An in vitro study of the action of immune bodies called forth in the blood of rabbits by the...

576.809.73 : 616-022. g (Bodo caudatus)

AN IN VITRO STUDY OF THE ACTION OF IMMUNE BODIES CALLED FORTH I N THE BLOOD OF RABBITS BY THE INJECTION OF THE FLAGELLATE PROTOZOON BOD0 CA UDA TUS.

MURIEL ROBERTSON. Division of Protozoology, Lister Institute, London.

INTRODUCTION.

DURING the last 20 years the study of the antibodies which appear in the blood of various animals in response to protozoal infections has received a good deal of attention. I n particular interesting varieties of manifestations of immune body have been described in the different forms of trypanosomiasis. These comprise (1) the various forms of adhesion phenomena (Rieckenberg 1917, Leupold 1928-29, Duke and Wallace 1930), (2) agglutination, (3) lysis and (4) the reproduction- inhibiting antibody or “ablastin ” described by Taliaferro and his colleagues in infections with T. lewisi in the rat. A fifth effect, that of irnmobilisation or paralysis in vitro, has been noted by Regendanz and Jurukoff (1930) for trypanosomes of mammalian origin and also by Schindera (1922) in the normal sera of mammals for the trypanoplasma of the snail.

This work on the immune reactions against flagellate protozoa has for the most part been based upon deductions and observations made in vivo, the counterpart of the intensive in vitro studies of bacteri- ologists into the serological reactions against bacteria having been relatively neglected. This has been due to the technical difficulties involved.

It has for instance been difficult (Taliaferro 1932) to get trypanosomes from the blood to survive long enough in vitro for the conditions of the test (in this instance in vitro lysis) to be fulfilled. Again, the antibodies against both free living and parasitic amebae in rabbits and man have been studied (Heathman 1932, Sherwood and Heathman 1932) but agglutination experiments were unsuccessful owing to the high degree of spontaneous clumping of the controls. A direct toxic effect was however noted with immune rabbit sera culniinating in the death of the homologous amcebte, a result which will be considered in

363

364 M. ROBERTSON

a later section. Lytic and toxic effects are also described by Sellards (1911) Rossle (1905) and Menendez (1932). I n dealing with the immune bodies in rabbits and man against amebce most of the workers cited, and Craig and Kagy (1933) working with dogs have used the method of complement fixation as being the most suitable technique, though precipitin tests have also been used. I n the case of coccidia, precipitin formation and complement fixation (usually with alcoholic or saline extracts as antigen) have been the methods of approach to the immunological relations (Bachman 1930).

The diversity of the manifestations in the serological reactions of flagellate protozoa and the difficulties encountered in the study of the response of protozoa in general to humoral antibodies suggested that an enquiry into the reactions t o the immune bodies called forth in the blood of rabbits by a flagellate which could be readily observed in vitro, might yield informalion as to the nature and effects of such bodies. The conditions, although presenting here also certain difficulties, permitted a study of the direct effect upon the living organisms of the immune bodies with and without the addition of complement. The effect of different salt concentrations upon their action could also be observed.

A further appositeness has been lent to this study by the fact that certain intestinal protozoa such as the trichomonads, genera usually living upon ingested bacteria, have of late been shown to include species which have invaded the tissues and become adapted to a life under truly parasitic conditions. The forms referred to are the trichomonads apparently associated with certain types of abortion in cattle (Witte 1933), and T. columbce (Bos 1933-34) which causes a disease in pigeons.

The object of the present study was t o find out how a flagellate of this type behaves in the presence of a homologous immune serum and particularly how its behaviour resembles or differs from that of other organisms under similar conditions.

Before passing to the description of the method used, it should be pointed out that in the work on the reactions of protozoa to immune body it is not as yet possible to obtain the extreme standardisation of method in the study of the behaviour of the different organisms which has become customary in bacteriology. This is due partly to the wider range in shape, size and physical consistency among the protozoa, partly to the variety of means of locomotion and nutrition and partly to the fragmentary state of our knowledge. The difficulties enumerated above have undoubtedly stood in the way of the study of immunity against the protozoa. The reactions of the protozoa to immune bodies have to be investigated for the present by methods appropriate to the particular organisms used and data must be gradually accumulated and elucidated before their exact relation t o the great body of detailed knowledge already existing in the case of the bacteria can be defined.

IMMUNE BODIES TO BOD0 365

The angle of approach in the present work is also slightly different from that usually obtaining in the highly developed bacterial immuno- logy. I n this study of bodo, as with many protozoa, the observations, in the present state of our knowledge, have to be made on the organism as a living cell.

MATERIALS AND METHODS.

Method of culture.

The flagellate Bodo caudatus is a free living organism with two flagella, a relatively short one, directed forwards when swimming and another, very much longer, which trails behind. It has a nucleus and, like practically all the trypanoplasms and trypanosomes, also a second body composed of dense chromatin which lies near the point of origin of the two flagella a t the anterior end of the body. This chromatin mass is the kinetonucleus, known in German literature as the blepharoplast. The flagella originate from the basal granules, which are non-chromatic structures lying one a t the base of each.

Bod0 has certain advantages as a test object as it can be very easily cultivated in an aqueous medium of appropriate salt content (see appendix), or in glass-distilled water in which a food bacillus, separately grown, is suspended. The bacillus (YP 74), a small motile gram-negative rod, was originally obtained along with the bod0 from hay infusion cultures of macerating tank material, and was grown over- night on Lemco-agar without peptone. The suspensioiis were made by washing the bacteria off the surface of the slopes with the water or salt solution selected and the opacity could be regulated by the use of the Wellcome” opacity tubes, opacity 3 being that usually chosen.

Healthy and vigorous cultures of bod0 could be obtained in fluids of very varying salt content and advantage was taken of this to have a series growing in a Ringer fluid of the same tonicity as mammalian blood. The salt tolerance of bod0 removed difficulties encountered for example by Sellards (1911) and Rossle (1905), who had t o carry out their work on the reaction of various protozoa to their immune sera in reduced salt concentration owing to the salt intolerance of the test object. On the other hand the good growth of this flagellate in distilled water and in low concentrations of salt permitted comparisons between the interactions of the protozoa and immune body under varying salt conditions. The convenient size of bod0 ( 5 to lop) permitted direct observation under the 8 in. objective during the whole course of the tests, which were carried out in small Petri dishes.

The disadvantage of bodo as an object for this type of study lies in its bacterial food supply, but as will be seen in the sequel, the immune body reactions of the bacteria do not prevent the observation of those caused by the protozoa in the direct method used. This was confirmed by the use of sera which had been absorbed with the bacteria, and with

2 D JOURN. OF PATE. - V O L XXXVIII.

366 M. ROBERTSON

sera made from the bacteria alone. The presence of bacteria, however, did render impossible macroscopic observation in tubes, the method used habitually in bacteriology, and they were also a serious hindrance in precipitin experiments.

Preparation of the sera. Two types of antigen were used for injection intravenously into

rabbits: (1) a washed suspension of bodos and of the bacteria carried down with them adjusted to 0.85 per cent. saline and containing 0.1 per cent. of formalin, and (2) a steamed antigen consisting of a washed suspension heated in distilled water in the steamer for one hour, the salt content being brought to 0.85 per cent. after heating. The immunisation has to he carried out with caution, but doses of formolised antigen commencing with 10 millions and rising gradually to 200 millions could be tolerated, and five to seven doses spaced a t 2 to 6 day intervals produced sera with a satisfactory content of immune body. The first sera were made with living bodos, but this was abandoned owing to difficulty in carrying the irnmunisation beyond the early doses. Larger quantities starting with the equivalent of 20 millions of the steamed antigen could be used.

As might be expected, all the sera possess immune bodies reacting not only with bod0 but also with the food bacillus (YP 74).

flpontaneous aggregation of jlagellutes.

Before describing the serum tests and considering the data they present it is necessary to give a brief account of the spontaneous aggregation of flagellates, as this is liable to cause confusion. There are two types of spontaneous aggregation. (I) Protozoa in general and flagellates in particular will aggregate in bands in response to chemotactic stimuli or respiratory needs. Bod0 species show particularly good examples of this in regard to CO, concentration, a phenomenon which has been studied by Fox (1920-21). This type of aggregation is not a source of error in the serum tests as the method permits the even distribution of all the soluble substances.

