A CONSIDERATION OF THE INFECTIVE GLZANULE IN THE LIFE-HISTORY OF PROTIST ORGAN- ISMS.‘

By HERBERT HENRY, M.D., B.S.(Lond.).

I+om the Ucpartnaent of Pathology, C’.Livcrsity of Shc$i~dol.

“ garurn senfenfiarum, quae uera sif, Beus afiquis uiberif.”

DURING recent years there have been recorded observations relative to a granule-shedding phase in the life-history of certain Protist organisms. With regard t o the significance of such a process there are a t the present time two diametrically opposite opinions.

On the one hand, there are those wlio hold that granule shedcling is a purely degenerative phenonienon ; that, therefore, it is a condition of little consequence and one that can be conveniently iguored. On the other hand, there are those who maintain that granule shedding is a vital phenomenon, that the so-called ‘( infective granule ” is in fact a propagative unit, and, as such, is the starting-point in the life-history of one individual or indeed of a whole series of individuals.

The main argument in support of this second view is furnished by the behaviour of certain blood-inhabiting spiroch&es, and in bringing this forward as evidence of the vita1 nature of granule shedding, oue is aware that one is approaching the subject from its most diificult standpoint, for the spirochztes, more particularly the blood-inhabiting spirochztes, have been assigned no very definite or secure position by the systematist. It is the tendency of all men, inherited perhaps, as Minchin (1 9 1 11’) has suggested, from an original father in Eden, to classify all the living objects met with in daily life into two main types-the aninial type and the vegetable type. Such a classification is obvious when one has to deal with the more highly organised forms of life, and i t is only when one comes to consider the more primitive forms that difficulties in the way of differentiation present themselves. I n the case of no group of organisms have these difficulties been so great as in the case of the blood-inhabiting spirochztes. At first these organisms were looked upon as bacteria of the genus spirillum. Then caiiie the

[Received August 1, 1913.1 Britain and Ireland, June 21-28, 1913.

Communicated t o the Pathological Society of Great

INFECTIVE GRANULE AND PROTIST ORGANISNS. 251

classical work of Schaudinn (1 9 04 23), in which spirochEtes were described as being possessed of kinetonuclear and trophonuclear apparatus together with flagellum and undulating membrane, a finding which naturally resulted in the transference of the blood-inhabiting spirochztes from the Bacteria to the Protozoa. However, with further investigations and the refutation of certain of Schaudinn’s findings, there has developed a tendency to expel the spirochztes from the sheep-fold of the Protozoa. Such an attitude springs, obviously, from the fact that the microbiologist has fallen into the primitive principle of classification above mentioned, it being assumed that if a spirochzte is not a bacterium it must be a protozoon, and vice versa. To obviate the difficulty in this case, certain investigators have placed these hapless organisms in an intermediate position, and in thus seeking to establish a compromise have very rightly refused to define a hard-and-fast boundary line between the Bacteria and Protozoa.

It is for this very reason that one has made use of the term I’rotista, for the term is one which would include all the Bacteria and the Protophyta on the one hand, all the Protozoa on the other hand, and also all intermediate organisms, such as the blood-inhabiting spirochztes and the Chlamydozoa.

The breaking up of the axial chromatic core of a spirochzte into granules was probably first described by Dntton and Todd (1907 ?) in the case of Spirochmta duttoni. ‘ I Sometimes, especially in preparations taken just as the parasites are about to disappear from the blood, this fragmentation proceeds until the whole chromatic core is broken up into granules.” Again, in regard to the phases of the organism as they occur in the tick, Ornithodorus nzoubata, they say : “ The most marked feature of the spirochcetz in the tick is, however, the extraordinary number of ways in which their chromatin may be fragmented.” ‘I The granules may be either coarse or fine. I n one or two instances the granules of a finely fragmented extracellular parasite are divided transversely so that its chromatin is represented by a row of paired granules. The granules of the encysted parasites are usually single. I n others, perhaps a t a more advanced stage, they are double, and the parasite then consists of a finely blue or reddish spherical matrix, about 5 to 7 p in diameter, in which are embedded perhaps a score of pairs of deep red granules. Many such bodies have been seen in preparations from the Malpighian tubules of an infected tick. The further develop- ment of these bodies is problematical; it is certain that some of them burst, and that their granules are thus freed. I n stained specimens it is apparently possible to trace the development of each single granule to a comma-shaped mass of chromatin, and these comma forms to small spirochztz.”

