IN INTESTINES IN OASES OF GOITRE fN ailiG IT. 723

Observations on the Amoebae in the Intestines ofPersons Suffering from Goitre in Gilgit.

ByRobert mtcCarrison, M.D., Itt.tt.C.P.L,oinl.,

Captain Indian Medical Service.

With 24 Text-figures.

MY researches on the astiology of endemic goitre have ledme to the conclusion that in all probability the organismwhich is responsible for the production of this disease isto be found in the intestinal tract. In is necessary, therefore,to give some account of the Protozoa which are so frequentlypresent in this situation. This paper deals only with theorganisms of the Amoeba group, and simply gives a briefdescription and figures of the amoebae found. It does notclaim to be a complete account of their life-histories. Whileno definite statement can be made as to the pathogen icity ofthese amoeba?, their possible importance is obvious when itis remembered that goitre is due to an oi'ganism cai'ried bywater.

The examination of the fresh fteces of goitrous individualswas undertaken solely for the purpose of determining thepresence or absence of Protozoa. This object was found tobe greatly facilitated by the addition of a small quantity ofiodine-water to the specimen, which caused the amoeba) tostand out clearly from surrounding objects. One hundredand three cases were examined in this way; amoeba) Averepresent in eighty-seveu. In forty-eight cases they werefound in large numbers, in twenty-seven in moderate numbers,and in twelve only after considerable time had been spent in

724 ROBERT MCCARRISOK".

searching for them. The fasces of 101 non-goitrous indi-viduals, living in the same locality, have also been examined.Amooba3 were present in twenty-nine of these; the infectionwas plentiful in eight, moderate in nine, and scanty intwelve. The typical cysts hereafter described were alsofound in the only case of goitre from another locality whichI have examined.

Two distinct amoebae are found in the fasces; these havenot been distinguished in the live state :

(1) A free amoeba which proceeds to encyst and developinto a typical 8-nucleated cyst.

TEXT-FIG. 2.TEXT-PIG. I.1

1.Text-fig. 1.—'Amoeba. I. Encysted amoeba, showing single

nucleus and port-wine staining area.Text-fig. 2.—Amoeba I. Encysted amoeba. Shows the kidney

shape of the povt-wine staining- area frequently observed.

(2) A free amoeba which does not form obvious cysts, butmultiplies by division and budding.

In addition, a third amoeboid body, enclosed in a charac-teristic capsule, is also present. Its affinities are not clear,and I can only note its occurrence.

1 [Figs. 1-7 and 23 are drawn from freehand sketches of the livinganimals. The preparations were treated with iodine water. Drawingsmade under Leitz -^ in. oil-immersion, ocular No. 4.

The remaining figures were drawn from fixed and stained preparations,under Zeiss 3 mm. apochromatic homog. oil-immersion, comp. oc. 12(X 2000). The drawings were made by Miss Rhodes, to whose skill inso accurately depicting the appearances observed I must pay a tribute.The preparations were stained with Haidenhain's hrematoxylin andDelafield's hoomatoxyliii.]

AMO?B/"E IN INTESTINES IN CASES OF GOlTKE IN GILGJ.T. 725

THE AuffiBiE IN THE FBESH STATE.

(a) The free amcebas.—I c:m ouly give a general accountof the free amoobas in the live state, an account which coversboth species. lu specimens treated with iodine solution theorganisms are more or less spherical. Their size variesbetween 12 /i and 20 /.i. Larger forms are occasionally seen.The protoplasm is granular, stains yellow with iodine, and isvery rarely vacuolated. A differentiation of the protoplasminto ectoplasm and endoplasm can only be made out in thoseanimals which show pseudopodin, and these are few. The

TEXT-FIG. 3.

TEXT-PIG. 4.

3 . 4 .Text-fig. 3.—Amoeba I. Encysted amoeba. Bi-nucleatod stage

with large port-wine staining area.Text-fig. 4.—Amoeba I. Encysted amoeba. A stage not

commonly met with, showing four nuclei and centrally placedport-wine staining area.

protoplasm contains food-vacuoles and other inclusions. Ihave never seen any evidence of the ingestion of blood-corpuscles or of epithelium. The nucleus., where observed,is spherical or oval, and is sometimes surrounded by a narrowhalo. In the larger organisms it measured 5 /i-8 fx. Thecharacters of the nucleus are very distinct in the two species;they have been studied only in stained specimens.

