EMBRYONATED EGGS FOR THE CHEMOTHERAPEUTIC STUDY OF GONOCOCCAL INFECTION TOMOJI TANI AND MINORU TASHIRO

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1 EMBRYONATED EGGS FOR THE CHEMOTHERAPEUTIC STUDY OF GONOCOCCAL INFECTION TOMOJI TANI AND MINORU TASHIRO Department of Microbiology, School of Medicine, Kanazawa University, Japan (Received : June 10th, 1955) Difficulties encountered in inducing experimental infection of animals with Neisseria gonorrhoeae have resulted in the achievement of numerous studies of varying devices (1, 2, 3, 4, 10, 11, 12), Some of those studies were accomplished by weakening the resistance of animals (for instance, mice) in order to facilitate the development of septicemia and some others by causing a localized infection (for instance, rabbit's eye) as it is observed in human cases. We investigated the availability of embryonated eggs, which have begun to be used extensively in various fields of studies on rickettsial, viral and bacterial infections. They were found to be highly susceptible to the gonococcal infection, and moreover, quite satisfactory as experimental animals for the investigation of the chemotherapy of gonorrhoea. Reported in the following are the findings of a series of studies accomplished in our laboratory. rhoeae: Experiment I Experimental infection of embryonated eggs with N. gonor Two strains freshly isolated from the patients suffering from gonorrhoeal urethritis were used. A culture grown for 24 hours on a blood-agar slant was suspended in the mixture of bouillon and physiological saline at a ratio of 1:4. Eggs on the 8th day of incubation at 39 Ž were used. Serial dilutions of the above suspension in a dose of 0.1 to 0.2 ml were inoculated into either the yolk sac, allantoic cavity, amniotic cavity or on the chorio-allantoic membrane of the embryonated eggs fib, 6, 8, 9). Then,they were incubated at 37 Ž for an observa tion period of 7 days. Six eggs were used for each dilution and some of the findings are shown in Table 1. Susceptibility of the eggs inoculated into the allantoic cavity was the lowest, all the eggs surviving the entire observation period of 7 days in spite of the inoculation in a dose of 15 mg of the organisms. The eggs inoculated on the chorio-allantoic membrane showed also a poor susceptibility all surviving the inoculation of 5 mg, but in this case, the inoculation of 10 mg caused a few deaths. While, the inoculation into the amniotic cavity caused the death of practically all the eggs received 10-3 mg within 5 days. Moreover, the inocula tion into the yolk sac killed all the eggs which had received 10-5 mg within 295

2 296 TANI et TASHIRO Vol. 8 Table 1. Virulence of N. gonorrhoeae inoculated into different sites of embryonated eggs (Experiment with Strain A) Control : Injection made with only the mixture of physiological saline and bouillon in a ratio of 4:1 4 days. The death of the eggs inoculated into the amniotic cavity are, however, inconsistent, and also the technique employed is much more complicated than the others. Intra-yolk-sac inoculation method, therefore, is considered to be best suited for the experimental study of the chemotherapy of gonorrhoea. Then we have investigated the distribution of gonococci in the yolk sac, in the blood and also in various organs of the embryo of the eggs received 10-2 mg of N. gonorrhoeae into the yolk sac. For observation of the organisms proliferated in the yolk, the content of the yolk sac was collected hourly by means of a syringe making a puncture through the side of the air sac. The material thus obtained was diluted 5 times with physiological saline and a loopful of this diluted material was smeared on a blood-agar plate. After an incubation at 37 Ž for 48 hours, the colonies formed were counted. For observation of the organisms proliferated in each organ of the embryo, prior to and after death,the organs were removed and washed with physiological saline for 3 times. Then, they were ground separately and a loopful of this ground material was likewise smeared on a blood-agar plate for the counting of the colonies grown after 48 hours incubation at 37 Ž. In the yolk, the number of organisms was found to increase following the elapse of time, while in the organs of the embryo, their proliferation was noted only in the organs of the died eggs especially in the blood.

