Virus multiplication and histopathologic changes were investigated in pancreatic tissue of youngadult mice inoculated intraperitoneally with coxsakie B viruses.
Further, in vitro virus multiplication in the pancreatic tissue infected with coxsackie B3 virus was tested by Rose's circumfusion organ culture. The following results were obtained.
1) Coxsackie virus B 1, B3, B5 and B6 have multiplied in mouse pancreatic tissue infected through intraperitoneal inoculation with the viruses and multiplied to the highest titers at the third day after the inoculation and then their titer decreased. Coxsackie virus B2 and B4 showed no multiplication.
2) In the pathologic findings in the pancreas infected with coxackie B viruses, B 1, B3 and B5 produced severe acinar cell necrosis and the necrotic lesions spread widely. But, the islets of Langerhans have maintained approximately the normal structure. Coxsackie virus B2, B4 and B6 produced no lesion.
3) The mouse pancreatic tissue infected with coxsackie B3 virus was cultivated by Rose's circumfution organ culture, but, there was no virus multiplication.

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We discussed the sensitivity to drugs of Streptococcus pyogenes Group A gathered from 1974 to 1975 as a yearly report after 1967 and drew a conclusion like the following:
1) All the supplied organisms had a sensitivity to Penicillins and Cephalosporins and not developed a tendency for tolerance at all.
2) The changing pattern of sensitivity to Chloramphenicol and Macrolides including Lincomycins is almost similar to each other. The inclination for tolerance originated from 1970 was decreased in both drugs in 1975.
3) Serotypes of the main organisms found from 1974 to 1975 were 12 and their isolated rate was approximately 70%. But, compared with the past data, it showed a decreased tendency this year.
4) Serotype 12 was often found in the resistant strains to many drugs of Chloramphenicol, Tetracycline, Macrolides and Lincomycins and its occurring rate was 72.4% in 4 years.
5) Serotype 4 was frequently seen in the resistant strains to Tetracycline alone and its occurring rate was 61.4% in 4 years.

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The drug sensitivity to 10 antibiotics of the 1, 353 strains of hemolytic streptococci consisting of 843 patient strains and 510 healthy-carrier strains (1, 176 strains of which were from Group A) which were collected in 1972-74 at 26 institutions scattersring throughout the country were subjected to the study. All the strains were found sensitive to PC, AB-PC, and CEX in contrast to Macrolide antibiotics such as EM, JM, OL and MDM together with LCM to which they were either sensitive or partly (33-38%) highly resistant and thus two peaks of the MIC distribution curve for each of Macrolides were depicted. The MIC of TC distributed widely ranging from sensitive area to highly resistant area (53% belonged to 100 μg) whereas in case of CP the distribution was within a relatively narrow range and 58% fell into 25-50μg.
Though the strain number was small, the sensitivity of the Groups B, C, and G to Macrolides was still retained. Among these three Groups Group B was least sensitive
Regarding the resistance pattern, 11 patterns appeared, of which sevenfold resistance such as TC/CP/EM/OL/JM/MDM/LCM occupied the first rank (427 strains), followed by TC single-resistance (278 strains), TC/CP double-resistance (243 strains), CP single-resistance (19 strains) and other minors in this order. The total number of strains up to the third rank from the first one occupied 96% of the whole number of resistant strains examined.
The 422 strains showing sevenfold-resistant pattern, with the exception of 5 untypable strains, fell into type 12. This was a marked contrast to the other patterns such as TC, CP, or TC/CP which covered a variety of types. Type 4 was siso outstanding in such context that all the 84 strains, with exception of 6 strains, were TC single-resistant. Further, type 3 was apparently differed from type 4 and showed TC/CP double resistance suggesting liability to the acquisition of CP-resistance. Since type 3 is an newly appeared epidemic type, any decisive conclusion can hardly be drawn about its resistance character, but type 4 has a long history of epidemic and therefore it can be mentioned that this type bears some barriers to the resistance acquisition to CP and Macrolides. Thus it was made clear for thefirst time that there exist some differences or orders among types in the liability of resistance acquisition to each of the antibiotics, i.e., the resistance pattern may differ according to type.
A similar finding relevant to resistance acquisition of type 12 was obtained between our epidemiological data and the experimental results on resistance transduction by bacteriophage performed by Ubukata, Konno, and Fujii (J. Antibiotics. 28, 681, 1975). Our data, as shown in Table 5, suggested that the sevenfold resistance was initially accomplished by three-step acquisition of type-12 strains through TC-single and then TC/CP-double resistance. Furthermore a difference in rapidity of the resistance acquisition between the three steps
was noted which was reflected by the time-lag between the year intervals, as can be postulated from those on the abscissa in Fig. 1, in whidh the steap upheaval of the percentage resistance started. This relative time-lag of the second-step resistance acquisition to the third one might be explained by the results of transduction experiment performed by them in which the transduction of TC-single resistance was, without exception, brought about independently and separately from other antibiotic resistance, whereas CP- and Macrolide resistance were transduced concomitantly.
The causative factor of the alteration of the “epidemic wave” from type 4 to type 12 occurring in the past has not been made clear up to now, however it may also be explained by the marked difference in resistance acquisition of these two types to CP and Macrolides.

