A method for screening dairy cows with high cellular immune response ability and its application

Abstract

The invention discloses a method for screening dairy cows with high cellular immune response ability and application thereof, comprising: collecting an initial blood sample of the dairy cow; determining the cellular immune index of the initial blood sample; intramuscularly injecting 2 ml of a pattern antigen mixture into the dairy cow on the day (day 0) after collecting the initial blood sample; intramuscularly injecting 2 ml of the pattern antigen mixture into the dairy cow again on the 14th day; collecting a final blood sample of the dairy cow on the 21st day; determining the cellular immune index of the final blood sample; calculating the increment of the cellular immune index after the injection of the pattern antigen; and screening dairy cows. Experiments have shown that the method can accurately screen dairy cows with high cellular immune response ability from a dairy cow herd, managers can clearly know the dairy cows with high cellular immune response ability and keep them as reserve cows for breeding, the heritability of the trait of high cellular immune response ability can reach 0.49-0.51%, the somatic cell count of the screened "high immunity cattle" is reduced by more than 100,000 / mL, the incidence of mastitis is reduced by 59.08%, the incidence of digestive system diseases is reduced by 100%, and the calf diarrhea is reduced by 88.08%.

Description

Technical Field

[0001] The invention belongs to the field of animal production, and specifically relates to a method for screening dairy cows with high cellular immune response ability and application thereof. Background Art

[0002] Improving the comprehensive disease resistance of dairy cows through disease resistance breeding is an important way to improve the health of dairy cows and reduce the incidence of diseases. Traditional single disease resistance selection and health trait selection have long cycles, little progress, and there are risks of reduced production performance and "one disease disappears, multiple diseases grow". Therefore, exploring disease resistance traits or markers that can reflect the comprehensive disease resistance of dairy cows and are easy to measure and apply is a major scientific issue that urgently needs to be solved in dairy cow breeding.

[0003] The development of research and new technologies related to animal immunology has provided new ideas for solving the above problems. The disease resistance of animals mainly depends on their own immune system. As the "last line of defense" of the body, the adaptive immune response is highly efficient, specific and has memory, which ultimately determines whether the animal is sick or not. Cellular immunity, as an important component of adaptive immunity, plays an important role in various common inflammatory diseases of dairy cows. The latest research shows that the distribution of T cell subpopulations in the peripheral blood of the body that maintains the balance of adaptive immunity and mediates cellular immune responses is greatly affected by genetics, and has great potential to become a trait or marker that reflects the comprehensive disease resistance of dairy cows. However, due to the limitations of livestock and poultry immune reagents and methods, it is currently impossible to conduct comprehensive, systematic and accurate phenotypic identification and functional analysis of each T cell subpopulation in the peripheral blood of dairy cows. In addition, there are many types of T cell subpopulations, and different cell subpopulations play different primary and secondary roles when different pathogens invade the body. It is still impossible to determine whether a certain cell subpopulation represents the comprehensive level of adaptive immunity. Therefore, an accurate and efficient marker of comprehensive disease resistance in dairy cows has not yet been identified. The existing alternative method is to use the classic delayed-type hypersensitivity reaction in immunology to measure the cellular immune response traits. These methods have been verified in chickens, pigs and cattle. The high immune response trait bulls selected by this trait have significantly reduced the incidence of common diseases such as mastitis, endometritis, and hoof disease in their daughters, and the quality of colostrum has been significantly improved, and production performance has not been affected. However, these detection methods require the cattle to be restrained, the process is cumbersome, the workload is large, and because the measurement process is mainly based on manual measurement, there are certain errors. As a result, it is currently only used in the selection of a very small number of bulls with high economic value, and cannot be widely used in other bulls and reserve cows.

[0004] Immunogenetic studies from pigs, dairy cows, etc. have shown that cell-mediated immune responses have quantitative trait characteristics with a heritability of 0.30%. A large number of modern medical practices have shown that the detection of delayed-type hypersensitivity reactions to candida albicans is an effective method for determining the function of cell-mediated immune responses. However, the above method is very difficult to apply to large livestock such as dairy cows, especially in order to detect delayed-type hypersensitivity reactions in animals, the animals need to be restrained, and the thickness of their skin folds before and after immunization needs to be measured. These operations are cumbersome and the manual measurement errors are large, which is not suitable for large-scale promotion. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for screening dairy cows with high cellular immune response ability.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: collecting an initial blood sample from a dairy cow;

[0009] The initial blood sample of the dairy cow is sterilely cultured, red blood cell lysis solution is added, and then centrifuged and the supernatant is discarded after standing at room temperature, and the above operation is repeated until no red blood cells are left, thereby obtaining a blood sample without red blood cells;

[0010] Use bovine serum albumin solution (0.5% BSA) to dilute fluorescein isothiocyanate-labeled WC1 antibody (anti-WC1-FITC), phycoerythrin conjugate composite fluorescein-labeled CD3 antibody (anti-CD3-PerCP-Cyanine5.5), phycoerythrin fluorescein-labeled CD8 antibody (anti-CD8-PE) and allophycocyanin fluorescein-labeled CD4 antibody (anti-CD4-APC) to prepare a combined dye dilution solution, add the combined dye dilution solution to the red blood cell-free blood sample, incubate at room temperature in the dark, add BSA, centrifuge and discard the supernatant, add PFA fixative, filter into a flow tube, and detect each T cell subset using a flow cytometer;

[0011] WC1 was selected in the lymphocytes of dairy cows + CD3 - and WC1 - CD3 + T cell population, WC1 - CD3+ Simultaneous selection of CD4 T cells + and CD8 + CD4 + CD8 + DP T cell population; WC1 + CD3 - The absolute number of T cells and their percentage in lymphocytes were used as cellular immunity indexes, CD4 + CD8 + Absolute numbers of DP T cell populations and in WC1 - CD3 + The percentage of T cell population was used as the cellular immunity index;

[0012] After collecting the initial blood sample, the cows were intramuscularly injected with the pattern antigen mixture on the same day; the cows were intramuscularly injected with the pattern antigen mixture again on the 14th day; the final blood sample of the cow was collected on the 21st day; the cellular immune index of the final blood sample was measured; the increment of the cellular immune index after the injection of the pattern antigen was calculated, and the calculation method was that the cellular immune index of the final blood sample was subtracted from the cellular immune index of the corresponding initial blood sample;

[0013] As a preferred embodiment of the method of the present invention, the blood samples collected from dairy cows are healthy cows without any diseases as determined by ranch veterinarians, and the blood samples are EDTA anticoagulated blood of dairy cows.

