GB/T 19915-2026Diagnostic techniques for swine streptococcosis (English PDF)

6.1 Epidemiology

6.1.1 It can occur year-round, but is more common in hot and humid seasons. Clinically, there are multiple serotypes of Streptococcus suis, and infections with Streptococcus suis are also observed. Mixed infection with other pathogens.

6.1.2 Pigs of different ages, breeds and sexes can be infected.

6.1.3 Sick pigs, carrier pigs, and dead pigs are the main sources of infection.

6.1.4 It is mainly transmitted through the respiratory tract, wounds, and digestive tract.

6.2 Clinical Symptoms

6.2.1 In the most acute form, the body temperature of the pig can reach above 42°C. When touched, the pig will cry out, its skin will turn bluish-purple, and it will die suddenly.

6.2.2 Acute cases in pigs present with a body temperature between 41°C and 43°C; widespread congestion and redness are observed on the head, neck, back, lower abdomen, and inner sides of the limbs. Purpura; neurological symptoms such as ataxia, circling, convulsions, swimming-like movements of the limbs, or coma; shortness of breath or difficulty breathing, with wheezing audible. Silence.

6.2.3 In chronic cases, the joints of the affected pigs are swollen and painful, causing lameness or inability to stand; the lymph nodes are swollen and hardened.

6.3 Pathological Changes During Autopsy

6.3.1 Pleural effusion, pulmonary hemorrhage, epicardial hemorrhage; fibrinous pneumonia, pericarditis.

6.3.2 Kidney and lymph node hemorrhage, splenomegaly.

6.3.3 Cerebral congestion and increased cerebrospinal fluid.

6.3.4 Joint swelling, suppuration, or degeneration and hyperplasia of the joint capsule tissue, congestion within the capsule, and turbid synovial fluid.

6.4 Result Determination Patients meeting the criteria in

6.1 and exhibiting the clinical symptoms described in

6.2 and the pathological changes observed during necropsy in

6.3 can be considered suspected cases of streptococcal infection in pigs. A definitive diagnosis should be made... Samples were collected for laboratory testing.

7.1 Instruments and Equipment

7.1.1 Scalpels, scissors, forceps, tissue homogenizers, mouth openers, tonsil collectors, syringes, etc.

7.1.2 Containers. Vacuum blood collection tubes (containing EDTA anticoagulant), centrifuge tubes (2mL, 10mL), self-sealing bags, sample preservation tubes, etc.

7.1.3 Personal protective equipment. protective clothing, protective goggles, protective caps, protective boots, masks, gloves, etc.

7.1.4 Sampling record supplies. sampling form, marker, waterproof label, etc.

7.1.5 Others. swabs, medical gauze, sealing film, etc.

7.2 Reagents and Materials 7.2.1

0.01 mol/L PBS (pH 7.2), prepared according to Appendix A, A.1.

7.2.2 Selective THB enrichment broth, prepared according to A.2.

7.3 Sample Collection and Processing

7.3.1 Sample Selection Samples such as joint fluid and anticoagulated blood can be collected from diseased pigs; tissues such as lymph nodes, brain, lungs, and spleen can be collected from dead pigs or from diseased pigs after necropsy. sample.

7.3.2 Joint fluid sample Collect joint fluid with a syringe, put it into a sample preservation tube, seal it, number it, and record it.

7.3.3 Anticoagulated blood samples Collect 5 mL of blood through the marginal ear vein using a vacuum blood collection tube (containing EDTA anticoagulant), seal it, number it, and record the results.

7.3.4 Tissue Samples Collect tissue samples from lymph nodes, brain, lungs, spleen, etc., using sterile scissors and forceps. Place them in self-sealing bags or sample preservation tubes, seal them, and label them. Good record.

7.4 Sample preservation, packaging and transportation Sample preservation, packaging, and transportation shall be carried out in accordance with the provisions of NY/T 541.

8.1 Instruments and Equipment

8.1.1 Class II biosafety cabinet.

8.1.2 Homogenizer or tissue grinder.

8.1.3 Electronic balance. sensitivity is 0.001g.

8.1.4 Incubator. Can be used for 37°C incubation.

8.1.5 Refrigerator (4°C, -20°C, -40°C and below).

8.1.6 Upright optical microscope (oil immersion).

8.2 Reagents and Materials

8.2.1 Unless otherwise specified, all reagents used are of analytical grade. 8.2.2

0.01 mol/L PBS (pH 7.2), prepared as in 7.2.1.

