Establishment of an animal model of poult proventriculitis induced by vomitoxin

Abstract

The present application belongs to the technical field of disease model construction, and discloses establishment of a vomiting toxin-mediated gosling adenomygatitis animal model, and specifically discloses a method for constructing a chicken adenomygatitis model, which comprises the following steps: 0-day-old goslings are infected with a mixed solution of mycoplasma gallisepticum and escherichia coli, and then the goslings are fed with starter feed containing vomiting toxin and full-price feed containing vomiting toxin, so that the chicken adenomygatitis model is constructed. The method for constructing the chicken adenomygatitis model is simple and fast, the constructed model has obvious clinical symptoms and typical autopsy characteristics, is suitable for research on drugs for preventing and treating chicken adenomygatitis, can be used for exploring the pathogenesis of chicken adenomygatitis, and can be used for screening and optimizing a treatment scheme, evaluating the effectiveness and safety of the treatment scheme, thereby providing an experimental basis for controlling the disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of disease model construction, and particularly relates to the establishment of a vomitoxin-mediated gosling adenomygatitis animal model. BACKGROUND

[0002] At present, chicken adenomygatitis characterized by enlarged and thickened glandular stomach, hemorrhage and erosion of glandular stomach papilla, necrosis, ulcer, perforation and shedding of the keratin layer of the muscular stomach has long plagued the poultry industry. The disease is highly prevalent in summer, leading to slow growth of animals, low feed conversion rate, 20%-90% morbidity, 10%-50% mortality, and huge economic losses of the poultry industry. Animal disease infection model is an important tool for us to carry out animal disease infection mechanism research and explore animal disease treatment.

[0003] The causes of chicken adenomygatitis are complex, and the main causes at present are two aspects, namely infectious factors and non-infectious factors. As for chicken adenomygatitis caused by infectious factors, it is believed that the pathogen is the cause of the disease. In cases showing typical pathological changes of chicken adenomygatitis, one or more pathogens are isolated, including coronavirus similar to infectious bronchitis virus, reticuloendotheliosis virus, reovirus and the like. Domestic and foreign scholars have not reached a consensus on the pathogen of chicken adenomygatitis. In addition, inoculation of the above-mentioned isolated pathogens into the animal cannot reproduce the pathological changes of chicken adenomygatitis, and the true pathogen has not been determined. Research on chicken adenomygatitis caused by non-infectious factors is less, and the main pathogenic factors include excessive biological amine in the diet, excessive mycotoxin and excessive addition of copper sulfate. Mycotoxin is a large class of secondary metabolites produced by fungi and molds, which poses a serious risk to human and animal health. Mycotoxin pollution widely exists in plant-derived feed, especially in cereals, fruits, hazelnuts, almonds, seeds, forages and other agricultural feed or food for human and livestock consumption. The common and more harmful mycotoxins in animal feed are aflatoxin, vomitoxin and zearalenone.

[0004] At present, there is no effective animal model that can reproduce the pathological changes of chicken adenomygatitis through vomitoxin, therefore, it is of great significance to develop a chicken adenomygatitis animal model that is simple to operate and has obvious symptoms, which is the basis for understanding the occurrence and development of mycotoxin-mediated chicken adenomygatitis. Establishing a scientific and reliable chicken adenomygatitis animal model can not only better study its pathophysiological changes and explore the pathogenesis, but also be used for screening and optimization of treatment programs and evaluation of the effectiveness and safety of treatment programs, thereby providing experimental basis for the control of the disease. SUMMARY

[0005] The present application establishes a vomitoxin-mediated chicken adenomygatitis animal model to provide a basis for the treatment or prevention of chicken adenomygatitis, thereby solving one of the problems in the prior art.

[0006] The first aspect of the present application aims to provide a method for constructing a chicken oesophagitis model.

[0007] The second aspect of the present application aims to provide a chicken oesophagitis model.

