Bactrian camel clostridium perfringens AD type bivalent inactivated vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a bactrian camel clostridium perfringens AD type bivalent inactivated vaccine as well as a preparation method and application thereof. The invention relates to a bactrian camel clostridium perfringens AD type bivalent inactivated vaccine, the bivalent inactivated vaccine comprises two inactivated strain antigens, and the antigens are a bactrian camel A type aerogenic bacteria clostridium perfringens strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.35194 and a double hump D type aerogenic bacteria clostridium perfringens strain with the preservation number of CGMCC No.35195. According to the application, a newly found bactrian camel clostridium perfringens strain is used as an antigen. The 1059 ST type clostridium perfringens are separated from intestinal tracts of young bactrian camels for the first time, and the 1059 type (A type) and 675 type (D type) clostridium perfringens are inactivated to prepare the bivalent inactivated vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a bivalent inactivated vaccine of Clostridium perfringens type AD from Bactrian camel, its preparation method and application. Background Technology

[0002] Bactrian camels are an important livestock resource in the arid northwest of my country, but their health is vulnerable to various pathogenic microorganisms. *Clostridium perfringens*, a Gram-positive anaerobic spore-forming bacillus, is widely found in soil, animal intestines, and the environment. It can produce various toxic agents (such as alpha toxins and ε toxins), which are particularly harmful to Bactrian camels. Clinically, *Clostridium perfringens* type A and D strains are the main pathogens causing sudden death syndrome and enterotoxemia in Bactrian camels. These diseases have a rapid onset and high mortality rate (up to 54.71%), severely hindering the healthy development of Bactrian camel farming.

[0003] Currently, there is a lack of inactivated Clostridium perfringens vaccines specifically for Bactrian camels. Existing vaccines are designed for other livestock species (such as cattle and sheep), and their direct application to Bactrian camels may lead to differences in immune response or safety issues. Therefore, there is an urgent need to develop a high-titer, safe inactivated vaccine against Clostridium perfringens for Bactrian camels. Summary of the Invention

[0004] To develop a new vaccine that can effectively prevent sudden death syndrome and enterotoxemia in Bactrian camels, this invention provides a bivalent inactivated vaccine of Clostridium perfringens type AD from Bactrian camels, its preparation method, and its application. This invention isolates and obtains exotoxins produced by Clostridium perfringens type A and D strains from Bactrian camels, which can be used to prepare vaccines and have good immunizing effects.

[0005] This invention provides a bivalent inactivated vaccine against Clostridium perfringens type AD from Bactrian camels, wherein the bivalent inactivated vaccine contains inactivated antigens of two strains.

[0006] Furthermore, the antigen is a type A aerogenes Clostridium perfringens strain from Bactrian humpsus with accession number CGMCC No. 35194 and a type D aerogenes Clostridium perfringens strain from Bactrian humpsus with accession number CGMCC No. 35195.

[0007] Furthermore, the bacterial count of the strains used in the bivalent inactivated vaccines is all 1×10⁻⁶. 9 CFU / ml.

[0008] Furthermore, the bivalent inactivated vaccine is detoxified using formaldehyde solution.

[0009] This invention provides a method for preparing a bivalent inactivated vaccine against Clostridium perfringens type AD from Bactrian camel. The method uses a Bactrian camel type A Clostridium perfringens strain with accession number CGMCC No. 35194 and a Bactrian camel type D Clostridium perfringens strain with accession number CGMCC No. 35195 as the inoculum. The bacterial culture of type A Clostridium perfringens with high α-toxin content is obtained by culturing in a special culture medium. The bacterial culture is then inactivated, sterilized, concentrated with α-toxin, and used to prepare the vaccine, resulting in the Bactrian camel type AD bivalent inactivated vaccine.

[0010] Furthermore, during seedling preparation, the culture solutions of Clostridium perfringens types A and D are mixed at a volume ratio of 1:1.

[0011] This invention provides the application of a bivalent inactivated vaccine in the preparation of products for preventing diseases caused by Clostridium perfringens.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. This application uses a newly discovered Clostridium perfringens strain from Bactrian camels as the antigen. For the first time, Clostridium perfringens strain 1059 ST was isolated from the intestines of young Bactrian camels. A bivalent inactivated vaccine was prepared by inactivating Clostridium perfringens strains 1059 (A) and 675 (D).

