The sera of all humans contain the isoagglutinins anti-A and anti-B, except when the corresponding agglutinogens are present in the red cells. Thus the sera of group O persons contain both anti-A and anti-B and the sera of group A persons anti-B alone. There are rare negative exceptions to this general principle, some of which are probably genetically determined (Race and Sanger, 1958) . The factors determining the development of the isoagglutinins are not fully understood. The infant is born with its mother's agglutinins, but commences to form its own in the first few months of life (Yliruokanen, 1958) . These ‘natural’ isoagglutinins are not associated with isohaemolysins.
Thomsen and Kettel (1929) determined the titres of the anti-A and anti-B agglutinins in the blood of Danish subjects and found that the anti-A titres were generally higher than the anti-B titres. The anti-A in group O sera was of higher titre than the anti-A in group B sera, but no difference was found with anti-B in group O and group A sera.
Cutbush, Falconer and Mollison (1950) found that about 20% of 125 group O sera contained α-haemolysins. These haemolysins arise following injection of human serum (Aubert, Boorman and Dodd, 1942), purified blood group specific substances from animal tissues (Witebsky, Klendshoj and McNeil, 1944) or from human sources (Loutit and Morgan, 1946), serum containing antitoxins and antibodies against bacteria (Davidsohn, 1938), prophylactic bacterial vaccines (Cutbush et al., 1950) and in cases of heterospecific ABO pregnancies (Smith, 1945) . Coombs and his.coworkers (Winstanley, Konugres and Coombs, 1957; Konugres and Coombs, 1958) have demonstrated that sera containing haemolysins agglutinate red cells from pigs with the A antigen and that the haemolysins, but not the ‘natural’ anti-A agglutinins, are removed by absorption of the sera with such pig red cells. They postulate that the haemolysins and the ‘immune’ agglutinins may act on an antigen called A^p present in human red cells and in A pig red cells.
The work to be reported in this paper had three objects. The first was to compare the titres of anti-A and anti-B in sera from group B and from group A subjects with the titres in sera from group O subjects. The second was to determine whether titration of anti-A and anti-B in a viscous medium (polyvinylpyrrolidone) increases the titre. It has been reported that the titre of immune sera is enhanced when the titration is performed using serum instead of saline as the diluent (Boorman, Dodd and Morgan, 1945) . The third purpose of the investigation was to determine, using freshly collected group AB serum, the incidence of immune haemolysins in the sera of group O, group A and group B Australian blood donors.

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In a previous publication (Nojima and Fukumi, 1957), an abortive infection of T2 bacteriophage in a mutant of E. coli B was reported. Such mutants were easily isolated by a usual procedure for picking up phage-resistant mutants, and the strain (B/2) ₅ was used as a representative for these mutants. In this system of abortive infection, as already reported, phage DNA containing hydroxymethylcytosine and phage specific complement-fixing protein are produced, but only quite a few proportion of them is successful to pass through the maturation process. In this publication more detailed informations will be presented about the protein components produced in the system of the abortive infection of T2 in (B/2) ₅cells.

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The trends of diphtheria cases and deaths per 100, 000 of population and the case-death rate during the past about 50 years in Japan are presented graphically in Fig. 1. After the termination of the World War II the diphtheria incidence in Japan decreased rapidly to the lowest level recorded in 1952 (9.8 per 100, 000 of population) . The highest figures that the epidemiological statistics in Japan experienced were 127.6 and 118.5 in 1944 and 1945, respectively. Towards the end of the year 1953, however, the first sign of increase in diphtheria cases was observed, and thereafter, the morbidity increased again and reached its peak in 1956 with a morbidity of 20.4 per 100, 000 of population, the figure being two times as much as that in 1952.
The age distribution underwent a remarkable change during this epidemic and outbreaks in elementary schools showed a tendency to rise. There have been several reports showing increase of the malignant diphtheria cases. However, no increase in the case-death rate has been observed during the period. Several factors have been considered concerning this epidemic.
It was already warned by one of the present authors, basing on the results of Schick test performed in several districts of Tokyo and in its neighborhood in the years 1952-1953, that Schick positive rate was very much high, particularly in young children, and that this fact showed the presence in Japan of a danger of new outbreaks of diphtheria (Kurokawa, 1954) . A survey carried out in 1955 by the Ministry of Health and Welfare for the feature of immunity to diphtheria of about 30, 000 children up to the age of 12 years throughout Japan showed approximately 80% of Schick positive rate in the children at the age of 3-5 years, confirming the results of similar surveys previously performed in a smaller scale.
On the other hand, the vaccination rate was not so high as expected. According to statistics, the vaccination rate among babies was 50-60% at most even in urban districts.
Hirayama (1957) ascribed the cause of the recent epidemics to the influence of the over all ban against general vaccination enforced in 1949 after a tragic accident resulted from preventive vaccination against diphtheria in Kyoto City in the beginning of November 1948 (See Addendum) .
As the factors causing an epidemic, not only those concerning host but also those of pathogenic agent must be considered.
McLeod and his collaborators (McLeod, 1943, 1950) offered an opinion that there were three cultural types in Corynebacterium diphtheriae and that these types were found related closely with the clinical severity of diphtheria, the outbreak of diphtheria epidemic and the effectiveness of preventive vaccination and serum therapy. Since their opinion was published, a great deal of literatures on the problem have appeared, but the above opinion has always been accepted.
During the years 1954-1956 approximately 200 strains of C. diphtheriae were isolated from patients and healthy carriers by various laboratories in various parts of Tokyo, and Kanagawa, Shizuoka, Ibaraki and Chiba Prefectures, all located in the eastern part of Honshu island. Most of them were forwarded to the National Institute of Health for more detailed bacteriological examinations. The present authors attempted to scrutinize the factors relating to the recent epidemics from the point of view of the pathogenic agent, using these strains.
In the present report, some of our examinations performed on several factors thought to be relative to the recent epidemics are to be described.

