Poultry clostridium perfringens and application thereof in constructing poultry necrotic enteritis animal model

Abstract

The invention provides clostridium perfringens and an application of the clostridium perfringens in construction of an animal model of necrotic enteritis of poultry. The preservation number of the clostridium perfringens is CGMCC (China General Microbiological Culture Collection Center) No.33593. The screened CP-G-17 strain is separated from the intestinal tract of a sick chicken suffering from necrotic enteritis, analysis is conducted in the virulence gene aspect, a CP-G-17 genome comprises a plurality of important genes for coding alpha toxin, hyaluronidase, sialidase, collagenase and adhesin, and the genes are beneficial to toxin generation, bacterium colonization and growth, and can be used for preparing a drug for treating necrotic enteritis. However, the G type strain CVCC 2027 preserved in a laboratory is lack of virulence factors such as nano I, nano J, nano J and nano L, and the pathogenicity of the G type strain CVCC 2027 is lower than that of CP-G-17. Animal test results also verify that the pathogenicity of the CP-G-17 is stronger than that of CVCC 2027, in a coccidium and clostridium perfringens mixed infection model, the chicken inoculated with the CP-G-17 isolate shows standard intestinal tract lesion, and the established model can be used for subsequent treatment application of avian necrotic enteritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopreparation, and particularly relates to a Clostridium perfringens of poultry and its application in constructing an animal model of avian necrotic enteritis. Background Art

[0002] Avian necrotic enteritis (NE) is an intestinal disease characterized by necrotic lesions of the small intestinal mucosa caused by Clostridium perfringens, often causing diarrhea, weight loss and even acute death. The average monthly mortality rate of broilers suffering from NE is 25-50% higher than that of non-diseased broilers, seriously affecting the production performance of animals and causing an economic loss of about $6 billion to the global aquaculture industry every year. Studies have shown that in 2018, the detection rate of Clostridium perfringens in broilers in Central China was 23.1%, in 2020, the detection rate in large-scale chicken farms in northern Guangdong was 21.33%, in 2021, the detection rates in broilers in Shanxi and Beijing were 23.4%, and in 2023, the detection rate in some broiler farms in Shandong was 22.75%.

[0003] As the main pathogenic bacterium of necrotic enteritis (NE), Clostridium perfringens type G is a newly discovered toxin type strain in recent years and is widely prevalent abroad. However, there are few studies on the pathogenic mechanism and pathogenic conditions of Clostridium perfringens type G in China. Therefore, constructing an ideal disease model is of great significance for deeply exploring the pathological mechanism of NE and developing prevention and control technologies.

[0004] The main inducing factors of avian necrotic enteritis NE include the use of high-protein feeds (such as fish meal and corn feed) and co-infection with coccidia. At present, studies have established animal models by jointly using high-protein feeds and inoculating Eimeria necatrix. However, these models have limitations. Firstly, it is impossible to directly determine whether the lesions are caused by Clostridium perfringens itself. Secondly, the establishment process of the challenge model is cumbersome and complex. Although some studies have tried to establish an animal model by challenging with Clostridium perfringens of poultry alone, the following problems still exist:

[0005] 1) Most existing studies use 1- to 7-day-old chicks and fail to successfully construct a challenge model for adult chickens, which greatly limits the research on adult chickens, especially the subsequent vaccine evaluation work, because it is impossible to evaluate the vaccine effect by challenging after immunizing chicks.

[0006] 2) The existing strains have a large inoculation amount and too many inoculation times, resulting in the established model presenting late symptoms and being not conducive to the study of early symptoms.

[0007] 3) The existing animal models have poor repeatability and it is difficult to ensure the stability and reliability of experimental results.

[0008] Therefore, in the current research on avian necrotic enteritis, there is an urgent need for effective model establishment methods to provide highly reproducible animal models. Summary of the Invention

[0009] The purpose of the present invention is to provide a Clostridium perfringens for poultry and its application in constructing an animal model of avian necrotic enteritis, so as to make up for the deficiencies of the prior art.

