GB/T 18651-2026Diagnostic techniques for bovine anaplasmosis (English PDF)

5.1 Popular Characteristics

5.1.1 Animals such as cattle, dairy cows, water buffalo, and yaks are all susceptible.

5.1.2 This disease is prevalent in areas where hard ticks are distributed.

5.1.3 This disease is most prevalent during the tick season, typically appearing in June, peaking from August to October, with occasional cases still occurring in November. In other seasons, sporadic cases may occur.

5.1.4 The older the cattle, the more severe the disease and the higher its pathogenicity. Local cattle show milder symptoms, while imported cattle, purebred cattle, and improved breeds often exhibit severe symptoms. Severe clinical symptoms, with visible hard ticks on the animal's body or a history of hard tick bites.

5.2 Clinical Symptoms

5.2.1 High fever retention. The body temperature of sick cattle can usually reach above 40°C.

5.2.2 Anemia, paleness of visible mucous membranes such as the conjunctiva.

5.2.3 Jaundice, yellowing of the skin and mucous membranes, and possible darkening of urine color.

5.2.4 Superficial lymph nodes such as the anterior shoulder lymph nodes and inguinal lymph nodes are swollen, firm in texture, and sensitive to palpation.

5.2.5 Weakness, slow walking, easy fatigue, rough and dull coat, pregnant cows are prone to abortion or giving birth to weak calves, etc.

5.3 Pathological Changes

5.3.1 Abnormal red blood cell morphology, reduced number, anemia.

5.3.2 The liver is enlarged, brittle, and exhibits hepatocyte degeneration and necrosis, with disordered lobular structure.

5.3.3 Splenomegaly, softened texture, red pulp hyperplasia, and increased macrophages.

5.3.4 Enlarged lymph nodes with indistinct boundaries between the medulla and cortex, dilated lymphatic sinuses filled with lymphocytes and plasma cells.

5.4 Result Determination If a patient meets the prevalence characteristics outlined in

5.1 and exhibits three or more of the conditions described in

5.2 and/or three or more of the conditions described in 5.3, they can be considered a suspected case of bovine erysipelas. Physical case.

6.1 Reagents and Materials

6.1.1 Medical protective clothing.

6.1.2 Sterilized scissors and tweezers.

6.1.3 Anticoagulation vacuum blood collection tubes.

6.1.4 Standard blood collection tubes.

6.1.5 Use a 6-gauge needle for tail vein blood collection and a 16-gauge needle for jugular vein blood collection.

6.1.6 Disposable syringes of various specifications.

6.2 Sample Collection Determine the blood collection site, either the jugular vein or the tail vein, and use sterile blood collection instruments to collect approximately 3 mL of anticoagulated blood and 3 mL of whole blood, respectively. Local disinfection should be performed before and after blood collection, and sample contamination should be avoided during the blood collection process. Each sample should be labeled with its number, name, animal origin, animal age, and species. Species, health status, sampling time, sampling location, and sampler, etc.

6.3 Sample preservation and transportation Collected blood samples, especially anticoagulated blood, should be stored at 2°C~8°C for no more than 24 hours. Whole blood should be allowed to coagulate naturally after collection. Serum can be isolated using this method, and long-term storage requires freezing at -20°C. Samples should be transported in a waterproof, leak-proof container, ensuring a complete seal. The container should be placed in a pressure-resistant, waterproof, and leak-proof environment. The sample is placed in the auxiliary packaging. Cryoprotectant is placed around the auxiliary packaging to keep the sample at a low temperature, ensuring the sample is delivered to the laboratory for testing within 24 hours.

7.1 Instruments and Equipment

7.1.1 Biological microscope.

7.2 Reagents and Materials

7.2.1 Glass slide. 7.2.2 1mL dropper.

7.2.3 Fixative. 95% methanol solution.

7.2.4 Giemsa staining working solution, prepared according to Appendix A, A.1.

7.2.5 Cedar oil.

7.3 Preparation of blood smears Using a micropipette, place a drop of anticoagulated blood (approximately 1 µL to 2 µL) onto one end of a glass slide. Hold the slide and align one end with the glass slide. The slide is brought into contact with a drop of blood at a 30°-45° angle, and the slide is smoothly pushed forward to spread the blood evenly onto the slide, forming a thin and uniform layer. Prepare a thin blood smear and dry it quickly.

7.4 Blood smear fixation Place the prepared blood smear horizontally, and use a dropper to draw 2 to 3 drops of fixative, then evenly add them to the blood smear, ensuring the fixative covers the entire surface. Cover the entire smear area and allow the fixative to act on the blood film for 3 to 5 minutes, then let it air dry.

7.5 Blood smear staining Place the prepared and fixed blood smear horizontally, add Giemsa staining working solution to cover the entire blood smear, and stain at room temperature for 15 minutes. 30 min. Gently rinse the blood smear with a slow-flowing stream of water to thoroughly remove any residual staining solution. Allow it to air dry or blot dry with filter paper. Inspection. During the staining process, it is important to prevent the staining solution from completely evaporating and forming precipitate particles.

