ON THE COMPOSITION OF B. WBLCHII TOXIN.

By HEEBERT HENRY, M.D.

Bacteriological Laboratory, Birmingham.

I. INTRODUCTION.

Is a previous paper it has been shown that B. rwelchii produces a hzmolysing substance which is of the nature of a toxin(l). The series of in-vitro experiments detailed in that report do not, however, give any indication of the pathogenic significance of this htemotoxin, nor is any evidence adduced as to the possible presence of other toxin-like substances in filtrates of cultures.

All observers who have worked experimentally with filtrates agree that one of the features of the illness which results in inoculated animals is hzmolysis(3). A minimal lethal dose when given to mice intramuscularly causes death in twenty-four to forty-eight hours, the illness being characterised by a progressively severe destruction of red corpuscles, accompanied by hamoglobinuria. If the amount of the inoculum be increased to five or ten minimal lethal doses the illness lasts no more than two to three hours; hamoglobinuria appears in twenty to thirty minutes after inoculation, and at death the red corpuscles are so completely destroyed that only small numbers of whole cells may be observed in thick blood smears. The same intensity of blood destruction is also frequently observed in pigeons treated intramuscularly. On the other hand, rabbits, which succumb to intravenous inoculation after an illness of two to three hours’ duration, show a t post-mortem a much slighter degree of hzmolysis. The urine is a t times blood-stained, but this is by no means constant, and tinting of the serum with haemoglobin is often absent. If, both in the mouse and the rabbit, the toxic dose be neutralised with antitoxin so that the mixture will not give in-vitro htemolysis, then the animal suffers no ill effect. It follows that the hzmotoxin which hzmolyses in vitro is probably identical with that which gives hzmolysis in the animal.

When we came to work with specimens of precipitated toxin it was found that mice and rabbits reacted rather differently although tested under the same conditions that obtained previously. Whereas, with fresh filtrates given intravenously, the rabbits died in two t o three hours or recovered completely, in the case of precipitated toxin there appeared to be a considerable number of late deaths occurring in twenty-four to forty-eight hours after inoculation. Moreover, it was

A Report to the Medical Besearch Counncil. From the City

JOIIRN. or PATK -POL. XXVI. 3 K

498 HERBERT HENRY

observed that many of these animals showed very little evidence of blood destruction. The same change was noted in the case of mice; the duration of the illness was lengthened and hwmoglobinuria, which was practically a constant finding in the case of filtrates, became a subsidiary feature.

It would thus appear from the data we have briefly summarised that filtrates differ from precipitates in that they cause greater intravitam hwmolysis and produce a more acute illness. If this difference in lethal capacity were due to difference in hzmotoxin content, then one must assume that in both products there is some other killing constituent, and that this too must vary in amount. Or alternatively, if there is no other killing element, then the hzmotoxin is responsible for the lethal capacity both of filtrates and of precipitates, though it must be altered in precipitates so as to be less active. The question then that emerges is as follows. Is the hamotoxin the only toxin present, or is there some additional lethal element in filtrates, just as tetanus filtrates contain tetanolysin and tetanospasmin ?

11. THE ItELATIONSHIY BETWEEK THE HWMOTOXIC TITRE OP u. WELCIIII

TOXIN AND ITS MINIMAL LETHAL DOSE IN MICE.

The lethal capacity of a number of culture filtrates freshly prepared and obtained from three different strains of B. wdchii was tested by the intramuscular inoculation of mice of 18 to 22 grams, the minimal lethal dose being taken as being the smallest amount of toxin which is capable of producing death within forty-eight hours. A t the same time the hEmolytic titre of each filtrate was estimated by the standardising method already described ('). The figures representing the hwniotoxic unitage are thus comparable throughout the whole series of filtrates.

With the two series of results, it is possible to arrive a t a figure which represents the hzmotoxin content of each mouse mininial lethal close (table I.). This figure is remarkably constant, and the result suggests that the lethal capacity of filtrates and their hxmolytic activity are due to one and the same substance. Or it may be that if the hzmolytic effect and the lethal effect are due to separate bodies then these substances are found in fresh culture filtrates in the same relative proportions. The evidence in regard to tetanus is against this second assumption, for tetanolysin would appear to bear no quantitative relationship to tetanospasniin : tetanolysin is, however, so unstable that it would be difficult to establish the exact relationship.

