STUDIES ON TETANUS TOXOID III. ANTITOXIC RESPONSE IN GUINEA PIGS IMMUNIZED WITH TETANUS ALUM-PRECIPITATED TOXOID FOLLOWED BY TET- ANUS SPORES F. G. JONES AND W. A. JAMIESON Lilly Research Laboratories, Indianapolis, Indiana Received for publication February 4, 1936 When an individual has been given one or more injections of tetanus toxoid and, at a later date, is injured and possibly infected with Clostridium tetani, what further precautions should be taken: (1) Administer antitoxin? (2) Give another injection of tetanus toxoid to accelerate the production of antitoxin? (3) Give no further treatment, depending on his present immunity to take care of the infection? The original idea in administering tetanus toxoid was to avoid the use of antitoxin. We have shown (Jones and Moss, 1936; Jones, 1936) that another injection of toxoid at the time of injury will quickly increase the antitoxin content twentyfold. This study was undertaken to ascertain whether a huge dose of tetanus spores would accelerate the production of additional antitoxin. Owing to the fact that there has been some difficulty in producing tetanus consistently in animals, we tried various methods. Gye and Cramer (1919) found that the ionizable salts of calcium injected together with the washed spores of Cl. tetani or of certain other anaerobes led to the development of the corresponding infections in their typical and fatal forms, while the washed spores alone showed no tendency to develop in the tissue after inoculation. The chlorides of sodium, potassium, ammonium, strontium, or manganese had no such effect. Later, Fildes (1927, 1929 a, 1929 b) showed that in the sub- 33 JOURNAL OF BACTERIOLOGY, VOL. 32, NO. 1
34 F. G. JONES AND W. A. JAMIESON cutaneous tissues of the living guinea pig the oxidation-reduction potential (Eh) at ph 7.0, is about +0.12 volt, while tetanus spores will not germinate readily at a potential more oxidizing than +0.01 volt at this ph. He further showed that the injection of solutions of calcium chloride led to the production of localized areas of oxygen deficiency, thus providing conditions suitable for the germination of tetanus spores. In our first experiment 1 cc. of a semisolid agar culture of Cl. tetani was mixed with 9 cc. of a 10 per cent solution of calcium chloride. Three guinea pigs were injected with this mixture as follows: GUINEA PIG NUMBER WEIGHT AMOUNT MIXTURE RESULTS CC. 5287 305 0.1 Dead 42 hours 5289 300 0.5 Dead 40 hours 5288 295 1.0 Dead 40 hours All pigs developed typical toxic symptoms of tetanus with a slight localized area of infection. They died, unquestionably, from the free toxin present. We next washed a culture twice and suspended the residue in 10 cc. physiologic salt solution. One-tenth cubic centimeter was injected intramuscularly. After 24 hours there was localized paralysis and at 82 hours complete paralysis, the pig dying after 96 hours. Again the reaction appeared to be due to free toxin. In the next experiment the culture of Cl. tetani was washed twice, the residue taken up in 10 cc. physiologic salt solution and then heated in a water bath at 560C. for 3 hours. One-tenth cubic centimeter was combined with 0.9 cc. of 10 per cent calcium chloride and 2 pigs injected intramuscularly, each with 0.1 cc. Both pigs showed localized symptoms with paralysis of the hind-leg that had been injected, but the symptoms did not spread, and both pigs recovered. We next increased the amounts of culture and calcium by combining 1 cc. of the washed and heated culture with 1 cc. of a 50 per cent solution of calcium chloride. Two guinea pigs were injected, one with 0.5 cc. and the other with 1 cc. Both de-
STUDIES ON TETANUS TOXOID 35 TABLE 1 Guinea pigs inoculated with C. tetani DATE GUINEA PIG WEIGHT AMOUNT RESULT NUMBERj 7/24/35 To 1 cc. semi-solid agar culture (not washed or heated) added 9 cc. of 10 per cent CaC12. Injected the following amount: 5287 305 gm. 0.1 cc. Dead 42 hours 5289 300 gm. 0.5 cc. Dead 40 hours 5288 295 gm. 1.0 cc. Dead 40 hours. Free toxin present 8/ 8/35 Culture washed twice and taken up in 10 cc. NaCl. Injected intramuscularly 4684 277 gm. 0.1 cc. Dead 96 hours with a typical tetanus toxemia Free toxin still present 8/12/35 Washed culture heated at 560C. for 3 hours. 