The test for the determination of viable Mycoplasma content of CBPP vaccines should be carried out on at least three vials of each freeze-dried or three bottles of each liquid vaccine batch.

The Test should be performed in parallel with a control vaccine preparation. For each test result to be valid the tire of the control vaccine preparation should not exceed 2 times the standard deviation of the calculated reference titre. Each titration shall be based on the use of ten-fold dilution series of the reconstituted vaccine and not less than 10 wells per dilution.

**NB:** *The minimum titre per (cattle) field dose should be at least 10 ^{7} viable mycoplasmas.
However taking into account the local storage and transportation conditions it is
recommended that production laboratories should endeavour to deliver vaccine with at
least 10^{8} mycoplasmas per dose.*

**I. Equipment**

- Sterile 96-well microtitration plates (Flat or U-bottomed)
- Plate sealer
- Glass of plastic bijoux bottltes (7 ml)
- Universal containers(30 ml)
- Pipetters. range 40 to 200 μl and 200 to 1000 μl
- Eppendorf multipette
^{(R)}4780 or other type of suitable repetitive pipette - Combitips, capacity: 2.5 ml, range 50–250 μl
- Microplate reader (viewer)

**II. Materials**

CBPP vaccine (freeze-dried or liquid)

Water, pyrogen free, high purity, ionic strength less than 2.0 Siemens

Counting Medium Base

DNA Stock Solution (0.2%)

Penicillin Stock Solution

Streptomycin Stock Solution

Complete Counting Medium x 2

**NB:***In case of testing streptomycin resistant strains (i.e. T*_{1}–SR or KH_{3}J–SR) streptomycin stock solution should be added to the complete counting medium to a final concentration of 0.1 mg/ml.Disinfectants (e.g. Hypochlorite, Quadralene 1000 or 2000 solutions. 70% Ethanol etc)

**III. Procedure**

All operations from step 1 to 3 should be performed using aseptic techniques in a Class II Laminar Air Flow Cabinet. All wastes generated in these operations should be thoroughly disinfected chemically or autoclaved before disposal. All equipment and containers used should be thoroughly disinfected and/or autoclaved before re-use.

Reconstitute the content of a vial of freeze dried vaccine in 10 ml of cold counting medium base. Shake thoroughly to ensure complete and uniform suspension of the vaccine.

Prepare 10–fold dilution series from 10

^{-10}to 10^{-10}of the reconstituted freeze dried vaccine or of the undiluted liquid vaccine in bijoux bottles containing 4.5 ml of counting medium base as follows:-Transfer 0.5 ml of vaccine suspension into the first bijoux bottle marked 10

^{-1}without touching the diluent meniscus. Discard the pipette. Mix the content of this bottle thoroughly by shaking vigorously. Using a different pipette, transfer 0.5 ml of the mixture into a second bijoux bottle marked 10^{-2}again without touching the meniscus of the diluent. Repeat this sequence of transfer and mixing until the tenth bijoux bottle marked^{-10}, each time using a fresh pipette for transfer.Dispense 100 μl of complete counting medium x 2 into each well of columns 1 to 10 of a microtitration plate.

Dispense 200 μl of complete counting medium into each well of column 12 (Counting Medium Control).

Using a different pipette dispense 200 μl of counting medium base into each well of column 11 (Base Medium Control).

Using an Eppendorf Micropipette or other suitable types of repetitive pipette, add 100 μl of vaccine dilution 10

^{-10}into each well of row H from columns 1 to 10. Add 100 μl of vaccine dilution 10^{-9}into each well of row G from columns 1 to 10. Repeat this process for each dilution towards row A (10^{-3}).Seal the microplate.

Incubate the plate at 37°C for 10 days.

Using a plate reading mirror, examine the cultures for evidence of growth on the third, sixth and finally on the tenth day of incubation. Record the results. Growth is indicated by change of colour of the medium from pink to yellow.

The test is valid if no colour change occurs in any of the medium controls.

**NB:** *Skimmed milk, the most frequently used stabilizer in freeze dried CBPP vaccines.
goes into uniform suspension slowly hence the use of a volume of reconstitution
fluid that is greater than the vaccine filling volume and then kept at 4°C for 10–20
minutes with intermittent shaking. Medium used as diluent should be precooled
in a refrigerator at 4°C. Vaccine dilutions should be kept on ice throughout the
dilution and inoculation processes. The same pipette tip can be used to inoculate
several dilutions of a series if the least concentrated suspension (highest dilution)
is inoculated first and the most concentrated (lowest dilution) last.*

**IV. Calculation of Titre**

The calculation of mycoplasma titre relies on the principle of quantal dose response
relationship. For a stimulus-subject system such as mycoplasma titration, measurement
of response is to record whether or not the subject manifests the expected reaction. The
quantal assay so used measures an “all-or-none” response, e.g. Medium colour change
as a manifestation of mycoplasma presence and growth. To measure such a quantal
response, the most frequently used system is the multiple serial dilution assay. In a
multiple serial dilution assay, each dilution is tested in replicates (at least five). The
end-point is the dilution of a substance at which a specified number of members of a
test group shows a defined effect. The most frequently used, and statistically useful endpoint
is 50%. It is the **Median Effective Dose**, which in mycoplasma viability titration
in broth culture medium is the Colour Changing Unit **(CCU _{50}).** Thus the median
effective dose is the dilution of the test population which will demonstrate response in
50% of the population, i.e. colour change in 50% of a large number of inoculated
cultures.

