Modeling method for inducing broiler immunosuppression and secondary bacterial infection

Abstract

The invention relates to the technical field of veterinary medicine and animal models, in particular to a modeling method for inducing broiler chicken immunosuppression and secondary bacterial infection. The method comprises the following steps: (a) starting to continuously feed broiler chickens with daily ration containing mycotoxin at the age of 6-10 days until molding is finished; (b) inoculating infectious bursal disease virus on the broiler chicken at the age of 12-16 days; and (c) inoculating avian pathogenic escherichia coli after inoculating the infectious bursal disease virus for a period of time. According to the modeling method, the clinical pathogenesis process is highly simulated, the mechanism fits the reality, the model success rate is high and stable, standardized operation is achieved, repeatability is good, simplicity, convenience and safety are achieved, and the biological safety risk of virulent strains is avoided; the application range is wide, and an excellent platform is provided for research and development; the method is low in cost and suitable for scientific research and large-scale drug screening popularization.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of veterinary medicine and animal model technology, in particular to a modeling method for inducing immune suppression and secondary bacterial infection of broilers. BACKGROUND

[0002] In modern intensive farming, broilers often have immune dysfunction and immune suppression due to high stocking density, multiple stress factors (such as transportation, changing feed, environmental changes), etc. Immune suppression is a core problem in broiler production, which can reduce the response ability of the flock to vaccines and greatly increase the susceptibility to opportunistic bacteria (such as E. coli, Salmonella, etc.), thereby causing severe secondary infection and causing huge economic losses.

[0003] Currently, the methods for establishing a broiler immune suppression model in laboratory research mainly include: (1) chemical drug induction method: such as using cyclophosphamide (CTX), dexamethasone, hydrocortisone, etc. immune suppressants. Among them, CTX is a commonly used alkylating agent antitumor drug, which has specific killing effect on rapidly proliferating lymphocytes and can inhibit cellular and humoral immunity. However, the use of CTX alone often only causes a decrease in immune function, and it is difficult to stably and efficiently simulate the common clinical pathological process of secondary bacterial infection caused by immune suppression. (2) Pathogen infection method: such as directly using infectious bursal disease virus (IBDV), chicken anemia virus (CAV) and other immunosuppressive viruses for infection. Although this method is close to natural infection in terms of pathological process, virus culture requires high requirements, has high biological safety risk, and the stability of the model is significantly affected by individual differences and virus titer, and it is not easy to combine with a specific secondary bacterial infection model. (3) Single bacterial challenge method: directly attacking healthy chickens without treatment or with slight stress, but since the immune system of healthy chickens is sound, a very high dose of bacteria is often required to cause disease, which does not conform to the actual situation, and the success rate and sensitivity to drugs of the model are not ideal.

[0004] Therefore, there is an urgent need in the art for a modeling method for broiler immune suppression and secondary bacterial infection that is simple to operate, short in cycle, low in cost, good in repeatability, clear in pathogenesis, and can highly simulate the clinical disease process, in order to meet the needs of scientific research and new drug development.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to provide a modeling method for inducing immune suppression and secondary bacterial infection of broilers. The method simulates the synergistic effect of the most common pathogenic factors in the farm through the sequential combination of "basic nutritional stress + mild viral infection + bacterial secondary challenge", which is more close to the actual production in mechanism.

[0007] In order to achieve the above-mentioned object of the present application, the following technical solutions are adopted: Another aspect of the present application relates to a modeling method for inducing immune suppression and secondary bacterial infection of broilers, comprising the following steps: (a) at the age of 6-10 days, continuously feeding the broilers with a daily ration containing mycotoxin until the end of modeling; (b) at the age of 12-16 days, inoculating infectious bursal disease virus; (c) inoculating avian pathogenic E. coli at a period of time after inoculating the infectious bursal disease virus.

[0008] The modeling method innovatively combines the "nutritional immune suppression factor (aflatoxin B1)", "immune suppression virus (IBDV)", and "conditional pathogenic bacteria (APEC)" in a specific time sequence, highly simulates the clinical disease process, and is more in line with the actual mechanism, avoiding the one-sidedness and limitations of single-factor modeling; the model is successfully established by first establishing deep immune suppression and then bacterial attack, has high success rate and is stable, and solves the problem of single bacterial attack method; the standardized operation is good in repeatability, simple and safe, and avoids the biosafety risk of virulent strains; the application range is wide, and the "prevention and health care" effect of single drug and comprehensive functional feed additive can be evaluated in multiple dimensions, providing an excellent platform for research and development; the cost is low, and the cycle is only about 3 weeks, which is suitable for scientific research and large-scale drug screening promotion.