The statements of Leishman (1 9 10 16) are practically identical with those of Dutton and Todd, the spiroch~tes breaking up into ‘‘ eoecoid granules )) in the ova and tissue cells of the tick.

Again, while investigating the spirochEtosis of Sudanese fowls, Balfour of Khartoum (1 90 8 z, discovered that theie occuired, from time to time, during the disease, a period which he has ciillecl the “ after-phase,” when the causal organisin as such disappeared from the peripheral circulation, and when, coincident with this disappearance, the erythroblasts were found to contain curious intracorpuscular bodies which he maintained were a stage in the life-history of the spirochzte in question. Such an interpretation was much disputed at the time, and still iiieets with opposition, but Balfour himself has since his original comniunications done much to throw light on the exact signi- ficance of these bodies (1 9 11 ”. Working with infected chickens, in which a natural crisis has occurred or in which an artificial crisis has been induced by salvarsan, he found by using dark ground illumination that the spirochztes break up into granules in the iiiteriial organs. These granules, to which has been given the iiiirne “ iufective gianules,” “ are discharged by the still living spirochcete from one or other end of its periplastic sheath or cell membrane, and would appear to con- stitute a stage in the non-sexual life-cycle of the parasite, for t1ic.y seemingly enter the red cells, develop in them, undergo a process akin to schizogony, and regain the liquor sanguinis as tiny riieroAoites, which vary in number and slightly also in size.”

The present position, then, with regard to granule shedding in the case of spiiochxrtes, is this. Certaiu spirochztes do undergo a process of fragmentation into granules, but that these granules are capable of initiating a further life-cycle is highly disputed. In discussing this question Miiicliin (1 9 1 2 18) remarks : “ A developriient of this type suggests, very strongly, affinities with bacteria, but none whatever with ProtoAoa of any class. The coccoid grains may be compared with the spore formation in bacteria, and with that described by Gross in Xn1mspil.a yra7Ldis. I n all cases throughout the series of living beings, whenever an organism presents, in its fully developed adult stage, peculiarities of a special kind, i t is above all t o bhe early develop- mental fornis that the natnrdist turns for indication of the true affinities of the organisin in question.”

When one conies to consider the question of granule shedding in the case of trypanosomes, one finds that the published observations are much scantier. Fry (1 9 11 lo) hm stated that 2‘rypnnosoma brzicei may throw off granules, and asserls that the liberated granules are distinctly motile. He niaintains, moreover, that the process is of a vital and not of a degenerative character. The same is true too, according to Ranken (1 9 1 2 2‘) , of Tyypnnosoma gawbiense. But, as far as one is aware, no further development of the shed granules has been described in either instance. Here again Minchin (1912 lo) refuses to recognise that these granules are other than products of degeneration, for he says : “ That a trypanosome or any other living cell might excrete grains which when set free could exhibit movenients

INFECTIVE GRANULE ALVD PROTlST ORGANISllrlS. 253

due to iiiolecular or other causes is highly probable ; but, that such grains represent a stage in the life-history of a trypanosoiiie is far from being so ; nor can analogy with spirochztes be considered as a legitimate argument in favour of any such conclusion.”

On the other hand, Buchanan (1911 6 ) , in describing certain cultural phases of Trypanosoma brucei, ends with a remark of consider- able significance. “ A marked feature, perhaps worthy of note, was the constant presence of free chromatin granules in nearly all the smears from the second day onwards. These were apparently derived from the nuclei of disintegrated ti-ypanosoines and possibly represented a first phase in development.”

In dealing with the hmnoflagellates one must not overlook Balfour’s assertion ( I 9 1 2 4, that Archibald has seen definite granule shedding in flagellates from cultures of Leishnzania tyopica by using a dark field method of examination.