A series of filtrates which had been kept in cold storage over long periods of time were tested as regards their lethal and hwmolytio capacity. At the same time a series of precipitated toxins were measured in the same manner. These latter had been subjected to the process which, as previously described, seemed to give the best

COMPOSITION OF B. WELCRII TOXIN 499

6 ’4 ...

6 ‘6 6 -4

6 *5 6 ’2 ...

6 *7

6 .O 6 ‘2 6 ’4 ... 6 ‘0

6.2 6 .i

...

...

...

yield of dried product, viz., a primary precipitation with ammonium sulphate followed by re-solution and a secondary precipitation with two

1740 1330 1160 870 800 800 616 264 280 200

1740 1660 1000 320

1000 1000 550 330

volumes of alcohol (2).

I Toxin. I Strain.

TABLE I. The hmotoxic titre per mouse minimal lethal dose of freshly-prepared

B. welchii culture filtrates.

Hamotoxic IC1.L.D. Hzmotoxic I Ago In weeks. I “;!$;pf‘ I e,c. I ytyzg 1

Ytrain. 1 Toxin.

267 617D 1 64 1160 0.25 378 B 85 24 870 0 ‘25

617 D 22 1250 0 ‘2 0 ‘8

383 617 D it: 1 0’6

396 403 386 387 402 394 395 399 389 405 A

397 398 401 c 406 B A

290 220 250 250 240

pH. 1 Haemotoxic units per 1.0 C.C.

397 327

400 ::: I 500 I t’i 1 1:; 1 B 85 617D

M.L.D. C.C.

0.1 0.1 0.15 0 -2 0.2 0 ‘25 0 ‘3 0.5 0 ’6 0 -8 0.1 0 ‘1 0 ‘2 0 ’5 0’17 0 ‘2 0 ‘3 0 ‘5

Earnotoxic units per M.L.D.

174 133 174 174 160 200 185 132 168 160 174 166 200 160 170 200 165 165

The results obtained (table 11.) show that specimens of the first series contain a considerably larger amount of hemolysin per minimal lethal dose than do fresh filtrates, and that those of the second series

TABLE 11. The hernotoxic titre per mouse, milzimal letha2 dose of stored

toxins amd of precipitated toxins.

Hzmotoxic Hamotoxic

1.0 C.C. 1 M.L.D. Ago In weeks. I u n i t * per I “$D I units per 1

I 1000 0’1 I 100 I I 0’08 90

,500 HERBERT HENR Y

Neutral mixture..

contain an amount of hzmolysin which is only about half that of filtrates. The lethal doses of fresh filtrate, of stored filtrate, and of precipitated filtrate are thus very different in regard to their hzmotoxin content. There must, therefore, be some other lethal factor as well as hzemotoxin. In the case of stored filtrate this lethal factor has degenerated niore rapidly than has the hzmotoxin, whereas in the case of precipitated filtrate the process of precipitation has resulted in loss of active hzemotoxin.

1

111. THE HBMOLYTIC CONTENT OF TOXIN-ANTITOXIN MIXTURES WHICH ARE NEUTRAL TO MICE.

These experiments show that under certain conditions the hzmolytic titre of filtrates affords a measure of their lethal capacity. There is also furnished some evidence as to the possible existence of another toxin, which tends to degenerate with the ageing of filtrates in cold storage, and which is more easily precipitated by the methods we have employed. If, then, there are two separate toxins, each of which is neutralisable by antitoxin, it may be possible by investigating toxin- antitoxin mixtures to find that the neutralisation of one does not coincide with that of the other.

The amounts of serum R173 necessary to protect 20-gram mice against a multiple of the minimal lethal dose of various toxins were

TABLE 111. The hamolytic content of toxin-antitoxk mixtures whicih ure

neutral to mice.

K 1 K11

383 384 329 329 329

Neutral mixtiira to mice. n m t n 1 i t . v

617D 0'0005 ' 0.0012 617D I i 1 0'0005 0'0014 617D 0'00045 0-0022 617D 0.0012 0,0035 SR 9 5 0'0005 0 '001 4 S R 9 1 i; I 0'0012 0'003 SR 9 0'003 0.006

A. B.

IIemotoxic iiiiits in

1 M.L.D.

C . 1 D. I I I I I 1

FILTILATES.

