A method for constructing a chicken deficiency diarrhea model

Abstract

The application discloses a method for constructing a chicken deficiency and cold diarrhea model, and takes Hailan brown chicks as samples, 1-2% sodium sulfite is added into the drinking water of the chickens, and the chickens are allowed to drink water and eat freely for 4-5 days, after the use of sodium sulfite is stopped, the chickens have deficiency and cold diarrhea for more than 7 days, and the chickens have no death, so that it is determined that the model construction is successful. The model construction method is simple and easy to operate, the constructed model has obvious clinical symptoms, rapid onset, high morbidity and long course, the model is stable and reliable, and can be suitable for the research of traditional Chinese veterinary drugs for preventing and treating chicken deficiency and cold diarrhea.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of chicken deficiency diarrhea model construction method, belong to the technical field of model construction of traditional Chinese veterinary medicine. BACKGROUND

[0002] Diarrhea is a common disease in poultry. Poultry diarrhea refers to the condition where poultry has increased frequency of defecation, watery stool, or stool like water, also known as diarrhea. In the treatment of diarrhea, traditional Chinese veterinary medicine can be divided into many different types according to different causes and symptoms, such as damp-heat diarrhea, deficiency diarrhea, etc. The treatment of diarrhea must be targeted to different causes and types, and different treatment methods and drugs should be selected flexibly to achieve good effect. Deficiency diarrhea is caused by cold accumulation in the spleen and stomach, and the cause is deficiency of the spleen and stomach, deficiency of the spleen and stomach, internal accumulation of cold and deficiency of the spleen and stomach.

[0003] Deficiency diarrhea in chickens has a great impact on the production performance of chickens, leading to malnutrition, growth stagnation and even death. There are many causes of chicken deficiency diarrhea, including improper feeding environment such as feed, temperature, infection of pathogenic microorganisms such as E. coli and coccidia, and physiological dysfunction caused by drug abuse. Due to the complexity of the causes, it is difficult to choose a targeted treatment method, and traditional Chinese veterinary medicine is based on disease types for targeted treatment, which helps to prevent and treat the disease. However, there is currently a lack of stable chicken deficiency diarrhea model, making it difficult to objectively evaluate the efficacy of traditional Chinese veterinary medicine. SUMMARY

[0004] To solve the above problems, the present application provides a method for constructing a chicken deficiency diarrhea model by screening a variety of pathogenic factors. The method successfully constructs a chicken deficiency diarrhea model by adding sodium sulfite to the drinking water of the chicken. The model construction method of the present application is simple and easy to operate, and the constructed model has obvious clinical symptoms, rapid onset, high incidence and long duration. The model is stable and reliable, and can be suitable for the study of traditional Chinese veterinary medicine for the prevention and treatment of chicken deficiency diarrhea.

[0005] The technical solution of the present application is: a method for constructing a chicken deficiency diarrhea model, characterized by: using Hailan brown chicks raised in SPF chicken isolators as samples, adding 1-2% sodium sulfite to the drinking water of the chicken, continuously for 4-5 days, during which the chicken is free to drink water and eat, and after stopping the use of sodium sulfite, the chicken has deficiency diarrhea for more than 7 days without death, which is determined as a successful model construction.

[0006] The standard of deficiency diarrhea syndrome is: chicken has cold accumulation in the spleen and stomach, weak spleen yang, and impaired transport function, with clinical symptoms such as diarrhea, low body temperature, and significant decrease in food intake, as well as decreased digestive capacity and immune-related indicators.

[0007] Preferably, 1% sodium sulfite is added to the drinking water of the chicken.

[0008] Preferably, the Hy-Line Brown chicks are 12-15 days old.

[0009] The present application constructs a chicken deficiency diarrhea model from the aspects of chicken feed, medicine, breeding environment, pathogenic microorganism, etc. It is accidentally found that the model constructed by adding 1-2% of sodium sulfite in the drinking water of the chicken has obvious clinical symptoms, rapid onset, high incidence and long course. The model is stable and reliable, thereby obtaining a simple and easy-to-operate model construction method. The constructed model can be suitable for the research of traditional Chinese veterinary medicine for preventing and treating chicken deficiency diarrhea. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The feces of the chickens in each experimental group of Example 1 after being treated for 4 days; Group I is a blank control group; Group II is a 2% dextran group; Group III is a 2% sodium sulfite group; Group IV is a 2% sodium sulfate group; Group V is a group of 1 mL of disease material grinding liquid per chicken; and Group VI is a group of 0.5 mL of disease material grinding liquid per chicken;

