THE MICROBIAL TOXINS.
There is but one way of characterizing the toxic poisons secreted
by microbes, and that is to apply to them the name of the microbes
generating them; thus the soluble and toxic poison of the tetanus
bacilli has received the name _tetanus toxin_.
In toxic microbial cultures it is necessary to distinguish the toxins
proper from the toxic alkaloids (ptomaines) which generally accompany
them; this is easily accomplished by evaporating the solution in a
vacuum at about 30°C., and then treating with alcohol and ether, in
which the alkaloids are soluble, while the true toxins are insoluble.
By fractional precipitation with alcohol it is easy to isolate the
peptones and true toxins.
The microbial toxins possess two essential properties; one the pyogenic
property, thanks to which the toxins first attract, then destroy the
white blood corpuscles or leucocytes, and transform them into pus, and
the other the pyretogenic property, which appears to belong only quite
indirectly to the pyogenic substance. The toxins in general retard the
heart action.
We will not speak of the distinctions it has been sought to establish
between the substances which possess these different properties, but
will at once take up the discussion of several of the microbial toxins.
=Anthrax Toxin=[100] (from Bacillus Anthracis).--We will describe the
preparation of this toxin as a type.
[100] ARLOING, CORNEVIN, THOMAS: _Le Charbon Symptomatique_, 1st edit.,
Paris; and LE DANTEC: _La Bactéridie du Charbon_, Masson, publ.;
STRAUS: _Le Charbon des Animaux et de l'Homme_, Paris, 1887.
The cultures of the bacillus are made in Liebig's bouillon, to which
has been added 0.1% of fibrin, the whole being carefully sterilized
for a long time at 110° C. The cultures medium is inoculated with a
drop of blood taken from the heart or spleen of an animal that has
died of anthrax. At the end of a week, the culture is filtered, and
the filtrate acidulated with a little acetic acid and precipitated
by adding powdered ammonium sulphate. The flocculent precipitate
is collected, washed, dissolved in distilled water, and dialyzed.
The dialyzed solution is concentrated in vacuo at 40-45° C., and
precipitated by adding to it alcohol. The precipitate formed is then
collected and dried.[101]
[101] HANKIN: _British Medical Journal_, Oct. 12, 1889, and July 12,
1890.
In this manner there is obtained a grayish-white substance which is
soluble in water, and which is fatal in large doses, but which, given
in repeated small doses, confers immunity against anthrax.
According to Hankin, it seems that the toxic property of this toxin is
due to an albumose.
Marchoux[102] has been able to confer immunity upon sheep by injecting
first small quantities of the filtered culture of the anthrax bacilli,
and then the virulent anthrax itself.
[102] _Annal. Instit. Pasteur_, IX, p. 785.
The animals thus rendered immune yield a serum which may be used as a
vaccin against anthrax, and which even possesses curative properties
under certain conditions.
In every case the acquired immunity is only temporary. We will recall
to recollection the method employed by Pasteur for vaccinating against
anthrax, using attenuated cultures, a method which is practiced daily
at the present time.[103]
[103] CHAMBERLAND: _Le Charbon et la Vaccination Charbonneuse_, Paris,
1887. PETERMANN: _Annal. Instit. Pasteur_, VI, p. 32.
From the cultures of symptomatic anthrax (Bacillus Chauvæ) Chauvée
extracted a very active toxin which can withstand without impairment
a temperature of 110°C.[104] Roux[105] has shown that the serum of
animals that have succumbed to the symptomatic anthrax is capable
of vaccinating against this disease; we have here a new proof that
the antitoxin is in fact a product of the defense of the cells of
the organism, and the author mentioned has been able to vaccinate
guinea-pigs by injecting into the peritoneum culture bouillon
sterilized by heating to 115° C. or by filtering through porcelain.
[104] DEUTSCHMANN: _Annal. Instit. Pasteur_, VIII, p. 403.
[105] _Annal. Inst. Pasteur_, Feb. 1888.