The granule shedding described in the previous coniniunication in the case of H. simoiicli (1913 14) is, as far as one is aware, thc only observation on the occurrence of such a phenomenon in a hmiio- sporidian, or, rather, it is the only observation of such a phenoinenon which has been interpreted as being of a vital nature.

If the intracorpuscular bodies described are indeed phases in the life-history of the hzmosporidian, then they niay legitimately be talien as representing stages in the evolution of the adult parasite, it., as being recapitulative in the phylogenetic sense (Minchiu, 1 9 1 2 lY).

Now, it is an established zoological maxim in the case of highly organised animals that community in embryonic structure reveals community in descent, and, further, that the einbryonic stages 1 weal more or less completely not only the condition of the progenitor of the whole group, but also the condition of that progenitor in its adult state. If the same m a s h be applied to the Protista, then the primitive pro- tist organieni, the progenitor so to speak of either a bactcriuin or a protozoon or a spirochzte, would consist of a minute granule of chromatin, which, so far as one can my, is quite devoid of protoplasm. A somewhat similar coiiclusion has of course already been leached by others on purely speculative grounds, and for this reason one is iiiuch at a loss to understand the mental attitude of those who seek on theoretical grounds to establish the existence of a chroinatinic primum vivens and who yet refuse to accept the infective granule as concrete laboratory evidence of the same.

What appears to me to be a strong argument in favour OP the interpretation I have put on the corpuscle inclusions found in Soleu is afforded by the fact that there are already known to exist separate and distinct hzemosposidia which conform morphologically to the phases I have described.

1. The coccoid bodies are represented by Theiler’s ilnaplasnza marginale (1910 25). The first description of such an organism

was probably given by Smith and Kilborne in their monograph on Texas fever (1893 z4). I n the acute form 01 the disease laige pyriforni :tnd spintile-shaped piroplasiiis were found in the corpuscles, wliereas in suli,iciite or chronic cases only “ periplierd coccus-like bodies ” were present. The view that these coccoid bodies were but phases in the life-history of the piroplasiu was supported by suhsequent worlters, and such a view was niorc or less accepted until Theiler in the course of investigations on Pircplasmosis in South Afiica came to thc conclusion that the I ‘ marginal points ” l i d no counection whatever with a piioplasni, but weie in fact a new and distinct paiasite to wliich he gave the name AiiapZasm,a m u g inale. Since then certain workers have supportetl Theiler, but there are others who have reverted to the older opinion and wlio assert their belief that the parasite represents none other than the young form of oue or other of the piroplusms. Certainly Thciler’s l’arasite has been desciibed S O frequently as existing side by side with a piroplasin, that this fact alone furnishes strong presumptive evidence of a possible relationship.

2. The bacillary phase is represented by the intmcorpusciilar bodies first described by Grahani-Smith in the blood of 2’aLpa e u ~ o p a , the coiimion niole (1 9 0 5 ll). Yiiiiilar bodies were found i n the Soudan by Balfour (I 9 0 6 l) in Jnculzis g o ~ d o 1 ~ i in association with a htcino- grcgarine ; and Franqa ( 1 9 11 9, has recorcled theiy presence in Portugal, not only in Tulpu, but also in X c r o t u s incertzis and in Eliornys q / i w r i n / ~ s . Laveran, who saw preparations sent by Graham-Smith, by Balfour, and by Franqa, in each case gave the opinion that the infected corpusclee were “ h h a t i e s niouchetdes,” i.e., that the corpnscles showed the granular basophilia which pathologists are accustomed to look for in c’ases of severe liuinan aneniia, and particnlaily in cases of plumbism. Fianga, being unwilling apparently to commit hiinself to any very definite opinion, described the bodies as “ pseudo-hbmatoaoaires cndoglubnlaires.” More recently these bodies have been the subject of study by Brurnpt (19115), who has come t o the conclusion that they cannot be basophilic granules iuasmuch as these latter are easily decolorised by Unna’s orange tannin. He suggests that they i m protozoan in nature, and lias given them the generic name, Grcchcmcllic. I have found similar bodies in Crossoptis fodiens and in Xicrotii, apest is , in each case in association with ii trypanosome. The following table in regard to the occurrence of G~ahnniella brings out a point of considerable interest in connection with the present discussion, niid it is this, in every case in which Grahnmlla lias been recorded it is associated with either a h:emosporidian or a trypanosome. Thornson’s parasite in the mole is the subject of a conin~nnic;ition which is, a t present, in process of preparation. A study of the organism sliows it to conform more closely to the hmiogregtrine type t h i to iiny other 11,~inosporidian type. Tlie adult trophomite is found to occur in two phases in the blood, a norinal phase with no extranuclear

