J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

321

THE DEVELOPMENT OF THE LEISHMAN BODY IN CULTIVATION.l By MAJOR W. B. LEISHMAN, AND CAPTAIN .T. C. B. STATHAM. Royal Army Medical Oorps. PART I. By CAPTAIN STATHAM. ADDITIONAL NOTES ON THE POST-MORTEM EXAMINATION. No ulcers on body, but two or three Plgmented scars due to quinine injections which had caused some sloughing .. Lungs.-Showed typical pneumonic consolidation in both lower lobes (stage of red hepatisation) ; some recent left pleurisy. Heart.-Normal; 10ozs. Liver.-Much enlarged-l06 ozs.-firm, and somewhat tough on section; had "nutmeg" appearance. Protected by copyright. Spleen.-Very much enlarged-87 ozs.-red on section; no capsulitis, no infarcts; substance friable but firm. Pancreas.-Eight ozs., firm; no noticeable changes in structure. Kidneys.-Six and a half ozs. each, somewhat dark and con­ gested, otherwise normal. Suprarenal bodies appeared normal. Gastro-intestinal tract examined from oosophagus to rectum­ no signs of ulceration in any part of tract; slight congestion of small intestine here and there; most congested part removed for microscopic examination; Mesenteric glands. - Not appreciably enlarged. http://militaryhealth.bmj.com/ Brain.-Forty-six ozs., appeared healthy; no congestion of pia mater or vessels on surface of brain; no petechire or hremorrhages. Bladder.-Contracted, but walls looked normal. Testicles.-Left somewhat enlarged, but this was found to be due to a hydrocele. Bone Marrow.-Normal in appearance. DISTRIBUTION OF THE PARASITES IN THE TISSUES. I. Liver.-A. Smears. Bodies found: (1) In two or three cells with polymorphonuclear nuclei and blue-staining protoplasm. on September 27, 2021 by guest. One contained 2 bodies, one 9 bodies, and a third 29 bodies.

1 A preliminary note by Captain J. C. B. Statham on the subject of this article appeared in THE JOURNAL OF THE ROYAL ARMY MEDIqAL CORPS, vol. iv., p. 13. January, 1905. J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

322 Development of the Leishman Body in Oultivation

(2) In several large cells with round or kidney-shaped nuclei, surrounded by considerable amount of protoplasm; often as many as 20 bodies found. No parasites were seen in typical liver-cells. B. Seotions. The parasites were abundant and occurred in the protoplasm of the endothelial cells lining the capillaries and lymphatic spaces. Il. Lung.-A. Smears. Leishman bodies found in: (1) Large mononuclear blood-cells. (2) Polynuclear blood-cells. (3) Several in a vacuolated mass of protoplasm, probably the remains of a cell. B. Seotions. Bodies found: (1) A few in faintly staining mono­ nuclear cells of alveolus detached. (2) In a cell from the alveolar lining. (3) A few. in polymorphonuclear cells in the exudation in alveolus. (4) One outside lung Gust outside pleura), in what looked like pl~mritic exudation; it was apparently lying free. (5) One or two in connective cells between alveoli. (6) In cells lining capillary;

Ill. Spleen.-A. Smears. Bodies found: (1) Many bodies present Protected by copyright. in large cells with faintly staining and abundant protoplasm. (2) A few in what looked'like large mononuclear blood-cells. (3) One or two bodies, suspiciously like Leishman bodies, in polymorpho;­ nuclear blood-cells. (4) In one or two cells a little smaller than large mononuclear blood-cells, with protoplasm of deeper blue and with single round nucleus. B. Sections. (1) In one or two instances bodies appeared to lie in the connective tissue cells forming the framework of the lymphatic space of the spleen. (2) The vast majority of the bodies seen were contained in very large pale­ staining cells of irregular shape. These cells appeared to line· the http://militaryhealth.bmj.com/ lymphatic spaces in some cases, a portion of the cell often jutting out into the space. Often these large cells, which had round or oval nuclei, appeared to be filling up the lymphatic space. (3) In fragments of which looked as if they had been detached from the cells above referred to. (4) In round cells, little larger than large mononuclear cells, with well-defined margins, of which the protoplasm stained. more deeply than the cells above referred to. (5) In what looked like endothelial cells lining the capillaries. IV. Suprarenals.-A. Smears. In one cell with a round nucleus,

qut in which the outline of the protoplasm could not be definitely on September 27, 2021 by guest. made out, three or four bodies were seen lying in the broken~up protoplasm .. B. Sections. (1) One or two in polygonal cells with a large oval nucleus, like cortical cells of suprarenal body; two pthers in endothelial cells lining a lymphatic space or capillary. (2) Two or three lying free in what appeared to be the fibrous tissue of the cortex. J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