( 2 ) Another and much more important form of aggregation from the present point of view is that which takes place round minute particles and bacterial felts in response to the stimulus of thigmotaxis. This is a sensitiveness of certain parts of the body to touch stimuli. The trailing flagellum of bod0 is positively thigmotactic and under a variety of conditions the tip becomes attached to the substratum or more usually to some minute aggregation of bacteria or to larger felts. The shorter flagellum is clearly negatively thigmotactic, the body of the organisu apparently indifferent to the stimulus except a t the extreme posterior end which may adhere temporarily to the glass or to the surface film of the water. The phenomenon of thigmotaxis

IiVMUNE BODIES TO BOD0 367

which occurs in some form or other in very many protozoa has not received much detailed study since Putter's paper in 1900 though Grass6 and Schindera have both considered it in flagellates.

I n the case of Bodo caudatus the phenomenon, which is particularly well-developed in the early and middle period of a culture, is very striking. The bodos collect in actively motile groups delicately attached by the tip of the trailing flagellum while the shorter is in perpetual movement, the anterior end of the body turning away from the object or centre of the group only t o be caught and forced to turn towards it again by the pull of the attached flagellum. Great balls of bodos may be formed and the bigger aggregates may attract the smaller and fuse with them. The organisms are however not attached by any part except the actual tip of the longer flagellum and, if the fluid containing the balls is drawn gently out and in with a Pasteur pipette, the elements separate and a dispersed culture is again produced. The bodos however gather up again into balls a t this stage while a t a later period the cultures show a spontaneous dispersal of the flagellates,

The exact conditions calling forth this reaction of thigmotaxis in the culture plates are extremely difficult to determine, but the accumulation of the products of metabolism of the culture as a whole tends to give rise to dispersed growth. These spontaneous aggregations of free bodos could be distinguished from the agglutinated masses about to be described by characteristic differences in appearance and, where any doubt existed, by the mixing up of the culture with a sterile pipette. The balls were then found to disperse completely while the agglutinated masses, even when the big clumps were broken up, showed the elements still tightly stuck.

Technique of tests.

The sera were diluted and placed in descending order of concentration in test tubes in quantities of 2 C.C. The living bodos from culture plates were centrifuged and washed once. The suspension was counted and then further diluted so as to give about 500,000 to 700,000 per c.c., 2 C.C. of this final suspension being added to each tube. The test tubes, containing now 4 c.c., were tipped out as soon as possible into small sterile glass Petri dishes 4 ctn. in diameter andwere marked with the final concentration of serum in the order of the tubes. The Petri dishes mere placed in a glass container at room temperature and shielded from direct light. All readings were made under a binocular microscope using a in. lens and 7 ocular and were spaced so as to give two or three in the first 9 to 10 hours. The plates were read again at about 24 hours, the sequel being observed where i t appeared still to be yielding data of interest.

The bodos are sufficiently large to be clearly seen a t this magnification and for their reactions to be recorded. Death could be recognised and agglutination assessed as even the faintest trace of sticking together could be distinguished. In the higher concentrations of serum, agglutination of the bacteria occurred but this did not prevent the behaviour of the protozoa from being followed. The control plates where the washed bodos were placed in the fluid used to dilute the serum showed the condition of the flagellates and also gave an account of the starvation effect as the test progressed. The paradox has to be borne in mind that bodos surviving in the higher concentrations of serum have a better food

368 M. ROBERTSON

supply owing to the bacteria upon which they feed having better means of growth in the greater amount of serum than in the lesser or in the control.

The observations were recorded in the following manner. The death of all the bodos in the plate in 24 to 48 hours was considered to be the killing titre of the serum, survival being always accompanied by some degree of multiplication of the bodos left alive.

The symbols used were : L L tr >> . . . for any minimal degree of agglutination, however slight or

rare ; + . . . for a small amount of clumping into groups of small size with

many free bodos ; + + . . . the frequent appearance among free bodos of clumps of small or

medium size, or the occurrence of a few clumps of large size ; + + + . . . good agglutination in clumps of large size with some bodos

still free ; + + + + . . . agglutination in very big clumps and cases where the culture

is completely clumped, but the occurrence of some individual free bodos cannot be excluded.

EXPERIMENTAL RESULTS.

I. Experiments with heated sera in salts isotonic with the blood.

(a) Xxperirnents with heated (55" fo r 30 minutes) immune sera made with formolised bodos.

The tests were set up in the manner described above, using washed bodos from cultures grown permanently in a mammalian Ringer solution and sera diluted either with this fluid or with 0.85 per cent. NaCl. The sera should be st,ained as little as possible with hEmo- globin as this substance seems to exercise some effect upon agglutination.

The reactions of living bodos to the presence of immune sera were (1) greater or lesser interference with motility, (2) agglutination of the bodies of the protozoa, and (3) death without lysis in the higher concentrations.

It is usual i n immunological studies with bacteria to give the t i tre of a serum in terms of macroscopic agglutination. In the present case the best estimate of the immune body content was the point a t which all the bodos were killed, the time limit being fixed between 24 and 48 hours. Sera with killing titres of from l/SOO to 1/3200 could be obtained. The reason for the use of death as a n estimate of the value of the sera rather than agglutination will become apparent in the description which follows immediately.

Bodos in the more powerful sera showed a considerable degree of immobilisation, although the flagella may be in feeble oscillation. It is a general observation among protozoa that the response to any form of attack not immediately destructive is followed by some degree of recovery. And this occurred here also, there being first a gradual or more rapid immobilisation followed by some degree of recovery.

In concentrations usually up to 11400 to ljSO0 agglutination was so

IMMUNE BODZES T O BOD0 369

powerful that it took place in from 2 to 5 hours, forming tightly stuck clumps with moving flagella no matter how little movement of the body was taking place. The beating of the flagella was often too feeble to cause any translation of the organism as a whole. I n concentrations below 1/800, but particularly from 11800 to l / lSOO, immobilisation was pronounced and was accompanied by a tendency to stick t o the glass, so that agglutination was very much reduced. Recovery of niotility to some slight extent occurred here too, but the death of the culture often supervened with only a very faint degree of agglutination.

I n still higher dilutions the recovery of motility coincided with a certain amount of agglutination and the growth and final survival of a proportion of the bodos. In cultures where the food supply permitted continued growth a delayed or secondary agglutination occurred in the surviving bodos in concentrations of 1125,000 or even less. This is due to thegreater sensitiveness of the bodos to agglutination a t different periods of their growth. The conditions of secondary agglutination were not considered to be adapted t o the appraisal of the immune body content of the sera.

With less powerful sera and with larger numbers of bodos particularly from younger cultures agglutination was not interrupted or only to a much slighter extent, and + + titres were shown a t 5 to 10 hours in dilutions up to 113200 or even 116400. Moreover, the death of the whole culture occurred only a t higher concentrations in these weaker sera.

The point in the dilution series a t which death of all the bodos took place was very constant whereas the degree of agglutination varied. Neither in the case of the clumps nor of single unagglutinated individuals was death accompanied by lysis or early breaking up of the body. Table I shows the reactions described.

(b) -Experiments with heated immune serum made with bodos steamed for one hour.

The study of sera of this type was not pushed very far. I n the most effective serum made, which was the result of the intravenous injection of a total of about '100 millions of bodos steamed for one hour, the killing titre was very much lower than in those made with unheated antigen, occurring a t a concentration between 1/100 and 11200. There was some effect on the motility of the bodos, but it was certainly much reduced. Agglutination was well developed and appeared up t o 113200 but the phenomenon of this reaction co-existing with active growth was shown very clearly in dilutions from 11400 onwards, indicating the much less toxic action of the seruni. Here also death was not accompanied by lysis.

It is not proposed to enter here, on the basis of these rather meagre results, into the intricate question of the possibility of a heat-labile

JOURN. OF PATH.-VOL. XXXVIII. 2 D 2

370 M. ROBERTSON

TABLE I. Reactiwn of living bodos in mammalian Ringer solution to heated immune

rabbit sera made with formolised bodos.

Expt. 115 Serum 30

Expt. 88 Serum 27

1/‘200.

