Table 2.

Quality control tests for leptospiral vaccine strains used as vaccines in China.

Test	Method and specification
Morphological and cultural characteristics	Leptospiral vaccine strains shall be inoculated into production medium at less than 5 %, and incubated at 28–32°C for 5–10 days. The cultures shall contain more than 100 organisms per high-power field at 400 ×. Leptospires appear as motile rods with regular shape and with both ends curved.
Serum agglutination	The microscopic agglutination test using serogroup reference serum shall be performed on leptospiral vaccine strains grown for 3–10 days and adjusted to 50–100 organisms per high-power field at 400 ×. The agglutination titer shall be no less than half of the titer of the reference serum.
Virulence	Six guinea pigs (180–220 g), divided into 2 groups, are subcutaneously injected with 2 mL of leptospiral vaccine strains grown for 5–10 days and adjusted to 50–100 organisms per high-power field at 400 ×. In 1 group, the blood shall be drawn from the heart 48 h after injection, and inoculated into the production medium or other appropriate media, and incubated for 14 days. The strain shall be considered low-virulence if growth is observed. In the other group, the guinea pigs are observed for up to 10 days after injection. The strain is considered highly virulent if at least 2 animals die of leptospirosis.
Immunogenicity	Three guinea pigs (120–220 g) are subcutaneously immunized with 0.5 mL leptospiral vaccine strains inactivated by heating at 56–58°C for 1 h or by adding 3.0 g/L phenol, adjusted to 70–100 organisms per high-power field at 400 ×, and diluted 3-fold with saline. A boost of 1 mL inactivated culture is administered at 5-day intervals. Three control animals are injected with equal volumes of saline. On days 10–12 after the last injection, the animals shall be challenged subcutaneously with 2 mL of the same strain grown for 5–10 days and adjusted to 50–100 organisms per high-power field at 400 ×. For highly virulent strains, the animals shall be observed for up to 10 days after challenge. All immunized animals shall survive, and have normal appearance, appetite, activity, and weight gain, but have no piloerection or jaundice. At least 2 control animals should die of leptospirosis. For low-virulence strains, blood will be collected from the heart 24 h after challenge, inoculated at about 1 % into Leptospira medium containing 5–8 % rabbit serum, and incubated for 14 days. The test shall be considered passed only if more than 2 cultures from immunized animals are negative, and all cultures from control animals are positive.
Antigenicity	Three healthy rabbits (2.0–2.5 kg) are intravenously immunized at 5-day intervals with 1, 2, and 5 mL leptospiral vaccine strains inactivated by heating at 56–58°C for 1 h or by adding 3.0 g/L phenol, and adjusted to 70–100 organisms per high-power field at 400 ×. On days 10–15 after the last injection, rabbit sera are collected, and tested by the microscopic agglutination test against the same strain. The test shall be considered passed if the serum titers of at least 2 rabbits are 1:10,000 or more.

*Three highly virulent (serogroups Icterohaemorrhagiae serovar lai, serogroup Grippotyphosa serovar linhai, and serogroup Autumnalis serovar autumnalis) and 4 low-virulence strains (serogroup Canicola serovar canicola, serogroup Pomona serovar pomona, serogroup Australis serovar australis, and serogroup Hebdomadis serovar hebdomadis) are currently used.