J R Army Med Corps: first published as 10.1136/jramc-06-06-07 on 1 June 1906. Downloaded from

653

THE LEISHMAN BODY, THE GREGARINE· STAGE OF A HERPETOMONAS.

By LIEUTENANT-COLONEL C. BIRT. Royal Army Medical Corps. IN September, 1904, Leishman1 called attention to the similarity which exists between the forms of herpetomonas, a parasite of the house-fly, and the organisms he discovered in the spleen of a man who succumbed to kala-azar. MesniF also shortly. afterwards' pointed out that the flagellates into which Leishman's bodies develop in sodium citrate solution were more closely allied to the herpetomonas than . to the trypanosome. This opinion, too, is shared by Rogers3 in his recent contribution to the Proceedings of the Royal Society, entitled" Further Work on the Development of the 'Hepatomonas ' (sic) of Kala-Azar and Cachexial Fever from Protected by copyright. Leishman-Donovan Bodies." A knowledge of this order has thus become requisite for the study of the epidemiology of" Dum-Dum" fever. This is attainable only in the form of isolated contributions published in foreign periodicals, inaccessible except in the Library of the Royal College of Surgeons of England. It will be remembered that when blood which contains the kala­ azar parasite is incubated at 22 0 C. with faintly acid sterile 2-5 per cent. sodium citrate solution in physiological saline fluid, actively motile flagellated organisms are developed which have a superficial resemblance to trypanosomes. Treated with Leishman's stain two http://militaryhealth.bmj.com/ spots become visible in them which are deeply tinted with the chromatin colour. The larger is termed the macronucleus, the smaller the micronucleus, blepharoplast or centrosome. A flagellum springs from the end where the micronucleus lies. The differences between the trypanosome and the flagellated stage of Leishman's body are these: (1) The trypanosome possesses an undulating membrane which is wanting in Leishman's flagellate; (2) the flagellum of the trypariosome arises from the end opposite to that in which the micronucleus is situated, while the flagellum of the on September 24, 2021 by guest.

1 W. B. Leishman. British Medical Journal, vol. iL, 1904, p. 644. 2 F ..Mesnil. Bulletin de l'Instit~~t Pasteur, T. ii., p. 957, December 15th, 1904. 3 L. Ragers. Proceedings of the Royal Society, February, 1906, Series B., val. 77. No. B.517, p.264. J R Army Med Corps: first published as 10.1136/jramc-06-06-07 on 1 June 1906. Downloaded from

654 The Grega1-ine Stage of a Herpetomonas kala-azar parasite has its origin from the extremity holding the micronucleus. The name" Herpetomonas" was first applied by Saville Kent! in 1881 to a flagellate found by Burnett thirty years previously in the digestive canal of the house-fly. Butschli next discovered a monodine much like the above in the intestine of a free nematode, Trilobus gracilis. Leger2 has made a very exact study of tl;18se and kindred organisms. Chatterjee3 had observed a flagellate in the abdominal cavity of Anopheles lItaculipennis. His description was meagre, and his figures drawn from specimens stained with 4 6 gentian violet show little Rtructure, Leger - pursued the subject and investigated a protozoon which he found in great numbers in the alimentary canal of a female A. maculipennis captured in Dauphine, France,' in 1902. He named this "Chrithidia jascicu­ lata." Two forms were noted: (1) A slender flagellated body, 8 to 14 f1- long, which resembled a trypanosome, except that an undulating membrane was absent and the flagellum was attached Protected by copyright. to the end near the micronucleus; (2) a shorter, somewhat oval structure, 3 to 8 f1- in length, possessing a macronucleus and a micronucleus. Multiplication took place by division. The micro­ nucleus and macronucleus divided i'n succession. His drawings of these are not unlike Leishman's flagellate and splenic body reRpec­ 7 9 tively. Leger - next traced the life history of a herpetomonas which he discovered in the intestine of Tabanus glaucopis, and of another from the alimentary canal of Nepa cinerea. The former he named H. subulata, and the latter H. Jaculum. Both occurred under'two aspects like the Chrithidi,a. (1) The "formes mona­ http://militaryhealth.bmj.com/ diniennes," very motile, 15 to 30 f1- long, devoid of an undulating membrane, and provided with a flagellum which arose from the micronucleus end. These were numerous. Many were attached to others by their flagella and so formed rosette-like clumps. This

1 Saville Kent. Manual of Infusoria, London,,1881-1882. 2 Leger. Comptes 1'end. de l'Academie des Sciences, March 17th, ,1902, p. 662. 3 B. C. Chatterjee. Indian Medical Gazette, xxxvi., p. 37.1, 1901. 4 Leger and Dubosq. "On the LarVal of Anopheles and their Parasites in