SP 1 B85 1 5 1 0.00017 1 0'0007 I 250 [ 166 o m 0 2 0~0008 166 2nn

143 125 125 100 100 125 100

100 59

100 166 50

100 166

COMPOSITZON OF B. WELCHTI TOXIN 501

determined by experiment. As a rule five niinimal lethal doses of toxin were mixed with graduated amounts of antitoxin, the total volume being made up to 1.0 C.C. and kept for one hour a t room temperature before inoculation. It was thus possible to select a series of toxin-antitoxin combinations which were just neutral to mice. The hzmotoxin content of these mixtures was then estimated. At the same time the amount of serum capable of completely inhibiting in-vitro hzmolysis by the corresponding amounts of toxin was determined. One thus had in the case of each toxin a series of connected observations (table 111.) :-

A. the amount of serum R 173 sufficient to protect mice against a

B. the amount of serum R 173 which gave complete neutralisation

C. the hzmolytic titre of the toxin used. D. the amount of uncombined hwmotoxin present in the toxin-

antitoxin mixtures neutral to mice. The amount of serum which mill protect mice is niuch less than

that which gives complete neutralisation of the hsmotoxin. With fresh filtrates, the protecting dose of serum is one-fourth to one-fifth of the full neutralising dose, and with precipitated toxins about one-half to one-fifth. It is also found that the amount of uncombined haemotoxin present in mixtures neutral to mice is approximately equivalent to the amount present in the minimal lethal dose. These results agree fairly closely with those previously recorded (I) in respect of the fractional saturation of B. welchii hzmotoxin with antitoxin. They show too, that, within the limits of the experiments herein described, the neutralisation of hEmotoxin by this particular antitoxin proceeds payi passu with the neutralisation of the full toxin. The findings with toxin 329, however, suggest that the protecting doses in mice for multiples higher than five minimal lethal doses deviate from the law of multiple proportions.

known amount of toxin.

of the haemolytic activity of the same amount of toxin.

IV. THE SATURATION OF TOXIN WITH CORPUSCLE SUSPENSIONS.

A toxin, SP, which killed mice in a dose of 0.1 C.C. intramuscularly, was diluted with an equal volume of washed rabbit and mouse corpuscles in normal saline, a control being put up with an equal volume of saline. Two sets of mixtures were made up, one series being incubated at 38" C. for one hour, the other series for two hours. The minimal lethal dose of these mixtures, which were not centrifuged, was then tested in mice intramuacularly. Although the actual quantity of mouse cells used was much less than that of rabbit cells, yet the results (table IV., A) show that mixture with mouse cells reduces the toxicity for mice, while rabbit cells have no appreciable effect in this direction. A few days later the same toxin was put up with cells as before, and after incubation the contents of each tube were divided, one half being tested without

JOURN. OF PATH.-VOL. XXVL 2 K 2

502 HERBERT HENRY

___ 11.

...

... 0.35 0.35

centrifuging (I.), the other half after centrifuging (11.). The results (table IV., B) show that saturatioii with mouse cells reduces the toxicity whether the mixture be centrifuged or not, whereas in the case of rabbit cells the reduction of toxicity is found only in the case of the centrifuged mixture. Experiments of a like nature were then carried out with two precipitated toxins and with one stored toxin. The findings show no reduction of toxicity for mice (table IV., C).

TABLE IV. Toxins sutzwated with corpuscle suspensions and then tested

intruwtzLsc3nrly on nzice.

___._____.

I . 11.

0.35 ... 0 '4 ... 0'35 0.4 0 '4 0 5

A. SP SP

B. SP SP

c. 383 2

267 2 384 1 2

1 2

1 2

Saline.

Toxin. , Curltrol. ! I

0.25 0.25

0'25 0 '3

0.25 0.17 0 5

Yinimal letlial dusr. after saturatioii with red cells.

llahhit. 1 Figeon.

-

Minimal lethal dose.

llabhit cells. I Mouue Cells.

I.

0 '3 0.25

0.25 0.25

...

...

...

0.25 ... 0 '25 0.17 ::: 1 0'2 0 d 0 5

Two filtrates and two precipitated toxins were mixed with suspensions of rabbit, pigeon, and mouse corpuscles, the mixtures being left in the cold room for twenty-four hours. The cells were then removed by centrifuging and the toxins tested in mice. The toxicity for mice was still further reduced (table V.) by admixture with mouse cells and slightly by saturation with pigeon cells; rabbit cells had no definite effect. The precipitated toxins were again practically unaffected by this proceclure.

TABLE V. Toxins saturated with corpuscle suspensions for tzoent?/-four hours in the

cold and then tested i n t ~ ~ ~ ~ s c ~ l a r ~ ? ~ on mice.

