576.809. 7 2 : 593.17 AN ANALYSIS OF SOME OF THE ANTIGENIC

PROPERTIES O F CERTAIN CILIATES BELONG- ING TO THE GLAUCOMA-COLPIDIUM GROUP AS SHOWN IN THEIR RESPONSE TO IMMUNE SERUM.

MURIEL ROBERTSON. From the Lister Institute, London.

IN the preceding paper (Robertson, 1939) the reactions of two species or varieties of Glaucoma to immune sera produced against them in the blood of rabbits were described and the earlier work of Bresslau on these organisms was reviewed. The reactions were studied on the living organisms and the sera were made by using the intact living creatures as antigen. The immunological responses were of a very characteristic type and in some degree conformed with those found in bacteria.

The antigenic properties of living matter, in the present state of our knowledge, are of rather particular interest and it seemed worth while to carry the investigation somewhat further by analysing the antigens by the now familiar method, inaugurated by Weil and Felix (1920), of subjecting the organisms to a variety of treatments before using them to evoke antibody in rabbits.

Certain circumstances were favourable for this study ; first of all the organisms were grown in sterile culture, the observations of the reactions to the sera could be carried out under the microscope and appreciated in detail and further these ciliates could be induced to give out one of the antigenic substances or complex of substances (namely the exudate) in solution.

This paper deals therefore with the immunological investigation (1) of the organism fixed by rapid heating to 73-75' C. for 1 minute 40 seconds, (2) of the separated exudate in solution as nearly free as possible from other cell constituents, (3) of the washed and heat-fixed bodies after the exudate had been extruded, (4) of the ciliates after treatment with alcohol and ( 5 ) after exposure to HC1 and (6) of the organisms exposed in distilled water to 100' C, for 1-2 hours. Precipitation and absorption experiments were also used for further elucidation of certain points and the changes in the effect brought about by the addition of complement to the various immune sera were observed.

323

324 M . ROBERTSON

Methods and material. The method used to obtain antisera was the injection of the antigen

however prepared into the ear vein of a rabbit. The tests wcre carried out in the manner described in tho previous paper, observations being made both macroscopically and microscopically in the small petri dishes whenever the two methods were applicable. The test antigen, except where expressly stated, was always the living organism suspended in a solution of peptone in 0.5 per cent. NaC1.

Reactions of sera made with heat-$xed cultures. The work dealing with the heat-fixed cultures, the exudate and

the washed bodies from which the exudate has been poured out can all be treated together.

Bresslau (1924) stated that if a culture of Glaucoma was centrifuged and washed and exposed to a temperature of about 35-36" C. for 8-1 minute the organisms poured out an exudate which he named " Tektin." Further very rapid heating to 71-73°C. caused fixation (heat coagulation) of the glaucoma bodies and solution of the exudate. If the material was now centrifuged a clear, faintly opalescent fluid was separated from the bodies. In practice this method required a little investigation and some further elaboration, but was carried out without any really material alteration.

The cultures used for this purpose should not be more than 48 hours old. The organisms were centrifuged down once, care being exercised to avoid as far as possible any damage or packing a t the bottom of the centrifuge tube. St.L. was particularly sensitive to this while alive, and samples were rejected if any considerable number of dead and burst individuals could be seen under the microscope. Seventy-five C.C. of peptone culture were treated a t once and resuspended in about 8 C.C. of 0.5 per cent. saline. Two small water-baths were prepared, one at 39-40' C. and one boiling vigorously a t 100" C. The test-tube with the living organisms was fitted with a thermometer passed through a rubber cork and was held in the bath at 39" until the thermometer registered 34-35' C. (usually for 1 minute and 15 to 30 seconds) and then was so manipulated that the temperature in the tube did not rise beyond 36" C. for a further 14 minutes. The sample taken a t this period and examined under the microscope showed living undamaged ciliates and a considerable amount of amorphous exudate causing a cloudy flocculent appearance in the tube. The tube fitted with the thermometer was then plunged into the bath of boiling water. Within 20 seconds the temperature usually registered 60" ; a few seconds later as the mercury column passed the 65" mark the tube was removed from the bath. The temperature continued to rise to 73-75' and after a total of 1 minute 40 seconds from the moment of plunging the tube into the heat it was opened. The examination a t this stage showed well-fixed glaucomas in fluid containing some cloudy material. The bodies were in good condition if the heating had not exceeded 78" and if the original rise in temperature had been sufficiently rapid, but a certain amount of shedding of clear hyaline discs or globules occurred without bursting or injuring the bodies. Their nature is not clear, but they disappear during the subsequent handling of the material. The still warm material was centrifuged again several times and a clear, faintly opalescent fluid finally obtained. The bodies together with some flocculent material remained as a deposit on centrifuging the heated contents of the tube.

