The relations between type c protein antigen of group B streptococci and new type protein antigens proposed by Denka-seiken were examined on immunoelectrophoresis, counter immunoelectrophoresis and immunodiffusion.
1. In 10 out of 14 new type strains (7 reference strains supplied by Denka-seiken and 7 group B isolates) were detected c^α and/or c^β antigen (s) on immunoelectrophoresis. Four strains contained non of c protein antigen.
2. The type c antiserum # 80 prepared by immunization with the cell of type Ia/c strain ss 700 contained c^α and c^β antibodies, and the other c antiserum Denka, which was immunized with extract of the same strain contained c^β antibody only.
3. Antisera prepared by immunization with new type strains (type Ia/Q strain Fz 345, type Ia/S Fz 301, type W strain Fz 450) shown type specificity on immunoelectrophoresis and immunodiffusion after absorption with type Ia/c strain ss 700.
4. Type Q protein antigen shown slower anodic migration than c^β protein antigen on immunoelectrophoresis and was susceptible to trypsin digestion.
5. Type S protein antigen shown as fast anodic migration as c^α protein antigen and was susceptible to pepsin and trysin digestion.
6. Type W protein antigen shown the same anodic migration as type Q protein antigen and was resistance to pepsin and trypsin digestion.

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One hundred group B Streptococci (Streptococcus agalactiae) isolated from clinical specimens investiaged for serotyping and drug susceptibility. Of these, 3 donor strains designated BTO 21, BTO 22 and BTO 23, transferred chloramphenicol (CP) resistance marker and chloramphenicol acetyltransferase (CAT) producing activity by conjugation into group B Streptococci recipient strains. The CP resistance appeared to be due to the presence of CAT mediated by a plasmid.
The molecular weight of a streptococcal conjugative R plasmaid, designanted pBTO 22, was investigated after transfer into recipient strains and was found to be similar to that the wild-type group B Streptococcus host (19-20×10⁶).
The products of chloramphenicol acetylation obtained by inactivation were identified as 1- acetoxy, 3-acetoxy and 1, 3-diacetoxy derivatives, respectively, by thin-layer chromatography.

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A new method of grouping beta-hemolytic streptococci by commercial latex agglutination reagent with nitrous acid extraction (Seroiden Streptokit “Eiken”: E-method) was examined. The E-method was compared with coagglutination test (Pharmacia Diagnostics: P-method) and slide-agglutination test (Denka Seiken Co. Ltd.: D-method). A total of 211 strains of beta-hemolytic streptococci isolated from the throats of the school-children were serologically grouped into A, B, C & G by D-method (slide-agglutination test with rabbit antiserum after trypsin digestion), P-method and E-method. Overnight procedure used Todd-Hewitt Broth (THB) cultures incubated overnight at 37°C for nitrous acid extraction was performed as E-method. The rate of agreement between D-method and E-method was 100%. However, cross-reactions were observed on the other groups strains when P-method was performed.
The E-method protocols permit four kind of procedures. The second modification of the E-method was performed with 34 clinical isolates. The suspension from overnight 5ml THB cultures were tested for reactivity with the latex reagent in this modification, called simplified method. The simplified method was compared with D-method, P-method and overnight method as E-method. The rate of the agreement between these four methods was 100%, on the other hand cross-reactions were observed in P-method and weak agglutination in the simplified method. A rapid four-hour THB cultures grouping method with nitrous acid extraction is the third modification of E-method. Of 21 strains tested, 12 were correctly grouped at four hours after examination of single colony from the primary isolation plates, and the remaining 9 strains were grouped at six hours. The minimal number of beta-hemolytic streptococci colonies were varied from 3 to 4 colonies, as determined the four-hour latex agglutination method.
Grouping directly on blood agar plates is the fourth modification of E-method. In this procedure, the latex agglutination test was performed on the isolated colonies after nitrous acid extraction step. The minimal number of streptococcal colonies cause a latex agglutination response was 3 to 7 colonies.

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During the 11-year period between 1975 and 1985, 4720 strains of β-hemolytic streptococci isolated from patients with streptococcal infection, mostly scarlet fever, and from some healthy children in Sapporo were typed by T-agglutination method.
The results were summarized as follows:
1. Serotyping of 4660 strains of group A streptococci were given as under; T12 (45.9%), T4 (23.3%), Ti (8.4%), T6 (4.3%), T18 (2.0%), T22 (1.7%), T3 (1.3%), T28, T11, T2, T8, T13 and B3264. Type T12 followed by T4 was the most popular type, and T1 or T6 were other predominant types. This pattern of type distribution on these four predominant types demonstrated in Sapporo was found on a nationwide scale. T28 or B3264 showed lower isolation lates than the general.
2. The predominant types of T12, T4 and T1 showed short epidemic waves having 6 or 7 year-period. Rare type of T18 was well as T22 and T3 showed a tendency to be in fashion with several-year interval.
3. An age-related difference was observed in type distribution; that is, as to predominant types, T12 and T4, the higher isolation rates were obtained from younger children and as to rare types and untypable strains obtained from older children

