Prevention of Anaphylactic Death in the Mouse Toxicity Prevention Test for Tric Agents

P R E V E N T I O N O F ANAPHYLACTIC D E A T H I N T H E MOUSE TOXICITY P R E V E N T I O N T E S T FOR TRIC AGENTS E. RUSSELL ALEXANDER,* M.D.,

AND J.

Classification of TRIC agent strains has been accomplished in mice using cross pro­ tection from toxic death after active immunization.1·2 Immunization and chal­ lenge are made by the intravenous route. In the conduct of this mouse toxicity preven­ tion test, increasing difficulty arose with a phenomenon of prompt death, occurring at an earlier time and having different charac­ teristics from the death ascribed to TRIC agent toxicity. Because of the similarity between this prompt death and mouse anaphylaxis,3'4 studies were made to define this phenomenon and to attempt its preven­ tion. MATERIALS AND METHODS

A. Conduct of the toxicity prevention test. The mouse toxicity prevention test was essentially the same as that developed by Wang and Grayston.1'2·6 Three- and fourweek-old N I H white Swiss mice were im­ munized intravenously with 0.5 ml of 1% yolk-sac suspension and challenged at five weeks of age by the same route. TRIC agents employed in these tests were TW-1, TW-3, TW-5, ND-3 1 and UW-1 2 . The chal­ lenge dose employed was estimated to be 1.5 LD50 by prior mouse toxicity titration and From the United States Naval Medical Re­ search Unit No. 2, Taipei, Taiwan, and the De­ partment of Preventive Medicine, University of Washington, Seattle. This study was supported in part by a United States Public Health Service research grant NB-03144, from the National In­ stitute of Neurological Diseases and Blindness, and by the Bureau of Medicine and Surgery, United States Navy. The opinions or assertions contained herein are the private ones of the au­ thors, and are not to be construed as official or reflecting the views of the Navy Department or the Naval Service at large. Reprint requests to: Dr. E. R. Alexander, De­ partment of Preventive Medicine, University of Washington, Seattle, Washington, 9810S. * Dr. Alexander is a Markle Scholar in Medi­ cal Science.

W.

FRESH,

M.D. (LCDR [MC] USN)

was given in 0.5 ml volume. Mouse toxicity titration was repeated in immunized mice on the day of challenge. In satisfactory tests the challenge was usually between 1.2 and 1.8 LD50.

B. Preparation of materials. The prepara­ tion of yolk-sac suspension of TRIC agent for toxicity tests was reported elsewhere.6 Pools were prepared by harvesting living eggs, after one-half to one-third of the inoc­ ulated eggs had died. Membranes of dead eggs were not used. A 40% yolk sac suspen­ sion (in terms of original yolk sac weight, 1.0 gm/ml = 100%) was made in sucrosepotassium-glutamate (SPG) by grinding and emulsifying with sterile mortars, sand, and pestles. (The method of grinding was changed to use of a Waring blender for one minute from August, 1965.) Materials were kept at low temperature during this process. Following light centri fugation (1500 rpm for 10 minutes), fat material floating on the surface of the supernate and gross debris were discarded. The centrifugation was made twice. The supernate emul­ sion was kept frozen' at — 65°C. Although heat killed or formalin killed yolk sac sus­ pensions may be used for immunization in this test, it is customary to use live orga­ nisms. With TRIC agents toxic or prompt deaths on the first or second immunization are rare. RESULTS

A. Toxic DEATHS AND PROMPT DEATHS Toxic deaths as a result of intravenous challenge were defined as those in which ill­ ness started not less than one hour after chal­ lenge and death occurred no later than 24 hours after challenge. Deaths rarely occurred after 24 hours in studies with usual TRIC agents. Prompt deaths (presumably nontoxic) always showed illness less than one hour after

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TABLE 1 EFFECT OF AQUEOUS EPINEPHRINE UPON THE PROMPT DEATH PHENOMENON IN THE MOUSE TOXICITY PREVENTION TEST