ANTIGENS OF PROTOZOA 325

The bodies of St.L. are not fragile after fixation and very little breaking up takes place a t this period, so that if the original centrifugation of the living culture has been successful there is very little if any of the cell constituents in the supernatant fluid after the final centrifuging except the soluble part of the exudate. The G1.R. strain, which is little affected by the first centrifuging if it is carefully done, is slightly more fragile after fixation and the final centrifuging and washing in the centrifuge must be done with some care. The best results are always got with young cultures growing in rapid subculture.

The antigenic qualities of the briefly heated material were investigated in three stages. (1) Rabbits were immunised with material from the boiling bath without any further treatment except cooling and some dilution with saline. In this state the inoculum was called the “ matrix ” and contained of course all the products of the culture other than those removed with the peptone water. It consisted of the heat-fixed organisms, the cloudy flocculent material and the soluble exudate, ( 2 ) Other rabbits were inoculated with the clear supernatant fluid obtained by centrifuging the “ matrix,” i.e. with the exudate. (3) Another group was inoculated with the many times washed bodies from which the soluble exudate had been removed. These washed bodies were also free from the amorphous flocculent material which remained after the heating and appeared to be exudate which had not gone into solution. The washed bodies when used were thus free from anything that had been poured out into the surrounding fluid. They were washed seven or more times in 0.5 per cent. saline.

(I) The sera made with the ‘‘ matrix ” material had the same reactions with the living glaucomas as those shown by a serum made with living antigen. The titre was a little lower in the examples tested but this was partly due to the fact that the immunising doses were rather lower. There were cross effects between the St.L. and G1.R. strains of the same nature as those already described (Robertson, 1939). There was no agglutination of the heterologous organisms, but a slight pouring out of exudate which seemed to combine with the serum, and formation and casting off of a sheath by a smaller or greater number of organisms could be observed. These cross effects were always slight and only to be seen in the highest concentrations ; they nearly always appeared late and were seen in the reading at 22 hours. They inight be so slight in the case of St.L. sera with the G1.R. suspension as to be difficult to observe. The reaction was clearer with a four-day growth than with younger cultures and it was usually a more evident reaction when a serum made from G1.R. was used with an St.L. suspension than in the converse experiment.

( 2 ) Sera made with the supernatant fluid or soluble exudate of St.L. when tested against the homologous living organism

JOURN. OR PATH.-TOL. XIVIII. x

326 M . ROBERTSON

produced the usual reactions, with however a slight alteration of the emphasis. Agglutination and the pouring out of exudate were well developed and appeared early, although the titre was not very high (1 : 160-1 : 320 for agglutination). Sheaths were also formed quickly, but were less solid looking and thick than those seen with a typical serum made with living organisms or with the unseparated “ matrix.” The sheaths were cast off rather rapidly and the agglutinated clumps dispersed, leaving the small plates in which they were observed filled with agglutinated masses of empty sheaths. These were still to be noted, though in reduced numbers and of shadowy appearance, at 1 : 640. In certain sera there was no detectable reaction at all, even in a concentration of 1 : 5, with the heterologous living strain G1.R. This was further tested and a very faint cross effect with G1.R. was detected in dilutions 1 : 5 and 1 : 10 of a serum made with a greater amount of antigen. These cross reactions were much more clearly shown in precipitation experiments dealt with later.

The sera made with the soluble exudate of G1.R. gave reactions which were along the usual lines, but the features were less distinctive than with similar sera made with St.L. There was a slight cross effect with the living heterologous strain St.L.

(3) The sera made with the washed bodies when used with the homologous organisms showed the familiar reactions but agglutination was delayed somewhat and, in the plates, developed rather poorly and irregularly; sheaths were formed in great numbers and on being cast off agglutinated into clumps and rafts.