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The classification of the subspecies of Pasteurella pestis has been made from a viewpoint of biological reactions on one hand and from a viewpoint of virulence on the other hand. The former viewpoint is, for example, represented by the classification employing the glycerol-fermentation test or by that depending on the host animal species from which a certain type of these organisms is preferentially recovered (Tumansky, 1957, 1958) . The latter viewpoint, which is thought to be a more practical one, has been taken by Bhatnagar (1940), Seal (1951 a, b) and Korobkova (1956), who classified P. pestis into virulent, avirulent protective and avirulent non-protective groups of strains on the basis of virulence and immunogenic properties to various experimental animals.
Jawetz and Meyer (1943) distinguished two groups of avirulent strains: the first group strains (A 1122, E.V. 51) possess a large amount of envelope antigen, i.e. Fraction I of Meyer (1950), and are also protective for mice; the second group strains (TRU, 14) are poor in envelope antigen, but protective for guinea pigs. Another group of strains which is avirulent and non-protective both for mice and guinea pigs was reported by Bhatnagar (1940), Seal (1951 a) and Amies (1951) . Kasuga (1945) described that an avirulent variant, strain MII40, had a protective power for guinea pigs and human beings from lethal infections with fully virulent plague bacilli, even though it had no detectable amount of envelope antigen.
Considering those results, the present author proposes at first the following scheme for classification of the groups or varieties of strains of P. pestis from the point of virulence and immunogenicity.
1. Virulent protective strains.
2. Avirulent protective strains with envelope antigen.
3. Avirulent protective strains without envelope antigen.
4. Avirulent non-protective strains.
As will be shown in the present series of papers, the interrelationship between these different groups of strains has been explained to a certain extent and some complemental corrections will be added in their conclusions. It is most conceivable from my results, that every strain of P. pestis can take any phase of the above varieties depending on their life history, in other words, they may represent each of phases of the evolutional process of plague bacilli, where the environmental factors seem to play important roles.
The present paper deals with an atypical form of P. pestis, which may be considered to occupy the fifth place in the above mentioned scheme of classification, with respect to lacking in the plague murine toxin which is contained in the other varieties. Evidences will be presented that this form has peculiar colonial and morphological characteristics. This form will be designated as the third colonial form of P. pestis hereafter, because it should be regarded as one of the phases of colonial variation such as classical smooth and rough forms, which have been customarily accepted as the representative colonial forms. Incidentally, it may be noteworthy to mention here that many practical laboratory workers dealing with plague bacilli have mistakenly recognized this form of organisms as contaminants other than P. pestis.