[0014] As a preferred embodiment of the method of the present invention, wherein: the red blood cell lysing solution is added and allowed to stand at room temperature, wherein the mass ratio of the cow blood sample to the red blood cell lysing solution is 2:3, and the standing time is 5 minutes.

[0015] As a preferred embodiment of the method of the present invention, the red blood cell lysis solution is added, allowed to stand at room temperature, and then centrifuged to discard the supernatant, the centrifugation speed is 1600 rpm / min, and the time is 5 min.

[0016] As a preferred embodiment of the method of the present invention, BSA is used to dilute anti-WC1-FITC, anti-CD3-PerCP-Cyanine5.5, anti-CD8-PE and anti-CD4-APC, wherein the mass fraction of BSA is 0.5%, and the dilution ratio is anti-CD3-PerCP-Cyanine5.5:1:300, anti-WC1-FITC:1:200, anti-CD8-PE:1:300, anti-CD4-APC:1:400.

[0017] As a preferred embodiment of the method of the present invention, the combined dye diluent is added to the red blood cell-free blood sample, wherein the mass ratio of the combined dye diluent to the red blood cell-free blood sample is 1:2.

[0018] As a preferred embodiment of the method of the present invention, BSA is added after incubation in the dark at room temperature, and PFA fixative is added after centrifugation and discarding the supernatant, wherein the incubation time in the dark is 20 minutes, the mass fraction of the added BSA is 0.5%, the centrifugation speed is 1600 rpm / min, the time is 5 minutes, the mass fraction of the PFA fixative is 0.5%, and the mass ratio of the added BSA to the PFA fixative is 3:4.

[0019] As a preferred embodiment of the method of the present invention, the model antigen mixture is prepared by adding inactivated Candida albicans and QuilA adjuvant in a phosphate buffer solution at a ratio of 2:1, and adjusting the concentration to 0.25 mg / ml.

[0020] As a preferred embodiment of the method of the present invention, the inactivation of Candida albicans comprises heating the standard strain of Candida albicans in a water bath at 88° C. for 1 hour.

[0021] As a preferred embodiment of the method of the present invention, the cows are intramuscularly injected with the pattern antigen mixture on the same day and again on the 14th day, wherein the mass ratio of the pattern antigen mixture injected on the same day to that on the 14th day is 1:1.

[0022] Beneficial effects of the present invention:

[0023] (1) T cells play an important role in the process of cell-mediated immune response. Therefore, the present invention systematically studied the T cell immune response of dairy cows to model antigens (inactivated Candida albicans) for the first time, detected a total of 216 indicators of 33 T cell subsets of dairy cows, and performed a kinetic analysis on them. The analysis results determined that 9 T cell subsets were involved in the immune response of dairy cows to model antigens (inactivated Candida albicans). Through correlation analysis with traditional delayed-type hypersensitivity reaction phenotypes, WC1 was finally accurately identified. + CD3 - γδ T cell population and CD4 + CD8 + DP T cell population 2 is a biomarker of cell-mediated immune response.

[0024] (2) The present invention is easy to operate and only requires two blood draws, two antigen injections, and two tests. Cows with high cellular immune response capabilities can be accurately screened out from a dairy cow herd within one month. Ranch managers can clearly identify cows with high cellular immune response capabilities and use the screened cows as reserve cows for artificial insemination.

[0025] (3) The present invention causes very little harm to the cows to be tested. Only one drop (200 μl) of blood sample is required, and the model antigen has been verified for a long time and in large quantities to have no effect on the health of the cows and the quality of milk. The detection cost of the present invention is low. According to calculations, the method of screening cows with high cellular immune response ability described in the present invention only costs 20 yuan in the laboratory application stage. After industrial application in the future, the cost can be further reduced.

[0026] (4) The present invention has high detection accuracy. It uses flow cytometry to detect the number of specific cell subpopulations in blood cells, which eliminates the errors caused by manual detection to the greatest extent. At the same time, the detection efficiency is significantly improved. The traditional method requires the cattle to be restrained, and it takes more than half a day to detect one indicator of one cow. In the method for screening cattle with high cellular immune response ability described in the present invention, one experimenter can complete the sample size detection of more than 200 cows in one day. The method for screening cattle with high cellular immune response ability described in the present invention has shown significant effects in the breeding practice of Chinese Holstein cattle. The determined heritability of the immune index can reach 0.49-0.51. The incidence of common dairy cow diseases such as mastitis and digestive tract diseases in the high immune response dairy cow group has decreased by more than 50%, and its somatic cell count has significantly decreased by more than 20%, and the milk production of high-immunity cattle has increased by more than 10% in 305 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. Among them:

[0028] Figure 1 It is the double skinfold thickness of the traditional skin test site for evaluating delayed hypersensitivity reaction of animals described in Example 1 of the present invention.

[0029] Figure 2 The dynamic changes of αβ and γδ T cell subsets induced by the model antigen described in Example 2 of the present invention ( Figure 2 A is the CD3 in bovine lymphocytes at different cellular immune response levels + WC1 - T cell percentage graph; Figure 2 B is CD3 in bovine lymphocytes at different levels of cellular immune response + WC1 - T cell number graph; Figure 2 C is CD3 in bovine lymphocytes at different levels of cellular immune response - WC1 +γδT cell percentage graph; Figure 2 D is a graph showing the number of CD3-WC1+γδT cells in bovine lymphocytes at different levels of cellular immune response).

[0030] Figure 3 It is a representative dot plot of the dynamic changes of CD4, CD8 and DP T cell subsets induced by the model antigen in Example 2 of the present invention.