While the distinctions were in no way sharply defined, there was a tendency in tests with antisera against the washed, heat-fixed bodies as compared with those in sera made with the supernatant fluid (soluble exudate), for the homologous organisms to show less complete agglutination in the plates and less free exudate. The sheath-producing effect on the other hand was very well developed, so that these shells were cast off after a time in such numbers that in dilutions of 1 : 160 and 1 : 320 the reaction involved practically all the organisms present and was the chief effect of the serum.

Reactions of sera prepared with acid-treated material. One hundred C.C.

of culture (3-4 days’ growth) were centrifuged and resuspended in 60 C.C.

of 0.5 per cent. saline ; 50 C.C. of a 10 per cent. solution of HC1 in 0.5 per cent. saline were added to the mixture and allowed to stand for 72 hours in the dark. The organisms were then washed five or more times in the centrifuge in saline. The pH of the suspension was tested and if found to be acid the washing was repeated. The acid treatment fixed the organisms in the histological sense quite well. The method as also that used later for alcohol treatment is essentially the same as that used by Felix and Pitt (1936) for bacteria of the Salmonella group.

The antigens for these sera were obtained as follows.

The rabbits showed no ill effects.

ANTIGENS OF PROTOZOA 327

The sera made with 31-32 million organisms showed when used with living glaucomas a reaction differing only in titre from that obtained with a serum prepared against a living antigen. The titre was uniformly lower but the character of the reaction was the same. The antigen had not apparently suffered any obvious qualitative change by treatment with acid. Cross effects with the heterologous organisms were present in a slight degree.

Reactions of sera prepared with alcohol-treated material. The preparation of the antigen for these sera was carried out as follows.

Seventy-five C.C. of a 2-3 day culture were centrifuged and the ciliates resuspended in 20 C.C. of 0.5 per cent. saline ; this was made up to 150 C.C.

with 96 per cent. alcohol. The flasks were corked and left for three days at room temperature in the dark. The organisms were removed from the alcohol and washed 3 or 4 times in 0.5 per cent. saline.

The organisms of both strains showed a characteristic appearance after exposure to alcohol in this way. The general fixation of the body was good ; the periphery however showed in a large proportion of the individuals a double line and even a delicate sheath-like appearance as though the cuticle and perhaps also some of the material from the alveolar layer had been precipitated by the alcohol so as to be separately visible. Neither acid nor heat- fixation appeared to have this selective action on the peripheral structures. The cilia incidentally were well fixed by all the methods. In the case of St.L. the centrifuging, as always, was apt t o produce some spontaneous agglutination and the extrusion of a certain amount of cloudy exudate ; this was much less pronounced with G1.R.

Sera made from rabbits by injecting alcohol-treated antigens had characteristic reactions with living organisms. The two strains differed slightly in their reaction to treatment with alcohol, St.L. being apparently more sensitive than G1.R. Serum St.L. with the homologous organisms showed very little agglutination and only a slight degree of immobilisation. These reactions were often extremely feeble. The sheath-forming reaction, however, was always very well developed so that after some hours these structures were discarded in masses with little interruption of the motility or other functions of the ciliates and they were usually agglutinated as empty shells before the 22-hour reading.

With serum G1.R. there was an appreciably greater degree of both immobilisation and agglutination of the living homologous organism. The sheath-forming reactions were always well developed, but it seemed clear that the antigen in G1.R. was slightly less affected by exposure to alcohol than that in St.L. The titres were lower than those of the sera made with living glaucomas but some discarded sheaths could be found in certain

328 M . ROBERTSON

Liring organism

Soluble exudate shed into saline

Washed bodies from which soluble exu- d a t e h a d b e e n obtained

Acid-treated

Alcohol-treated

of these sera at 22 hours in dilutions up to 1 : 600-1 : 800. There were faint cross effects in the more potent of these sera, reduced as usual to the casting ofa few sheaths on the part of the heterologous organisms.

Living glaucoma

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Reactions of sera m,ade with steamed material.

For the preparation of the sera from steamed organisms, the latter were grown in peptone water without sodium chloride and were centrifuged and suspended in neutral distilled water and steamed for 1-2 hours as desired. Salt was added to bring the concentration up to 0-88 per cent. after the heating was concluded.

