Method for evaluating anti-brucellosis character of sheep

Abstract

The invention discloses a method for evaluating the anti-brucellosis character of sheep, and the method specifically comprises the following steps: respectively infecting the sheep with an equal amount of pathogens according to the weight ratio of the sheep in an eyeshade manner; the method comprises the following steps: collecting serum 30 days after infection, measuring the serum Brucella antibody level and serum IL-12 and IL-17 cell factors, taking left parotid gland lymph nodes and right parotid gland lymph nodes of an infected object for general pathological semi-quantitative evaluation, and measuring the bacterium loading amount of the left parotid gland lymph nodes and left submandibular lymph nodes; and carrying out standardization processing on the measured data, and calculating by using a stepwise regression analysis method to obtain an anti-brucellosis index R of the sheep so as to evaluate the disease resistance of the sheep. The index for comprehensively evaluating the anti-brucellosis character of the sheep is constructed, so that the evaluation result is more accurate, and the limitation and uncertainty of judging the disease resistance character by a single immune index are overcome.

Description

Technical Field

[0001] The invention belongs to the technical field of disease resistance trait evaluation, and particularly relates to a method for evaluating sheep resistance to brucellosis. Background Art

[0002] Brucellosis in sheep is a global zoonotic infectious disease caused by Brucella infection, which affects a wide range of countries and regions. Common pathogens that cause brucellosis in sheep include Brucella melitensis and Brucella ovis. In recent years, Brucella abortus and Brucella suis have also been isolated and identified in clinical diagnosis. Brucella melitensis, Brucella bovis and Brucella suis can all cause human infection, seriously threatening the health of related practitioners. At present, comprehensive brucellosis prevention and control measures are mainly based on vaccination, supplemented by quarantine, culling and harmless treatment. The intracellular parasitic characteristics of Brucella and the subclinical infections it often causes increase the complexity of disease prevention and control. Through molecular breeding technology, combined with the selection of anti-brucellosis traits, sheep strain selection can be carried out to enhance the resistance of individuals to Brucella at the genetic level and achieve effective accumulation of disease resistance. Through continuous optimization over generations, the overall disease resistance of the group will be gradually improved, and ultimately the goal of raising the upper limit of disease resistance will be achieved.

[0003] Disease resistance mainly refers to the ability to reflect the body's defense function and immune response to diseases, and is a low heritability trait. Since the immunity of animal bodies includes two categories: innate immunity and adaptive immunity. Therefore, the evaluation of disease resistance needs to comprehensively cover innate immunity and adaptive immune responses. Among them, innate immunity, as the first line of defense for the body to resist the invasion of pathogens, is gradually formed during the long-term evolution of the population. It is already present when an individual is born, has a wide range of effects and is not targeted at specific antigens. Innate immunity is affected by the combined influence of genetics and the environment, reflecting the body's extensive defense function against diseases. Adaptive immunity is a specific immune response produced after an individual comes into contact with a specific antigen. It is a highly specific and memory-based defense mechanism established by the body through continuous struggle with invading pathogenic microorganisms from the outside world. Adaptive immunity has the characteristics of targeting specific pathogens and can provide accurate and lasting protection for the body. By detecting the relevant disease resistance indicators of sheep against brucellosis and determining the strength of the body's disease resistance traits, a theoretical basis can be provided for subsequent disease-resistant breeding work. Currently, methods for evaluating sheep's resistance to brucellosis include serological testing, cellular immune response assessment, bacterial load determination, etc., but these methods have limitations such as insufficient specificity, lack of unified operating standards, complex testing and high cost. A single method is difficult to comprehensively and accurately evaluate disease resistance traits. Summary of the invention

[0004] The purpose of the present invention is to provide a method for evaluating the brucellosis resistance of sheep, aiming to solve the problem of low accuracy of judging the disease resistance by using a single index in the prior art.

[0005] The technical scheme adopted by the present invention is a method for evaluating the anti-brucellosis trait of sheep, which specifically comprises the following steps: infecting sheep with equal amounts of Brucella pathogens according to the weight ratio of the sheep, and the infection method is eye drop; collecting sheep serum 30 days after infection for the determination of serum Brucella antibody level and serum IL-12 and IL-17 cytokines, taking the left parotid lymph node and the right parotid lymph node of the infected object for semi-quantitative evaluation of gross pathology, and determining the bacterial load of the left parotid lymph node and the left submandibular lymph node; standardizing the measured data, and calculating the anti-brucellosis index R of the sheep by stepwise regression analysis, so as to evaluate the disease resistance of the sheep.