[0031] Figure 4 The dynamic changes of CD4, CD8 and DP T cell subsets induced by the model antigen in Example 2 of the present invention ( Figure 4 A is the different levels of cellular immune response of bovine CD3 + WC1 - CD4 + CD8 - T cell percentage graph; Figure 4 B is the different levels of cellular immune response of bovine CD3 + WC1 - CD4 - CD8 + T cell percentage graph; Figure 4 C represents different levels of cellular immune response of bovine CD3 + WC1 - CD4 + CD8 + T cell percentage graph; Figure 4 D is the different levels of cellular immune response of bovine CD3 + WC1 - CD4 + CD8 - T cell number graph; Figure 4 E represents different levels of cellular immune response of bovine CD3 + WC1 - CD4 - CD8 + T cell number graph; Figure 4 F represents the different levels of cellular immune response of bovine CD3 + WC1 - CD4 + CD8 + T cell numbers (Figure ).

[0032] Figure 5 This is the gating strategy for detecting the activation status of T cell subsets and identifying memory cells in Example 2 of the present invention.

[0033] Figure 6 The activation changes of CD4, CD8 and γδT cell subsets induced by the model antigen of Example 2 of the present invention ( Figure 6A represents the different levels of cellular immune response of bovine CD4 + CD8 - CD44 in T cells + High T cell percentage; Figure 6 B is the different levels of cellular immune response of cattle CD4 - CD8 + CD44 in T cells + High T cell percentage graph; Figure 6 C represents different levels of cellular immune response of bovine CD3 - WC1 + CD44 in γδ T cells + High T cell percentage graph; Figure 6 D is the different levels of cellular immune response of cattle CD4 + CD8 - Mean fluorescence intensity of CD44 in T cells; Figure 6 E represents the different levels of cellular immune response of bovine CD4 - CD8 + Mean fluorescence intensity of CD44 in T cells; Figure 6 F represents the different levels of cellular immune response of bovine CD3 - WC1 + Mean fluorescence intensity of CD44 in γδ T cells).

[0034] Figure 7 The dynamic changes of naive and memory T cell subsets induced by the model antigen in Example 2 of the present invention. Figure 7 A represents the different levels of cellular immune response of bovine CD4 + CD8 - CD27 in T cells + CD45RO + T cell percentage graph; Figure 7 B is the different levels of cellular immune response of cattle CD4 - CD8 + CD27 in T cells + CD45RO + T cell percentage graph; Figure 7 C represents the different levels of cellular immune response of bovine CD3-WC1 + CD27 in γδ T cells + CD45RO + T cell percentage graph; Figure 7 D is the different levels of cellular immune response of cattle CD4 + CD8 - CD27 in T cells + CD45RO - T cell percentage graph; Figure 7E represents the different levels of cellular immune response of bovine CD4 - CD8 + CD27 in T cells + CD45RO - T cell percentage graph; Figure 7 F represents the different levels of cellular immune response of bovine CD3 - WC1 + CD27 in γδ T cells + CD45RO - T cell percentage graph).

[0035] Figure 8 The CD4 + Pattern of antigen-specific cytokine expression secreted by T cells ( Figure 8 A represents the different levels of cellular immune response of bovine CD4 + CD8 - IFN-γ in T cells + T cell percentage graph; Figure 8 B is the different levels of cellular immune response of cattle CD4 + CD8 - TNF-α in T cells + T cell percentage graph; Figure 8 C represents different levels of cellular immune response of bovine CD4 + CD8 - IL-2 in T cells + T cell percentage graph; Figure 8 D is the different levels of cellular immune response of cattle CD4 + CD8 - Mean fluorescence intensity of IFN-γ in T cells; Figure 8 E represents the different levels of cellular immune response of bovine CD4 + CD8 - Mean fluorescence intensity of TNF-α in T cells; Figure 8 F represents the different levels of cellular immune response of bovine CD4 + CD8 - Mean fluorescence intensity of IL-2 in T cells.

[0036] Fig. 9 The CD8 described in Example 2 of the present invention + Pattern of antigen-specific cytokine expression secreted by T cells ( Fig. 9 A represents the different levels of cellular immune response of bovine CD4 - CD8 + IFN-γ in T cells + T cell percentage graph; Fig. 9 B is the different levels of cellular immune response of cattle CD4 - CD8+ TNF-α in T cells + T cell percentage graph; Fig. 9 C represents different levels of cellular immune response of bovine CD4 - CD8 + IL-2 in T cells + T cell percentage graph; Fig. 9 D is the different levels of cellular immune response of cattle CD4 - CD8 + Mean fluorescence intensity of IFN-γ in T cells; Fig. 9 E represents the different levels of cellular immune response of bovine CD4 - CD8 + Mean fluorescence intensity of TNF-α in T cells; Fig. 9 F represents the different levels of cellular immune response of bovine CD4 - CD8 + Mean fluorescence intensity of IL-2 in T cells.

[0037] Fig.10 This is a correlation analysis diagram of the dynamic changes of T cell subsets and delayed-type hypersensitivity reactions described in Example 3 of the present invention.

[0038] Fig.11 This is the correlation analysis between T cell subset activation and memory changes and delayed-type hypersensitivity reactions described in Example 3 of the present invention.

[0039] Fig.12 This is the correlation analysis between the dynamic changes of T cell subsets and delayed-type hypersensitivity reactions described in Example 3 of the present invention.

