495 TRANSACTIONSOF THE ROYAL SO~UErYOF TROPIC&L MEDICINE AND HYGIENE. Vol. 40. No. 4. March, 1947. OBSERVATIONS ON LEPTOMONAS CTENOCEPHALI (FANTHAM, 1912). BY

ALFRED J. GIBBS.* Downloaded from https://academic.oup.com/trstmh/article/40/4/495/1936312 by guest on 29 September 2021

"ITae parasite was discovered in the dog , Ctenocephalus canis, by BAsllm (1910), who believed he was dealing with a species of . FmTXtAM (1912) placed it in genus Her#etomonas, and provisionally gave it the specific name Ctenocephali. Since, however, and trypanosomes are not found at any stage, it was later transferred to the genus Leptoraonas.

DgVELOPMENT IN LARVA. Infection of the flea takes place during the larval stage, and is the result, as will be shown, of the ingestion of resistant bodies of l~ishmm~ia form which are present in the faeces of infected . The faeces consists chiefly of partially digested blood which can hc dissolved in saline under a cover-glass for the study of the bodies in the living state. They are spherical and without apparent internal detail. When fixed and stained by Leishman's method they are ovoid in form and measure some 3 /~ by 2"2 /~. The nucleus and kinetoplast are situated near one end, while the rhizoplast extends from the region of the kinetoplast to the opposite end of the body. No cyst wall can be seen either when stained or examined in the fresh state in saline. (Fig. 1, p. 497.) Larvae feed readily on the faeces of the flea, which, when dry, is found in tho form of granules on the body of the dog. Newly hatched specimens, which have been experimentally fed on the faecal blood of infected fleas, are often found to harbour leptomonads on tho day following hatching. Multi- plication does not take place in the pre-flagellate form as the infective bodies develop into flagellates. At first the parasites are confined to the mid-gut (stomach) and are very few in number, probably representing individuals which have developed directly from the infective bodies. These earliest found forms are highly motile leptomonads about 21/~ long with flagella of the same length. There are usually one or more twists in the body. (Fig. 2.) A' constant characteristic of the larger leptomonad forms is that the nucleus is located within the anterior third of the body, and the nucleus and kineto plast are situated in close proximity. Multiplication takes place, and the o It is desired to express appreciation of invaluable assistance given throughout the course of this work by Dr. ANDREWROBERTSON (formerly lecturer, Department of Proto- zoology, London School of Hygiene and Tropical Medicine), Dr. H. S^NDO~ (Department of Zoology, University of Cape Town), and Professor J. T. IRVINO (Department of Physiology, University of Cape Town). 496 I,I~PTOMON/I..S G'.I'tcNOfJEPI.IALI

mid-gut of the larva becomes heavily infected with long, slender leptomonads Later the lengd~ of the flagellum diminishes to about one quarter of the body-length and its power of movement is reduced to a series of periodic jerks with little or no travel. At a still later stage the flagellum practically disappears, but the body remains elongated and twisted. (Fig. 3.) Under natural conditions larvae attain the pupal stage in about 4 Downloaded from https://academic.oup.com/trstmh/article/40/4/495/1936312 by guest on 29 September 2021 days, but under the laboratory conditions available they failed to progress beyond the first moult. It is possible, thereh~re, that subsequent phases of the life-cycle of the parasite and its disposition in the host, although described as occurring in the larva, may normally take place after the pupal stage is attained. About seven days after hatching, the infection travels to the hind-gut which first harbours a few active leptomonads similar to those first found iu the mid-gut. The body is twisted and the flagellum is long and active, but is always attached near its tip to the gut-wail. Free forms arc not found in the hind-gut at any time. This adherent condition of the flagellate has never been observed among leptomonads in the mid-gut. Although the mid-gut and hind-gut are occasionally infected concurrently (the former with lepto~ monads which have practically lost their flagella, and the latter with a few adherent leptomonads with long flagella), it is usual to find that the parasites in the mid-gut disappear when the hind-gut becomes infected. The adherent flagellates soon become rounded and take on leishmania-like form. Multipli- cation then becomes rapid and isolated dusters of leishmanial parasites can be found at various points in the hind-gut, but they are never found in the rectum. Later, the hind-gut becomes packed with parasites. The Malpighian tubules of larvae are never found to be infected.