W. B. Leishman a.nd J. O. B. Statham 323 V. Kidneys.-No definite bodies found in smears or sections, but fragmentation of nuclei made decision hard to arrive at. VI. Bone Marrow.-Smears. Bodies found: (I) Chiefly in large m,yelocytes (?) with neutrophile granules and large round or kidney­ shaped nuclei (Cornil's myelocytes). (2) In smaller myelocytes with round nuclei and deeper staining protoplasm (Ehrlich's myelocytes). VII.. Intestines.-In sections of the small intestine (congested patches) no bodies were seen. VIII. Mesenteric Glands.-Smears. No bodies found. Sections. IN 0 definite bodies found. IX. Brain.-No bodies found in sections. X Pancreas.-No bodies found in sections or smears. XI. Basilar Artery.---.,.No bodies found. XII. Aorta.-No bodies found.

THE CULTIVATION OF THE PARASITES.

December 5th, 1904.-A number of splenic and liver punctures Protected by copyright. were made under aseptic precautions, and the blood placed in ­ ~ubes, each containing about 1 cc. of a 4 per cent. sodium citrate solution. These were incubated in an extemporised incubator, the ~emperature of which oscillated between 15° C. and 24° C. Next lllorning some fresh sterile human blood was added to four of the cultures. December 6th. - (Thirty-five hours' development). Examined tllbes and found-Hanging drop: A' few Leishman bodies, which ~ppeared enlarged (clear refracting, lightly greenish bodies); non­ motile. Stained specimen: Leishman bodies present and mostly http://militaryhealth.bmj.com/ increased in size. Most· of the bodies were free, but a few were .s~ill contained in the large splenic cells. The protoplasm of the parasites stained blue and was much vacuolated, the· enlarged ~acronucleus consisted of a loose skein of chromatin, staining light red. The slightly enlarged micronucleus was rod-shaped and stained gensely. (All specimens were stained by Leishman's modification of Romanowsky.) . December 7th. - (Fifty-seven hours' development.) Hanging 4rop: Bodies still further enlarged-mostly free. Stained speci­ men: Bodies enlarged to the size of, or a little smaller than, a red on September 27, 2021 by guest. l?100d-cell-5-7 ",. Appearance of protoplasm and nuclei as found, on December 6th. . December 8th.-(Eighty-one hours' development.} Great change fpund to have taken place. Hanging drop: Shows large round J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

324 Development of the Leishman Body in Oultivation bodies, 5-8 f/,; oval bodies, 7.:.9 f/,; and long trypanosoma-like forms, 12-20 f/,. Some of the oval and all the long forms were seen to be flagellated; they were motile and moved slowly across the field of the microscope, flagellated end forward, the. lashing some­ what slowly from side to side ahead of the advancing parasite. The parasites were very transparent. Stained specimen: Showed round, oval and long forms, as in hanging drop. Protoplasm stained blue and was much vacuolated. Macronuclei consisted of a loose skein of chromatin lightly stained; micronuclei rod-shaped and ruby-red to purple in colour. Many oval and all the long trypanosome forms flagellated, and many of these showed division in all s'tages; some commencing to divide, macro- and micronuclei dividing first. Other forms had completed division, and .were united only by anterior or posterior extremities. The condition was evidently one of. division and not conjugation, for like parts were in apposition in the early dividing forms- to macronucleus, micro­