These sera had a very feeble action with the living organisms, showing that the antigen had been very much altered by the treatment. There was no agglutination of the ciliates and no immobilisation, but after 10-22 hours a certain proportion were seen to have cast their sheaths in dilutions up to about 1 : 40-1 : 80 and there was usually a little exudate shed into the surrounding medium. Although these reactions were so very faint there was a just recognisable trace of cross effect with the heterologous strain. Sera made with antigen steamed for 2 hours were even more feeble and a definite reaction could not always be registered in the experiments with the living organisms. Table I gives the reactions to the various types of sera in tabular form.

TABLE I .

Reactions of sera prepared with various antigens.

Antigen. 1 Test object. 1 Reaction.

Steamed for 1 hour 1 9 , ,,

Production of clear exudate, immobilisation, agglutination : formation and casting of sheaths : potent sera may cause death in higher concentratj ons

Exudate, immobilisation, agglutination, slight reduction in the formation and casting of sheaths

Exudate, immobilisation and agglutination slightly reduced: emphasis is on the formation and casting of sheaths

Reaction as for serum from living antigen but titre much reduced

Exudate, immobilisation and agglutination much reduced, but formation and casting of sheaths well developed

No immobilisation or agglutination ; only a slight degree of formation and casting of sheaths

ANTIGENS O F PROTOZOA 329

Summary of the foregoing sections.

The soluble exudate poured out from the body of the glaucomas under the conditions described seems to contain both agglutinogen and sheath substance and evokes a serum which shows both the associated reactions with the homologous organisms. The emphasis is, however, on the agglutination and less on the sheath-inducing and sheath-combining effect. The washed bodies from which the exudate has been obtained also shows both substances, but the serum made with them gives a set of reactions with the emphasis on the sheath-forming effect.

Treatment with acid considerably weakens the antigen quantitatively but leaves it apparently unchanged qualitatively, and the serum reacts with the homologous living organism as a low titre serum made with living antigen.

Alcohol treatment very markedly alters the effectiveness of the antigens which call forth the immune bodies associated with agglutination, immobilisation and the outpouring of the amorphous exudate, and these reactions are very much reduced in consequence in tests with the resulting sera. The sheath substance appears to be very little affected by the action of alcohol and the sera made from antigen treated with this reagent have well developed sheath- combining immune bodies. There is here evidence of two elements in the antigenic make-up of Glaucoma which are affected by alcohol in different degrees.

Exposure to 100°C. for 1 hour destroys the antigens except a certain amount of the basic sheath element which produces a very low-titre serum, causing with the living glaucoma apparently no other reaction than a somewhat delayed formation and discarding of a sheath by a proportion of the ciliates. The data here set out seem to indicate that the antigenic substance of these ciliates has a heat-labile and a heat-stable element.

Precipitation experiments. Precipitation experiments were carried out using very carefully

controlled solutions of exudate obtained by the method already described. These were practically free from cell elements other than those poured out into the saline during life. They were used lllvll W~vIIv&€ui i i i i t ; i I i (6.25 per ceric. j and gave identical results, but the formolised samples kept better and could be used over a period of several weeks.

The precipitin tests were much less sensitive in distinguishing between the strains than those made with the living organisms so that the common factors in the antigens were much emphasised. Controls with normal sera or with sera made against other protozoa were uniformly negative. The low-titre sera made with acid-

--24.L - 2 --.:Ll. -

JOURN. OF PATH.-VOL. XLVIII. x2

330 M . ROBERTSON

1:z. ~~

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treated antigen reacted poorly with the soluble exudate, but these were 18 months old when tested. Sera made with steamed antigen (1 hour) reacted with the soluble antigen and when tested with living organisms showed only the late sheath effect, the deduction being that a certain amount of sheath-forming substance was poured out into the saline as well as the clear exudate which seemed to be concerned with agglutination. This conclusion was borne out also as shown already by the effect of the serum made from the exudate.

Quantitatively the precipitin reaction could not be considered to give an accurate account of the amount of overlapping, as there was no means of standardising the antigenic content of the exudate solution and there was reason to suppose that the more prolific growth of G1.R. probably yielded a greater amount of exudate.

Table I1 gives an abridged account of some of the precipitin reactions with various types of sera.

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TABLE 11.

Abridged table of precipitin reactions with soluble exudate.

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ANTIGENS OF PROTOZOA 33 1

Absorption experiments. In the preceding paper it was shown that the living glaucomas

could absorb antibodies from homologous sera made with living antigen. In the present study alcohol-treated and steamed (1 hour) antigens were used to absorb various sera.