[0040] Fig.13 Comparison of production performance of dairy cows with different cellular immune response levels identified by the present invention as described in Example 4 of the present invention ( Fig.13 A is the comparison of the difference in milk production at 305 days in cows with different cellular immune response levels; Fig.13 B is a comparison of differences in milk fat rates of cows with different cellular immune response levels; Fig.13 C is a comparison chart of the difference in milk protein rate at different cellular immune response levels; Fig.13 D is a comparison of the differences in somatic cell scores of cattle with different cellular immune response levels; Fig.13 E is a comparison of the differences in urea nitrogen in cattle at different cellular immune response levels; Fig.13 F is a comparison of the differences in endurance of cattle at different cellular immune response levels; Fig.13 G is a comparison chart of the peak milk production of cows with different cellular immune response levels; Fig.13 H is a comparison of the differences in peak milk production days in cows with different cellular immune response levels). DETAILED DESCRIPTION

[0041] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0042] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0043] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0044] The inventors of the present invention randomly selected 135 lactating cows of the second parity from about 3,500 cows in a cattle farm in Jiangsu Province. Multi-parameter flow cytometry was used to comprehensively analyze the T cell response in the peripheral blood of cows injected with pattern antigens, and the delayed-type hypersensitivity reaction of cows was detected. New immune evaluation indicators were screened out through correlation analysis to replace the traditional delayed-type hypersensitivity reaction indicators. Finally, the immune index of cows was calculated and screened using the method of the present invention, and the production performance, disease resistance traits, and natural antibody levels and growth and development of the offspring calves of the screened cows were detected to verify the method of the present invention.

[0045] The method for screening Chinese Holstein cows with high immunity according to the present invention is specifically described below through specific examples.

[0046] Main reagents in the embodiment of the present invention: PBS1× (HyClone: ​​SH30256.01); RPMI-1640 culture medium (GIBCO: 11875127); red blood cell lysis solution (brosharp: 22195356); 4% paraformaldehyde (brosharp: 2227051); bovine serum albumin (source leaf: N11GS); BrefeldinA (Biolegend: 420601); Candida albicans (ATCC: 8878977); fetal bovine serum (CYTIVA: DG29922724); BD Cytofix / Cytoperm Kit (BD: AB_2869009); PerCP / Cy5.5 Conjugation Kit-Lightning-Link (Abcam: GR3355432-1); PE / Cy7Conjugation Kit-Lightning-Link (Abcam: GR3355432-2); DMSO (Source: R21950); other reagents were all domestic analytical grade products.

[0047] Antibodies used for flow cytometry are shown in Table 1:

[0048] Table 1

[0049]

[0050] Reagent preparation: 0.5% BSA: add 0.5g bovine serum albumin to 100mL PBS; 0.5% PFA: dilute 4% PFA 8 times with PBS; BFA diluent: the purchased BFA (5mg / mL) is 1000×, and needs to be diluted to a 96-well plate at a final reaction concentration of 1:1 using RPMI-1640 culture medium and protected from light; 1× permeabilization solution: the permeabilization solution in the BD Cytofix / Cytoperm Kit is diluted 10 times with ddH2O; Ionom (ionomycin): a powdered drug, prepared with cell-grade DMSO at a concentration of 500ug / mL, and the prepared reagent is 1000×; PMA: a powdered drug, prepared with cell-grade DMSO (dimethyl sulfoxide) at a concentration of 50ug / mL, and the prepared reagent is 1000×.

[0051] Main instruments: LSRFortessa flow cytometer (BD, USA); cell counter (Invitrogen, USA); high-speed refrigerated centrifuge (Thermo, USA); LX-B50L high-pressure sterilizer (Shen'an Medical, Hefei, Anhui); ultra-clean operating table (Purification Biology, Suzhou, Jiangsu); Forma-80℃ ultra-low temperature refrigerator (Thermo, USA).

[0052] Example 1

[0053] Model Antigen Immunization Protocol

[0054] 135 lactating Chinese Holstein cows of second parity were injected with 2 ml of model antigen mixture into the gluteal muscle on day 0 and day 14 of the experiment. The model antigen was prepared by adding inactivated Candida albicans and QuilA adjuvant in a 1:1 ratio in phosphate buffer solution and adjusting the concentration to 0.25 mg / ml. The inactivated Candida albicans was heated in a water bath at 88°C for 1 hour using the Candida albicans standard strain (ATCC10231).

[0055] Delayed hypersensitivity test

[0056] The traditional method for detecting the characteristics of cellular immune response is to evaluate the delayed hypersensitivity reaction of the host to the antigen. The specific detection method is as follows: On the 21st day of model antigen immunization, 0.1 ml of inactivated Candida albicans with a concentration of 1 mg / ml was intradermally injected on one side of the cow's neck, and 0.1 ml of PBS was injected on the same site on the other side of the neck as a negative control. The injection was administered using a 28-gauge needle. Before injection and 24 and 48 hours after injection, the double skinfold thickness of each injection site was measured using a vernier caliper (repeated 3 times). The difference between the double skinfold thickness 24 and 48 hours after injection and before injection is the trait indicator of cellular immune response. Quartiles were used to divide the test cattle into three groups with "high, medium, and low" cellular immune response levels.

[0057] The results are as follows Figure 1 As shown, the skinfold thickness at 24 hours and 48 hours after skin test antigen injection was significantly higher than that before injection (P<0.01), while the difference in skinfold thickness between 24 hours and 48 hours was not significant (P>0.05).

[0058] Example 2

[0059] Isolation of Cow Peripheral Blood Mononuclear Cells (PBMC)