Motility much

reduced. Agglut. + + + +

All dead. Not lysed. Agglut. + + + +

...

Motility much

reduced. Agglut. + + + + All dead. Not lysed. Agglut. + + + +

...

2 C.C. serum diluted in Ringer + 2 C.C. washed bodos.

11400.

Motility much


A11 dead. Not lysed.

*..

Motility reduced. Agglut. + + + +

Many dead Agglut. + + + +

All dead. Not lysed.

1jsoo.

Almost immobile. Agglut. + +

1. few alive No lysis. Agglut. + +


Motility reduced. Agglut. + + +

Many still alive.

Agglut. + + +


1/1600.

Immobile. Agglut. “ tr.”

Few alive. No lysis. Agglut. “ tr.”


Motility reduced. Agglut. + +

Survival. Growth. Agglut. “ tr.”

...

(flagellar) and heat-stable (somatic) antigen.

113200.

Immobile. Agglut. ‘‘ tr.”

Recovery. Agglut.

“tr.”

Survival. Growth.

Agglut. +

Motility fair.

Agglut. + +

Survival. Growth. Agglut. ‘‘ tr.”

...

1/6400.

Almost immobile. Agglut. “ tr.”

Recovery. Survival. Growth.

Agglut. + ...

Motility fair.

Agglut. + +

Survival. Growth. Agglut. +

...

Time (hours).

1 to 10.

22.

After 24.

1 to 10.

22.

After 24.

Suffice it to say that the enormously greater amount of body substance as compared with the flagellar material would, as with 7? cholerat: (Balteanu 1926), probably demand special methods to demonstrate a particular flagellar element in the tests. Moreover the degree of heating was not sufficient, on the analogy of bacteria (Weil and Felix 1920), t o preclude the survival of some heat-labile antigen. A further point of interest lies in the morphology ; the flagella are organelles anchored in the endoplasm, out-growths originating from the basal granules in this position and, whatever their actual substance, they are not prolongations of the periplast or ectoplasm.

The matter brought forward here is simply the observation that heating the antigen to 100” C. for one hour permits ‘the production of an immune body capable of bringing about (1) some degree of injury to motility, (2) agglutination, and (3) death of all the bodos in the higher concentrations. Both the reduction in the death titre and the more feeble effect upon the motility were striking in comparison with

1MMUNE BODIES T O BOD0 371

Motility normal. Agglut. + + Growth.

the action of a serum made from an unheated antigen. the reactions described.

Table I1 shows

TABLE 11. Reaction of living bodos in mammalian Ringer solution to heated immune

rabbit serum made with antigen. heated to 100" f o r 1 hour.

Motility normal. Agglut. " tr."

Growth.

-

Expt. 94 Serum 33

Motility normal. Agglut. + + + Growth. Agglut. + +

2 C.C. serum diluted in Ringer + 2 C.C. bodo suspension.

Motility normal. Agglut. + + Growth. Agglut.

+ +

Time (hours).

1/3200. 1/25. 1poo. 1/200. 1/800.

Motility good.

Agglut. + + + Growth. Agglut. + + + +

...

Motility much

reduced. Agglut. + + + + Many dead.

Agglut. + + + + All dead. Not lysed.


Many dead.

Agglut. + + + + All dead. Not lysed.

Motility good.

Agglut. + + + +

Few dead. Agglut. + + + +

Dead. Not lysed.

Motility good.

Agglut. + +

Growth. Agglut. " tr."

...

1 to 10.

22.

4fter 24.

(c) Experiments with heated normal sera of rabbits and guinea-pigs. The reactions of bod0 in heated normal sera diluted with mammalian

Ringer solution were the same in the case of rabbits and guinea-pigs (table 111). I n these tests the bodos showed no loss of motility and

TABLE 111. Reaction. of living bodos in Ringer solution to heated normal guinea-pig and

rabbit seru.

2 C.C. serum diluted with Ringer + 2 r.c. bodo suspension. Time

(hours). 1/20. I jSO. ~ 1/320. 115. Expt. 103

Guinea-pig serum 242, old, heated 55°C. for 45 minutes

1/40.

Motility normal. Agglut.

+ + Growth. Agglut. + +

Motility normal. Agglut. + + +

Growth. Agglut.

+ +

Motility normal. Agglut. + + Growth. Agglut. + +

1 to 10.

20 to 24.

1 to 10.

20 to 24.

Motility normal. Agglut. + + + + Growth. Agglut. + + + +

Motility normal. Agglut. + + + Growth. Agglut. + + + +

Motility normal. Agglut. + + 4-

Growth. Agglut. + + +

Expt. 107 Normal rabbit serum 4

hours old, heated 55°C. for 30 minutes

372 M. ROBERTSON

agglutinated by their bodies in clumps of all sizes, though there were always a few free individuals and multiplication went on continuously. I n heated normal rabbit serum which had been shed only 4 hours previously, an agglutination titre of + + in 11640 was obtained but 11160 to 11320 was more usual for sera which had been kept some time. The agglutination titre of heated guinea-pig serum is usually + + in about 11160.

Even in a concentration of 115 there was no case where heated normal sera were able to bring about the death of all the bodos; some individuals and some clumps did indeed die in the highest concentrations, but active growth and the production of numerous cultures always resulted. The contrast between the paralysing effect of immune sera in agglutination and immobilisation and the continued activity and immediate growth in heated normal sera in spite of the collection into clumps was very striking. The action exerted by these sera wa8 that of a normal agglutinin.

Bodos in immune sera made with the formolised and heated food bacillus as antigen behaved exactly as in heated normal sera, thus showing that the effect on the bacteria was not an obvious contributory cause of the reaction noted in the immune sera made with the bod0 as antigen .

(d) Lethal e f ec t of heated immune sera.

It has been shown that in heated immune sera used with mammalian Ringer solution there is a powerful killing capacity which is absent in the normal sera tested. Death may follow upon agglutination, but single unagglutinated individuals are also killed in these concentrations. This killing titre was found to be remarkably constant and death of all the cells took place in dilutions of 1jSOO to 113200 according t o the degree of immunisation in sera made with formolised bodos, and in dilutions of 1/50 t o l/200 in sera made with bodos steamed for 1 hour. Death of the protozoa in these circumstances was not connected with nor followed by lysis. There were no data as to exactly what necessary function was being interfered with. I n many cases the degree of recovery of motility of the flagellar organelles in individuals which afterwards died did not suggest any particular attack upon these structures themselves. While the actual resistance of a few living bodos prolongs the final moment a t which the contents of a test plate can be described as ‘I all dead ” to between 24 and 36 hours, the great majority of the organisms in the higher concentrations were dead within 10 hours.

The fundamental importance of the lethal effect as an immune reaction arises out of its absence or very feeble development in normal sera and from its stability of titre in the different circumstances about to be described.

IMMUNE BODIES T O BOD0 373

11. Reactions in the presence of complement in salts isotonic with the blood.

(a) Normal guinea-pig and rabbit serum with the natural conylenwnt intact.

Before considering the effect of the addition of fresh guinea-pig serum as complement to the immune sera in Ringer solution, the reaction produced by normal guinea-pig serum alone must be recorded.

I n the tests with fresh guinea-pig serum (24 hours in the ice chest) in concentrations up to and including 1/20, the bodos were immobilised, the body of the flagellates became rounded in shape and, after a brief period during which they appeared as shadowy transparent cells, they were completely lysed in 2 to 4 hours. This rapid death was accompanied only by a very small amount of agglutination (table IV,

TABLE IV. Reaction of living bodos in Ringer solution to fresh guinea-pig serum (24 hours

old) and jresh rabbit serum (4 hours old).

3xpt. 102 Fresh guinea - pig

serum 24 hours old

Expt. 105 ?resh rabbit serum

4 hours old

2 C.C. serum diluted with Ringer + 2 C.C. bodo suspension.

115.

All dead. Agglut. '' tr." Lysis.

...

...

All dead.

tr. Lysis.

4%:g'$

...

...

1/20.

All dead. Agglut. +

Lysis.

...

...

[mmobilised Agglut. " tr."

Nearly all Ipsed.

All dead. Lysis.

...

1/40.

Nearly all dead.

igglut. + + Lysed.

Recovery. Growth.

Growth. igglut. + +

Motility reduced.