[0006] The present invention is also characterized in that:

[0007] The formula for calculating the anti-brucellosis index R of sheep is as follows:

[0008] R = 0.38803 × X 1 +0.29724×X 2 -0.24259×X 3 -0.54804×X 4 +0.64594×X 5

[0009] +0.54346×X 6 +0.48047×X 7 +0.23634×X 8 +0.48696×X 9 +3.5151×10 -6

[0010] Among them, X 1 -X 9 They respectively represent the standardized results of serum antibody determination by SAT method, serum antibody determination by cELISA method, serum antibody determination by iELISA method, serum IL-12 cytokine content determined by ELISA method, serum IL-17 cytokine content determined by ELISA method, bacterial load of left parotid lymph node, bacterial load of left submandibular lymph node, semi-quantitative evaluation of gross pathology of left parotid lymph node and semi-quantitative evaluation of gross pathology of right parotid lymph node.

[0011] Method for determining serum Brucella antibody levels: Serum was collected from infected subjects 30 days after infection with the pathogen, and the serum Brucella antibody levels of sheep were detected using tube agglutination test (SAT), Brucella competitive enzyme-linked immunosorbent assay (cELISA), and Brucella indirect enzyme-linked immunosorbent assay (iELISA).

[0012] The method for determining the content of serum IL-12 cytokine is as follows: 30 days after the infected subject is infected with the pathogen, the serum is collected and the level of serum IL-12 cytokine is detected using a sandwich ELISA kit.

[0013] The method for determining the serum IL-17 cytokine content is as follows: the serum of the infected subject is collected 30 days after being infected with the pathogen, and the level of serum IL-17 cytokine is detected using a sandwich ELISA kit.

[0014] Method for semi-quantitative evaluation of gross pathology of left parotid lymph nodes: 30 days after the infected subjects were infected with the pathogen, the left parotid lymph nodes were aseptically collected and semi-quantitative evaluation of gross pathology was performed according to the semi-quantitative evaluation criteria of gross pathology.

[0015] Method for semi-quantitative evaluation of gross pathology of right parotid lymph nodes: 30 days after the infected subjects were infected with the pathogen, the right parotid lymph nodes were aseptically collected and semi-quantitative evaluation of gross pathology was performed according to the semi-quantitative evaluation criteria of gross pathology.

[0016] Method for determining bacterial load: 30 days after the infected subject was infected with the pathogen, the left parotid lymph node and the left submandibular lymph node were aseptically collected, the left parotid lymph node and the left submandibular lymph node were weighed respectively, ground with sterile PBS buffer, and the number of bacteria in the tissues of the infected subject was calculated by the plate count method.

[0017] The beneficial effects of the present invention are:

[0018] Compared with other evaluation methods, the present invention is the first to be proposed in this field; the present invention establishes a highly accurate comprehensive evaluation index for sheep anti-brucellosis traits by measuring multiple biological parameters such as serum antibody levels, cytokine content, semi-quantitative evaluation of gross pathology, and bacterial load, using standardized processing and stepwise regression analysis. This method comprehensively considers innate immunity and adaptive immune responses, and is verified by correlation analysis and ROC curve analysis. The present invention overcomes the limitations and uncertainties of single indicator evaluation, significantly improves the accuracy of evaluation results, and provides a reliable scientific basis for disease-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a correlation analysis diagram of the average bacterial load of the present invention;

[0020] Figure 2 It is the ROC curve diagram of the present invention. DETAILED DESCRIPTION

[0021] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Example 1

[0023] The method for evaluating the anti-brucellosis trait of sheep of the present invention specifically comprises the following steps: infecting sheep with equal amounts of Brucella pathogens according to the weight ratio of the sheep, and the infection method is eye drop; collecting sheep serum 30 days after the infection for the determination of serum Brucella antibody level and serum IL-12 and IL-17 cytokines, taking the left parotid lymph node and the right parotid lymph node of the infected object for semi-quantitative evaluation of gross pathology, and determining the bacterial load of the left parotid lymph node and the left submandibular lymph node;

[0024] After the values ​​of the nine indicators, including serum Brucella antibody level, serum IL-12 content, serum IL-17 content, bacterial load of left parotid lymph node, bacterial load of left submandibular lymph node, semi-quantitative evaluation of gross pathology of left parotid lymph node and semi-quantitative evaluation of gross pathology of right parotid lymph node, were standardized, the anti-brucellosis index R of sheep was calculated by stepwise regression analysis; the smaller the R value, the stronger the disease resistance trait, and the larger the R value, the weaker the disease resistance trait;