[0014] 2. This application addresses the shortage of a specific vaccine for Clostridium perfringens infection in Bactrian camels.

[0015] Biological Preservation Instructions:

[0016] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0017] Accession number: CGMCC No. 35194;

[0018] Deposit date: July 28, 2025;

[0019] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0020] Taxonomic name: Clostridium perfringens.

[0021] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0022] Accession number: CGMCC No. 35195;

[0023] Deposit date: July 28, 2025;

[0024] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0025] Taxonomic name: Clostridium perfringens. Attached Figure Description

[0026] Figure 1 This is a diagram showing the autopsy results of a diseased camel that died in Example 1.

[0027] Figure 2 This is a diagram showing the autopsy results of a diseased camel that died in Example 1.

[0028] Figure 3 This is a pathological section of the internal organs of a dead camel in Example 1.

[0029] Figure 4 This is a pathological section of the internal organs of a dead camel in Example 1.

[0030] Figure 5 This is a stained image of a liver smear from Example 1.

[0031] Figure 6 This is a diagram showing the results of TSC culture in Example 1.

[0032] Figure 7 This is a diagram showing the Gram staining results in Example 1.

[0033] Figure 8 This is a diagram of the milk fermentation experiment in Example 1.

[0034] Figure 9 This is a diagram of the dynamic-nitrate puncture experiment in Example 1.

[0035] Figure 10 This is a diagram of the lactose-gelatin puncture experiment in Example 1.

[0036] Figure 11 This is a diagram showing the multiplex PCR detection results of Clostridium perfringens isolated in Example 1.

[0037] Figure 12 The graph shows the growth curves of Clostridium perfringens types A and D in Example 2.

[0038] Figure 13 This is a picture of the dead mice in Example 2.

[0039] Figure 14 This is a Gram staining result of the visceral smear in Example 2.

[0040] Figure 15 This is a comparison of the internal organs of the dead mice and healthy mice in Example 2.

[0041] Figure 16 This is a diagram of the pathological sections of mouse internal organs in Example 2.

[0042] Figure 17 This is a graph showing the results of culturing 0.5% vaccine at TSC for 24 hours in Example 2.

[0043] Figure 18 This is a diagram of the bivalent inactivated Clostridium perfringens vaccine prepared in Example 3.

[0044] Figure 19 The image shows the results of mouse α-toxin C antibody detection in Example 4.

[0045] Figure 20 The image shows the detection results of α-toxin C antibody in young camels in Example 5. Detailed Implementation

[0046] Example 1: Isolation, identification, and screening of Clostridium perfringens strains of type A and D from Bactrian camel.

[0047] 1. Autopsy of a camel that died of disease

[0048] Methods: Autopsies were performed immediately after the young camels died to observe pathological changes.

[0049] Results: Autopsy revealed that the dead young camels had bent heads and necks, severe dehydration, significantly distended abdomens, and their perianal area was contaminated with loose stools. Some of the dead camels also showed bleeding from their eyes. Figure 1 A); Enlarged anterior shoulder lymph nodes with petechiae or ecchymoses ( Figure 1 B); tracheal congestion or bleeding points, containing a large amount of white frothy exudate ( Figure 1 C); The abdominal cavity contains pale yellow exudate, the intestinal mucosa is congested or hemorrhagic and flushed, the mesenteric lymph nodes are congested, hemorrhagic or edematous, the intestines are filled with gas, and the intestinal wall is thin and transparent, indicating hemorrhagic enteritis (C). Figure 1 D); edema of the rumen and abomasum mucosa with petechiae; curd-like masses in the stomach ( Figure 1 E); The liver is enlarged, with petechiae, fragile, and dark red in color, with scattered yellowish-brown lesions of varying sizes on the surface ( Figure 1 F); pericardial effusion, partial myocardial necrosis, petechiae or hemorrhages, engorged blood vessels, dendritic pattern (F); Figure 1 G); Splenomegaly, hemorrhage (G); Figure 2 A); In most dead camels, the lungs adhered to the thoracic cavity, darkened in color, swollen and congested, and contained a large amount of white foamy exudate in the bronchi ( Figure 2 B), enlarged kidneys, darker in color, with congestion or hemorrhage; longer-term cases show nephrosoftening, cortical dissolution, and renal papillary necrosis. Figure 2 C), some of the dead camels showed necrosis of the bladder and ureters, and their urine was dark yellow. Figure 2 D), meningeal congestion and edema, dilation of meningeal blood vessels, scattered hemorrhages; in chronic cases, the pia mater is thickened and milky white, brain tissue is edematous, gyri are flattened, and sulci are shallowed. Figure 2E)