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For the purpose of investigating the present condition of streptococcal infection in a typical pediatric outpatient clinic, this observance was undertaken at a private clinic in Matsuyama City, Ehime Pre., Shikoku Island.
A total of 2373 cases of streptococcal infection, confirmed bacteriologically, were studied from May, 1975 to March, 1985. Results were as follows:
1) Group A streptococci were identified in 96.2% of these cases; 68.1% of the patients had been diagnosed as pharyngitis and 31.2% as scarlet fever.
2) Usually two epidemic peaks were observed in each year, which was measured as the period from the beginning of April to the end of the following March. One was a small peakoccuring in early summer (mainly May, June and July) and the other was a large peak occuring in winter (Oct., Nov. and Dec.). From the analysis of age distribution of the patients and serological types of group A streptococcal isolates, it seems that the typical epidemic is caused by onetype of streptococcus. The largest number of cases is seen in the winter, because of the patients' lowered resistance, andthe beginning of the school year in April exposes susceptible youngsters toinfection, resulting in a small epidemic peak in early summer.
3) Prevalent serotypes of group A streptococci were types 12, 4 and 1. It was observed that the peak frequency of isolation could be found for each strain every 5 to 6 years.
4) T-typable rate of group A streptococcal isolates was 99.6% and M-typable rate was 67.9%. Coincidence between T and M types was as follows; Type 3; 97.9%, Type 6; 97.7%, Type 4; 92.4%, Type 1; 91.3%, Type 12; 79.9%.
5) M12 strain had a more statistically significant tendency to induce scarlet fever than did the other types of group A streptococci.
6) 12.9% of the patients in this study were confirmed to be reinfected cases. It is speculatedthat this result suggests that the use of antibiotics in the early stages of infection surpressed the production of antibodies and was responsible for the later reinfection.

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Hemolytic streptococci isolated from clinical specimen in Toyama Prefecture in 1985 were investigated for the relationship between the difference in the source of specimen and the distribution of serological groups or types. The patterns of antibiotic sensitivity of the isolates were also examined in relation to the serological groups or types. This data is compared with those of isolates from 1980 to 1984.
1. Grouping of 241 isolates in 1985 by both agglutination (antisera produced by Denka Seiken) and gel diffusion (antisera produced by Wellcome Diagnostics) revealed that 143 (59.3%) isolates belonged to group A, 82 (34.0%) to group B, 14 (5.8%) to group G and 2 to unknown groups except for A, B, C, and G. The ratio of group B strains increased in 1985 in comparison to that of isolates from 1980 to 1984. With regard to type distributions of group B strains by agglutination test (antisera produced by Denka Seiken), little changes were observed for the 6 year period from 1980 to 1985, type specific polysaccharides of both Ia and III being always predominant.
2. As to type distributions of group A strains by agglutination test (antisera produced by Denka Seiken), the ratio of type 12, which had been dominating in Toyama Prefecture from 1970s to 1984, decreased in 1985, 31 (21.7%) out of 143 group A isolates belonging to this type. In turn, type 3, which occupied only 7 (1.2%) out of 591 group A isolates for the past 5 years, became predominant in 1985, 55 (38.5%) belonging to this type.
3. Characteristics of these type 3 strains were followings. Colony morphologies on the blood agar plate are variable and hemolysis is rather strong in group A strains, clear s hemolytic rings around the colonies being observed after overnight cultivation at 37°C. Type 3-specific M protein is abundantly detected by the gel diffusion analysis between the acid-extracted antigen and antiserum against the M protein (self-produced). This and the fact that, in some cases that the children from whom type 3 strains were isolated developed typical symptoms of scarlet fever, imply that the pathogenicity of type 3 strains is comparable to that of type 12 strains, if not exceeding. In contrast to type 12 strains, which often showed resistance to antibiotics such as tetracycline, chloramphenicol and macrolides, all of these type 3 strains, except one, are sensitive to these antibiotics, not to mention penicillin and cephalosporin.

