Search

CN-121569776-B - Animal immunity evaluation method based on Chlamydia cat flea infection model

CN121569776BCN 121569776 BCN121569776 BCN 121569776BCN-121569776-B

Abstract

The invention provides an animal immunity evaluation method based on a Chlamydia cat flea infection model, which comprises the steps of S1, preparing and screening experimental animals, S2, preparing and quality verifying Chlamydia cat flea, S3, infecting the experimental animals by the Chlamydia cat flea, S4, detecting immune related indexes, S5, evaluating animal immune functions, S6, repeatedly verifying and summarizing data. The invention realizes the systematic and objective evaluation of the effect of the immune candidate drug.

Inventors

  • ZHANG LU
  • CAO YUYING
  • QIU JICHENG
  • GENG XIANGFEI
  • XU FENGLIN

Assignees

  • 北京远大星火医药科技有限公司

Dates

Publication Date
20260508
Application Date
20251128

Claims (10)

  1. 1. A method of evaluating animal immunity based on a model of infection by a cat chlamydia, comprising: s1, preparing and screening experimental animals, namely selecting healthy domestic cats, screening qualified individuals through body surface parasite screening, clinical symptom evaluation, blood routine detection, blood biochemical detection and toxoplasma antibody detection, recording health baseline data, and constructing a standardized host model; s2, preparation and quality verification of Chlamydia felis, namely screening positive cats with the number of live fleas on the body surface being more than or equal to 4 and toxoplasma antibody negative, carrying out morphological identification and molecular biological identification on fleas collected from the body surface of the positive cats, and screening female adult fleas with standard movement capacity, morphological integrity and blood sucking potential to form a standardized infection source; S3, randomly dividing the healthy cats screened in the step S1 into a blank control group, an infection control group and an immune candidate drug group, wherein the blank control group is independently fed in an environment without flea pollution, the infection control group and the positive cats in the step S2 are mixed in a cage, the immune candidate drug group is fed with the immune candidate drug according to weight 24 hours before being mixed with the positive cats, and then mixed in the mixed culture period; s4, immune related index detection, namely collecting blood samples of all groups of cats and immune organ samples at partial time points at preset time points after infection, detecting humoral immunity indexes, cellular immunity indexes and infection control effect indexes, recording original detection data and performing quality control; S5, evaluating animal immune functions, namely firstly extracting a principal component with a cumulative variance contribution rate of more than or equal to 85% through principal component analysis to reduce the dimension and remove the noise of the detection data in the step S4, then carrying out nonlinear correction on the extracted principal component by adopting a quadratic polynomial surface fitting model to obtain a comprehensive principal component score, distributing objective weights to the corrected comprehensive principal component score and the extracted principal component by adopting a analytic hierarchy process based on the variance contribution rate and a correlation coefficient, calculating a corrected immune comprehensive score, and finally mapping the corrected immune comprehensive score into an immune grade by adopting a support vector machine model; S6, repeating verification and data summarization, namely performing 3 independent repeated experiments according to the steps S1-S5, controlling experimental animals, chlamydia cat, reagent instruments and operator variables, performing consistency verification on immune grade results of the 3 experiments, and summarizing whole-flow data to form an evaluation report.
  2. 2. The method of claim 1, wherein in step S1, the blood routine test indicators comprise 14 red blood cells, hemoglobin, white blood cells, lymphocyte ratio, hematocrit, average red blood cell volume, average red blood cell hemoglobin content, average red blood cell hemoglobin concentration, neutrophil count and ratio, monocyte count and ratio, eosinophil count and ratio, basophil count and ratio, platelets; the biochemical blood detection indexes comprise total protein, albumin, glucose, urea, alanine aminotransferase, aspartic acid aminotransferase and creatinine, 7 items in total, and the detection of toxoplasma antibodies adopts a commercial ELISA kit, and is qualified when the OD value is less than 0.3.
  3. 3. The animal immune evaluation method based on the Chlamydia cat infection model according to claim 1, wherein in the step S2, molecular biology identification is carried out to detect the Chlamydia cat mitochondrial COI gene by PCR, specifically comprising the steps of extracting the flea genomic DNA by using a blood/cell/tissue genomic DNA extraction kit, constructing a 25 mu L PCR reaction system by using an upstream primer 5'-AGA ATT AGG TCA ACC AGG A-3' and a downstream primer 5'-GAA GGG TCA AAG AAT GAT GT-3', carrying out PCR according to a procedure of pre-denaturation at 94 ℃ for 5 minutes, denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 35 cycles for 40 seconds, and final extension at 72 ℃ for 10 minutes, carrying out agarose gel electrophoresis on the product, and confirming species homozygosity if a band conforming to the size of a target fragment appears and the sequence result is more than or equal to 99% of the sequence homology of the Chlamydia cat COI gene in GenBank.