[0060] Collect bovine peripheral blood before (0 day) and on the 14th and 21st days after the injection of the model antigen, add 5 mL of fresh anticoagulated whole blood (EDTA, sodium citrate or heparin anticoagulation) or defibrinated blood to a 15 mL centrifuge tube, let it stand at room temperature, and then add an equal volume of whole blood diluent or PBS to dilute the whole blood. Add 5 mL of separation solution to a new centrifuge tube (when the blood volume after dilution is less than 3 mL, add 3 mL of separation solution; if it is greater than or equal to 3 mL, add an equal volume of separation solution. The total volume cannot exceed two-thirds of the centrifuge tube), draw 5 mL of diluted blood and spread it on the surface of 5 mL of separation solution, and pay attention to keep the interface between the two liquid surfaces clear. Centrifuge at room temperature, 650g horizontal rotor, and centrifuge for 30 minutes. (Low temperature may cause increased red blood cell contamination in the buffy coat. The centrifuge speed increase and deceleration time should be adjusted to the slowest). After centrifugation, obvious stratification will appear; the top layer is the diluted plasma layer, the middle layer is the transparent separation liquid layer, the flocculent white film layer between the plasma and the separation liquid is the lymphocyte layer, and the bottom of the centrifuge tube is the red blood cells and granulocytes. Carefully aspirate the lymphocyte layer (second layer) cells into a 15mL clean centrifuge tube (more aspirates are needed, and the same samples are combined into 1 tube of 15mL clean centrifuge tube), and then add 10mL PBS or cell washing solution to wash the lymphocyte layer cells. 2000rpm, centrifuge for 10min. (If there is no time for centrifugation, the cells added with the washing solution need to be placed on ice). Discard the supernatant, add 5mL PBS or washing solution to resuspend the cells, and centrifuge at 2000rpm for 10min. Discard the supernatant, add 3mL red blood cell lysis solution, lyse for 5min, and add 3mL stop solution (PBS). Centrifuge at 1600rpm for 5min and discard the supernatant. According to the number of cells obtained, add an appropriate amount of RPMI-1640 cell culture medium and resuspend the cells. Generally, 0.5-1 mL of the culture medium containing 10% fetal bovine serum is added. Finally, 10% of the volume of the cell suspension is added with DMSO (dimethyl sulfoxide), and the cells are placed in a cell freezing box for 4 hours and then placed in liquid nitrogen for storage.

[0061] Flow cytometry detection of T cell subsets

[0062] The present invention uses flow cytometry to analyze the dynamic changes of T cell subsets in three cattle groups with high, medium and low levels of cellular immune response traits identified by traditional methods after immunization with model antigens. The specific detection method is as follows:

[0063] (1) Adjust the PBMC cell concentration to 10×10 6 Then, 200 μL of PBMC cell solution was added to each well of a 96-well V-bottom plate, so that each well had 2×10 6 PBMC cell samples.

[0064] (2) Use 0.5% BSA to dilute various antibodies used for T cell subset staining to prepare combined dye dilution solution. The dilution ratio is WC1:1:200, CD8:1:300, CD3:1:300, CD4:1:400, CD44:1:400, CD27:1:200.

[0065] (3) After adding the cells, the 96-well V-bottom plate was centrifuged at 1600 rpm / min for 5 min and the supernatant was discarded.

[0066] (4) Add 50 μL of the combined dye dilution for cell subpopulation staining to each sample well of a 96-well V-bottom plate and incubate at room temperature in the dark for 20 min. Corresponding single-staining and blank wells need to be designed.

[0067] (5) Add 150 μL of 0.5% BSA to each sample well of a 96-well V-bottom plate, then centrifuge at 1600 rpm / min for 5 min and discard the supernatant.

[0068] (6) Finally, 200 μL of 0.5% PFA fixative was added to each sample well of the 96-well V-bottom plate to fix the cells, and the cells were filtered into a flow tube using a nylon membrane at 4°C for testing. Data were collected using a flow cytometer, and Flowjo (10.1.1) software was used for data analysis, and the percentage and total number of cells in each specified T cell subset were calculated.

[0069] The results are as follows Figure 2 As shown, 21 days after immunization with the model antigen, WC1 + The absolute number and percentage of γδT cells increased significantly, and the cows with high cellular immune response levels were slightly higher than those with medium and low levels, indicating that WC1 + γδT cells may play an important role in the delayed-type hypersensitivity reaction in cattle, which is a candidate marker of cellular immune response. - CD3 + The percentage of T cells did not change significantly. Cell count results showed that 14 days after immunization with the model antigen, WC1 of cattle with high cellular immune response levels - CD3 + The absolute number of T cells increased significantly, and the level of cellular immune response in cattle WC1 was high on day 21. - CD3 + The absolute number of T cells is significantly higher than that of cattle with low levels. The changes in this cell subset have also been included in the subsequent screening of candidate markers for cellular immune response traits.

[0070] In order to further analyze the differences in the changes of T cell subsets in cattle with different cellular immune response abilities after model antigen immunization, the present invention was carried out in WC1- CD3 + Three T cell subsets (CD4 + 、CD8 + and CD4 + CD8 + ), the representative dot plots of three T cell subsets of cattle with different cellular immune response traits are shown in Figure 3 The analysis results are shown in Figure 4 As shown, CD4 + 、CD8 + The percentage of T cells did not change significantly. Cell count results showed that 14 days after immunization with the model antigen, the CD4 + 、CD8 + The absolute number of T cells increased significantly, but there was no significant difference between the high and low levels of cellular immune traits. It is worth noting that the present invention first discovered that the model antigen caused the CD4 + CD8 + The percentage and number of DP cells increased significantly, and the cows with high cellular immune response levels were significantly higher than those with medium and low levels. + CD8 + DP cells play an important role in the delayed-type hypersensitivity reaction of dairy cows, which is an important marker of cellular immune response.

[0071] Gating strategy for detecting activated T cell subsets and memory cells

[0072] The present invention uses three groups of dairy cows with high, medium and low cellular immune response ability identified by delayed-type hypersensitivity reaction as the research objects, and uses multicolor flow cytometry to detect the dynamic changes of various T cell subsets in the peripheral blood of the three groups of dairy cows on the 0th, 14th and 21st day of model antigen immunization. The gate strategy is as follows: Figure 5 As shown. T cells can be divided into two subgroups based on TCR: αβ and γδT cells. Bovine WC1 is a widely recognized marker for bovine γδT cells. This study used bovine WC1 and CD3 antibodies to identify αβ and γδT cells in bovine lymphocytes. Bovine CD4 and CD8 antibodies were used to define αβT cells as CD4 T cells (CD4 + CD8 - ), CD8 T cells (CD4 - CD8 + ) and CD4 + CD8 +Double positive (DP) T cells. CD44 molecule is a typical activation marker of bovine T cells. Therefore, this study used CD44 antibody to analyze the activation status of the above-defined γδT cells, CD4 T cells, and CD8 T cells. The activation status was defined as CD44 according to the intensity of antibody fluorescence. - 、CD44 low and CD44 High Memory cells play a very important role in the process of cellular immune response, so this study attempted to further analyze the dynamic changes of memory T cells. CD27 is a marker for identifying the differentiation state of T cells, and CD45RO has been used as a marker for memory T cells many times in previous studies. Therefore, this study used CD27 and CD45RO antibodies to identify memory and naive T cells in γδT cells, CD4 T cells, and CD8T cells, and analyzed the dynamic changes after model antigen immunization.

