GB/T 34750-2025Diagnostic techniques for Glässer's disease (English PDF)

1 Scope

This document describes the clinical diagnosis of Glässer's disease and the laboratory diagnostic methods of sample collection, handling and storage, bacterial isolation and identification, nested PCR detection, real-time fluorescent PCR detection and indirect ELISA.

This document applies to the diagnosis and the monitoring of Glässer's disease.

4 Abbreviations

The following abbreviations apply to this document. bp, base pair; Ct value, cycle threshold; DNA, deoxyribonucleic acid; dNTPs, deoxyribonucleoside triphosphates; ELISA, enzyme linked immunosorbent assay; IgG, immunoglobulin G; MGB, minor groove binder; NAD, nicotinamide adenine dinucleotide; PBS, phosphate buffer solution; PCR, polymerase chain reaction; Taq enzyme, Taq DNA polymerase; TSA, tryptic soy agar; TSB, tryptic soy broth.

5 Clinical diagnosis

Epidemiology. Glässer's disease is a bacterial infectious disease caused by infection with Glaesserella parasuis. The organism infects only pigs, and sick pigs and carrier pigs are the main source of infection. Pigs of every age, sex and breed are susceptible, and the disease occurs mainly in pigs around weaning and in the nursery stage. It can occur in any season of the year, and is most frequent in early spring and late autumn. Infection is mainly by the respiratory route, and may be acute or chronic, chronic infection being the more common.

Acute cases. Fever, with the body temperature rising to 40.5 degrees Celsius to 42 degrees Celsius, dullness and reduced appetite. Laboured breathing, with mucous and serous discharge from the nostrils. Reddening of the skin early in the infection and cyanosis of the margins of the ears later. Lameness, trembling and nervous signs.

Chronic cases. Falling appetite, wasting and weakness, a rough coat and poor growth. Recurrent fever, coughing and laboured breathing. Swollen joints, weakness of the limbs or lameness.

Pathological changes. Inflammation of the pericardium, the pleura, the peritoneum and the joint synovium is characterised by serous and fibrinous exudate. The joints are swollen and contain serous or fibrinous exudate or a jelly-like material. The thoracic cavity contains a large quantity of pale yellow or slightly reddish fluid together with clots of fibrinous exudate. There is pericardial effusion, the pericardium is thickened and often contains caseous exudate adhering to the heart so as to form a villous heart; there are haemorrhagic spots in the myocardium and abundant fibrinous exudate on its surface. The lungs are congested and oedematous, covered on the surface with a fibrin film and adherent to the chest wall. There is serous or fibrinous peritonitis, with ascites or adhesion of the internal organs. The lymph nodes throughout the body are enlarged, and the liver, the spleen and the kidney show congestion and focal haemorrhage.

Interpretation. A case that meets 5.1 and meets three or more of the signs in 5.2, or that meets one or more of the pathological indicators in 5.3, or both, is judged a suspect case of Glässer's disease; confirmation calls for laboratory diagnosis.

6 Sample collection, handling and storage

Samples for microscopy. Fresh tissue such as lung, heart and brain, thoracic, abdominal or pericardial effusion or joint fluid, and anticoagulated blood are collected aseptically from pigs that have died of suspected Glaesserella parasuis infection.

Serum samples. Blood is taken aseptically from the ear vein or the anterior vena cava, not less than 5 mL from each animal; the serum is separated aseptically into a 2 mL centrifuge tube, numbered, and used for ELISA detection.

Tissue samples. Fresh tissue samples with obvious lesions, such as lung, heart and brain, are taken. About 0.5 g of the sample to be tested is cut out with sterile scissors and forceps into a tissue homogeniser or a mortar and thoroughly homogenised or ground; 0.3 mL to 0.5 mL of PBS is added and mixed, and the tissue suspension is transferred into a sterile centrifuge tube, numbered and set aside.

Effusion. Between 2 mL and 3 mL of thoracic or abdominal effusion or of joint fluid is collected with a sterile syringe, transferred into a sterile centrifuge tube, numbered and set aside.

