CONTAGIOUS BOVINE PLEUROPNEUMONIA (CBPP) LIVE ATTENUATED VACCINE

S.O.P.1: FORMULATION OF MEDIA AND SOLUTIONS

The preparation of media and solutions should be separated from the other laboratory activities. For this purpose a laboratory has to be set aside and used exclusively for preparation and testing of such media and solutions. Such a laboratory should have its own dedicated equipment and the operations therein carried out under aseptic conditions. Within such a laboratory storage areas should be provided for materials that have already undergone and passed all quality control testing and other areas for those substances that are still undergoing tests. Clear and accurate records of all quality control tests carried out and the results must be maintained. All materials must be clearly labelled indicating nature of the substance, date prepared and batch number.

I. EQUIPMENT

• Class II Laminar air flow cabinet

• Incubator set at 37 +/- 0.5°C

• Autoclave

• Precision balance, spatulas and weighing boats

• pH meter, range 0 to 14 pH with accuracy of +/- 0.05 pH units

• Magnetic stirrer

• Seitz or membrane filter assembly, EKS 2 filter pads or membrane filter discs (pore size 0.2μm)

• Vacuum or Peristaltic pump

• Water bath

• Water still machine or Water purifier

• Inverted microscope or Binocular dissection microscope

• Microplate viewer/Reading mirror

• Bench centrifuge

• Refrigerator

• Freezer -20°C

• Pipette aid/Controller

• ELISA plate reader and accessories

• Pipetters, Range 10–40 μl; 40–200 μl and 200–1000 μl

• Multichannel pipetter, range 40–200 μl

• Gel punchers (stainless steel), diameters 3.5, 4 and 5 mm

• Gel punching grid

• Anaerobic system (Oxoid or Difco) or Candle jar

• Dessicator or Moisture Analyser(e.g. Sartorius MA 40)

• Eppedorf pipette model and tips

II. Media for Growth, Titration and Characterisation

A. PANVAC Counting Medium

1. Counting Medium-Base

   Heart Infusion Broth (Difco)	25 g
   or
   Brain Heart Infusion	37 g
   Yeast Extract (Oxoid or Difco)	5 g
   Glucose	4 g
   Phenol red	0.02 g
   Distilled or purified water	890 ml

   Dissolve the ingredients thoroughly by magnetic stirring. Adjust pH to 7.8 using 1M NaOH then sterilize by filtration through a membrane filter (pore size 0.2μm) or through EKS 2 Seitz filter pads. Test for sterility and store in a refrigerator at 4°C.

2. Complete Counting Medium^*

   Base Counting Medium (sterile)	88 ml
   Horse Serum (sterile)	10 ml
   DNA Stock Solution (sterile)	1 ml
   Penicillin Stock Solution	1 ml
   Streptomycin Stock Solution	0.5 ml
   (only for streptomycin resistant strains)

3. Complete Counting Medium × 2^*

   Base Counting Medium	76 ml
   Horse serum	20 ml
   DNA Stock Solution	2 ml
   Penicillin Stock Solution	2 ml
   Streptomycin Stock Solution	1 ml
   (only for streptomycin resistant strains)

^* Test for sterility and keep in a refrigerator at 4°C. Use completed medium within 30 days.

B. Gourlay Medium

(Gourlay medium may be used as alternative for titration or vaccine production)

Dissolve the ingredients by stirring on a hot plate. Adjust pH to 7.6 using 1M NaOH then sterilize by filtration through EKS 2 Seitz filter pads or through membrane filter of pore size 0.2 μm. Add:

Test for sterility, aliquot in 100 ml amounts and store at 4°C. Use medium within 14 days.

Ref: Brown, R.D, Gourlay, R.N., and MacLeod, A.K. (1965).
The production of T₁ broth culture contagious bovine pleuropneumonia vaccine.
Bull. Epizoot. Afr., 13, 149–155

C. Medium F66

Medium F66 has been recommended for bulk vaccine production because it is relatively cheap and easy to prepare.

1. Preparation of Heart Infusion

   Bovine Heart Infusion is obtained using either of the procedures outlined below:

   1. Procedure I

      • Remove fat from 5 kg of bovine heart.
      • Chop (macerate) the defatted heart muscle into small pieces.
      • To the chopped pieces add 10 litres of distilled water.
      • Heat the heart muscle-water mixture to 50°C and then hold at this temperature for 1 hour.
      • Raise the temperature of the mixture to boiling point.
      • While hot, clarify the suspension by passing it through a coarse filter paper.

   2. Procedure II

      An alternative method of preparing bovine heart infusion which has been used by some laboratories with good results is via the use of the enzyme Papain to aid in the extraction of the infusion. Briefly:

      • To 5 kg of defatted and chopped bovine heart add 10 L distilled water.

      • Add 50 g Papain (Difco)

        .

        Agitate the mixture by magnetic stirring while raising its temperature as described below:

      • Initially raise temperature of mixture to 50–60°C then maintain at that point for 15 minutes. Heat to 60–70°C then hold for 15 minutes. Heat to 70–80°C then maintain at that temperature for 15 minutes. Finally heat to 80–85°C and hold for 15 minutes.

