GB/T 18649-2024Diagnostic techniques for contagious bovine pleuropneumonia (English PDF)

Introduction Introduction

Contagious bovine pleuropneumonia (CBPP) is an infectious disease of bovine animals caused by Mycoplasma mycoides subsp. mycoides (Mmm), also known as cattle lung plague, marked by serous exudative fibrinous inflammation of the interlobular lymphatics of the lung and by serofibrinous pleurisy. The World Organisation for Animal Health lists it as a notifiable animal disease, and the list of class one, two and three animal diseases last revised by China's Ministry of Agriculture and Rural Affairs in 2022 places it among the class one animal diseases.

China developed an effective attenuated vaccine in 1960 and, through wide and dense vaccination combined with slaughter, quarantine inspection and other control measures, saw no further clinical case after 1989 and finally eliminated the disease. In 2011 China received the WOAH certificate of freedom from CBPP and became an internationally recognised CBPP-free country.

The laboratory detection methods of the document, being pathogen isolation, the polymerase chain reaction (PCR), the complement fixation test (CFT) and the competitive enzyme-linked immunosorbent assay (ELISA), are each applied in the different situations set out in Annex A.

1 Scope

The document describes the epidemiology, clinical signs, pathological changes, sample collection, storage and handling of CBPP, together with the diagnostic methods of pathogen isolation and identification, PCR detection, CFT detection and competitive ELISA.

It applies to the diagnosis, quarantine inspection, surveillance and epidemiological investigation of CBPP.

2 Normative references

Three undated references are cited: GB/T 6682 on water for analytical laboratory use, GB 19489 on general requirements for laboratory biosafety, and NY/T 541 on technical specifications for the collection, storage and transport of veterinary diagnostic samples.

3 Terms and definitions

The document states that it has no terms or definitions that need defining.

4 Abbreviations

CBPP, contagious bovine pleuropneumonia; CFT, complement fixation test; DNA, deoxyribonucleic acid; ELISA, enzyme linked immunosorbent assay; Mmm, Mycoplasma mycoides subsp. mycoides; OD value, optical density; PBS, phosphate buffered saline; PCR, polymerase chain reaction.

5 Laboratory biosafety measures

Once cattle are judged to be suspected cases of CBPP infection, the relevant material is sent to a laboratory approved by the agricultural administrative authority for further pathogen isolation and identification. Laboratory diagnosis of CBPP is carried out under GB 19489 and, following the Detailed Rules on Biosafety Requirements for Experimental Activities with Animal Pathogenic Microorganisms, pathogen isolation and identification are to be carried out in a biosafety level three laboratory. Sample collection, storage and transport follow NY/T 541. Water used to prepare the reagents of the document shall meet GB/T 6682. A laboratory that has not obtained the approval of the agricultural administrative authority shall not carry out isolation and culture of the pathogen.

6 Clinical diagnosis

6.1 Epidemiological features. Infected cattle are the chief source of infection. Inhalation of droplets from infected cattle by healthy cattle after direct contact is the chief route of transmission, the droplets being the vehicle. The disease may also be transmitted vertically through the semen and embryos of infected cattle. Under natural conditions yellow cattle, dairy cattle, yaks and dzo are the most susceptible, buffalo are less so; deer and antelope are also fairly susceptible, and goats and sheep may be infected.