0.1 cc. taken up in 0.9 cc. of 10 per cent solution CaCl2 I Guinea pig 2663 had received 0.25 2663 } cc. 460 gm. 0.1 alum toxoid one month cc. pre- 5943 460 gm. 0.1 cc. viously. Guinea pig 5943 was normal. Neither pig developed tetanus 9/ 5/35 To 1 cc. tetanus culture (washed and heated) added 1 cc. of 50 per cent CaCl2 9313 415 gm. 0.5 cc. Dead 100 hours Normal pigs. Both 9034 90340 410 gm. gin 1.0 cc. c. Dead 88 hours hurs typical pigs died tetanus with 9/ 9/35 Used tetanus culture as prepared 9/5 for the balance of the experiments 2663 Treated 0.5 cc. Localized tetanus. No general symptoms. Recovered 5943 Control 0.5 cc. Dead 90 hours, typical tetanus 9/13/35 9336 Treated 0.5 cc. Localized tetanus 9337 Treated 0.5 cc. Localized tetanus 9339 Treated 0.5 cc. Localized tetanus 9033 Control 0.5 cc. Dead 88 hours, typical tetanus 9/23/35 2650 Treated 0.5 cc. Localized tetanus 2655 Treated 0.5 cc. Localized tetanus 2661 Treated 0.5 cc. Localized tetanus 9338 Treated 0.5 cc. Localized tetanus 9380 Control 0.5 cc. Dead 112 hours, typical tetanus
36 F. G. JONES AND W. A. JAMIESON veloped a severe localized infection with paralysis; on the third day they showed a generalized paralysis. The pig receiving 1 cc. died at 88 hours and the one receiving 0.5 cc. at 100 hours. This appeared to be an ideal mixture and in all other experiments it was used in a 0.5 cc. dose. This will be known as T. C. M. (tetanus spores and calcium chloride mixture). E-4 z 04 P4 04 5. K I II III {' TABLE 2 Potency of guinea pigs treated with tetanus alum precipitated toxoid IV l! 9337 300 7/15 4 4, 8/26 9/13 8 8 V R~ M ~ a0 ~. - '0 8.0.0.0 a'80.0 9339 290 tc o F R to co COD LO co m ez a 40 8/1 00 41 co cm4 cm m 4'- 93 300 7 m4 82 0 Oa E 5. 229 } 4 0 01 9 0 12 1985 units 19556 units units 1935 units 1935 units units units units unite units 2663 315 7/15 4 2 4 9/9 4 4 265 31 }7/5 40 8/1 00 9/2 4-2 - 9336 290 7/15 4 8/12 16 9/13 4 { 2650 315 7/15 4 8/12 16 9/23 4-2 -2 2655 315j 2661 295 7/5 4 8/26 16 9/23 12 12 12 9338 290J'71 EXPERIMENT I. INJECTION OF THE SPORES GUINEA PIGS INTO IMMUNIZED On 7/15/1935, guinea pig 2663 was given 0.25 cc. of tetanus alum-precipitated toxoid subcutaneously. On 8/12/1935 (4 weeks later) its serum tested 4 units antitoxin per cubic centimeter. On 8/26/1935 (6 weeks later) it again tested 4 units per cubic centimeter. On 9/9/1935 this pig was inoculated intramuscularly into the right hind-leg with 0.5 cc. of T. C. M. Normal guinea pig 5943 was injected with the same volume of T. C. M. Results. Guinea pig 2663 developed a localized infection in 72 hours with some local paralysis which gradually disappeared, l 12 45 2 8
STUDIES ON TETANUS TOXOID and the infection cleared up at the end of 2 weeks. The pig was bled 7 days after being infected and the serum still showed 4 units. It was again bled after 18 days and the titer remained 4 units. The control pig, 5943, developed a localized infection with paralysis in 48 hours which became generalized in 72 hours and the pig died at 90 hours with typical tetanus. 37 EXPERIMENT II On 7/15/1935, 2 guinea pigs, each weighing 290 grams, were injected subcutaneously with 0.25 cc. tetanus alum precipitated toxoid. Four weeks later they were bled, and their combined sera showed a potency of 4 units per cubic centimeter. Immediately after bleeding, they received a second injection of 0.25 cc. tetanus toxoid. They were bled 2 weeks after the second injection and their combined sera had a potency of 16 units per cubic centimeter. On 9/13/1935, both pigs, together with a normal pig, received 0.5 cc. of T. C. M. The control died in 88 hours with typical tetanus. One week later the treated pigs were bled and tested separately. Each serum had a potency of 4 units per cubic centimeter. A week later they were again bled and tested. One pig had a titer of 8 units antitoxin per cubic centimeter and the other of 4 units. EXPERIMENT III In experiment II the potency was much lower when tested after injecting the spores. Not knowing whether the potency had dropped before or after the spores were administered, we decided to bleed just before giving the spores in this experiment. On 7/15/1935, two guinea pigs, each weighing 315 grams, were injected subcutaneously with 0.25 cc. tetanus alum-precipitated toxoid. Bleedings 4 weeks later tested 4 units. The pigs received a second injection of 0.25 cc. toxoid. Two weeks later their sera tested 16 units, but after 4 weeks the potency had dropped to 4 units. Then the 2 pigs together with a normal pig were injected