Agglut. +. Some lysis.

Recovery. Growth. Agglut. I' tr."

Growth. Agglut. f f

1/80.

Motility reduced.

Agglut. + Some lysis.

Recovery. Growth.

...

Motility normal.

Agglut. +. A little lysis.

Growth. Agglut. +.


1jSZO.

Motility normal. Agglut. '' tr."

Growth.

...

Motility normal. Agglut. + + + No lysis.


...

Time (hours).

1 to 10.

20.

Ifter 24.

1 to 10.

20.

ifter 24.

expt. 102). I n 1/40 the great majority of the flagellates were lysed in the same way, but the survivors recovered and produced a vigorous growth which then agglutinated a t about 48 hours ; in higher dilutions a very slight amount of clumping occurred. All samples of complement caused death in a dilution of 1/20 in these tests but the degree of agglutination varied considerably in the higher dilutions.

374 M. ROBERTSON

The action of fresh normal rabbit serum, used within 3 or 4 hours of shedding in order to obtain the natural complement intact, was very similar to that of 24-hour-old guinea-pig serum and for the sake of brevity is included in table IV (expt. 105). Immobilisation was a little more gradual than with guinea-pig serum, but was followed by death and rapid lysis in exactly the same way up to 1/20. I n 1/40 agglutination was very much delayed, not showing until 41. hours ; 1/80 and 1/160 showed agglutination after 20 hours while the next two dilutions showed immediate agglutination.

The only difference between the 4-hour-old rabbit serum and the guinea-pig serum in the condition in which it is usually used as complement (24 hours old in the ice chest) was the slightly less rapidity and activity of the lysis and the slightly greater degree of agglutination in the rabbit serum.

Having seen that heated normal guinea-pig sera produced agglutination but neither immobilisation nor lysis in the conditions of the experiment, and that when unheated (i.e. when containing the complement) there was an active development of lysis killing the whole culture up to 1/20, it was suggested that the normal agglutinin was functioning as a lysin in the presence of the undisturbed complement. It did not seem necessary to suppose that another sensitising agent was present.

(b) Addition of guinea-pig complement to heated immune sera.

From the data in the immediately preceding paragraphs it was clear that in adding complement to heated immune serum it wa,s necessary to choose such a concentration that the total amount of guinea-pig serum in the test plates was not in itself lethal.

The content of the tubes which was poured into the little Petri dishes was made up of 2.0 C.C. of the dilution of the immune serum, t o which was added 0.5 C.C. of 1/10 complement and finally 1.5 C.C. of the bod0 suspension in Ringer solution, the controls without complement having 0.5 C.C. of the Ringer solution added instead of the 1/10 complement. The final concentration of guinea-pig serum in any of the observed plates was therefore 1/80.

I n expt. 99 with the addition of complement there was a much reduced motility; the bodies became gradually rounded, swollen and transparent and death of all the bodos with lysis of the dead cells occurred i n concentrations up t o 1/800. The flagella here also did not appear to be specifically attacked and cells which were manifestly affected by the serum still showed faint movements of these organelles. Agglutination was poorly developed. The attack on the body of the cells is quite unmistakable and while the actual killing titre is not raised beyond 11800, which is also in this type of dilution the death point of the serum without the complement, the reaction is strikingly different. Death is more

Table V shows the result of such a test.

IMMUNE BODIES TO BOD0

TABLE V.

375

Effect of addition of guinea-pig complement to immune rabbit serum in Ringer solution. (The titration of the sample of complement is shown in table IV., expt. 10%)

Expt. 99. Serum 27. Immune rabbit +

complement.

Expt. 98. Serum 27. Immune rabbit with

out complement.

2 C.C. 8emm diluted with Ringer + 0.6 C.C. 1/10 complement + 1.5 C.C. bod0 suspension.

I j l O O .

All dead. Lysed.

...

All dead. Lysed.

...

1/400.

Motility much

reduced. Many dead and lysed.

Agglut.

All dead. Lysed.

+

...

1/800.

Motility much

reduced. Some lysis.

Agglut. +

Nearly all dead. Lysis.

All dead. Lysis nearly complete.

lj1600

Motility reduced.

Some lysis. Agglut.

+ +

Many dead. Partial lysis.

Survival. Agglut. +

Motility reduced. Agglut.

+ +

Some dead. Some lysis.

Survival. Agglut. " tr."

2 C.C. serum diluted with Ringer + 0.5 C.C. Ringer solution and 1.6 C.C. bod0 suspension.

Motility much


All dead. No lysis.

...


Many dead. No lysis. Agglut. + + + +


Motility reduced. Agglut. + + +

Some still alive.

Agglut. + + +


Motility fair.

Agglut. + +

Some dead. Survival. Agglut. " tr."

...

1/3300

Motility fair.

Agglut. + +

Survival. Agglut. '' tr."

...

Time (hours).

1 to 10.

20.

Yfter 24.

1 to 10.

20.

After 24.

rapid and destruction of the cells is complete so that in the higher concentrations hardly any remains of the protozoa can be recognised. I n the lower concentrations there is a considerable amount of killing off of individual bodos but the cultures survive.

There is apparently no necessary agglutination of the cells before lysis and this reaction was not developed to anything like the degree of the control without complement, as illustrated in table V, expt. 98. Nevertheless if the complement was weak and if the number of bodos was great, good agglutination sometimes occurred prior t o lysis.

The control of the complement in 1/80 had to be considered in reading expt. 99. The titration of the sample of complement used is given in table IV, expt. 102, and shows that in 1/80 immobilisation and death of some of the individual bodos occurred but there was vigorous growth as well.

376 M. ROBERTSON

It should be pointed out that in none of these tests could the lysis be due t o an osmotic effect as the cultures had been growing in the Ringer solution for weeks and there was no difference in the tonicity of any of the fluids. It appeared therefore that the addition of guinea- pig complement to immune sera in mammalian Ringer solution produced an active lysis of the bodos.

111. Absorption of normal rabbit sera with bod0 and the food bacillus.

A small number of absorption experiments were carried out with normal rabbit sera to see if the agglutinin observed could be removed (table VI). Motility was not affected in any of the tests and is therefore not indicated in the table.

TABLE TI. Absorption of normal serum (heated to 55°C. for 30 minutes) with living bodos and with

the Eiviny food bacillus, in Ringer solution. Motility was normal throughout.

Expt. 177. Normal rabbit serum.

Expt. 118. Normal rabbit serum

absorbed with living bodos.

Expt. 114. Normal rabbit serum

absorbed with the living food bacillus.

2 C.C. serum diluted with Ringer + 2 C.C. bodo suspeusion.

1/10. ~

Agglut. + + + + Growth. Agglut. + + + Growth. Agglut. + + +

Not affected.

Slow growth.

Very active growth.

Agglut. + + + + Growth. Agglut. + + + + Growth. Agglut. + + +

- 1/20.

Agglut. + + + Growth. Agglut. +f+


Not affected.

Slow growth.

Very active growth.

Agglut. + + +



1/40.

Agglut. + + + Growth. Agglut.

+ + Growth. Agglut.

+ +

Not affected.

Not affected.

Very active growth.

Agglut. + + + Growth.

+ + Agglut.

...

1/320.

Agglut.

Growth, Agglut. +

+

...

Not affected.

Not affected.

Very active growth.

Agglut.

Growth. Agglut. +

+

..-

Time (hours).

1 to 10.

20.

After 24.

1 to 10.

20.

After 24.

1 to 10.

20.

After 24.

Normal heated serum absorbed in saline with living bodos (washed three times) showed (expt. 118) that the normal agglutinin had been completely removed even from the 1/10 dilution, while treatment with the thrice-washed living food bacillus (UP 74) gave a test in which no

IMMUNE BODlES TO BOD0 377

appreciable loss of the normal agglutinin for bod0 could be detected (expt. 114).

The specificity of the antibodies in normal sera has been asserted and denied in the recent literature (Mackie and Finkelstein 1931 and 1932, Finkelstein 1933, Gordon and Carter 1932, Gordon 1933). I n the present instance the normal antibody which reacts with bod0 is specific to the extent that it can be absorbed by the bod0 suspension, but not by the food bacillus. No experiments t o test its further specificity have been made.