nucleus to micronucleus. No evidence of an undulating membrane Protected by copyright. was found, and the micronucleus was in all cases near the base of the flagellum. December lOth.-(Five days seven hours.) Specimens as seen in hanging drop and stained smears practically similar to those of December 8th. Some Leishman bodies were seen developing' in a large splenic cell, showing that'the bodies may be considerably developed before becoming free. December 13th.-(Eight days.) In addition to forms as seen on December 8th and 10th, very small, thin, trypanosome-like forms, almost approximating to spirilla, were noticed. Agglomerated parasites were also seen. http://militaryhealth.bmj.com/ December 17th.-(Twelve days.) Only a few parasites obtained from culture tubes. Hanging drop: One very motile, small form was seen; its presence was at first noted by the commotion caused among the blood-cells, and the nature of the moving body was only ascertained when it was brought to a halt for an instant by a block of cells: it was then seen to be a long, thin and small spirillum-like form. Stained specimen: Showed very 'few parasites, the same in character as on December 13th.

December 19th.-(Fourteen days.} Very few parasites found. on September 27, 2021 by guest. Hanging drop: Parasites not clearly defined and not motile. Stained specimens: Showed that the parasites were much broken up; they stained badly, and were evidently degenerating. After this date only degenerated forms were found. The para., sites had, therefore, lived for about fourteen or fifteen days in the J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

W. B. Leishman and J. O. B. Statham 325 citrated splenic and liver blood tubes. Some difficulty was ex­ perienced in obtaining satisfactory films, as the parasites appeared to develop in a jelly-like substance (probably broken-down splenic pulp) at the bottom of the tubes, and it was only when one of these jelly-like masses could be hooked up through the supernatant layer of citrated plasma that a good film could be obtained. If the cul­ tures were previously shaken up parasites were often found, but they were not numerous. An impression was gained that the parasites developed better in the splenic cultures to which sterile human blood had been added.

NOTES ON SUB-CULTURES. (1) In Fresh Sterile Human Blood.-On December 9th, 1904, about 4 cc. of human blood were placed in sterile tubes containing 1 cc. of a sterile 4 per cent. solution of sodium citrate. Into this medium four or five loopfuls of the splenic cultures were placed, Protected by copyright. and the tubes incubated at 20° C. Three days afterwards (i.e., on December 12th) some evidence of a growth in the tubes was noticed. The growth consisted of small, greyish, granular masses, about the size of a small pin's head, lying on the surface immediately above the layer of white blood-cells, which formed a whitish-pink bed beneath them. Three such granular growths were found in one tube, separate from each other; two in another tube; while in the third culture there was a larger lump, which appeared to consist of two or three small masses heaped together. The granular, round growths were somewhat transparent looking in parts by transmitted http://militaryhealth.bmj.com/ light; while by reflected light the growth looked more opaque and grey. The difference may have been due to part of the growth beirig the material from which the parasites were growing, while the clearer portions consisted of the zooglea colonies of the parasite itself. From these growths parasites were obtained exactly similar to those found in the citrated splenic blood tubes. By December 16th (i.e., eight days after the inoculation of the tubes) growth appeared to have reached its maximum, although the size of the colonies was but slightly increased from that noted on December

13th (i.e., four days after inoculation of the tubes). On December on September 27, 2021 by guest. 16th parasites were again obtained from these growths. After December 16th the growths began to melt away, and the parasites obtained were found to be degenerated. The parasite had, there­ fore, lived and grown in the culture of human blood for eight or nine days. J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

326 Development of the Leishman Bodly in Oultivation

(2) Ascitic Fluid.-A tube of sterile ascitic fluid was inoculated on December 10th, and examined four days after, the fluid being centrifuged to obtain any parasites that might be present. A few bodies which looked like degenerated parasites were recovered, but none stained well. ' (3) Tubes of (i.) sterile tap water, (ii.) sterile pond water, (iii.) ordinary pond water, were inoculated on December 10th from the splenic cultures. On December 14th, four days after inoculation, a portion of each of these tubes 'Was centrifuged and the deposit spread on slides. No parasites were found in the sterile tap water; a few broken-down parasites were recovered from the sterile pond water, but these stained badly. In the ordinary pond water manyani.mal­ culre were found, but nothing that resembled the Leishman body in any of the stages of ita development. In hanging drops from the tubes of sterile tap and pond water no motile organisms were found.

From this it appears that the parasite does not thrive well in water Protected by copyright. at 20° C.