8.2.3 Selective THB enrichment broth, prepared in the same way as 7.2.2.

8.2.4 Selective THB sheep blood plate, prepared according to A.3.

8.2.5 Commercially available Gram staining solutions.

8.3 Test Procedure

8.3.1 Isolation of pathogens from tissues Aseptically, 0.1g of tissues from lymph nodes, brain, lungs, spleen, etc., were taken and homogenized in.200µL of 0.01mol/L PBS (pH 7.2). Add the homogenate to a test tube containing 5 mL of sterile selective THB enrichment broth, incubate at 37°C for 8-10 hours, then streak inoculate onto the selected culture medium. Selective THB sheep blood agar plates were incubated at 37°C for 10 hours; if colony growth was slow, the incubation period could be extended to 20 hours for observation. For liquid culture, samples were picked... Single colonies were inoculated with selective THB enrichment broth and incubated at 37°C for 10 hours.

8.3.2 Isolation of pathogens from anticoagulated blood and synovial fluid, etc. Aseptically, add

0.5 mL of anticoagulated blood or synovial fluid to a test tube containing 5 mL of sterile selective THB enrichment broth, and incubate at 37°C. After incubation for 8-10 hours, streak the culture onto selective THB sheep blood agar plates and incubate at 37°C for 10 hours. If colony growth is slow, the incubation period can be extended. Observe after 20 hours. For liquid culture, pick a single colony and inoculate it with selective THB enrichment broth, then incubate at 37°C for 10 hours.

8.4.1 Isolation and Identification of Streptococcus suis

8.4.1.1 Bacterial Culture. Streptococcus suis forms round, slightly convex, smooth, moist, and well-defined patterns on selective THB sheep blood agar plates. Semi-transparent colonies, approximately 1 mm in diameter; most strains exhibit grass-green alpha-hemolysis, while some strains, or those with prolonged incubation, may produce transparent beta-colony. Hemolytic colonies. Colony morphology is shown in Figure B.1 of Appendix B.

8.4.1.2 Staining and microscopic examination. Gram-positive Streptococcus suis, with a cell diameter of 0.5 µm to 1 µm. Solid cultures showed predominantly short chains, while liquid cultures showed... Nutrients are mainly long-chain organisms.

8.4.1.3 Those that meet the criteria of

8.4.1.2 can be identified as suspected Streptococcus suis.

8.4.2 Isolation and Identification of Streptococcus equi Subsp. porcupine

8.4.2.1 Bacterial Culture. Streptococcus equi subsp. veterinaria forms a 3mm-4mm mucus-like substance with a smooth surface on selective THB sheep blood agar plates. Large, slippery colonies exhibit significant beta-hemolysis; some strains present as non-mucous colonies, approximately 1 mm in size. See Figure B.2 for colony morphology.

8.4.2.2 Staining and microscopic examination. Gram-positive Streptococcus equi subsp. porphyria, with a cell diameter of 0.5 µm to 1 µm. Fresh cultures show spherical cell formation. In older cultures, the bacterial cells appear oval. Microscopic observation of clinically collected tissue samples (touch slides) and colonies picked from solid culture media can reveal [the presence of these cells]. The bacteria were observed to exist singly or in short chains. When smears were taken from liquid culture medium and examined under a microscope, long chains of bacteria were visible, which were difficult to centrifuge. collect.

8.4.2.3 Those that meet the criteria of

8.4.2.2 can be identified as suspected Streptococcus equi subsp. veterinary.

9.1 Instruments and Equipment

9.1.1 Class II biosafety cabinet.

9.1.2 Centrifuge.

9.1.3 Vortex oscillator.

9.1.4 Fully automated nucleic acid extractor.

9.1.5 Electronic balance. sensitivity is 0.001g.

9.1.6 Micro-adjustable pipettes (with specifications of 1µL~10µL, 20µL~200µL, and 100µL~1000µL).

9.1.7 Fluorescent PCR amplification instrument.

9.2 Reagents and Materials

9.2.1 Bacterial genomic DNA extraction kit.

9.2.2 Nuclease-free water. Purchase commercially available nuclease-free water or prepare your own, following the preparation method in A.4.

9.2.3 Fluorescent PCR enzyme mixture (containing DNA polymerase, dNTPs, Mg2+, etc.).

9.2.4 Primer and probe sequences were prepared to a working concentration of 10 µmol/L by adding nuclease-free water according to C.1~C.5 in Appendix C.

9.2.5 The positive control for Streptococcus suis fluorescent PCR is genomic nucleic acid extracted from Streptococcus suis strains; if strains are difficult to obtain, Streptococcus suis strains can be used. The recN gene fragment of cocci or a plasmid carrying this gene fragment.