[0011] Figure 2 The detection results of the contents of α-amylase (α-AMS) and pepsinogen (PG) in the serum of the experimental groups of Example 1 after being treated for 4 days and after recovery for 7 days; A is the content of α-AMS in the serum of each group after being treated for 4 days; B is the content of α-AMS in the serum of each group after recovery for 7 days; C is the content of PG in the serum of each group after being treated for 4 days; D is the content of PG in the serum of each group after recovery for 7 days; Group I is a blank control group; Group II is a 2% dextran group; Group III is a 2% sodium sulfite group; Group IV is a 2% sodium sulfate group; Group V is a group of 1 mL of disease material grinding liquid per chicken; and Group VI is a group of 0.5 mL of disease material grinding liquid per chicken;

[0012] Figure 3 The detection results of the content of secretory immunoglobulin (SIgA) in the serum of the experimental groups of Example 1 after being treated for 4 days and after recovery for 7 days; A is the content of SIgA in the serum of each group after being treated for 4 days; B is the content of SIgA in the serum of each group after recovery for 7 days; Group I is a blank control group; Group II is a 2% dextran group; Group III is a 2% sodium sulfite group; Group IV is a 2% sodium sulfate group; Group V is a group of 1 mL of disease material grinding liquid per chicken; and Group VI is a group of 0.5 mL of disease material grinding liquid per chicken;

[0013] Figure 4 The feces of the chickens after being treated with sodium sulfite in drinking water for 4 days in Example 2; A: blank group; B: 0.5% sodium sulfite group; C: 1% sodium sulfite group; D: 1.5% sodium sulfite group; and E: 2% sodium sulfite group;

[0014] Figure 5 The lesions of the glandular stomach and the muscular stomach of the chickens after being treated with sodium sulfite in drinking water for 4 days in Example 2;

[0015] Figure 6 The results of the detection of the contents of α-amylase (α-AMS) and pepsinogen (PG) in serum after 4 days of treatment and 7 days of recovery of the experimental groups of Example 2: A is the content of α-AMS in serum of each group after 4 days of water treatment; B is the content of α-AMS in serum of each group after 7 days of recovery; C is the content of PG in serum of each group after 4 days of water treatment; D is the content of PG in serum of each group after 7 days of recovery;

[0016] Figure 7 The results of the detection of the contents of secretory immunoglobulin (SIgA) in serum after 4 days of treatment and 7 days of recovery of the experimental groups of Example 2: A is the content of SIgA in serum of each group after 4 days of water treatment; B is the content of SIgA in serum of each group after 7 days of recovery;

[0017] Figure 8 The feces situation after 7 days of recovery of Example 3; the left graph is the blank group, and the right graph is the 1% sodium sulfite group. DETAILED DESCRIPTION

[0018] The effects are illustrated below in combination with examples.

[0019] Example 1: Drug screening for constructing Hailan brown chick asthenia and diarrhea model

[0020] 1.1 Chickens, 12-day-old Hailan brown chickens raised in SPF chicken isolators; SPF chicken isolators produced in Jiangsu Changshu.

[0021] 1.2 Drugs: Dextran, sodium sulfite, sodium sulfate: analytical pure, purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0022] 1.3 Disease material grinding liquid, taken from a breeding farm in Yishui, Shandong, 20g of duodenal tissue of sick chickens with asthenia and diarrhea was added to 20mL of normal saline, ground with a tissue grinder, and then centrifuged to obtain the supernatant.

[0023] 1.4 Test grouping and treatment method: the chicks were randomly divided into groups I-VI, 10 in each group. Group I was the blank control group, normal free drinking and feeding; group II was added with 2% dextran in drinking water for 4 consecutive days; group III was added with 2% sodium sulfite in drinking water for 4 consecutive days; group IV was added with 2% sodium sulfate in drinking water for 4 consecutive days; group V was orally administered with 1mL of disease material grinding liquid per chicken for 2 consecutive days; group VI was intraperitoneally injected with 0.5mL of sterile filtered disease material grinding liquid per chicken for 2 consecutive days. Groups I, V and VI were normally drinking throughout the process, and groups II, III and IV were normally drinking and feeding for 7 days after 4 days of treatment. Clinical symptoms were observed, weight was measured, cloacal temperature was measured, and blood was collected to separate serum for detection of digestive and immune indicators.