7.6 Microscopic examination Place the stained and dried blood smear on the microscope stage and secure it with slide clips. Add cedarwood drops to the blood smear. The oil was then gradually switched to an oil immersion lens (100×) for observation.

7.7 Result Determination

7.7.1 Suspected. Numerous, densely stained, round, and compact typical small bodies, with a diameter of 0.3 µm, are visible within red blood cells and white blood cells in the blood smear. A 1.0 µm lesion located at or near the edge of a red blood cell is considered a suspected marginal apoplastic infection (see Figures B.1 and B.2 in Appendix B).

7.7.2 Negative. If the result of

7.7.1 is not present, the blood smear test is considered negative.

8.1 Instruments and Equipment

8.1.1 Micro-adjustable pipettes and matching pipette tips (0.1µL~2.5µL, 0.5µL~10µL, 10µL~100µL, 100µL~ 1000µL).

8.1.2 Microplate reader.

8.2 Reagents and Materials

8.2.1 ELISA plate. 8.2.2 96-well plates are used for adsorption/transfer of serum to reduce background staining.

8.2.3 Water, GB/T 6682, Grade II water.

8.2.4 Phosphate buffer (PBS),

0.01 mol/L pH 7.2, prepared according to A.2.

8.2.5 Washing solution, prepared according to A.3.

8.2.6 Recombinant marginal plasmaless MSP5 antigen, the preparation method is described in Appendix C.

8.2.7 Recombinant edge-free plasmaless MSP5 protein polyclonal antibody, the preparation method is described in Appendix D.

8.2.8 Preparation of recombinant marginal plasmaless MSP5 protein monoclonal antibody, the preparation method is described in Appendix E.

8.2.9 Horseradish peroxidase-labeled MSP5 antibody, preparation method is given in Appendix F.

8.2.10 Blocking solution. PBS solution containing 1%~5% bovine serum albumin.

8.2.11 Substrate liquid.

8.2.12 Termination solution.

8.2.13 Positive control. Bovine serum with no positive plasma cells at the edge, indicating an infection rate of over 5%.

8.2.14 Negative control. Bovine serum without infection margins and no plasma.

8.3 Antigen coating and blocking

8.3.1 Dilute the edge-free, plasma-free recombinant MSP5 antigen to a concentration of 10 µg/mL with phosphate buffer and add it to the wells of the ELISA plate. 100 µL per well, incubated at 37°C for 1-2 hours.

8.3.2 After coating, discard the liquid in the wells, wash the microplate 3 to 5 times with washing buffer, soaking for 1 to 2 minutes each time, then... Pat dry.

8.3.3 Add blocking solution, 100 µL per well, and incubate at 37°C for 1 to 2 hours.

8.3.4 Discard the blocking solution, wash the ELISA plate 3 to 5 times, and pat dry.

8.4 Sample Testing

8.4.1 Add 70 µL of undiluted serum sample to a 96-well plate and incubate at room temperature for 30 min.

8.4.2 Transfer 50 µL of the adsorbed serum from each well of the 96-well plate to a microplate coated with recombinant MSP5, and set up a negative control at the same time. (Known negative bovine serum), positive control (known positive bovine serum), and blank control (diluent added only), add 1.5000 dilution to each well immediately. Add 50 µL of MSP5 antibody with high concentration of peroxidase, mix well, cover with an enzyme-labeled plate, and incubate at 37°C for 30-60 minutes.

8.4.3 Discard the liquid in the wells, wash the microplate 3 to 5 times, and pat dry.

8.4.4 Add 50 µL of substrate solution to each well and incubate at 37°C in the dark for 10-30 minutes. The specific time should be determined based on the color development. When positive... The reaction can be terminated when a significant color change is observed in the control well.

8.4.5 Add 50 µL of stop solution to each well to terminate the colorimetric reaction.

8.4.6 Use an ELISA reader to read the absorbance (OD value) of each well at a specific wavelength (e.g., 450 nm).

8.5 Inhibition rate

8.6 Conditions for the Experiment to be Successful The test is valid when the negative control OD value is between

0.40 and

2.10 and the positive control PI is >=30%. Otherwise, the test should be repeated.

8.7 Result Determination

8.7.1 When the inhibition rate (PI) of the sample to be tested is >=30%, it is judged as a positive result for edge aplasma antibody.

8.7.2 When the inhibition rate of the sample to be tested is < 30%, it is judged as negative for edge aplasma antibody.

Note. When using commercially available reagent kits, follow their instructions for operation and judgment.