[0025] R = 0.38803 × X 1 +0.29724×X 2 -0.24259×X 3 -0.54804×X 4 +0.64594×X 5

[0026] +0.54346×X 6 +0.48047×X 7 +0.23634×X 8 +0.48696×X 9 +3.5151×10 -6

[0027] Among them, X 1 -X 9 They represent the standardized results of serum antibody determination by SAT method, serum antibody determination by cELISA method, serum antibody determination by iELISA method, serum IL-12 cytokine content determined by ELISA method, serum IL-17 cytokine content determined by ELISA method, bacterial load of left parotid lymph node, bacterial load of left submandibular lymph node, semi-quantitative evaluation of gross pathology of left parotid lymph node and semi-quantitative evaluation of gross pathology of right parotid lymph node respectively;

[0028] Method for determining serum Brucella antibody levels: serum was collected from infected subjects 30 days after infection with the pathogen, and the serum Brucella antibody levels of sheep were tested using tube agglutination test (SAT), Brucella competitive enzyme-linked immunosorbent assay (cELISA), and Brucella indirect enzyme-linked immunosorbent assay (iELISA);

[0029] The method for determining the levels of serum IL-12 and IL-17 cytokines is as follows: 30 days after the infected subjects were infected with the pathogen, their serum was collected and the levels of serum IL-12 and IL-17 cytokines were detected using a sandwich ELISA kit;

[0030] Method for semi-quantitative evaluation of gross pathology: 30 days after the infected subjects were infected with the pathogen, the left and right parotid lymph nodes were aseptically collected and semi-quantitative evaluation of gross pathology was performed according to the semi-quantitative evaluation criteria of gross pathology;

[0031] Method for determining bacterial load: 30 days after the infected subject was infected with the pathogen, the left parotid lymph node and the left submandibular lymph node were aseptically collected, the left parotid lymph node and the left submandibular lymph node were weighed respectively, ground with sterile PBS buffer, and the number of bacteria in the tissues of the infected subject was calculated by the plate count method.

[0032] In order to avoid the influence of the indicator dimension, the data is standardized; the specific method is to subtract the mean value of the same variable and then divide it by the standard deviation, so that the standardized data becomes comparable;

[0033] The formula used is Y ij =(X ij -X j ) / S j (i=1,2,···,n; j=1,2,···,p).

[0034] Among them, Y ij is the value of the original variable after standardization, X ij is the original data of the jth indicator of the ith sample, X j is the average value of the jth index of n samples, S j is the standard deviation of the sample; p represents the total number of indicators.

[0035] Example 2

[0036] The present embodiment used experimental animal is sheep.Each experimental animal is divided into infection group and control group, and infection group is injected with M28 brucella according to body weight ratio and infected sheep, and injection dosage is 5,000,000 CFU / every 25kg, and control group injects equivalent PBS.Infected in latter 30 days, gathered serum and was used for the mensuration of serum brucella antibody level and serum IL-12, IL-17 cytokine, separately got the left side parotid lymph node of infected object, right side parotid lymph node and carried out gross pathology semiquantitative evaluation, and left side parotid lymph node, left side submandibular lymph node were carried out to bacterial load measurement.

[0037] Example 3

[0038] Serum Brucella antibody level: Serum was collected from each sheep 30 days after infection with Brucella and tested by tube agglutination test (SAT), Brucella competitive enzyme-linked immunosorbent assay (cELISA), and Brucella indirect enzyme-linked immunosorbent assay (iELISA). Brucella standard positive serum was used as positive control, and Brucella standard negative serum was used as negative control.

[0039] Serum IL-12 and IL-17 cytokine levels: Serum was collected from each sheep 30 days after infection with Brucella, and the levels of serum cytokines IL-12 and IL-17 were detected using sandwich ELISA kits.

[0040] Semi-quantitative evaluation of gross pathology: 30 days after each sheep was infected with Brucella, the left and right parotid lymph nodes were taken for semi-quantitative evaluation of gross pathology. The semi-quantitative evaluation criteria of gross pathology are shown in Table 1;

[0041] Table 1 Semi-quantitative evaluation criteria for gross pathology

[0042]

[0043] Note: This table is intended to semi-quantify gross pathological changes in order to facilitate equation calculations.

[0044] Pathogen clearance: 30 days after each sheep was infected with Brucella, the left parotid lymph node and the left submandibular lymph node were taken, the samples were weighed aseptically, and ground with sterile PBS buffer. The number of bacteria in the tissues of the infected objects was calculated by the plate counting method. CFU index is the logarithmic value of the average bacterial load of 9 tissue samples, where the 9 tissue samples are the left parotid lymph node, the right parotid lymph node, the left submandibular lymph node, the right submandibular lymph node, the left anterior shoulder lymph node, the right anterior shoulder lymph node, the left inguinal lymph node, the right inguinal lymph node, and the spleen. The calculation formula of CFU index is,

[0045] CFU index = Log 10 (Mean CFU+1) .