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In November 1975, we observed 5 cases of scarlet fever from a classroom of the first grade in an elementary school in Sapporo. In this classroom and other one as control, throat cultures for 13-hemolytic streptococci were carried out. This examination was attempted 3 times for the period from December 1975 to February 1976.
The results were as follows:
1. In this classroom the isolation rate of P-hemolytic streptococci was 64.1%, on the other hand in another classroom which had no scarlet fever patient was 19.1%.
2. The higher level of isolation rate in this classroom was observed during 3 months with 3 times examinations.
3. Oral administration of antibiotics (ABPC) which was parformed to only the children who carried f3-hemolytic streptococci was effective to decrease the number of the carriers.
4. Serotyping of the strains isolated from the children in this classroom revealed T28, T6, T4, T3, and T12, among them T28 and T6 were the predominant types, especially T28 was observed in 56.0%. In this connection, however, we couldn't obtain T28 but found T6 from 3 cases out of 5 cases of scarlet fever. Fom 2 remainder patients any p-hemolytic streptococci was not revealed.
5. 5 strains isolated from 3 patients showed the same pattern on drugs susceptibility tests against 7 kinds of antibiotics, but we couldn't find out this pattern among 65 strains isolated from the children in concerned 2 classrooms.
6. As for isolation rate we found out little difference between the streak plate method and the pour plate method, and could elevate the isolation rate by employing these 2 methods at the same time.

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Children in M nursery school in Kurobe City, Toyama Prefecture, where a small epidemic of reumatic fever caused by an erythromycin-resistant M type 12 streptococcus of group A occurred from November to December in 1973, were followed up both bacteriologically and serologically for approximately 1 year after the epidemic.
1. The carrier rate of hemolytic streptococci in this population was dramatically decreased 6 months after the epidemic, no M type 12 streptococcus carriers being detected at this time (June, 1974). The rate, however, re-increased in November 1974, 1 year after the epidemic, and the numbers of M type 12 streptococcus carriers outnumbered those in the epidemic period.
2. The individual carrier state of M type 12 strains between the 3rd and the 4th follow-up examinations (November 26, 1974 and January 16, 1975 respectively) was mostly unchanged, indicating the sedentariness of this type in the population of M nursery school.
3. Among the individuals who possessed the ASO antibody titer of 333 or more when the epidemic occurred, the average antibody titer decreased by 3 dilution tubes after 6 months and by 1 tube after another 6 months. Two cases of fresh streptococcal infection were suspected by a significant increase in ASO antibody titer during the follow-up examination period, one being a new carrier of M type 12 strain in the winter 1 year after the epidemic.

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One class each of the 2nd grade in two primary schools from northern part of Toyama city (school A and D in industrial zone) and two from southern part (school B and C in agricultural zone) were randomly chosen, and the carrier state of hemolytic streptococci in these school children was, sequentially and quantitatively, investigated.
1. Carrier rates of hemolytic streptococci were, in general, significantly high, although variations were observed depending on the geographical or seasonal changes.
2. Both erythromycin-resistant M type 12 and erythromycin-sensitive T type 12 (M untypable) strains of group A, dominant in shhools B and D respectively, were sendentary in each population, the individual carrier states seeming to be also stationary.
3. Quantitative examinations revealed that many of the 102 carriers totally observed in this investigation have extremely low ratio of hemolytic streptococci to the total bacterial counts (either aerobic or anaerobic) in pharyngial swabs. Thus 95% of the carriers had the ratio of 1/100 or less, 65%, 1/10, 000 or less, and only 3 to 4 carriers showed the ratio of 1/10.
4. The carrier group in schools A and B, in general, possessed higher levels of both ASO and ASK in comparison to the non-carrier group which showed significant deviation in these antibody levels.
5. Two among 102 carriers, who were carrying, in significant quantity, erythromycin-registant M type 12 strains, seemed to have a potential to become an important source of an epidemic by these organisms. These two may be the most important object if any measure is to be done for the eradication of these organisms. The virulence of T type 12 (M untypable) strains is to be clarified