NOTES ON SOME OTHER WORK IN CONNECTION WITH CASES OF DUM-DuM FEVER. (1) F(J3ces of the above case were during life highly diluted and then incubated at 20° C.; films made from a portion of this dilution after centrifugalisation were examined every day for six days. This examination was carried on intermittently for two or three months, and some 150 slides examined, but nothing found. Dilutions of http://militaryhealth.bmj.com/ urine were similarly incubated and examined, with negative results. Freces and urine of another case of Dum-Dum fever in hospital were similarly incubated and examined, and nothing found. (2) Purpuric spots and small blebs which· appeared on the leg of an undoubted case of Dum-Dum fever were examined, smears being taken from the ulcerated surface left after bursting of the blebs. No Leishman bodies were seen. (3) Finger blood of a third case of Dum-Dum fever was drawn off into a small quantity of citrate of soda solution and centrifuged so as to get a layer of white blood-cells. No Leishman bodies were on September 27, 2021 by guest. recognised in slides prepared by smears from the layer of leuco­ cytes. The centrifugalised blood after incubation at 20° C. showed no flagellated bodies. . (4) Ascitic fluid, drawn off from a case of undoubted Dum-Dum fever in the last stages, and centrifugalised to obtain white blood- J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

W. B. Leishman and J. O. B. Statham 327 cells, showed only two bodies in some hundreds of cells. Both these bodies were in large mononuclear cells. The fluid drawn off asep­ tically and incubated for two days showed no parasites. My thanks are due to Lieutenant A. H. Proctor, LM.S., for his valuable assistance and co-operation in much of the work recorded above. PART n. By MAJOR LEISHMAN. THE DEVELOPMENT OF THE PARASITES IN CULTURES. The first sign of change is seen in the blue-staining protoplasm of the spleen parasites, which loses its transparent hyaline appearance and becomes more granular and opaque (fig. 1,2, and plate, fig. 3). The parasites then commence to enlarge, retaining, however, their original oval or circular shape; . this initial enlargement appears to be due, at least in part, to the enlargement of the macronucleus, which from occupying about a quarter of the parasite comes to fill

half or more (fig. 1, 3 and 4, and plate, figs. 4 and 5), This enlarge­ Protected by copyright. ment of the macronucleus is soon followed by an increase in the . quantity of protoplasm, which now takes a deeper blue tint with Romanowsky's stain. The micronucleus appears to be somewhat enlarged, but retains its characteristic shape, usually that ofa short, slightly curved rod, and it displays the same intense affinity for this stain, appearing almost black even after short staining. A marked feature in the process of development in all our films was the early appearance of in the protoplasm; these were always circular in outline and fairly uniform in size, having an

average diameter of '5-1 #; very rarely larger ones were seen of http://militaryhealth.bmj.com/ a maximum diameter of 3 11-' These vacuoles, as will be noticed from the forms figured in the coloured plate, persist throughout all the stages of development, and comparatively few parasites were seen in which they were absent. As to their nature and function, it seems probable that they are nutrition vacuoles concerned in the rapid process of growth, but as Rogers' did not mention them in connection with his culture, it is possible that they may be due to some difference in the tonicity of the citrated plasma employed in his cultures and in ours.

Growth at this stage is very rapid, the quantity of vacuolated on September 27, 2021 by guest. protoplasm increasing, while the macroneuc]eus becomes greatly enlarged and its chromatin network loosened, while its staining reaction is much feebler; the micronucleus, however, at this stage, appears to undergo no further enlargement or alteration. From the earliest stages of growth of the spleen parasites multi­ plication by occurs; and even in very young cultures large J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

DESCRIPTION OF COLOURED PLATE.

FIG. 1.-Large splenic cell containing parasites. 2.-Various forms of the parasites as seen in smear preparations from the sple?n. 3.-Early stages of development of the parasites in cultures. Protected by copyright. " 4.-Further developed forms, some showing stages of division. 5.-Group of young parasites, some flagellated. " 6.-Small group of oval, non-flagellated forms . ." 7.-Young flagellated parasites. 8-16.-Various forms of fully-developed flagellated parasites . ." 9, 1O.-Division forms. " H.-Group of flagella,ted forms. 12.-Form showing the disposition and shape of the chromatin granUles. 17.-Small, flagellated" spirillar forms." All the figures have been drawn from specimens stained by Leishman's stain. http://militaryhealth.bmj.com/ on September 27, 2021 by guest. J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