6.2 Clinical signs. The incubation period averages 7 d to 28 d, being as short as 7 d and as long as several months. By course the disease is divided into acute, subacute and chronic forms. In the acute form the signs are marked, typical and characteristic: the body temperature rises to 40 °C to 42 °C in a persistent fever; breathing is laboured and shallow, and abdominal; coughing grows frequent; the chest is painful so the animal is unwilling to move or lie down; expiration is long and inspiration short, with the neck stretched out and gasping, often with groaning; there is a serous or purulent nasal discharge; rumination and milk production stop and appetite is lost; the visible mucous membranes are cyanotic and the coat rough and disordered; percussion of the chest may give a dull or flat area on the affected side; auscultation over the affected part gives moist rales, with the vesicular sound weakened or lost and replaced by bronchial breathing and at times a friction sound. In the late stage the heart weakens and the pulse becomes thready and fast, reaching 80 to 120 beats per minute. Death generally follows in 8 d to 10 d, the whole course lasting about 15 d to 30 d. The subacute form resembles the acute but is somewhat milder and runs a rather longer course; when the temperature rises to 40 °C breathing is markedly laboured, the animal stands with its neck stretched and head lowered and moves with difficulty, refuses feed and is weak in spirit, and the prognosis is poor. The chronic form mostly follows from the acute or subacute; the temperature is irregular with transient fever, often resembling bovine tuberculosis; the animal wastes, digestion is disordered and appetite fluctuates. Given good nursing and proper treatment such an animal may recover gradually and become a carrier; where the lesions are extensive it grows weaker by the day and the prognosis is poor.

6.3 Pathological changes. The characteristic changes are chiefly in the lung and the thoracic cavity, the typical change being a marbled appearance of the cut surface of the lung and serofibrinous pleuropneumonia. Early in the course the picture is lobular bronchopneumonia, and at the middle stage a typical serofibrinous pleuropneumonia, mostly on the right side and at times on both. The lesions are most often in the diaphragmatic lobe of the lung, and may also occur in the cardiac or the caudal lobe. The pleura commonly shows haemorrhage and thickening and adheres to the diseased part of the lung. The pulmonary pleura and the pericardium carry fibrinous deposits on their surface.

6.4 Judgement of the result. Where an animal shows some or all of the clinical signs and pathological changes above, it may be judged a suspected case of CBPP. Confirmation calls for further sampling and laboratory diagnosis.

7 Sample collection, storage and handling

7.1 Reagents and materials: medical protective clothing; sterilised scissors and forceps; sterile cotton swabs; disposable syringes of various sizes, sterile collection bags and collection tubes; PBS solution at pH 7.4, prepared under B.1 of Annex B; and PBS solution containing penicillin and streptomycin, prepared under B.2.

7.2 Sample collection. Blood samples: 10 mL of blood is drawn aseptically with a syringe and serum separated after clotting, or anticoagulated blood is collected and plasma separated by centrifugation; anticoagulated blood or plasma may be used for isolation of Mmm and for PCR, and serum for detection of antibody to Mmm. Tissue samples: a block of lung about 2 cm square is collected aseptically from an animal that has died or been killed for examination, taken at the boundary between diseased and normal tissue, together with part or all of the hilar lymph nodes, and placed sealed in a sterile collection bag or other sterilised container for isolation of Mmm and for PCR; 3 mL to 5 mL of thoracic effusion, bronchial lavage fluid, pericardial fluid or, where there are signs of arthritis, joint fluid, is collected aseptically into sterile collection tubes for the same purposes. Nasal swab samples: a sterilised cotton swab is put into the nasal cavity and turned to wipe the mucosa three to five times; once mucus adheres, the swab is placed in a sampling tube holding 2 mL of PBS solution at pH 7.4 containing penicillin and streptomycin, capped and sealed, for isolation of Mmm and for PCR.

7.3 Storage. Samples are placed in an insulated box as soon as possible with dry ice or pre-cooled ice packs, sealed, kept in an unbroken cold chain and sent to the laboratory within 24 h. Samples awaiting examination are handled as soon as possible and held for not more than 24 h at 4 °C, or frozen at -20 °C for not more than 6 months. For long storage they should be held at -70 °C. Repeated freezing and thawing is to be avoided as far as possible.

7.4 Handling. Serum samples are centrifuged at 2 000 r/min for 5 min and the supernatant taken. Tissue samples: about 2 g of the lung or lymph node sample is broken up cold in a tissue grinder, 10 mL of PBS solution containing penicillin and streptomycin added to make a tissue suspension, the suspension shaken and mixed thoroughly and centrifuged at 6 000 r/min for 5 min at 4 °C, and the supernatant taken. Swab samples are mixed on a vortex shaker and centrifuged at 6 000 r/min for 15 min at 4 °C and the supernatant taken. Body fluid samples, being thoracic effusion, bronchial lavage fluid, pericardial fluid and joint fluid, are centrifuged at 6 000 r/min for 5 min at 4 °C and the supernatant taken.

