The smooth mucoid part of the colony was subinoculated every other day for approx. eighteen months. The following experiment was carried out to see if the deaths in the guinea pigs inoculated with the crude variant were due to contaminating virulent forms. Three guinea pigs which had survived the inoculation of the rough variant (Table III) were each inoculated with one-sixth of an agar slant of the smooth mucoid variant two weeks later.
Thus guinea pigs that had received two or more injections of the rough variant were completely immune to a very large smooth mucoid strain test. It was clear that the serum of immune pigs afforded considerable protection against a very large dose of the mild mucoid variant. The dose of the proximate variant given to the sheep was, however, less than the dose given to the guinea pigs.
Anthrax A.—Six guinea-pigs which had survived inoculation with smooth mucoids and close variants ('fable XIII) received two more injections of the same strains, and the immunity was then tested as follows:-. Tests with variant strains 568.-Three P-ach guinea pigs received two inoculations of the smooth mucoid strain at an interval of 10 days. Three guinea pigs were each inoculated with l/3 slope of the 4th subculture of a rough variant.
Drummond.-The five guinea-pigs which survived the virulence test, together with three uninoculated controls, each received 1/6 of a slant of the smooth mucoid variety from virulent Boshoff. Six guinea pigs were each injected twice with t-agar slants of the crude variant of the Pretoria North strain. The two injections of the coarse variants from strain IV previously gave guinea-pigs a much increased resistance to the smooth mucoid Boshoft straiu, but a solid immunity was not established.
The low residual virulence of the rough variant was probably due to contaminating smooth forms. Three guinea pigs "·er each inoculatorl with irrl of a slant of the rough v:nia nt. At the same time another tube was inoculated from the smooth mucoid part of the eulture and At the same time there was a subculture of the smooth mucoid part of the growth of carbon dioxide (ubatecl in air and this was called the "smooth" variant). Three guinea pigs were each exposed to a slope of the rough strain derived from the smooth mucoid growth of carbon dioxide. Three guinea pigs were each exposed to a bit of the rough strain derived from the rough growth of carbon dioxide. Two of the immunized guinea pigs lived significantly longer than the controls and one survived. Immunity tests on sheep.-The results of the immunity tests on guinea pigs justified a test on sheep. At the same time, eight sheep received a subcutaneous injection of the crude variant of strain XVIII. A recalculation of the dilution later showed that a greater closure of the avirulent strain had been applied. Guinea pigs that had previously received an injection of the avirulent variant from the XX strain showed a much higher resistance to anthrax than normal pigs. A culture was made from one of the guinea pigs that had received the close variant .and died ·On the 5th day. The results of the immunity test performed 4 to 5 weeks after inoculation of the avirulent strain are summarized below. Subcultures from both the smooth mucoid and the rough portion of the gro"·th in carbon dioxide were made very tough in air and injected into guinea pigs after 48 hours. Sheep were inoculated with a small enclosure of the nonvirulent type XXVI developed a better immunity than those inoculated with the usual vaccine strain XVII The enclosure of the avirulent strain was less than that usually given in vaccines, so that the terms were slightly weighted against it. In the last part of the work (Part D) it was shown that avirulent coarse dissoci.ants, obtained from virulent strains growing smooth mucoid in carbon dioxide, were very efficient immunizers, unless the smooth mucoid culture. In the case of the weakened cultures the smooth mucoid variant grew faster, and the rough dissociants could not establish themselves. In the experiment noted in Table LII, a comparison was made of the immunity produced in sheep by the avirulent variant of strain XVIII and an attenuated strain. There was evidence that the ability of the rough variants to immunize might be a function of the virulence of. However, there was no doubt that the avirulent variant from Boshoff's ability to immunize was not an extraordinary case, as the crude variants from. Transplants from the smooth mucoid growth in carbon dioxide to ordinary agar in air showed an immediate return to the typical very rough growth of the original virulent strains, as did grafts from the rough h variants. Therefore, the appearance of virulent anthrax strains grown on serum agar in carbon dioxide may be a completely "normal" appearance of the virulent strain. As shown in the preceding part of the discussion, there is evidence that the immunizing power of the rough avirulent variants does depend on the virulence of the smooth mucoid from which they are derived.