[0010] The present invention first provides a Clostridium perfringens CP-G-17 strain for poultry, which was deposited on February 20, 2025 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33593.

[0011] Another aspect of the present invention also provides a use of the above-mentioned Clostridium perfringens for poultry, which is an application in constructing an animal model of avian necrotic enteritis;

[0012] As a specific record of the embodiment, the animal model is a chicken necrotic enteritis animal model.

[0013] Furthermore, the chicken is an adult SPF chicken.

[0014] Another aspect of the present invention also provides a method for constructing an animal model of avian necrotic enteritis, and the method is to use the above-mentioned Clostridium perfringens for poultry as the challenge strain to construct the animal model;

[0015] Furthermore, in the method, the number of bacteria for challenge is 1×10 8 CFU;

[0016] Furthermore, in the method, coccidia can also be selected for co-challenge to construct the animal model.

[0017] The CP-G-17 screened in the present invention was isolated from the intestine of diseased chickens with necrotic enteritis. From the analysis of virulence genes, the CP-G-17 genome contains many important genes encoding alpha toxin, hyaluronidase, sialidase, collagenase, adhesin, and these genes contribute to toxin production, bacterial colonization and growth. The G-type strain CVCC 2027 preserved in the laboratory lacks virulence factors nanI, nanJ, nagJ, nagL, and its pathogenicity will be lower than that of CP-G-17. The animal test results also confirm this point. The pathogenicity of CP-G-17 is stronger than that of CVCC 2027. In the model of mixed infection with coccidia and Clostridium perfringens, the chickens inoculated with the CP-G-17 isolate showed standard intestinal lesions, and the established model can be used for subsequent treatment applications of avian necrotic enteritis. Brief Description of the Drawings

[0018] Figure 1 : Colony morphological characteristic diagrams of the selected strains on selective media, where 1. TSC (containing egg yolk) medium; 2. TSC (without egg yolk) medium; 3. Chromogenic medium; 4. Blood agar medium; 5. Iron-containing milk medium;

[0019] Figure 2 : Results diagram of multiplex PCR genotyping detection of Clostridium perfringens isolates, where M. DL2000 Marker; 1. Negative control; 2. Isolated strain CP-G-17; 3. G-type representative strain CVCC 2027;

[0020] Figure 3 : Growth curve diagram of strain CP-G-17, where blue represents CP-G-17; yellow represents CVCC 2027;

[0021] Figure 4 : Intestinal lesion diagrams of each experimental model group, where 1. Blank control; 2. Group challenged with CVCC 2027 alone; 3. Group challenged with CP-G-17 alone; 4. Attenuated EM control group; 5. SPF chickens, group challenged with CVCC 2027 + attenuated EM mixture; 6. Broiler chickens, group challenged with CVCC 2027 + attenuated EM mixture; 7. SPF chickens, group challenged with CP-G-17 + attenuated EM mixture; 8. Broiler chickens, group challenged with CP-G-17 + attenuated EM mixture; 9. Virulent EM control group; 10. SPF chickens, group challenged with CVCC 2027 + virulent EM mixture; 11. Broiler chickens, group challenged with CVCC 2027 + virulent EM mixture; 12. SPF chickens, group challenged with CP-G-17 + virulent EM mixture; 13. Broiler chickens, group challenged with CP-G-17 + virulent EM mixture.

[0022] Figure 5 : Intestinal score difference analysis diagram, where 6-1. Intestinal score difference analysis between the blank control group and the group challenged alone; 6-2. Intestinal score difference analysis between the virulent EM control group and the group challenged with Clostridium perfringens + virulent EM mixture; 6-3. Intestinal score difference analysis between the attenuated EM control group and the group challenged with Clostridium perfringens + attenuated EM mixture; 6-4. Analysis of the influence of virulent and attenuated EM on the intestinal score differences in different challenged groups.

