively. Mix by pipetting 5 times with an eight-channel pipette, and then add 25 μL to each well corresponding to wells A2-H2. Mix by pipetting 5 times with each well, and then add 25 μL to each well corresponding to wells A3-H3. Continue this process, diluting each of the seven bovine, sheep, and goat serums and the positive serum of Histophilia hygroscopicus standard by 2-fold to wells A10-H10. Mix by pipetting 5 times with each well in wells A10-H10, and then discard 25 μL from each well. Wells A11-H11 are used as negative controls without serum. Wells A12-H12 are used as blank controls with 25 μL of 0.05 mol / L pH=7.2 sodium phosphate buffer.

[0164] ③ Add 25 μL of the above-verified 4 units of Histophilic hemagglutination antigen to each of wells A1 to H11 (without the pipette tip touching the liquid in the well), place on a microplate shaker and shake for 1 min, then let stand at 30°C for 30 min to react.

[0165] ④ Add 1% aldehyde-treated sheep erythrocytes as described in Example 2 to each well (without the pipette tip touching the liquid in the well), suspended in the air, and shake on a microplate shaker for 1 min. Incubate at 30°C for 60 min. Visually observe the hemagglutination inhibition titer to determine its effectiveness. The results showed that the aldehyde-treated sheep erythrocytes in the negative control wells (A11-H11 wells) were completely agglutinated, while the aldehyde-treated sheep erythrocytes in the blank control wells (A12-H12 wells) were completely precipitated at the bottom center of the well. The standard positive serum control hemagglutination inhibition titer was 512 (a standard positive serum hemagglutination inhibition titer of 256, 512, or 1024 indicates a valid standard positive serum control). Figure 9 All controls were valid, and the hemagglutination inhibition titers of N3 dairy bovine serum, MY2 sheep serum, and SY1 goat serum were less than or equal to 8 (Table 3). Figure 9The result indicates that the serum is negative for Histophilia serovar anti-antibody, meaning there are no Histophilia serovar anti-antibody antibodies in the serum.

[0166] The hemagglutination inhibition titers of N1 bovine serum, N2 bovine serum, MY1 sheep serum, and SY2 goat serum were 512, 128, 64, and 256, respectively (Table 3). Figure 9 The results showed that the serum of N1 dairy cows was positive for Histophilia hygrophila antibodies, with the highest relative concentration of antibodies in N1 dairy cows, a relatively high relative concentration in SY2 goats, a relatively low relative concentration in N2 dairy cows, and the lowest relative concentration in MY1 sheep (Table 3). Figure 9 ).

[0167] This invention utilizes the unique characteristic of *Histoplasma dormica* bacteria—the presence of filamentous hemagglutinin proteins on their surface that can agglutinate animal erythrocytes—to screen for the Yunnan isolate ZY25055, which exhibits high hemagglutination activity. Hemagglutination antigens were prepared from *Histoplasma dormica* (especially the Yunnan isolate ZY25055) bacterial suspensions inactivated with β-propiolactone. *Histoplasma dormica* standard positive serum was prepared by immunizing goats with the Yunnan isolate ZY25055. Sheep erythrocytes were prepared from sheep blood and aldehyde-treated with glutaraldehyde to prepare 1% aldehyde-treated sheep erythrocytes. Using the prepared *Histoplasma dormica* hemagglutination antigen, *Histoplasma dormica* standard positive serum, and 1% aldehyde-treated sheep erythrocytes, a detection method and kit for detecting *Histoplasma dormica* hemagglutination inhibition antibodies were established for the first time both domestically and internationally. This detection method has advantages such as high specificity, simple operation, and no need for complex equipment (only a common incubator is required). It can be applied to the detection of *Histoplasma dormica* antibodies in bovine serum, sheep serum, and goat serum.

Claims

1. A strain of hibernating histophilic bacteria, characterized in that, The somnophilic bacteria was isolated from a goat, strain number ZY25055, and deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20251686. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit date is July 23, 2025.

2. A histophilic hemagglutination antigen, characterized in that, The Histophilic hemagglutination antigen was prepared by culturing Histophilic bacteria and inactivating them with β-propiolactone; the Histophilic bacteria were: 。 3. The histophilic hemagglutination antigen according to claim 2, characterized in that, The sleep-loving histophile is the strain of sleep-loving histophile as described in claim 1.

4. A histophilic hemagglutination antigen according to claim 2 or 3, characterized in that, Methods for inactivating histophilic bacteria include: at 2 × 10 9 Add β-propiolactone to a final concentration of 0.2% in the CFU / mL bacterial culture and inactivate at 25℃ for 5 h.