[0046] The bacterial load data of the 9 tissue samples and the calculated data of CFU index are shown in Table 2 ;

[0047] Table 2 Bacterial load data of 33 sheep tissue samples

[0048]

[0049]

[0050] Note: S stands for parotid lymph nodes, H stands for submandibular lymph nodes, J stands for anterior shoulder lymph nodes, F stands for inguinal lymph nodes, L stands for left side, R stands for right side, and B stands for bacterial load.

[0051] Example 4

[0052] The results of serum antibody determination by SAT method, serum antibody determination by cELISA method, and serum antibody determination by iELISA method were expressed as 0 for negative and 1 for positive. The semi-quantitative evaluation of gross pathology of left parotid lymph nodes and the semi-quantitative evaluation of gross pathology of right parotid lymph nodes were expressed according to the established scoring criteria. The measured values ​​of the 9 shape indices are shown in Table 3.

[0053] Table 3 The measured values ​​of various indicators of 33 sheep

[0054]

[0055]

[0056] Note: S represents parotid lymph node, H represents submandibular lymph node, L represents left side, R represents right side, B represents bacterial load, and A represents semi-quantitative evaluation of gross pathology.

[0057] In order to avoid the influence of indicator dimension, the original data is standardized. The specific method is to subtract the mean value of the same variable and then divide it by the standard deviation, so that the standardized data becomes comparable.

[0058] The formula used is Y ij =(X ij -X j ) / S j (i=1,2,···,n; j=1,2,···,p).

[0059] Among them, Y ij is the value of the original variable after standardization, X ij is the original data of the jth indicator of the ith sample, X j is the average value of the jth index of n samples, S j is the standard deviation of the sample; p represents the pth indicator.

[0060] The data after standardization are shown in Table 4;

[0061] Table 4 Standardization of raw data

[0062]

[0063]

[0064] Note: The Z before each index represents standardization, S represents parotid lymph node, H represents submandibular lymph node, L represents left side, R represents right side, B represents bacterial load, and A represents semi-quantitative evaluation of gross pathology.

[0065] Example 5

[0066] SPSS25.0 was used for stepwise regression analysis, using the stepwise backward method. Since 27 of the 36 indicators were not significant for the average bacterial load, these 27 independent variables were gradually eliminated and 9 independent variables were retained. The coefficients, significance and confidence intervals of each variable are shown in Table 5; since the trait indicators retained by the stepwise regression analysis were all significant or extremely significant for the average bacterial load, the multiple regression equation was established using 9 indicators, including the results of serum antibody determination by the SAT method, the results of serum antibody determination by the cELISA method, the results of serum antibody determination by the iELISA method, the level of serum IL-12 by the ELISA method, the level of serum IL-17 by the ELISA method, the bacterial load of the left parotid lymph node, the bacterial load of the left submandibular lymph node, the semi-quantitative evaluation of the gross pathology of the left parotid lymph node, and the semi-quantitative evaluation of the gross pathology of the right parotid lymph node.

[0067] Table 5 Coefficients, significance, confidence intervals and collinearity statistics of individual variables

[0068]

[0069] Note: The analysis method is backward stepwise regression analysis.

[0070] The multiple regression equation obtained by stepwise regression analysis is:

[0071] R = 0.38803 × X 1 +0.29724×X 2 -0.24259×X 3 -0.54804×X 4 +0.64594×X 5

[0072] +0.54346×X 6 +0.48047×X 7 +0.23634×X 8+0.48696×X 9 +3.5151×10 -6

[0073] Among them, X 1 -X 9 They are respectively the standardized SAT serum antibody determination results, cELISA serum antibody determination results, iELISA serum antibody determination results, ELISA serum IL-12 determination levels, ELISA serum IL-17 determination levels, left parotid lymph node bacterial load, left submandibular lymph node bacterial load, left parotid lymph node gross pathology semi-quantitative evaluation, and right parotid lymph node gross pathology semi-quantitative evaluation.

[0074] By comparing the coefficients of individual variables, it was found that the five indicators, including the ELISA serum IL-12 level, ELISA serum IL-17 level, the bacterial load of the left parotid lymph nodes, the bacterial load of the left submandibular lymph nodes and the semi-quantitative evaluation of the gross pathology of the right parotid lymph nodes, had a greater impact on the average bacterial load after Brucella infection.