[0009] Compared with the prior art, the present application has the following beneficial effects: 1. Highly simulates the clinical disease process, and is more in line with the actual mechanism: The present application innovatively combines the "nutritional immune suppression factor (aflatoxin B1)", "immune suppression virus (IBDV)", and "conditional pathogenic bacteria (APEC)" in a specific time sequence of "basic stress → immune stimulation → disease outbreak", perfectly reproduces the classic disease process of secondary bacterial infection on the basis of "background disease" (such as mycotoxin poisoning and subclinical viral infection) in a breeding farm. The model can stably reproduce the progressive and complex pathological process from immune dysfunction to typical E. coli disease outbreak, avoiding the one-sidedness and limitations of single-factor modeling.

[0010] 2. High success rate and stability of the model: By first establishing a deep immune suppression state by using nutritional toxins and viruses in cooperation, and then attacking bacteria at the most vulnerable period of the immune system, the success rate of secondary infection is greatly improved. The implementation case shows that the mortality rate of the model group is as high as 42%, and all of them show typical complex pathological changes, proving the reliability and stability of the method, and solving the problems of low success rate and large individual differences of single bacterial attack method.

[0011] 3. Good repeatability, simple and safe operation: The application adopts standardized diet addition, virus inoculation of attenuated strain and specific dose bacterial challenge, and all operations can be completed under conventional experimental conditions without complex virus culture or high-risk pathogen operation, thereby avoiding the biological safety risk caused by the use of virulent strain. The doses and action times of various factors are clear, so that the experiments between different batches have good repeatability and comparability.

[0012] 4. Wide application range and comprehensive evaluation dimension: The model can not only be used for evaluating a single drug (such as an antibiotic), but also can effectively evaluate a feed additive (such as the compound plant extract in the examples) with comprehensive functions of immune enhancement, liver protection and detoxification, and antiviral effect. Through monitoring of multiple-dimension indexes such as mortality, production performance, immune organ index, antibody level and inflammatory factor, it can be clearly distinguished whether the tested substance focuses on "prevention of immunosuppression" or "treatment of bacterial infection", thereby providing an excellent evaluation platform for the research and development of new drugs and new feed additives.

[0013] 5. Low cost and short cycle: The entire modeling cycle can be completed within 3 weeks, which is much shorter than some natural infection models of viruses, and the cost of the reagents (such as AFB1, IBDV attenuated vaccine and APEC strain) is relatively low, which is very suitable for popularization and application in scientific research and large-scale drug screening. DETAILED DESCRIPTION

[0014] The technical solutions of the present application will be described clearly and completely in combination with specific embodiments, but those skilled in the art will understand that the following described embodiments are part of the embodiments of the present application, not all the embodiments, and should not be regarded as limiting the scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. The specific conditions are not specified in the embodiments, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments not specified by the manufacturer are all conventional products that can be purchased on the market.

[0015] Another aspect of the present application relates to a modeling method for inducing immune suppression and secondary bacterial infection in broilers, comprising the following steps: (a) Start to continuously feed the diet containing mycotoxin at the age of 6-10 days (for example, it can be but is not limited to any one of 6 days, 7 days, 8 days, 9 days or 10 days, or a range value between any two of them) of the broiler, and continuously feed until the end of modeling; this step continuously attacks by low dose of mycotoxin, simulates the common feed mold problem in breeding, damages the liver function and preliminarily suppresses the immune function; (b) at the age of 12-16 days (for example, it can be but is not limited to any one of 12 days, 13 days, 14 days, 15 days or 16 days, or a range value between any two of them), inoculate infectious bursal disease virus (IBDV); inoculate a moderately weak virulent infectious bursal disease virus, simulate wild virus infection, mainly attack the central immune organ bursa of Fabricius, cause severe acquired immunosuppression characterized by B lymphocyte depletion; (c) a period of time after inoculation of the infectious bursal disease virus, inoculate avian pathogenic E. coli.

[0016] The present application can stably replicate the disease process from mild to severe and progressive development by controlling the dosage and action time of each factor, has a high model success rate, and provides an excellent evaluation model for evaluating nutritional regulators, antiviral drugs, antibiotic substitutes and feed additives with comprehensive health care functions.

[0017] The pathological process of the present application highly simulates the clinic, innovatively combines the nutritional toxin, immunosuppressive virus and conditional pathogenic bacteria in a specific sequence, perfectly reproduces the real disease process of secondary infection on the basis of "background disease" in the farm, and avoids the one-sidedness of single-factor modeling.