[0008] The third aspect of the present application aims to provide the method for constructing a chicken oesophagitis model of the first aspect of the present application or the chicken oesophagitis model of the second aspect of the present application.

[0009] The fourth aspect of the present application aims to provide the use of vomitoxin in constructing a chicken oesophagitis model.

[0010] To achieve the above-mentioned object, the technical solution adopted by the present application is as follows:

[0011] The first aspect of the present application provides a method for constructing a chicken oesophagitis model, which comprises the following steps: infecting 0-day-old chicks with a mixed solution of Mycoplasma gallisepticum and Escherichia coli, and feeding the chicks with starter feed containing vomitoxin and complete feed containing vomitoxin before and after, so as to construct a chicken oesophagitis model.

[0012] In some embodiments of the present application, the mixed solution is used to infect the chicks by means of nose drops and eye drops.

[0013] In some embodiments of the present application, the chicks are infected with the mixed solution twice a day, and the mixed solution is used in an amount of 0.2-0.4 mL each time.

[0014] In some embodiments of the present application, the starter feed contains 3-15 mg / kg of vomitoxin.

[0015] In some preferred embodiments of the present application, the starter feed contains 5-15 mg / kg of vomitoxin.

[0016] In some more preferred embodiments of the present application, the starter feed contains 5-10 mg / kg of vomitoxin.

[0017] In some embodiments of the present application, the complete feed contains 3-15 mg / kg of vomitoxin.

[0018] In some preferred embodiments of the present application, the complete feed contains 5-15 mg / kg of vomitoxin.

[0019] In some more preferred embodiments of the present application, the complete feed contains 5-10 mg / kg of vomitoxin.

[0020] In some embodiments of the present application, the chicks are infected with the mixed solution for 2-4 days.

[0021] In some preferred embodiments of the present application, the time for infecting 0-day-old chicks with the mixed solution is 3 days.

[0022] In some embodiments of the present application, the chicks are fed with open feed containing vomitoxin during the infection.

[0023] In some embodiments of the present application, the time for feeding the chicks with the complete feed containing vomitoxin is 4-7 days.

[0024] In some preferred embodiments of the present application, the time for feeding the chicks with the complete feed containing vomitoxin is 5 days.

[0025] In some embodiments of the present application, the concentration of Mycoplasma gallisepticum in the mixed solution is 1-9x10 8 CCU / mL.

[0026] In some embodiments of the present application, the concentration of Escherichia coli in the mixed solution is 1-9x10 9 CFU / mL.

[0027] In some embodiments of the present application, the criteria for judging the success of the model construction are: enlarged and thickened glandular stomach, hemorrhagic glandular stomach papilla, necrosis and ulcer of the keratin layer of the muscular stomach.

[0028] In some embodiments of the present application, in addition to the above symptoms, the model chickens generally also have symptoms such as reduced feed and water intake, slow growth, listlessness, and inability to stand.

[0029] In a second aspect of the present application, a chicken Gumboro disease model is provided, which is constructed by the construction method of the first aspect of the present application.

[0030] In a third aspect of the present application, the construction method of the first aspect of the present application or the chicken Gumboro disease model of the second aspect of the present application is used in any one of (1)-(3):

[0031] (1) screening drugs for preventing or treating chicken Gumboro disease;

[0032] (2) evaluating the effectiveness and safety of a treatment regimen for chicken Gumboro disease;

[0033] (3) studying the pathogenesis of chicken Gumboro disease.

[0034] In a fourth aspect of the present application, vomitoxin is used in the construction of a chicken Gumboro disease model.

[0035] The present application has the following beneficial effects:

[0036] The chicken adenomygatitis model provided by the application has simple and rapid construction method, obvious clinical symptoms, typical dissection characteristics, is suitable for the research of drugs for preventing and treating chicken adenomygatitis, can be used for exploring the pathogenesis of chicken adenomygatitis, and can be used for screening and optimizing treatment programs and evaluating the effectiveness and safety of the treatment programs, thereby providing experimental basis for the control of the disease. The method also provides a unique method and idea for the construction of the chicken adenomygatitis model. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The gizzard of the chick in the challenge group is swollen.