Several approaches for calculating the median effective dose are available, including the Reed-Muench and Spearman-Karber methods. The Reed-Muench method is so simple in structure that it is still often used and regarded as standard, yet it has no sound theoretical basis. The Reed-Muench method is not recommended (Finney, 1978, Statistical Method in Biological Assay) because it does not permit assessment of precision from the data of a single assay; it gives no validity test and even under the most favourable conditions it is less precise than the Spearman-Karber method. The Spearman-Karber method, which is statistically markedly superior and involves relatively simple calculation, is described below:

**Calculation using the Spearman-Karber Formula**

The test sample is diluted in a geometric series, that is, with a constant ratio between successive dilutions, and a constant volume (usually 0.1 ml) of each dilution is inoculated into each of at least five broth cultures. The most commonly used dilution factor is 10-fold.

For the Spearmann-Karber formula to be applicable, it is necessary to use constant
number of test broth culture inocula per dilution (n_{i}), a constant dilution factor and a
range of dilutions wide enough to bracket both the dilutions at and below which 100%
of n_{i} subjects (i.e. broth culture inocula) tested will respond positive and the dilutions
at and above which 100% of n_{i} subjects tested will be negative.

If one or more of these conditions is not met, it is sometimes assumed that, for a constant dilution factor, the next higher or lower dilution to the last one tested would have produced the desired result. The “fabrication” of data in this way is without any theoretical basis, but if applied with suitable caution it may do little harm. However, it is preferable to repeat the titration with more appropriate range of dilutions, and this is essential if there are serious shortcomings in the data.

According to the Spearmann Karber formula:

Log_{10} Median Dose = (X_{0} -(d/2) + d(∑ r_{i}/n_{i})

where: | X_{0} | = | log_{10} of the reciprocal of the lowest dilution at which all
test inocula are positive. |

d | = | log_{10} of the dilution factor (i.e. the difference between the
log dilution intervals) | |

n_{i} | = | number of test inocula used at each individual dilution (after discounting accidental losses) | |

r_{i} | = | number of positive test inocula (out of n_{i}). | |

∑(r_{i}/n_{i}) | = | ∑(P) = sum of the proportion of positive tests beginning at the lowest dilution showing 100% positive result. |

Summation is started at dilution X_{0}.

Example I: Mycoplasma Titre of Vaccine - Equal Numbers per Dilution

Log_{10}dilution | n_{i} | r_{i} | Proportion Positive(P) | 1-P |
---|---|---|---|---|

- 3 | 10 | 10 | 1 | 0.00 |

- 4 | 10 | 10 | 1 | 0.00 |

- 5 | 10 | 10 | 1 | 0.00 |

- 6 | 10 | 10 | 1 | 0.00 |

- 7 | 10 | 7 | 0.70 | 0.30 |

- 8 | 10 | 3 | 0.30 | 0.70 |

- 9 | 10 | 1 | 0.10 | 0.90 |

- 10 | 10 | 0 | 0.00 | 1.00 |

X_{0} = 6.0 ; d = 1.0 Log_{10} 50% end-point dilution | = | (6 -0.5)+1*(10/10+7/10+3/10+1/10) |

= | (6-0.5)+2.1 | |

= | 7.6 | |

Log_{10} CCU_{50} per volume inoculated (0.1 ml) | = | 7.6 |

Therefore, Mycoplasma Titre per ml of the reconstituted vaccine sample | = | 8.6 log_{10} CCU_{50} |

and Titre per vial | = | 9.6 log_{10} CCU_{50} |

**NB:** *When using the Spearmann-Karber method, it has to be borne in mind that random
variation in the number of positive culture inocula will cause small but unknown
deviations from the true values of the end-point dilutions. These deviations will be large
if only a small number of inocula per dilution is used. There may also be slight
inaccuracies resulting from the method of estimation itself, but it has been shown that
on the whole such inaccuracies are smaller with the Spearman-Karber method than with
other comparable methods (e.g. using the Reed-Muench formula).*

*If the value of n _{i} (number of test broth culture inocula used at each individual dilution)
has been reduced by accidental losses (e.g. contamination, etc.), it is still possible to
obtain a valid estimate of the median effective dose using the Spearman-Karber formula.*

Example II: Mycoplasma Titre of Vaccine - Unequal Numbers per Dilution.

Log_{10}dilution | n_{i} | r_{i} | Proportion Positive(P) | 1-P |
---|---|---|---|---|

- 3 | 10 | 10 | 1 | 0 |

- 4 | 10 | 10 | 1 | 0 |

- 5 | 10 | 10 | 1 | 0 |

- 6 | 8 | 8 | 1 | 0 |

- 7 | 9 | 9 | 1 | 0 |

- 8 | 9 | 3 | 0.33 | 0.67 |

- 9 | 10 | 1 | 0.10 | 0.90 |

- 10 | 10 | 0 | 0 | 1.00 |

X_{0} = 7.0; d = 1.0 | ||

Log_{10} CCU_{50}/inoculum | = | (7-0.5) + 1*(9/9/+3/9+1/10+0/10) |

= | (7-0.5)+1.43 | |

= | 7.93 | |

Log_{10} CCU_{50}/ml vaccine | = | 8.93 |

Log_{10} CCU_{50}/Vial of Vaccine | = | 9.93 |

The estimated Standard Error is calculated using the following formula: | ||

Log Std. Error | = | d* √ (∑(p*(1-p)/(n_{i}-1))) |

Using values from Example II: | ||

Log St Error | = | 1* √ (((0.33*0.67)/8)) +((0.10*0.90/9))) |

= | 0.19 | |

Thus the mycoplasma titre may be quoted as 8.93 ± 0.19 Log_{10} CCU_{50}/ml |

**NB:** *In calculating the titre of Mycoplasma per vial account should be taken of the diluent
volume used for reconstitution of the vaccine.*