8 Pathogen isolation and identification

8.1 Equipment: a CO2 incubator; tissue homogenising equipment; a refrigerated bench-top high speed centrifuge; a class II biological safety cabinet; an optical microscope; and an autoclave.

8.2 Reagents and materials: Martin broth prepared under B.3; Martin broth agar prepared under B.4; sterilised scissors, cotton swabs and syringes; and adjustable micropipettes with matching tips.

8.3 Dilution of the sample. Under sterile conditions 0.5 mL of the liquid-handled sample is drawn and inoculated into 4.5 mL of Martin broth medium, marked as the 10 to the minus one tube, and a ten-fold dilution series continued to the 10 to the minus three tube. The clause cites the handled sample as coming from 7.5, a subclause that does not exist in the document.

8.4 Inoculation of the sample. From each of the three dilution steps, 0.5 mL is inoculated into 4.5 mL of Martin broth medium and 0.2 mL onto Martin broth agar plates; these are held in a CO2 incubator at 37 °C +/- 0.5 °C with 5 % carbon dioxide by volume and colony growth observed daily.

8.5 Cloning and purification. The inoculated material is observed for 7 d in succession, mycoplasma growth being checked daily. Where fried-egg colonies appear on the Martin broth agar plate and mycoplasma growth is suspected, a single colony together with its block of agar is cut out and inverted onto the agar surface of a fresh plate and the block moved gently to clone and purify it; after two successive rounds of cloning and purification the culture of the single colony is identified under clause 9. Where the Martin broth turns yellow and slightly turbid, 0.2 mL is inoculated onto a plate and incubation continued, and cloning and purification carried out under 8.5.2 once fried-egg colonies appear. Where after 7 d neither the liquid medium nor the solid plate shows suspected growth of Mmm, the Martin broth agar plate is incubated on to 21 d and discarded if there is still no suspected growth, and 0.5 mL of the liquid culture is subcultured blind into 4.5 mL of fresh liquid medium at most twice and observed and handled under 8.5.2, being discarded if there is still no growth. Where bacterial contamination appears during culture, the culture fluid is filtered through a microporous membrane of 0.45 µm pore size and 0.5 mL of the filtrate inoculated and observed under 8.4.

8.6 Judgement of the result. The cloned and purified culture is further tested by PCR, the result being judged under 9.4.2.

9 PCR detection

9.1 Equipment: a PCR instrument; a refrigerated bench-top high speed centrifuge; a mini centrifuge; and a vortex mixer.

9.2 Reagents and materials: a commercial genomic DNA extraction kit; the primers of C.1 in Annex C; thin-walled PCR tubes or eight-tube strips; 2 x PCR reaction premix containing Taq polymerase and dye; the positive control, plasmid pUC-TM, prepared as described in C.2; the negative control, nuclease-free water; a DNA molecular mass marker; and 1 % agarose gel prepared under B.5.

9.3 Procedure. Genomic DNA is extracted from the nasal swab, blood, tissue and body fluid samples with a commercial genomic DNA extraction kit, the steps following the kit instructions; here too the clause cites the samples as coming from 7.5, a subclause that does not exist in the document. The reaction tubes and the negative and positive control tubes are labelled and the reagents added in turn according to the reaction system of Table 1. Table 1 lists six components, being 2 x PCR reaction premix, 10 µmol/mL primer 1, 10 µmol/mL primer 2, template DNA, nuclease-free water and the total volume, against a column of volumes in µL; the volumes listed do not add up to the stated total, so they are not reproduced here. 2 µL of sample DNA template is added to the prepared reaction system, mixed thoroughly, and amplification carried out. The amplification programme is pre-denaturation at 95 °C for 3 min; 35 cycles of 94 °C for 45 s, 57 °C for 45 s and 72 °C for 1 min; and extension at 72 °C for 10 min. The products are examined by electrophoresis on 1 % agarose gel.