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The mechanism of the immunity against plague has been one of the important and interesting problems in the study of plague. In general, there have been employed two kinds of antiplague vaccines for active immunization of human beings in the world, that is, the dead vaccine and the live vaccine.
The problem which is more effective and safer is regarded as important, but it has not been solved yet to give a clearcut conclusion. It seems that this problem may comprise three criteria to be fulfiled, first of all, before being answered.
The first point is whether there may be any probability that the avirulent protective strains employed for live vaccine can revert to the virulent bacilli.
The second point is which antigenic component (or components) plays the main and determinant role in the enhancement of virulence and in protective power. If such antigen were found, it may be possible to protect against plague by vaccinating with the antigen.
The third point is the presumption that the vaccination with live avirulent bacilli may have such potentialities on the site of interactions with host cells that may not be expectable from the vaccination with killed bacilli. Namely, it would be assumable that the live avirulent bacilli can affect host cells in some way, while they are multiplying and disseminating in the host.
Concerning the second point, the studies of Meyer and his associates on the envelope antigen, i. e., Fraction I, (Meyer, 1950; Baker et al., 1951) and the studies of Burrows et al, on the V and W antigens (Burrows and Bacon, 1956) and the antigen responsible for the pigmentation of colonies on heamin or Fecontaining media (Jackson and Burrows, 1956a, b) seem to represent two main directions of the research.
According to Meyer, Fraction I was decisively protective for mice and rats against infection of fully virulent plague strains. Later reports confirmed the effectiveness of Fraction I for the protection of monkeys (Meyer et al., 1948, cited in Girard, 1955) .
Fraction I had nest been believed to protect guinea pigs, until the report appeared in 1958 (Spivack et al., 1958) informing that a relatively small amount of Fraction I protected guinea pigs against infection of fully virulent bacilli but too much Fraction I did not, and such a phenomenon could be interpreted by immunoparalysis.
On the other hand, Kasuga (1945) reported that live vaccine made of strain MII40 protected guinea pigs completely against infection of fully virulent plague strains and its prophylactic applications to the human proved striking effectiveness in the epidemic of plague in Manchuria (Kasuga, 1946 b) .
This strain, MII40, was isolated from the iliacal lymph node of a killed Citellus mongolicus 25 days after inoculation of an envelope-rich and fully virulent strain, Mieguchi. The animal had hibernated when inoculated with strain Mieguchi, and was awaked 11 days after inoculation.
According to Kasuga we have possibility to reproduce the isolation of bacilli resembling to strain MII40 by inoculating virulent bacilli into immunized animals. Thus it would be reasonable to regard strain MII40 rather as a representative strain of such a group or a variety of strains of P. pestis as has characteristics described by Kasuga than as a particular strain. Kasuga also stated that strain MII40 was lacking in the envelope antigen when examined by India ink method and by the cross precipitation test with the absorbed sera.
If this is true, the remarkably strong protective power of strain MII40 for guinea pigs cannot be explained by Fraction I.
In the preceding report (Wake, 1959 a) was described the third colonial form which had neither Fraction I nor toxin.

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Since Matumoto (1957) demonstrated that JBE virus could be readily grown in 1-day eggs, it had been a focus of our interest whether it was possible to isolate this virus by the 1-day egg inoculation, until an opportunity was given to test this possibility in 1957, when a brain material was supplied from an autopsy case of fatal JBE, and a strain was isolated which was identified as JBE virus.
Subsequently serial passages of this strain were carried out by inoculation into 1-day eggs. A special purpose, thereby, was to obtain a variant possessing a low infectivity to nervous tissue. This was attained by applying limiting dilution passage in the serial 1-day egg passages. The decrease in infectivity to nervous tissue was evidenced by lowering of its infective titer for mice intracerebrally inoculated in contrast to consistently high infective titers for 1-day eggs.
The present report describes in detail the changes in the mouse infectivity which ensued during the 1-day egg passages following the isolation of this strain and discusses the feasibility of the method employed to develop an attenuated live virus vaccine.

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Experiences in the tissue culture of animal cells have indicated that prolonged serial passage of cells can be a cause for the occurrence of variation in the cellular morphology, malignancy and nutritional requirements (Sanford et al., 1954; Earle and Nettleship, 1943; Goldblatt and Cameron, 1953; Sanford et al., 1950; Chang, 1957; Haff and Swimm, 1957; Perry et al., 1955; Puck and Fischer, 1956) . In the field of virology, it has attracted attention that the properties of cells subject to such variation include the spectrum of viruses to which they are susceptible. Bang et al. (1952) reported that the variation which occurred in a stable cell line of normal rat fibroblast, 14p, altered not only its malignancy but also its susceptibility to eastern equine encephalitis virus. A recent finding by Sheffield (1957) revealed that the changes of rabbit embryo kidney cells after a serial cultivation in vitro rendered them susceptible to polioviruses.
The widespread use of strain HeLa cells (Gey et al., 1952) has led to the finding that there exist cell lines with different abilities to propagate viruses. Scherer (1955) demonstrated that the HeLa strain supplied from“Microbiological Associates”differed from an original line of HeLa cells in their cytologic responses to polioviruses. A similar note was made in studies on adenovirus about different susceptibilities to viruses among HeLa cell lines used (Graystone et al., 1958) . The HeLa cell strain employed by Quersin-Thiry (1958) was inferior to the one used by Lennox et al. (1957) in susceptibility to diphtheria toxin, and insusceptible to the cytopathogenic effect of ECHO viruses in contrast to the one used by Arcketti (1957) . Of interest is the success in producing“resistant resistant culture”to poliovirus by Vogt et al. (1958) by means of repeated inoculations of Type 3 poliovirus into the culture of the S3 clone of HeLa cells (Puck and Marcus, 1959) .
In the course of our study on the adaptation of ECHO viruses to HeLa cells, several cell lines possessing varying degrees of resistance to the cytopathogenic effect of virus have been obtained from HeLa cells surviving an exposure to a massive amount of virus. They are similar, in many respects, to the above-cited“resistant resistant culture”of Vogt et al. (1958), but exhibit no morphological changes as compared with the parent cells. The purpose of this paper is to describe some characteristics of these resistant cultures.

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