[0050] 2. Histopathological observation of diseased and dead camels

[0051] Methods: Tissues ranging in size from 0.5 to 1.0 cm, including those from the brain, heart, liver, spleen, lung, kidney, anterior shoulder lymph nodes, rumen, reticulum, small intestine, and bladder, were collected and fixed in 4% paraformaldehyde tissue fixative (Guangzhou Yongjin Biotechnology Co., Ltd., Guangzhou, China). After trimming, dehydration, embedding, sectioning, staining, and mounting, the tissues were observed using a panoramic slide scanner (3DHISTECH (Hungary) DESK / MIDI / 250 / 1000) and a CaseViewer 2.4 (3DHISTECH (Hungary)).

[0052] Results: Microscopic observation revealed a large number of lymphocytes evenly distributed and densely arranged, numerous lymphoid nodules, and extensive interstitial vascular congestion and dilation. Figure 3 (A orange arrow), occasional brownish-yellow pigment deposition, no medulla ( Figure 3 (A blue arrow). The atria of the heart are richly supplied with blood; the myocardial fibers are finer than those of the ventricles, loosely and irregularly arranged, with a small number of atrophied fibers. Figure 3 (B, blue arrow) Small in size, with widened intercellular spaces; multifocal interstitial edema, loose connective tissue, accompanied by a small amount of lymphocyte infiltration ( Figure 3 B (red arrow), often seen as capillary congestion ( Figure 3 B (orange arrow). The liver tissue shows indistinct lobule boundaries, with a central vein in the center, surrounded by hepatocytes and sinusoids arranged in a roughly radial pattern. A small number of hepatocytes are edematous. Figure 3 C (blue arrow), with loose, pale-stained cytoplasm and extensive hepatic sinusoidal congestion and dilation ( Figure 3 C (black arrow); occasional small-scale lymphocyte infiltration in the portal area ( Figure 3 C (red arrow), extensive vascular congestion ( Figure 3 C (orange arrow). The splenic capsule is composed of dense connective tissue of uniform thickness, rich in elastic and smooth muscle fibers. The capsule connective tissue extends into the spleen to form trabeculae, which are well-developed and show no obvious abnormalities. The splenic parenchyma consists of red pulp and white pulp. The white pulp is composed of the lymphatic sheath around the central artery, lymphatic nodules, and the marginal zone. It has a clear structure, is relatively few in number, and has a regular shape. The number of lymphocytes around the central artery in the white pulp is small and loosely arranged (3D black arrow). The red pulp is distributed in a large area under the capsule, around the trabeculae, and lateral to the marginal zone of the white pulp. It is composed of splenic cords and splenic sinuses, and is clearly demarcated from the white pulp. It is extensively congested. Figure 3 D (blue arrow), a small number of granulocytes scattered throughout ( Figure 3D (red arrow). The surface of the lung tissue is covered with a smooth serous membrane, with no obvious abnormalities; the alveolar walls are composed of a single layer of epithelial cells, with a clear structure, and multifocal congestion of alveolar wall capillaries ( Figure 3 E (orange arrow) indicates alveoli of varying sizes, with a small amount of eosinophilic material scattered throughout the alveolar cavities. Figure 3 E (black arrow); diffuse bleeding ( Figure 3 E (red arrow), a small number of red blood cells can be seen in the alveoli and bronchiolar cavities; occasionally, flattening of bronchiolar epithelial cells is observed. Figure 3 E (blue arrow); occasionally, intravascular cell debris, unstructured eosinophils, and leukocytes are observed. Figure 3 E (green arrow). The renal parenchyma consists of a superficial cortex and a deep medulla, with a clear cortical-medullary boundary; glomeruli are evenly distributed in the cortex, and the number of cells and matrix within the glomeruli are uniform, with extensive edema of the renal tubular epithelial cells. Figure 3 F (blue arrow), cytoplasm is loose and lightly stained, with a small amount of eosinophilic material visible in the renal tubules ( Figure 3 F (red arrow); the connective tissue between the urinary tubules is the renal interstitium, with no significant interstitial proliferation and extensive interstitial vascular congestion (…). Figure 3 (F Orange Arrow); no obvious inflammatory cell infiltration was observed. Extensive loss of intestinal villi was observed in the duodenum. Figure 3 (G - Black arrow) The lamina propria is exposed, the intestinal gland structure is unclear, and focal infiltration of numerous