The sera made with steamed antigen in experiments with the living homologous organisms had shown a poor effect restricted to a relatively slight and late sheath-combining reaction and causing no agglutination of the living ciliates. It seemed reasonable therefore to suppose that the agglutinogen was destroyed by heating at 100" C. for 1 hour. Treatment with alcohol, however, judging from the action of sera made with this antigen, had left intact the sheath- forming substance and only partially reduced the agglutinogen. Table I11 gives some of the representative results of the absorptions.

The absorptions require large quantities of organisms and should be done in stages, the serum being drawn off and mixed with further doses of the absorbing antigen. It was found that the sheath-combining element could be completely removed from sera made with living antigen by absorption with alcohol-treated glaucoma, with only a relatively slight reduction in agglutination titre and in immobilising effect.

Complete absorption of the sheath-combining antibody was never actually obtained with antigen steamed for 1 hour in the case of sera made from the living organisms, but a considerable reduction in this reaction was brought about without damage to the agglutination titre (expts. 432 and 434, table 111). A serum (1 05) made from alcohol-treated G1.R. could, however, be deprived of practically all the sheath-combining antigen, in the dilution used, by this treatment (expts. 436 and 438).

The inability of the steamed glaucomas to absorb agglutinins was striking, but was to be expected from the facts already observed. It was also clear that the alcohol-treated glaucomas could affect to a certain extent the agglutination and immobilisation titre (expts. 443 and 444, table 111) with the serum made from the soluble exudate of G1.R. With a good serum made from the intact living organisms, however, it was remarkable how the alcohol- treated antigen could, upon repeated exposure, remove a very highly developed sheath-combining antibody while only very slightly lowering the agglutination titre (expts. 413 and 414). Here again the evidence for the existence of the two elements in the antigenic complex seemed to be clear.

Effect of addition of complement on the action of various sera. It now remained to see if a study of the sera made from antigen

treated in various ways and of the absorbed sera would throw

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ANTIGENS OF PROTOZOA 333

any light on the combination of the antibody with complement in effecting the death of the organisms with or without lysis. It has been shown in the previous paper that dilute guinea-pig serum with its complement intact, added to immune serum made with the living organisms, increased the titre of the serum very markedly, and the curious fact was observed that the most intense phase or peak of the reaction as regards death followed by almost complete lysis took place in an unexpectedly high dilution of the immune body. In the higher concentration of the serum death might occur in very large numbers but lysis did not follow, and in many cases the immune serum plus complement was actually less lethal where the concentration of the immune serum was higher. This suggested either that some factor of optimum proportions was operative or that there was some competition of antigens for the complement, or both, or even that there was some antagonism between the action of the immune body and complement.

Experiments in which the absorbed sera were used in conjunction with complement formed the basis of an analysis of these questions.

It should be noted that experiments of this type were somewhat elaborate and presented technical difficulties. The complement as has already been remarked has of itself a very acute action and must be suitably diluted; at least two concentrations should be used. The complement was placed in contact with the serum and allowed to stand at 3 7 ° C . for 15 minutes and after cooling to room temperature the suspension was added. Even with every care experiments must sometimes be discarded owing to the dilution of the somewhat variable complement being unsuitable and causing an independent attack upon the organisms or alternatively to its being too weak to combine effectively. Moreover some sera-fortunately only very few-were found to be anticomplementary.

The analysis resolved itself into determining the effect of adding complement (1) to an immune serum deprived of the sheath- combining antibody but still possessing the agglutinin and allied antibodies, and (2) to an immune serum containing the sheath- combining body but none, or as little as possible, of the immobilising and agglutinating antibody.

(1) Sera made with living organisms and absorbed with alcohol- treated or steamed antigen were used with added complement. The absorbed serum without complement showed as already described reduced sheath formation but practically unaltered agglutination. With complement it produced lysis in a much higher concentration than did the unabsorbed serum. Thus in one experiment the absorbed serum showed lysis at 1 : 400 while in the unabsorbed the lysis did not appear in any marked degree until the dilution of the immune serum was about 1 : 6400. In these potent sera, made with the living organisms, the absorption of the sheath-combining antibody was seldom if ever quite complete and the clearest picture of the action was got from experiments

334 M . ROBERTSON

with sera such as that made from the soluble exudate of strain G1.R. and absorbed with the homologous alcohol-treated antigen.