IT. Experiments carried out in reduced salt concentration and in distilled water.

(a) Experiments in distilled water with heated i,mmune sera. The action of immune ‘sera in reduced salt concentration has

received attention because of its theoretical interest. I n bacterial agglutination tests, this method is frequently used for the bacterial suspensions (Arkwright 1921) in order to obviate spontaneous agglutination of the controls.

In the present work, as the first cultures were grown in low salt concentrations (Peters’ salt sol. ; see appendix), the early tests were carried out with bodos in this solution with sera diluted with 0.85 per cent. saline. The possibility of an osmotic effect contributing to the action of the serum was however present in such a test. Experiments were therefore set up with reduced salt to dilute the serum, and finally when certain interesting differences were observed in the reaction under these conditions, tests were also carried out using distilled water for diluting both culture and serum.

The necessity for the presence of ealt (electrolyte) to effect the agglutination of a suspension of organisms by a specific immune body is generally accepted (Bordet 1899, Arkwright 1931). The process is considered to be carried out in two stages; first the coating of the cell or part of the cell with the serum and then the agglutination by means of the electrolyte. I n the absence of significant amounts of salt, the cells are still coated by the immune body, but the second stage, i.e. the actual agglutination, is postponed until the salt is added.

It should be noted that in the experiments about to be described where distilled water or very low salt concentrations were used, the sera must not be stained with hEmoglobin as even very small amounts of this substance apparently take the place of the salts and act a8 electrolyte.

In tests set up with heated sera made with living or formolised bodos in which distilled water was used as diluent and with cultures growing permanently in distilled water, the reactions of the bodos were as follows. I n concentrations from about 1/100 to l l800 or 111600 the bodos were completely immobilised in about 30 to 60

378 M. ROBERTSON

minutes, although here as in the cultures with salt the flagella did not seem to be injured and there was also some degree of recovery of the power of movement, particularly in the lower concentrations. The protozoa become rounded and swollen and, after a time during which they can be distinguished as transparent shadowy cells, they are completely lysed in the course of 5 to 10 hours. This occurred in typical sera from concentrations of 1/100 to l/SOO or 1/1600. There was, in sera not stained with hemoglobin, practically no agglutination in dilutions from 1/100 downwards, but if salt were cautiously added to any of these plates agglutination appeared in a short time.

The faint degree of agglutination sometimes produced in these distiIled water tests varied a little according to conditions which were not apparent, the slightest staining of the serum with hzmoglobin however always greatly increasing it. I n the highest concentrations such as 1/12 to 1/50 there was imniobilisation and bursting of the bodos, but agglutination, usually of the already dead cells, took place t o some degree owing to the action of the salts in the serum; the clumped masses moreover were not completely lysed.

Sera prepared with bodos heated to 100°C. for one hour showed no significant difference in the distilled water tests from those made with formolised antigen exceph in the matt)er of the titre, which was reduced to lj200.

The lrilliug titres of immune sera were, within the limits of accuracy of the test, the same in the distilled water as in Binger solution, though the death of all the bodos occurred much more rapidly in distilled water.

It should be observed again here that the lysis is not an osmotic effect as the whole system is in distilled water.

Tcsts in intermediate degrees of salt concentration showed an intermediate effect, so that the bodies were less completely lysed and there was more agglutination as the salt coricentration was increased.

In tests with distilled water, therefore, two actions of the heated immune sera were apparent, ( a ) immobilisatioii and (b ) lysis. Death did not follow inevitably upon imniobilisation but once the body of the protozoon was attacked by the serum so as to show any appreciable degree of swelling death always followed.

The interpretation that suggested itself was that the immune body, coating the cells in the absence of salt, attacked the protoplasm producing death and lysis. It is known that immune body can coat bacteria in the absence of salt, but they do not seem to be broken up by this process (Bordet 1899). It was not perfectly clear from these experiments whether the coating of the cells which ended in their death was brought about by the agglutinin, though it seemed highly probable from their behaviour when salt was added. The ifollowing absorption experiment was therefore carried out.

Table V I I illustrates the reactions described.

ZIMMUNE BODIES T O BOD0 379

TABLE VII. Reaction of living bodos from distilled water culture i n heated immune sera

diluted with distilled water. In ex@. 116 the serum was made with formolised bodos, in ex@ 95 with bodos heated to 100°C. f o r 1 hour.

Expt. 116 Serum 30,

made with formolised antigen

Expt. 95 Serum 33,

made with a n t i g e n s t e a m e d one hour

1/50.

mmobilised All dead. Aggli;;. “ tr.

Incomplete lysis.

Incomplete lysis.

mmobilised Agglut. “ tr.”

All dead. Incomplete

lysis.

Incomplete lysis.

...

2 C.C. serum diluted with Ringer + 2 C.C. bodo suspension.

1poo.

Immobilised All dead. Almost

complete lysis.

Complete lysis.

Immobilised Agglut. +

Very many dead.

Incomplete lysis.

All dead.

1/400.

Immobilised. All dead. Complete

lysis.

...

Immobilised. Agglut.

“tr.”

Many dead but survival.

Feeble survival.

I j lOOO.

Motility much

reduced. &any dead tnd lysed.

All dead. :ompletelq

lysed.

Motility fair.

Agglut. Ib tr.”

Growth. Agglut. ‘‘ tr.”

...

113200.

Motility much

reduced. Some dead.

tecovery.

Growth. Not

agglut.

Motility good.

Agglut. “tr .”

Growth not

affected.

...

1/6400.

Motility much

reduced. A few

lead. Not agglut.

Growth not

affected.

...

...

...

Time [liuurs).

to 10.

22.

. to 10.

22.

After 24. __

(b) Absorption of the immune serum in. distilled water.

A large amount of thrice-washed bod0 culture in distilled water was placed in contact with heated seruni no. 30 (made with formolised bodos) in a concentration of l / l O O in distilled water. This concentration was chosen so that the salt content niight be sufficiently low ;lot to produce agglutination. The centrifuged absorbed serum was then divided into two portions to one of which hypertonic salt was added t o bring the content t o 0.85 per cent. Expts. 124 and 123 (table VIII) shorn the results with the absorbed sera,expts. 122 and 121 the unabsorbed controls in which the serum was treated in exactly the same way except for the absorption. In the tests with the absorbed serum it was found that there was no attack upon the motility in either the salt or the distilled water series, and there was no swelling of the bodos nor any sign of lysis in the distilled water experiment in 10 hours, although in the salt test there was a little agglutination during this period in the first two dilutions. The controls show the characteristic reactions for a salt and a distilled water series respectively.

This result suggested that the immune body called forth in the blood of the rabbit by the bodo cell-substance is able, when the salt

380 M. ROBERTSON

TABLE VIII. Absorption of a heated immune serum made from formolised bodos with living

bodos i n distilled water. The salt concentration in expts. 122 and 124 was brought to 0'85 per cent. after the absorption had been carried out in the portion used in expt. 124. Distilled water cultures were used in expts. 121 and 123, and cultures in Ringer solution for expts. 122 and 124.

Serum dilutions. Time

(hours). 1jzoo. 11400. l / l G O O . 1/3200. 1/6100. Expt. 122.

Serum 30, not ab. sorbed, diluted with Ringer solution. Motility

much reduced. Agglut. + + + +


...

Motility much



...

:mmobilised A11 dead. Complete

lysis.

...

...


+

Growth. Agglut. '' tr."

...

Immobilised

L' tr. Agg"t!.

Many dead. Agglut. "ti."

Not lysed.


Immobilised All dead. Complete

lysis.

...

...

Immobile Agglut. '' tr. "

Some deac Agglut.

+

Survival. Growth. Agglut. " tr."

Almost immobile Recovery Agglut. " tr."

Survival. Growth. Agglut. +

...

1 to 10.

22.

ifter 24

Expt. 121. Serum 30, not ab-

sorbed, diluted with distilled water.

hmobilisec All dead. Complete

lysis.

...

...


Not agglut.

...

Almost immobile. Uany deai snd lysed

Recovery. Survival.

Survival. Growth.

\Tot agglui

Almost immobile. Some deac Recovery

Survival. Growth.

Survival. Growth.

I to 10.

22.

Lfter 24,

Expt. 124. Serum 30, absorbed

with living bodos in distilled water, salt then added ; diluted with Ringer salt solution.