lymphocytes and macrophages is visible. Figure 3 G (blue arrow); small-scale edema in the submucosal layer ( Figure 3 (G, orange arrow) The connective tissue is loose, with occasional lymphocyte infiltration; the muscle layer is uneven in thickness, and the muscle fibers are irregularly arranged. Extensive shedding of intestinal villi is visible in the ileum. Figure 3 H (black arrow) indicates exposed lamina propria, loosely arranged intestinal glands, extensive shedding of intestinal gland epithelium, and widening of the distance between the glands and the basement membrane. Figure 3 H (orange arrow); the submucosa consists of loose connective tissue, the muscle layer is uneven in thickness, with multifocal edema, and the muscle fibers and connective tissue are loosely arranged, accompanied by lymphocyte infiltration ( Figure 3 H (blue arrow); massive vascular congestion ( Figure 3 H (red arrow). The cecal tissue mucosal epithelium is incomplete and has extensive sloughing. Figure 4 A black arrow); the lamina propria contains numerous intestinal glands, which are short tubular and densely arranged, and necrosis of intestinal glands is frequently observed. Figure 4 (A green arrow), nuclear condensation, cytoplasmic disintegration, and indistinct small areas of intestinal gland structure ( Figure 4 A (orange arrow), significant vascular congestion ( Figure 4 (A red arrow) The muscularis mucosae separates the lamina propria from the submucosa; the submucosa is loose connective tissue, with a thick muscular layer, irregular muscle fiber arrangement, and focal infiltration of macrophages (A red arrow). Figure 4 A blue arrow). The glandless portion of the rumen is covered by stratified squamous epithelium, with a small number of epithelial cells having loosely stained, pale cytoplasm. Figure 4 B (blue arrow); extensive shedding of the mucosal epithelium is visible in the glandular area. Figure 4 (B, black arrow) The lamina propria is rich in gastric glands, which are tightly arranged, with a small amount of gastric gland epithelium sloughed off. Figure 4 (B, purple arrow) Local infiltration of numerous lymphocytes and macrophages is visible. Figure 4 B (red arrow), significant vascular congestion ( Figure 4 B (orange arrow); uneven muscle layer thickness, irregular muscle fiber arrangement, and occasional focal aggregation of lymphocytes and macrophages ( Figure 4 B (green arrow). The reticulosal mucosa forms folds inward into the gastric lumen, and extensive mucosal epithelial shedding is visible. Figure 4 C (black arrow) – the lamina propria is exposed, and numerous gastric glands are visible within the lamina propria. The cytoplasm of many gastric gland epithelial cells is loosely stained and pale. Figure 4 C (blue arrow) – a small amount of gastric glandular epithelium sloughed off, with increased distance between the epithelium and the basement membrane. Figure 4 C (purple arrow), massive vascular congestion ( Figure 4 C (orange arrow); the submucosa is loose connective tissue, the muscle layer is thick, and the muscle fibers are irregularly arranged. The bladder mucosa shows intact mucosal epithelium, with edematous mucosal cells. Figure 4 D (blue arrow), cytoplasm is loosely stained and lightly dyed, nuclear chromatin is marginalized ( Figure 4 (D, red arrow) A small amount of vascular congestion in the lamina propria, with a small number of white blood cells observed within the vessels ( Figure 4 (D, black arrow) The muscle layer is uneven in thickness, and the muscle cells are arranged irregularly. The brain tissue shows a low number of neurons and signs of shrinkage. Figure 4 (E, red arrow) Cells are smaller, irregularly shaped, with indistinct nucleus-cytoplasm boundaries, and darker staining; numerous glial cells are scattered throughout. Figure 4 E (blue arrow); numerous small vacuoles are visible within the brain tissue. Figure 4 E (orange arrow); massive vascular congestion ( Figure 4 (E, black arrow) A small number of lymphocytes and macrophages are visible infiltrating around the blood vessels, forming a ring and creating a vascular sleeve. Figure 4 E (purple arrow). The cerebellar tissue is covered by meninges, with no obvious abnormalities observed; the cortex shows clear molecular layer, Purkinje cell layer, and granular layer structures, with few glial cells in the molecular layer. Figure 4 (F, red arrow) The number of granular layer cells is relatively small; the medullary tissue is loosely arranged, and numerous small vacuoles are visible. Figure 4 F (blue arrow), extensive vascular congestion ( Figure 4 (F - Orange Arrow)