=Tubercular Toxin.=--The culture bouillons of Koch's bacillus contain
one or more active substances which constitute, and which is at the
present designated as, tuberculin.[106] Koch's therapeutic tuberculin
is obtained by evaporating to one-tenth its volume a culture bouillon
of Koch's tubercle bacilli prepared from a 4-per cent. glycerinic
mutton bouillon, and filtering through porcelain. By fractional
precipitation it is possible to obtain from the crude tuberculin so
prepared a product which is considered as pure tuberculin, and which
possesses considerable activity.
[106] AUCLAIR: _Thèse de doctorat_, Paris, 1897; and _Arch. de
Médecine_, exp. 1898.
Prolonged boiling on the water-bath completely destroys the activity
of this tuberculin, which moreover hardly ever keeps longer than three
weeks. It has been found possible to preserve it for an indefinite
period, however, by adding to it 30 to 40 per cent. of glycerin. It
possesses all the general reactions of albuminoids.
Tuberculin is not toxic in the proper sense of the word. Injected in
small quantities into the healthy human being[107] and into healthy
animals, it exerts no effect; on the other hand, however, in tubercular
organisms, even in incipient stages of the disease, even where it is
almost impossible to make a clinical diagnosis, the injection of very
small quantities develops a lively and characteristic reaction.[108]
[107] KOCH: _Deutsch. Med. Woch._, Nov. 13, 1890-1897, No. 14, p. 209.
[108] _Annal. de l'Instit. Pasteur_, V, p. 191; _Arch. de la Soc. Biol.
de Saint-Pétersbourg_, I, p. 213.
Grasset and Vedel consider the tuberculin as an excellent means of
diagnosing tuberculosis in man, but in such a case it is necessary
to operate with the greatest caution, with very small quantities of
the tuberculin, and to feel, in some sort, the sensitiveness of the
patient, particularly in the case of children.
It is chiefly for the diagnosis of tuberculosis in cattle, however,
that tuberculin is valuable. Thanks to Nocard, the procedure has to-day
become a common practice. The injection of a fairly large dose, 0.3
to 0.4 Gm., according to the size of the animal, causes, in about ten
hours or so, if the animal is tuberculous, a strong febrile reaction
with an elevation of temperature of 1.5 to 3° C., whereas if the animal
is not tuberculous no such reaction takes place.
Cases in which tuberculosis is far advanced, and in which the organism
is impregnated with tuberculin, do not react after the injection of
tuberculin.[109]
[109] NOCARD and LECLAINCHE: _Les Maladies Microbiennes des Animaux_.
Tuberculin does not confer immunity, and the bacillus retains all its
virulence, even in injected tissues; nevertheless, the return to health
of animals in which injections have been recently made may be due to
the action of large doses of the serum; and on the other hand the
tuberculin, in large quantities, may render the location unsuitable for
the development of the tubercle bacilli.
=Diphtheria Toxin.=--The most characteristic property of the diphtheria
bacillus is the production, in culture media, of a special toxic
substance which has been named _diphtheritic toxin_; this name,
however, has come to be also extended to a liquid in which the bacilli
have lived, and which has been sterilized by filtration or by any other
suitable process.
Roux and Yersin[110] were the first to affirm that diphtheria is an
autointoxication caused by a very active poison formed by the microbe
in the restricted locality where it develops. In order to obtain this
toxin[111] a culture of the bacillus is first made in a mutton bouillon
made strongly alkaline with sodium carbonate (10 grams per liter), and
with the addition of 2 per cent. of peptone. At the end of about one
month, the culture being kept at about 37° C., the liquid is filtered
through porcelain. It is indispensable to employ a very virulent
bacillus; it is hence frequently advantageous to increase the virulence
and toxigenic power of the bacilli it is desired to use.
[110] _Annal. de l'Instit. Pasteur_, II, p. 632, and VIII, p. 611.
[111] See SPRONK: _Annal. de l'Instit. Pasteur_, IX, p. 785; _Ibid._,
X, p. 333; MARTIN, _Ibid._, XII, p. 26; SPRONK, _Ibid._, XII, p. 711.