A typical experiment with this serum is shown in table IV. Here all the sheath-combining antibody was absorbed and the agglutination and immobilising titres were considerably reduced in the serum when tested without the addition of complement. When complement was added in a suitable dilution the death of about one half of all the glaucomas occurred, with complete lysis in a serum dilution of 1 : 40, the effect diminishing rather sharply with further dilution of the serum until only a trace was found in 1 : 320-1 : 640. The important point here is that the much weakened immune body consisted of the agglutinating and immobilising elements alone and when combined with complement death with lysis was produced.

In the unabsorbed control (expt. 449, table IV), death increased in the dilutions from 1 : 40 to 1 : 1280 with diminution of the immune body, partial lysis appearing at 1 : 320 and a good degree of lysis only at 1 : 1280. With further dilution the number of dead diminished but those that were killed were also lysed. The sheath- reacting antibody apparently competed for complement or inhibited its union with the agglutinin.

Here again, as in the tests described in the previous paper, the lytic power of very small amounts of antibody when combined with Complement is very striking, but it must be borne in mind also that the organisms exposed are alive and the action of a lytic agent on a small portion of the surface would probably suffice to destroy the complex balance of the peripheral system and disrupt the organism.

(2) The elucidation of the effect of the addition of complement to a serum containing only the sheath-combining antibody presented some technical difficulties also in getting a proper balance of complement and immune serum, and particular care had always to be taken to guard against the independent action of too large an amount of complement. The reaction was studied in sera made with steamed antigen and with alcohol-treated organisms. The results are shown in table V.

In experiments 340 and 343 serum 63 made with G1.R. steamed for 1 hour showed no agglutination or immobilisation of the living homologous organisms and was completely innocuous as regards death, but produced the sheath-forming and discarding reaction in a moderate number of organisms in the later readings in a dilution of 1 : 5, diminishing to a trace in 1 : 20. In further dilutions there was no registrable effect. The addition of a suitable dilution of complement to the serum produced an interesting result. A proportion of the organisms were killed and agglutinated, but although the contents of the ciliates were sometimes half extruded

TA

BL

E IV.

Effe

ct of

addi

tion

of co

mpl

emen

t to

abs

orbe

d an

d un

abso

rbed

ser

um.

I I

Exp

t. n

o.

I

448.

Se

rum

93,

una

bsor

bed

1 : 8

0-1 : 3

20.

No

effe

ct

449.

Se

rum

93,

not

abs

orbe

d,

+com

plem

ent

1 : 3

20

1 : 6

40-1

: 25

60.

No

effe

ct

445.

Se

rum

93

abso

rbed

with

al

coho

l-tr

eate

d G

1.R

.

...

1:4

0,

1:80

, 1

:160

.

Agg

lutin

atio

n +

( +)

to +

; + t

o f

Tra

ce o

f ef

fect

, a

very

few

dea

d de

ad,

no l

ysis

; a

few

dis

card

ed

but

not

lyse

d sh

eath

s

Tra

nsito

ry a

gglu

tinat

ion

+ + +

to

++ .

mas

ses

disc

ard

shea

ths ; '

none

dea

d

Agg

lutin

atio

n +

to +

+ . $

to

!z. de

ad,

not

lyse

d ;

&as

ses

disc

ard

shea

ths

Tra

nsito

ry a

gglu

tinat

ion

+ +

to

tr..

m

otili

ty

redu

ced.

ex

uda&

; n

o sh

eath

s ;

uond

de

ad

Agg

lutin

atio

n + +

+ to

+

. + d

ead

1:4

0;

few

de

ad

1 :

160 ;

com

plet

e ly

sis

No

effe

ct

1 : 3

20-1

: 64

0.

No

effe

ct

Agg

lutin

atio

n ++

to

+ ;

thin

dis

card

ed s

heat

hs

1 : 2

0.

Non

e de

ad ; n

o ob

viou

s ag

glnt

ina-

tio

n; m

any

disc

arde

d sh

eath

s

Agg

lutin

atio

n + +

+ to

+

++

(+).

; f

to n

earl

y al

l de

ad, p

artia

lly l

ysed

No

effe

ct

1 : 4

0-1 : 8

0.

A f

ew d

isca

rded

she

aths

Ver

y sm

all n

o. d

ead

and

lyse

d

Agg

lutin

atio

n +

+ .