Motility normal. Faintly sticky.

Not agglut.

Not affected.

...

Motility normal.

Not affected.

...

...

Motility normal.

Not affected.

Motility normal.

Not affected.

Motility not

affected.

1 to 10.

22.

,fter 24.

Expt. 123. jerum 30, absorbed

with living bodos in distilled water; diluted with distilled water.

Motility normal.

Growth not

affected.

Motility normal.

Growth not

affected.

Not affected.

...

Not affected.

...

Not affected.

...

to 10.

22.

IMMUiVE BODIES TO BOD0 381

concentration is sufficiently low, to combine directly with the living protoplasmic antigen and cause it t o break up, but that when sufficient salt is present, while the combination takes place, it produces agglutination and gradual death but the body is not broken up. The conclusion seems warranted that the agglutination in the salt and the lysis in the distilled water are brought about by the same antibody.

(c) Experiments with normal rabbit and guinea-pig sera, heated and fresh, in distilled water.

In work with normal rabbit and guinea-pig sera in distilled water it must be remembered that staining of the sera with haemoglobin was of greater importance owing to the high concentrations used than in tests with immune sera. Normal rabbit sera could be got which were satisfactory in this respect but guinea-pig serum was always stained to ahgreater or less extent even when obtained by heart puncture and placed immediately in the ice chest. The bodos were always agglutinated in the distilled water series in sera stained with hamoglobin and the clumps had a somewhat more fused appearance than in agglutination in the presence of salt. As already stated the explanation appeared to be that the haemoglobin took the place of the salt as electrolyte in effecting the reaction.

I n tests set up with bodos from distilled water cultures and with unstained rabbit serum diluted with distilled water, there occurred in serum which though heated was quite fresh, a trace of direct lysis followed by a slight degree of agglutination in concentrations up to 1/10. The culture however was not killed off in any case. Thereafter no further clumping occurred. I n older normal sera there was usually only a little agglutination up to 1/20 with perhaps faint traces in higher dilutions (table IX).

Heated normal guinea-pig serum 24 hours old used in distilled water tests showed variation in the degree of agglutination in relation apparently to the hamoglobin staining. Expt. 56 (table IX) is a typical example and in this the culture was not killed in 1/10 although there was some death of individual bodos. Agglutination appeared up to 11320 but the flagellates were unaffected a t 11640.

Experiments with fresh rabbit and guinea-pig serum in distilled water. As already mentioned, rabbit serum in order to have its complement intact must be used within a few hours of shedding. In a test with rabbit serum 4 hours old, with distilled water, gradual immobilisation followed by death and lysis of all the bodos took place with faint traces of agglutination in dilutions of 115 and 1/10, but in 1/20 and greater dilutions there was only a little stickiness. It would appear that the normal immune body was able, in the amount of salt present in the serum itself, to produce lysis of the cells in conjunction with the

Heated normal rabbit serum.

Heated normal guinea-pig serum.

JOURN. OF PATH. -VOL. XXXVIII. 2 E

30 2 M. ROBERTSON

TABLE IX.

Action. of noi,mal ratbit and guinea-pig serum, heated and fresh, in. distilled water, with culture of bodo in. distilled water.

Time :hours).

Serum 2 G.C. dilutrd with distilled water + bodo 2 C.C. in distilled water. __-

1/320. 1/5. 1/20. 1/80. 1/640. lxpt. 108. Tormal rabbit serum 4 hours old, heated.

1/10.

Motility normal.

Agglut. + l race of

direct lysis.

Growth. Agglut.

+ Growth. Agglut.

+ +

mmobilised Nearly

all dead. Lysed.

All dead.

...


-t

Growth. Agglut.

Growth. A gglut.

+ +

+


' * tr."

Growth. Agglut.

Growth. Agglut.

+ +

+

Motility normal.

Not agglut .

Not affected.

...

Motility normal.

Not agglut.

...

...

Not affected.

Not affected.

Not affected.

Not affected.

Not affected.

Not affected.

I to 10

20.

After 24.

Motility normal.

Trace of direct lysis.

Growth. Agglut. ' ' tr. "

Growth. Agglut.

igglut. +

+ + ~~

All dead. Agglut. '' tr."

Lysed.

...

...

...

...

...

%xpt. 106. Jormal rabbit serum 4 hour: o l d , w i t h natural complement.

Motility normal.

'' tr."

Not affected.

Not affected.

Agglut.

Motility normal.

'' tr.

Not affected.

Not affected.

Agg'$.

Motility normal.

" tr.

Not affected.

...


Growth.

...

... I 20.

... I Afte 24.

__ 1 to1

20.

Aft( 24.

~

1 to :

20

Aft, 24

-

Motility normal. Agglut. +i

Growth. Aggl2t. '' tr.

Growth.

Not affected.

Not affected.

...

Motility normal. Agglut. + + + + Growth. Agglut. + + Growth.

Motility normal.

Growth. Agglut. + + Growth.

Sxpt. 56. Ieated giiinea. pig serum 24 hours old.

Zxpt. 127. ?resh guinea pig serum 2! hours old.

Motility slightly lowered.

Growth. Agglut. + + + Growth. Agglut. + +

Agglut. -

Motility. normal.

Growth. Agglut.

Growth.

+


Growth.

...

IMMUNE BODIES TO BOD0 383

natural complement, but that in the higher dilutions the weak immune body and the very low salt content gave only the trace of stickiness noted. It is very important t o distinguish between the thigmotactic balls, which are particularly well developed in these richly fed cultures in the very high concentrations of normal serum, and true agglutination. The test of drawing the fluid in and out with a pasteur pipette will always make the difference clear.

Fresh guinea-pig serum was used after 24 hours in the ice chest in order that the results might be comparable with those of the serum as used for complement. I n these tests in distilled water with fresh guinea-pig serum there was in some samples though not in all, death with lysis of all the flagellates in a concentration of 115.. This was never found in 1/10, where survival was the invariable rule, and in general throughout the series the visible reaction apart from the death of a fow individual bodos consisted in a variable amount of agglutination.

It should be recalled that Sachs and Teruuchi (1907) and Sachs and Altmann (1916-17) showed that complement was inactive in hzmolysis in very low salt concentrations and the results just recorded in bod0 in normal fresh guinea-pig sera with distilled water afford another example. In tests with Ringer solution lysis was complete in 1/20 and marked in 1/40 though the culture recovered, while in distilled water it appeared only in 115 and was not always effective a t that concentration.

A few tests were carried out with immune sera with the addition of fresh guinea-pig serum, the whole system being in distilled water. The results as might be expected from the data just considered gave no increase in the high degree of lysis of the bodos which occurred without complement.

DISCUSSION. The results detailed above have shown that in heated immune sera

made with living or formolised bodos an immune body is produced which, acting in Ringer solution, gives an agglutination of the bodies of the flagellates with a greater or lesser degree of paralysis of their movement. The agglutinin has the power t o bring about the death of the protozoon although the action is not very rapid. The conception of the action of an agglutinin as primarily a coating of the surface or part of the surface of the cell by the homologous element in the serum, put forward by Bordet (1899) and Arkwright (1931) and restated in the light of more modern work by Shibley (1926)) is generally accepted.

I n the present case this coating of the surface which precedes aggllltination under the influence of the satt, appears when sufficiently complete to have a definitely toxic effect ending in death but the cell is not broken up or burst. The addition of complement in the presence of salt brings about a lysis of the cell preceded in some cases by agglutination, but the lytic action may be so abrupt that death and

384 M. ROBERTSON

bursting of the cell have occurred before agglutination could take place. This is also in general agreement with the accepted views upon cytoly sis.

Lysis in high concentrations of quite fresh normal sera is an example of the same action, normal immune body and normal complement supplying the two elements. I t was here assumed that there was only one normal sensitising immune body (Gordon and Wormall 1928), namely that which was demonstrated as the agglutinin in the heated (55" C.) samples. Some authors however seem to describe the nornial sensitising amboceptor which acts in conjunctionljwith complement as coexisting with a normal agglutinin (Mackie and Finkelstein 1931).