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Samples of 414 sera were collected from patients with scarlet fever and healthy carriers of group A streptococci, and were examined for antibodies to group A streptococcal T antigens. T antigens were prepared by digesting type T strains of group A streptococci with trypsin. After absorption of group specific agglutinin in tested serum specimens with heated type T 6 and 25 streptococci, type specific antibodies (agglutinins) to T antigens were detected by agglutination test of microtiter method, using 0.85% saline containing 0.1% bovine serum albumin as diluent and T antigens at the concentration of 0.35OD₂₈₀. The obtained results were summerized as follows.
1. Thirty eight (90.5%) out of 42 patients with scarlet fever in Tokyo and Akita in 1973-1974 from whom type T 3, 4, 6, 12, 18, and 22 group A streptococci, were recovered, respectively, showed no or poor antibody response against T antigen of the isolate. It was difficult to develope the antibody response to T antigen of the isolate in cases of such patients who received antibiotic therapy.
2. All of 22 healthy children examined with paired sera from whom type T 12 or 22 streptococci were isolated revealed a significant antibody response to T antigen of the isolate, during the epidemic of scarlet fever caused by type T 12 streptococci in Kakunodate town, Akita, 1973. They had no antibiotic therapy. Therefore, it will be possible to utilize T antibody as indicator for seroepidemiological surveys on group A streptococci.
3. Of 77 healthy carriers from whom type T 4, 6 and 12 streptococci (first group) were recovered, respectively, in Nishime and Jyumonji towns, Akita, 1972-1974, 94.8% showed antibody titer of 1:≥20 to T antigen of the isolate in their sera after three months from the recovery. However, of 79 healthy carriers from whom type T 5 or B 3264 streptococci (second group) were recovered, only 51.9% revealed the antibody response to the isolate. On the basis of the result, it was suggested that T antibody responses to streptococci in first group might differ from them in second group.
4. T antibody positive rates to type T 1, 3, 4, 11, 12, 14, 27, 44, 49, and B 3264 streptococci, respectively, were 30.6-77.8% in Akita (Nishime and Jyumonji, 1972-1974), but no T antibodies to type T 8, 25 and Imp. 19 were detected. On the other hand, although T antibody positive rates to type T 1 and 12 streptococci, respectively, were 16.1-21.0% in Tokyo (1974), the rates to other type T antigens were less than 10% or zero. It was suggested that contamination of streptococci in Akita might be more remarkble than in Tokyo.

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ASO titre (hemolysis inhibition reaction), Blue ASO titre (passive agglutination reaction) and ASK titre (Kinase test) of the paired serum specimens which were taken from 40 patients with scarlet fever were measured.
A rise in antibody titre by more than 2 tubes in the course of illness was observed in 47.5% by ASO, in 75.0% by Blue ASO and in 42.5% by ASK.
In 16 cases by ASO and 18 cases by ASK in which no change in antibody titre was observed, Blue ASO titre showed significant rise by more than 2 tubes in the couse of illness in 68.7%(11 in 16 cases) and in 66.7%(12 in 18 cases), respectively. However hard we tried to decrease negative diagnosis rate by combining the above 3 tests, we could not down it below 20%.

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The third and fourth complement components (C₃, C₄) were studied in the patients with scarlet fever without any complications. In the initial stage of scarlet fever, the values of C₃ and C₄ in the serum of the patients were significantly elevated than the values in one to five weeks after onset, but the fluctuations of C₃ and C₄ levels were always within normal ranges.

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Separation and identification of erythrogenic toxin produced by group A hemolytic streptococci, which included strain NY 5, T 18 and five isolated strains from scarlet fever patients, were carried out by DEAE-Sephadex column equilibrated to 0.05 M Tris-HCl buffer (pH 8.6). Linear gradient elution was carried out by 0.05M Tris-HCl buffer (pH 8.6) containing 0.4M NaCl.
A and B toxin produced by strain NY 5 was respectively eluted from the column by 0.14M and 0M NaCl, and C toxin produced by strain T 17 was eluted by 0.07M NaCl. Concentrated culture filtrates of five isolated strains were chromatographed with this procedure and separated toxin fractions were identified serologically. In this procedure the eluate by 0 M, about 0.08M, and about 0.15M NaCl was respectively neutralized by anti-B, abti-C, and anti-A rabbit serum. Furthermore, unknown toxin type eluted by 0.25M NaCl was recognized in one of isolated strains. From these results, NaCl concentration in the eluted toxin fraction is usable for presumptive identification of streptococcal erythrogenic toxin type. All of these isolated strains produced C toxin and it was major toxin component, and except for one strain they produced one or more other minor toxin type.

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The method of comparative clinical evaluation on various antibiotics was investigated either by open-study or double-blind study with the patients sufferring from scarlet fever.
The results were summarized as follows.
1. In comparison of alleviating effect to fever after initation of the medication, the difference of effectiveness of drugs was signifiant only when comparing drugs have marked difference in activity in general.
2. When the drugs of similar effectiveness are compared, the difference hardly appeares in the comparison of alleviating effect to fever, but the difference is significant in eradicating effect on pharyngeal H. S.
3. When the disappearing rate of pharyngeal H.S. is used as the marker in the judgement of drug effectiveness, open-study and double-blind have almost the same value in evaluation of drugs used.
4. The improvement of clinical findings and disappearing rate of pharyngeal H. S. are most significantly correlated.

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