Prompt Deaths in Homologous Immunization— Challenge

Prompt Deaths in Heterologous Immunization— Challenge

P/T P/T* Control Challenge (no prior epinephrine) 27/51 21/51 1/24 0/24 Epinephrinef 1 Minute Before Challenge Epinephrine 30 Minutes Before Challenge 11/24 14/24 (Drug control epinephrine only in unimmunized and unchallenged mice P/T=0/10)

Prompt Deaths in Total Tests P / T (%) 48/102(47) 1/48 ( 2) 25/48 (52)

* P / T = No. of prompt deaths/total mice tested t Epinephrine administered in 0.2 ml volume intraperitoneally—0.01 mg/mouse

challenge, although they were never observed to start less than 10 minutes after challenge. In this way, and in the clinical appearance of the mice, the prompt death phenomenon could readily be differentiated from particle embolization, which occurred within seconds after immunization. Some of the prompt deaths could occur in the second hour, but in all cases unmistakable symptoms began in the first hour. During an interval of 10 minutes to one hour after intravenous challenge, mice that were later to die in less than three hours (most often in less than one hour) were noted to have a brief and transient episode of hyperactivity, being unusually responsive to external stimuli. They often scratched their noses, and swelling of the nose and lips was occasionally seen. This stage was followed by a quiet phase of less than 20 minutes where the mice would lie huddled and then prone. Breathing, initially difficult and shallow, became progressively deeper and slower, the fur was ruffled and there was often diarrhea. In the final phase the mice became unresponsive to pain, were prostrate, and had irregular gasping respi­ ration. Terminally, there were convulsive movements and the breathing character­ istically ceased before beating of the heart. This picture should be contrasted with the slow onset of toxicity in mice, with no hy­ peractive phase. The terminal phase of toxic death cannot be distinguished clinically from that of prompt death, however, and

the primary difference is in the timing and characteristics of onset. On pathologic examination, the distinctive findings in prompt deaths were marked pul­ monary vascular congestion and alveolar distension. Often there were pooling of blood in the viscera and some ischemia of liver and kidneys. The usual appearance of toxic death was one of focal or generalized atelectasis of the lungs, with prominent findings of liver, spleen and renal conges­ tion. There was often toxic hepatitis, and the general pathologic picture was of a toxic death. Thus, clinically and pathologically the phenomena of prompt death and toxic death could be differentiated. Furthermore, in both these respects, prompt death was very similar to mouse anaphylaxis.3'4'7 B. RESPONSE OF PROMPT DEATHS TO EPI­ NEPHRINE

The next step was to study the influence of drugs which affect mouse anaphylaxis upon this prompt death phenomenon. The first tried was epinephrine, which has been shown to be effective in prevention of mouse anaphylaxis only if it is used imme­ diately before challenge.8 In Table 1 and in subsequent tabulations of the effect of drugs upon the prompt death phenomenon, we have usually summa­ rized the results of a number of experi­ ments. We selected as immunization and challenge antigens those strains with which

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TABLE 2 EFFECT OF DOSE OF AQUEOUS EPINEPHRINE UPON THE FREQUENCY OF PROMPT DEATH IN THE MOUSE TOXICITY PROTECTION TEST

Control Challenge (no prior epinephrine )

Prompt Deaths in Homologous Immunization— Challenge

Prompt Deaths in Heterologous Immunization— Challenge

Prompt Deaths in Total Tests

(P/T)* 20/33

(P/T) 22/37

(P/T) (%) 42/70 (60) Drug Control (epinephrine in unimmunized mice) P / T

Prior Epinephrine (dose in mg/mouse) 0.2 0.1 0.05 0.025 0.0125 0.00625t 0.001

16/19 15/31 8/38 0/24 2/21 4/21 5/7

15/19 13/32 1/39 0/26 1/21 3/21 4/7

31/38 28/63 9/77 0/50 3/42 7/42 9/14

(82) (44) (12) ( 0) ( 7) (17) (64)