]~'ORMS ~'OUND IN ADULT FLEA. As the larvae failed to attain the pupal stage, investigation has been limited to the examination of the larva and the adult flea. Parasites are never found in the mid-gut of the fea, the infection always being confined to the hind-gut and rectum, while occasionally the Malpighian tubules are involved. Usually, parasitization commences abruptly behind the pyloric opening and extends downwards according to the intensity of the infection. The forms found in the Malpighian tubules are long, active leptomonads, similar to those first fo,nd in the mid-gut of larvae; they are about 23/~ long and exhibit twists. "l~ey arc usually found near the distal extremities of the tubules. The rounded forms described by P^rro~~ and RAo (1921) as occur. ring in the Malpighian tubules of the human flea, PuIex irritans, have not been found in the present species. In order to explain the presence of immotile forms in the tubules, these authors suggest that they have probably been carried there by the active flagel|ates. WES~'ON (1926) regards the theory ALFPJ~D J. GIHBS 497

as improbable, but an instance has been noted recently (G,~,s, 1942), in which leptomonads and crithidia carried about with them a number of leishmania like forms adherent to the flagella and never seen to become detached. The forms found in the gut and rectum are (a) sttimpy leptomonads, (b) rounded leishmaniadike forms, and (c) smaller non-flagellate bodies. Lcptomonads about 5"5 ~ long with short flagella constitute the majority of Downloaded from https://academic.oup.com/trstmh/article/40/4/495/1936312 by guest on 29 September 2021 the parasites in the gut and rectum. (Fig. 4.) They are never found free within the lumen, anchorage always being afforded by the flagellum, but there is frequently a pendulum-like swaying of the body. They arc sluggish

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8 when liberated into saline; there is movement of the flagella but they do not travel and are never found with the twists which ate characteristic of lepto- monads which infect the mid-gut of larvae. The larger rounded forms measure about 5 ~t across and are often fmmd with short flagella, indicating development to or from leptomonad for::;. The nucleus is usually eccentrically situated and the kinetoplast is large; the rhizoplast is very distinct. (Fig. 5.) They are ;requently found in a sta:c of division and rosette formation is common. The small, ovoid leishmania-like forms are never fomld dividing. These are the resistant, infective bodies which are present in the dejecta of infecte6 ~eas. WF,NYON (1926) states that when there is little nourlshnlent ia ti,e gut practically all the flagellates are in the attached condition, but after a long 498 LEPTOMONA$; C'TENOf2EPIIALI

meal of blood, many active forms can be seen within the gut contents. During the present investigation free forms have never been observed within the gut, even after a meal. Wz~-rON also states that "towards the posterior end of the intestine the attached flagellates, and also those free within the cavity, become smaUer, till finally little ovoid leishmania forms are pro- duced." It has not been possible to confirm this statement as no consistent differentiation has t~en noted between the parasites situated just behind Downloaded from https://academic.oup.com/trstmh/article/40/4/495/1936312 by guest on 29 September 2021 the pyloric opening and those found at any other part of the hind-gut oI within the rectum. It will subsequently be shown, also, that the smaller leishmanial forms (the resistant bodies) which are described by SHORTT (1923) as occurring only in the rectum, are also to be found at the upper end of the hind-gut.

DEVI.;LOP/VIENT OF RESISTANT BODIES IN SAIA'NI,:. WE~YoN (1914) noted that in some form the parasite is resistant to desic- cation, as he obtained growth in culture from material which had been dried for 24 hours, and concluded that the resistant forms are the small leishmania-like bodies. It has been found that the development of the resistant body takes place in normal saline, and it is thus possible to follow directly the subsequent flagellation and change of form of the organism. The period of viability of the bodies while contained in dried faecal blood is considerable; an interval of 2 raonths does not in any way appear to affect development. Resistant bodies of Leptoraonas ctenocephali can be obtained for study in two ways. Firstly, they can be found in the faeces of infected fleas which has been dissolved in saline; and, secondly, they can be obtained directly from the hind-gut and rectum by distortion. In the first case the faecal blood can be dissolved under a cover-glass and observation commenced immediately. If the saline is considerably hypotonic the bodies disintegrate owing to osmotic pressure differences, while if die blood is dissolved in water disinte. gration is immediate and complete. In the second case the gut and rectum can be removed, split lengthwise and laid on a slide to dry. Under these conditions the bodies survive for about 5 hours, even when gentle warmth has been applied for a short, time to ensure thorough drying. When examined later in saline during the period of viability they can be easily distinguished from all other types by. their smooth appearance, whidl contrasts sharply with the vacuolated condition of the dead flagellates and larger leishmania-likc forms. It is found that the resistant bodies obtained from the flea by dissec- tion survive for a period of 3 days when they are dried in a solution o1 saline and abattoir blood. The development of the resistant bodies in saline takes the same forn~ whether passed out naturally with the faeces, or obtained from the gut or rectum of the insect by dissection. At first they lie at the bottom of the saline ALFI~ED.I. GIBBS 499 and it is not until the flagellum is fairly active that they commence to rise. In a period varying from 10 to 30 minutes the first indications of the growth of a flagellum can be detected. Fig. 6 shows a stained specimen in which the flagellum has commenced to protrude from the body. At first the fila- ment is non-motile, but when it attains a length equal to one-half of the body-length, it begins a jerky, side to side movement which increases in Downloaded from https://academic.oup.com/trstmh/article/40/4/495/1936312 by guest on 29 September 2021 vigour with growth. Simultaneously the body elongates and the parasite assumes leptomonad form. Fig. 7 shows a stained leptomonad after 30 to 60 minutes in saline. In 60 to 120 minutes the body becomes twisted (Fig. 8), and soon afterwards it attains a length of approximately 13/L with, a long flagellum. At this stage the parasite, although smaller, is similar to the first forms which infect the mid-gut of the larva, and is highly motile. Dividing forms can be found after some hours. Development then ceases, although the flagellates may continue to be active for 24 hours. The resistant bodies can be found at the upper end of the hind-gut as well as in the posterior portion and in the rectum. This has been established by the following often repeated experiment. The alimentary tract was removed from an infected flea and the hind-gut cut as closely as possible to the pyloric opening. The remaining length of gut and the rectum was dis- carded, while the short length taken from just behind the mid-gut was placed on a dean slide and dried. When examined later in saline many of the infective bodies could be found, and these afterwards developed into motile forms. It has been found that when artificially removed parasites are dried for a period not exceeding about 15 minutes, a few flagellate forms revive within a minute or two in saline, while the resistant bodies flagellate later.