  4. 4. The animal immunity evaluation method based on the Chlamydia cat infection model according to claim 1, wherein in the step S3, the group differences of the basic weight and the blood baseline index of 3 groups of cats are verified after grouping, the group balance is considered when the difference is judged to have no statistical significance by adopting single factor analysis of variance, the infection model verification standard comprises that the infection rate of 4 th to 7 th weeks of an infection control group is more than or equal to 75%, 87.5%, 100% and 100%, the average number of live fleas of 7 th week is more than or equal to 14, the pruritus of more than or equal to 70% of cats of 7 th week is more than or equal to 25% of cats is more than or equal to red rash, at least 3 blood indexes of 7 th week are abnormal, and the blank control group is free of flea infection in the whole course and has normal indexes.
  5. 5. The animal immunity evaluation method based on the Chlamydia cat infection model according to claim 1, wherein in the step S4, the humoral immunity index is the specific IgG titer of the Chlamydia cat, the detection is carried out by adopting an indirect ELISA method, the supernatant of the Chlamydia cat subjected to ultrasonic cleavage and centrifugation by the RIPA lysate is used as an antigen, the carbonate buffer is diluted to 2 mug/mL to coat an ELISA plate, a plasma sample diluted according to a gradient of 1:100-1:12800 is added after the sealing, then an HRP-marked goat anti-cat IgG secondary antibody is added, and the maximum dilution factor when the OD value is more than or equal to 0.2 and is 2 times of that of a negative control OD value is used as the IgG titer after the color development.
  6. 6. The animal immunity evaluation method based on the Chlamydia cat infection model according to claim 1, wherein in the step S5, in the main component analysis, 5 immune related indexes are firstly constructed into an original matrix and standardized, a covariance matrix of the standardized indexes is calculated, a characteristic value and a corresponding characteristic vector of the covariance matrix are solved, the main component is screened according to the accumulated variance contribution rate of more than or equal to 85%, and the 5 immune related indexes comprise specific IgG titer of the Chlamydia cat, CD4 + /CD8 + T cell ratio, IFN-gamma concentration, flea survival rate and blood absorption rate.
  7. 7. The animal immunity evaluation method based on the flea cat infection model according to claim 1, wherein in step S5, the quadratic polynomial expression of the surface fitting model comprises a constant term, a first term of the principal component, a second term of the principal component and a cross term between the principal components, and the model coefficients are solved by a least square method to determine the coefficient R 2 to be equal to or greater than 0.85, and the fitting effect is judged to be qualified.
  8. 8. The animal immunity evaluation method based on the cat flea infection model according to claim 1, wherein in step S5, when the analytic hierarchy process is weighted, the comprehensive principal component score, the variance contribution rate of each principal component and the Pearson correlation coefficient of the immune effect true value are calculated, the variance contribution rate and the Pearson correlation coefficient are respectively standardized, and the standardized variance contribution rate and the Pearson correlation coefficient are fused according to equal weights to obtain objective weights of all evaluation objects, and the immune effect true value is calculated based on the standardized value of 5 immune correlation indexes and the weight determined by the information entropy.
  9. 9. The animal immunity evaluation method based on the cat comb flea infection model according to claim 1 is characterized in that in step S5, a support vector machine model adopts a radial basis function, a grid search is combined with 5-fold cross validation optimization punishment coefficient C and a nuclear parameter gamma to determine optimal parameters with classification accuracy rate not less than 90%, a one-to-one strategy is adopted to construct a class II classifier, a voting method is adopted to determine final immunity grades, the immunity grades are classified into four grades of excellent, good, medium and bad, and a sample label is marked based on objective threshold values of 5 immune related indexes.
  10. 10. The animal immunity evaluation method based on the Chlamydia cat flea infection model according to claim 1, wherein in the step S6, the consistency test of the repeatability test adopts Kappa coefficient, when the Kappa coefficient is more than or equal to 0.80, the consistency of 3 test results is judged to be excellent, if the test results of a certain batch are inconsistent with other batches, a plurality of results are taken as final evaluation results after adding 2 times of repeated tests, the reagents used in the 3 times of repeated tests are the same batch, the instrument is calibrated before each use, and the core operation is completed by a fixed personnel.