+ de

ad,

DO

ly

sis ;

man

y di

scai

ded

shea

ths

Seru

m m

ade

with

sol

uble

exu

date

;

Agg

lutin

atio

n +

( +) ; + d

ead

part

ly

A s

mal

l no.

dea

d an

d ly

sed,

rest

not

ly

sed .

mas

ses

of

disc

arde

d af

fect

ed

shea

th

1 : 1

280-

1 : 2

500.

Agg

lutin

atio

n +

to -

; tr

. ex

udat

e ;

no s

heat

hs

Agg

lutin

atio

n + +

+ + ; n

earl

y al

l dea

d an

d ly

sed

No

effe

ct

No

effe

ct

sts

on li

ving

Gla

ucom

a.

1 : 5

000-

1 : 4

0,00

0.

Tr.

exud

ate

to 1

: 50

00

Agg

lutin

atio

n+ +

+ +

to +

(+);

di

min

ishe

d no

. of

de

ad

and

lyse

d

No

effe

ct

No

effe

ct

Con

trol

.

Sal

ine;

no

ef

fect

Com

plem

eui

1:32

0; nc

ef

fect

Sali

ne;

nc

effe

ct

Com

plem

enl

1 : 3

20 ; nc

ef

fect

TA

BL

E V.

Eff

ect

of

addi

tion

of c

ompl

emen

t to

ser

a pr

epar

ed w

ith s

team

ed a

nd a

lcoh

ol-tr

eate

d an

tigen

s.

Exp

t. n

o.

I 1

: 5.

I 1

: 10-

1 : 2

0.

I 1

: 40.

343.

Se

rum

63

with

co

mpl

e-

men

t 1 3

60

... ...

297.

Se

rum

81,

alc

ohol

-tre

ated

301.

Se

rum

81,w

ith c

ompl

emen

t

St.L

.

1 : 4

20

1 : 1

60-1

: 320

.

No

effe

ct

1 : 6

40-1

: 1

0,00

0.

No

effe

ct

KO

effe

ct

I

Salin

e an

d co

mpl

emen

t con

trol

s w

ere

in p

erfe

ct c

ondi

tion

and

show

ed n

o ef

fect

.

336 M . ROBERTSON

from the solid-looking sheath the substance of the more or less disrupted organism was in no way attacked or lysed. A rapidly diminishing number of dead were found up to a dilution of 1 : 40, still without lysis, and in further dilutions there was no effect a t all. Here the sheath-forming antibody, by itself quite innocuous in this concentration, combined with complement to produce death with some degree of agglutination but without lysis. The effect has a perfectly simple relation to the dilution and is most pronounced in the highest concentration of the antiserum.

In experiments 297 and 301 (table V) a low-titre serum (81) made from alcohol-treated ciliates of strain St.L. was tested with the homologous living organism with and without complement. I n this serum the sheath-combining antibody was present in sufficient amount to produce only the characteristic sheath-casting reaction up to a titre of about 1 : 80, while the agglutinin and the immobilising antibodies, which are always present to some extent in sera of this kind, were not powerful enough to be readily recognised in the reaction of the ciliates to the serum without complement. The addition of complement produced a combination with the sheath-reacting antibody in the higher concentrations which was lethal, killing large numbers of the organisms without lysis, while the trace of agglutinin and of immobilising antibody combined with the complement to produce a much restricted trace of lysis in 1 : 160-1 : 320 where the sheath-reacting antibody was not operative. Dilutions from 1 : 640 to 1 : 10,000 were without any effect.

Attention is drawn to the small amount of lysis (disappearing at the dilution 1 : 320 of the immune serum) correlated with the very slight development of agglutinin and immobilising antibody in this experiment. It is in sharp contrast to the very high titre of the lytic process in sera derived from the living antigen in the same circumstances.

Summary of .the foregoing sections.

In those experiments where sera containing only, or little but, the sheath-forming antibodies were combined with complement, there was no increase in the lethal action in the higher dilutions and the effect disappeared with diminution in the concentration of the antibody. This seemed to be linked with the fact that there was little or no competition for the constant amount of complement between the two different types of antibodies. In the absence of the agglutinin and its allied elements there was no lysis and the sheath-combining antibody in conjunction with complement produced death but not lysis. Where, as in the absorbed sera, the agglutinins (using this to mean the antibodies derived from the

ANTIGENS OF PROTOZOA 337

heat-labile antigens) were freed from the competition for complement and the antagonistic action of the sheath-forming antibody, they combined even in minute quantities to cause death with agglutina- tion and actual lysis or dissolution of the living elements of the body of the organisms.