The toxic liquid obtained is exceedingly powerful: 0.1 Cc. kills a
rabbit in forty-eight hours. This toxin is very sensitive to the
effects of heat. When heated to 65° C. it loses almost all its
toxicity; at 70º C. it becomes innocuous; and it only requires to be
heated to 100° C. for fifteen minutes in order to lose all immediate
activity even in large doses. Nevertheless toxins thus weakened are
capable of proving fatal to an animal even after five or six months.
Light, oxygen, ozone and all oxidizers destroy the active principle of
the diphtheria toxin, which is, moreover, rendered almost inactive by
organic acids.
This toxin is capable of diffusing through animal membranes, a fact
that is in agreement with the toxic effect seen in a subject attacked
with diphtheria, and due to the toxin passing through the mucosa. In
spite of this property, however, the diphtheritic poison may be taken
into the stomach without any pernicious results.
Roux and Yersin have shown that, like all the diastases, it may be
precipitated from its solutions by the development, within these,
of certain precipitates, particularly calcium phosphate. It is
precipitated from its solutions by alcohol, as has been observed
also in the case of diastatic solutions. All the toxic substance
is contained in the albuminous precipitate thus obtained; but the
prolonged action of alcohol, or repeated successive precipitations,
alter it finally. Diphtheria toxin is likewise precipitated by the
reagents for albumoses, particularly sodium sulphate in saturated
solution. This procedure has been utilized by Brieger and Fraenkel
for preparing the pure toxin, which they obtained in the form of very
light, brilliant white, amorphous flocks, affording all the principal
reactions of the soluble albumoses (biuret, xanthoproteic, Millon's),
and which they characterized as a toxalbumin.
On injecting into healthy animals this diphtheria toxin attenuated
by sufficiently heating at 70° C, employing at first small doses,
and gradually increasing, it is possible to immunize them against
diphtheria, as was first demonstrated by Carl Fraenkel.
Roux and Martin, who have specially studied this procedure,[112] have
shown that a horse may be easily immunized by injecting into the animal
the toxin diluted with a third of its volume of Gram's iodine solution,
and in successively increasing doses. The initial dose is 0.25 Cc.;
then, after two days, 0.5 Cc. of the same toxin is injected, and in
like manner the dose is increased up to the eighteenth day, when the
pure toxin is injected, at first in small doses, which are gradually
increased so that at the end of two or three months injections of 80
Cc. of the pure toxin may be given without danger; the animal is then
completely immunized.
[112] Contribution à l'Étude de la Diphtérie. _Annal. de l'Instit.
Pasteur_, VIII, p. 609; _Ibid._, p. 640.
The serum of an animal rendered immune in this manner contains a
diphtheria antitoxin which possesses high power. A guinea-pig which
has received an injection of 0.01 Cc. of the antitoxin is perfectly
capable of withstanding a lethal dose of 0.5 Cc. of the toxin. The
antidiphtheria serum thus obtained, and in almost limitless quantities,
from an immunized animal, is capable of saturating the therapeutic
diphtheritic toxin, and has to-day taken rank in therapeutics as the
most efficacious remedy in diphtheria. Injected in varying doses, it
confers a temporary but immediate immunity.
Nevertheless antidiphtheria serum must not be considered as an
antidote; and in pathological diphtheria, the more serum is required
the later it is used.[113] In certain cases, if employed too late, it
may prove ineffective.
[113] BAYEUX: _Thèse de Doctorat_, Paris, 1899.
The preventive action of the serum is remarkable. In 10 000 inoculated
cases Behring and Ehrlich have had but 10 cases of diphtheria, and
these were, moreover, of a benign character. The duration of the
immunizing action appears to be from three weeks to two months.