Description

Animal immunity evaluation method based on Chlamydia cat flea infection model Technical Field The invention relates to the technical field of parasite immunology and pharmacology evaluation, in particular to an animal immunity evaluation method based on a Chlamydia cat flea infection model. Background The traditional animal immunity evaluation method has a plurality of limitations, on one hand, the standardization degree of an infection model is low, the species purity and activity of an infection source and the physiological consistency of a host are lack of strict control, so that the model stability is insufficient, on the other hand, the immunity evaluation is mostly dependent on single index or subjective judgment, nonlinear association among immunity indexes is difficult to capture, the problems of one-sided evaluation and poor result reliability exist, and accurate and systematic support cannot be provided for research and development of immune candidate medicaments. Disclosure of Invention The invention provides an animal immunity evaluation method based on a Chlamydia cat infection model, which comprises the steps of firstly screening healthy experimental cats with consistent physiological states, constructing a standardized host model, secondly preparing and verifying the Chlamydia cat infection model through morphology and molecular biology to ensure the purity and activity of an infection source, then grouping the experimental cats, constructing a standardized infection model through mixed culture with positive cats, simultaneously setting a comparison group, collecting samples at key time points, detecting multidimensional indexes related to humoral immunity, cellular immunity and infection control, then utilizing PCA dimensionality reduction, curved surface fitting nonlinear correction, AHP objective weighting and SVM classification prediction to fuse four algorithms, converting original indexes into immunity comprehensive scores and immunity grades, finally verifying the stability of results through repeated experiments, and summarizing data to form a standardized evaluation report, thereby realizing the system and objective evaluation of immune candidate medicine effects. In order to achieve the above purpose, the invention adopts the following technical scheme: a method of evaluating animal immunity based on a model of infection by a cat chlamydia, comprising: S1, preparing and screening experimental animals, namely selecting 1-3 year old healthy domestic cats, screening qualified individuals through ectoparasite screening, clinical symptom evaluation, blood routine detection, blood biochemical detection and toxoplasma antibody detection, recording healthy baseline data, and constructing a standardized host model; S2, preparing and verifying quality of Chlamydia felis, namely screening positive cats with the number of live fleas on the body surface being more than or equal to 4 and toxoplasma antibody negative, carrying out morphological identification and molecular biological identification on fleas collected from the body surface of the positive cats, screening female adult fleas with standard movement capacity, morphological integrity and blood sucking potential, forming a standardized infection source and providing a quality verification report; S3, randomly dividing the healthy cats screened in the step S1 into a blank control group, an infection control group and an immune candidate drug group, wherein the blank control group is independently fed in an environment without flea pollution, the infection control group and the positive cats in the step S2 are mixed in a cage, the immune candidate drug group is fed with the immune candidate drug according to weight 24 hours before being mixed with the positive cats, and then mixed in the mixed culture period; s4, immune related index detection, namely collecting blood samples of all groups of cats and immune organ samples at partial time points at preset time points after infection, detecting humoral immunity indexes, cellular immunity indexes and infection control effect indexes, recording original detection data and performing quality control; S5, evaluating animal immune functions, namely firstly extracting a principal component with a cumulative variance contribution rate of more than or equal to 85% through principal component analysis to reduce the dimension and remove the noise of the detection data in the step S4, then carrying out nonlinear correction on the extracted principal component by adopting a quadratic polynomial surface fitting model to obtain a comprehensive principal component score, distributing objective weights to the corrected comprehensive principal component score and the extracted principal component by adopting a analytic hierarchy process based on the variance contribution rate and a correlation coefficient, calculating a corrected immune comprehensive score, and finally mapping the corrected immune comprehens