38 F. G. JONES AND W. A. JAMIESON intramuscularly with 0.5 cc. of T. C. M. The normal pig developed typical tetanus and died in 112 hours. The treated pigs had only a localized infection with complete recovery. They were bled 2 days after receiving the spores and tested -2 units per cubic centimeter; after 2 more days they tested -2 units and after 3 more days just 2 units. A month after the second injection of toxoid, these pigs had dropped quite a bit in potency. After giving the spores there was another temporary drop, probably owing to a large amount of the available antitoxin being used to neutralize the massive infection, but within a week they were returning to the potency which was present before the infection. EXPERIMENT IV One guinea pig inoculated on 7/15/1935 with 0.25 cc. tetanus alum-precipitated toxoid when bled 4 weeks later tested 4 units, and at 6 weeks still tested 4 units. It was again injected with 0.25 cc. toxoid. Eighteen days later, this pig and a normal one were injected intramuscularly with 0.5 cc. T. C. M. The control pig developed typical tetanus and died in 88 hours. One week later the treated pig tested 8 units and after another week it still tested 8 units. EXPERIMENT V Two other pigs inoculated on 7/15/1935 with 0.25 cc. tetanus alum-precipitated toxoid showed 4 units of antitoxin 4 weeks later and the same after 6 weeks. On this date they were again inoculated with 0.25 cc. toxoid. Two weeks after their combined sera contained 16 units antitoxin per cubic centimeter, but after another 2 weeks they had dropped to 12 units. On this date both pigs together with a normal pig were injected intramuscularly with 0.5 cc. T. C. M. The normal pig developed typical tetanus and died at 112 hours. The treated pigs were bled 48 hours after receiving the culture and their combined sera still tested 12 units. After 96 hours they were still 12 units, but after 7 days had dropped to 8 units.
STUDIES ON TETANUS TOXOID 39 DISCUSSION The massive infective dose used in these tests may be considered unfair, but we wanted to obtain the extreme value. It is our intention in a further study to attempt to standardize an infective dose that will prove fatal in from 10 to 12 days. The protocols would indicate that with the present infective dose, which proves fatal to a guinea pig in from 88 to 112 hours, it requires from 2 to 3 units of antitoxin per cubic centimeter of blood in the immunized pig to neutralize the toxin produced from the infection. None of the treated pigs receiving the infective dose showed any marked increase in antitoxin within 18 days, but we shall continue to bleed these pigs at intervals and test their serum. It is possible that a smaller infective dose would permit an acceleration in antitoxin production, while the huge infective dose produced such a rapid infection that no time was allowed for acceleration in antitoxin. Also, our tests were made for 2, 4, 8, 12, and 16 units which are at very wide intervals, being as much as 50 to 100 per cent. In testing human subjects after administering diphtheria toxoid, only enough antitoxin is required to neutralize a Schick test dose, which is 1/50th M.L.D. Contrasted with this one must consider that 1000 M.L.D. of tetanus toxin are required to neutralize 1 unit of antitoxin, so we may be expecting too much when we test for a titer of such a wide range as from 2 to 4 units of antitoxin. These experiments would indicate the advisability of administering another dose of tetanus toxoid at the time of injury. CONCLUSIONS Guinea pigs that have received one or more injections of tetanus alum-precipitated toxoid, and are infected within 2 months, are protected against a massive dose of tetanus spores which kills normal pigs in from 88 to 112 hours. A massive dose of tetanus spores does not markedly accelerate the production of antitoxin in guinea pigs that have been previously immunized with tetanus alum-precipitated toxoid.
40 F. G. JONES AND W. A. JAMIESON REFERENCES FILDES, P. 1927 Tetanus. VI. The conditions under which tetanus spores germinate in vivo. Brit. Jour. Exper. Path., 8, 387. FILDES, P. 1929a Tetanus. VIII. The positive limit of oxidation-reduction potential required for the germination of spores of B. tetani in vitro. Brit. Jour. Exper. Path., 10, 151. FILDES, P. 1929b Tetanus. IX. The oxidation-reduction potential of the subcutaneous tissue fluid of the guinea pig; its effect on infection. Brit. Jour. Exper. Path., 10, 197. GYE, W. E., AND CRAMER, W. 1919 Sixth Sc. Rep. Imp. Cancer Res. Fund, 40, 57. JONES, F. G. 1936 Studies on tetanus toxoid, II. In press. JONES, F. G., AND Moss, J. M. 1936 Studies on tetanus toxoid, I. Jour. Immunol., 30, 115. Downloaded from http://jb.asm.org/ on October 13, 2018 by guest