Discussion.

From the foregoing it will be seen that the main characteristics of the reactions of these organisms to the homologous antibodies depend on the cell substances lying just below the cuticle. From this area is poured out the exudate which appears at the moment when the glaucoma first reacts to an effective serum made from the living antigen and it is the effect upon this part too which is apparently responsible for the immobilisation.

Agglutination, immobilisation (without, however, direct injury to the cilia) and the extrusion of exudate have been classed together here as allied reactions to immune sera. It seems reasonable to conclude that they are concerned with one complex of antigens which are connected with the periphery of the organism and in particular with the soluble exudate obtainable from the living organism in saline by the kind of treatment described earlier in this paper. It is not claimed that any further analysis of the relation of this antigen complex to the immune body has been made. It has, however, been made clear that certain elements in this exudate are the antigen or antigens responsible for the antibodies, which may for convenience be called the agglutinins, giving the particular reactions detailed above and for the combination with complement which produces death and lysis of the organisms. The antigenic properties of these elements have been shown to be destroyed by heat (steaming for 1 hour) and their effectiveness is greatly reduced by exposure to alcohol. The nature of the alcohol effect has not been precisely determined and it has not been ascertained whether it is due to denaturation of the protein or to some other action.

It appears also that the actual sheath-forming substance is present, at least to some extent, in the exudate poured out into the saline as well as in the washed bodies themselves. This second element is much more resistant to the action of heat and of alcohol. It is not perfectly clear if the antibody evoked by it is able to produce any degree of agglutination in addition to causing the appearance and final casting off of the sheath. There was no agglutination of khe living bodies in such a serum, but the empty sheaths sometimes showed the process to a varying extent.

The sheath-forming antibody produced so massive a reaction when the ciliates were treated with sera against the living organism that it constituted a very striking part of the total effect. The

338 M . ROBERTSON

addition of complement to this antibody enhances its action in causing the death of the ciliates and the antibody interferes with the combination of complement with agglutinin (or antibody against the heat-labile elements), which alone is the active lytic agent in the immune serum.

The division of the elements into heat-labile and heat-stable does not appear to be so sharp here as in the bacteria, for although antisera can be made with the steamed antigen they are of very low efficiency. The steamed glaucomas are capable of absorbing the sheath-combining element from the antisera, but it is very difficult if not impossible to remove all this antibody by this means from a serum made with the intact living organism.

The conclusion is that these ciliates contain antigenic substances which share many of the characters of those already known in bacteria and that they contain both relatively thermostable elements analogous with the 0 or heat-stable antigen and more labile elements analogous with the H or heat-labile antigens of bacteria.

The very active response of the surface of the organism is particularly striking and certain resemblances to bacteria suggest themselves, such as the marked swelling of the capsules of pneumo- cocci when acted upon by a specific antiserum. Moreover the defensive mechanism of the sheath in these protozoa is perhaps of interest in connection with the greater virulence of certain capsulated bacteria (Topley and Wilson, 1936).

Summary.

The antigenic properties of two varieties of ciliate belonging to the Glaucoma-Colppidium group have been investigated and sera prepared in rabbits from antigens obtained by treating the living glaucoma in various ways. From a study of the reactions of the living organisms to these sera, correlated with those found in sera absorbed by steamed and alcohol-treated glaucomas, it has been shown that the antigenic substances contained in these protozoa can be divided into two groups possessing different powers of resistance to heat (100' C. for 1 hour) and to alcohol.

Lysis of the ciliates results from the combined action of complement and the agglutinin evoked by the heat-labile antigen.

REFERENCES.

BRESSLAU, E. . . . . . 1924. Verhundl. Dtsch. 2001. Gas., xxix. 91. FELIX, A., AND PITT, R. M. . 1936. Brit. J. Exp. Path., xvii. 81. ROBERTSON, M. . . . . 1939. this Journal, xlviii. 305. TOPLEY, w. W. c., AND 1936. The principles of bacteriology and

WILSON, G. s. immunity, 2nd ed., London, pp. 444, 445 and 657.

WEIL, E., AND FELIX, A. . . 1920. 2. Irnrnunitatsforsch., xxix. 24.