17/22 2/13 0/8 0/4 0/5 0/5

All epinephrine administered in doses of 0.2 ml intraperitoneally one to five minutes before challenge * P/T—No. of prompt deaths/total mice tested t This is a composite of 28 mice given 0.00625 and 14 mice given 0.005 mg

prompt death was more often associated. 30 minutes before challenge the frequency Certain pools of TW-5 with homologous of prompt death was the same as in the con­ TW-5 challenge or heterologous TW-3 chal­ trol. lenge were particularly apt to yield many The effect of dose size of aqueous epi­ prompt deaths. TW-1 and UW-1 strains nephrine upon the frequency of prompt were also often employed. Besides the pro­ death in the mouse toxicity prevention test pensity for certain strains to show this phe­ was studied (table 2). Once again, about nomenon, certain pools of these strains 50% of the control mice that received no more often resulted in prompt deaths. In prior drug died promptly, as did mice re­ this and the following tables, we have cumu­ ceiving as little epinephrine as 0.001 lated all tests using the same strains, and mg/mouse. In this series of tests 0.025 tests using different strains for immuniza­ mg/mouse seemed to offer the best preven­ tion and challenge, and present each sepa­ tion of prompt death (and with subsequent rately. We have not included data on the experience we now routinely use 0.02 toxic deaths or protection from them. Of mg/mouse). Many mice who received 102 mice tested with either heterologous or higher doses of epinephrine died promptly. homologous challenge, 40-50% of the mice This was an epinephrine toxicity death. died with prompt death before they could be That this toxicity is increased in challenged used for the mouse toxicity prevention test. animals can be seen by comparing results When 0.01 mg epinephrine per mouse was with the drug control mice (table 2 ) . In the inoculated intraperitoneally,* one minute be­ few mice studied pathologically "epinephrine fore challenge, only one of 49 mice died a toxicity" is similar to prompt death but the prompt death. When epinephrine was given deaths can be differentiated by time of death. Nearly all epinephrine toxicity deaths occur »Epinephrine U.S.P. 1:1000 aqueous solution in less than 10 minutes, but prompt deaths (Gotham Pharmaceuticals) diluted to 0.1 mg/ml, due to mouse anaphylaxis begin later. with 0.85% NaCl.

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TRACHOMA AND ALLIED DISEASES TABLE 3 EFFECT OF DOSE OF CHLORPROMAZINE AND PROMETHAZINE UPON THE FREQUENCY OF PROMPT DEATH IN THE MOUSE TOXICITY PROTECTION TEST

Control Challenge (no prior drug)

Prompt Deaths in Homologous Immunization— Challenge

Prompt Deaths in Heterologous Immunization— Challenge

P/T*

P/T

P/T

(%)

7/14

14/29

21/43

(49)

Prompt Deaths in Total Tests

Drug Control (in unimmunized mice)

Prior Chlorpromazine (dose: mg/mouse) 0.25 0.125 0.0625 0.03 0.015

0/14 2/14 6/14

0/15 0/15 0/29 4/14 3/14

0/15 ( 0) 0/15 ( 0) 0/43 ( 0) 6/28 (21) 9/28 (32)

0/6 0/6 0/10 0/4

Prior Promethazine (dose: mg/mouse) 1.0 1/13 0/14 1/27 ( 4) 0.5 0/13 0/13 0/26 ( 0) 0.25 1/13 4/29 5/42 (12) 0.125 — 2/15 2/15 (13) 0.0625 — 2/15 2/15 (13) Chlorpromazine and promethazine administered in 0.5 ml doses intraperitoneally :