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330 Development of the Leishman Body in Q"ltivation be more diffuse and less definite in outline, appearing to mel'ge in the blue - staining protoplasm of thc parasite. In two or three instances this pink zone, whieh may be termed the "flagellar ," without prejudice to its real nature and function, attained a size equal to half thc diameter of the parasite itself (fig. 1, 5). The micronucleus at this stage may remain apparently unaltered, or may, as in the example figured, change in shape to a small circnlar mass of deep-staining chromatin; in one case no trace of the micronucleus was seen, but the margin of the vacuole showed Protected by copyright.

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lhG. I.-STAGES IN TliE D.lGVl!:LOl'Mll:NT Ob' ~'nE FLAGELLUM. (1) Ordinary form of spleen pllrasite; (2) slightly clllu·rged parasite, from a youog culture; (3) further stage of growth, va.cuolation of the protoplasm; (4) development: of the" flagellar vacuole" close to micronucleus; (5) increase in size of "flagella.r vacuole"; (6) rupture of 11(1.Ouole and protrusion of the young flagellum in the form of n. tuft or bunch of pink-sta.ioing threws; (7) growth of the filtgellum, the thickened http://militaryhealth.bmj.com/ ba.se being iuoorted in the collapsed flagella.r vacuole; (8) trypano8omu.l form with fully· developed flagellum. a deeper tint of chromatin than the pink homogeneous contents of the vacuole itself, Following on this enlargement of the vacuole the next stage appears to be the rupture of the thin rim of proto­ plasm or o[ cuticular substance which forms its externnJ border, and the protrusion of part of the contents of the vacuole in the form of a fringed proce~s or tuft of the same staining reaction as

the contents of thc vacuole, and also of a fully-developed flagellum on September 27, 2021 by guest. (fig. 1, 6). Such [arms were only rarely seen, but in all of them the same picture presented itself of a bunch or tuft of pale pink threads starting from the neighbomhood of the micronueleus, passing outwards through the partially collapsed flagellar vacuole, and projecting for a distlLnce of 3-5 JL clear of tbe body of the parasite. Of the next stage examples were rather more common; here the J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

W. B. Leishman and J~ O. B. Statham 329 numbers of fission forms are to be seen in all stages (plate, fig. 4). As a rule, the first sign of this fission process is seen in the macro­ nucleus, which becomes in succession elongated, constricted in the middle and, finally, separated into two distinct parts. Similar division of the micronucleus follows, but occasionally this may precede the division of the macronucleus forms being met with containing two micronuclei and but one macronuclens. As a general rule the process is completed by simple cleavage of the parasite into two independent parasites, but at times a single parasite may give rise to three or even to four new individuals, by a process of further subdivision of the macro- and micronuclei. As this fission goes on considerable masses of young parasites are formed; they are circular or oval in shape and of an average diameter of 6-7 1'-, or approximately equal in size and shape to red blood corpuscles (plate, fig. 5). These developmental changes,

up to and even beyond this stage, may also take place while the Protected by copyright. parasites are still embedded in the protoplasm of the splenic " macrophages." Once the above stage has been reached further development appears to proceed with varying degrees of rapidity, as, twenty­ four hours later, a great variety of forms were seen, many of them fully-developed , while among them were to be found all the earlier stages as well as the intermediate forms now to be described. The next step towards form consists in an alteration of the shape of the parasites; from being circular or oval they become elongated, usually thicker at one end than the other, and often http://militaryhealth.bmj.com/ distinctly pyriform, the protoplasm and the macro- and micronuclei preserving the characters seen in the circular forms. At this stage, also, multiplication by simple longitudinal fission occurs. By this time a flagellum has in most instances appeared, and it is of interest to note that this structure may even be developed while the parasite is still definitely circular in torm. The development of the flagellum is, apparently, a very rapid process, as the enormous majority of the parasites examined were

either definitely non-flagellated or were in possession of a fully­ on September 27, 2021 by guest. developed flagellum of the usual type and staining reaction. By prolonged study of the films, however, forms have been met with which appear to illustrate some of the stages of the developmental process; a few of these forms are illustrated in fig. 1. The first change which appears to take place is the development of a circular, pink-staining area in contact with one side of the micronuclear rod (fig. 1,4). At times, however, this pink zone may J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

