Normal virulent B. anthracis all possess the genetical potentiality of producing capsules, but the capsule is only produced in vivo or under special cultural conditions suitable for capsule formation, especially in the presence of CO2.

During multiplication the potentially capsulated cells regularly produce a small number of a stable non-capsulated avirulent variant. All laboratory stock cultures of B. anthracis examined were found to contain from 0·14 to 32·4% of this avirulent variant. The variant has also been demonstrated in hair and wool from anthrax endemic areas, which suggests that the dissociation may also occur in nature.

Both the normal potentially capsulated cell and the variant incapable of producing capsules grow on plain agar in air as rough colonies with apparently non-capsulated cells. But when they are grown on serum agar in the presence of CO2, the normal potentially capsulated cell gives rise to a smooth mucoid colony with fully capsulated cells, while the variant still produces a rough colony with non-capsulated cells. If we regard the smooth mucoid colony produced by normal virulent B. anthracis on serum agar in CO2 as the normal colony form of B. anthracis, then the dissociation of the avirulent non-capsulated variant with the rough colony form falls in line with the usual S-R dissociation of other bacteria.

The more widely known variation of B. anthracis, which had long been held as an exception to the usual S-R variation of other bacteria, refers to colonies grown on plain agar in air. This variation is different from the one just described and is not so clear-cut and significant. Its underlying cause is not quite clear except that the chain length of the growth appears to play some part.

A summarized picture showing the whole range of variation in colony appearance and cell structure of B. anthracis is presented.

1. Treatment of washed sheep erythrocytes with dilute solutions of tannic acid rendered them capable of adsorbing diphtheria toxin from solution. Diphtheria toxoid was not adsorbed under similar conditions.

2. Cells thus sensitized with diphtheria toxin were agglutinable by a proportion of children's sera and human cord sera; sera of rabbits, mice and horses, immunized with soluble or adsorbed diphtheria toxoid, also agglutinated sensitized cells, as did also the sera of children suffering from diphtheria and treated with antitoxic serum. Clear-cut results were usually obtained, but the end-point of titrations on horse sera was, in some cases, difficult to determine.

3. Using intradermal toxin neutralization tests, a highly significant linear relationship was demonstrated between the haemagglutinating titres of children's sera for the sensitized red cells, and the antitoxin content of their sera. The antitoxin content corresponding to the smallest detectable amount of haemagglutinating antibody was 0·008 unit per ml. serum.

4. The haemagglutinating titre was an accurate measure of the mean antitoxin level of a group of sera and a close measure of the antitoxin content of most specimens in individual tests.

An epidemic of epidemic hepatitis occurred in the district of a rural Central School in Yates and Ontario Counties of Central New York. The epidemic comprised 176 cases of which 135 were jaundiced, and extended from September 1949 to May 1950, with most cases in March 1950.

No common source could be demonstrated and the transmission was believed to be due to human contact alone.

The overall morbidity rate was about 5%, attack rate among school children 22%, and among the adults and pre-school family contacts of infected school children the attack rate was 18%, with no significant age difference in attack rate.

Differences in attack rate in the various school grades were explained by higher number of siblings of children in certain grades.

Household attack rates among non-school persons were related to number of sick school children, family size, and hygienic standard of the home. The first of these factors seemed most important.

More jaundiced cases occurred in families where the exposure was heaviest.

A technique is developed by which minimal changes in haemagglutinin inhibitors can be detected.

It is shown that the inhibitor index of normal human nasal secretions is 33·2 with a standard deviation of ±7·4. This value is independent of the donor, of the absolute amount of mucus collected, of the time of extraction, and of the period of storage.

Minimal amounts of influenza viruses (less than one agglutinating dose) are capable of causing significant alteration in the inhibitor index upon interaction in vitro for 60 min. at 37° C.

Changes in the inhibitory properties of human nasal mucus were found to be positively correlated with objective signs of influenza virus infection during the 1950 epidemic. In approximately 80% of the cases the inhibitor index was significantly increased during the acute stage of the disease, and reverted to normal during convalescence.

Various respiratory diseases other than epidemic influenza did not cause similar changes.

Examination of the inhibitory pattern of nasal mucus is proposed as a presumptive test for influenza virus infection, and its merits are discussed under the headings of reproducibility, specificity and sensitivity.

1. The susceptibility of the Henzerling strain of R. burneti to phenol, formalin, ‘Lysol’, ‘Dettol’, ‘Cetavlon’, and ether was investigated.

2. Preliminary investigation of techniques showed that, with one passage, guinea-pigs, mice and fertile hens' eggs were of similar susceptibility to small numbers of viable organisms, but the greater feasibility of sub-passage from the eggs increased the sensitivity and made this the technique of choice.

3. Under the condition of testing, ‘Lysol’ was the most active disinfectant. Treatment of the rickettsial seed with 1% ‘Lysol’ at 37° C. was in fact the only effective method for destroying completely the infectivity in 3 hr.

4. Some living organisms survived contact for 3 hr. with each of the other chemicals in concentrations up to 1%. A rough quantitative comparison of these agents was made.

5. The failure of 1% phenol to destroy R. burneti raised the possibility of this organism surviving as a contaminant in vaccine lymph. This problem was investigated and is discussed.

The labile Vi antigens of Salmonella typhi, Salm. paratyphi A and B, and of Salm. typhi-murium were compared, especially in their response to various chemical and physical agents.

1. Inactivation by dilute acid:

(a) The TVi antigen is the most resistant to acid treatment, the BVi antigen is most readily inactivated and the AVi antigen holds an intermediate position.

