Canine Distemper Vaccine, Live is a freeze-dried preparation of a strain of canine distemper virus that has been attenuated for dogs and is grown either in suitable cell cultures or SPF eggs. 

Production 

The virus is propagated in suitable cell culture or SPF eggs. The cell culture or SPF eggs complies with the requirements for cell culture or egg for production of veterinary vaccines (2.7.13). The viral suspension is harvested, titrated and may be mixed with a suitable stabilizing agents. The vaccine is then freeze-dried and can be used with any suitable diluent or used after reconstitution with licensed liquid vaccine components. 

Choice of vaccine strain 

A reference strain obtained from an authentic source shall be used for the vaccine production. The master seed which has been established as pure, safe and immunogenic for the species for which it is intended shall be used for vaccine production. 

Identification 

1. Either of the test A or B may be carried out. 
2. The vaccine reconstituted as stated on the label and mixed with a mono-specific serum against canine distemper virus no longer infects chorioallantoic membranes of SPF embryonated eggs.
3. The vaccine reconstituted as stated on the label and mixed with a mono-specific serum against canine distemper virus no longer provokes cytopathic effects in susceptible cell cultures.

Tests 

Mycoplasma (2.7.8). Complies with the test for freedom from mycoplasmas. 

Extraneous agents. Either of the test A or B may be carried out. 

 Neutralize the vaccine virus with a suitable mono-specific antiserum against canine distemper virus and inoculate into cell cultures known for their susceptibility to viruses pathogenic for the dog. Carry out 2 passages with an interval of 6 to 8 days. The vaccine complies with the test if no cytopathic effect develops for 10 days.

1. Use a sufficient number of mice, not less than ten, each weighing between 11 and 15 g and administer each mouse intracerebrally with 30 µl of the vaccine. Observe for 21 days. Not more than two mice die within the first 48 hours. If more than two mice die within the first 48 hours, repeat the test. From the third day to 21 days after the injection, the mice do not show any abnormalities attributable to the vaccine.

Sterility (2.2.11). Complies with the test for sterility. 

Safety. Reconstitute the vaccine as recommended on the label and carry out the following tests.

1. For chicken embryo-adapted vaccine only.Inject 30 µl intracerebrally into each of a group of eight mice, between 3 and 4 weeks old, and 0.5 ml intraperitoneally into each of another eight mice of the same age group. Observe both the groups for 7 days. Not more than one mouse in either group shows any abnormal local or systemic reaction attributable to the vaccine.

1. The test is carried out for each recommended route of administration. Use five susceptible puppies of the minimum age recommended for vaccination and that do not have antibodies against canine distemper virus. Administer to each puppy by a recommended route a quantity of virus corresponding to not less than ten times the maximum titre that may be expected in a dose of vaccine. Observe the puppies for 42 days. The puppies remain in good health and there is no abnormal local or systemic reaction.

If the vaccine is intended for use in pregnant bitches, administer the virus to five bitches at the recommended stage of pregnancy or at a range of stages of pregnancy according to the recommended schedule. Prolong observation until 1 day after parturition. The bitches remain in good health and there is no abnormal local or systemic reaction. No adverse effects on the pregnancy or the offspring are noted. 

Increase in virulence. Administer by a recommended route to each of two puppies, 5 to 7 weeks old and which do not have antibodies against canine distemper virus a quantity of virus corresponding to one dose of vaccine. Kill the puppies 5 to 10 days later, remove nasal mucosa, tonsils, thymus, spleen and the lungs and their local lymph nodes from each puppy and pool the samples; administer intranasally 1 ml of the pooled organ suspension to each of two other puppies of the same age and susceptibility; carry out these operations at least five times; verify the presence of the virus at each passage by direct or indirect means. If the virus has disappeared, carry out a second series of passages. Inoculate virus from the highest recovered passage level to puppies, observe for 42 days and compare any reactions that occur with those seen in the test for safety described above. There is no indication of an increase of virulence as compared with the non passaged virus. 

Immunogenicity. Inject each of five susceptible dogs, between 8 and 14 weeks old, that have been previously tested and shown to be free from canine distemper virus neutralizing antibodies with a volume of the reconstituted vaccine containing a quantity of the virus equivalent to the minimum titre and by stated on the label. Use another two dogs of the same age group as unvaccinated controls. Observe the animals for a further 21 days. Inject intravenously each of the seven animals with a quantity of virulent strain of canine distemper virus sufficient to cause death or produce typical signs of the disease in a susceptible dog. Observe the animals for a further21 days. The vaccinated animals survive and show no clinical signs of canine distemper. The test is not valid unless the control dogs die or show symptoms typical of canine distemper. If one of the control animals does not show any sign of canine distemper, repeat the test. The vaccinated animals of the second group remain in normal health and the control animals die from canine distemper or show symptoms typical of canine distemper. 

If the potency test has been performed with satisfactory results on a representative batch of the vaccine from the seed lot, it may be omitted as a routine control test during production on other batches of the vaccine prepared from the same seed lot. 

Virus titre. Not less than 10³  TCID₅₀/CCID ₅₀/EID ₅₀ of the virus per dose, determining the titre of the vaccine in a suitable cell culture with suitable medium. 

Manufacturer’s tests 

Virus titre. Virus titre is determined in final bulk harvest in a suitable cell culture with suitable medium. 

Identification. Vaccine complies the requirements of the test mentioned under production. 

Extraneous agents. Vaccine complies the requirements of the test mentioned under production. 

Mycoplasmas (2.7.8). Complies with the test for freedom from mycoplasmas. 

Sterility (2.2.11). Complies with the test for sterility. 

Batch tests 

Identification 

Vaccine complies the requirements of the test mentioned under production. Alternatively, identification on the final lot by molecular techniques is acceptable and can be used in the routine batch release tests after proper validation (2.8.1).   

Water (2.3.43). Not more than 3.0 per cent. 

Extraneous agents. Vaccine complies the requirements of the test mentioned under Production. Alternative, molecular techniques for detection of pathogenic viruses of dog are acceptable batch release test after proper validation (2.8.1). 

Mycoplasmas (2.7.8). Complies with the test for freedom from mycoplasmas. Alternatively, molecular techniques for detection of mycoplasma nucleic acid are acceptable batch release test after proper validation (2.8.1). 

Virus titer. Not less than 10³ TCID₅₀ of the virus per dose, determining the titre of the vaccine in a suitable cell culture with suitable medium or one dose of vaccine contains not less than quantity of virus equivalent to the minimum virus titre stated on the label. 

Sterility (2.2.11). Complies with the test for sterility. 

Safety. Inject intramuscularly 10 times the minimum dose stated on the label into each of two dogs of the minimum age recommended for vaccination. Observe the animals for 21 days. None of the dogs shows abnormal local or systemic reactions or dies of any causes attributable to the vaccine. 

Potency. The vaccine complies with the requirements of test mentioned under immunogenicity when administered by a recommended route and method. It is not necessary to carry out the potency test for each batch of the vaccine if it has been carried out on a representative batch of the vaccine using a vaccinating dose containing not more than the minimum titre stated on the label. The virus titer is considered for a routine batch release provided the traceability of the vaccine strains used is from the same master seed. 

Labelling. The label states (a) the minimum dose; (b) the recommended routes of administration; (c) storage temperature (d) virus titer (e) expiry period.