[0038] Figure 2 The gizzard papilla of the chick in the challenge group disappears.

[0039] Figure 3 The chick in the chicken adenomygatitis animal model is listless and cannot stand.

[0040] Figure 4 The gizzard papilla of the chick in the chicken adenomygatitis animal model disappears, fuses, bleeds, and the gizzard keratin layer necroses and ulcerates.

[0041] Figure 5 The chicken adenomygatitis clinical pathological changes are displayed. DETAILED DESCRIPTION

[0042] The content of the application will be further described in detail through specific examples.

[0043] It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application.

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme in the embodiments of the application will be clearly and completely described below. The specific conditions not noted in the embodiments 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.

[0045] The features and performances of the application will be further described in detail below in combination with examples.

[0046] Example 1

[0047] 1. Culture of Mycoplasma gallisepticum (Mycoplasma gallisepticum S6 strain, referred to as S6)

[0048] (1) Preparation of culture medium: 7.5 mL fetal bovine serum, 5 mL yeast extract, 0.5 mL glucose solution, 0.5 mL thallium acetate solution, 0.25 mL penicillin solution, 1 mL phenol red solution were taken into a 15 mL sterile centrifuge tube, mixed uniformly, adjusted to pH 7.8, filtered, mixed into 35 mL PPLO liquid medium, and stored at 4°C for standby.

[0049] (2) Revival: When reviving Mycoplasma gallisepticum, take one test tube containing Mycoplasma freeze-dried powder, add 3 mL of culture medium, mix well by blowing, then transfer 1 mL of bacterial solution to 3 sterile 15 mL centrifuge tubes containing 4 mL of culture medium, seal with sealing film, and place the centrifuge tubes vertically in a 37°C, 5% CO2 incubator for about 72 hours. When the color of the culture medium changes from dark red to orange yellow, it can be used as seed liquid for continuous subculture.

[0050] (3) Subculture: 4.5 mL of culture medium was taken into 5 sterilized test tubes, and 0.5 mL of Mycoplasma gallisepticum (MG) seed liquid was added to each test tube. 7.2 mL of culture medium was taken into 2 10 mL EP tubes, and 0.8 mL of MG seed liquid was added to each tube. After sealing with sealing film, they were incubated at 37°C in a 5% CO2 incubator. When the color of the culture medium changes from dark red to orange yellow (usually 48 hours), the MG is in the logarithmic growth phase, with the best activity and highest concentration, and is stored at 4°C for standby.

[0051] (4) Detection of concentration: The liquid culture of MG in the logarithmic growth phase was diluted 10 times with culture medium to 1×10 -1 ~ 1×10 -12 times dilution concentration (a total of 12 tubes, the 13th tube as a control), and incubated at 37°C in a 5% CO2 incubator. The highest dilution when the culture medium changes from red to orange yellow was taken as the color change unit (CCU), and the concentration of the bacterial solution was calculated as CCU / mL. The concentration of the bacterial solution was adjusted to 5×10 9 CCU / mL for standby.

[0052] 2. Culture of avian pathogenic Escherichia coli (avian pathogenic Escherichia coli O78 strain, abbreviated as APEC)

[0053] (1) Preparation of culture medium: 10 g of proteose peptone, 10 g of NaCl, and 5 g of yeast extract were weighed into a 1 L beaker, about 800 mL of deionized water was added, and the mixture was stirred to dissolve. NaOH solution was added to adjust the pH to 7.0, and deionized water was added to bring the medium to 1 L. After high-temperature high-pressure sterilization, it was stored at 4°C for standby.

[0054] (2) Take 2.5 mL of frozen E. coli solution, melt at room temperature, add to the above medium at a ratio of 1:100, incubate overnight at 37°C, adjust the concentration to 1x10 7 CFU / mL using a McFarland turbidimeter, and store at 4°C for later use.