This diphtheria antitoxin was first prepared by Guérin and Macé[114] by
adding to the antidiphtheria serum a large volume of alcohol, washing
the precipitate, and drying it in a vacuum. It is soluble in water, and
loses its activity when heated to 65° C. Wassermann[115] has proposed
to extract it from the milk of immunized animals, by first coagulating
the milk by rennet in the presence of sodium chloride, filtering, and
removing the fat from the clear liquid by means of chloroform. After
decanting, the clear solution obtained is precipitated by adding to it
30 to 33 per cent. of ammonium sulphate. The precipitate is dried in a
vacuum on a polished porcelain slab after having first been strongly
expressed. It is then dissolved in water.[116]
[114] _Compt. rend. de l'Acad. des Sc._, Apr. 5, 1895.
[115] _Zeitschr. für Hygiene_, XVIII, p. 235.
[116] ROUX and MARTIN: Contribution à l'Étude de la Diphtérie. _Annal.
de l'Instit. Pasteur_, VIII, p. 512.
=Tetanus Toxin.=--The fact that the tetanus bacillus never penetrates
to the interior of the organism enabled us long ago to foretell that
it secretes a very powerful toxin capable of dialyzing and diffusing
through the economy. Kuno Faber was the first to fully recognize the
fact that the culture bouillon of this bacillus, fully sterilized by
filtration through porcelain, possesses an exceedingly high toxicity,
and exerts a toxic effect on 50 000 000 times its own weight of living
organism. Brieger had previously, however, extracted three ptomaines
from the cultures of the bacillus--_tetanin_, _tetanotoxin_, and
_spasmotoxin_.[117] In order to obtain a highly active liquid, the same
culture medium is inoculated several times in succession, but filtering
each time before the new inoculation; the microbes greatly increase in
number after each fresh inoculation, and the toxic substance developed
by them accumulates.[118]
[117] Die Pathogenese des Tetanus. _Berlin. Klin. Wochenschr._, 1890,
No. 31.
[118] NAILLARD: _Compt. rend. de l'Acad. des Sciences_, CXX, p. 1181.
Experiment has shown that the culture bouillon thus obtained contains
two kinds of toxic substances[119]--highly toxic alkaloidal bases
(ptomaines, tetanin, tetanotoxin, etc.), and a true toxin, possessing
diastatic properties, and of almost incredible toxic power.
[119] _Annal. Instit. Pasteur_, V, 15.
This toxin had already been isolated by Kitasato. It is a toxalbumin,
and is very sensitive to the action of heat. A temperature of 65° C.,
maintained for 30 minutes, renders it quite inactive; and it becomes
oxidized and is destroyed by the action of the air in the presence of
light.
Brieger and Boer,[120] by precipitating with zinc chloride the filtered
culture bouillon, obtained a pure, amorphous tetanus toxin, which they
also considered as a toxalbumin, and which possesses exceedingly toxic
properties.
[120] _Deutsche Med. Wochenschr._, No. 49, Dec. 3, 1896.
If a precipitate be caused to form in these toxic solutions, as, for
instance, a precipitate of calcium phosphate, this carries down with it
all the toxin present in the liquid. 0.0005 Gm. of this precipitate is
surely fatal to a guinea-pig.
Dozon and Cournemont have observed that even in doses of 300 to 400 Gm.
of the filtered culture liquid, this toxin is not immediately toxic
to a horse, but kills the animal only after a period of incubation
of at least twenty-four hours. The blood of such an animal, however,
is immediately and directly fatal to animals into which it is
injected.[121]
[121] _Compt. rend. Soc. Biol._, 1893, p. 294; _Ibid._, 1894, p. 878.
Experiment has shown that animals that have been cured of tetanus
possess no immunity whatever against tetanus; nevertheless Behring and
Kitasato[122] first, and Wassermann and Kitasato later on, succeeded
in preparing a _tetanus antitoxin_. To obtain this, the immunization
of the animal, horse or cow, is effected by injecting increasing
quantities of the toxin, more or less attenuated by mixing it with
Gramm's iodine solution; the immunization is easily and rapidly
accomplished by the process devised by Roux and Vaillard.[123]
[122] _Deutsch. Med. Wochenschr._, 1890.
[123] _Annal. Instit. Pasteur_, VII, p. 64.