Anticoagulated blood. Between 2 mL and 3 mL of anticoagulant, that is Alsever's solution, is first drawn into a sterile syringe; an equal volume of blood is taken from the ear vein or the anterior vena cava, mixed quickly and transferred into a sterile centrifuge tube, numbered and set aside.

Enrichment culture. 500 microlitres of the enrichment culture is taken with a sterile pipette tip into a sterile centrifuge tube, numbered and set aside.

Storage. After collection the samples are placed in an insulated box with pre-cooled ice packs and sealed, and should be sent to the laboratory within 24 h. Samples kept at 2 degrees Celsius to 8 degrees Celsius shall be held for not more than 24 h. Where they have to be kept for a long time they shall be placed at minus 70 degrees Celsius.

7 Bacterial isolation and identification

Main apparatus. Biological safety cabinet; constant-temperature incubator; optical microscope; automated microbial identification system; refrigerator at 2 degrees Celsius to 8 degrees Celsius.

Main reagents and materials. TSA solid medium and TSB liquid medium prepared in accordance with A.1 and A.2 of Annex A. NAD stock solution prepared in accordance with A.3 and kept at 2 degrees Celsius to 8 degrees Celsius. Gram staining reagents, comprising crystal violet with ammonium oxalate, Lugol's iodine, 95 % alcohol and safranin, kept at room temperature. Sheep blood plates without NAD, and micro biochemical identification tubes for glucose, sucrose, fructose, lactose, galactose and xylose, kept at 2 degrees Celsius to 8 degrees Celsius. Nitrate reduction test reagents and 0.1 % dimethyl-para-phenylenediamine dihydrochloride solution prepared in accordance with A.4 and A.5 and kept at room temperature.

Isolation culture. In the biological safety cabinet the sample is picked up aseptically with an inoculating loop and streaked onto TSA solid medium, then incubated at 37 degrees Celsius for 24 h to 48 h so that colonies form for identification. Pinpoint colonies of about 1 mm to 2 mm in diameter, round, raised, smooth and moist, colourless, transparent and with an even edge are picked from that culture and streaked onto TSA solid medium for pure culture; after 24 h to 48 h at 37 degrees Celsius the same small colonies appear, as shown in Figure B.1 of Annex B.

Smear, staining and microscopy. One or two drops of sterile distilled water are placed on a slide, a colony of the pure culture is picked into it, mixed and spread as a thin film of bacteria. The smear is left to dry naturally in air. The dried slide is held film upward and fixed by passing it five or six times through the flame of a spirit lamp. It is stained by the Gram method, the operation following the instructions of the Gram staining kit, and examined under an optical microscope at 1 000 times magnification, that is 10 by 100. Glaesserella parasuis appears as Gram-negative short rods or coccobacilli of uneven size, about 0.5 micrometres by 1.5 micrometres to 2.0 micrometres, mostly slender rods, without flagella and not forming spores, as shown in Figure B.2.

Satellite growth test. In the biological safety cabinet a single colony from the purified culture is picked aseptically and streaked in a line on a sheep blood plate without NAD, and Staphylococcus aureus is then streaked at right angles to that line. After 24 h to 48 h at 37 degrees Celsius the growth of the colonies is examined; Glaesserella parasuis does not haemolyse and shows the satellite growth phenomenon, that is the colonies of Glaesserella parasuis close to the staphylococcus are larger and those further away from it are smaller.

Enrichment culture. In the biological safety cabinet a single non-haemolytic colony showing the satellite growth phenomenon is picked aseptically and purified once more on TSA solid medium; a single colony is then inoculated into 5 mL of TSB liquid medium and incubated at 37 degrees Celsius for 24 h to 48 h. If the liquid becomes turbid the enrichment culture is judged successful.

Biochemical identification, sugar fermentation test. In the biological safety cabinet the biochemical identification tubes are opened aseptically and NAD is added to each tube to a final concentration of 10 micrograms per millilitre; with an inoculating needle the enriched culture is inoculated aseptically into micro biochemical identification tubes of glucose, lactose, galactose, mannitol and xylose, which are incubated at 37 degrees Celsius for 24 h and then read. Fermentation of glucose and galactose without fermentation of lactose, mannitol or xylose is positive; otherwise the result is negative.