5. A method for detecting hemagglutination inhibition antibodies against histophiliae, characterized in that, Includes a Histotrophozoites hemagglutination antigen as described in any one of claims 2-4, and further includes 1% aldehyde-treated sheep erythrocytes and Histotrophozoites standard positive serum; In the method for detecting the hemagglutination inhibition antibody of Histophilia dormans, the conditions for the reaction between the Histophilia dormans hemagglutination antigen and the antibody in the serum of the tested cattle and sheep are: 30℃ for 30 min, and the conditions for the hemagglutination inhibition reaction after adding 1% aldehyde-treated sheep red blood cells are: 30℃ for 60 min.

6. The method for detecting hemagglutination inhibition antibodies against histophiliacs according to claim 5, characterized in that, The 1% aldehyde-treated sheep red blood cells were prepared by collecting sheep blood, adding glass beads and slowly shaking to remove fibrin, washing with sodium phosphate buffer (pH 7.2, 0.05 mol / L), adding glutaraldehyde to a final concentration of 1% and aldehyde-treating at 4°C for 30 min, and then adding sodium thimerosal to a final concentration of 0.005%.

7. The method for detecting hemagglutination inhibition antibodies against histophiliacs according to claim 5, characterized in that, The Histotrophozoites standard positive serum was prepared by immunizing goats with Histotrophozoites bacterium solution inactivated by β-propiolactone, separating the serum from the blood, and adding sodium thimerosal to a final concentration of 0.005%.

8. The method for detecting hemagglutination inhibition antibodies against histophiliacs according to claim 5, characterized in that, After the reaction is complete, the antibody detection results are accurately determined by directly observing the aggregation or precipitation of aldehyde-modified sheep red blood cells at the bottom center of the micro-reaction plate wells with the naked eye.

9. The method for detecting hemagglutination inhibition antibodies against histophiliacs according to claim 8, characterized in that, Under the conditions that aldehyde-treated sheep erythrocytes in the negative control well without serum completely agglutinate and aldehyde-treated sheep erythrocytes in the blank control well without serum and hemagglutination antigen completely precipitate at the center of the bottom of the well, the hemagglutination inhibition titer of the test serum is determined by the maximum dilution factor that completely inhibits the agglutination of aldehyde-treated sheep erythrocytes. When the hemagglutination inhibition titer of the test serum is less than or equal to 8, it is judged as negative for Histoplasma dormatum antibody; when the hemagglutination inhibition titer of the test serum is greater than or equal to 16, it is judged as positive for Histoplasma dormatum antibody. The higher the hemagglutination inhibition titer, the higher the concentration of Histoplasma dormatum antibody in the serum.

10. A method for detecting hemagglutination inhibition antibodies against histophiliacs according to claims 5-9, characterized in that, It can specifically detect serum antibodies against Histophilia hygrophila and shows no cross-reactivity with positive sera of common bacteria and viruses in cattle, sheep, and goats; Positive sera for common bacteria and viruses in cattle, sheep, and goats include: Brucella abortus, Pasteurella multocida, Clostridium perfringens, Hemolytic Mansonia, Corynebacterium pseudotuberculosis, Escherichia coli, Salmonella enterica, Staphylococcus aureus, Streptococcus multifiliis, Cryptobacterium pyogenes, Mycoplasma bovis, Mycoplasma caprineis, peste des petits ruminants virus, sheep pox virus, and foot-and-mouth disease virus.

11. The application of a method for detecting Histoplasmosis hemagglutination inhibition antibodies according to any one of claims 5-10 in the detection of Histoplasmosis antibodies in bovine serum, sheep serum and goat serum.

12. The application according to claim 11, characterized in that, This includes laboratory testing or on-site testing, with on-site testing including testing at cattle farms, sheep farms, and goat farms.

13. The application of a method for detecting Histoplasmosis hemagglutination inhibition antibodies according to any one of claims 5-10 in the qualitative or relative quantitative detection of Histoplasmosis antibodies in bovine serum, sheep serum, and goat serum.

14. A detection kit for hemagglutination inhibition antibody against histophiliae, characterized in that, The kit includes a Histotrophozoites hemagglutination antigen as described in any one of claims 2-4, and also includes 1% aldehyde-treated sheep erythrocytes and Histotrophozoites standard positive serum as described in any one of claims 5-7 in a Histotrophozoites hemagglutination inhibition antibody detection method.

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