[0018] Further, the content of the mycotoxin in the daily ration containing the mycotoxin is 1.0-1.5 mg / kg, including but not limited to any one of 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg or 1.5 mg / kg, or a range value between any two of them. The setting of this content range is the key to realizing stable and controllable basic immunosuppression. The aflatoxin B1 at this dose can effectively damage the detoxification function and protein synthesis of the liver and continuously inhibit the immune function, but also avoids acute poisoning and a large number of deaths caused by too high a dose, ensures the survival of the animals during the modeling process and the smooth development of the subsequent disease process, and makes the model have both significant pathological effects and good operability.

[0019] Further, the mycotoxin includes aflatoxin. Aflatoxin, especially B1, is selected because it is one of the most common and toxic mycotoxins in the breeding industry, and can highly simulate the immunosuppression problem caused by feed mold in real production. Its specific damage to the liver and strong inhibition of cellular immunity provide a precise pathological basis for establishing a "background disease" model that conforms to the clinical reality.

[0020] Further, the inoculation amount of the infectious bursal disease virus is 100-500 ELD 50 / each, including but not limited to 100 ELD 50 / each, 150 ELD 50 / each, 250 ELD 50 / each, 300 ELD 50 / each, 350 ELD 50 / each, 400 ELD 50 / each, 450 ELD 50 / each, 450 ELD 50 / each, or 500 ELD 50 / each, or a range value between any two of the foregoing. This inoculum range ensures that the virus effectively replicates in the meat chicken and successfully attacks the bursa of Fabricius, causing significant depletion of B lymphocytes and suppression of humoral immunity, while avoiding premature collapse of the model caused by a virulent strain or an excessively high dose. This dose provides a reliable guarantee for achieving reproducible, severe acquired immunodeficiency.

[0021] Further, the inoculation route of the infectious bursal disease virus includes at least one of nasal instillation, eye instillation, or oral administration. Using these mucosal routes for inoculation accurately simulates the main mode of infection of IBDV under natural conditions. This not only improves the success rate of viral infection and ensures that the virus targets the bursa of Fabricius, but also makes the model establishment process closer to the epidemiological rules, enhancing the authenticity and persuasiveness of the model.

[0022] Further, the period of time is 3 to 6 days, including but not limited to a point value of any one of 3 days, 4 days, 5 days, or 6 days, or a range value between any two of them. At this time, the viremic peak has passed, and the immune system is at its most vulnerable. Setting the bacterial attack time point at 3 to 6 days after viral inoculation is based on the kinetic characteristics of viral infection. This time period is exactly the "opportunity window" after the viremic peak, when the immune organs (especially the bursa of Fabricius) are most severely damaged and the overall immune defense ability of the body is at its lowest. Performing bacterial attack during this window maximizes the success rate and consistency of secondary infection, perfectly simulating the internal logic of clinical secondary infection.

[0023] Further, the inoculum of the avian pathogenic E. coli is 10 7 ~10 9 CFU / each. This dose range ensures that in an immune-suppressed body, typical E. coli disease pathological changes (such as pericarditis and perihelitis) can be stably induced without the use of supraphysiological doses of bacteria. This not only more accurately reflects the pathogenic characteristics of opportunistic pathogens in immunodeficient hosts, but also makes the model more sensitive and accurate for evaluating the efficacy of antibacterial drugs or immune enhancers.

[0024] Further, the inoculation route of the avian pathogenic E. coli comprises intraperitoneal injection and / or aerosol inhalation. Intraperitoneal injection can reliably induce systemic sepsis, and the model is highly reproducible. Aerosol inhalation can simulate the respiratory infection route and replicate the common air sac inflammation lesions in clinic. The setting of the two routes provides flexibility for researchers to choose the appropriate challenge method according to different research purposes (such as evaluating systemic antibacterial drugs or respiratory mucosal immune enhancers), thereby broadening the application range of the model.

[0025] Further, the modeling method for inducing immune suppression and secondary bacterial infection in broilers further comprises: (d) Model evaluation.

[0026] Further, the model evaluation is performed within 85-96 hours (for example, it can be but is not limited to any one of 85 hours, 88 hours, 90 hours, 93 hours or 96 hours, or a range value between any two of them) after inoculation of the avian pathogenic E. coli. The setting of the uniform evaluation in the specific time window of 85 to 96 hours after challenge is an optimized choice based on the disease development rule. At this time, the typical lesions and clinical symptoms caused by bacterial infection have fully appeared, but complete evaluation data can be captured before the animals die due to infection. This provision ensures the standardization of model success determination, the synchronization of data collection and the comparability of results between different batches of experiments.