9.1 Reagents and Materials

9.1.1 Black agglomeration reaction plate.

9.1.2 Water, GB/T 6682, Grade II water.

9.1.3 Recombinant marginal non-plasmic MSP5 antigen, same as 8.2.6.

9.1.4 Physiological saline.

9.1.5 Polystyrene latex particles.

9.1.6 Positive control, same as 8.2.13.

9.1.7 Negative control, same as 8.2.14.

9.1.8 Recombinant MSP5 latex agglutination particle antigen, the preparation method is described in Appendix G.

9.2 Preparation of Latex Agglutination Antigen Reaction Plates Use a pipette to draw 25 µL to 50 µL of latex agglutination antigen and add it to a black agglutination reaction plate.

9.3 Serum dilution Use a pipette to add 500 µL of physiological saline to a clean centrifuge tube, then add 50 µL of the serum sample to be tested to physiological saline. Dilute the solution at a volume ratio of

1.10 and mix thoroughly.

9.4 Sample addition and testing

9.4.1 Using a pipette, aspirate the diluted sample serum and negative and positive control serum, and add them to the recombinant MSP5 latex agglutination antigen. Add 25 µL to 50 µL to each well in the reaction plate.

9.4.2 Gently shake the reaction plate containing the antigen to mix the serum and antigen thoroughly, and observe the reaction results within 2 to 5 minutes.

9.5 Conditions for the Experiment to be Successful The positive control area showed clear particle aggregation, while the negative control area showed no identifiable aggregated particles; therefore, the test was deemed a failure. Established.

9.6 Result Determination

9.6.1 If obvious agglutination particles are found in the tested serum, it is considered to be antibody positive.

9.6.2 If no agglutination occurs in the tested serum, it is considered to be antibody negative.

9.6.3 If the result is between positive and negative, it is considered suspicious. 10 PCR detection methods

10.1 Instruments and Equipment

10.1.1 Micro-adjustable pipettes and matching pipette tips (0.1µL~2.5µL, 0.5µL~10µL, 10µL~100µL, 100µL~1000µL).

10.1.2 Vortex Oscillator.

10.1.3 High-speed centrifuge.

10.1.4 Constant temperature incubator.

10.1.5 High-pressure steam sterilizer.

10.1.6 PCR gene amplification instrument.

10.1.7 Nucleic acid electrophoresis apparatus.

10.1.8 Nucleic acid electrophoresis tank.

10.1.9 Gel Imaging Analysis System.

10.2 Reagents and Materials

10.2.1 PCR tube.

10.2.2 Water, GB/T 6682, Grade II water.

10.2.3 Phosphate buffer, same as 8.2.4. 10.2.4 1×TAE buffer, prepared according to A.4.

10.2.5 Safe and non-toxic nucleic acid dyes.

10.2.6 Blood Genomic DNA Extraction Kit.

10.2.7 PCR amplification kit (commercially available product; alternative products of the same type may be selected).

10.2.8 Standard molecular mass of DNA. 10.2.9 1% agarose gel, prepared according to A.5.

10.2.10 Positive control. Anticoagulated bovine blood with no positive plasma at the margins, indicating an infection rate of over 5%.

10.2.11 Negative control. Anticoagulated bovine blood without infection margins and no plasma.

10.2.12 Specific primers for PCR amplification of the Msp4 gene locus at the edge of the plasma nucleus, all with a working concentration of 25 µmol/L.

10.3 Pathogen DNA Extraction

10.3.1 Use a commercially available blood DNA extraction kit to extract DNA from blood samples, strictly following the kit's instructions. This process ensures the quality and purity of the extracted DNA.

10.3.2 Extracted DNA samples should be immediately subjected to PCR amplification or stored at -20°C for later use.

10.3.3 When extracting DNA from samples, at least one positive control and one negative control sample should be included.

10.4 PCR reaction system The plasma-free PCR detection reaction system was prepared according to Table 1.

10.5 PCR Amplification Program Initial denaturation at 94°C for 30 seconds, followed by denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 68°C for 1 minute, for a total of 35 cycles. Extend the temperature to 68°C for 1 minute.

10.6 Agarose gel electrophoresis and imaging of PCR products Prepare a 1% agarose gel using 1×TAE buffer. Perform electrophoresis on a 1% agarose gel with 10 µL of the PCR product under constant voltage. Electrophoresis was performed at 120V for 35 minutes, and the results were observed and imaged using a gel imaging analysis system.

10.7 Conditions for the Experiment to be Successful When the positive control without plasma at the edge shows the corresponding specific band (see Appendix H), and the negative control shows no amplification band, the test is considered successful.

10.8 Result Determination

10.8.1 The presence of a specific band at the corresponding target fragment (see Appendix H) is sufficient to determine a positive nucleic acid result, further confirming genomic variations. Then, PCR products were sequenced (see Appendix I) and BLAST compared. If sequence homology exceeded 98%, it was determined to be marginal non-plasma infection and predicted. Genomic variation.

10.8.2 If no specific band is found at the corresponding target fragment, it is determined to be a negative result for edge-free plasma nucleic acid.