      • Clarify the infusion as described for procedure I.

2. Preparation of Medium F66

   To prepare medium F66, heart infusion and the other medium ingredients are mixed and processed as outlined below:

   Bovine heart infusion	900 ml
   Bacto-tryptose (Difco)	10 g
   Glucose	2 g
   Na2HPO4 (Anhydrous)	2.5 g

   Dissolve by heating to 80°C cool to about 50°C then add:

   Yeast extract (25% w/v)	50 ml
   Glycerol	0.3 g
   Oleic acid	0.015 g
   Palmitic acid	0.010 g
   Penicillin	1 000 000 IU
   Streptomycin	100 mg
   (only for streptomycin resistant strains)
   Horse serum	100 ml

   Adjust pH to 7.6 using 1M NaOH then sterilize by filtration through EKS 2 Seitz filter pads. Test for sterility then store at 4°C. Use medium within 14 days.

   Ref: Provost, A., Borredon, C., and Queval, R. (1970) Recherches immunologiques sur la pèripneumonie VI. Un vaccin vivant mixte antibovipestique-antiperi-pneumonique inocute en un seul temps. Conception, Production, contrôles. Rev. Elev. Mèd. Vèt. Pays trop. 23(2): 143–162.

   NB: In the preparation of Complete Counting, Gourlay or F66 media, Horse, Donkey, or Pig serum may be used depending on local availability and/or price. Frequent thawing and freezing of serum leads to deterioration of its quality hence sterile bulk serum batches are best aliquoted in 100 ml amounts and then stored frozen at -20°C so that only the amount required at any one given time can be thawed out.

D. Mycoplasma Agar Medium for Growth Inhibition, Digitonin Sensitivity and Colony Characterisation

1. Agar Medium Base

   Mycoplasma broth base (Oxoid)	2.55 g
   or
   Brain heart infusion broth (Difco)	3.7 g
   D-glucose	0.1 g
   Agar No. 1 (Oxoid)	0.9 g

   • Add brain heart infusion (or Mycoplasma broth base), glucose and agar to 85 ml of distilled water.
   • Stir the mixture using a magnetic stirrer and adjust pH if necessary to 7.6 using 1M NaOH.
   • Autoclave at 121°C, Pressure 15 lb/sq.inch (1.06 bar) for 15 minutes. Remove from autoclave while still hot and place in a water bath at 50–55°C.

2. Complete Agar Medium

   • Add 10 ml horse serum, 5 ml of a 25% w/v yeast extract and 1 ml of penicillin stock solution in a separate container and place the container in the water bath at 50–55°C.
   • Allow all media components to equilibrate at 50–55°C.
   • In a laminar air flow work station add the horse serum, yeast extract, and penicillin into the 84 ml of the molten agar base medium. Swirl the complete medium to ensure homogeneous mixing of the medium components.
   • Dispense 5 ml of medium into each 60 × 15 mm Petri dish. Allow the agar medium to set, wrap plates in aluminium foil to avoid drying and store at +4°C till needed. Use prepared agar medium plates within 14 days.

   NB: Dispensing should be done uniformly to avoid formation of air bubbles but quickly because agar solidifies at 45°C.

E. Preparation of Agar Gel for the Test of Identity of M. mycoides subsp. mycoides by Agar Gel Immunodiffusion (AGID)

1. Add 1 g of Noble agar powder (Difco) to 100 ml PBS containing 0.1% sodium azide in a bottle. Dissolve by autoclaving (110°C for 10 minutes), or in a waterbath (100°C). When the agar is completely dissolved it should be clear and without any trace of cloudiness.

   NB: Sodium azide is poisonous! Care must be taken when handling it to avoid inhalation, ingestion or skin contact. Sodium azide can also form explosive compounds with metals hence material containing azide should NOT be disposed of through metal sinks.

2. Add 3 g of polyethylene glycol (PEG) molecular weight 6000, and allow the mixture to dissolve completely by heating in a water bath while shaking intermittently.

3. The molten agar can be used directly to prepare agar gel immunodiffusion plates or it can be dispensed in 20 ml volumes into glass universal bottles, capped and stored at 4°C until needed. Agar prepared and stored this way can be used for up to 6 months.

   NB: Bottles containing agar should NOT be frozen.

F. Media for Biochemical Characterisation

1. Glucose Fermentation Medium, pH 7.8

   Heart Infusion Broth (Difco), 2.5% w/v solution	180 ml
   Horse Serum (inactivated)	40 ml
   Yeast Extract (25% w/v)	20 ml
   DNA Stock Solution (0.2% w/v)	2.6 ml
   Glucose (50% w/v solution)	2 ml
   Phenol red (1% w/v solution)	5 ml

   Dispense into bijoux bottles at 2.25 ml per tube and store at 4°C. Use dispensed medium within 30 days.