[0027] Further, the success of the model is evaluated by the following indicators: (1) Clinical performance observation: the model successful broilers exhibit typical clinical symptoms such as depression, significant decrease in feed intake, diarrhea and lameness; (2) Necropsy lesion examination: Immune organ lesions: atrophy, hemorrhage or jelly-like edema can be observed in the bursa of Fabricius; Liver lesions: the liver presents the characteristic lesions of mycotoxicosis, such as enlargement and yellowing of the color; E. coli disease lesions: typical pathological changes of avian pathogenic E. coli infection such as pericarditis, perihepatitis and air sac inflammation; (3) Immunological index detection: Immune organ index: the bursa of Fabricius index and the thymus index are significantly reduced; Humoral immune function: the serum Newcastle disease (NDV) antibody titer is significantly reduced as detected by hemagglutination inhibition (HI) test.

[0028] (4) Pathogen examination: Virus isolation: infectious bursal disease virus (IBDV) can be isolated and identified from the bursa of Fabricius tissue; Bacterial isolation: the E. coli challenge strain can be recovered from the liver and heart.

[0029] When the experimental animal meets the above clinical manifestations, characteristic necropsy lesions, significantly abnormal immunological indexes, and positive etiological examination simultaneously, the model of immune suppression and secondary E. coli infection of broilers is determined to be successfully established.

[0030] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not noted in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not noted by the manufacturer are all conventional products that can be obtained by market purchase.

[0031] Example 1 The modeling method for inducing immune suppression and secondary bacterial infection of broilers provided in the present embodiment comprises the following time sequence steps: (1) Nutritional stress period: to establish a basic immune suppression state; Select 1-day-old healthy commercial broilers, and feed them under standard conditions until 7-day-old; from 8-day-old, feed the test group of broilers with daily feed containing 1.2 mg / kg of aflatoxin B1 (AFB1) until the end of modeling; (2) Immune stimulation period: to apply viral attack; At 14-day-old of the broilers, inoculate infectious bursal disease virus by nasal drop, and the inoculation amount is 200 ELD 50 / each; (3) Disease outbreak period: to trigger secondary bacterial infection; On the 4th day after inoculation of virus in step (2) (i.e. 18-day-old of the broilers), at this time, the viremia peak has passed, and the immune system is at the most vulnerable period, inoculate 2×10 8 CFU / each of avian pathogenic E. coli (APECO 78 strain) by intraperitoneal injection; (4) Model evaluation: within 96 hours after bacterial challenge, whether the model is successfully established is comprehensively evaluated by the following indexes: Clinical manifestations: depression, decreased feed intake, diarrhea, lameness, etc.; Necropsy lesions: atrophy, hemorrhage or jelly-like edema of bursa of Fabricius; liver enlargement, yellow color (AFB1 toxicity characteristics); pericarditis, perihelitis, air sacculitis (E. coli disease characteristics); Immunological indexes: significantly reduced bursa index and thymus index; significantly lower serum Newcastle disease antibody titer (HI test) than the blank control group; Etiological examination: IBDV can be isolated from the bursa of Fabricius; E. coli challenge strain can be isolated from liver, heart and other parts; When the experimental animal meets the above clinical manifestations, characteristic necropsy lesions, significantly abnormal immunological indexes, and positive pathogen examination at the same time, it is determined that the broiler immune suppression and secondary E. coli infection model is successfully established.

[0032] Example 2 The modeling method for inducing broiler immune suppression and secondary bacterial infection provided in this example includes the following time sequence steps: (1) Nutritional stress period: establish a basic immune suppression state; Select 1-day-old healthy commercial broilers, and raise them under standard conditions until they are 6 days old; from 7 days old, feed the test group of broilers with a daily ration containing 1.5 mg / kg of aflatoxin B1 (AFB1) until the end of modeling; (2) Immune challenge period: apply viral attack; At 12 days of age, the broilers are inoculated with infectious bursal disease virus by oral route, with an inoculation amount of 500 ELD 50 / each; (3) Disease outbreak period: trigger secondary bacterial infection; On the 6th day after inoculation of the virus in step (2), 10 7 CFU / each of avian pathogenic E. coli (APECO78 strain) is inoculated by intraperitoneal injection; (4) Model evaluation, same as Example 1.

[0033] Example 3 The modeling method for inducing broiler immune suppression and secondary bacterial infection provided in this example includes the following time sequence steps: (1) Nutritional stress period: establish a basic immune suppression state; Select 1-day-old healthy commercial broilers, and raise them under standard conditions until they are 10 days old; from 11 days old, feed the test group of broilers with a daily ration containing 1.0 mg / kg of aflatoxin B1 (AFB1) until the end of modeling; (2) Immune challenge period: apply viral attack; At 16 days of age, the broilers are inoculated with infectious bursal disease virus by eye drop, with an inoculation amount of 100 ELD 50 / each; (3) Disease outbreak period: trigger secondary bacterial infection; On the 3rd day after inoculation of the virus in step (2), 1×10 9 CFU / each of avian pathogenic E. coli (APECO78 strain) is inoculated by air inhalation; (4) Model evaluation: same as Example 1.