Catalase test. One drop of the enriched culture is taken onto a clean slide, one drop of 3 % hydrogen peroxide is added and mixed, and the reaction is read within 30 s; abundant bubbles appearing at once are judged positive and no bubbles negative. Nitrate reduction test. 0.2 mL each of 0.8 % sulfanilic acid in glacial acetic acid and 0.5 % alpha-naphthylamine in ethanol are mixed, and 0.1 mL of the mixed reagent is added to the enriched culture; the result is read at once, a red colour being judged positive and no red colour negative. Urease test. 100 microlitres of the enriched culture is inoculated into a tube of urease test liquid medium, shaken, and incubated at 37 degrees Celsius, the results being read after 10 min, 60 min and 120 min; a pink colour is judged positive and no change of colour negative. Oxidase test. One drop of the enriched culture is taken onto a clean slide and one drop of dimethyl-para-phenylenediamine dihydrochloride solution is added; the result is read within 2 min, a bright blue colour being judged positive and no change of colour within 2 min negative. Identification with the automated microbial identification system is carried out on the enriched bacteria following the manual of the instrument.

Interpretation of the biochemical tests. A culture whose sugar fermentation results are fermentation of glucose and galactose and no fermentation of lactose, mannitol or xylose, whose catalase and nitrate reduction results are both positive and whose urease and oxidase results are both negative, is judged positive for the general type of Glaesserella parasuis; a culture identified as Glaesserella parasuis by the automated microbial identification system is likewise judged positive for the general type; otherwise it is judged negative for the general type.

Interpretation. A culture that meets 7.4 or 7.5, or both, is judged a suspected positive for Glaesserella parasuis infection; a culture positive in any one of the biochemical identifications in 7.7 is judged positive for the general type of Glaesserella parasuis.

8 Nested PCR detection

Main apparatus. Automated nucleic acid extractor; PCR thermal cycler; refrigerated benchtop high-speed centrifuge able to reach a maximum centrifugal force above 12 000 g; regulated electrophoresis power supply and horizontal electrophoresis tank; gel imaging system or ultraviolet transilluminator; adjustable micropipettes of several ranges.

Main reagents and materials. An inactivated reference strain of Glaesserella parasuis is used as the positive control and sterile TSB medium as the negative control. The forward and reverse primers for nested PCR amplification are synthesised in accordance with C.1 of Annex C.

Preparation of the sample DNA. Nucleic acid is extracted from the various samples with a DNA extraction kit, or from the various samples with an automated nucleic acid extractor. Where the test is to be carried out within 2 h the extracted nucleic acid may be kept on ice; otherwise it shall be kept in a freezer at minus 20 degrees Celsius.

First round of PCR amplification. Reaction system I is made up as follows: 2.5 microlitres of ten times PCR buffer containing magnesium ions; 2 microlitres of dNTPs at 2.5 mmol/L; 0.5 microlitres of primer P1; 0.5 microlitres of primer P2; 0.25 microlitres, that is 5 U, of ExTaq DNA polymerase; and 17.25 microlitres of sterile double-distilled water, giving a total volume of 23 microlitres. After thawing at room temperature the tubes are spun briefly so that all the liquid collects at the bottom, 2 microlitres of the corresponding sample DNA is added to each tube, and after thorough mixing the tubes are spun again so that all the liquid collects at the bottom.

Amplification conditions. Pre-denaturation at 95 degrees Celsius for 5 min; then 35 cycles of denaturation at 94 degrees Celsius for 45 s, annealing at 58 degrees Celsius for 45 s and extension at 72 degrees Celsius for 45 s; and a final extension at 72 degrees Celsius for 10 min. When the reaction has finished the tubes are taken out and kept at 2 degrees Celsius to 8 degrees Celsius.