[0023] Figure 6 : Pathological tissue section diagrams (40×), where 1. Blank control; 2. Group challenged with CVCC 2027; 3. Group challenged with CVCC 2027 + virulent EM mixture; 4. Attenuated EM control; 5. Group challenged with CP-G-17 + attenuated EM mixture; 6. Group challenged with CP-G-17 + virulent EM mixture. Specific implementation manners

[0024] A special strain of Clostridium perfringens in poultry was screened and obtained in the present invention, and this strain was used as a challenge strain to prepare an animal model of avian necrotic enteritis.

[0025] The present invention will be described in detail below in conjunction with specific embodiments and drawings.

[0026] Example 1: Screening of strains

[0027] In a laminar flow hood, take the small intestine lesion site and its contents of diseased chickens collected from a chicken farm in 2021 into a 2 mL centrifuge tube, add PBS buffer, 4 - 5 small magnetic beads, grind, centrifuge, take a part of the supernatant and add it to FTG medium, add a layer of liquid paraffin on the top to seal and isolate oxygen, and place it in a constant temperature incubator for culturing at 42 °C for 8 h - 10 h. Pipette 100 μL of the cultured bacteria solution after FTG culture onto a TSC medium plate for inoculation, and place it in an anaerobic chamber containing an anaerobic gas generator for anaerobic culture at 42 °C for 24 - 48 h. During autopsy, there is a strong putrid smell in the abdominal cavity, the intestines are enlarged, inflated, fragile, congested, with thin walls and filled with gas, and there are bleeding points or bleeding patches after the intestines are opened.

[0028] For the collected samples, scrape about 2 g of the contents of the severely diseased site, fecal samples or swab samples suspected of being infected with Clostridium perfringens and put them into a 50 mL centrifuge tube. Add 10 mL of PBS solution to each centrifuge tube, vortex and mix well, and let it stand for 5 min.

[0029] Take 10 mL centrifuge tubes, add 5 mL of FTG medium to each tube, take 100 μL of the supernatant / swab leachate from the previous step and add it to the centrifuge tube, seal it with a sealing film and make good marks, and place it in an incubator at 37 °C for culturing for 8 h - 12 h.

[0030] Shake the cultured bacteria after enrichment, take 100 μL and add it to a 1.5 mL centrifuge tube, dilute it with ultrapure water at different concentration gradients to prepare dilutions of 10 2 、10 3 、10 4 at three different concentrations; respectively take 100 μL and spread it evenly on the TSC solid medium with a spreading rod; after the bacteria solution dries, cover it with a layer of TSC fixed medium; after solidification, invert it in an anaerobic incubator or anaerobic jar for anaerobic culture at 37 °C for 16 h - 24 h. Expand the culture of single colonies of Clostridium perfringens, take out the plate, and colonies with black dots in the milky white round spots appear on the plate, which are initially judged as Clostridium perfringens spots.

[0031] Pick a single colony from the TSC solid medium, dilute it with normal saline, smear it on a glass slide, fix it by heating with an alcohol lamp, and use a Gram rapid staining kit to stain and identify the suspected Clostridium perfringens, or stain and examine the bacterial solution after FTG culture under a microscope. It was observed that the isolated strain was a Gram-positive bacterium, with a morphological appearance of thick bacilli, straight edges, blunt and relatively neat ends, arranged singly, in pairs, or in short chains, which was consistent with the microscopic examination results of Clostridium perfringens.