[0055] 3. Culture of mixed E. coli and mycoplasma challenge solution

[0056] Inoculate the E. coli solution after overnight culture at a ratio of 1:10 into the mycoplasma culture medium after 24 hours of subculture, and incubate overnight at 37°C in a 5% CO2 constant temperature incubator. Adjust the concentration to 1x10 9 CFU / mL (wherein the S6 chicken mycoplasma contains 10 8 CCU / mL), and store at 4°C for later use.

[0057] Example 2

[0058] A chicken adenomygatitis animal model is constructed by the following method: select 0-day-old K996 eucalyptus chickens, inoculate 0.2 mL (0.2 mL of nose drops and 0.2 mL of eye drops) of mixed solution of chicken mycoplasma and avian pathogenic E. coli (prepared in Example 1) by nose drops and eye drops, inoculate twice a day (10 am and 3 pm), and continue for 3 days. Feed the eucalyptus chickens with millet starter containing 5 mg / kg of vomitoxin (DON) when they are 0-3 days old, and feed them with full-price feed containing 5 mg / kg of vomitoxin (i.e., 181 compound feed, purchased from Guangzhou Nongzhidao Feed Co., Ltd., with product number 20231107) when they are 4-9 days old. End the experiment when the eucalyptus chickens are 10 days old, and obtain the chicken adenomygatitis animal model.

[0059] Example 3

[0060] A chicken adenomygatitis animal model is constructed by the following method: select 0-day-old K996 eucalyptus chickens, inoculate 0.2 mL (0.2 mL of nose drops and 0.2 mL of eye drops) of mixed solution of chicken mycoplasma and avian pathogenic E. coli (prepared in Example 1) by nose drops and eye drops, inoculate twice a day (10 am and 3 pm), and continue for 3 days. Feed the eucalyptus chickens with millet starter containing 5 mg / kg of vomitoxin (DON) when they are 0-3 days old, and feed them with full-price feed containing 5 mg / kg of vomitoxin (i.e., 181 compound feed, purchased from Guangzhou Nongzhidao Feed Co., Ltd., with product number 20231107) when they are 4-9 days old. End the experiment when the eucalyptus chickens are 10 days old, and obtain the chicken adenomygatitis animal model.

[0061] Effect example

[0062] A chicken adenomygatitis animal model is constructed according to Example 2 and Example 3, and an inoculation group (compared with Example 2, the difference is that the eucalyptus chickens are fed with millet starter when they are 0-3 days old, and with 181 compound feed when they are 4-9 days old) and a blank control group (compared with the inoculation group, the difference is that the eucalyptus chickens are not inoculated) are set. The specific test grouping and treatment methods are shown in Table 1, and there are 10 replicates for each treatment. During the experiment, the feed intake and body weight are recorded, and the average daily feed intake, average daily weight gain, and feed conversion ratio are calculated at the end of the experiment.

[0063] The results are shown in Table 2. Compared with the blank control group, the average daily weight gain and average feed intake of the chicks infected with Mycoplasma gallisepticum and avian pathogenic E. coli decreased. On the basis of the infection of the chicks with Mycoplasma gallisepticum and avian pathogenic E. coli, feeding of vomitoxin (10 mg / kg) further reduced the average daily weight gain of the chicks (by 16.42%) and increased the feed-meat ratio (by 20.75%).