[0034] Experimental Example The health care effect of a composite plant extract was evaluated using the model provided in Example 1.

[0035] Two hundred 1-day-old AA broilers were raised to 7 days old and then randomly divided into 4 groups: The blank control group was fed with the basic diet; The model group was subjected to the modeling method of Example 1; The prevention group was subjected to the modeling method of Example 1, and 0.1wt% plant extract was added to the basic diet from 1 day old, the main active ingredients of the plant extract being astragalus polysaccharide, chicoric acid and andrographolide; The treatment group was subjected to the modeling method of Example 1, and after inoculation with avian pathogenic E. coli, enrofloxacin was administered in drinking water at a concentration of 50mg / L for 5 days.

[0036] All groups (except the blank group) were fed with a diet containing 1.2mg / kg AFB1 from 8 days old, and at 14 days old, all groups (except the blank group) were inoculated with 200ELD 50 / each of IBDV CE strain attenuated virus, and at 18 days old, all groups (except the blank group) were injected intraperitoneally with 2x10 8 CFU / each of APEC O78.

[0037] The test results are shown in Table 1 below.

[0038] Table 1 Effect of composite plant extract on production performance, immune organs, pathology and serological indicators of model broilers

[0039] The model group showed typical composite pathological changes, with a mortality rate of 42%, and upon dissection, atrophic bursa of Fabricius, yellowish and oily liver, severe pericarditis and perihepatitis, and extremely significantly reduced immune organ index.

[0040] The prevention group showed significantly reduced clinical performance and pathological damage, with a mortality rate of 12%, and significantly higher immune organ index than the model group.

[0041] The mortality rate of the treatment group was lower than that of the model group, but the immune organ damage was still severe, indicating that the drug mainly played an antibacterial role, but could not repair the previously caused immune suppression.

[0042] From the above, it can be seen that the present experimental example successfully verified the stability and effectiveness of the modeling method, and could clearly distinguish the different effects of the test substance "prevention and health care" and "treatment".

[0043] Although the present application has been described and illustrated with a certain degree of particularity, it is understood that the present application has been made by way of examples only and that numerous changes in the details of execution can be made by those skilled in the art without departing from the spirit and scope of the application.

Claims

1. A method of modeling immune suppression and secondary bacterial infection in broiler chickens, comprising, The method comprises the following steps: (a) at 6-10 days of age, continuously feeding the broiler with a mycotoxin-containing diet until the end of modeling; (b) at 12-16 days of age, inoculating the broiler with infectious bursal disease virus; (c) inoculating the broiler with avian pathogenic E. coli at a time period after the inoculation of the infectious bursal disease virus.

2. The method of claim 1, wherein the broiler chickens are induced to be immunosuppressed and secondary bacterial infection is modeled, characterized in that, The content of mycotoxin in the mycotoxin-containing diet is 1.0-1.5 mg / kg.

3. The method of claim 1, wherein the broiler chickens are induced to be immunosuppressed and secondary bacterial infection is modeled, characterized in that, The mycotoxin comprises aflatoxin.

4. The method of claim 1, wherein the method is characterized by, The inoculation amount of the infectious bursal disease virus is 100-500 ELD 50 / emu.

5. The method for inducing immunosuppression and secondary bacterial infection in broilers according to claim 1, characterized in that, The inoculation route of the infectious bursal disease virus comprises at least one of nose drop, eye drop and oral administration.

6. The method of claim 1, wherein the broiler chickens are induced to be immunosuppressed and secondary bacterial infection is modeled by, The time period is 3-6 days.

7. The method of claim 1, wherein the broiler chickens are induced to have immune suppression and secondary bacterial infection. The inoculation amount of the avian pathogenic E. coli is 10 7 ~10 9 CFU / each.

8. The method of claim 1, wherein the broiler chickens are induced to have immune suppression and secondary bacterial infection. The inoculation route of the avian pathogenic E. coli comprises intraperitoneal injection and / or aerosol inhalation.

9. The method of claim 1-8, wherein the method is characterized by, Further comprising: (d) model evaluation.

10. The method of claim 9, wherein the broiler chickens are induced to be immunosuppressed and secondary bacterial infection is modeled by, The model evaluation is performed within 85-96 hours after the inoculation of the avian pathogenic E. coli.

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