[0024] 1.5 Detection of digestive and immune indicators: The levels of pepsinogen (PG), α-amylase (α-AMS), and secretory immunoglobulin (SIgA) in serum were detected according to the ELISA kit method.

[0025] 1.6 Judgment criteria: The presence of symptoms of deficiency-cold diarrhea.

[0026] 2 Experimental Results

[0027] 2.1 Clinical Symptoms: On the second day of the experiment, all chicks in the experimental group exhibited symptoms of deficiency-cold diarrhea, which gradually worsened with the duration of medication. Fecal results on the fourth day of the experiment are shown in [the table / document / etc.]. Figure 1 The sodium sulfite group (Group III) had the thinnest feces and the most severe feed-induced diarrhea, along with symptoms of lethargy. The group receiving intraperitoneal injection of the ground feed showed significant intragroup variation, with only a portion (40%) exhibiting symptoms of weakness and diarrhea. Seven days after recovery, the sodium sulfite group still experienced diarrhea and incomplete digestion, but their feces were partially formed, and they remained lethargic. The symptoms of weakness and diarrhea in the other groups largely disappeared, but intragroup variation was significant, indicating poor homogeneity.

[0028] 2.2 The average body weight and weight gain rate of chickens in each group were statistically analyzed, as shown in Table 1. The weight gain rate of chickens in each experimental group was lower than that of the control group, with the sodium sulfite group showing the slowest weight gain and the lowest weight gain rate.

[0029] Table 1. Average body weight and weight gain rate of chickens in each group.

[0030]

[0031] 2.3 Changes in body temperature

[0032] The average cloacal temperature of chickens in each group was recorded 4 days after administration of the medication (groups I, V, and VI received normal drinking water for 4 days, the same below), as shown in Table 2. The average temperature of chickens in each experimental group was lower than that of the control group, with the sodium sulfite group and sodium sulfate group having a body temperature more than 0.2℃ lower than that of the control group.

[0033] Table 2. Average temperature of chickens in each group after 4 days of drinking water with medication.

[0034]

[0035] 2.4 Results of Digestive and Immune Indicators Tests

[0036] The results of serum α-AMS and PG levels are as follows: Figure 2The results were analyzed by Tukey multiple comparison method. The a-AMS content of each experimental group was lower than that of the blank group after 4 days of treatment, and the sodium sulfite experimental group was significantly different from the blank group (p<0.05). After 7 days of recovery, the a-AMS content of each experimental group recovered, and the sodium sulfite experimental group was still significantly lower than the blank group. After 4 days of treatment, the PG content of the sodium sulfite and sodium sulfate groups was significantly lower than that of the blank group. After 7 days of recovery, the PG content of the sodium sulfite group was significantly lower than that of the blank group (p<0.05), and the other experimental groups were not significantly different from the blank group.

[0037] The results of the SIgA content in serum are shown in Figure 3 After 4 days of treatment, the SIgA content of each experimental group was lower than that of the blank group, and the difference between group III and group I was significant (p<0.05). The difference between group IV and group V and group I was extremely significant (p<0.01). After 7 days of recovery, the SIgA content of each experimental group was significantly lower than that of the blank group (p<0.01).

[0038] In summary, each experimental group can cause diarrhea, reduce weight gain rate, and significantly reduce some indicators reflecting digestive capacity and immune level. The body temperature of some chickens significantly decreased. The indicators of the sodium sulfite drinking group were most significantly different from the blank group, indicating that each treatment method in this experiment can cause the virtual cold and diarrhea syndrome, and drinking sodium sulfite is the optimal method to construct the virtual cold and diarrhea model.

[0039] Example 2: Construction of a virtual cold and diarrhea model of Hyline brown chicks

[0040] 1. Materials and methods

[0041] 1.1 Chicks, 15-day-old Hyline brown chickens raised in SPF chicken isolators; SPF chicken isolators produced in Jiangsu Changshu.

[0042] 1.2 Sodium sulfite: analytical pure, purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0043] 1.3 Grouping: chicks were randomly divided into sodium sulfite different concentration test groups (0.5%, 1%, 1.5%, 2%) and a blank group, 10 in each group.

[0044] 1.4 Treatment method: the blank group freely drank and ate normally; the test groups added 0.5%, 1%, 1.5%, and 2% sodium sulfite in the drinking water for 4 days, and then normally drank and ate for 7 days of recovery. Clinical symptoms, weight, cloacal temperature, blood sampling, serum separation, and detection of digestive and immune indicators were observed.