[0075] The anti-brucellosis index R of 33 sheep was calculated, as shown in Table 6;

[0076] Table 6 Anti-brucellosis index R of 33 sheep

[0077]

[0078]

[0079] Example 6

[0080] In order to verify whether the results predicted by the above model are accurate, a correlation analysis was conducted between the score results and the average bacterial load. The results showed that P<0.01, and the correlation between the two was extremely significant. Figure 1 and Figure 2 The ROC curve was drawn, and the sheep were divided into bacteria-carrying sheep and non-bacteria-carrying sheep according to whether the average bacterial load was 0 (bacteria-carrying sheep were recorded as 1, and non-bacteria-carrying sheep were recorded as 0). As shown in Table 7, the area under the ROC curve was 0.974, which was greater than 0.9, and the asymptotic significance was less than 0.01.

[0081] Table 7 ROC test results

[0082]

[0083] The present invention constructs an index for comprehensively evaluating the anti-brucellosis trait of sheep, so that the evaluation result is more accurate and overcomes the limitation and uncertainty of judging the disease resistance trait by a single immune index.

Claims

1. A method for evaluating the brucellosis resistance of sheep, characterized in that: Specifically: the sheep were infected with equal amounts of Brucella pathogens in proportion to their body weight, and the infection method was eye drop; serum was collected 30 days after infection for the determination of serum Brucella antibody levels and serum IL-12 and IL-17 cytokines, and the left parotid lymph nodes and right parotid lymph nodes of the infected subjects were taken for semi-quantitative evaluation of gross pathology, and the bacterial load of the left parotid lymph nodes and left submandibular lymph nodes was determined; the measured data were standardized, and the anti-brucellosis index R of the sheep was calculated by stepwise regression analysis to evaluate the disease resistance of the sheep.

2. The method for evaluating the anti-brucellosis trait of sheep as claimed in claim 1, characterized in that: The calculation formula of the anti-brucellosis index R of the sheep is as follows: R=0.38803×X1+0.29724×X2-0.24259×X3-0.54804×X4+0.64594×X5 +0.54346×X6+0.48047×X7+0.23634×X8+0.48696×X9+3.5151×10 -6 Among them, X1-X9 respectively represent the standardized serum antibody determination results of the SAT method, the serum antibody determination results of the cELISA method, the serum antibody determination results of the iELISA method, the serum IL-12 cytokine content determined by ELISA, the serum IL-17 cytokine content determined by ELISA, the bacterial load of the left parotid lymph nodes, the bacterial load of the left submandibular lymph nodes, the semi-quantitative evaluation of the gross pathology of the left parotid lymph nodes, and the semi-quantitative evaluation of the gross pathology of the right parotid lymph nodes.

3. The method for evaluating sheep anti-brucellosis traits as claimed in claim 1, characterized in that Method for determining serum Brucella antibody levels: Serum is collected from infected subjects 30 days after infection with the pathogen, and the serum Brucella antibody levels of sheep are detected using the test tube agglutination test SAT, Brucella competitive enzyme-linked immunosorbent assay cELISA, and Brucella indirect enzyme-linked immunosorbent assay iELISA.

4. The method for evaluating sheep anti-brucellosis traits as claimed in claim 1, characterized in that The method for determining the serum IL-12 cytokine content is as follows: serum is collected from the infected subjects 30 days after infection with the pathogen, and the level of serum IL-12 cytokine is detected using a sandwich ELISA kit.

5. The method for evaluating sheep anti-brucellosis traits as claimed in claim 1, characterized in that The method for determining the serum IL-17 cytokine content is as follows: serum is collected from the infected subject 30 days after infection with the pathogen, and the level of serum IL-17 cytokine is detected using a sandwich ELISA kit.

6. The method for evaluating sheep anti-brucellosis traits as claimed in claim 1, characterized in that Method for semi-quantitative evaluation of gross pathology of left parotid lymph nodes: 30 days after infection, the left parotid lymph nodes were obtained from the infected subjects for semi-quantitative evaluation of gross pathology.

7. The method for evaluating sheep anti-brucellosis traits as claimed in claim 1, characterized in that Method for semi-quantitative evaluation of gross pathology of right parotid lymph nodes: 30 days after infection, the right parotid lymph nodes were obtained from the infected subjects for semi-quantitative evaluation of gross pathology.

8. The method for evaluating sheep anti-brucellosis traits as claimed in claim 1, characterized in that Method for determining bacterial load: 30 days after the infected subject was infected with the pathogen, the left parotid lymph node and the left submandibular lymph node were aseptically collected, the left parotid lymph node and the left submandibular lymph node were weighed respectively, ground with sterile PBS buffer, and the number of bacteria in the tissues of the infected subject was calculated by the plate count method.

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