0/4 0/10 0/6 0/6

P / T = N o . of prompt deaths/total mice tested

C. R E S P O N S E O F P R O M P T D E A T H TO CHLOR­

PROMAZINE AND PROMETHAZINE

Chlorpromazine8"10 and promethazine9·10 were also evaluated (table 3 ) . Prompt death in unprotected mice was about 50%. Chlorpromazine* was effective at a dose of 0.625 mg or above, and promethazine1, at a dose of 0.5 mg/mouse. Each of these drugs was given 0.5 ml intraperitoneally one-half hour before challenge. With chlorpromazine inoculation there were severe symptoms, even in protected mice. Within 10 minutes after injection, all mice lay prone. Then, breathing became difficult. The mice ap­ peared very weak, and the hind legs tended to show involuntary contractions. These * Chlorpromazine : Thorazine hydrochloride (Smith, Kline and French Laboratories) 25 mg/cc, diluted in 0.85% NaCl. t Promethazine : Promethazine hydrochloride (Phenergen) (Wyeth Laboratories) U.S.P. for in­ jection. 25 mg/cc, diluted in 0.85% NaCl.

symptoms lasted three to seven hours ac­ cording to dose of drug used (0.06 mg last­ ed three hours, 0.25 mg lasted seven hours). Mice injected with promethazine at 0.5 or 1.0 mg/ mouse were deeply sedated. D. RESPONSE OF PROMPT DEATHS TO RESERPINE AND CORTISONE

Reserpine8·10·11 was effective at doses of 0.06 mg or above (table 4 ) . This dose was given subcutaneously 18 hours before challenge.* Marked sedation with diarrhea was present in reserpine inoculated mice given 0.1 mg per mouse. Cortisone5 was clearly effective in preven­ tion of prompt death if given 24 hours be­ fore challenge10 (table 5 ) . It is fully effec­ tive only at a dose of 1.0 mg/mouse. t Reserpine : Serpasil 1.0 ml containing 1.0 mg for injection (Takeda Chemical Industries Ltd). § Cortisone : Cortisone acetate suspension. Sterile U.S.P. 25 mg/cc (Coast Chemical Company).

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TABLE 4 E F F E C T OF DOSE OF RESERPINE UPON THE FREQUENCY OF PROMPT DEATH IN THE MOUSE TOXICITY PROTECTION TEST

Prompt Deaths in Homologous Immunization— Challenge

Prompt Deaths in Heterologous Immunization— Challenge

P/T*

P/T

P/T

6/17

5/17

11/14 (79)

Control Challenge (No prior drug)

Prompt Deaths in Total Tests (%) Drug Control in Unimmunized Mice)

Prior Reserpine (dose: mg/mouse)

0.1 0/7 0/7 0/14 ( 0) 0/5 0.06 0/7 0/7 0/14 ( 0) 0/5 0.02 _ 1/7 2/7 3/14 (21) 0/5 Reserpine administered in 0.5 ml doses. For 0.1 mg dose, reserpine given intraperitoneally 48 and 24 hours before challenge. For 0.06 mg and 0.02 mg doses reserpine given subcutaneously 18 hrs before challenge * P / T = No. of prompt deaths/total mice tested

E.

RESPONSE OF PROMPT DEATHS IN

DBA/2

MICE

Tokuda and Weiser noted that the inbred strain of black mice DBA/2 were highly re­ fractory to anaphylaxis.8 They exhibited only mild symptoms following injection of three times the usual lethal dose of antigen, and even higher doses did not cause death. A colony of DBA/2 mice is being estab­ lished at NAMRU-2 and only the results of one preliminary experiment will be present­ ed, but the clear-cut nature of these results prompted their inclusion in this report (table 6 ) . There were not enough mice to study both mouse toxicity and toxicity pre­

vention, and therefore this preliminary ex­ periment was confined to toxicity alone. It is clear that with DBA/2 mice epinephrine was not necessary, as they did not exhibit the prompt death phenomenon and yet they did exhibit toxic death. In comparison, the N I H Swiss strain of mice required epineph­ rine to retard or inhibit the prompt death phenomenon. It must be noted that it is not yet established whether mouse toxicity can be prevented by immunization in these mice, nor whether there are quantitative differences in antigen needed for protection. Parenthetic­ ally, it should be noted that DBA/2 mice are harder to inject intravenously because of their

TABLE 5 E F F E C T OF DOSE OF CORTISONE UPON THE FREQUENCY OF PROMPT DEATH IN THE MOUSE TOXICITY PREVENTION TEST

(Heterologous Immunization—Challenge with T R I C Agents)

