: “ 45 I #{149}5S

FURTHER OBSERVATIONS ON THE IMMUNISATION

OF ANIMALS TO THE POISONS IN FUNGI

WILLIAM W. FORD, M.D.

From the Bacteriological Laboratory, Johns Hopkins University

. Received for publication, August 19, 1910

It has previously been pointed out (1) that it is possible to

immunise animals to haemolytic extracts of the poisonous fungus
Amanita phalloides, and that in the course of this immunisation
the animals develop sera which have both antihaemolytic and
antitoxic properties. The antihaemolytic strength of such
sera is frequently 1-1000 or more but the antitoxic value is always
low, one cubic centimeter neutralising but three or four fatal
doses of the poisonous extract. At that period of our investiga-
tions upon fungi, it was believed on the basis of Kobert’s earlier
publications (2) that the blood-laking substance in the “deadly
aanita”was the active principle, and that the problem of obtain-
ing an antitoxic serum of a higher potency lay in the immunisa-
tion of larger animals and in the production of more powerful
antihaemolytic sera. When, however, the attempts which were
made to immunise goats and horses demonstrated that the treat-
ment could be pushed only to a certain point and that the animals
succumbed to large doses, it was surmised that the production
by animals of an antihaemolytic serum was no sure proof that
they would also produce an antitoxic serum and the suspicion
arose that there might be other factors concerned than those first
considered. A further study of the action of the fungi demon-
strated (3) that in addition to the blood-dissolving substance,
which is destroyed by heating to 60-65#{176}Centigrade there is also
present in A manila phalloides a heat-resistant poison to which we
gave the name Amanita-toxin. An examination of the fungus
rr-

: 146 WILLIAM W. FORD

extract by chemical methods was now undertaken and it has been

‘ 55 #{149} shown by Abel and myself (4) that the two poisons may be sep-
S arated by alcohol, basic lead acetate and other reagents, and that
the Amanita-ha’molysin solutions when freed of proteid give
the reactions of a glucoside containing a pentose. We have sub-
r sequently developed a method for the isolation of this body and
S a number of occasions have obtained highly active products
which were of a sufficient degree of purity to permit preliminary
analysis (5). Our final material, still highly hamolytic, gave
the same reactions as those originally ascribed to it, namely, those
of a pentose-containing glucoside, while its analysis gave a per-
centage composition of C 48.93, H = 6 . 08, N 10.83,
= S = =

; 1 . 94, 0 32.322.
= We could only conclude therefore that the
Amanita-hamo1ysin must belong to the group of hamo1ytic
glucosides of which we have a number of examples in saponin,
1 sapotoxin, solanin, etc., and not to the group of hamo1ytic pro-
teids. More knowledge of the constitution of this complex body
4 may reveal to us that particular group in its molecule which effects
the solution of the red blood cells.
:S S It has also been shown in association with Schlesinger (6) and
with Prouty (7) that the Amanila-toxin may be isolated by the
use of phosphotungstic acid (10 per cent phosphotungstic acid in
5 per cent sulphuric acid) and that when freed from impurities,
it gives neither proteid, alkaloidal, glucosidal nor conjugate sul-
phate reactions. A further study of this poison, to determine
its more exact chemical characterisation, is now in progress.
Since the Amanita-toxin is resistant to the temperature to which
these poisonous fungi may be submitted without losing their
deadly properties, and is of extreme toxicity for a great variety
of animals, it is believed on these and on other grounds (8) that
it and not the haemolytic body is the active principle. It was
moreover suspected that the presence of this poison in extracts
4 of the plant might be the factor which prevented the immunisa-
tion of animals to such a degree that their sera contained efficient
antitoxic as well as antihaemolytic substances. At the same
time the fact that the haemolysin apparently belonged to the
group of glucosides proved to be of great theoretical interest in
454454i y ‘ . ‘${44\. ..S#{149} S.. S S 51 ;: S4

IMMUNISATION OF ANIMALS TO POISONS IN FUNGI 147

the subject of immunity and a further study of the action upon

animals of the chemically separated poisons was therefore under-
taken.
The relative amount of haemolysin and toxin in different ex-
tracts of Amanita-phalloide.s varies greatly, or rather, since the
former body deteriorates rapidly both in the dried plant and in
solution, the amount of active blood-laking material is greater
in fresh than in old preparations although the absolute quantity
of the glucoside may be the same in the two cases. Since both
the haemolysin and the toxin kill animals, the extent to which the
action of any particular preparation must be referred to either
ingredient will depend upon the age of the fungi employed and the
length of time the poisons have been in solution. Fresh extracts
which contain powerful haemolysins will owe more of their toxicity
to the activity of this body than will old ones in which the gluco-
side has partially lost its strength while the toxin remains unim-
paired. In the same way a serum which has a powerful anti-
haemolytic and a low antitoxic action will neutralise fresh far
better than old extracts. Upon animals the action of the two
poisons differs radically. The toxin kills acutely, the animals
dying in 24-48 hours and showing no changes beyond a fatty
degeneration of the internal organs. The haemolysin kills slowly
in 3-10 days, the animals apparently succumbing to the extensive
blood destruction. At the site of inoculation is found a huge
gelatinous oedema, there is frequently a blood stained serous
exudate in the peritoneal cavity, and the urine in the bladder is
reddish in color but shows no intact blood cells, a true haemoglo-
binurea. On microscopic examination the pigment in the various
organs, especially in the spleen and liver, is greatly increased.
The immunisation of animals with these individual poisons, freed
as far as possible from foreign admixture, has been undertaken
at various times and the properties of a number of antisera have
beei investigated. Another glucoside found in fungi, in this
case an agglutinin, has also been studied in the same connection.
The results obtained with these various poisons may be briefly
summarised.
 ;S4; 55 55 S ‘ 4 4 .#4454
{149}