Second round of PCR amplification. Reaction system II is made up in the same way as system I except that primers P3 and P4 take the place of P1 and P2; 2 microlitres of the corresponding product of the first round is added to each tube in place of the sample DNA. The amplification conditions are the same as for the first round.

Electrophoresis of the amplification products. 1.2 g of agarose is weighed into 100 mL of nucleic acid electrophoresis buffer and heated to boiling; when it has fully dissolved it is cooled to about 50 degrees Celsius, 10 microlitres of nucleic acid stain is added and mixed, and the mixture is poured into the gel tray to form the gel plate. One times TAE nucleic acid electrophoresis buffer is added to the tank until the liquid just covers the gel. 10 microlitres of the amplification product of each sample and of the negative and positive controls is mixed with 2 microlitres of six times loading buffer and loaded into the wells of the gel, and 5 microlitres of DNA molecular mass standard is loaded into one well. Electrophoresis is run at a constant 5 V/cm for about 30 min, and the gel is then placed on an ultraviolet transilluminator or in a gel imaging system, the result read and the test record made.

Interpretation. The test is valid where the nested PCR product of the Glaesserella parasuis positive control gives a specific band at 312 bp after electrophoresis and the PCR product of the negative control gives no band at all, as shown in Figure D.1 of Annex D; otherwise the result is not valid. Where the test is valid and the PCR product of a sample gives a specific band at 312 bp after electrophoresis, the sample is judged positive in the general type nucleic acid test for Glaesserella parasuis. Where the test is valid and the sample gives no specific band at 312 bp, it is judged negative in the general type nucleic acid test for Glaesserella parasuis.

9 Real-time fluorescent PCR detection

Main apparatus. Automated nucleic acid extractor; real-time fluorescent PCR instrument; refrigerated benchtop high-speed centrifuge able to reach a maximum centrifugal force above 12 000 g; adjustable micropipettes of several ranges. The forward and reverse primers and the probe for real-time fluorescent PCR amplification of Glaesserella parasuis are synthesised in accordance with C.2, and the sample DNA is prepared as in 8.3.

Reaction system. The real-time fluorescent PCR reaction system is made up as follows: 2.5 microlitres of ten times PCR buffer containing magnesium ions; 2 microlitres of dNTPs at 2.5 mmol/L; 0.5 microlitres of primer FQ-P1; 0.5 microlitres of primer FQ-P2; 0.5 microlitres of MGB probe; 0.25 microlitres, that is 5 U, of ExTaq DNA polymerase; and 16.75 microlitres of sterile double-distilled water. Two microlitres of the corresponding DNA is added to each tube, mixed thoroughly and collected at the bottom of the tube, and the reaction tubes are set out in the order in which the samples are to be loaded.

Reaction parameters. First stage, pre-denaturation at 94 degrees Celsius for 5 min. Second stage, 40 cycles of denaturation at 94 degrees Celsius for 30 s and extension at 60 degrees Celsius for 30 s, the fluorescence signal being collected at the end of the extension step of each cycle, with the fluorescence mode set to FAM and NONE dual labelling.

Interpretation. The test is valid only where the negative control gives no specific amplification curve and a Ct value above 32.0 or none at all, and the positive control gives a Ct value of 29.0 or less together with a specific amplification curve, as shown in Figure D.2; where the negative and positive controls do not satisfy these conditions the run is regarded as invalid. A sample with a Ct value of 29.0 or less that also gives a specific amplification curve is judged positive for the general type of Glaesserella parasuis. A sample with a Ct value above 32.0 or none at all and with no specific amplification curve is judged negative for the general type of Glaesserella parasuis. A sample with a Ct value above 29.0 and up to 32.0 that gives a specific amplification curve shall be tested again; where the repeat result is positive it is judged positive for the general type of Glaesserella parasuis, otherwise negative.

10 Indirect ELISA

Main apparatus. Microplate reader; incubator; plate washer or wash bottle; 96-well microplates; U-bottom 96-well dilution plates; adjustable micropipettes; reagent reservoir.