[0053] 3. Collect liver and intestinal tissue from the dead young camels in step 2, and store them at 4℃ for later use. Gram stain the collected liver tissue slides to observe the bacterial infection status. Use sterile cotton swabs to collect intestinal contents and inoculate them into 5 ml of liquid thioglycolate broth (FTG, Qingdao Haibo Biotechnology Co., Ltd., Qingdao, China) and anaerobically incubate at 37℃ for 15 h. Take 100 µl of enriched FTG broth and spread it on tryptone-sulfite-cycloserine agar medium (TSC, Qingdao Haibo Biotechnology Co., Ltd., Qingdao, China) containing 0.5% D-cycloserine and anaerobically incubate at 37℃ for 15 h. Gram stain the obtained single colonies for identification, and confirm them by milk fermentation, dynamic nitrate and lactose gelatin tests according to the test standard of Clostridium perfringens (GB 4789.13—2012). Store the isolated bacteria in 50% glycerol at -80℃. DNA from *Clostridium perfringens* isolated using a bacterial genomic DNA extraction kit was extracted. The extracted DNA was amplified by PCR using primers designed by Baums et al. (Table 1). The genes plc (cpa), cpb, ext, ipa, cpe, NetB, and cpb2 were amplified in a 50 μl volume. The PCR reaction system consisted of: 12.5 μL of 2×Taq Master Mix (Beijing TransGen Biotech, Beijing, China), 1 μL each of forward and reverse primers (10 μmol / L), 2 μL of DNA extract, and ddH2O to a final volume of 50 μL. After the reaction, the PCR products were detected by 2.0% agarose gel electrophoresis, and the products were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for Sanger sequencing. The sequencing results were compared using NCBI-BLAST.

[0054] The relevant methods for milk fermentation, dynamic nitrate and lactose gelatin experiments refer to the national standard: Food Microbiology Examination - Clostridium perfringens Examination GB 4789.13—2012.

[0055] The results are as follows Figure 5-11 As shown: After Gram staining of the collected liver tissue slides, a large number of Gram-positive bacilli were observed, forming round or oval spores with a diameter larger than the bacterial cell, located in the center, extremes, or sub-extremes of the bacterial cell, with the bacterial cell swollen and spindle-shaped. Figure 1 The isolated strains appeared as round or oval black colonies on TSC medium, with a milky white opaque ring around the colonies. The colony edges were neat, and the surface was moist and smooth. Figure 2 Gram staining of single colonies reveals Gram-positive bacilli with blunt ends, occurring singly or in pairs, without flagella. Figure 3In the milk fermentation experiment, all strains exhibited vigorous fermentation after 2 hours, with the milk curd breaking down and rapidly forming a sponge-like substance that rose to the surface of the culture medium. Figure 4 The dynamic-nitrate stab test revealed that all strains diffused along the stab line, and the culture medium turned red, indicating that nitrate was reduced to nitrite. Figure 5 Gas was produced during lactose-gelatin puncture, the culture medium turned from red to yellow, indicating that the lactose was fermented and produced acid. After 1 hour at 5°C, the gelatin liquefied. Figure 6 Molecular identification showed that the Plc gene was detected in all 8 isolated strains, and the etx and Cpe genes were detected in 2 strains. Six strains of Clostridium perfringens type A and two strains of Clostridium perfringens type D were detected in this study. Figure 7 ).