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We studied the yearly changes of the serological types of the 605 group A streptococci and 640 group B which were obtained from clinical subjects since 1978 to 1985. And we examined the drug sensitivity of the 142 group A stains and 282 group B stains which were isolated from 1983 to 1985.
In group A, one of the most frequently isolated type was T-12 or T-4, and T-1 and T-13 came after, before 1984, while T-3 which was rare before 1984 increased in number rapidly and its occurance rate reached to nearly fifty per cent in 1985. However, in group B, the most frequent type of strains was steadily Ia. And III was the next. In the drug sensitivity, following antibitics were studied, chloramphenicol, tetracycline, penicillin G, ampicillin, cefazoline, ceftizoxime, erythromycin, josamicin and oleandomycin. All group A and B were sensitive to the antibiotics of the penicillin-group and the cephem-group both. In group A, some strains which were resistant to tetracycline, erythromycin, josamycin and oleandomycin were related to the serotypes respectively. Most of T-4 and all of T-13 were resistant to tetracycline and T-12 was resistant to multiple drugs including MLs.
Group A was mainly isolated from throat swabs and group B from vaginal swabs and urine. We also obtainted group B strains from two neonates with septicepmia or arthritis, five infants with meningitis, and six adults with septicemia. Blood and throat swabs from neonatal septicemia and blood and cerebrospinal fluid from infantile meningitis simply yielded the type III strain. Blood and pus from neonatal arthritis yielded type Ib. However, six strains from adult septicemia were Ia, IaQ, IaS, Q and R individually.

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Antibiotic sensitivity of a total of 2, 499 strains of group A hemolytic streptococci isolated in 1983-1985 from clinical specimens such as throat swab, sputum, urine, vaginal discharge and pus, as well as from throat swabs of healthy pupils were studies, 2, 242 strains among them were also studied for their T-types.
Antibiotic sensitivity test was carried out by a plate dilution method using 5% horse blood agar (Sensitivity Disc Agar-N, Nissui). Antibiotics used include benzylpenicillin (PCG), ampicillin (ABPC), cephaloridine (CER), cephalexin (CEX), tetracycline (TC), chloramphenicol (CP), erythromycin (EM), oleandomycin (OL) and lincomycin (LCM).
The most prevalent serotype was T-12 and it was followed by T-4, T-3 and T-28, both in clinical and pupil isolates.
No resistant strains to f3-lactams was found. However, 53.3% of the clinical isolates were resistant to TC, 12.2% to CP and 6.6% to OL, and 36.3% of pupil strains were resistant to TC, 12.4% to CP and 3.4% to OL.
Multi-resistant strains were 3.3% in clinical isolates and 1.9% in pupil isolates. Incidence of multi-reistant strains in this study period was lower than those in 1978-1979 and 1980-1982 periods, which were 23.1% and 31.7%, respectively.
In type 12, multi-resistant strains were encountered at a relatively high rate compared with other types, representing 9.5% in clinical strains and 7.5% in pupil strains, although these figures were smaller than those in the past.
In type 12, strains resistant to only TC were most prevalent and they were followed by those resistant to TC·CP and TC·MLs.
While, in type 4, incidence of TC-resistant strains remained at a high level, representing about 90%, as same as in the past. Multi-resistant strains were very rear in this type.

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Experiment in which mice were challenged with strains of Streptococcus sanguis derived from patients yielded pathohistologic findings similar to those of mucocutaneous lymph node syndrome (MCLS), i. e., systemic perivasculitis and/or vasculitis. PHA titers of paired sera from 14 patients against polysaccharides (PS) of S. sanguis and S. mitis showed, in general, no change during a 30-day hospital course, suggesting immunosuppression. There was one exception, however, whose titer on discharge exhibited apparent two scores increase only against sanguis. The titer against mitis didn't change at all. Since he was the only case who exhibited prodromal rash around the BCG vaccination sites, a reflection of immunopotentiation, the specific rise of the titer would indicate that S. sanguis must have been the trigger agent for MCLS.
Analysis of 141 colonies of viridans streptococci randomely taken from isolation plates of throat swabs from 10 selected patients showed negative results in cultures of S. sanguis around the preeruption stage, Of the 10 patients 4 were negative; their post-birth months were either at the preeruption or around the first eruption of the primary teeth (8 months). Since S. sanguis were isolated from all the tested mothers of culture negative infants, we suggest, as it has also been approved generally among oral microbiologists, that the organisms which usually reside and propagate on the tooth surface were transmitted from their mothers through nearest contact, that they directly attacked the mucous membrane, by secreting sialidase and protease like the strains from infectious endocarditis, because of the absence or scanty growth of teeth, or later directly through eruption opening sites during the eruption age, and that they invade and, after reaching the adjacent organs, and/or attaching to the endothelial surface of blood vessels by elaborating dextran, as demonstrated by our model experiment, they propagate there and produced toxins and/or proteases consecutively which may result in MCLS. We are now in the process of testing the above hypothesis.