0.25 i 0.3 I 0.5 1 0'6 I 'E 1 0'4 0 '4 0% 0'8

3R3 j 0'25 0.25 384 ' 0.17 1 0.2

~

Two filtrates and two precipitated toxins were saturated for twenty- four hours in the cold with thick suspensions of rabbit cells and then

COMPOSZTZON OF B. WELCHII TOXZN 503

given to rabbits intravenously (table VI.). The filtrates were slightly diminished in toxicity by this means, whereas the precipitates were unaffec tecl.

TABLE VI. Toxins saturated with rabbit cells in the cold and then given

Toxin.

~

F Control . . Saturated . .

G Control . . Saturated , .

383 Control . . Saturated . .

384 Control . . Saturated . .

intravenously to rabbits.

Lethal dose, C.C.

5.0. - + + - I - -

+ + _ _

__

3’0.

+ - -

i-

--

__ 2.5. 2.0. -

+ + + + +

1’5.

_ _

f ti-

- 1’0.

__

-

V. THE SATURATION OF TOXIN WITH MUSCLE.

I t has been shown by numerous workers that brain tissue taken from certain animals not only neutralises tetanus toxin but is also capable of combining with much more toxin than it neutralises. It has also been demonstrated that this property of brain matter depends on the susceptibility of the particular animal for tetanus : the brains of horse, man, and mouse have a stronger action than those of rabbit, pigeon, and chicken. Since, then, B. tetani produces a lysin which can be absorbed with red cells or with corpuscle stroma and also tetanospasmin which can be absorbed with brain tissue, it appeared to be not improbable that B. welchii, which produces a lysin capable of partial neutralisation by susceptible red cells, might also make another toxin capable of neutralisation or of physical absorption by some tissue taken from a susceptible animal, muscle being suggested by the occurrence of muscle necrosis in inoculated animals. If sterile pieces of muscle from rabbit, guinea-pig, or pigeon be incubated in contact with strong solutions of toxin, it is found that after several hours the muscle swells up and becomes opaque. Microscopically the appearance presented by these swollen bits of muscle agrees very closely with that which has been described in human gas gangrene.

The experiments showed that the toxicity of B. welchii toxin can, under certain conditions, be reduced by muscle, and that the amount of hzemotoxin present is unaffected.

Experiment 1.-Eight grams of guinea-pig muscle, cut up into small pieces and freed from blood as far as possible by washing in saline, were incubated with toxin for two and a half hours. The control

501 HERBERT HENRY

specimen of toxin was incubated for the same period. Both the saturated specimen after centrifuging and the normal specimen were then inoculated intravenously into rabbits with the following results :-

Dose, C.C. i I Toxin M'3.

Normal . . . Saturated. . .

~

Experinmat 2.-A mixed toxin in 20 C.C. amounts was saturated with 8 grains each of fresh muscle, liver, and kidney from a guinea-pig, and also with a similar amount of autoclaved horse muscle. The mixtures were incubated for two hours then centrifuged and tested as before. Fresh muscle alone was effective.

Normal . . . . Saturated fresh muscle .

,, fresh brain. . ., fresh kidney . ,, autoclaved horse

Dose, C.C. ._"I 3.0.

+ + + - _ _

+

.Experime./zt 3.-The next experiment was devised to ascertain whether dried muscle had the same function, or whether strong muscle extracts in saline coulcl also reduce the toxicity. A specimen of rabbit muscle chopped up into small bits was partially dried in large Petri dishes for two days a t 38°C. The desiccation was completed over sulphuric acid in a partial vaciiuni. It was then ground to powder in a mortar. Another specimen of rabbit muscle finely chopped was steeped in saline overnight in the cold room. The solution from this

Dose, C.C.

Toxin WE. 5.0.

-

Normal control. . . + Saturated fresh muscle . Saturated dried muscle . +

Toxin, Muscle extract, Result.

5 '0 5 '0 + 2 '0 4 '0 + 2 '0 8 .O +

10.0 -

C.C. C.C.

...

COMPOSITION OF B. WELCH11 TOXIN 505

B

6 *5 6 '2 6'3 6 '2 ...

6 '5

6 -5 6 .O

was then expressed, centrifuged, and passed through a Berkfeld filter. The test consisted in placing 20 C.C. lots of toxin in contact with 4 grams of dried muscle and 8 grams of fresh muscle respectively. Various mixtures of toxin and muscle extract were also made. All these preparations and a toxin control were incubated a t 38" C. for two hours and then given intravenously to rabbits.