[0056] Table 1 PCR primer sequences and annealing temperatures

[0057]

[0058] Example 2

[0059] 1. Determination of growth curves of Clostridium perfringens types A and D from Bactrian camels

[0060] The growth of Clostridium perfringens within 24 hours was determined using a spectrophotometer, and the results are as follows: Figure 12 As shown, the growth of Clostridium perfringens types A and D reached its peak at 16 h.

[0061] 2. Virulence tests of Clostridium perfringens types A and D from Bactrian camels

[0062] Sixty Kunming mice were randomly divided into 6 groups. Each group (n=5 mice / group) received an intraperitoneal challenge dose equivalent to 1 live bacteria count. 10 7 CFU / mL, 1 10 6 CFU / mL, 1 10 5 CFU / mL, 1 10 4 CFU / mL, 1 10 3 CFU / mL, with one group receiving intraperitoneal injection of thioglycolate culture medium as a negative control. Mice were isolated and housed separately after different gradient challenges. Survival and mortality were observed after challenge, and the median lethal dose (LD50) of Clostridium perfringens types A and D was calculated using the Reed-Muench method. The results are shown in Table 2: LD50 of type A was determined. 50 2.4 × 10 6 D-type LD 50 3.2×106 .

[0063] Table 2. Virulence test of Clostridium perfringens in mice.

[0064]

[0065] 3. Autopsy of mice that died after being challenged with the virus

[0066] Methods: Collect samples from dead mice ( Figure 13 Gram staining was performed on visceral tissue slides of mice to observe bacterial infection; pathological changes in dead mice were also observed.

[0067] Results: After staining of visceral slides, a large number of Gram-positive rods were observed. Figure 14 Upon necropsy, the challenged mice showed significant enlargement of the heart, liver, spleen, lungs, and kidneys, with hemorrhage in the liver and spleen. The intestinal wall was thinned and showed inflammation. Figure 15 ).

[0068] 4. Histopathological observation of mice that died after being challenged with the virus

[0069] Methods: Heart, liver, spleen, lung, kidney, and other tissues were collected from dead mice and fixed in 4% paraformaldehyde tissue fixative (Guangzhou Yongjin Biotechnology Co., Ltd., Guangzhou, China). After trimming, dehydration, embedding, sectioning, staining, and mounting, the tissues were observed using a panoramic slide scanner (3DHISTECH (Hungary) DESK / MIDI / 250 / 1000) and CaseViewer 2.4 (3DHISTECH (Hungary)).