3 1955 N. GONORRHOEAE 297 The strains successively transplanted on the blood-agar as long as for 6 months demonstrated a considerable decrease in their virulence, and when expressed in terms of 50% lethal dose (LD 50), the decrease was from 1/4 to 1/5 of the original LD50 in the mild case and from 1/200 to 1/3,300 in the severe case as shown in Table 2. Table 2. Comparison of virulence in terms of the 50% lethal dose on the 5th day after inoculation into the yolk between the freshly isolated strain and that after 6 months of successive transplantations on blood agar The embryonated eggs on the 8th day of incubation were found to be best suited for the present purpose, the M.L.D. being 10-5 mg. The resistance being increased following the advance in the days incubated, the M.L.D. against the eggs on the 14th day of incubation was found to advance up to 10-2 mg. Experiment II Resistance originally possessed by the strains of N. gonorrhoeae against various chemotherapeutic preparations: Employing 10 strains of N. gonorrhoeae, their in vitro resistance was determined against 12 kinds of sulfa-drugs; sulfamine, acetosulfamine, sulfapyridine, sulfathiazol, sulfamethylthiazol, sulfadiazine, sulfamerazine, marfanil, 3-4-dimethyl-5-sulfamidoisoxazol (trade name of the product used, thiazine-yamanouchi), 6-sulfamidopyrimidine (trade name, domian-dainippon), N1-3-4-dimethylbenzoyl-sulfanilamide (trade name, irgafen-fujisawa), supronal (Beyer), and 5 kinds of antibiotics; penicillin (Meiji), terramycin (Pfeizer), aureomycin (Lederle), chloromycetin (Park Davis), Streptomycin (Takeda). The medium used was Ishiwara's fluid medium (7), and the minimum growth inhibition concentrations of the above mentioned preparations were as shown in Table 3. The resistance of those strains against sulfa-drugs was found to be extremely variant ; for instance, the inhibition zone of sulfamerazine was from 1:4,000 to 1:2,048,000 depending on the strain used. Generally speaking, however, sulfathiazol and sulfadiazine revealed the strongest growth inhibition among the

4 298 TANI et TASHIRO Vol. 8 Table 3. In vitro growth inhibition zone of various chemotherapeutic preparations against 10 strains of N. gonorrhoeae sulfa-drugs tested. Likewise, the growth inhibition of antibiotics was variant depending on the strain tested and in the case of penicillin the minimum inhibition concentration was from 0.04 to u/ml. Growths of the 3 strains of N. gonorrhoeae isolated from the 3 cases of gonococcal infection resistant to penicillin treatment were found to be inhibited by penicillin in vitro at the concentration of 0.04 u/ml showing that they had not acquired any particular resistance. Experiment III Experimental chemotherapy with various antibacterial preparations : A freshly isolated strain was used (Strain D). The chemotherapeutic preparations used were 8 kinds of sulfa-drugs; sulfathiazol, sulfadiazine, sulfamerazine, marfanil, 3-4-dimethyl-5-sulfamido-isoxazol (thiazine), 6-sulfamidopyrimidine (domian), N1-3-4-dimethylbenzoylsulfanilamide (irgafen), supronal and 4 kinds of antibiotics; penicillin, terramycin, aureomycin and chloromycetin. Embryonated eggs on the 8th day of incubation were used. Prior to the therapeutic experiment, the 50% lethal dose (LD50) of each preparation was determined by the injection of varying concentrations on the chorio-allantoic membrane each in a dose of 0.5 ml after observation for 5 days and was found to run parallel approximately to the LD54 dose determined on mice.

5 1955 N. GONORRHOEAE 299 In the therapeutic experiment, the organisms in a dose of 10-2 mg were inoculated into the yolk sac and the eggs received the treatment in the same manner as in the toxicity test of drugs, and in one series simultaneously with the bacterial inoculation, in other series 6 hours after the inoculation. After the observation for 5 days, the 50% curative dose (CD 50) of each drug was found and the ratio between CD50 and LD 50 (chemotherapeutic coefficient) was determined. The results are shown in Table 4. Table 4. Comparison of CD50 and LD50 of various chemotherapeutic preparations by the injection on the chorio-allantoic membrane Out of the sulfa-drugs tested, irgaf en, thiazine, sulfathiazol revealed excellent therapeutic results and sulfamerazine, domian, sulfadiazine followed. As for the antibiotics, all were found with excellent therapeutic effect, particularly, penicillin was the best of all, its therapeutic coefficient being less than 1/1,000. This finding completely agrees with that reported on human cases, suggesting the excellent reliability of the embryonated egg method in the experimental chemotherapy of gonorrhoea. There are several reports on the use of embryonated eggs in the experimental chemotherapy of gonorrhoea (2, 3, 4), but all of them were not satisfactory as the amount of the organisms inoculated on the chorio-allantoic membrane was too large. The present authors, as the result of systematic investigations, discovered the intra-yolk-sac method which will enable the infection of embryonated eggs with a minute amount of the organisms, and moreover, this method was found to be fully satisfactory for the experimental chemotherapy of gonococcal infection.