Corsica." Comptes rend. Congo de Montauban, 1902, p. 703. on September 24, 2021 by guest. 5 Leger. Comptes rend. de l'Acad. des Sciences, April 7th, 1902, p. 781. 6 Leger. Comp. rend. Soc. de Biologie, 1902, p. 354. 7 Leger. qomp. rend., de l'Acad. des Sciences, April 7th, 1902, p. 781. 8 Leger. Comp. rend. Soc. de Biol., 1902, p. 399. 9 Leger. Sur quelques Cenomonadines nouvelles ou peu connus parasites. Archiv. fur Protistenkunde, B. iL, Heft. 2, 1903. S.181. J R Army Med Corps: first published as 10.1136/jramc-06-06-07 on 1 June 1906. Downloaded from

0, Birt 655 process ,of aggregation into rosettes is a common phenomenon in flagellates. rrrypanosomes in blood and culture display it. The "formes monadiniennes" multiply by longitudinal division and are rapidly reproduced; (2) "formes gregariniennes." These may be regarded as developed from the former by an indrawing of the flagellum. The anterior end then fixes itself to the epithelium of the digestive canal of the host. They multiply by binary division of the micronucleus. Leger points out their striking resemblance to young gregarines, to the gregarine stage of p. jasciculata and C. minuta, an inhabitant of Tabanus tergestinus, and to piro­ plasma. The plates which accompany his paper, although pub­ lished two years before Rogers cultivated Leishman's bodies in vitro, would serve to illustrate Rogers's report even to the rosette formation. His drawings of the" formes gregariniennes " resemble closely the splenic parasites. Prowazek1 has devoted himself to the study of the herpeto-' monas of the house-fly, discovered by Bm'nett in 1851, and to that Protected by copyright. of an allied species he found in the blow-fly. He has confirmed Leger's observations. He, like him, distinguishes a flagellate stage and a gregarine stage, In some of the flagellated bodies, besides a macronucleus and a micronucleus near the flagellum, he has seen two extra chromatin spots which he calls the" diplosome." Similar dots are depicted in some of Leishman's drawings. He has observed longitudinal division and rosette formation in which the flagella are in the centre. He finds that there are male and female monodines which form by their union, after loss of . their flagella and reduction of their nuclei, a structure much like a http://militaryhealth.bmj.com/ Leishman body which he cells a "dauerzyst" or resting stage'. The conveyance of the parasite from fly to fly can be made by cysts of two kinds, viz., those which result from conjugation and those which ao not, and by infection of the ova with gregarine forms, Cysts sometimes undergo a degeneration of the principal nucleus which he terms" etheogenesis." His pictorial illustrations of this process recall the appearance of a human endothelial cell crowded with kala-azar bodies. Pfeiffer2 has made observations on a herpetomonas inhabiting on September 24, 2021 by guest. th~ stomach of a pediculus-Melophagus ovinus-a common para­ site on sheep. His description follows that of H. subulata given above. The gregarine forms are ,so numerous that they appear

1 Prowazek. Arb. a'us d. Kais. Gesundheitsamte, xx., 1904, p ..440-445. 2 Pfeiffer. ZeitschTift f. Hyg. und Infect. B.L., May 19th, 1905, p. 324-328. 48 J R Army Med Corps: first published as 10.1136/jramc-06-06-07 on 1 June 1906. Downloaded from

656 The G1'egarine Stage of a Herpetomonas

as a thick layer lining the digestive tube. The process prints of his photographs are blurred and, not very instructive. Levaditi1 has investigated a herpetomonas discovered in the ccelomic cavity of the silkworm-Bombyx mori-and has noted flagellate and gregarine stages. His drawings of the former correspond with those of Leishman's flagellates. The following enumeration of the invertebrates in which species of herpetomonas have been hitherto found is, I believe,

complete. House7fly, Blow-fly, Trilobus, Anopheles, Chironomus, T. glaucopes, N. cineTea, Ha3matopota italica, M. ovinus (sheep louse), Pollenia, Fucillii, Silkworm. Leger,2 while remarking on the ancestry of herpetomonas, reiterates his opinion, expressed in 1902, that certain species of this order are stages in the life history of hffimoflagellates of the vertebrates. This view is supported by Schaudinn's3 discovery that the HalteTidiu1}v noctUa3 multiplies in the Culex in the form Protected by copyright. of a herpetomonas. IJ8ger thinks that the ancestors of herpeto­ monas passed their whole existence in one insect host. Such a life cycle is still completed in the non-suctorial insects ClJ1~usca, SaTcophaga, Po llinia , FllCillii, Bombyx). In the blood-suckers the richer pabuluin constituted by the blood of the vertebrate would cause an enormous multiplication of the parasites, and at the same time would prepare them for an existence in the vertebrate, whither they would ultimately be compelled to seek sustenance on account of the small size of their insect host. This would amount to a partial adaptation of a protozoon, primitively parasitic in the intestinal or entero-ccelomic cavity http://militaryhealth.bmj.com/ of an invertebrate, to, a vertebrate host. It would explain why the herpetomonas would return to its primitive invertebrate host to effect its sexual multiplication. This conception is supported by the life history of the Plasmodiu1}v malaTia3. Schizogeny alone takes place in the blood of man. Sexual reproduction requires. the return of the parasite to the mosquito. Rogers considers that the bug (Reduvius, seTratu8 61' cruentus) may be the intermediary in the transmission of kala-azar, since the