9.4 Judgement of the result. The test is valid where the negative control gives no band at all and the positive control gives a fragment of about 300 bp; where the positive control gives no fragment of the target size, or the negative control gives the target fragment, the PCR is void. A sample is judged Mmm nucleic acid positive where it gives a single specific band of about 300 bp matching the molecular mass of the positive control band, and Mmm nucleic acid negative where it gives no specific band.

10 CFT detection

10.1 Equipment: an electronic balance; a water bath; a vortex shaker; a bench-top centrifuge; and a constant temperature incubator.

10.2 Reagents and materials: PBS solution at pH 7.4 prepared under B.1; positive control serum and negative control serum, which may be supplied by a specialist CBPP laboratory; CBPP complement fixation test antigen, which may be supplied by a specialist CBPP laboratory, its titration being given in D.1 of Annex D; commercial haemolysin, its titration given in D.2; sensitised sheep red blood cells prepared as in D.3; and commercial complement, stored in a freezer at -20 °C for generally not more than 30 d, its titration given in D.4.

10.3 Procedure. The test serum and the positive and negative control sera are all diluted 1 in 10 with PBS solution at pH 7.4 and inactivated in a water bath at 56 °C for 30 min. The test then follows the procedure of Table 2, with an anticomplementary control, a positive serum control, a negative serum control, a haemolysin control and a red blood cell control set up alongside. Table 2, headed as a microtitre method, lists the components antigen, serum, complement and sensitised sheep red blood cells in µL against the several controls and the test serum, with the mixing and incubation steps at 37 °C for 30 min and a 10 min tilted standing before reading; in the extracted text the component labels and the volume figures are separated from one another and cannot be paired to the columns with certainty, so the volumes are not reproduced here. A note to the table gives the symbols: a solidus means the reagent was not added; four plus signs mean all the red cells have settled; three plus signs mean 75 % have settled; two plus signs mean 50 % have settled; one plus sign means 25 % have settled; and a minus sign means the red cells are wholly haemolysed.

10.4 Judgement of the result. The test stands where the controls give the following results, failing which it is repeated: the anticomplementary control, red cells wholly haemolysed; the positive serum control, red cells wholly settled; the negative serum control, red cells wholly haemolysed; the haemolysin control, red cells wholly haemolysed; and the sheep red blood cell control, red cells wholly settled. A test serum is judged positive where its red cells have wholly settled, and suspect where 25 % to 75 % have settled; a suspect sample calls for fresh serum and a repeat test, and where the repeat is still suspect, pathological and pathogenic examination is to be carried out. A test serum is judged negative where its red cells are wholly haemolysed.

11 Competitive ELISA

11.1 Equipment: a microplate reader; a constant temperature incubator; a shaker; and a plate washer.

11.2 Reagents and materials: PBS solution at pH 7.4; PBST wash buffer prepared under B.6; serum diluent and enzyme-labelled antibody diluent, together called the diluent, prepared under B.7; positive and negative control sera, which may be supplied by a specialist CBPP laboratory; goat anti-mouse IgG horseradish peroxidase conjugate, called the enzyme-labelled antibody, a commercial reagent; TMB substrate solution, a commercial reagent; stop solution prepared under B.8; the ELISA antigen-coated plate, which may be supplied by a specialist CBPP laboratory, being a 96-well ELISA plate coated with Mmm membrane protein and blocked; and an ELISA reagent trough.