Control Challenge (No prior drug) Prior Cortisone (i.p. dose: mg/mouse) 1 mg 0.5 mg 0.25 mg 0.125 mg

Prompt Deaths P/T 9/23

(%) (39)

Prompt Deaths (cortisone 24 hr before challenge) P/T 0/23 4/23 15/22 14/23

(%) ( 0) (17) (65) (61)

Prompt Deaths (cortisone $ hr before challenge) P/T 5/23 5/23 5/23 7/23

(%) (22) (22) (22) (30)

TRACHOMA AND ALLIED DISEASES

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TABLE 6 COMPARISON OF PROMPT DEATH AND MOUSE TOXICITY IN DBA-2 AND NIH WHITE SWISS MICE

Immunization : 1% yolk-sac suspension ND-3 at 3 and 4 wk of age (i.V.) Challenge : 24% yolk-sac suspension (i.V.) Mouse Strain

Pretreatment

Prompt Deaths

Toxic Deaths

DBA-2

Epinephrine* None

1/19 0/18

18/18 18/18

NIH Swiss

Epinephrine None

1/12 5/11

11/11 6/6

* Epinephrine 0.02 mg/mouse inoculated i.p. 1-3 minutes before challenge

color, and they are very much more nervous and jumpy and hard to handle. At present, it takes about twice the time to test the same number of DBA/2 mice as it does N I H Swiss. DISCUSSION

The mechanism of mouse toxicity of TRIC agents following intravenous inocula­ tion of live organisms remains unknown.12 A similar phenomenon has been described for Rickettsiae,13 for lymphogranuloma venereum14 and for psittacosis.15 In these descriptions and in the original adaptations of toxic protection tests to TRIC agents by Bell5 and Wang and Grayston6 anaphylactic deaths are mentioned as rare occurrences, but little attention is given to them. In sub­ sequent reports by Bell16 it was stated that in many experiments there were clusters of deaths from 20 to 40 minutes after inocula­ tion which were considered to be anaphylac­ tic. An antihistamine, Thenfadil, was ad­ ministered intradermally before challenge at a dose of 0.32 mg/mouse, but was stated to be of questionable value. Although the im­ portance of the phenomenon was not stressed in early reports, in personal com­ munications between laboratories conducting these tests it became apparent that the phe­ nomenon was a major hindrance to the con­ duct of these tests. Even if the phenomenon had no effect on the toxicity of intravenous TRIC agent, or its prevention, in many in­ stances the recurrence of the phenomenon

required the use of twice the number of mice per test that would be required if it were eliminated. At NAMRU-2, the phenomenon occurred more frequently as changes were made in the methods of antigen preparation and immunization.1·6 Which factors were most important are still not clear. The incidence of the phenomenon is not dependent upon im­ munity, because no increase in the incidence is seen with increasing dosage of antigen in potency tests.6 Nor is it different in heterologous or homologous prevention tests. Also it is not directly related to the antigenic mass of egg yolk antigen, as the strains with which it is most often associated are not those requiring larger doses of challenge an­ tigen. This study had theoretical and practical aspects. Although the mechanism of the prompt death phenomenon deserves further study, it is clear from these experiments that it is a phenomenon of mouse anaphylaxis. In the earlier studies of Bell5 it was suggested that the phenomenon could be minimized by use of purer preparation of antigens. It has been our experience that this might apply to challenge antigen which, in most cases, is crude yolk-sac suspension, but use of purified vaccine preparation as immunizing antigen does not alter the oc­ currence of anaphylaxis. The practical aspect of these findings is the selection of a method for minimizing the interference of anaphylaxis with the mouse