Leishman body developes most readily in a faintly acid sterile on September 24, 2021 by guest. medium at a temperature of 22° C. Pediculi, fleas, mosquitoes, and

1 Levaditi. Comp. rend. de l'Acad. des Sciences, T. cxli., p. 631·634, October 16th, 1905. . 2 Leger. Comp; rend. Soc. de Biol., T. 58, Deceinber 24th, 1904, p. 613·617. 3 Schaudinn. Arb. aus. d. Kais. Gesundheitsamte, xx., 3, 1904, p. 387.439. J R Army Med Corps: first published as 10.1136/jramc-06-06-07 on 1 June 1906. Downloaded from

O. Birt 657 other blood-sucking flies cannot escape suspicion. Leeches are thought by some to be i'mplicated. The only evidence I can find in favour of this. theory is that Bentley~ stated kala-azar is more frequent in children than infants. Children who can walk would be more likely to encounter those pests than those of more tender age. Whatever, the mediate host may be, a perusal of the litera­ ture given and a comparison of the illustrations in the text- with the figures portrayed by Leishman, Rogers, Christophers, Statham, Chatterjee, render the conclusion irresistible that the authors are dealing with closely-allied organisms. That the Leishman body is a gregarine stage in the life history of a herpetomonas which completes its sexual cycle· as a flagellate in some blood-sucking insect, therefore appears probable. At the same time, it would be rash to assert that all these flagellates are of one variety. There. also seems no sufficient reason to assume the identity of the splenic paras\tes with forms· morphologically similar found in tropical sore (Aleppo or Delhi boils). The fact that the former Protected by copyright. are always highly virulent and productive of a general infection which the latter do not cause, is in itself a significant difference. The chief objectiori to the theory of infection by blood suckers has been the scarcity of' the parasite in the peripheral blood. ' Success has been achieved only by examining several hundred leucocytes. One point, however, seems to have been overlooked. No systematic examinations of the blood have been made at stated intervals during the day and night over considerable periods. On post-mortem examination the endothelial cells of the liver and spleen are so distended with the specific bodies that they readily http://militaryhealth.bmj.com/ burst and discharge their contents. The inference is that this too must occur at times during life. It is well-known that Filaria; nocturna; appear in the blood of the extremities about 6 p.m. and vanish some six hours later-a periodicity which has been evolved to permit the filaria to adapt itself to the habits of the Culex, in the body of· which it must sojourn to complete its cycle of existence. Thus the gregarine of kala-azar may also escape intermittently into the peripheral circulation solely during the hours of the night for the purpose of gaining the alimentary canal of some blood-sucking on September 24, 2021 by guest. insect in which it must go through its sexual phases.

SUGGESTIONS FOR FU'TURE RESEARCH. (1) Periodical nightly examinations of the blood of kala-azar patients during a wave of high fever. Bentley! states they are

l Bentley. Brit. Med. Jour" val. ii., 1902, p. 875, and val. ii., 1904, p: 655. J R Army Med Corps: first published as 10.1136/jramc-06-06-07 on 1 June 1906. Downloaded from

658 The Gregarine Stage of a Herpetomonas more infectious at this time. The cultural method, by mixing several c.mm. of the blood with sterile 5 per cent. sodium citrate solution and incubating at 22" C., would probably be a more delicate test than staining of blood films. (2) Search for herpetomonas in all suctorial invertebrates in .endemic areas. Where tropical sores prevail, flies and blow-flies should be examined for endo-parasites. (3) Feeding experiments with the above on kala-azar sufferers, and on tropical sores, with subsequent seatch for herpetomonas in the intestinal canal. In the case of Delhi boil, however, direct transference of the infective material without the agency of an intermediate host is possible. 'Fleming1 proved this in 1868, when he successfully inoculated himself with what he ascertained were the specific cells of the ulcer. (4) Eogers found that freedom from bacterial contamination was an essential condition of success in the cultivation of .the Leishman body. Toxins seemed inimical to its growth. Terminal infections Protected by copyright. with staphylococci, streptococci, pneumococci, dysentery bacilli, are the immediate cause of death for the most part in kala-azar. By subcutaneous injection of killed cultures of some of these pathogenic agents, the immunity of the individual against that particular infection would be raised, arid simultaneously the toxin might prove antagonistic to the kala-azar invasion. A trial of this mode of treatment seems justifiable.

REFERENCES. http://militaryhealth.bmj.com/ For a Comprehensive Summary of the Work on the" Parasites of Culieidal," .scc Dye, Archives de Parasitologie, T. ix., 1905, p. 1. . Some reference to the herpetomonas may be found in Laveran and Mesnil's ," Trypanosomiasis," and S. Prowazek, Arb. aus d. Kais. Gesundheitsamte 22, 1905, p. 351·395. . on September 24, 2021 by guest.

J J. Fleming. Army Medical Reports jor 1869, vo!. xi., p. 511.