[0070] Results: The endocardium and epicardium of the heart tissue were clearly defined, with no obvious abnormalities observed; a small number of cardiomyocytes showed increased eosinophilicity in their cytoplasm. Figure 16 A (yellow arrow); no obvious abnormalities were observed in the interstitium; the liver capsule was composed of dense connective tissue of uniform thickness rich in elastic fibers, the lobule boundaries were indistinct, the central vein was located in the center of the lobule, and extensive hepatocyte edema was visible. Figure 16 B (green arrow), with loose, lightly stained cytoplasm and swollen cells; pigment deposition is frequently observed. Figure 16 B (brown arrow); the spleen's capsule is composed of dense connective tissue of uniform thickness, rich in elastic and smooth muscle fibers. This connective tissue extends into the spleen to form trabeculae, with no obvious abnormalities. The spleen parenchyma consists of red and white pulp. The white pulp is composed of a lymphatic sheath surrounding the central artery and lymphatic structures, with numerous white pulp adhesions (…). Figure 16 C (black arrow), irregular in shape; the red pulp is distributed in a large area under the capsule, around the trabeculae, and lateral to the white pulp, and is composed of splenic cords and splenic sinuses. The red pulp structure is loose, and a small number of granulocytes are visible infiltrating ( Figure 16 C (yellow arrow); occasional pigment deposition ( Figure 16C (brown arrow). The lung tissue surface is covered with a smooth capsule, with no obvious abnormalities; numerous granulocyte infiltrations are visible in the alveolar walls ( Figure 16 (D, blue arrow) Extensive moderate thickening of alveolar walls, widening of alveolar septa, and compensatory dilation of numerous alveoli; numerous fragments of necrotic cells are visible. Figure 16 D (black arrow); homogeneous eosinophilic material is visible in a small area of ​​alveolar cavities ( Figure 16 D (light green arrow); many bronchiolar epithelial cells are arranged irregularly ( Figure 16 (D, green arrow) Eosinophilic material is visible within the lumen; white blood cells are visible within the blood vessel lumen. Figure 16 D (yellow arrow). The renal capsule is composed of dense connective tissue of uniform thickness; the renal parenchyma consists of a superficial cortex and a deep medulla, with a clear boundary between the cortex and medulla; glomeruli are evenly distributed in the cortex, and the number of cells and matrix in the glomeruli are uniform; eosinophilic material is occasionally seen in Bowman's capsule. Figure 16 E (light green arrow); a small number of renal tubular epithelial cells showed nuclear pyknosis ( Figure 16 (E, black arrow); The connective tissue between the urinary tubules is the renal interstitium, and there is no significant interstitial proliferation.

[0071] 5. Optimize formaldehyde detoxification conditions

[0072] Six bottles of *Clostridium perfringens* were cultured overnight for 15 h, and then formaldehyde was added to achieve final concentrations of 0.3%, 0.4%, 0.5%, 0.6%, and 0.7%, respectively. The bottles were then placed in an anaerobic incubator at 37 ℃. Twenty-five mice per group were removed at 24 h, 48 h, and 72 h and administered an intraperitoneal injection of 1 mL per mouse. Mice were observed for mortality to determine the optimal formaldehyde detoxification conditions, i.e., the minimum formaldehyde concentration and the shortest detoxification time. Simultaneously, 200 μL of the solution was plated onto TSCs (Tetracycline Chloride Strain Tablets) for colony observation. The results are shown in Table 3. Figure 17 As shown, after mice were injected with inactivated vaccines of different formaldehyde concentrations, no mice injected with 0.3%, 0.4%, and 0.5% vaccines died after 24 hours of incubation. However, the vaccines with formaldehyde concentrations of 0.3% and 0.4% showed a small number of colonies on TSC medium. The optimal conditions for formaldehyde detoxification, namely the minimum formaldehyde concentration and the shortest detoxification time, were determined to be 0.5% and 24 hours, respectively.

[0073] Table 3. Optimization results of inactivation conditions for Clostridium perfringens cultures

[0074]

[0075] Example 3

[0076] Preparation of inactivated vaccine against Clostridium perfringens

[0077] 1. Bacterial culture: The standard strains of Clostridium perfringens type A and type D were inoculated into FTG and anaerobic cultured at 37 ℃ for 24 hours. The colony morphology and hemolysis were observed (type A colonies had a β hemolytic zone and type D colonies had a θ hemolytic zone). After confirming that there was no contamination by other bacteria, the bacterial culture was collected.

[0078] Bacterial inactivation: Dilute type A and type D bacterial suspensions with PBS buffer (pH 7.2-7.4) to a bacterial count of 1×10⁻⁶. 9 CFU / ml, then mix the two in a 1:1 ratio. Add a 0.5% formaldehyde solution (volume ratio) to the final concentration, and inactivate at 37°C for 24 hours, shaking every 4 hours to ensure complete inactivation.

[0079] Emulsification and adjuvant addition: Take 1 mL of bacterial culture and inoculate it into 100 mL of FTG. After culturing at 37℃ for 24 h, add formaldehyde to the final concentration of 0.5% and continue culturing for 24 h. Add aluminum gel adjuvant at a volume ratio of 9:1 to the inactivated bacterial culture and store at 4℃.