INFECTIVE GRANULE AND PROTIST ORGANISMS. 255

chromatin granules, and a granule - shedding phase comparable to H. simondi.

TABLE.-Graham-Snl.ith’s In fracorpuscdar Bodies, which have been desci-ibetl 6y FTanpa as (‘ pseudo-heinatozoaires endoglobulaires,” and again by BTl6z;pt as (( G’rahanaella,” have been found to occur in-

(a) Thomson’s intracorpusnular parasite (1906 2’j), which bas been named by Franp, Elloipsisoma thomsmni (1911 y). It is nearly related to the hcemogregarines.

(a) A trypanosonie, first seen by Gros in Russia (184512), then described in turn by Petrie in England (1905*0), by Franqa in Portugal (1911g), and by Wrublewski in Russia (1912 9.

2. ~ ~ c z ~ Z u s ~ a e u Z u s , the jerboa or desert rat, along with a hsmogregarine, first described by Halfour (1906 l), and then by Laveran, who has named it H. balfOwi (1905 15).

3. Microtus incertus, a vole, along with a piroplasm which occuls in Portugal and which has been named Smithiffi microti by Franpa (1910s).

4. Micrutus ayrestis, the ordinary field vole. Specinlens taken in the neighbourhood of Shefield have been found to harbour Crahamella and a trypanosome, which latter shows a resernbla.nce to but is larger than Z’r. Zewisi.

Only two specimens of this little animal have been obtained. One of these showed the presence of CrnhameZZa in large numbers, the other showing a scanty infection with a trypanosome which closely resembles that found in TffiZpa.

1. Talpn europma, the common mole, along with

5. Cros$opLs fudiem, the water shrew.

Are we to assume, then, that Gyahamella has nothing whatever to do with the parasites along with which it has been found to exist ? A consideration of the above Lable, I believe, goee far to dispel such an assumption, and I venture to suggest that Graham-Smith’s bacillary bodies are none other than the infective granule stage of a hmiosporidian.

( a ) The parasite which occurs in Cottus and which is described in the present Journal (1913 13) ;

(b j A piroplasm described by Franqa, Xnzithia naicroti (1910 ”. “La forme jeune de cet H4matozoaire est ovalaire et a

1.5 p de long. Sa chromatine est disposde en calotte A l’extrkmit8 grossie.

“La forme la plus commune est ronde ou ovalaire, de 2 Q 3 p de dianibtre. La cliromatine des formes rondes est rbduite ti une petite granulation b la pkriphkrie. Dans les fornies ovalaires, elle se dispose comnie un capuchon B l’un des pales du parasite. Du reste la disposition de la chrotnatine est trbs variable, nGme dans des formes identiques, comme on le voit bien dans les figures qui aceompagnent ce travail.

“ On observe aussi, trbs souvent, des parasites presentant la forme d‘une ellipse trts allongQe, ayant sa chromatine en filament le long de l’un des bords de 1’HQmatozoaire et dans une certaine Qtendue.

“ On peut, enfin, observer des QlQments pirifornies, trks intehessants, occupant les uns seulement une partie de l’h6mntie . . . les autres toute le diamktre de l’hkniatie.