[0073] Results of T cell activation Figure 6 As shown, CD4 + and CD8 + CD44 in T cells +High The percentage of T cells and mean fluorescence intensity (MFI) increased significantly, but there were no significant differences between cows with different cellular immune response traits. + The activation state of γδT cells showed a significant increase on the 14th day and a recovery trend on the 21st day, but there was no significant difference between cattle with different cellular immune response traits. + 、CD8 + T cells and WC1 + The dynamic change trend of γδT cells is consistent, because CD44 is closely related to lymphocyte migration. CD44 plays an important role in participating in lymphocyte homing, T cell activation and promoting cell migration.

[0074] T cell naive and memory phenotype results Figure 7 As shown, WC1 + The Tcm produced by γδT cells did not change significantly on the 14th day after the first immunization with the model antigen, but increased significantly on the 21st day. This indicates that the Tcm induced on the 14th day after the first immunization with the model antigen may have homed to the lymph nodes and was recalled to the peripheral blood when stimulated by the model antigen again. + Produced by γδT cells The percentage of T cells decreased significantly on the 14th day after the first immunization with the model antigen, while there was no significant change in cattle with high cellular immune response traits. + Produced by γδT cells The percentage of T cells increased significantly to the pre-immunization level. + 、CD8 + The Tcm produced by T cells increased significantly on the 14th day after the first immunization with the model antigen, and there was no significant difference between cattle with different levels of cellular immune response traits. There was no significant change in the percentage of T cells.

[0075] Flow cytometry detection of cytokines in T cells

[0076] The present invention collects blood 21 days after immunization to separate PBMC, and then uses model antigens for in vitro stimulation and culture. After stimulation, intracellular staining of each T cell subset is performed to detect the expression of IFN-γ, TNF-α, and IL-2. The specific method is as follows:

[0077] (1) PBMC samples of cow model antigens with different immune response traits were taken on the 21st day of immunization for intracellular cytokine staining experiments. The concentration of the cell samples was adjusted to 15×10 6 / mL, and then the cells were plated in a 96-well U-shaped plate at 200μL per well. All samples were divided into antigen stimulation group and blank control group. In addition, three samples of "high-immunity cattle" were selected to set up two positive control groups, with 3 samples in each group. The two positive control groups were PMA stimulation group and Candida albicans peptide stimulation group.

[0078] (2) Add 50 μL of IonoM+PMA to the PMA stimulation group, 50 μL of mixed and diluted Candida albicans peptide to the Candida albicans peptide stimulation group, and 50 μL of inactivated Candida albicans with a final concentration of 5 μg / mL to the antigen stimulation group. Add 50 μL of RPMI-1640 medium to the blank group and place in a 37°C CO2 incubator.

[0079] (3) After the cells in the positive control group were cultured for 2 h, 20 μL of BFA diluent was added to each sample well. After 4 h of culture, the cells were transferred to a 96-well V-shaped plate and stored at 4°C for staining at the same time as the other two groups.

[0080] (4) After culturing the cells in the antigen stimulation group and the blank control group for 8 h, 20 μL of BFA diluent was added to each sample well and the cells were transferred to a 96-well V-shaped plate and then returned to a CO2 incubator at 37°C for further culturing for 4 h.

[0081] (5) After the culture was completed, each group of cells was centrifuged at 1600 r / min for 5 min and the supernatant was discarded.

[0082] (6) Use 0.5% BSA to dilute various antibodies used for extracellular staining and prepare a combined dye dilution solution. The antibody ratio is CD8: 1:300, CD3: 1:300, CD4: 1:400.

[0083] (7) Add 50 μL of the above-prepared surface antibody mixture to each sample well, incubate at room temperature in the dark for 20 min, then add 150 μL of 0.5% BSA, centrifuge at 1600 r / min for 5 min, and discard the supernatant.

[0084] (8) Add 100 μL of 4% PFA to each sample well for fixation and incubate on ice for 7 min in the dark. Centrifuge at 2000 r / min for 5 min and discard the supernatant.

[0085] (9) Add 150 μL of diluted permeabilization solution to each sample well to permeabilize the cells and facilitate the entry of antibodies into the cells. Incubate on ice for 20 min in the dark. Centrifuge at 2000 rpm for 5 min and discard the supernatant.

[0086] (10) Use permeabilization buffer to prepare intracellular antibodies. After preparation, add 50 μL to each sample well and incubate on ice for 30 min. Protect from light. The antibody ratio is 1:200 for TNF-α, 1:200 for IFN-γ, and 1:200 for IL-2.

[0087] (11) Add 150 μL / well permeabilization buffer to each sample well, centrifuge at 1600 r / min for 5 min, and discard the supernatant. Then add 50 μL of BV421 secondary antibody to each sample well and incubate on ice for 20 min. Keep away from light.

[0088] (12) Add 150 μL of permeabilization buffer, stop staining, centrifuge at 2000 r / min for 5 min, and discard the supernatant.

[0089] (13) Finally, add 200 μL of 0.5% PFA fixative to each sample well and mix well by pipetting.

[0090] (14) The mixed sample was filtered with nylon cloth before being added to the flow cytometer and then loaded onto the flow cytometer for testing. Data were collected using a flow cytometer and analyzed using Flowjo (10.1.1) software, and the percentage and total number of cells in each specified T cell subset were calculated. The gating strategy during the analysis was based on FMO.