148 WILLIAM W. FORD

THE AMANITA-HAEMOLYSIN

I Animals treated with whole extracts of Amanita phalloides

: develop antihaemolytic sera which may have a strength of 1-

1000, 1-2000, or even more. Such sera are obtained from rab-
I bits after 6-8 weeks treatment. Similar sera are produced by
S large animals, and some time ago Dr. Kinyoun immunised a horse
for me obtaining an antiserum in which 1 cubic centimeter

k .
neutralised
guinea pig.
four fatal
This serum
doses
despite
of the whole extract
its low antitoxic
for
value
a 500-gram
was power-
fully antihaemolytic and tests have been made with it now for
nearly four years. When first examined it had a strength of
k
.
over 1-1000,
neutralising
1 cubic
a haemolytic
centimeter of a 1-1000 dilution
unit, that is, the amount
completely
of the glucoside
which will give solution of 1 cubic centimeter of a 5 per cent blood

suspension in 18 hours. The strength of this serum remained

: almost stationary for a long period of time, but recently it has
shown considerable deterioration. When such sera are precipi-
tated by ethyl alcohol, the filtrate shows no antihtemolytic action,
455 S while a saline solution of the precipitate neutralises the glucoside
S to the same extent that the original serum does. This antibody

. is therefore either directly precipitable by ethyl alcohol, or it is

S so tied to the proteid fraction of the serum as to be carried down
with it. In the same way it may be precipitated by basic lead
S 45 acetate.
S If extracts of Amanita phalloides be concentrated and treated
with ethyl alcohol, or if the dilute solution of the plant juice be
treated with basic lead acetate, the haemolysin may be obtained
free from the toxin. If the fungi be first extracted with alcohol
to dissolve out the toxin an aqueous solution of the residue will
be found to contain the glucoside, together with a considerable
amount of toxic material which may be removed by dialysis.
Toxin-free haemolysins may thus be prepared and their strength
accurately determined. Animals treated with small doses of this
material quickly develop an immunity to its poisonous action. At
first a little oedema develops at the site of inoculation and the ani-
inals lose in weight, but theoedemasoon disappears and theweight
 S- #{149}S
S

IMMUNISATION OF ANIMALS TO POISONS IN FUNGI 149

is gradually restored to normal. Such animals produce an effec-

tive antiserum and they are moreover absolutely immune, recover-
ing completely from the treatment and showing no late conse-
quences. Animals so immunised have been bled, usually after
their sera show an antihaemolytic strength of about 1-1000, this
being considered a satisfactory serum to study.
Finally since some doubt has been expressed as to the possi-
bility of our antihaemolysin being in reality an antibody to
a glucoside, the small amount of plant proteid in our original
solutions being supposed to exert some influence upon the reac-
tion we have described, haemolysins have been prepared both
toxin-free and proteid-free and the treatment of animals under-
taken with such preparations. These haemolysins have either
been freed of proteid by uranyl acetate in alkaline solution and
from the toxin by precipitation with alcohol or basic lead acetate
or they have been obtained from the fungi by aqueous solution
after the toxifl has been taken out with alcohol. In the latter
case the proteid has been removed in the usual way and the solu-
tions then dialysed for 24-48 hours to rid them of the salts and
the toxin residue. The immunisation of animals with this pro-
teid-free solution of our haemolytic glucoside proceeds exactly
as the immunisation of animals to the toxin-free haemolysins
just described. The animals first develop a sub-cutaneous oedema
and suffer a loss in weight but the swelling of the tissues at the
point of inoculation disappears as the treatment is continued and
the weight soon becomes normal. After 6-8 weeks treatment the
serum of these animals is found to be powerfully antihaemolytic,
the animals having responded to the introduction of this proteid-
free material and having produced a characteristic neutralising
serum. Thus a number of rabbits were immunised with proteid-
free haemolysins in 1909. They developed typical antihaemoly-
tic sera; in one instance, a serum having a strength of 1-1000 and
in another, a value of 1-800. These sera subsequently deterio-
rated in strength, when last tested having apotency of about 1-200.
As was to be expected, the proteid-free haemolysins have the same
action as the solutions containing traces of plant proteid. This
latter substance seems to be quite inert, exerting no influence upon
‘5q5 5SS T44 I 84

150 WILLIAM W. FORD

the response of the animals to the actively haemolytic glucoside.