“ La chromatine des parasites piriformes, plus petits, se prksente comme une granulation plac6e dans la partie plus

3. The peripheral chromatin phase is found in

He says of i t :

356

voluniineuse ou acolde ix 1’un dos borcls. Les poires lcs plus glandes ou t I(. plus, souvent la chromatine condens6e en un giiiiiieau sit& b la partic ktroite du corps; dans tin grand nornbie cl’exemplaires cependant elle se trouve situk le long tit: l’uii dcs b o d s dn parasite, soit comme un filament, soit coiriine Line sdrie de granulations. I’armi les formes en poire n u u b en avons trouvde une o h la chromntine se niontre en cleux filsnients, un Lie chaque cot& du p:irasite.”

Cei taiii parts of this description of Snzitlzicc micmti might very well be applied to the Cottiics parasite, and indeetl a reference to Fiaiqa’s illate of the foirner and t o lriy own figures of the latter will show the close morphological resemblance between these two organisms.

It is not improbable that a recognition of the coccoid, bacillary, :nid periplieral chroinatin types of parasite as being evolutionary phases in the fashion I have suggested, will ultimately help to dispel iiimj iliIliculties in the way of a proper classification of the 13;eiiiosporidia. If one accepts the existence of a primordial cliioniatin giannle in the case of the lilclniosporidia and its developnient along the lines I have indicated, then one is, I believe, furnished with a simple classification founded on phylogenetic principles. The iiiost primitive h~mosporiiliari organism would be represented by such a parasite as Theilcr’s AwLplnsma mcuyinale, if indeed this is to be considered as distiiict fioiii some piroplasm. In the same category would come the bacillary bodies of (:rahani-Smith. The acquisition of protoplasm by the coccoicl or bacillary type would be a further stage in evolution, such bodies Leing represented by the piroplasms. Again, the peripheral chromatin phase, as seen in Si?2ithia mic ro t i , would represent :in interiiiediate or transition phase between the piroylasiiis and the limnogregarines, or indeed any other adult hzmosporidian of the celliilar type. The coccoid, l~acillai y, and peripheral chroniatin types then would be but phases in the upward evolution of a cellular individual. I n this connection a question which naturally presents itself is this, why does tlevelopiiient become retarded aL any one given htage ? or in other words, why does the granule in one case proceed through the bacillary and peripheral chromatin phases to the cellular type, whereas in another case it stops a t an earlier stage, as for example in the case of a piroplasm? The obvious answer to this question is-that retardation of development is likely to occur just in that stage wliere icproduction comes possible. Further, one must bear in mind this possibility, that if a parasite becomes capable of reproduction a t nn early age, before acquiring adult characters, then d c ~ elopiiierit tends to beconie arrested a t this adolescent stage, and the adiilt stage would in consequence, sooner or later, become lost. I n other wolds, the power of reproduction of a parasite is the deterniinant uf its stability in any givcn phase.

1NFZCTIV.E GRANULE AND PROTIST ORGANISMS. 257

REFERENCES.

1. BALFOUR . . . . . .

. . . . . . 2. ,,

3. ,, * . . . . .

4. ,, . . * . . .

5 . BRUMPT . . . . . .

6. BUCHANAN . . . . .

7. DUTTON AND TODD. . .

8. FRAN~A . . . . . .

9. PRAN~A . . . . . .

10. F R Y . . . . , . .

11. GRAHAM-SMITH . . . .

12. GROS . . . . ~ . .

13. HENRY. . , . . . .

14. ,) . . . . . . .

15. LAVBRAN . . . . . .

16. LEISHMAN . . . . . .

17. MINCHIN . . . . . .

“ A New Hamogreprine of Narnmal~,” See- ond Rqi. Wellcome Researck Laboratortes, Khartoum, 1906, p. 97.

“ SpirochBtosis of Sudanese Fowls,” Tl~ir(7 Rep. FVellconae Research Laboratories, Khartoum, 1908, p. 38.

“ Spirochatosis of Sudanese Fowls,” Fourth Rep, W’eflcome Research Laboratories, Khartoum, 1911, p. 76.

Discussion on Patton’s “The Kala azar Pro- blem,” Brit. M P ~ . Journ., London, 1912, vol. ii. p. 1197.