(b) The encys ted forms.—Live cysts of definite contourare very commonly seen; they represent stages in the life-history of Amoeba I, and show different appearances dependenton the length of time the animal has been encysted. Thesecysts are, as a rule, perfectly spherical, but they may be oval.

726 HOBERT MCCABRISOK.

Their size varies from 14 f.i-20 /.t, the latter beiug the almostconstant diameter of the 8-nucleated forms. The cyst-wallvaries in thickness, and sometimes it is made out with diffi-culty. It encloses a yellow staining granular protoplasm, oneor more nuclei, and a characteristic port-wine staining areawhen treated with iodine (text-figs. 1 to 4). The nucleus isspherical, very clearly defined, and shows retractile granuleson its surface and in its interior. A well-marked karyosomeis usually present. The nucleus varies in size from 2 ft—3 ftin the 8-nucleated cysts to 6/u-S^u in the single-nucleated

TEXT-FIG. 5. TEXT-FIG. 6.

If.•J

5. 6.Text-fig. -r>.—Amoeba I. Encysted amoeba. Tlie nuclear

division lias resulted in the formation of five nuclei; comparetext-fig. 17.

Text-fig. 6.—Aniceba I. Encysted aniceba, 8-nucleated stage,cyst-wall ill-defined; a centrally placed remnant of the port-wine staining area is seen—a very uncommon appearance atthis stage.

form. The nuclei vary from one to eight in number, dependenton the phase of development of the cyst (text-figs. 1 to 7).The commonest phases met with are those where the cystcontains one, two, or eight nuclei. Cysts showing four nucleiare much less commonly found. The port-wine staining areais present in the majority of all cysts containing one to fournuclei (text-figs. 1 to 4). It is not, as a rule, present in the8-nucleated cyst; I have only met with it at this stage ofdevelopment in one instance (text-fig. 6). I t is oval, spherical,or kidney-shaped in form, and occupies about one half of the

AMCK1!* IN INTESTINES IN OASES Of GOITBE IN GILGIT. 727

cyst. It usually lies iu one hemisphere while the protoplasmis situated in the other, but it may be centrally placed. Itappears sometimes to alter its position relative to the nucleiand to the cyst-wall. The port-wine reaction with iodinemarks off the ai*ea from the rest of the protoplasm in a mostdistinctive way, for while the protoplasm is granular andstains yellow this area appears to be structureless.

I have never been able to observe a division of the proto-plasm around the nuclei in the large 8-nucleated cysts.

TEXT-FIG. 8.

TEXT-PIG. 7.

TEXT-FIG. 9.

- 9.o.

Text-fig. 7.—Amoeba I. E n c y s t e d amoeba. The nucleardivision has resulted in the formation of five nuclei; comparetext-fig. 17.

Text-fig. S.—Amoeba I. F r e e amoeba. Sideview of organismshowing finely granular appearance of protoplasm. The nucleusis seen surrounded by a narrow halo, and shows chromatm moreor less evenly distributed with slight massing at four points ofthe periphery. A central karyosome is seen.

Text-fig, y.—Amoeba I. U n e n c y s t e d amoeba. Probably astage in simple fission of free amoeba. Chromatm massed atperiphery of nuclei, which have beaded appearance. Karyo-some is seen ; nature of included bodies not known.

(c) The third organism referred to is less commonly found.The following are its main characteristics iu both fresh andstained specimens. In the living state it is seen as a pear-shaped, oval, or spherical body, having a well-defined clearcapsule. The protoplasm is granular and stains yellow withiodine. It is split up by a median fissure, on either side of

728 ROBERT JfCOAliBISON.

which aud approximately at right angles to it is a shorterfissure, which further divides up the protoplasm (text-fig. 23).Movements of the protoplasm aud alterations in the positionof the nuclei within the capsule are sometimes seen. Thenuclei, four in number, usually lie clumped together or in theposition shown iu the figure (text-fig. 23). They are clear,spherical bodies of uniform size, very sharply defined, andsometimes showing a central dot, features which are wellbrought out by staiuing (text-fig. 21). When the organism is

TEXT-FIG. 10.

TEXT-FIG. 12.TEXT-FIG. 11.

1 2 .Text-fig. 10.—Amoeba I. E n c y s t e d amoeba. Protoplasm filled

with spherical hyaline masses of variable size. Two chrouiatinmasses are seen in the protoplasm. Chroniatin heaped up atopposite poles of nucleus ; karyosome well marked.