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toxicity prevention test. Although chlorpromazine, promethazine and reserpine affect mouse anaphylaxis, their profound effect on the mice made their choice for rou­ tine use unsuitable. Cortisone was only effective in high doses. T h e inbred strain of D B A / 2 mice might be considered, but the mice were hard to inject and to handle. T h e inoculation of epinephrine remained the simplest and safest method. T h e transitory value of the protection from anaphylactic shock requires that epinephrine be given to mice intraperitoneally within five minutes of the challenge. F u t u r e studies will evaluate the use of epinephrine in oil to extend this interval, which if successful would facilitate the practical conduct of the test. SUMMARY

1. T h e phenomena of prompt death and toxic death of immunized mice when chal­ lenged with intravenous T R I C agents may be distinguished clinically and pathological­ ly. I n both respects the prompt death phe­ nomenon is similar to mouse anaphylaxis. 2. This prompt death phenomenon m a y be prevented by various drugs that prevent mouse anaphylaxis. 3. Aqueous epinephrine, given immedi­ ately before challenge, is effective in preven­ tion of mouse anaphylaxis, as is chlorpromazine. Promethazine and reserpine are effective when given at longer intervals be­ fore challenge. T h e last three drugs have considerable side-effects on experimental an­ imals. 4. Cortisone is effective in large doses. 5. T h e inbred strain of mice D B A / 2 are refractory to prompt death, while they do exhibit toxic death to intravenous T R I C agents. 6. O n the basis of these findings we now routinely employ aqueous epinephrine (0.02 mg L P . per 15-gm mouse less than five min­ utes before challenge) in the conduct of the mouse toxicity prevention test.

MAY, 1967

ACKNOWLEDGMENTS

We particularly wish to acknowledge the valuable technical assistance of Mr. C. H. Liu and his lab­ oratory associates at NAMRU-2 in the conduct of these investigations. REFERENCES

1. Wang, S. P. and Grayston, J. T. : Clas­ sification of trachoma virus strains by protection of mice from toxic deaths. J. Immunol. 90:849, 1963. 2. Alexander, E. R., Wang, S. P. and Grayston, J. T.: Further classification of TRIC agents from ocular trachoma and other sources by the mouse toxicity prevention test. Am. J. Ophth. 63: 1469, 1967. 3. Weiser, R. S., Golub, O. J. and Hamre, D. M. : Studies on anaphylaxis in the mouse. J. In­ fect. Dis. 68:97, 1941. 4. McMaster, P. D. and Kruse, H.: Peripheral vascular reactions in anaphylaxis of the mouse. J. Exper. Med. 89:583, 1949. 5. Bell, S. D., Snyder, J. C. and Murray, E. S. : Immunization of mice against toxic doses of homologous elementary bodies of trachoma. Science. 130 :626, 1959. 6. Wang, S. P. and Grayston, J. T. : A potency test for trachoma vaccine utilizing the mouse tox­ icity protection test for TRIC agents. Am. J. Ophth. 63:1443, 1967. 7. Schultz, W. H. and Jordan, H. E. : Physiolog­ ical studies in anaphylaxis. III. A microscopic study of the anaphylactic lung of the guinea pig and mouse. J. Pharmacol. 2:375, 1911. 8. Tokuda, S. and Weiser, R. S. : Studies on the role of serotonic and mast cells in anaphylaxis of the mouse produced with soluble antigen antibody complexes. J. Immunol. 86:292, 1961. 9. Halpern, B. N., Neven, T. and Spector, S. : On the nature of the chemical indicator involved in anaphylactic reactions in mice. Brit. J. Phar­ macol. 20:389, 1963. 10. Fox, C. K., Einbruder, J. M. and Nelson, C. T. : Comparative inhibition of anaphylaxis in mice by steroids, tranquilizers and other drugs. Am. J. Physiol. 192:241, 1958. 11. Gershon, M. D. and Ross, L. L. : Studies on the relationship of 5-hydroxy-tryptomine and the enterochromaffin cell to anaphylactic shock in mice. J. Exper. Med. 115:367, 1962. 12. Wang, S. P., Kenny, G. E. and Grayston, J. T. : Characterization of trachoma antigen protec­ tive against mouse toxicity. Am. J. Ophth. 63 : 1454, 1967. 13. Bengston, I. A., Topping, N. H. and Hen­ derson, R. G. : Studies of typhus fever. Epidemic typhus: Demonstration of a substance lethal for mice in the yolk sac of eggs infected with Rîckettsiae Prowazeki Nat. Inst. Health Bull. 183:25, 1945. 14. Rake, G. and Jones, H. P. : Studies on lymphogranuloma venereum: II. The association of