[0091] The results are as follows Figure 8As shown, in cattle with high cellular immune response levels, their CD4 + T cells expressed antigen-specific TNF-α and significantly increased cellular immune responses in high and moderate bovine CD4 + The expression of antigen-specific IL-2 by T cells was significantly reduced. + There was no significant change in antigen-specific cytokines expressed by T cells. Fig. 9 As shown in the figure, different cellular immune response traits of cattle after CaWC antigen stimulation, CD8 + The expression of antigen-specific IFN-γ by T cells was significantly reduced. After CaWC antigen stimulation, CD8 + The antigen-specific TNF-α expressed by T cells was significantly increased.

[0092] Example 3

[0093] Correlation analysis between T cell immune changes and immune response traits

[0094] In order to screen new cellular immune response trait markers and develop a convenient and efficient new cellular immune response trait determination method, this study used the traditional delayed-type hypersensitivity reaction determination value as a reference. At 0d, 14d and 21d after immunization with model antigens in 135 Chinese Holstein cattle, the corresponding immune response traits were determined and the dynamic changes of T cells mediating cellular immune responses were detected. The number and percentage of each T cell subset were used as candidate indicators of new immune response traits. All measured candidate indicator data were log-transformed, and the logarithm of the initial candidate indicator data was subtracted from the candidate indicator data measured after immunization to determine the values ​​of several candidate markers of new immune response traits. The candidate markers of each T cell subset can obtain two values ​​on the 14th day and the 21st day. The values ​​of the candidate markers of each T cell subset were correlated with the delayed-type hypersensitivity reaction at the 24th hour to screen new cellular immune response trait markers. The correlation analysis was performed by calculating the Pearson correlation coefficient between the values, and the new cellular immune response trait markers were finally determined based on the obtained Pearson correlation coefficient. The correlation analysis formula (1) is as follows:

[0095]

[0096]

[0097] The above formula defines the overall correlation coefficient, with ρX,Y as the representative symbol. Estimating the covariance and standard deviation of the sample can obtain the Pearson correlation coefficient, represented by r, where and σ X are the sample mean and sample standard deviation of the sample X respectively.

[0098] The results of correlation analysis are as follows Figures 10 to 12 As shown, CD4 + CD8 + DP T cells and WC1 + CD3 + The increase in the percentage and number of T cells relative to before immunization was most strongly correlated with the delayed-type hypersensitivity reaction 24h and 48h after the model antigen skin test immunization, showing a significant positive correlation. + CD8 + DP T cells and WC1 + CD3 + T cells play an important role in the delayed-type hypersensitivity reaction induced by model antigens in Chinese Holstein cattle. + CD8 + DP T cells and WC1 + CD3 + The changes in T cells after model antigen immunization can be used as an important marker of cellular immune response traits.

[0099] Example 4

[0100] Verification of the effect of the present invention on screening dairy cows with high cellular immune response ability

[0101] 686 healthy lactating cows with similar body condition and lactation period and 367 young cows with similar age and body condition were randomly selected as research subjects in a large-scale dairy farm in Jiangsu, China. The feeding management level, breeding method, pasture environment, feeding diet, etc. of the farm are similar to the above conditions. The experimental cows all have complete population genetic improvement (DHI) records, and all DHI records are measured and generated by Nanjing Dairy Cow Genetic Improvement Center. The method of the present invention is used to evaluate the cellular immune response ability of the experimental cows, and to group the "high, medium, and low" cellular immune response ability cows, and compare the production performance, disease incidence and other indicators of different cellular immune response ability cows to verify the effect of the present invention on screening high cellular immune response ability cows. The present invention identified 224, 617, and 212 cows with "high, medium, and low" cellular immune response ability, respectively.

[0102] The genetic parameters of the cellular immune response index were estimated using the half-sibling assay. + CD8 + The heritability of DP T cells was 0.49, and WC1 + The heritability of γδT cells is 0.52. This indicates that the two traits are stable and can be used as new traits for the selection and breeding of Chinese Holstein cattle for cellular immune response ability. + CD8 +DP T cells were positively correlated with 305d milk production, milk fat rate, milk protein rate, urea nitrogen and other indicators, but negatively correlated with SCC. + CD3 - γδT cells were positively correlated with 305d milk production, milk fat percentage, urea nitrogen, endurance, peak milk, peak day and other indicators, and negatively correlated with SCC.

[0103] Table 1 Heritability of cellular immune response index and its genetic correlation with production traits

[0104]

[0105]

[0106] Note: The bold black values ​​in the table are heritability, the other values ​​are genetic correlation, and the values ​​in brackets are standard deviations.

[0107] The present invention compares the production performance differences between the screened dairy cows with different cellular immune response abilities. Fig.13 It can be seen that the cows with high cellular immune response levels identified by the present invention have significantly lower SCC than cows with low cellular immune response levels, and their milk production on day 305 has increased. The selection of cellular immune response ability has no significant effect on other production performance indicators. This shows that the screening of cellular immune response ability of dairy cows using only the present invention will not affect the production performance of dairy cows.

[0108] The cellular immune response trait markers screened by the present invention are mainly used for the identification of high-immunity cattle. In order to determine the effectiveness of the trait markers, this study conducted a statistical analysis of the incidence records of cattle with different immunity levels identified by the new markers. As shown in Table 2, the incidence of mastitis, digestive system diseases and hoof diseases in "high-immunity cattle" identified by the cellular immune response trait markers screened by this study is significantly lower than that in "low-immunity cattle". The incidence of diarrhea and respiratory diseases in "high-immunity cattle" among young cattle is significantly lower than that in "low-immunity cattle".

[0109] Table 2 Statistics of common diseases in cattle with different immune response traits

[0110]

[0111]

[0112] In order to further determine the impact of the cellular immune response traits identified by the new markers in this study on the risk of cattle disease, the bovine disease traits were defined by binary method, and the risk of common diseases in cattle with different immune response traits was analyzed by logistic regression analysis. The results are shown in Table 3. The immune response traits have a significant impact on the risk of mastitis in adult cows and diarrhea in calves. The risk of common diseases in all "high-immunity cattle" is lower than that in "low-immunity cattle". The risk of mastitis in "high-immunity cattle" in adult cows is only 29.4% of that in "low-immunity cattle". The risk of respiratory diseases in "high-immunity cattle" in young cattle is only 6.7% of that in "low-immunity cattle". It can be seen that the cellular immune response trait markers determined in this study can effectively identify "high-immunity cattle" with strong comprehensive disease resistance, which has important production application value.