[0032] The selected and identified Clostridium perfringens strain on the selective medium TSC (containing egg yolk) plate can decompose lecithin and form a milky white reaction ring around the black colony; on the TSC (without egg yolk) plate, since the medium contains sulfite and iron salts, the strain can reduce sulfite to sulfide, react with iron to form a black colony, and at the same time lecithinase decomposes lecithin to form an opaque halo, but it is not easily seen; on the chromogenic plate, orange-red, raised, smooth, round colonies with neat edges can be seen; on the blood agar plate, gray-green colonies with a smooth surface and a transparent inner circle will appear, and a double hemolysis ring will be formed, with the inner ring being completely hemolyzed (θ hemolysis) and the outer ring being incompletely hemolyzed (α hemolysis), that is, a double hemolysis ring; a large amount of gas is produced in the iron-containing milk medium, and the coagulated casein is washed into a honeycomb shape ( Figure 1 ).

[0033] Use the PCR amplification method to detect the α, β, ε, ι, cpe, and NetB genes of the selected and purified Clostridium perfringens strain. Design primers using the genomic DNA of the isolated strain as a template.

[0034] Reaction system (25 μL): 2×Taq Master Mix 12.5 μL, DNA template 2 μL, mixed primers 3 μL (primers in the system are mixed in equal proportions), ddH2O 7.5 μL.

[0035] Reaction program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 30 s; 30 cycles; extension at 72°C for 10 min; store at 4°C.

[0036] Take 5 μL of the PCR product and perform electrophoresis on a 1% agarose gel at 200 V for 20 min, and observe it with a gel imager using DL 2000 DNA Marker as a reference.

[0037] Table 1: Sequence information table of primers

[0038]

[0039] The multiplex PCR method was used to detect six toxins (α, β, ε, ι, cpe, NetB) of Clostridium perfringens isolates from poultry. The results showed that a fragment with the expected size of the NetB toxin gene was detected in this strain, and the results were as Figure 2 shown. According to the PCR toxin identification results of the strain, referring to Table 2, Figure 2 the strain CP-G-17 isolated by typing the strain into types A-G was a type G strain.

[0040] Table 2: Toxins contained in each type of Clostridium perfringens and typing table

[0041]

[0042]

[0043] The virulence factors widely present in the Clostridium perfringens isolated in the present invention, namely nagL, nagK, ploA, colA, nanH, plc, nagH, nanJ, nagJ, nanI, cloSI, nagI, were analyzed. Other strains isolated in the experiment, such as strains CP28, CP29, and CP31, showed the phenomenon of virulence gene deletion. The analysis results showed that all virulence factors were detected in CP-G-17, while the other type G representative strain CVCC 2027 lacked the virulence factors nanI, nanJ, nagJ, and nagL, and the virulence factors showed differences.

[0044] Example 2: Determination of the growth curve of the screened strain CP-G-17

[0045] Take the strain CP-G-17 and the typical strain CVCC 2027 of Clostridium perfringens. Inoculate 0.1 mL of the well-cultured culture in 10 mL of FTG medium, inoculate 12 tubes, and culture in an incubator at 42 °C. Take 2 mL of the culture solution from 1 test tube every 2 h, zero the spectrophotometer with the FTG medium without inoculating CP, and measure the OD600nm value of the culture solution to determine the optimal culture time.

[0046] From Figure 3 it can be seen that the logarithmic growth phases of the strains CP-G-17 and CVCC 2027 are 2-8 h, the stable growth phase is 8-14 h, and the decline phase begins after culturing for 20 h. Moreover, the bacterial viability of the isolated strain CP-G-17 is significantly higher than that of the typical strain CVCC 2027 of Clostridium perfringens.

[0047] Example 3: Construction of an animal model of Clostridium perfringens in poultry

[0048] Using the isolated Clostridium perfringens type G (CP-G-17) of the present invention and the typical strain CVCC 2027 of Clostridium perfringens preserved in the laboratory as control strains, animal experiments were carried out, and Eimeria maxima (EM) was selected as the strain.