6 800 DANI et TAESHIRO Vol. 8 CONCLUSIONS 1. The inoculation of N. gonorrhoeae in such a small dose of 10-5 mg into the yolk sac of the embryonated eggs on the 8th day of incubation could kill them within 3 to 4 days. The organisms were recovered from the blood and the organs of the embryo of the died eggs and the organisms were found to proliferate most abundantly in the blood. Whereas, the culture from the organs of surviving embryos was negative. 2. Following the above mentioned method, the inoculation into the amniotic cavity showed also a high infectivity. 3. The virulence of a strain freshly isolated was much stronger than that of a strain successively transplanted on the artificial culture media. The comparison of the LD50 between a freshly isolated strain and that after transplantations repeated for 6 months on culture media revealed a decrease in virulence from 1/4 to 1/5 of the original LD50 in mild case and from 1/200 to 1/3,300 in severe case. 4. In vitro resistance of freshly isolated strains of N. gonorrhoeae against various chemotherapeutic preparations was considerably different depending on the strain tested. In the case of sulfa-drugs, the minimum growth inhibition concentration was from 1:4,000 to 1:2,048,000. Sulfathiazol and sulfadiazine were found to have comparatively strong growth inhibitory influence. The growth inhibitory influence of penicillin was about 0.04 to u/ml. 5. The in vitro resistance of the 3 strains of N. gonorrhoeae isolated from the patients resistant to penicillin treatment was found to be only 0.04 u/ml. 6. After inoculation of 10-2 mg (100 M.L.D.) of N. gonorrhoeae into the yolk of embryonated eggs, treatments were attempted with varying kinds of chemotherapeutic preparations administering them on the chorio-allantoic membrane. Better results were obtained from the use of antibiotics than those responded to sulfa-drugs. Penicillin was the best of all, its therapeutic coefficient being below 1/1,000. REFERENCES 1) Bradford, W.L. and Titzler, R.: Experimental gonococcal infection in the chick embryo, Proc. Soc. caper. Biol. and Med., 34, , ) Hill, J.H. and Piths, A.C.: The growth of Neisseria gonorrhoeae on the chorio - allantoic membrane of the chick embryo, J. of Urol., 41, 81-83, ) Morrow, G. and Berry, G.P.: The cultivation of Neisseria gonorrhoeae on the chorio-allantoic membrane of the chick embryo and the use of this technique for the study of sulfanilamide, J. Bacter., 36, , ) Bang, F.B. and Bang, B.: Sufanilamide, Sulfapyridine and sulfathiazol therapy of gonococcal infection of the chorio-allantoic membrane, Proc. Soc, exper. Biol. and Med., 46, , 1941

7 1955 N. GONORRHOEAE 301 5) Goodpasture, E.W.: Some uses of the chick embryo for the study of infection and immunity, Amer. J. Hyg., 28, , 1938 The developing egg as a culture medium, J. Labor. and clin. Med. 26, , ) Fukuzumi, S.: On the cultivation of Rickettsia prowazeki in the embryonated egg, Saikingaku-Zasshi, 544, , 1941 (in Japanese) 7) Ishiwara, T.: Studies on N. gonorrhoeae, Seiby, 36, , , , , 1952 (in Japanese) 8) Inoue, S.: Studies on the experimental chemotherapy by embryonated eggs, Nihon-Saikingaku-Zasshi, 4, 73-76, , , 1950, (in Japanese) 9) Ikeno, K.: Studies on the chemotherapy by embrynated eggs, J zen-igakkai- Zasshi, 56, , 1954 (in Japanese) 10) Miller, C.P., Drell, M.J., Moeller, V. and Bohnhoff, M.: Experimental gonococcal infection of the rabbit's eye, J. Infect. Dis., 77, 193,-200, , , ) Miller, C.P.: Experimental gonococcal infection of the rabbit's eye, Amer. J. Syphilis, Gono. and Vener. Digs., 32, , ) Miller, C.P.: The enhancement of virulence of the gonococcus for the mouse, Amer. J. Syphilis, Gono. and Vener. Dis. 28, , 1944