[0064] Further, after the end of the test, all animals were dissected to observe the clinical pathological changes of the gizzard and the muscle stomach, and the pathological changes were scored according to the scoring standard of chicken gizzard-muscle stomach disease (Table 5). The results are shown in Tables 3 and 4, and the clinical pathological changes of the chicken gizzard-muscle stomach disease of each treatment group are shown in Figure 5 The test results show that, compared with the blank control group, after the chicks are infected with Mycoplasma gallisepticum and avian pathogenic E. coli, the gizzard will show obvious clinical pathological changes (P<0.05), such as gizzard swelling Figure 1 ), disappearance of gizzard papilla Figure 2 ), thickening of the stomach wall, but no pathological changes in the muscle stomach (P>0.05). On the basis of the infection of the chicks with Mycoplasma gallisepticum and avian pathogenic E. coli, feeding of vomitoxin (5 mg / kg) causes the chicks to be listless, and the pathological changes in the gizzard have no significant difference (P>0.05) compared with the infection group, but the muscle stomach will show obvious pathological changes (P<0.05) such as keratin layer necrosis and ulceration. Feeding of vomitoxin (10 mg / kg) causes the chicks to be listless and unable to stand Figure 3 ), and on the basis of the obvious pathological changes in the gizzard, the muscle stomach will show clinical pathological changes (P<0.05) such as keratin layer necrosis and ulceration Figure 4 .

[0065] Table 1 Test grouping and treatment method

[0066]

[0067] Table 2 Production performance results of chicks in different treatment groups

[0068]

[0069] Table 3 Scoring results of gizzard, muscle stomach and intestinal tract of chicks

[0070]

[0071] Note: The superscript letters represent significant differences (P<0.01).

[0072] Table 4 Raw data of pathological change scoring of chicken gizzard-muscle stomach disease

[0073] Number Glandular stomach score Glandular stomach lesion Muscular stomach score Muscular stomach lesion Duodenum Jejunum Ileum Rectum Note 1-1 2 Fusion of papilla 1 Local whitening of gizzard- gizzards 0 0 0 0 1-2 1 Glandular stomach swelling 1 Local whitening of gizzard- gizzards 0 0 0 1 Rectal distension 1-3 1 Glandular stomach swelling 2 Muscular stomach ulcer 0 0 0 0 1-4 3 Glandular stomach like globular swelling, fusion of papilla 1 Local whitening of gizzard- gizzards 0 0 0 0 1-5 2 Glandular stomach swelling 1 Local whitening of gizzard- gizzards 0 0 0 1 1-6 1 Glandular stomach swelling 1 Local whitening of gizzard- gizzards 0 0 0 0 1-7 1 Glandular stomach swelling 1 Local whitening of gizzard- gizzards 0 0 0 0 1-8 1 Glandular stomach swelling 1 Muscular stomach ulcer 0 0 0 0 1-9 1 Glandular stomach swelling 1 Local whitening of gizzard- gizzards 0 0 0 0 Thymus enlargement with bleeding, bleeding point 1-10 1 Glandular stomach swelling 2 Muscular stomach ulcer 0 0 0 1 2-1 3 Glandular stomach swelling, basal bleeding 3 Muscular stomach blackening, bleeding 0 0 1 1 2-2 2 Glandular stomach swelling, basal bleeding 1 Local whitening of gizzard- gizzards 0 0 0 0 2-3 1 Glandular stomach swelling 2 Muscular stomach ulcer 0 0 0 1 2-4 3 Fusion of papilla 1 Local whitening of gizzard- gizzards ulcer 0 0 0 0 Unsteady keel protrusion 2-5 1 Glandular stomach swelling 1 Local whitening of gizzard- gizzards, ulcer 0 0 0 0 2-6 2 Fusion of papilla 2 Muscular stomach ulcer 0 0 0 1 2-7 1 Glandular stomach swelling 1 Local whitening of gizzard- gizzards 0 0 0 0 2-8 2 Basal bleeding 2 Muscular stomach ulcer 0 0 0 0 Thymus deep red, congestion 2-9 3 Fusion of papilla 1 Local whitening of gizzard- gizzards 0 0 0 1 2-10 2 Glandular stomach swelling fusion of papilla 1 Local whitening of gizzard- gizzards 0 0 0 1 Thymus bleeding 3-1 2 Basal bleeding 2 Local whitening of gizzard- gizzards, ulcer 0 0 0 0 3-2 2 Glandular stomach swelling, fusion of papilla 1 Muscular stomach ulcer 0 0 0 0 Gallbladder enlargement 3-3 3 Glandular stomach swelling, fusion of papilla 1 Local whitening of gizzard- gizzards 0 0 0 0 3-4 3 Glandular stomach swelling fusion of papilla 1 Local whitening of gizzard- gizzards 0 0 0 0 3-5 2 Glandular stomach swelling 1 Local whitening of gizzard- gizzards 0 0 0 0 Gallbladder enlargement 3-6 2 Glandular stomach swelling 1 Local whitening of gizzard- gizzards 0 0 0 0 3-7 2 Glandular stomach swelling 1 Local whitening of gizzard- gizzards, ulcer 0 1 0 0 3-8 2 Glandular stomach swelling 2 Muscular stomach ulcer 0 0 0 0 3-9 1 Glandular stomach swelling 2 Local whitening of gizzard- gizzards 0 0 0 0 3-10 1 Thymus enlargement 1 Local whitening of gizzard- gizzards 0 0 0 0 Local whitening of gizzard- gizzards 4-1 0 1 Muscular stomach ulcer 0 0 0 0 4-2 0 0 0 0 0 0 4-3 0 0 0 0 0 0 4-4 0 0 0 0 0 0 4-5 0 1 Muscular stomach abnormality 0 0 0 0 4-6 0 0 0 0 0 0 4-7 0 1 Local whitening of gizzard- gizzards 0 0 0 0 Muscular stomach ulcer 4-8 0 0 0 0 0 0 4-9 0 1 ​ 0 0 0 0 4-10 0 1 ​ 0 0 0 0