[0045] 1.5 Digestive and immune indicators: The contents of pepsinogen (PG), α-amylase (α-AMS) and secretory immunoglobulin (SIgA) in serum were detected according to the method of ELISA kit.

[0046] 1.6 Judgment criteria: the occurrence of cold diarrhea syndrome.

[0047] 2 Results

[0048] 2.1 Clinical symptoms: the test group chickens showed diarrhea symptoms on the second day of the test, and the diarrhea symptoms of the chickens in each test group gradually worsened over time. After 4 days of drinking water, the feces of the chickens in each group were Figure 4 Within the range of less than 1.0% of sodium sulfite concentration, sodium sulfite can cause chickens to have diarrhea, incomplete digestion, and gizzard ulcers. After 7 days of recovery, the chickens still had diarrhea, but the feces were formed, and the feed intake and body weight were not significantly affected. When the concentration was greater than 1.5%, sodium sulfite caused the chickens to reduce water intake and feed intake by 50%, and the chickens were depressed and had less feces.

[0049] 2.2 The average body weight and weight gain rate of each group of chickens were calculated, as shown in Table 3. Within the range of less than 1.0% of sodium sulfite concentration, the body weight increased slowly when drinking sodium sulfite, and increased rapidly during the recovery period. When the concentration was greater than 1.5%, the body weight decreased, and the body weight increased slowly during the recovery period. This indicates that the addition of sodium sulfite can significantly reduce the weight gain rate of chickens, and the weight gain rate decreases with increasing concentration of sodium sulfite.

[0050] Table 3 Average body weight and weight gain rate of each group of chickens

[0051]

[0052] 2.3 After 4 days of drinking sodium sulfite, the gizzard keratin layer was rough and had different degrees of ulceration, the keratin layer was thin and easy to peel off, the glandular stomach was enlarged, the glandular stomach wall was thin, and individual chickens had glandular stomach hyperemia and mild bleeding. The lesions in the 1.5% and 2% sodium sulfite groups were significantly more severe than in the other groups, and the epithelium of some tissues at the junction of the glandular and muscular stomachs was shed and necrotic Figure 5 The glandular and muscular stomachs of the blank control group were normal.

[0053] 2.3 Body temperature changes

[0054] The average temperature of the cloaca of each group of chickens after 4 days of drinking the drug was calculated, as shown in Table 4. The body temperature of each experimental group was lower than that of the blank group, and was more than 0.2°C lower than that of the blank group.

[0055] Table 4 Average temperature of each group of chickens after 4 days of drinking the drug

[0056]

[0057]

[0058] 2.4 Digestive and immune index detection results

[0059] The results of the contents of α-AMS and PG in serum are shown in Figure 6 Tukey multiple comparison method. After 4 days of sodium sulfite drinking water Figure 6 A) and 7 days of recovery Figure 6 B), the α-AMS content of each experimental group was lower than that of the blank group, and the difference between the 1%, 1.5%, and 2% sodium sulfite experimental groups and the blank group was significant (p<0.05). After 4 days of sodium sulfite drinking water Figure 6 C) and 7 days of recovery Figure 6 D), the PG content in the serum of each group of chickens was lower than that of the blank group, and the difference between the 2% sodium sulfite experimental group and the blank group was significant (p<0.05), and the difference between the other experimental groups and the blank group was not significant.

[0060] The results of the content of SIgA in serum are shown in Figure 7 After 4 days of drinking water Figure 7 A) and 7 days of recovery Figure 7 B), the SIgA content of the 1%, 1.5%, and 2% sodium sulfite experimental groups was significantly lower than that of the blank group (p<0.05), and the difference between the 0.5% experimental group and the blank group was not significant.

[0061] In summary, adding more than 1% sodium sulfite in drinking water can cause diarrhea and reduce the weight gain rate of chickens, and some indicators reflecting the digestive capacity and immune level of the chickens decrease significantly, and the body temperature of the chickens decreases. After 7 days of recovery, the indicators of the 1.5% and 2% sodium sulfite groups were significantly different from those of the blank group, but some chickens showed growth stagnation and could not recover. Therefore, adding 1% sodium sulfite in drinking water for 4 consecutive days is selected as the method for constructing the model of the cold and diarrhea syndrome.

