INTRODUCTION.*

By THEOBALD SMITH, M.D.

This inquiry was suggested by the following important problems bearing upon the restriction of diphtheria:

1. Is there any difference in the pathogenic power of diphtheria bacilli from different localities?

2. Is the pathogenic activity of bacilli producing diphtheria in the summer season different from that of those producing disease in winter?

3. Is there any reduction in the pathogenic power of bacilli in cases in which they persist in the throat after recovery?

4. Are there any differences noticeable between the bacilli of mild and those of severe cases?

The third and the fourth questions have been attacked by other observers, while the first and the second have not been especially investigated. The answers to the third and the fourth questions have been, as a rule, negative. Observers have found little or no difference in bacilli from mild and severe cases, nor have they been able to show any recognizable loss of virulence in the bacilli persisting in the throat after recovery.

The reasons for entering upon this subject again were the opportunity we have had of examining cultures from different towns within the State, and more especially certain improved methods of cultivation by which the maximum toxin-producing capacity of each bacillus could be brought out and measured more accurately than had been done heretofore.

The selection of cultures for the study of the questions stated above has not been entirely satisfactory, mainly because much of the clinical information necessary to a proper choice was not accessible

The writer wishes to acknowledge the faithful assistance of J. R. Stewart, to whom the preparation of the culture media was chiefly entrusted. As will be seen from what follows, this is not a simple task.

at the time the cultures were received, and in some instances obtainable only with difficulty at the last moment when the final results were tabulated. We hope, however, that the material at hand may be supplemented by more in the near future.

THE MODE OF ACTION OF DIPHTHERIA BACILLI.

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It is now a generally accepted theory that diphtheria bacilli act in the main through the toxins which they produce, and which are rapidly diffused into the fluids containing the vegetating bacilli. The contents of the bacilli themselves seem to be of little moment as pathogenic factors. Park and Williams allowed the washed diphtheria bacilli to soak for a week in a 0.5 per cent. alkaline carbolic solution." The injection of one cubic centimeter did not "produce any marked reaction in a 500-gramme guinea-pig," although the bacilli themselves were powerful toxin-producers. Kossel† collected the bacillar membranes from cultures, washed the bacilli repeatedly by centrifugalizing with 0.5 per cent. sodium chloride; then, after killing them with vapors of chloroform, he extracted them for several days in a few cubic centimeters of weakly alkaline fluids. The extract was only feebly poisonous, for it required 5 cubic centimeters to kill a 360-gramme guinea-pig in fortyeight hours.

Brieger and Boer ‡ found that shaking diphtheria bacilli with ammonium chloride and allowing them to stand for eighteen to twenty hours removes the toxin from the bodies of the bacilli. The bacilli after extraction were fatal to a 500-gramme guinea-pig, in doses of 0.01 gramme of bacillar substance. They acted by producing local necrosis. Brieger states that antitoxin had no effect upon this action of the dead bacilli, and that immunization towards it by gradually increasing doses failed. The poison itself withstood an hour's boiling.

These experimental observations, taken together, show that the toxin in the culture fluid and not the body substance of the bacilli themselves is to be looked upon as the disease agent. The success following the prompt application of antitoxin in sufficient doses is an additional support to this view. Moreover, the bacilli themselves do not penetrate into the body in large numbers, hence need not be specially considered as adding to the toxic effect of their products.

*"Journal of Exp. Med.," I., page 174.

"Deutsche Med. Wochenschrift," 1896.

+"Centralblatt f. Bakteriologie," XIX., page 977.

We may, for convenience, regard the disease-producing power of diphtheria bacilli as made up of two elements, toxicity and virulence. The former represents the rate of accumulation of toxin in culture fluids, and is easily measured; the virulence, on the other hand, which may be regarded as the behavior of diphtheria bacilli toward living tissue, is as yet an unknown quantity. This distinction between the toxic product of diphtheria bacilli and their inherent vital power to cope with living tissue seems to be established, at least experimentally, by the increase in virulence of diphtheria bacilli in their passage through a series of guinea-pigs, which has been reported by various observers. Thus, Aronson* states that a culture which was at first fatal to guinea-pigs of medium weight, in 0.1 cubic centimeter doses, was fatal, after some serial inoculations, in doses of 0.008 cubic centimeter. That is to say, its virulence was augmented twelve times. This experiment evidently means not that the toxin formed in the sub-cutis of guinea-pigs became twelve times stronger in quality at the end of the series, but that the bacilli injected were capable, by an adaptation of some sort, to multiply much more abundantly toward the end of the series, and hence produce more toxin. The other explanation, that the toxin itself had become more potent in quality, could only gain confidence if the bouillon culture produced much more toxin at the end of the experiment than at the beginning, the conditions remaining precisely the

same.

To compare the disease-producing power of diphtheria bacilli from different sources, it was, therefore, thought best to study the relative accumulation of toxin in bouillon, and eliminate the bacilli by filtration before the test upon animals. The writer is fully aware of the fact that but an instrument of pathogenic power is here dealt with, and under artificial conditions, since we do not know the nature of the nutritive fluid which the bacilli make use of on mucous membranes, nor, as a consequence, whether the toxin production in bouillon is a true index of the production of toxin on mucous membranes. The problem is, in fact, very complex, as with all infectious diseases, and all we can hope to do at a time is to examine one factor of disease as carefully as possible, while eliminating all the others for the time being. The use of living cultures upon animals is of no service in these experiments, because it introduces at once three variable factors: (1) the bacilli as potential toxin-producers after

"Berl. klin. Wochenschr.," 1893, Nos. 25 and 26.

injection; (2) the poison of their bodies after destruction; and (3) the toxin pre-formed in the culture fluid injected. As a consequence, all who have used cultures find them uncertain in their action, as compared with the toxin alone. The bacilli injected as nearly free from fluid as possible are equally unreliable as measures of toxicity, as the following tests show :

Two cultures of diphtheria bacilli are selected, which differ considerably in toxin-producing power, as is shown in Table I., where the toxin-producing power of one (No. 14) is about three times that of the other (No. 40). Inclined agar cultures are prepared from each, and after six days' growth the bacilli are removed with a platinum wire, the amount of moist bacilli weighed and stirred in 5 cubic centimeters sterile bouillon, making a moderately cloudy suspension. One cubic centimeter contained by weight about 0.0007 grammes of moist bacilli.

Bacillus No. 14. - Five-tenths cubic centimeter of the suspension, injected subcutaneously into a guinea-pig weighing 313 grammes, is fatal in five days; 1 cubic centimeter is fatal to a 330-gramme pig in six days.

Bacillus No. 40. Of the suspension made in the way described, 1 cubic centimeter is injected into a guinea-pig weighing 315 grammes. Animal just escapes death, and is chloroformed on the sixth day. Another, weighing 330 grammes, receives 0.5 cubic centimeter. A slough forms at the place of injection. The guinea-pig remains in fair condition.

Though these tests show a greater activity on the part of Bacillus No. 14, yet we miss here not only the sharp definition in the results obtained by varying the dose of the same culture, but also in comparing the effect of the same doses of cultures from different sources.

A prolonged study of the relative production of toxin in bouillon under certain uniform conditions has shown such remarkably uniform results with the same culture, even after long intervals of time, that the results obtained in this way may be accepted as showing an inherent difference in the various bacilli studied.

THE METHOD EMPLOYED IN COMPARING THE TOXIN PRODUCTION OF DIFFERENT CULTURES.

In a former publication* the writer has given the conditions which must be fulfilled in order that a maximum accumulation of toxin may take place in bouillon cultures. The facts there considered and

* "Trans. Association American Physicians," for 1896.