2. Arginine Hydrolysis Medium, pH 7.3

   Heart Infusion Broth(Difco, 2.5% w/v solution)	180 ml
   Horse serum	40 ml
   Yeast Extract (25% w/v suspension)	20 ml
   DNA Stock Solution (0.2% w/v)	2.6 ml
   L-Arginine monohydrochloride (30% w/v solution)	8.5 ml
   Phenol red (1% w/v solution)	5 ml

   Dispense 2.25 ml into bijoux bottles and store 4°C. Use dispensed medium within 30 days.

3. Medium for Testing Reduction of Tetrazolium, pH 7.5

   Heart Infusion Broth	180 ml
   Horse serum	40 ml
   Yeast Extract (25% w/v suspension)	20 ml
   DNA Stock Solution (0.2% w/v)	2.6 ml
   Triphenyltetrazolium chloride (2% w/v solution)	5 ml

   Dispense 2.25 ml of medium into bijoux bottles and store at 4°C until needed. Use prepared medium within 30 days.

4. Medium for Testing Phosphatase Activity, pH 7.8

   Agar Base Medium	74 ml
   Horse Serum (inactivated at 60°C)	20 ml
   25% w/v Yeast Extract	5 ml
   Penicillin (200 000 IU/ml)	0.2 ml
   Phenolphthaleine sodium
   diphosphate (1% w/v solution)	1 ml

   NB: The serum supplement of the phosphate activity test medium should be decomplemented (inactivated) in order to eliminate the activity of serum phosphatase. This is achieved by heating the serum at 60°C for at least 30 minutes.

5. Medium for Liquefaction of Coagulated Serum

   Horse Serum	80 ml
   Counting Medium Base	20 ml

   Sterilize by filtration (if necessary) and then dispense medium into test tubes at 5 ml per tube. Place at an inclined position in a waterbath and heat at 85°C for 30 minutes to coagulate the serum. Store medium tubes at 4°C overnight. Decant off any condensation fluid in the tubes that may have formed during storage before performing the test for serum liquefaction. Prepared media tubes are used within 30 days.

G. Yeast Extract

1. Yeast extract

   25% Yeast extract suspension is prepared by adding 25 g of Oxoid or Difco yeast extract powder into 100 ml of distilled water and dissolved by magnetic stirring. This is then sterilized by passing the suspension through a membrane filter of pore size 0.2 μm. The filtrate is tested for sterility then aliquoted in 20 ml quantities into universal bottles and then stored frozen at -20°C until needed. Yeast extract stored at -20°C may be used for up to one year.

2. Weybridge Yeast Extract

   This is prepared by suspending 50 g of baker's yeast in 100 ml of 0.2M KH₂PO₄ and heated at 80°C for 20 minutes. It is then clarified by centrifugation at 2000 rpm for 20 minutes and then sterilized by filtration through Seitz EKS 2 filter pads and then tested for sterility. Prepared yeast extract is dispensed and stored as described for 1. above.

H. DNA Stock Solution (0.2% w/v)

Place the DNA container at 37°C for 1 to 2 hours while shaking intermittently to ensure complete dissolution. Sterilize by filtration through a membrane filter of pore size 0.2 μm. Test for sterility, dispense in 1 ml aliquots and store at -20°C till needed. The DNA solution stored at -20°C can be used for up to 1 year.

I. Antibiotic Stock Solutions

1. Penicillin stock solution

   Penicillin G (benzylpenicillin)	106 IU
   Sterile distilled water	20 ml

2. Streptomysin Stock Solution

   Streptomycin sulphate	1 g
   Sterile distilled water	50 ml

   Dissolve the content of the vial of the antibiotic in the stated volume of sterile distilled water. Sterilize by filtration through a membrane filter of pore size 0.2 μm. Dispense 1 ml aliquots into sterile containers and store at -20°C. Use antibiotic solution within 1 year.

J. Phosphate Buffered Saline (PBS), pH 7.2

Dissolve the ingredients by magnetic stirring. Adjust pH to 7.2 using 1M NaOH. Make up to 1000 ml with distilled water and sterilize by autoclaving for 15 minutes at 15 lb/sq.in. or 1.6 bar. If at the end of the sterilisation process evaporation has occured, make up to the 1000 ml mark by adding sterile distilled water.

K. Preparation of Digitonin Sensitivity Discs

1. Add 0.15 g Digitonin to 10 ml absolute ethanol. Shake thoroughly to give a homogeneous suspension.
2. Sterilize by filtration through a syringe membrane filter of pore size 0.2 μm.
3. In a laminar air flow work station, place sterile antibiotic discs (size 6 mm diameter) in a sterile Petri dish and add 30 μl of the digitonin suspension on to each disc. Seal the Petri dish with an adhesive tape.

   NB: While dispensing the digitonin suspension should be shaken continuously to ensure its homogeneity.

4. Dry the discs by placing the Petri dish container in an incubator at 37°C overnight.
5. In a laminar air flow cabinet aseptically transfer the discs into a sterile screw-capped universal container and store at 4°C until needed. The digitonin discs stored at 4°C may be used for up to one year.