(b) Pure Vi sera may be obtained by absorption with acid-treated bacilli, but this technique gives irregular results.

(c) Contrary to Kauffmann's statements, the TVi antigen is not ‘destroyed’ by acid treatment.

2. Inactivation by dilute alkali:

(a) All the Vi antigens so far tested are susceptible to dilute alkali.

(b) Complete inactivation, or extraction, of the Vi antigens is ensured when the yield of alkali-treated bacteria represents no more than 25% of the original bacterial count.

(c) The most reliable method of preparing a pure Vi serum is by absorption of the Vi + O serum with alkali-treated organisms of the same strain as that employed in immunization.

(d) Immunization with alkali-treated bacilli results in the elaboration of pure 0 antisera entirely devoid of Vi antibody.

3. Inactivation by heat (in aqueous suspensions):

(a) The TVi antigen is the one most readily inactivated, the BVi antigen is the one most resistant and the AVi antigen holds an intermediate position.

(b) These differences are only of minor significance, since similar differences have been established for the TVi antigen itself when it is contained in the two varieties of Vi strain of Salm. typhi.

4. Treatment with alcohol:

(a) Most of the properties of the three Salmonella Vi antigens remain unimpaired by alcohol treatment.

(b) The physico-chemical behaviour of alcohol-treated or heated bacilli, as exemplified by agglutinability by pure Vi antiserum, depends on the presence of other constituents of the bacterial cell.

5. Treatment with formalin:

(a) The Vi agglutinability of the different Salmonella is preserved undamaged for long periods of time. The 0 inagglutinability of Salm. typhi is also well maintained.

(b) It is not yet known whether formolized AVi and BVi antigens undergo an alteration that leads to ‘functional deficiency’ of the corresponding antibody similar to that of the altered T Vi antibody.

6. Treatment with phenol produces a reversible inactivation of the agglutinogenic activity of the T Vi antigen but not of the A Vi and B Vi antigens.

7. The inadequacy of the symbols employed by Kauffmann for expressing the changed reactivity of differently treated antigens is emphasized.

1. Salmonella paratyphi A possesses a specific Vi antigen essentially similar to that of the Vi antigen of Salm. typhi.

2. O-insensitive strains of Salm. paratyphi A contain more Vi antigen, and are more virulent for mice, than O-sensitive strains.

3. The most suitable strains for the preparation of paratyphoid-A vaccine are those that develop both the Vi and the O antigens in maximum quantities. The methods of selecting the vaccine strain of Salm. paratyphi A and routinely examining the cultures are essentially the same as those applied to the virulent Vi+O form of Salm. typhi.

4. Pure A Vi antiserum is destined to assume importance in the identification of Salm. paratyphi A strains.

5. Kauffmann's erroneous conclusion that Salm. paratyphi A does not develop Vi antigen is critically reviewed.

1. Salmonella paratyphi B possesses a Vi antigen essentially similar to the Vi antigens of Salm. typhi and Salm. paratyphi A.

2. The biological function of the Vi antigen of Salm. paratyphi B is to protect the O antigen against the action of the natural or immune O antibody, thereby increasing the virulence of the microorganism.

3. The most suitable paratyphoid-B vaccine strains are those that develop both the Vi and the O antigens in maximum quantities. The methods of testing vaccine strains of Salm. paratyphi B are essentially the same as those applied to the virulent Vi+O form of Salm. typhi.

4. Evidence is brought to show that Kauffmann's conclusion that Salm. paratyphi B does not develop Vi antigen is unfounded. The so-called O-factors V and V obviously cannot be classified as O antigens.

5. It is suggested that the symbol V of the Kauffmann-White schema be abandoned and this antigenic component be classed as the Vi antigen of Salm. paratyphi B.

1. Four different Bacteriaceae possessing the same Vi antigen showed, after exposure to heat, striking differences in the physico-chemical behaviour of the Vi antigen. The most noticeable differences observed were those in the changes in Vi-agglutinability and O-inagglutinability of the bacteria; lesser differences were noted in the agglutinogenic activity of the Vi antigen, and none in its agglutinin-binding capacity.

2. Treatment with alcohol altered the TVi antigen of one of the species (Bact. coli 5396/38) in a way different from that seen in the other three species.

3. On the other hand, dilute acid or alkali produced the same chemical changes in the TVi antigen of all four Bacteriaceae.

4. The TVi antigen present in the four Bacteriaceae appears to be one and the same substance; it cannot be differentiated by the customary serological methods. Its different physico-chemical state after exposure to heat or alcohol is, therefore, conditioned by other constituents of the bacterial cell, which may, or may not, be antigenic.

5. The simultaneous O- and Vi-inagglutinability resulting from heating at 75° C. is particularly impressive since it does not appear to be specially related to any one of the known antigenic components.

6. These findings invalidate the basis on which the L, A and B antigens of Bact. coli have been differentiated.

7. There is also no valid reason for designating the labile somatic antigens of Salmonella and other Bacteriaceae as K antigens. These antigens have the general characters of the Vi antigen of Salm. typhi, are demonstrated by methods developed in the study of the typhoid Vi antigen, and are not associated with typical capsules.

8. The M (mucoid) antigens of Salmonella and of Bact. coli are in many respects different from the Vi antigens and should be classified separately.

I gratefully acknowledge the valuable technical assistance received from Mr F. J. Flynn throughout the work reported in this and the preceding papers of this series.