Some of the rabbits immunised to this proteid-free body were
kept under observation for long periods of time to determine
whether the immunity induced in them is characteristic in the
sense that theydo not develop late lesions and die of a chronic form
of intoxication. In two instances rabbits were watched for over a
year during which time they remainedinperfect health. After the
lapse of 7-8 months the samples of serum removed from the ear
vein were found to be almost devoid of antihaemolytic action.
One of these animals was subsequently killed by a dose of haemoly-
sin and toxin while the other was employed for immunisation
with another lot of proteid-free haemolysin and developed a
serum of the strength of 1-400. This animal was purposely
destroyed 18 months after the original treatment. The artificial
immunity induced in animals by small doses of this haemolytic
glucoside seems to correspond in all essential particulars to the
artificial immunity produced by the true toxins of bacterial origin.

THE AMANITA-TOXIN

Since the Amanita-toxin is apparently the active principle of

A manila phalloides or at least is the more important of the two
poisons in human intoxications, the haemolysin acting possibly
only when poisoning follows consumption of the raw fungus, it is
important to determine how far animals can be immunised to the
pure toxin, free from admixture with the blood-laking material.
Thus far no success has been met with in this attempt. Animals
will withstand the introduction of low multiples of a fatal dose
and apparently have a heightened resistance to the action of the
poison but in no instance has a definite artificial immunity been
established. It is evident that it was the presence of this power-
ful toxin in our original extracts which was the source of the dis-
crepancy already referred to, namely that a powerful antihaemo-
lytic serum may show but a low degree of antitoxic action, and
that immune animals succumb to the introduction of high mul-
tiples of a fatal dose even when their serum is endowed with neu-
tralising substances for the haemolytic glucoside. A curative
 S S 55 S5 5S ; C ‘ ‘

IMMUNISATION OF ANIMALS TO POISONS IN FUNGI 151

serum for this variety of fungus intoxication can thus be prepared

to the same degree that the haemolysin acts as an etiological
S agent, unless further investigation shall prove that active immu-
nity towards the Amanita-toxin can be brought about by some
other methods.

THE MU5CARIA-AGGLUTININ

Amanita muscaria, the “ yellow or fly agaric” contains a heat-

resistant agglutinin which may be isolated from infusions of the
plant by chemical methods and which also gives the reactionsof a
glucoside (9). It is apparently the first substance with this action
upon blood corpuscles which has been shown to belong to this
group. This agglutinin is seemingly devoid of toxic action upon
animals, and large quantities can be administered subcutaneously
without appreciable effect. In the animals thus far studied,
where the substance was given in small doses over a period of
6-8 weeks the serum showed no increase of antiagglutinating
action, inhibiting the agglutination of the corpuscles by this
agent to practically the same degree as normal serum.

CONCLUSIONS

Three different substances in fungi, the Amanita-haemolysin,

the A manila-toxin and the Muscaria-agglulinin, have now been
tested at some length in regard to their power of stimulating ani-
mals to antibody formation. But one of these, the A manila-
haemolysin can be said to act like a true toxin in this respect, but
with this poison the i.mmunisation of animals has now been car-
ried out on so many different occasions and the sera produced
have such definite and lasting antihaemolytic properties as to
leave little doubt of the definiteness of the reaction. The fact
that our chemical investigations indicate that this haemolysin
must be classed as a glucoside, and that animals may be immu-
nised to it after it has been freed of proteid, raises important ques-
tions in regard to immunity production, and suggests that the
study of other substances than the toxic proteids may throw
5S5S484S S S 4 5 5 54

152 WILLIAM W. FORD

some light upon that remarkable phenomenon inwhich the tissues

and cells of the animal organism throw out protective substances
when certain poisons come in contact with them, but fail to react
in this way under the influence of other poisons.

BIBLIOGRAPHY

(1) FORD: The Toxins and Antitoxins of Poisonous Mushrooms. Journal of

Infectious Diseases, vol. iii, no. 2, April, 1906, pp. 191-224.
(2) KOBERT: Ueber Pilzvergiftung. St. Petersburger med. Wehschr., 1891,
16, pp. 463, 471.
(3) FORD: Toxicological
The Constitution of Amanita Phalloides. Journal of
Experimental Medicine, vol. viii, no. 3, May 26, 1906, pp. 437-450.
(4) ABEL AND FORD: On the Poisons of Amanita Phalloides. Journal of Biologi-
cal Chemistry, vol. ii, no. 4, January, 1907, pp. 273-288.
(5) ABEL AND FORD: Further Observations on the Poisons of Amanita Phal-
bides. Arch. f. exp. Path. u. Pharm., Schmiedeberg Festschrift, 1908, p. 8.
(6) SCHLESINGER AND FORD: On the Chemical Properties of the Amanata-toxin.
Journal of Biological Chemistry, vol. iii, 1907, pp. 279-283.
(7) FORD AND PROUTY: Note on the Amanita-toxin. Journal of Pharmacology
and Experimental Therapeutics, vol. 1, no. 3, October, 1909, p. 389.
(8) ABEL AND FORD: (1. c.)
(9) FORD: The Distribution of Poisons in the Amanitas. Journal of Pharma-
cology and Experimental Therapeutics, vol. 1, no. 3, August, 1909, p. 275.