[0074] Note: No. 1-1 ~ 1-10 is the attack group, No. 2-1 ~ 2-10 is the example 2 group, No. 3-1 ~ 3-10 is the example 3 group, No. 4-1 ~ 4-10 is the blank control group.

[0075] Table 5 chicken pathological changes of glandular stomachitis score criteria

[0076]

[0077] In summary, the present application successfully established a vomit toxin mediated chicken glandular stomachitis animal model (construction method as in example 2-3). Using Mycoplasma gallisepticum S6 strain and avian pathogenic E. coli O78 strain by way of nasal eye drops inoculation of Ephedra chicks, can make the chicks appear glandular stomachitis clinical pathological changes, on this basis, feeding vomit toxin, not only will make the chicks' glandular stomach appear pathological changes, but also will make the muscular stomach appear pathological changes such as erosion, ulcer.

[0078] The above embodiments of the present application are described in detail in combination with the drawings, but the present application is not limited to the above embodiments, and within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for constructing a chicken proventriculitis model, characterized in that, The construction method includes the following steps: 0-day-old chicks are infected with a mixture of Mycoplasma gallisepticum and Escherichia coli for 2-4 days, and during the infection process, they are fed starter feed containing vomitoxin; then they are fed complete feed containing vomitoxin for 4-7 days, thus constructing a chicken proventriculitis model.

2. The construction method according to claim 1, characterized in that, The mixture is administered to chicks via nasal drops and eye drops.

3. The construction method according to claim 2, characterized in that, The starter feed contains 3–15 mg / kg of vomitoxin.

4. The construction method according to claim 2, characterized in that, The complete feed contains 3–15 mg / kg of vomitoxin.

5. The construction method according to any one of claims 1 to 4, characterized in that, The concentration of Mycoplasma gallisepticum in the mixture is 1–9 × 10⁻⁶. 8 CCU / mL; and / or, the concentration of Escherichia coli in the mixture is 1–9 × 10⁻⁶. 9 CFU / mL.

6. The construction method according to claim 5, characterized in that, The criteria for successfully constructing the model are: enlarged and thickened proventriculus, bleeding from the proventricular papillae, and necrosis and ulceration of the gizzard cuticle.

7. The application of the construction method according to any one of claims 1 to 6 in any one of (1) to (2): (1) Screening for drugs to prevent or treat proventriculitis in chickens; (2) Study the pathogenesis of chicken proventriculitis.

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