The immunized animals yield a serum which, mixed with tetanus cultures,
renders these innocuous, and which enjoys an antitoxic power that
borders on the marvelous.[124] A quintillionth of a cubic centimeter
of the serum per gramme weight of a live mouse suffices to protect the
animal from an otherwise fatal quantity of tetanus toxin.[125]
[124] NOCARD: _Bull. de l'Acad. de Médecine_, Oct. 22, 1895.
[125] NAILLARD: _Compt. rend. de l'Acad. de Sciences_, CXX, p. 1181.
This serum is nevertheless powerless to preserve man in cases of acute
tetanus; it confers an immediate, but only transitory, immunity.
As to its mode of action, it appears to cause a permanent condition
of excitation or of nutritive reaction of the cells, which makes
these resistant to the poison. As in the case of the other toxins,
the quantity of antitoxin necessary to protect an organism is so much
greater the later the treatment is applied.
=Mallein (Toxin of Glanders).=--Among the soluble products secreted
in the culture media by the glanders bacilli, there are found true
toxins to which are ascribed certain symptoms of glanders infection.
These toxins have been isolated and designated by the name _mallein_.
First prepared by Helman and Kalmino, mallein was later on specially
studied by Roux and Nocard, and, in consequence of the researches of
the last-mentioned scientist, it has acquired great importance.[126] It
is obtained by sterilizing at 110° C. cultures of the glanders bacillus
made with mutton bouillon with the addition of salt, glycerin, and
peptones. To isolate the toxin the culture bouillon is first sterilized
by heating for half an hour in an autoclave at 100° C. It is then
filtered, concentrated to one-tenth its volume on a water-bath, and
filtered through a Chardin filter. The mallein is thus obtained in the
form of a brown syrupy liquid containing half its weight of glycerin.
[126] NOCARD: _Les Maladies microbiennes des animaux_, Paris.
This solution keeps well when kept from air, light, and heat. In
practice it is employed in 10-per cent. solution in phenolated water
(5:1000). The mallein may be precipitated from the crude solution by
the addition of alcohol, as recommended by Foth. Foth's mallein occurs
as a white, light powder, very easily soluble in water.
Mallein enjoys a very important rôle in veterinary therapeutics, a rôle
analogous to that of tuberculin, permitting the diagnosis of incipient
glanders.[127]
[127] STRAUSS: _Arch. de Médic. expériment_, 1886.
Experience has shown that in animals already attacked by glanders,
even if ever so slightly, the thermic reaction never fails when 0.25
Cc. of the mallein solution is injected. In healthy animals, however,
the injection of mallein, even in much larger quantities, causes no
apparent effect. In animals attacked by glanders the reaction attains
its maximum in twelve hours, and several days are required for the
temperature to return to normal.[128]
[128] CADIOT and ROGER: _Compt. rend. Soc. Biol._, 1895, p. 770;
WLADIMIROW: _Arch. des Sciences Biol. de St.-Pétersbourg_, IV, p. 30;
BOURGES and MÉRY: _Soc. de Biol._, Feb. 5, 1878.
According to Nocard, mallein possesses no immunizing properties
whatever.[129]
[129] GALTIER: _Compt. rend. de l'Acad. des Sciences_, XCII, p. 303;
STRAUSS: _Arch. de Médic. expériment_, I, p. 489.
=Typhoid Toxin.=--This is obtained, like the other microbial toxins,
from a culture, prepared with more or less difficulty, from Eberth's
typhoid bacillus. This toxin, injected into guinea-pigs, develops in
them typhoid fever.
In the solution there occurs a ptomaine, which has been isolated by
Brieger, and which gives rise to almost all the phenomena of typhoid
fever; this ptomaine is called _typhotoxin_.[130]
[130] BRIEGER: _Microbes, Ptomaïnes et Maladies_, Doin, publ., Paris,
1887; LUFF: _Brit. Med. Journ._, 1889.
The same author, in collaboration with Fraenkel,[131] later on isolated
a toxalbumin from the culture bouillon of the typhoid bacillus.