Text-fig. 11.—Amoeba I. E n c y s t e d amoeba. Protoplasmshows hyaline masses of larger size. Nucleus has divided intotwo. Retieular structure of nucletis and karyosome seen.

Text-fig. 12.—Amoeba I. E n c y s t e d amoeba. Protoplasmshows single hyaline mass. Nuclei as in Text-fig. 11, but oflarger size.

stained the capsule is uot coloured, and it appears to be openat its broader end. The granular protoplasm sometimesstains so deeply that no structure can be made out. In themore faintly stained animals the protoplasmic fissures referredto can be seen, but not with such distinctness as in the livinganimal (text-fig. 24). Sometimes the protoplasm, with its con-tents, is seen contracted up at one side of the capsule. Thesize of this animal in its longest diameter is fairly constant,and measures in the live state 14^-15//.

AMCEB/E IX INTESTINES IN CASES OF GOITltK IN GILG1T. 729

THK FIXED AND STAINED AHO:BA;.

The material used for this part of the investigation wassent from Gilgit to England in Schaudinn's fixing reagent(saturated watery solution of corrosive sublimate two parts,alcohol one part). The methods of stainiug employed wereDelafield's and Haidenhain's hasmatoxylin. The examinationof: the material was carried out in Professor Minchin'slaboratory at the Lister Institute, to whom I am indebtedfor help and advice.

TEXT-FIG. 13.

TEXT-PIG. 14

Text-fig. 13.—Amoeba I. E n c y s t e d amcubu. Shows well-marked cyst-wall. A large hyaline mass occupying about onehalf of the cyst is seen. Cbromatin musses lie above and belowthis area. Nuclei are seen lying at either side of cyst, eachsurrounded by halo. Note the reticular structure and granularappearance of nuclei. No karyosome is seen in either nucleus.

Text-fig. 14.—Amoeba I. E n c y s t e d amoeba. One nucleus hasproceeded to second division before the other. A commonappearance of the nuclei —wheel-like—at this stage is well seen.Two chromatiii masses in protoplasm.

A study of the stained amoebae shows thai; there are twodistinct species present. In view of the many opinions heldwith regard to intestinal amcebse I hesitate to describe thoseorganisms under specific names. Nevertheless, I am inclinedto think that Amoeba I, which forms the 8-nucleatedcysts, is the Entamoeba coli, Schaudinn, and that AmoebaII corresponds to the Entamoeba h i s t o ly t i c a , Schaudinn.Certain points in which they appear to differ from the two

730 KOBKRT MCCAREISON.

species of Schaudinn will be referred to in the course of mydescription.

Amoeba I.The unencysted amoeba usually appears as a spherical

body of variable size, ranging up to 20 fx in diameter. Theprotoplasm is finely and evenly granular (text-fig. 8). I candetect no differentiation into ectoplasm and endoplasm.

In some the protoplasm contains food-material and variousinclusions, while in others it appears to be free from

TEXT-FIG. 15.TEXT-FIG. 16.

15. 16.Text-fi . 15.— Amoeba I. Encysted amoeba. The nuclear

division has resulted in the formation of five nuclei.Text-fit;. 16.—Amoeba I. Encysted amoeba. Typical8-nucle-

ated cyst, of very common occurrence in fasces. Each nucleusis ring-like with a small karyosome in its interior. A few smallchromatin masses are seen.

extraneous matter. Such inclusions as blood-corpuscles orepithelium have never been met with. The protoplasm rarelyshows a vacuole. The nucleus is very distinct, and is usuallycentrally placed (text-fig. 8). It is commonly surrounded bya narrow but distinct halo. In text-fig. 8 the nucleus appearsto lie in a cavity lined by a membrane. Since it has beenpresei ved in a sublimate mixture the appearance may be dueto shrinkage of the protoplasm. The nucleus stains deeply;it is rich in chromatin, which in the adult unencysted animalis more or less uniformly distributed throughout this structure.There is often a slight tendency for the chromatin to be

AMCEB/E IN INTESTtNKS IN CASKS Oi1 GOITRE IX GILGIT. 731

massed irregularly at the periphery of the nucleus. It isgenerally reticulate in character, and, as a rule, shows adistinct karyosome. I have not been able to satisfy myselfas to whether division of the nucleus takes place in theunencysted organism or not. Test-fig. 9 represents an orga-nism of a type only occasionally met with, in which it wasimpossible to make out the existence of a cyst-wall. This isprobably a st;ige in simple fission of the free amoeba. Never-theless I hesitate to offer au opinion on the point, and simplydraw attention to the figure. One point is certain, that in all

TEXT-FIG. 17.