11.3 Procedure. All reagents are brought to 18 °C to 26 °C before use, shaken gently or vortexed to mix, a separate tip used for each sample, and the controls may be placed anywhere on the microplate. The monoclonal antibody detection solution is made by diluting the monoclonal antibody 500-fold in the diluent; the standard gives as an example that one plate needs 12 mL, made by adding 24 µL of monoclonal antibody to 11.976 mL of diluent. An unused 96-well plate is taken and the diluent, controls, serum samples and monoclonal antibody detection solution added in the volumes shown in Table 3. Table 3 sets out the pre-dilution scheme for the positive serum, the negative serum, the monoclonal antibody control, the blank control and the test serum, in columns of diluent, serum and monoclonal antibody detection solution in µL, each row totalling 120 µL. From each well of the pre-reaction plate 100 µL is transferred to the corresponding well of the ELISA antigen-coated plate, which is covered with sealing film and incubated at 37 °C for 1 h. The liquid is discarded, 250 µL of PBST wash buffer added per well, left 3 min and discarded, the wash repeated four times and the plate finally patted dry on absorbent paper. 100 µL of enzyme-labelled second antibody is added per well and the plate incubated at 37 °C for 45 min, then washed again as before. 100 µL of TMB substrate solution is added per well and the plate incubated at 37 °C in the dark for 10 min. 100 µL of stop solution is added per well, mixed by gentle shaking, and the OD value read in the microplate reader at a wavelength of 450 nm; the reading is to be completed within 5 min of adding the stop solution.

11.4 Judgement of the result. The inhibition percentage of a sample is calculated by formula (1), whose legend gives IPS as the inhibition percentage of the test serum sample, OD450-Mab as the OD value of the monoclonal antibody control at 450 nm, OD450-CC as the OD value of the second antibody control at 450 nm, and OD450-S as the OD value of the test serum sample at 450 nm. The test stands where the controls give the following results, failing which it is repeated: the mean OD 450 of the Mab control lies between 0.2 and 2.0; the inhibition percentage of the positive control sample lies between 55 % and 110 %; and the inhibition percentage of the negative control sample is below 40 %. A sample with an inhibition percentage of 50 % or more is judged positive for antibody to Mmm, and one below 50 % negative.

12 Overall determination

A preliminary diagnosis may be made from the clinical signs and pathological changes; where some or all of those in 6.2 and 6.3 are met, the case may be judged suspected CBPP. An animal without clinical signs that tests positive by any one of the methods of clause 9, clause 10 and clause 11 is judged suspected CBPP. An animal without clinical signs that tests positive by clause 8 may be judged CBPP. An animal judged suspect on clinical diagnosis that also tests positive by any one of the methods of clause 9, clause 10 and clause 11 may be judged CBPP.

Annex A Situations in which the laboratory detection methods for CBPP are applied (informative)

Table A.1 sets out the applicability of the laboratory detection methods against the situations in which they are used. The method rows are grouped as pathogen detection and identification, covering pathogen isolation and identification and PCR, and serological detection, covering CFT and competitive ELISA; the situation columns are quarantine inspection, confirmation of a clinical case, epidemiological surveillance and eradication. A note gives the symbols: three plus signs mean the method is recommended; two plus signs mean it suits a very limited set of circumstances; one plus sign means it is recommended but has limitations; and a minus sign means it does not suit that purpose. In the extracted text the method labels, the situation labels and the symbol strings are printed apart from one another and cannot be paired into cells with certainty, so the matrix is not reproduced here.

Annex B Preparation of reagents (normative)

B.1 PBS solution, 0.01 mol/L, pH 7.4: 8 g NaCl, 3.6 g Na2HPO4 12H2O, 0.27 g KH2PO4 and 0.2 g KCl are weighed out and dissolved in 900 mL of double distilled water; once fully dissolved the pH is adjusted to 7.4 with 1 mol/L HCl and the volume made up to 1 L with double distilled water. The solution is sterilised by filtration through a 0.22 µm filter or autoclaved at 121 kPa for 30 min, and kept at room temperature, 20 °C +/- 5 °C.

B.2 PBS solution containing penicillin and streptomycin: 1 000 000 U of penicillin and 1 000 000 µg of streptomycin are made up with sterile deionised water to 10 mL of stock solution of the two antibiotics, at 100 000 IU/mL and 100 000 µg/mL respectively; the stock is dispensed into small bottles and kept at -20 °C. The penicillin and streptomycin stock is added to PBS solution at pH 7.4 to give penicillin and streptomycin contents of 2 000 U/mL and 2 mg/mL respectively.