In turning now to the lysis of the bodos in distilled water and in low concentrations of salt in the heated immune sera, this stands for the moment as an observation apparently not exactly paralleled in the literature. The coating of the cell however in the absence of the electrolyte (Bordet 1899) takes place and in the case of bod0 this apparently is capable of bursting the cell if i t is not protected by the presence of salt.

A reaction that bears some analogy to this lysis by immune body in distilled water was described in detail by Topley (1914-15) and has been repeatedly observed (Sachs and AItmann 1916-17, Georgi 1920). Topley showed that if red cells were exposed to guinea-pig serum (complement) in a system where the salt is replaced by sugar the cells are lysed by the guinea-pig serum without the presence of antibody. The case of bod0 is the converse, in that a reaction (lysis) which in the presence of sufficient salt requires complement as well as specific immune body to bring it about, can take place without the complement if the salt concentration is sufficiently reduced.

The state of the surface of bod0 in its relation to the fluids surrounding i t is no doubt different in the distilled water cultures from what it is when grown in Ringer solution. The possibility of the sensitisation (i.e. the coating of the cells) in the absence of salts is not disputed; the nature of the cell in this case appears to supply some condition which makes it unable to remain intact when so coated in the absence of salt. That electrolyte affects the reaction of cells in the production of lysis is shown again by the fact that while sensitisation of the red cells can occur under very various conditions (Cloulter 1920-21 and 1921-22 and Brown and Broom 1929) actual lysis by the addition of complement can be inhibited either by too low or by too high a concentration of salts (Wright and MacCallum 1922).

The question as to whether the immune body is the same substance in the agglutination reaction with salt and in lysis in the distilled water series cannot be settled by these expepiments, but its identity appears to be highly probable. The experiment in which the immune body was absorbed in distilled water and the absorbed serum then adjusted to 0.85 per cent. saline and tested with living bodos would

IMMUflE BODZES TO BOD0 386

along the lines of Bordet’s conception be an indication that the great reduction in agglutination and in lethal effect was due to a true absorption of agglutinin. The agglutination in the control serum which was not absorbed showed that the sojourn in distilled water and subsequent addition of hypertonic salt had not destroyed the immune body, but i t might be argued that in the absorbed serum the lysed bodo substance was interfering in some way other than by a true absorption with the development of the reaction.

The least clear element in the action of the serum in the work described is the interference with motility. The picture given by Arkwright (1927) of the microscopic appearances in agglutinated bacteria is only reproduced here in so far as agglutination of the body (i.e. ‘‘ somatic ” agglutination) can be accompanied by continued activity of the flagella. The possibility of a particular flagellar antigen is suggested, but its demonstration is not clear and from the analogy with bacteria, notably ?? choler@!, it seems probable that owing to the great quantitative difference between body and flagella, its action might be masked (Balteanu 1926). Reduction in motility in this type of organism could moreover be just as well due to a general action on the body as to a particular one on the flagellum.

As already stated the qualitative analysis into heat-labile (flagellar) and heat-stable (somatic) antigen was not investigated. Boiling the antigen for one hour produced a great diminution in killing titre and a reduction in immobilisation, but some interference with the movement of the flagellate undoubtedly occurred.

A comparison of the results obtained with bod0 with those shown in serological work with other protozoa as recorded in the literature reveals many points in common. Agglutination of trypanosomes and Leishmania has been carried out with sera from infected or specially immunised animals by various workers. Mattes (1912) used formolised suspensions of trypanosomes as his test antigen and in spite of difficulties in getting good controls he was able to investigate a number of different species. He used sera from heavily infected animals, mostly rabbits, and obtained agglutination titres up to 1/12,000. The formolised trypanosomes became tightly agglutinated and even fused together, the flagellum resisting the disintegrating action of the serum longer than the protoplasm. The titre was unaffected by inactivation of the sera. Normal sera had an agglutination titre of 1/100.

Noguchi (1926) and Kligler (1926-26) produced immune sera in rabbits by the injection intravenously of living Leishmania from cultures. Kligler describes immobilisation followed in two or three hours by agglutination in 115 with degeneration of the flagellum and finally of the nucleus. Noguchi gives as the direct effect of adding 0.05 C.C. of immune serum to 1 C.C. of a saline suspension of living culture (i.e. a 1/20 dilution) an agglutination which he considers differed in

JOURN. OF PATH.-VOL. XXXVIII. 2 E 2

386 M. ROBERTSON

appearance from the rosettes so general in cultures of Leishmania. His description is as follows: “The organisms become sluggish or motionless, the bodies swollen and uneven in contour, the flagella twisted irregularly and adherent to whatever comes in contact with them and finally the bodies are broken up.”

Eoth these authors therefore find immobilisation, agglutination, death and breaking up of the bodies (lysis) in inimune sera without added complement. I t is not clear in either account if the sera used in agglutination were inactivated by heating or not. The reactions were strictly specific and could be used to distinguish the various species of Leishmania. Both Noguchi and Kligler finally used methods of culture in immune sera as a better means of distinguishing the different species, and Kligler states that growth can be prevented completely in tubes having 20 per cent. of 115 immune serum while growth with agglutination occurs in higher dilutions. Adler and Theodor (1926) were also able to distinguish between the different species of Leishmania by agglutination tests.

Sonie quite recent work by Reiner and Chao (1933) gives results which are in very close accord with those obtained with bodo. The authors immunised rats by injecting trypanosomes (2”. eqzc+erdum) killed with para-benzoquinone. In order to avoid the very serious difficulties in getting test suspensions that could be relied upon as controls, they used fresh but inactivated normal rat serum in which i t was found that the trypanosomes lived well for 24 hours. They considered that this procedure probably lowered the agglutination titre of the immune sera. l n concentrations up to 1/16 OY 1/32 they found rapid agglutination and described the adherence of the bodies into clumps while the flagella were still capable of movement. Some degree of agglutination was registered up to a dilution of 1/480. When complenient was added they obtained agglutination of the trypanosomes ; the masses were then inimobilised and finally completely lysed. The authors found that agglutination was more rapid than lysis, but they suggest that the sensitising and agglutinating immune bodies are the same, as they found a parallelism between the two actions. If no complement was added and the serum was inactivated agglutination only occurred and no lysis. This is in exact agreement with the results recorded for bodo in the tests with full salt concentration.

Taliaferro (1932) who has made a very complete study in wivo of the immune bodies in the blood of rats against T. lewisi, described the well-known sudden disappearance of the trypanosonies from the blood and brought evidence that this is due to the action of a specific trypanocidal body. There was great difficulty in denionstrating the Iytic action in vitro, partly because the controls could not be induced t o live for any length of time. He did however demonstrate that in a proportion of cases the trypanosomes were killed and lysed in tubes

IMMUNE BODIES TO noDo 387

containing inactivated immune serum with complement while they remained alive in the serum alone and in the complement controls.

Nothing resembling ablastin, the reproduction inhibiting antibody, was found in the work with bod0 and the conditions were not suitable for its investigation.

The adhesion phenomena in trypanosomes present a particularly interesting set of immune body reactions. Their interest lies partly in the circumstance that they emphasise the fact that the larger size and the nature of the body surface in protozoa give a wider range of reactions than appears among the bacteria. The adhesion phenomena are considered by Regendanz and Jurukoff (1930) to be due to the action of agglutinins and in their view it depends upon the condition and number of trypanosomes whether agglutination or adhesion results, also upon the strength of the agglutinin. These authors also describe the immobilisation of trypanosomes i n vitro under the influence of immune sera. They consider it t o be due to the action of an immune body and in their adhesion tests they adjust the dilution so as to avoid its manifestation. They do not however further elucidate its nature.

Leupold (1928-29) found that if she used the relatively powerful immune serum, obtained by the curative action of neosalvarsan i n heavily infected mice or other animals, for the blood platelet adhesion test, agglutination occurred instead of adhesion. She therefore diluted the immune body content of tho serum by the ingenious method of injecting it into other mice who were then the passive carriers of a suitable amount of immune body for the adhesion reaction.

It seems now to be clear that complement is necessary for the adhesion reaction to take place (Brussin and Kalajev 1931), certainly for the red cell adhesion (Wallace and Wormall 1931). While these studies reveal the presence of an agglutinin and of some agent which immobilises the trypanosomes, i t is not clear what relation the group of antibodies known as “adhesins” bears t o the agglutinins and lysins.