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In order to investigate statistical relation between numbers of patients with streptococcal infectious disease and meteorological data, we carried out multiple regression analysis on numbers (dependent variable) of the patients per a hospital and ten days and meteorological data (independent variables) of mean temperature (°C), max. temperature (°), min. temperature (°), relative humidity (%), mean vapor pressure (mb), wind speed (m/sec), total radiation on a horizontal surface (MJ/m2), no. of hours with sunshine (h) and precipitation (mm) per ten days, during the period from September in 1976 through August in 1977 and from September in 1978 through February in 1985. Namely, they were used as mean data per ten days from period No.1 of the first ten days of January to period No.36 of the last ten days of December, respectively. Meteorological data were also analyzed after numerical and/or logarithmic transformations. The personal computer of type Canon CX-1 and its programs for the analysis were used in this studies.
Out of obtained multiple regression equations, two equations with high multiple correlation coefficients and high proportions were statistically significant: that is, one of them, with 0.946 of multiple correlation coefficient and 0.895 of proportion, was Y=5.216 logT_ME-0.017T_MAX+2.108logRH+1.990 logNHS-3.652 logTRH-0.558logP_R-5.548, and the other with 0.941 of multiple correlation coefficint and 0.885 of proportion was Y=1.595 logT_ME+0.005RH-0.010MVP+2.639logNHS-4.126 logTRH+2.407. Although some small errors were observed between estimated and surveyed no. of the patients from April to October, it was thought that the multiple regression equations were useful in estimation of no. of the patients.

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The cross-reactivities of T antibodies against types T3, T13 and TB3264 antigens and types T8, T25 and TImp. 19 antigens of group A streptococci in rabbit antisera and human sera were studied. The obtained results were as follows.
1) When T antibody titers of against homologous and heterologous antigens in rabbit antisera were compared, respectively, it was presumed that the cross-reactivities of anti-T3 serum with T13 and TB3264 antigens were comparatively low, but the cross-reactivities of anti-T13 and anti-TB3264 sera with heterologous antigens in T3/T13/TB3264 group were remarkable. On the other hand, anti-T8 and anti-T25 sera exhibited low corss-reactivities with heterologous antigens in T8/T25/TImp. 19 group.
2) When 250 serum specimens collected from people of Honjyo City, Akita, and Ecuador were tested, 22 (34.9%) out of 63 samples which possessed one or more of T3, T13 and TB3264 antibodies, showed antibody positive pattern of three types. Two (4.4%) out of 45 samples which possessed one or more of T8, T25 and TImp. 19 antibodies, also revealed antibody positive pattern of three types. However, we could not discriminate clearly in this report that these antibodies were produced against streptococci with homologous antigens or detected as cross-reactivities of heterologous antibodies in the same group, with the exception of comparison of their antibody titers.
3) On the other hand, 31 (49.2%) out of above 63 samples possessed only one of T3, T13 and TB3264 antibodies, and 32 (71.1%) out of above 45 samples possessed only one of T8, T25 and TImp. 19 antibodies. It was presumed that they were antibodies against homologous antigens, because group specific antibodies were removed previously by absorbents of heated types T6 and T25 streptococci before the tests.

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Group A streptococci were isolated from 690 (22.1%) out of 3, 128 of patients with various infectious diseases including scarlet fever, streptococcal infectious disease, tonsillitis, pharyngitis, upper respiratory infectious disease, etc. in the surveillance system of pathogenic microbes in Akita during the period from April, 1976 to March, 1986. The highest positive rate of the isolation wa 71.6% from patients with scarlet fever and the lowest rate was 0.9% from patients with diarrhea. Of 690 strains of the streptococci isolated, 608 (88.1%) were typed to 14 serotypes; type T12 (40.1%), T4 (19/3%), T6 (7.7%), Ti (4.8%), T13 (3.2%), T3 (3.0%), T28 (2.6%), TB3264 (2.5%), T25 (2.0%), T22 (1.4%), T14 (1.0%), T5 (0.1%), T9 (0.1%) and T23 (0.1%). Types T2, T8, T11, T18 and TImp. 19 of the streptococci, however, were not detected.
Although yearly changes were observed in serotype distributions of many types, the trend in recent years in these serotypes was summarized to next three points; namely, (1) types T12 and T4 were still predominant, (2) types T3 and T28 increased, and (3) types T1, T6 and T13 decreased. It was noticed especially that the streptococci of type 3 isolated hardly in Akita until 1982 began to increase from 1983.
Serotype distributions of types T12, T13, T25, T28 and TB3264 isolated from scarlet fever and streptococcal infectious disease of group I differed significantly from those from other infectious diseases of group III, with the exception of eruptive infectious disease, respectively. When serotype distributions of the isolates in Akita were also compared with those from prefectural and municipal public health institutes and health centers in Japan, the distributions of types T4, T12, T14 and T25 in Akita and types T1, T11, T13, T18, T22, T28 and TB3264 in Japan were significantly higher than those in Japan or in Akita, respectively.

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