[0113] Table 3 Analysis of the risk of common diseases in cattle with different immune response traits

[0114]

[0115]

[0116] Immunogenetic studies from pigs, dairy cows, etc. have shown that cell- and antibody-mediated immune responses have quantitative trait characteristics with a heritability of 0.30%. A large number of modern medical practices have shown that delayed-type hypersensitivity detection for candida albicans is an effective method for determining the function of cell-mediated immune responses. However, the above method is very difficult to apply to large livestock such as dairy cows, especially in order to detect delayed-type hypersensitivity reactions in animals, the animals need to be restrained, and the thickness of their skin folds before and after immunization needs to be measured. These operations are cumbersome and the manual measurement errors are large, which is not suitable for large-scale promotion. The present invention team has noticed that T cells play an important role in the process of cell-mediated immune response. Therefore, the present invention systematically studied the T cell immune response of dairy cows to the model antigen (Candida albicans) for the first time, and detected 216 indicators of 33 T cell subpopulations of dairy cows, and conducted a kinetic analysis. The analysis results determined that 9 T cell subpopulations participated in the immune response of dairy cows to the model antigen (Candida albicans). The changes in the number or percentage of each cell subpopulation participating in the immune response before and after immunization accumulated 44 indicators. The above indicators were used to perform correlation analysis with the delayed-type hypersensitivity reaction indicators, and finally the biomarkers in the blood that can evaluate the cellular immunity of dairy cows were screened, and the cellular immune index was defined based on the biomarker. Cows were screened according to the determined cellular immune index, and the production performance and disease resistance of the screened dairy cows and their offspring were compared to verify the screening method. The results showed that the milk production of the high cellular immune response dairy cow group was significantly increased in 305 days, the somatic cell score was significantly reduced, the incidence of common dairy cow diseases such as mastitis and digestive tract diseases was reduced by more than 50%, and the trait heritability of the immune index reached 0.49. The application of the method of the present invention can significantly improve the comprehensive disease resistance of dairy cows, providing the possibility for breeding new dairy cow strains with high immunity.

[0117] The method can accurately screen out dairy cows with high cellular immune response ability in a dairy cow herd. Managers can clearly know the dairy cows with high cellular immune response ability and breed them as reserve cows. The heritability of the trait of high cellular immune response ability can reach 0.49-0.51. The somatic cell count of the screened "high-immunity cows" is reduced by more than 100,000 / mL, the incidence of mastitis is reduced by 59.08%, the incidence of digestive system diseases is reduced by 100%, and the diarrhea rate of calves is reduced by 88.08%.

[0118] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for screening dairy cows with high cellular immune response ability, characterized in that: The method comprises selecting a specific T cell population in peripheral blood to measure the cellular immune index, and screening dairy cows according to the increment of the cellular immune index after injection of the model antigen; Among them, the specific T cell population in peripheral blood is WC1 in the peripheral blood of dairy cows. + CD3 - T cell populations and CD4 + CD8 + Double-positive T cell population; WC1 + CD3 - The absolute number of T cells or the percentage of lymphocytes in lymphocytes is used as the cellular immunity index, CD4 + CD8 + The absolute number of DP T cell population or in WC1 - CD3 + The percentage of T cell population was used as the cellular immunity index; The model antigen is prepared by adding inactivated Candida albicans and Quil A adjuvant in a ratio of 2:1 into a phosphate buffer solution, and adjusting the concentration to 0.25 mg / ml.

2. The method according to claim 1, characterized in that: The method for calculating the increment of the cellular immune index after injection of the pattern antigen is as follows: after collecting the initial blood sample of the cow, the pattern antigen mixture is injected for the first time, the pattern antigen mixture is injected again two weeks later, and the final blood sample is collected in the third week. The cellular immune index of the final blood sample is subtracted from the cellular immune index of the corresponding initial blood sample to obtain the increment of the cellular immune index.

3. The method according to claim 2, characterized in that: After collecting the initial blood sample from the dairy cow, a red blood cell lysing solution is added and allowed to stand at room temperature, wherein the mass ratio of the initial blood sample from the dairy cow to the red blood cell lysing solution is 2:3, and the standing time is 5 minutes.

4. The method according to claim 3, characterized in that: The red blood cell lysis solution is added, allowed to stand at room temperature, and then centrifuged to discard the supernatant to obtain a red blood cell-free blood sample, wherein the centrifugal speed is 1600 rpm / min and the time is 5 min.

5. The method according to claim 4, characterized in that: The red blood cell-free blood sample is mixed with a combined dye diluent, wherein the mass ratio of the combined dye diluent to the red blood cell-free blood sample is 1:

2.

6. The method according to claim 5, characterized in that: The combined dye diluent is a combined solution of bovine serum albumin solution diluting fluorescein isothiocyanate labeled WC1 antibody, phycoerythrin conjugate composite fluorescein labeled CD3 antibody, phycoerythrin fluorescein labeled CD8 antibody and allophycocyanin fluorescein labeled CD4 antibody.

7. The method according to claim 6, characterized in that: The combined solution is prepared by diluting anti-WC1-FITC, anti-CD3- PerCP-Cyanine5.5, anti-CD8-PE and anti-CD4-APC using BSA, wherein the mass fraction of BSA is 0.5%, and the dilution ratio is anti-CD3- PerCP-Cyanine5.5: 1:300, anti-WC1-FITC: 1:200, anti-CD8-PE: 1:300, anti-CD4-APC: 1:

400.

8. The method according to claim 1, characterized in that: The inactivation of Candida albicans is performed by heating the Candida albicans in a water bath at 88° C. for 1 hour.

9. The method according to claim 2, characterized in that: The mass ratio of the first injection of the model antigen mixture and the second injection of the model antigen mixture two weeks later is 1:1.

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