9.2.6 The positive control for fluorescent PCR of *Streptococcus equi* subsp. *porphyria* is the genomic nucleic acid extracted from the *Streptococcus equi* subsp. *porphyria* strain; if the strain is difficult to... To obtain this gene fragment, the *Streptococcus equi* szM subsp. *porphyria* gene fragment or a plasmid carrying this gene fragment can be used.

9.2.7 The positive control for Streptococcus suis type 2 fluorescent PCR is the genomic nucleic acid extracted from Streptococcus suis type 2 strain; if the strain is difficult to obtain, it can be... The cps2J gene fragment of Streptococcus suis type 2 or a plasmid carrying this gene fragment was used.

9.2.8 The positive control for Streptococcus suis type 7 fluorescent PCR is the genomic nucleic acid extracted from Streptococcus suis type 7 strain; if the strain is difficult to obtain, it can be... The cps7H gene fragment of Streptococcus suis type 7 or a plasmid carrying this gene fragment was used.

9.2.9 The positive control for Streptococcus suis type 9 fluorescent PCR is the genomic nucleic acid extracted from Streptococcus suis type 9 strain; if the strain is difficult to obtain, it can be... The cps9H gene fragment of Streptococcus suis type 9 or a plasmid carrying this gene fragment was used.

9.2.10 The negative control is sterile, nuclease-free water.

9.3 Nucleic Acid Extraction Bacterial nucleic acids were extracted from each sample using a bacterial genomic DNA extraction kit; alternatively, a fully automated nucleic acid extractor could be used. Bacterial nucleic acid in the sample. If testing is to be performed within 2 hours, the extracted nucleic acid can be stored on ice; if testing is to be performed after 2 hours, it should be stored at -40°C or below. Store for later use, and avoid repeated freeze-thaw cycles.

9.4 Procedure for Fluorescent PCR of Streptococcus suis

9.4.1 Reaction System The reagents required to prepare a 20 µL reaction system are as follows:

---10µL fluorescent PCR enzyme mixture;

---4.4µL of nuclease-free water;

---1.5 µL upstream primer (10 µmol/L);

---1.5 µL of downstream primer (10 µmol/L);

---0.6 µL probe (10 µmol/L);

---2µL of sample nucleic acid. Positive and negative controls should be set up for each fluorescent PCR amplification.

Note. The reaction system may vary depending on the reagents used.

9.4.2 Reaction Conditions Centrifuge the reaction tube briefly, place it in a fluorescent PCR amplification instrument, edit the sample table, select FAM as the reporter group, and select BHQ1. Quenching group. Amplification conditions. 95°C for 2 min; 95°C for 15 s, 53°C for 1 min, with fluorescence collected in the second step of each cycle (53°C for 1 min). Optical signal, 40 cycles in total.

9.5 Procedure for Fluorescent PCR of Streptococcus equi Subsp. veterinaria

9.5.1 Reaction System The reagents required to prepare a 20 µL reaction system are as follows:

---10µL fluorescent PCR enzyme mixture;

---3µL nuclease-free water;

---0.5 µL upstream primer (10 µmol/L);

---0.5 µL of downstream primer (10 µmol/L);

---1µL probe (10µmol/L);

---5µL of sample nucleic acid. Positive and negative controls should be set up for each fluorescent PCR amplification.

Note. The reaction system may vary depending on the reagents used.

9.5.2 Reaction Conditions Centrifuge the reaction tube briefly, place it in a fluorescent PCR amplification instrument, edit the sample table, select FAM as the reporter group, and select BHQ1. Quenching group. Amplification conditions. 95°C for 10 min; 95°C for 15 s, 60°C for 45 s, with fluorescence collected at the second step of each cycle (60°C for 45 s). The signal has 40 cycles.

9.6 Procedure for Fluorescent PCR of Streptococcus suis Type 2

9.6.1 Reaction System The reagents required to prepare a 20 µL reaction system are as follows:

---10µL fluorescent PCR enzyme mixture;

---5µL nuclease-free water;

---1.2 µL upstream primer (10 µmol/L);

---1.2 µL of downstream primer (10 µmol/L);

---0.6 µL probe (10 µmol/L);

---2µL of sample nucleic acid. Positive and negative controls should be set up for each fluorescent PCR amplification.

Note. The reaction system may vary depending on the reagents used.

9.6.2 Reaction Conditions Centrifuge the reaction tube briefly, place it in a fluorescent PCR amplification instrument, edit the sample table, select FAM as the reporter group, and select BHQ1. Quenching group. Amplification conditions. 95°C for 2 min; 95°C for 15 s, 55°C for 1 min, with fluorescence collected in the second step of each cycle (55°C for 1 min). Optical signal, 40 cycles in total.