W. B. Leishman and J. O. B. Statham 331 flagellum tapers slightly from its apparent origin at the bottom of the flagellar vacuole towards its free extremity, while its length is now about half that of the parasite (fig. 1, 7). After this the flagellum apparently increases very rapidly in length, as very few forms were seen between the last stage and that of the fully-developed flagellum, which is usually of about the same length as the parasite to which it is attached and uniform in thickness throughout (fig. 1,8). In many instances it may still be seen to have its origin in the remains of the pale pink vacuole, which apparently persists for some time; but in some of the most fully-developed parasites it appears to be inserted directly into the protoplasm of the parasite, its point of origin being invariably in the immediate neighbour..; hood of the micronucle1J.s, though it does not appear to be directly connected wIth this structure. The flagellum always arises from the rounded end of the parasite,

and projects at once clear of the body into the surrounding Protected by copyright. fluid, and does not course down the body and project beyond the pointed extremity, as in the case of a trypanosome. This point will be referred to again. The motility of the flagellated forms is, as a rule, sluggish and the parasites advance with the flagellar end foremost. The most active individuals were the thin spirillar forms described below. Considerable variations were met with in the shape and structure of the macronucleus. A few young flagellated forms were seen in which the nuclear chromatin appeared brightly red and nearly homogeneous, and in these the protoplasm was usually more darkly stained than usual and free from vacuoles. As a rule; however, the http://militaryhealth.bmj.com/ nucleus appears granular, or in the form of an irregular network of chromatin threads. The granular ones showed considerable irre­ gularity both in the numbers and the arrangement of the granules, the most frequent appearance being that of a wreath, or ring, of chromatin granules, enclosing a non-granular central zone. These granules are most probably chromosomes, and from the variability of their numbers, as well as from other variations noted in the shape, size and staining reactions of the parasites, it appears possible that

there may be a sexual differentiation among these flagellated forms. on September 27, 2021 by guest. Further experience and more extended observation are, however, necessary, before this point can be definitely affirmed. . In many of the parasites small chromatin dots, distinct from the niacro- and micronuclei, were found in the protoplasm. These varied much in size, but were usually definitely circular in outline and tended to occur in pairs resembling small diplococci (plate, J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

332 Development of the Leishman Body in Oultivation

fig. 12). They were most numerous in the younger stages prior to exflagellation, but were also found in many of the mature parasites. From their appearance, arrangement and staining reactions, these -granules appear to represent various stages in the of a. blepharoplast or accessory nucleus, such as has been described by Schaudinn,2 in connection with Trypanosoma noctuCl3, it was not, however, possible to detect any filament-representing the central thread of the spindle-connecting the pairs of chromatin dots to one another or to the parent nucleus. As far as could be seen with the' magnification employed (1,000 diameters), there was a slight difference in size between the individuals of each pair, so that if it is the case they are concerned in a mitotic process, the resulting spindle is probably heteropolar. Occasionally these chromatin dots were seen in the neighbourhood of the micronucleus, and it isnot impos­ sible that such may have been concerned in the development of the flagellum, but they were much more frequently found close to the Protected by copyright. macronucleus, and ,no further details were detected pointing to flagellar development occurring upon the lines described by Schau­ dinn.' The two cases further present the following difference, that in the case of TrypanosomanoctuCB, there is at first only the single nucleus of the ookiriet of the halteridium, from which ablepharo­ plast has to be formed prior to the elaboration of the flagellar apparatus; whereas, in the case of the parasites under discussion, the micronucleus, which is usually accepted as being a blepharo­ plast, is already in existence. Multiplication by longitudinal fission apparently occurs during the flagellated as well as during the non-flagellated stage, but in the http://militaryhealth.bmj.com/ case of many of the twin parasites it appeared likely that fission had taken place at an earlier stage,and that the twins had grown to maturity side by side. Mature forms showing a preliminary division of the macro- and micronuclei, though met with at times, were rare. A curious process of unequal longitudinal fission was observed in some of the mature flagellated forms, and in this process the nuclei of the ,parent did not appear to play any part. The result of this process was the splitting off of a thin spirillum-like parasite from one side of the mature flagellate, and ,sketches of parasites showing on September 27, 2021 by guest. various stages in .this method of multiplication are given in fig. 2. Occasionally a form was seen showing that more than one such form might be split off from the same parent (fig. 2, 6 and 7.) These thread-like forms have usually two or more chromatin dots in their substance, and- often show as well the characteristic vacuolation of the protoplasm, but' no further details could be made out owing to their minute size. When newly-separated from the J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