TEXT-PIG. 18.

18Text-fig. 17.—Amoeba I. Encysted amoeba ; Smicleated cyst.

The cyst-wall is thicker than in text-fig. 16. The nuclei arevery clearly defined and each shows a karyosome.

Text-fig. 18.—Amoeba I. Encysted amoeba. Abnormal formshowing twelve nuclei.

organisms in which there were more than two nuclei a cyst-wall was always obvious. Unfortunately I have not noted asimilar appearance in the living state, but, as I have said, myobservations were then only diagnostic.

The encys t ed amoeba.—The earliest stages of enc}rst-ment which I have observed are shown in text-fig. 10. Thechromatin is heaped up at the periphery of the nucleus, andhere and there in the protoplasm dark masses presentingstaining reactions similar to those of chromatin are occa-sionally found. The appearance of the protoplasm is dis-

732 ROBERT MCCARRISON.

tinctive; it seems to be filled with spherical hyaline massesof variable size. It is difficult to offer an opinion as to thenature of these spherical masses. Such text-figures as 11 and12 suggest tliat they gradually fuse, with the ultimate forma-tion of a large, clear hyaline body, as seen in text-fig. 13. Thisfusion appears to take place in those stages which representthe division of the nucleus after eacystation. I am convincedthat the clear area (text-fig. 13) is that which gives rise tothe characteristic port-wiue reaction with iodine water in theliving animal. The hyaline body becomes less marked as

TEXT-FIG. 19.

\ TEXT-FIG. 20. TEXT-FIG. 21.

19. 2 0 2 1

Text-fig. IS).—Amoeba II. Two typical organisms. Shows palestaining nuclei surrounded by narrow halo.

Text-fig. 20.—Aniceba II. Organism showing three nuclei andtwo darkly staining bodies. The precise nature of the latterobjects is unknown ; probably food-material.

Text-fig. 21.—Amceba II. A multi-nucleated organism. Nucleiof varying sizes. A stage of multiple division.

the further development of the organism proceeds to thetypical 8-imcleated form; ultimately it completely disappears.This hyaline body is at its highest development in the latebi-nucleated cyst. I regard this spherical mass as being ofthe nature of food material, a view which is upheld byJurgens (2). A similar appearance has been described byWenyon (3) in Entamcoba muris , and he has consideredit to be "oE the nature of food products which have not beenthrown out of the animal." It is questionable, however,whether the body seen by Wenyon is identical with that

AMCBB2E IN INTESTINES IN GASES OF GOITKE IX GILGIT. 733

which I am describing. Wenyon has found that the refractilebody of B. muris "stains feebly, and shows a coarse reti-cular structure/' and that on breaking up "the separate partsshrink to form masses which stain deeply with hasinatoxylin."The hyaline body I have described has not these characters.Schaudinn (4) has described the protoplasm of the encystedE. coli as "divisible into an outer, and denser layer con-taining the nucleus and an inner and more liquid portion/'

TEXT-HG. 22.

TEXT-FIG. 23. TEXT-FIG. 24

23. : 24.Text-fig. 22.—Amoeba II. Group of amoeba), the result of

multiple division; a common appearance.Text-fig. 23.—The third amoeboid body. Shows the characteristic

capsule and fissured protoplasm as observed in the fresh state.The drawing also shows a common position cf the fourspherical nuclei.

Text-fig. 24.—The third amoeboid body. Shows the typicalappearance of this organism when stained. Note the slightlybeaded appearance of the nuclei, the presence of karyosomes,the position of the nuclei, and the manner in which they lieclumped together. The unstained capsule and the fissuredprotoplasm is well seen.

and Wenyon considers that the more liquid portion probablycorresponds to the refractile body of E. muris. It does not,however, correspond to the hyaline body of the amoeba underconsideration. The characteristic port-wine reaction differen-tiates it from the yellow-staining granular protoplasm. Thishyaline body is a conspicuous feature of the great majority

734 BOBERT MCCABRISON.

of encysted amoebae during the earlier phases of theirdevelopment.