[0049] Twenty-two-day-old chickens were used, and they were grouped as evenly as possible according to body weight and transferred to the wire netting in the coccidia isolation unit for feeding. The coccidia control group and the mixed infection group were orally gavaged with 10,000 virulent and attenuated coccidia vaccines respectively; CP-G-17 screened by the present invention was orally gavaged with 1×10 8 CFU / mL of fresh bacterial solution once a day from 26 to 27 days of age, and challenged twice continuously; CVCC 2027 preserved in the laboratory was orally gavaged with 1×10 9 CFU / mL of fresh bacterial solution once a day from 26 to 29 days of age, and challenged four times continuously; and high-protein fish meal was started to be added to the diet, and a diet with a crude protein content of 30% was fed, and they were allowed to eat and drink freely.

[0050] In this experiment, in order to ensure the correctness of the experiment, SPF chickens with clear background information were selected as the main experimental animals. Since Clostridium perfringens is prone to disease in broilers clinically, in order to ensure the integrity of the experiment, a broiler experimental group was set up in parallel. It was divided into 13 groups with 5 chickens in each group: Group 1 was 5 SPF chickens, the group of single challenge with bacterial solution CP-G-17; Group 2 was 5 SPF chickens, the group of mixed infection with virulent coccidia and bacterial solution CP-G-17; Group 3 was 5 SPF chickens, the group of mixed infection with attenuated coccidia and bacterial solution CP-G-17; Group 4 was 5 broilers, the group of mixed infection with virulent coccidia and bacterial solution CP-G-17; Group 5 was 5 broilers, the group of mixed infection with attenuated coccidia and bacterial solution CP-G-17; Group 6 was the group of single challenge with bacterial solution CVCC 2027; Group 7 was 5 SPF chickens, the group of mixed infection with virulent coccidia and bacterial solution CVCC 2027; Group 8 was 5 SPF chickens, the group of mixed infection with attenuated coccidia and bacterial solution CVCC 2027; Group 9 was 5 broilers, the group of mixed infection with virulent coccidia and bacterial solution CVCC 2027; Group 10 was 5 broilers, the group of mixed infection with attenuated coccidia and bacterial solution CVCC 2027; Group 11 was the virulent coccidia control group; Group 12 was the attenuated coccidia control group; Group 13 was the blank control group. The specific grouping and bacteriophage challenge are shown in Table 3.

[0051] Table 3: Table of experimental grouping and bacteriophage challenge

[0052]

[0053]

[0054] Intestinal lesions: The lesions of the bacteria-challenged group were compared with those of the blank group and the coccidia control group. The virulence and pathogenicity of Clostridium perfringens strains were determined according to the lesion scores. Lesions greater than 2 points and above were necrotic positive lesions. The specific lesion scoring criteria are shown in Table 4.

[0055] Table 4: Intestinal lesion scoring criteria

[0056]

[0057] Weigh the chickens at 33 days of age, perform necropsy, observe the intestinal lesions and conduct intestinal scoring.

[0058] The intestinal necropsy lesions of different groups are as Figure 4 , among which the intestinal thickness of the blank control group was uniform and there were no lesions.

[0059] The dose of the CP-G-17 strain screened in the present invention for the single bacteria-challenged group was 1×10 8 CFU / ml, 1 ml per chicken, and the starting age of inoculation was 26 days old. Obvious typical symptoms such as obvious flatulence, thinning of the intestinal wall, and bleeding appeared after continuous inoculation twice on the 26th and 27th days of age; the dose of the single bacteria-challenged group of the reference strain CVCC 2027 was 1×10 9 CFU / ml, 1 ml per chicken, and certain flatulence, thinning of the intestine, and a small number of bleeding points appeared after continuous inoculation 4 times from the 26th to the 29th days of age; different degrees of flatulence, thinning of the intestinal wall, bleeding points, erosion, necrosis and other symptoms appeared in the chicken intestines of the mixed bacteria-challenged group with coccidia added. According to the results, the single bacteria-challenged group of the CP-G-17 strain screened in the present invention could successfully induce disease, with more obvious symptoms compared to the reference strain CVCC 2027 and in line with the actual clinical lesions. The inoculation bacteria amount was 10 times lower and the inoculation times were significantly reduced.