Main reagents and materials. The coating antigen is the recombinant P2 protein of Glaesserella parasuis, whose preparation and identification are given in Annex E. The enzyme-labelled antibody is rabbit anti-pig IgG conjugated to horseradish peroxidase. For the controls, the preparation and identification of the antibody positive control, that is the positive serum, are given in F.1 of Annex F and those of the negative control, that is the negative serum, in F.2. The coating solution, the washing solution, the blocking solution, the sample dilution solution, the conjugate dilution solution and the stopping solution are prepared in accordance with G.1 to G.6 of Annex G. The substrate solution is a commercial ready-to-use TMB substrate solution.

Preparation of the plate coated with the recombinant P2 protein. Into each well of a 96-well microplate is added 100 microlitres of Glaesserella parasuis recombinant P2 antigen diluted in coating buffer to its working concentration, that is 0.5 micrograms per millilitre, and the plate is left at 4 degrees Celsius overnight for 16 h to 22 h. The coating solution is discarded from the plate, 300 microlitres of washing solution is added to each well for one wash, the washing solution is shaken out and the residual liquid tapped dry on absorbent paper; 120 microlitres of blocking solution is added to each well and the plate is placed in an incubator at 37 degrees Celsius for 2 h, or at 4 degrees Celsius overnight for 16 h to 22 h. When blocking has finished, the blocking solution is discarded from the plate, the plate is tapped dry on absorbent paper, dried at room temperature and then kept dry at 4 degrees Celsius.

Addition of the test sera, the positive control serum and the negative control serum. The test sera, the positive control serum and the negative control serum are each diluted 1 in 100 with dilution solution and 100 microlitres is added to each well, each control serum being run in two duplicate wells; the plate is placed in an incubator at 37 degrees Celsius to react for 60 min, then taken out, the reaction liquid discarded, and 300 microlitres of washing solution added to each well for five washes.

Addition of the enzyme-labelled antibody. The rabbit anti-pig IgG horseradish peroxidase conjugate is diluted 1 in 5 000 with sample dilution solution to its working concentration and 100 microlitres is added to each well; the plate is placed in an incubator at 37 degrees Celsius to react for 30 min, then taken out, the reaction liquid discarded, and the plate washed three times.

Addition of the substrate solution and stopping. 100 microlitres of TMB substrate solution is added to each well and the plate is placed in an incubator at 37 degrees Celsius to react in the dark for 15 min. 50 microlitres of stopping solution is then added to each well to stop the reaction. The absorbance of each well is read on the microplate reader as the optical density at a wavelength of 450 nm.

Data analysis. The test is valid where the mean absorbance at 450 nm of the positive control wells is above 0.5 and the mean absorbance at 450 nm of the negative control wells is below 0.2. The mean absorbance of the positive control wells is the mean of the absorbances of positive control wells 1 and 2, and the mean absorbance of the negative control wells is the mean of the absorbances of negative control wells 1 and 2. The sample to positive ratio of a test serum is calculated as the absorbance of the sample minus the mean absorbance of the negative control wells, divided by the mean absorbance of the positive control wells minus the mean absorbance of the negative control wells.

Interpretation. Where the sample to positive ratio of a sample is 0.40 or more, the sample is judged positive for antibody to Glaesserella parasuis; where it is below 0.40, the sample is judged negative for antibody to Glaesserella parasuis.

11 Comprehensive determination

Suspicion. A case that meets any one of the clinical diagnosis in 5.4, the smear staining and microscopy in 7.4 or the satellite growth phenomenon in 7.5 is a suspected Glaesserella parasuis infection.

Confirmation. A case judged a suspected Glaesserella parasuis infection that is positive in any one of the bacterial isolation and identification in 7.8, the nested PCR detection in 8.6 or the real-time fluorescent PCR detection in 9.5 may be judged a Glaesserella parasuis infection.

A case judged a suspected Glaesserella parasuis infection in an animal that has not been vaccinated against Glaesserella parasuis, and in which antibody is detected as positive by indirect ELISA, may also be judged a Glaesserella parasuis infection.