At the time of encystment there is oue nucleus present (text-fig. 10). This nucleus divides into two daughter-nuclei (text-figs. 11, 12). I have not been able to trace the complicatedseries of nuclear changes described by Schaudinn in B . col iand by Wenyon in E . m u r i s as occurring at this stage,though I have seen a large number of amoeba). The twodaughter-nuclei lie most commonly side by side (text-figs. 11,12). At this stage they vary greatly in size. I have seen themso large their diameter almost equalled halE that of the cystcontaining them. I have observed in one case a division-figure corresponding closely to fig. 73 of DobelPs paper (5) ;unfortunately the specimen could not be drawn. In text-fig.

13 the two nuclei have separated to either side of the cyst.Division of the two daughter-nuclei takes place, and text-fig.14 shows that one nucleus has proceeded to the second divisionbefore the other. I t has been difficult to find examples show-ing four nuclei j text-fig. 15 shows a further division of thenuclei into five. The ultimate division into eight is shown intext-fig. 16. The cyst-wall eventually becomes thickened as intext-fig. 17. Division of the protoplasm around the nuclei hasnever been seen. Very rarely a form showing more thaneight nuclei is met with (text-fig. 18), but it is so rare thatwe must regard it as abnoruaal.

I t is evident that the animal here described correspondsvery closely to the En t amoeba col i of Scliaudinnand to theE n t a m c e b a m u r i s of Wenyon. The descriptions, however,do not correspond as regards the hyaline body, which is socharacteristic of this organism; nor have I been able to tracein it the nuclear changes described by these observers.

Amoeba II .

Amoebae of this species are exceedingly plentiful in somecases; as many as three or four are often found in one fieldof the microscope. They may occur alone or in association

AMCEB2E IN INTESTINES IN CASES OF CiOITKE IN GILC1T. 735

with one or other of those described in the precedingsections.

The animal is usually spherical and of variable size, theforms most commonly met with averaging 15 yu—20/.i in dia-meter. The protoplasm is sometimes divisible into a granularendoplasrn with a thin layer of ectoplasm sm-rounding it.The ectoplasm is most obvious in these rare cases in whichprotrusions in the form of pseudopodia are seen. Often it isnot very clearly marked off. The endoplasm is finely granularand contains extraneous matter; such inclusions as blood-corpuscles or epithelium have never been seen. I have notobserved vncnoles. The nucleus in some cases is very difficultto see, presenting as it does staining reactions differing onlya little from those of the protoplasm. It is frequently sur-rounded by a clear halo (text-fig. 19). It is very poor in chro-matin and does not appear to possess a karyosome. There isnothing distinctive in the position of the nucleus; it may becentral, but is more frequently excentric. It varies very con-siderably in size. Forms are commonly seen containing twoor more nuclei (text-figs. 20, 21). Multiplication is apparentlyvery rapid, and takes place either by a process of simpledivision or by multiple division of the nuclei with budding ofthe protoplasm, examples of which processes are shown intext-figs. 19 and 22). This organism does not encyst as doesAmoeba I. I have been unable to find that it developscysts such as Schaudinn has described in E. histolytica.It is true that I have frequently found small, dark brown,spherical bodies in preparations where this was the onlyamoeba present; their structure could not be made out, norcould it be shown that they had any connection with thisamoeba.

This organism resembles closely the E. his tolyt ica ofSchaudinn. There was, however, no evidence that patientswhose faeces swarmed with this Amoeba were suffering fromdysentery.

I would also like to point out that the amoeba here des-

736 BOBERT MCCARKISON. *

cribed is much smaller in size than the Bntamceba histo-lytica of Schaudinn.

LISTER INSTITUTE,March f)th, 1909.

REFERENCES.

1. McCarrison.—"A Summary of Further Reseaches on the ^Etiologyof Endemic Goitre," 'Royal Society,' B. vol. lxxxi, 1909.

2. Schaudhm.—'Arbeiten aus dem Kaiserlichen Gesundheitsamte,"Bd. xix, Heft 3,1903.

3. Jurgens.—" Zur Kenntniss der Darni-anioben tind der Amoben-Enteritis " (Veroff, 'Peb. Militiir-Sanitsitswesens,' Heft 20).

4. Wenyon, 0. M.—" Observations on the Protozoa in the Intestinesof Mice," 'Arch. Protistenk.,' suppl. i, 1907.

5. Dobell, C. C.—"Researches on the Intestinal Protozoa of Frogs andToads," ' Quart. Journ. Micr. Sci.,' vol. 53, part 2, January, 1909.