9.7 Procedure for Fluorescent PCR of Streptococcus suis Type 7

9.7.1 Reaction System The reagents required to prepare a 20 µL reaction system are as follows:

---10µL fluorescent PCR enzyme mixture;

---5.1µL of nuclease-free water;

---1.2 µL upstream primer (10 µmol/L);

---1.2 µL of downstream primer (10 µmol/L);

---0.5 µL probe (10 µmol/L);

---2µL of sample nucleic acid. Positive and negative controls should be set up for each fluorescent PCR amplification.

Note. The reaction system may vary depending on the reagents used.

9.7.2 Reaction Conditions Centrifuge the reaction tube briefly, place it in a fluorescent PCR amplification instrument, edit the sample table, select FAM as the reporter group, and select BHQ1. Quenching group. Amplification conditions. 95°C for 2 min; 95°C for 15 s, 58°C for 1 min, with fluorescence collected in the second step of each cycle (58°C for 1 min). Optical signal, 40 cycles in total.

9.8 Procedure for Fluorescent PCR of Streptococcus suis Type 9

9.8.1 Reaction System The reagents required to prepare a 20 µL reaction system are as follows:

---10µL fluorescent PCR enzyme mixture;

---5.5µL of nuclease-free water;

---1 µL upstream primer (10 µmol/L);

---1 µL of downstream primer (10 µmol/L);

---0.5 µL probe (10 µmol/L);

---2µL of sample nucleic acid. Positive and negative controls should be set up for each fluorescent PCR amplification.

Note. The reaction system may vary depending on the reagents used.

9.8.2 Reaction Conditions Centrifuge the reaction tube briefly, place it in a fluorescent PCR amplification instrument, edit the sample table, select FAM as the reporter group, and select BHQ1. Quenching group. Amplification conditions. 95°C for 2 min; 95°C for 15 s, 56°C for 1 min, with fluorescence collected in the second step of each cycle (56°C for 1 min). Optical signal, 40 cycles in total.

9.9 Conditions for the Experiment to be Successful Positive controls had a Ct value <= 30 and exhibited a specific amplification curve; negative controls had no Ct value and no specific amplification curve.

9.10 Result Determination

9.10.1 Interpretation of Streptococcus suis test results The tested sample had no Ct value and no specific amplification curve, and was therefore determined to be negative for Streptococcus suis nucleic acid; the tested sample had a Ct value <=35 and showed specific amplification curves. A heterogeneous amplification curve indicates a positive result for Streptococcus suis nucleic acid; if the tested sample has a Ct value of 35 < 40 and exhibits a specific amplification curve, it is determined to be Streptococcus suis nucleic acid. The streptococcal nucleic acid result is questionable. Suspicious samples should be retested. If the retest still shows a Ct value < 40 and a specific amplification curve, the sample is classified as swine. The result is positive for Streptococcus nucleic acid; otherwise, it is considered negative for Streptococcus suis nucleic acid. The interpretation of Streptococcus suis fluorescent PCR detection results is as described in Appendix D. Figure D.1.

9.10.2 Determination of Detection Results for Streptococcus equi Subspecies of Veterinary Disease The tested sample had no Ct value and no specific amplification curve, therefore it was determined to be negative for Streptococcus equi subsp. porcupine nucleic acid; the tested sample's Ct value... <=35 and the appearance of a specific amplification curve indicate a positive result for Streptococcus equi subsp. porcupine nucleic acid; samples with a Ct value < 35 and a Ct value < 40, and the appearance of a specific amplification curve, are considered positive. The heterogeneous amplification curve indicates a questionable result for the nucleic acid of *Streptococcus equi* subsp. *porphyria*. Suspicious samples should be retested. If the retest still shows a Ct value < 40... If a specific amplification curve is observed, it is determined to be positive for Streptococcus equi subsp. veterinaryis nucleic acid; otherwise, it is determined to be negative for Streptococcus equi subsp. veterinaryis nucleic acid. Refer to Figure D.2 for the interpretation of the results of fluorescent PCR detection of Streptococcus equi subsp. veterinaria.

9.10.3 Interpretation of Streptococcus suis type 2 test results The tested sample had no Ct value and no specific amplification curve, indicating it was negative for Streptococcus suis type 2 nucleic acid; the tested sample had a Ct value <=35 and showed no specific amplification curve. The current specific amplification curve indicates a positive result for Streptococcus suis type 2 nucleic acid; the tested sample has a Ct value of 35 < 40 and exhibits a specific amplification curve. The result was deemed suspicious for Streptococcus suis type 2 nucleic acid. Suspicious samples should be retested. If the retest still shows a Ct value < 40 and specific amplification occurs... An increasing curve indicates a positive result for Streptococcus suis type 2 nucleic acid; otherwise, it indicates a negative res...