W. B. Leishman and J. O. 13. Statham, 333 parent they are destitute of a flagellllm, but this is subsequently developed (plate, fig . 17), and these spirillar flagellated forms showed much more active motility than the larger parasites. The possession of chromatin by these minute thread-like forms suggests the possibility that they Illay undergo a further process of longitudinal division and, if this be so, it is conceivable that the pl'oducts of such further division might be of ultra-microscopic size, and we should then have a pos~iblc explanation of some of the

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.FTO. 2, -FT~ AOI:LLA '1'ED P AILASITES 01\'11>0 H.lSE 'la 8PIRILLAR FORMS BY A PnOCESS OF UNEQUAL LONGITU D1NAL FISSION. (1-5) Clcn;\'o.go of sioglc spirillar forIDs from the parent; (6) para.!lite giving rise to two spirillar Corms; (7) two spirillar forms completely separated from the parent. puzzling features in the etiology of the disease to which they give rise. The resemblance, aga.in, between these minute forms and the on September 27, 2021 by guest. spirochrutes is obviolH:; and at the same time suggestive, in the li ght of Sehaudinn's work on Spi,·oclu.te Ziemanni. Nothing of the nature of '" conjugation process was observed, and although [arms similar to those described and figured by Rogers as conjugation forllls were seen, they appeared in all cases to he divisjon forms in which Sepal'i1tion was incomplete. 23 J R Army Med Corps: first published as 10.1136/jramc-04-03-05 on 1 March 1905. Downloaded from

334 Development of the Leishman Body in Oultivation

With regard to the biological position of the parasite, it will be seen from the above description and from the figures that the most fully developed of the flagellated forms differ from true trypanosomata in two points only, the course taken by the flagellum and the absence of an undulating membrane. It has been proved, however, that trypanosomata in cultures may differ considerably from the form in which they occur in their host, and it is not till such cultural forms are once more introduced into a suitable host, that they resume their classical form. For instance, Smedley 3 has recently shown that in the case of cultures of Trypanosoma Lewisi the parasites are destitute of an undulating membrane, and their flagella project at once clear of the parasite instead of coursing along the body and projecting beyond the tapering extremity of the parasite. It appears, then, from the facts at present available, that the spleen parasites, or "Leishman bodies," are undoubtedly a stage in the development of a flagellated organism and, further, that nothing has yet been observed in the flagellated forms inconsistent with their Protected by copyright. eventually proving to be trypanosomata. It should not, however, be concluded that the last word upon the nature of the parasite has been said when it has been assigned its proper place among the flagellata. In view of the striking pleomorphism which it has been shown to exhibit under cultural conditions, it would be rash. to a~sume that the forms figured and described in this article and elsewhere, exhaust its possible changes of form, and it may yet be found to exist outside the body of man in a shape as widely different from those at present known as the

flagellated forms themselves differ from the spleen parasites. http://militaryhealth.bmj.com/ I need add little to Captain Statham's description of the cultural characters of the parasite. The cultures which I obtained from him, however, retained their vitality somewhat longer than his, the last motile parasite being seen on the twenty-first day, and evidence of growth and development was observed in a first and second sub­ culture made in citrated human blood. A number of animal experi­ ments were undertaken with the cultures, both by inoculation and by feeding, so far with negative results. Some of these experiments are, however, not yet concluded, and should they prove of interest they will form the subject of a further communication. on September 27, 2021 by guest.

REFERENCES.

1 L. Rogers, Quarterly Journal of Micr08copical Science, new Series, No; 191, p. 367, November, 1904. • F. Schaudinn, Arbeiten aU8 dem Kaiserlichen Ge8undheit8o,mte, vol. xx., p. 387, 1904. 3 R. D. Smedley,Journal of Hygiene, vol. v., p. 24, January, 1905.