The results show that neither dried muscle nor saline extract of muscle can take the place of fresh muscle.

Experiment 4.-The method of testing toxin by the intravenous inoculation of rabbits is a ready one in that it gives a positive or negative result in a short space of time, but it has the disadvantage of providing no more than an approximate titration. I t is also expensive. We sought therefore t o transfer our observations in regard t o toxin absorption by muscle to the intramuscular test in mice. It was found to be a difficult matter to get sterile muscle from rabbits or guinea-pigs. I n the case of an intravenous dose the presence of a few organisms makes no difference to the result which is obtained almost immediately. With mice, however, treated intramuscularly, where the effect is not complete for twenty-four to forty-eight hours, a few stray B. welchii or streptococci may give rise t o a fatal infection which invalidates the experiment. Practically all our earlier results in mice had to be discarded for this reason.

Muscle in a sterile condition can be easily removed from the pectoral region of pigeons, and in the following series of experiments this muscle was used. Chopped pigeon muscle freed from blood by washing was added to toxin in the proportion of 8 grams of muscle to 20 C.C. of toxin. After incubation for the periods indicated the centrifuged toxins were injected into mice. I n addition the reaction of each toxin was estimated colorirnetrically before and after muscle saturation, and the amounts of hzemotoxin present in the normal and the saturated toxins were determined (table VII.). The results were

TABLE VII. Toxins saturated with pigeon muscle and then tested om mice.

A-before saturation. B-after saturation.

A

16bO 1250

400 2000 1250

1660

1430 1660

Contact I Toxin' 1 hours.

386 387 389 396 402

267

383 384

2 2 2 3 3

24

3 3

I I

2000 1430

330 2000 1000

2000

1430 1660

P.H. I Hernotoxic units. I M.L.D. I

0.15 0.25 0 '2 0 *5 0 % 1 '0 0'1 0.17 0 '2 0 '3

0.25 0'35

A

6 '6 6.4 6 -7 6'4

6.5

6 *5 6 '0

...

506 HERBERT HENRY

that filtrates which have been saturated with pigeon muscle show a slight decrease in toxicity, which is not apparent with precipitated toxins (383, 384). It is unlikely that this is due to alterations in reaction which are quite small. The hzemotoxin content remained practically unaltered.

TI. SUMMARY.

1. B. welchii produces a hzmotoxin andeanother toxin which may be designated a myotoxin.

2. There is some evidence that the susceptibility of an experimental animal t o fresh filtrates or to precipitated preparations depends on the vulnerability of the individual tissues to these components. Mice would seem to be very susceptible to the hzmotoxin, while it is suggested that in the rabbit succumbing to intravenous inoculation death is to a greater degree attributable to the myotoxin being brought into direct contact with the heart muscle. Those who have had clinical experience of gas gangrene in man may recall the suddenness with which many apparently favourable cases terminate by heart failure.

3. The in-vitro titration of the hzmotoxin as it exists in fresh filtrates afEords a measure of full toxicity, so that it also affords a relative measure of the myotoxin. The titration of toxin-antitoxin mixtures by a hzmolysis test show that a t the point at which neutrality for mice occurs the amount of free hzmotoxin present is roughly equivalent to that contained in one minimal lethal dose. This finding opens up the possibility of titration of antitoxin by a hamolytic test.

4. Under certain conditions there is partial dissociation of these two lethal components each from the other. But the methods we have employed have not been successful in effecting complete separation. They may be linked together in such a way that disruption is not possible.

5. We have used only one antitoxin, R173, for investigation. It was produced in a horse by repeated inoculation of culture filtrates and showed as much anti-myotoxin as aiiti-hzmotoxin when used against sinall multiples of the minimal lethal dose. The increased amount of anti-myotoxin as compared with anti-hzmotoxin requisite for neutralisa- tion in the case of larger amounts of toxin (toxin 329, table 111.) may depend on a, time factor rather than on a quantitative disparity. The combination of hmnotosin and anti-hEmotoxin occurs very rapidly, while the neutralisation of myotoxin may be much slower.

REFERENCES. 1. HENRY . . . . . . . . .7ourn. Path. and Bacteriol., 1922, vol. xxv. p, 1. 2. HENRY AND LACEY . . . Ibid., 1920, vol. xxiii. p. 273. 3. ANAEROBE C O M M I T T E E . . Medical Research Council, Special Report Series,

1919, No. 39, p. 104.