1. The virulence of vaccine virus for the testicular tissues increases until its maximum is finally reached. The selective increase is not associated with any loss, reduction, or modification in its virulence for the skin. A highly potent testicular vaccine is also highly active for the skin.
2. The testicular strain of vaccine virus has no more tendency to localize in various organs than the ordinary skin strain. Both may localize in adjacent lymph nodes when introduced intravenously, subcutaneously, or intratesticularly in sufficiently large quantities, but other organs are not involved.
3. Intravenous inoculation of an excessive amount of a powerful vaccine virus (1 to 2 cc. of undiluted stock emulsion), irrespective of whether it is from the testis or the skin, will result in a generalized eruption over the entire body surface of rabbits. The eruption may be confluent on mucous membranes of the mouth, nostrils, genitalia, etc. Intratesticular or subcutaneous inoculations of the same virus fail to produce this effect.
4. Subcutaneous or intravenous introduction of much smaller quantities of the virus does not cause an appreciable local or general reaction in the rabbit. But the animals which have once received these injections become refractory to a subsequent vaccination as applied to the skin. It seems probable that an active immunity has been conferred.
5. Experiments on the viability and resistance of the testicular strain of vaccine virus indicate that the virus is best preserved when emulsified with Ringer's solution or 0.9 per cent saline solution. Distilled water, while apparently one of the best diluents, fails to keep the virus active as long as Ringer's or saline solutions. As would be expected, the lower the temperature is, the longer the virus retains its viability. At 18° or 37°C., the deterioration of the virus proceeds rapidly. However, a small part of the virus survives after many weeks' standing at 37°C.
6. Of the two most commonly employed chemical agents for the ripening (eliminating bacteria) process of the green vaccine pulp, glycerol and phenol, the latter is the less injurious. Phenol in concentration above 2 per cent destroys the virus within 24 hours at any temperature, but it has almost no injurious effect when used in 0.5 to 1 per cent. On the other hand, glycerol is a powerful vaccinicide. When used in full strength it destroys the virus within 24 hours, even at 4°C. In a concentration of 40 per cent, that ordinarily recommended for the ripening, the virus retains some of its virulence for about half a year at 4°C., while at higher temperatures the same concentration kills the virus within 1 to 2 months. The virus preserved in distilled water or Ringer's solution under similar temperature conditions remains more active during this period. From this it may be concluded that glycerol is not an indifferent agent, as is assumed by many, but a powerful vaccinicide when used in high concentrations. The injurious effect is markedly accelerated at 18° or 37°C.
7. The vaccine virus retains its virulence better in a sealed tube containing either hydrogen, nitrogen, or air than in an open receptacle. The virus deteriorates when placed in a sealed tube with oxygen or carbon dioxide.
8. Desiccation decreases to a considerable degree the virulence of the vaccine virus. In the dried state the virus retains its viability about as long as does the emulsion, but it is not protected from the deterioration caused by age under various conditions.
9. Iodine is a powerful disinfectant for the vaccine virus, but its sodium and potassium salts have no effect. Various bile salts destroy the vaccine virus when employed in sufficient concentration.