Sanarelli[132] obtained an active toxin by macerating for several days
at 60° C. a month-old culture of the typhoid bacillus made with a 2-per
cent. glycerin-bouillon. Chantemesse has also published a process which
yields a highly virulent toxin.[133]
[131] _Berlin. Klin. Wochenschr._, 1890.
[132] _Annal. de l'Instit. Pasteur_, VIII, p. 103.
[133] _Compt. rend. Soc. de Biol._, p. 232, Jan. 30, 1897. _Congrès
d'Hygiène de Madrid_, 1898.
Chantemesse and Widal[134] have shown that on injecting into an
organism increasing quantities of the sterilized cultures of Eberth's
Bacillus, it is possible to fully immunize an animal against the
bacillus itself, and even also against the Bacillus coli communis. The
operation, however, is tedious and painful. The serum of immunized
animals possesses preventive and curative properties respecting the
effects of typhoid bacilli.
[134] _Annal. l'Instit. Pasteur_, VI, p. 755; SANARELLI: _Ibid._, p.
721.
A dose of the filtered culture, which is fatal to a guinea-pig,
becomes innocuous when mixed with 0.5 Cc. of the serum of a vaccinated
guinea-pig; 6 Cc. of the serum injected six hours after an injection
of the virulent culture, hence when this is in full action, suffice
to save the animal.[135] So far as the human being is concerned, the
results obtained have not been sufficiently satisfactory.
[135] FUNCK: _La Sérothérapie de la Fièvre Typhoïde_, I, Brussels, 1896.
The culture bouillon of the Bacillus coli communis, which is closely
allied to Eberth's bacillus, also contains soluble toxic substances
which have been named coli-bacillus toxin. This substance, which is
produced only in small quantity by the microbe, is fatal only in very
large doses.
=Cholera Toxin.=--Very little is known regarding the toxic products of
the spirillium choleræ; nevertheless, the fact that typical cholera
exhibits every symptom of the action of a toxic agent demonstrates
quite clearly the elaboration of some toxic substance within the
cultures of this microbe.
Villiers[136] found in it a liquid ptomaine; Klebs[137] found another
and crystallizable ptomaine; while Pitai discovered in it a toxin
unalterable by heat, and which he considered as a toxopeptone.
According to Gamaleia[138] there is present a true toxin, alterable
by heat, and the reactions of which entitle it to be considered as a
nucleo-albumin; he has also found in it a toxic nuclein.
[136] _Compt. rend. de l'Acad. des Sciences_, Jan. 12, 1885.
[137] KLEBS: _Allgem. Wien. Med. Zeit._, 1887.
[138] _Arch. de Méd. Expérim._, IV, p. 173.
These toxic substances are found, according to Gamaleia, Pfeiffer,
and Sanarelli,[139] confined during the life of the microbe within
its cellular envelope, and does not diffuse through this. Metchnikoff
and Roux are of the contrary opinion,[140] however, and they have
prepared a toxin almost insensitive to a temperature of 100° C., and
precipitable from its solutions by ammonium sulphate or strong alcohol;
the toxin is a toxalbumin. This toxin is quite toxic; one-third of a
cubic centimeter suffices to kill 100 Gm. of guinea-pig in 18 hours;
with larger doses, death is almost immediate.
[139] _Annal. de l'Instit. Pasteur_, IX, p. 129.
[140] _Ibid._, X, p. 257.
By immunizing guinea-pigs, rabbits, and horses with this cholera toxin,
Metchnikoff and Roux obtained a serum which is distinctly antitoxic for
rabbits. Nothing absolutely certain has been found as to its action on
man.[141]
[141] HAFFKINE: _Compt. rend. de l'Acad. des Sciences_, 1892;
METCHNIKOFF: _Annal. de l'Instit. Pasteur_, VII, p. 403; and ROUX:
_Ibid._, X, p. 253.
* * * * *
We will not dwell longer here on the toxins of microbial origin. It
appears evident, however, from what has been stated above, that the
great majority, if not all, of the virulent microbes manifest their
virulence by means of toxic secretions. Almost every one of these
toxins has been the subject of study. They would otherwise not have
interested us here, where our main object was but to dwell upon the
general properties.