[0062] Example 3: Construction of a diarrhea model by adding 1% sodium sulfite in the drinking water of Hyline Brown chicks

[0063] 1.1 Chicks, 12-day-old Hyline Brown chicks raised in SPF chicken isolators; SPF chicken isolators produced in Changshu, Jiangsu.

[0064] 1.2 Sodium sulfite: analytical pure, purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0065] 1.3 Grouping: chicks were randomly divided into experimental and blank groups, 10 in each group.

[0066] 1.4 Treatment method: the blank group was given normal free drinking water and food; the experimental group was given 1% sodium sulfite in drinking water for 4 days, and then normal drinking water and food were given for 7 days of recovery. The clinical symptoms of the two groups of chickens were observed, the weight was measured, the cloaca temperature was measured, and the serum was collected for detection of digestive and immune indicators.

[0067] 1.5 Digestive and immune indicators: The contents of pepsinogen (PG), α-amylase (α-AMS), and secretory immunoglobulin (SIgA) in serum were detected by referring to the method of ELISA kit.

[0068] 1.6 Judgment criteria: diarrhea and loose stool were regarded as the onset of the disease.

[0069] 2 Results

[0070] 2.1 Clinical symptoms: The chickens in the test group all showed loose stool symptoms on the second day of the test. The diarrhea symptoms of the chickens in the test group gradually worsened over time, and the digestion was incomplete, with a muscle stomach ulcer. After 7 days of recovery, the chickens still had diarrhea, but the feces could be shaped (Fig. 2), and the feed intake and body weight were not significantly affected. Figure 8

[0071] 2.2 The average body weight and weight gain rate of the chickens in each group were statistically analyzed after 7 days of recovery, as shown in Table 5. It can be seen that after adding 1% sodium sulfite in the drinking water, the body weight gain of the chickens was significantly inhibited.

[0072] Table 5 Average body weight and weight gain rate of chickens in each group

[0073]

[0074] 2.3 Body temperature changes

[0075] The average cloaca temperature of the chickens in each group after 4 days of drinking the drug was statistically analyzed, as shown in Table 6. The average body temperature of the chickens in the experimental group was 0.21℃ lower than that of the chickens in the blank group.

[0076] Table 6 Average temperature of chickens after 4 days of drinking the drug

[0077]

[0078] 2.4 Digestive and immune indicator detection results

[0079] The detection results of α-AMS, PG, and SIgA in serum after 4 days of drinking water and after 7 days of recovery are shown in Table 7. The results show that at two detection time points, the contents of α-AMS, PG, and SIgA in the serum of the sodium sulfite experimental group chickens were significantly lower than those of the blank group (t-test, p<0.05).

[0080] Table 7 Detection results of α-AMS, PG, and SIgA in serum after 4 days of drinking water and after 7 days of recovery

[0081]

[0082] ​In conclusion, the model of deficiency-cold diarrhea syndrome was successfully established by adding 1.0% sodium sulfite in drinking water for 4 days, which caused diarrhea, decreased weight gain rate and body temperature, and significantly decreased some indexes reflecting the digestive capacity and immune level.

Claims

1. A method for constructing a chicken deficiency diarrhea model, characterized in that, Taking Hailan brown chicks as samples, 1-2% sodium sulfite is added in the drinking water of the chicks for 4-5 days, during which the chicks are allowed to drink and eat freely, and after the use of sodium sulfite is stopped, the chicks are continuously observed for more than 7 days to see whether there is deficiency-cold diarrhea syndrome and whether the chicks die, and if so, the model is determined to be successfully constructed.

2. The method for constructing a chicken deficiency diarrhea model according to claim 1, characterized in that, The deficiency-cold diarrhea syndrome is that the chicks have cold and excessive spleen and stomach, weak spleen yang, and disordered transportation and transformation function, and have clinical symptoms of diarrhea, decreased body temperature and significantly decreased food intake, and the digestion capacity and immunity related indexes decrease.

3. The method for constructing a chicken deficiency diarrhea model according to claim 1, characterized in that, 1% sodium sulfite is added in the drinking water of the chicks.

4. The method for constructing a chicken deficiency diarrhea model according to claim 3, characterized in that, Sodium sulfite is added in the drinking water of the chicks for 4 days.

5. The method for establishing a chicken deficiency diarrhea model according to any one of claims 1-4, characterized in that, The Hailan brown chicks are bred in a SPF chicken isolator.

6. The method for constructing a chicken deficiency diarrhea model according to claim 5, characterized in that, The Hailan brown chicks are 12-15 days old.

Images