[0060] The results showed that when establishing an animal model with the CP-G-17 strain screened in the present invention, the inoculation bacteria amount and the inoculation times were both reduced, and the disease symptoms were more obvious. An effective model could be established in both adult SPF chickens and broilers.

[0061] The intestinal scores of each experimental group were analyzed, and the results were as Figure 5 follows: There were significant differences between the single bacteria-challenged group and the blank control group, and the difference in the CP-G-17 group was more significant; there was a significant difference between the CP-G-17 + attenuated EM mixed bacteria-challenged group and the attenuated EM experimental group (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001); there was no significant difference between broilers and SPF chickens with the same bacteria and coccidia; when only the strong and weak coccidia were used as a single variable, there was no significant difference in each group. The above results indicated that the score of the single bacteria-challenged group of CP-G-17 was more obvious and effective.

[0062] Intestinal tissue pathological sections were prepared for the significantly diseased parts of different groups, and the results were as follows Figure 6 It can be seen that intestinal tissue damage of varying severity occurred in the intestines of each challenged group, with intestinal villi broken and shed, and the outer epithelial cells of the intestinal villi necrotic and disappeared; there was infiltration of inflammatory cells in the stroma, and symptoms of necrosis of some intestinal villus cells (yellow arrows indicate infiltration of inflammatory cells, red arrows indicate shedding of villi, green arrows indicate cell necrosis, and black arrows indicate normal villi and glands).

[0063] The results showed that the CP-G-17 experimental group challenged alone could cause typical pathological changes in the intestinal tissue.

[0064] The present invention successfully established an animal model of Clostridium perfringens in poultry. Under the condition of no inducing factors, infection with the CP-G-17 strain could cause severe intestinal damage (lesions greater than 2 points), providing an ideal animal model for the pathogenesis of avian necrotic enteritis. In the establishment of this model, compared with single factors (fish meal or coccidia), intestinal lesions caused by multiple factors were more obvious. The finally established animal model used 26-day-old SPF chickens, the challenged strain was CP-G-17, coccidia were not added, the challenge dose was 1×10 8 CFU / mL, and intestinal observation was carried out after continuous challenge twice. When the score was greater than 2 points, it was judged as diseased, providing a stable model for the subsequent research and screening of Clostridium perfringens vaccines and drugs in poultry.

[0065] The reported results showed that when Clostridium perfringens was used alone, it could not cause ideal lesions, and the coccidia vaccine could play a key inducing role. The weight gain rates of chickens in both the group challenged alone with Clostridium perfringens and the group co-infected with coccidia and Clostridium perfringens were lower than those of the control group. These results strongly indicated that these two infections jointly promoted the pathogenesis of avian necrotic enteritis. Moreover, the CP-G-17 strain had strong pathogenicity, with reduced challenge dose and number of challenges, reducing the cumbersome operation steps in the clinical application process, and being able to induce obvious lesions in adult chickens, providing a basis for the evaluation of subsequent vaccines.

Claims

1. A poultry Clostridium perfringens, characterized in that The deposit number of the avian Clostridium perfringens is CGMCC No.33593.

2. Use of the avian Clostridium perfringens described in claim 1 in constructing an animal model of avian necrotic enteritis.

3. The use according to claim 2, characterized in that The animal model is a chicken necrotic enteritis animal model.

4. The use according to claim 3, characterized in that The chickens are adult SPF chickens.

5. A method for constructing an animal model of avian necrotic enteritis, characterized in that: The method is to construct an animal model using the avian Clostridium perfringens described in claim 1 as a challenge strain.

6. The method according to claim 5, characterized in that The number of the virus-challenging strain is 1×10 8 CFU.

7. The method according to claim 6, characterized in that The method also uses coccidia to challenge the animal model.

8. An animal model, characterized in that: The animal model is constructed using the method described in claim 5.

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