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TRACHOMA AND ALLIED DISEASES specific toxins with agents of the lymphogranuloma-psittacosis group. J. Exper. Med. 79:463, 1944. IS. Manire, G. P. and Meyer, K. F.: The tox­ ins of the psittacosis-lymphogranuloma group of agents: I. The toxicity of various members of

the psittacosis-lymphogranuloma venereum group. J. Infect. Dis. 86:226, 19S0. 16. Bell, S. D. and Theobald, B.: Differentia­ tion of trachoma strains on the basis of immuniza­ tion against toxic death of mice. Ann. N.Y. Acad. Sci. 98:337,1962.

F U R T H E R CLASSIFICATION O F TRIC AGENTS FROM OCULAR TRACHOMA AND O T H E R SOURCES BY T H E M O U S E TOXICITY P R E V E N T I O N T E S T E. RUSSELL ALEXANDER,* M.D.,

SAN P I N W A N G , M.D.,

T. THOMAS GRAYSTON,

In previous reports by Wang, Grayston and Chang,1·2 the classification of trachoma strains was described using the mouse toxic­ ity prevention test, ( M T P T ) as devised by Bell3'4 and modified by Wang. This report will present the results of cross protection tests on 50 strains of TRIC agent from var­ ious countries and various sites. Combining these observations with the previous reports of these laboratories, we have classified 80 TRIC agent strains. Although our experi­ ence is limited in terms of the number of strains studied, the fact that antigenic differences exist, and that they correlate with cross protection studies of ocular tra­ choma in monkeys,5 makes it advisable to construct a working classification of strains. Knowledge of these antigenic relationships From the United States Naval Medical Re­ search Unit No. 2, Taipei, Taiwan, and the Depart­ ment of Preventive Medicine, University of Wash­ ington, Seattle. This study was supported in part by a United States Public Health Service re­ search grant, NB-03144, from the National Insti­ tute of Neurological Diseases and Blindness, and by the Bureau of Medicine and Surgery, United States Navy. The opinions or assertions contained herein are the private ones of the authors, and are not to be construed as official or reflecting the views of the Navy Department or the Naval Service at large. Reprint requests to: Dr. E. R. Alexander, De­ partment of Preventive Medicine, University of Washington, Seattle, Washington, 98105. * Dr. Alexander is a Markle Scholar in Medi­ cal Science.

AND

M.D.

is essential for full interpretation of protec­ tion experiments in primates or in man. MATERIALS AND METHODS

The detailed description of the methods presently used is contained in a companion paper.6 Yolk sac suspensions of TRIC agents are prepared from 40% yolk-sac sus­ pensions in sucrose-potassium glutamate ( S P G ) . Pools are prepared by harvesting live eggs after one-third of the eggs have died. Membranes of dead eggs are not satis­ factory in this test. A typical cross protection test is outlined in Table 1. Three-week-old N I H Swiss Webster mice were immunized intravenous­ ly with 0.5 ml of a \% yolk-sac suspension of a TRIC agent. In the test illustrated in Table 1, mice were immunized against G-62, TW-5 and ND-3 strains. One week later, this immunization was repeated. After one further week, mice were challenged intrave­ nously with 0.5 ml of a crude yolk-sac sus­ pension, which was estimated on the basis of previous toxicity titration to be approxi­ mately 1.5 LD 50 . This toxicity titration was repeated again on the day of challenge. Epinephrine (0.02 mg/mouse) was injected intraperitoneally five minutes before challenge in order to prevent mouse anaphylaxis.7 The toxicity of intravenous TRIC agent for mice, although quantitatively reproduci­ ble, is very sensitive to slight changes in