“Note eur le parasite des HQmatirs de la Taupe,” Bull. Sor. Path. Ezot., Paris, 1911, vol iv. p. 514.

“Some Observations on Trypanosoniu brucei(l),” Fourth Rep. Je l lcome Research Laboratories, Khartoum, 1911, p. 57.

“ A Note on the Morphology of SpirochztR duttoni,” Lancet, London, 1907, vol. ii. p. 1523.

“ Sin In classification des Piroplasnies et description de deux formes de ces parasites,” Arch. do Real. Instit. Bad. Cam. Pest., Lisbon, 1910, tonie iii. fasc. i. p. 11.

“ Sur les Hhatozoaires des Taupes,” Inst. de Bad. Cam. Pest., Lisbon, 1911, tome iii. fasc. iii. p. 271.

“The Extrusion of Granules by Trypanosomes,” Proc. Roy. Soc. London, (B), 1911, vol. Ixxxiv. p. 79.

“ A New Kind of Parasite found in the Red Elood Corpuscles of Moles,” Journ. Hyg., Cambridge, 1905, vol. v. p. 453.

“ Observations et inductions microscopiques sur quelques parasites,” Bu71. de la Soc. Inaper. des Natiiralisfes de Moscou, 1845, tome xviii. p- 424.

‘‘ An Intracorpuscular Parasite from the Blood of Cottus budalis and Cottus sco~pius,” Journ. Path. and Bacferiol., Cambridge, 1913, vol. xviii. p. 224.

“ The Granule Shedding of Hmniogregamha siinondz,” Ibid,, 1913, vol. xviii. p. 240.

“ Sur uno h6mogregarine des gerboiyes,)’ Con@, rend. Acad. d . Sc., Paris, 1905, tome cxli. p. 295.

“ Mechanism of Infection in Tick Fever and Hereditary Traiismission of Spirorhctfa duttoni in the Tick,” Trans. Sac. [Z’rop. &fed. Hyg., London, 1910, vol. iii. p. 77.

“ Some Problems of Evolution in the simplest Forms of Life,” Journ. QueXett Micr. Club, London, 1911, vol. xi. No. 68, p. 165.

258 INFECTIVE GRANULE AND PROTIST ORGANZSMS.

18. MINCHIK . . . . . . “ A n Introduction to the Study of the

19. J , . . . . . . Ibad., p. 306. 20. PETRIE . . . . . . “Observations relating to the Structure and

Geographical Distribution of certain Try- pmosomw,” Journ. of H?yg., Cambridge, 1905, vol. v. p. 191.

21. RANREN . . . . . . “Granule Shedding i n Trypanosonin gam- biense,” Brit. Ned. Journ., London, 1912, vol. ii. p. 408.

22. K o s ~ , MOORE, AND WALKER “ Centroeoines and other Structures in the Red Blood Corpuscles of Vertebrates,” Trans. Path. Soc. London, 1907, vol. lviii. p. 107.

33. SCHAUDINN . . . . . “Generations-und Wirtvechsel bei Try- panosoma und Spirochzte,” Arb. aus den kaiserlichen Geswm’neitsmibe, Berlin, 1904, vol. xx. S. 387.

‘‘ Southern Cattle Fever,” U.S. Dept. of-ilgric., Eighth and Ninth Reports Bureau Aninial Inclusfry, Washington, 1891, 1892, p. 77.

25 THEILER . . . . . . “Texasfieber. Rota asser, und Gallenkrankheit der Binder,” Zeitschr. f. Infectionskrunk- heiten der Haustiere, 1910, vol. viii. S. 39.

26. THOXSO~ . . . “ Blood Parasites of the Mole, including a new form of Intracorpuscular Parasite,” Journ. of H:jg., Cambridge, 1906, vol. vi. p. 574.

27. WRUBLEWSKI . . . “ D i e Rlutpnrasiten dcs Maul\nurfee,” Cen- fralbl. -6 Eucferiol. u. Parasitenk., Jena, 1912, Ahth. I., Orig. Ed. Ixii. S. 140.

Protozoa,” London, 1912, p. 468.

‘34. SMITH AND KILBORNE. .