[0080] Dispensing and Quality Inspection: Dispense the vaccine into vials (2.0ml / vial) (e.g.) Figure 18 As shown), each batch of vaccines must undergo the following quality tests:

[0081] Property testing: After standing, observe the color of the upper liquid and the state of the lower sediment. After shaking, check the uniformity of suspension.

[0082] Sterility test: Meets the requirements of the sterility test method in the 2020 edition of the Chinese Pharmacopoeia.

[0083] Example 4

[0084] Mouse protection experiment of inactivated vaccine against Clostridium perfringens from Bactrian camels

[0085] One hundred mice were selected and divided into four groups of 25 mice each. Mice in groups 1, 2, and 3 were intraperitoneally injected with 0.1 ml, 0.2 ml, and 0.3 ml of inactivated vaccine, respectively. Mice in group 4 were intraperitoneally injected with 0.2 ml of thioglycolate culture medium without bacterial culture. Mice were re-inoculated after 14 and 21 days. Serum samples were collected on days 0, 14, 21, and 28 to detect α-toxin C antibodies. On day 21, mice were intraperitoneally injected with 0.5 ml of Clostridium perfringens culture to observe mortality. Results are as follows: Figure 19 As shown, the level of α-toxin C antibody in mouse serum gradually increased after immunization. On day 28, mice in the 0.3% group were intraperitoneally injected with 0.5 ml of Clostridium perfringens solution. No mice died within 3 days after intraperitoneal injection of 0.5 ml of Clostridium perfringens solution into groups 1, 2, and 3.

[0086] Example 5

[0087] Protection of young camels with inactivated vaccine against Clostridium perfringens from Bactrian camels

[0088] Eight 60-day-old juvenile camels were selected, and each camel was injected with 1.5 ml of the solution. Re-inoculation was performed at 14 and 21 days later. Serum samples were collected on days 0, 14, 21, and 28 to detect α-toxin C antibodies. The presence of stress symptoms in the juvenile camels was observed. Results are as follows: Figure 20 As shown: In the eight vaccinated young camels, the serum α-toxin C antibody level gradually increased with the number of immunizations and time. Body temperature rose by 0.3-0.8 ℃ 12-24 hours after vaccination, returning to normal within 48 hours; slight swelling with a diameter of 0.5-1.0 cm appeared in the subcutaneous part of the neck, which subsided spontaneously within 3 days; there were no serious abnormalities such as lethargy or respiratory distress, and no deaths occurred.

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A bivalent inactivated vaccine against Clostridium perfringens type AD from Bactrian camels, characterized in that, The bivalent inactivated vaccine contains two inactivated bacterial antigens, namely, Clostridium perfringens type A aerogenes strain from Bactrian humpsus with accession number CGMCC No. 35194 and Clostridium perfringens type D aerogenes strain from Bactrian humpsus with accession number CGMCC No. 35195.

2. The bivalent inactivated vaccine as described in claim 1, characterized in that, The bacterial count of the strains in the bivalent inactivated vaccines is 1×10⁻⁶. 9 CFU / ml.

3. The bivalent inactivated vaccine as described in claim 1 or 2, characterized in that, The bivalent inactivated vaccine was detoxified using formaldehyde solution.

4. A method for preparing a bivalent inactivated vaccine against Clostridium perfringens type AD from Bactrian camels, characterized in that, The strain of Clostridium perfringens type A, with accession number CGMCC No. 35194 and the strain of Clostridium perfringens type D, with accession number CGMCC No. 35195, as described in claim 1, are used as the inoculum for vaccine preparation. The inoculum culture of type A and type D Clostridium perfringens with high α-toxin content is obtained by culturing in a special culture medium. The inoculum culture is then inactivated, sterilized, concentrated with α-toxin, and used to prepare the vaccine to obtain the bivalent inactivated vaccine of Clostridium perfringens type AD.

5. The preparation method according to claim 4, characterized in that, During seedling preparation, the culture solutions of Clostridium perfringens types A and D were mixed at a volume ratio of 1:

1.

6. The use of the bivalent inactivated vaccine as described in claim 1 in the preparation of products for the prevention of diseases caused by Clostridium perfringens.

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