B.3 Martin broth, pH 7.8 to 8.0: commercial Martin broth medium is dispensed into sterilised tubes at 4.5 mL per tube and 0.5 mL of sterile horse serum added.

B.4 Martin broth agar: 1.3 % to 1.5 % agar powder is added to commercial Martin broth medium and autoclaved at 121 kPa for 30 min to give Martin agar; when it has cooled to 55 °C to 60 °C, 10 mL is taken, 1 mL of sterile horse serum added and mixed evenly by gentle shaking, and the mixture left to set, giving Martin agar medium.

B.5 1 % agarose gel: 1 g of agarose is put into 100 mL of 1 x TAE electrophoresis buffer and melted with heat; when the temperature has fallen to about 60 °C a suitable nucleic acid stain is added and the gel poured evenly to a thickness of 3 mm to 5 mm.

B.6 PBST wash buffer: 1 mL of Tween 20 is added to 1 L of PBS and mixed thoroughly. It is made up fresh for use, being used within 7 d of preparation, and kept at room temperature, 20 °C +/- 5 °C.

B.7 Serum diluent and enzyme-labelled antibody diluent: 0.5 mL of healthy horse serum is added to 99.5 mL of PBS and mixed, for diluting serum and enzyme-labelled antibody.

B.8 Stop solution, 2 mol/L sulfuric acid: 109 mL of sulfuric acid at 18.4 mol/L is dropped slowly into 800 mL of double distilled water; once cool the volume is made up to 1 L with double distilled water and the solution kept at room temperature, 20 °C +/- 5 °C.

Annex C PCR primer sequences and construction of the positive control plasmid (informative)

C.1 PCR primer sequences. SC1: ATATACTTCTGTTCTAGTAATATG. SC2: CTGATTATGATGACAGTGGTCA.

C.2 Construction and identification of the positive control plasmid. With the primers listed in C.1, the genomic DNA of Mmm strain Ben-1 is amplified by PCR, the amplification product recovered with a commercial gel recovery kit and ligated into the pUC-57 vector, and the ligation transformed into DH5 alpha competent Escherichia coli cells to give the recombinant. The plasmid is extracted with a plasmid extraction kit and identified by PCR and sequence analysis as the recombinant positive plasmid pUC-TM. The plasmid and the positive bacteria carrying the recombinant are kept at -20 °C and -80 °C respectively.

Annex D Titration of complement fixation test reagents (informative)

D.1 Titration of the CBPP complement fixation test antigen. The antigen is diluted with PBS solution at pH 7.4 in two-fold steps from 1 in 10 to 1 in 640, and the positive serum in two-fold steps from 1 in 10 to 1 in 1 280. The antigen is titrated in a chequerboard on a 96-well microtitre plate as set out in Table D.1: to each well are added 25 µL of antigen at the corresponding dilution, 25 µL of positive serum at the corresponding dilution and 25 µL of complement at 2.5 units; the plate is shaken to mix and held in a water bath at 37 °C for 30 min; 25 µL of sensitised sheep red blood cells is added per well, the plate shaken to mix and held again at 37 °C for 30 min, then centrifuged at 1 000 r/min for 2 min and read. A negative serum control at each antigen dilution, a sensitised red cell control and an anticomplementary control are set up at the same time. Table D.1 is an example of the titration result, giving a grid of plus and minus symbols against antigen dilutions from 1 in 10 to 1 in 640 and serum dilutions from 1 in 10 to 1 in 1 280 plus three control rows; in the extracted text the symbol strings cannot be aligned to the grid with certainty, so the grid is not reproduced here. Where all the controls stand, the antigen titre is the highest antigen dilution corresponding to the highest dilution of the positive control serum that fixes complement fully, shown by four plus signs. The worked example in the standard states that where the antigen-antibody complex formed by positive control serum diluted 1 in 160 and antigen diluted 1 in 80 fixes complement fully, the antigen titre is 1 in 80, and that two units are used in testing, the antigen being diluted 80 divided by 2, that is 40-fold.

