S.O.P.7: THE TEST FOR STREPTOMYCIN RESISTANCE FOR IDENTIFICATION OF STREPTOMYCIN RESISTANT VARIANTS OF M. MYCOIDES subsp.MYCOIDES.

Two strains of M. mycoides subsp. mycoides have been used for the production of live attenuated vaccines for CBPP in Africa. These are T₁/44 and KH₃J; each of which has a streptomycin resistant variant namely T₁-SR and KH₃J-SR respectively. The use of strain KH₃J and KH₃J-SR has now been abandoned due to its poor immunogenicity. Hence T₁/44 and T₁-SR are the only seed strains currently used for the manufacture of CBPP vaccines in Africa.

I. Materials

• Complete Counting or Gourlay medium containing Streptomycin at a final concentration of 1 mg/ml
• CBPP vaccine
• Sterile distilled water

II. Test Procedure

All batches of T₁SR-based CBPP vaccines should be tested for streptomycin resistance.

1. Dispense 2.5 ml complete Counting or Gourlay medium containing 1 mg/ml streptomycin into 5 ml screw capped tubes or Bijoux bottles.

2. Reconstitute the contents of a vial of CBPP vaccine with a volume of cold, sterile distilled water which is equal to the filling volume of the vaccine.

3. Inoculate 250 μl of reconstituted vaccine suspension into each of two tubes of the test medium. Inoculate 250 μl of the reconstitution fluid into two tubes of test medium.

4. Incubate all inoculated tubes at 37°C for 10 days.

5. Visually observe and record changes in the colour of the incubated media on day 3, 6 and finally on day 10.

III. Interpretation of the result

The vaccine batch passes the test for streptomycin resistance identity if the colour of the medium in the tubes inoculated with the vaccine suspension changes from pink to yellow and the colour of the medium inoculated with the reconstitution fluid remains pink. If the colour the medium inoculated with the reconstitution fluid changes the test is not valid and must be repeated.