Schindera (1922) found immobilisation and agglutination of Trypano- plasma helicis in concentrations up to 1/20 of the normal sera of various animals, but he considered these normal agglutinins to be non-specific.

Menendez (1932) working with Entamaba histobytica described the lytic properties of the sera from immunised rabbits and his results, as he points out, are in general agreement with those of Sellards (1911) on ameba and Rijssle (1905), who worked with ameba and a ciliate showing that “ the sera of animals immunised with protozoa con- tained immobilising and lytic antibodies which were more or less species-specific.”

Sellards noted the interesting point that he could obtain no reaction with ammbic cysts. Serum made by injecting cysts intravenously into

388 M . ROBERTSON

rabbits failed to produce any effect upon cysts, and further, a serum made with the ameboid phase as antigen was also quite ,inactive with the cysts.

Heathman (1932) described the toxic effect of an homologous immune rabbit serum against the ameba Chaos difluens. The salt concentration used was very low, 0.085 per cent. She noted death in a l / l O O O dilution in 8 hours and found that heating the serum to 56°C. did not alter the effect, which was independent of the action of Complement, a result which is in general agreement with that obtained with bod0 in very much reduced salt. The toxic effect, which Heathman does not describe in further detail, is specific.

SUMMARY. (1) The preparation of immune sera in rabbits against a flagellate

protozoon Bodo caudatus is described and an account is given of the method of culture of the organism and the particular technique used for the serum tests.

(2) The reactions of immune heated sera made with living or formolised bodos in tests in mammalian Ringer solution with living bodos were found to comprise ( a ) some degree of inimobilisation often considerable, ( b ) agglutination, and (c) the gradual death without lysis of all the bodos. The killing titre of the sera ranged from l/SOO to l/lSOO or 113200 and reasons are given why this very constant effect was used as a measure of the immune body content rather than agglutination, which showed very considerable variation.

(3) The extent of the action of heated and fresh normal rabbit and guinea-pig serum is described. A normal agglutinin wa8 found and in sera with the natural complement intact lysis was observed up to a dilution of 1/20.

(4) The addition of fresh guinea-pig complement to heated immune sera brought about the lysis of the bodos, preceded in some cases by agglutination, but more often the lysis was almost complete before agglutination had occurred. The killing titre of the sera was not, in the type of dilution used, raised by the addition of complement, but the time required to cause the death of all the protozoa was reduced.

(5) Experiments using distilled water instead of salt solution were made and it was found that in heated immune sera agglutination, a8 was t o be expected, did not take place. The immune body caused, however, (a ) a diminution or abolition of motility, and ( b ) death by lysis of the bodos, the killing titre being the same as in the circumstances detailed above. This effect is considered in relation to the work of various authors on the action of sera in reduced salts.

(6) The implications of the results are discussed in the light of existing knowledge in this field.

IMMUNE BODIES TO %OD0 389

REFERENCES. ADLER, s., AND THEODOR, 0. . 1926. ARKWRIGHT, J. A. . . . . . 1921.

. . . . . 1927. . . . . . 1931.

BACHMAN, G. W. . . . . . 1930. BALTEANU, I.. . . . . . . 1926. BORDET, J. . . . . . . . 1899.

,, ,,

Bos,A. . . . . . . . . 1933-34. BROWN, H. c., AND BROOM, 1929.

BRUSSIN, A. M., AND KALAJEV, 1931. J. C.

A. W. COULTER, C. B. . . . . . 1920-21.

. . . . . . 1921-22. CRAIG, c. F., AND KAGY, E. . 1933. DUKE, H. L., AND WALLACE, 1930.

9 ,

J. M. FINKELSTEIN, M. H. . . . . 1933.

GEORGI, w. . . . . . . . 1920. GORDON, J. . . . . . . . 1933. GORDON, J., AND CARTER, H. 1932.

GORDON, J., AND WORMALL, A. 1928. GRASSI~, P. P. . . . . . . 1926. HEATHMAN, L. . . . . . . 1932. KLIGLER, I. J. . . . . . 1925-26.

LEUPOLD, F. . . . . . . 1928-29. MACKIE, T. J., AND FINKEL- 1931.

MACKIE, T. J., AND FINKEL- 1932.

FOX, M. H. . . . . . . 1920-21.

S.

STEIN, M. H.

STEIN, hil. H. M A T T E S , ~ . . . . . . . . 1912. MENENDEZ, P. E. . . . . . 1932. NOGUCHI, H. . . . . . . . 1926. PUTTER,A. . . . . . . . 1900.

REGENDANZ, P., AND JURUKOFF, 1930.

REINER, L., AND CHAO, 8. 8. . 1933. B.

RIECKENBERG, H. . . . . . 1917. ROSSLE,R.. . . . . . . . 1905.

SCHINDERA, M. . . . . . . 1922. SELLARDS, A. W. . . . . . 1911.

SHIBLEY, a. S. . . . . . . 1926. TALIAFERRO, W. H. . . . . 1932. TOPLEY, w. w. C. . . . .

SACHS, H., AND ALTMANN, K. 1916-17. SACHS, H., AND TERWCHI, Y. . 1907.

SHERWOOD, N. P., AND HEATE- 1932. MAN, L.

1914-15.

Ann. Trop. Med. and Parasitol., xx. 355. this Journal, xxiv. 36. Ibid., xxx. 566. A system of bacteriology, Med. Res.

Amer. J. Hyg., xii. 624. this Journal, xxix. 251. Ann. Inst. Past., xiii. 225. 261. Bakt., Abt. I., Orig., cxxx. 220. Brit. J. Exper. Path., x. 387.

Z. Immunitatsf., Ixx. 497.

J. Gem. Ph?ysiol., iii. 513. Ibid., iv. 403. Amer. J. Hyg., xviii. 202. Parasitology, xxii. 414.

this Journal, xxxvii. 359. J . Gen. Physiol., iii. 483. Z. Imrnunitat'tsf., 1. Orig., xxix. 92. this Journal, xxxvii. 367. Ibid., xxxv. 549.

Ibid., xxxi. 753. Arch. de 2001. Exp. Gdn., Ixv. 345. Amer. J. Hyg., xvi. 97. Trans. Roy. Soc. Trop. iMed. Hyg., xix.

2. Hyg. Infektionkr., cix. 144. J. Hyg., xxxi. 35.

Ibid., xxxii. 1.

Zbl. BLtkt., Abt. I., Orig., Ixv. 538. Arner. J. Hyg., XV. 785. J, Exper. Med., xliv. 327. Arch. Anat. Physiol., Physiol. Abt.,

2. hiamunitatsf., Ixvi. 32.

Council, vi. 381, London.

330.

Suppl., p. 243.

Amer. J. Trop. Med., xiii. 525. 2. ImmunitBtsf., xxvi. 53. Arch. Hyg., liv. 1. Biochem. Z., lxxviii. 46. Bed. klin. Woch., xliv. 467, 520, 602. Arch. f. Protistk., xlv. 200. Philipp. J. Sci., B. Xed. Sci., vi. 281. Amer. J. Hyg., xvi. 124.

J. Exper. Med., xliv. 667. Arner. J. Hyg., xvi. 32. Proc. Roy, SOC. Ser. B., Ixxxviii. 396.

390 M . ROBERTSON

WALLACE, J. &AND WOXMBLL, 1931. PCM"asitOlOgv, XXi i i . 346. A.

WEIL, E., AND FELIX, A. . . 1920. 2. Inzmunitatsf., xxix. 24. WITTE, J. . . . . . . . . 1933. Zbl. Bakt., Abt. I., Orig., cxxviii. 188. WRIGHT, H. D., AND MAC- 1922. this Jourml, xxv. 316.

CALLUM, P. Appendix.

Peters' salt solution. Sodium chloride . . 0 6 grm. Potassium chloride . 0'01 ,, Calcium chloride . 1 0'02 ', Magnesium sulphate . . 001 ,,

Glass-distilled water to 1 litre.

Ringer solution used. Sodium chloride . . 9'0 grms. Potassium chloride . . 0.075 grin. Calcium chloride . , 0'1 grm.

Glass-distilled water to I litre.

An in vitro study of the action of immune bodies called forth in the blood of rabbits by the...

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