D.2 Titration of haemolysin. 0.1 mL of haemolysin is diluted in ten-fold steps to 1 in 1 000 to serve as the base solution, by the dilution method of Table D.2, and D.3 then followed. Table D.2 gives, for eight tubes, the dilution, the volume of haemolysin base solution in mL, the volume of PBS in mL and the total: tube 1, 1 in 2 000, 1.0 and 1.0 giving 2.0; tube 2, 1 in 3 000, 1.0 and 2.0 giving 3.0; tube 3, 1 in 4 000, 1.0 and 3.0 giving 4.0; tube 4, 1 in 5 000, 1.0 and 4.0 giving 5.0; tube 5, 1 in 6 000, 1.0 and 5.0 giving 6.0; tube 6, 1 in 8 000, 1.0 and 7.0 giving 8.0; tube 7, 1 in 10 000, 1.0 and 9.0 giving 10.0; and tube 8, 1 in 15 000, 1.0 and 14.0 giving 15.0. Table D.3 records the haemolysin titration itself, with columns for the tube number, the haemolysin dilution, the volume of haemolysin, the volume of 1 in 20 complement, the volume of 6 % sheep red blood cells and the volume of PBS in mL, and a result column read after shaking and 30 min in a water bath at 37 °C, together with complement, 1 in 100 haemolysin and red cell control rows; in the extracted text the numeric columns of Table D.3 run together and cannot be split into fields with certainty, so its values are not reproduced here. The unit of haemolysis is the highest dilution of haemolysin that gives complete haemolysis; the standard states that in Table D.3 this is 5 000-fold, and that since two units of haemolysin are used in the test proper, the haemolysin in that example is diluted 5 000 divided by 2, that is 2 500-fold.

D.3 Preparation of sensitised sheep red blood cells. Venous blood is collected aseptically from a healthy ram, defibrinated, suspended in PBS solution at pH 7.4 and washed three times at 2 000 r/min, 10 min each time. The red cell deposit is collected and made up to a 6 % suspension with PBS solution at pH 7.4 and used after standing 48 h at 4 °C; the cell suspension can be kept at 4 °C for at most 21, the unit of that figure being missing in the original text. Two units of haemolysin are mixed with an equal volume of the 6 % sheep red blood cells, shaken once every 5 min, held in a water bath at 37 °C for 30 min and then kept at room temperature for use.

D.4 Titration of complement. Complement is diluted with PBS solution at pH 7.4 as set out in Tables D.4 and D.5. Table D.4, headed complement dilution I, gives six tubes with the volume of PBS in mL, the volume of complement in mL and the dilution: 1.45 and 0.05 giving 1 in 30; 1.70 and 0.05 giving 1 in 35; 1.95 and 0.05 giving 1 in 40; 2.20 and 0.05 giving 1 in 45; 2.45 and 0.05 giving 1 in 50; and 2.70 and 0.05 giving 1 in 55. Table D.5, headed complement dilution II, gives six tubes with the complement dilution, 0.5 mL of PBS and 0.5 mL of complement in each, and the final complement dilutions 1 in 60, 1 in 70, 1 in 80, 1 in 90, 1 in 100 and 1 in 110. For the titration, 25 µL of complement at each dilution from 1 in 30 to 1 in 110 is placed in a 1.5 mL centrifuge tube, 25 µL of PBS solution at pH 7.4 added to each tube and then 25 µL of sensitised sheep red blood cells; the tubes are shaken to mix, held in a water bath at 37 °C for 30 min, centrifuged at 1 000 r/min for 2 min and read. The complement titre is the highest dilution of complement that haemolyses the sheep red blood cells completely, and 2.5 units of complement are used in testing; the standard's worked example states that where complement at 1 in 60 haemolyses the sheep red blood cells completely, the complement titre is 60 and the complement is used at a dilution of 60 divided by 2.5, that is 24-fold.