SURVIVAL UNDER ADVERSE CONDITIONS OF TEMPERATURE~ ETC. Experiments. were undertaken to study the effect of various temperatures on the resistant bodies contained in the faeces of fleas. No effect on develop- ment is to be noted after refrigerating the material at a temperature of 0 ° C. for 24 hours. After a similar period at 45 ° C. there is a lengthening of the time taken to become motile (I hour), while certain individuals fail to become as active as usual. Larvae which feed on the blood become infected. When the blood is heated to 65' C. for 24 hours only about 1½ per cent:, of the bodies develop in saline, while the time taken to become motile is increased to 5 hours. They are very feebly active and are incapable of rising from the bottom of the liquid. Development does not progress beyond the very stumpy leptomonad form. Larvae fed upon this material do not become infected. Twenty-four hours at a temperature of 70 ° C. was found to be lethal. Granules of faecal blood which have been immersed in absolute alcohol for 30 minutes will yield active flagellates when dissolved in saline. ~01} LEI,'I"OMONtffl Ug'IgNOC~Ptt~II..I

DIScusSION. The resistant bodies appear to ewflve directly l!rom odml forms, ratimr than result from a special division, as neither the larger leishmanial parasites nor the leptomonads are found dividhlg in a way which might give ri~ to them. Unless the gut contains substances unfavourahle to their development,

there appears to be no obvious reason why they should not flagellate within Downloaded from https://academic.oup.com/trstmh/article/40/4/495/1936312 by guest on 29 September 2021 the body of the host as readily as they do in saline, tt is reasonable to assume, therefore, that the resistant phase in the host is a temporary one, and that unless passed out of the gut with the faeces, the resistant body reverts to flagellate or larger leishmanial form. The prolonging of the period of viability of artificially removed bodies by drying in abattoir blood, indicates that the faecal blood, which envelops naturally voided resistant bodies, assists in the maintenance of viabihty over long periods by preventing too great a loss of moisture. :Yhe blood appears to serve the purpose of the more usual cyst-wall. The rapid development of the bodies in saline and the complete disintegration when immersed in water, add further evidence of the non-existence of any tough protective covering.

SUMMKRY. l, The developmental cycle of Leptomonas ctenocephali in the larval and adult stages of the , Ctenocephalus canis, is described. 2. Resistant forms of the parasite are contained in the dried faeces of infected fleas, and these, when ingested by larvae, flagellate and multiply in flagellate form in the mid-gut. 3. In the adult flea the infection is confined to the hind-gut, rectum and occasionally the Malpighian tubules. The forms found are leptomonad, leishmania-like and smaller leishmanial resistant bodies. 4. The resistant bodies develop in saline after a period o[ 2 months in dried faecal blood. When artificially removed from the flea by dissection they develop in saline after being dried for not longer than about 5 hours. 5. The development of the resistant body into a long, higlfly motile lepromonad takes places within about I~- hours in saline. 6. Experiments regarding the survival of the resistant bodies under adverse conditions are described.

REFERENCES. BAS~L~.,C. (1920). Parasitology, 12, 366. FANTHA~, II. 13. (1912). Brit. med..~., ~,, lig& GxaBs, A.J. (1942). S. Afr. y. raed. Sci., 7, Biol. Supp',., 50. PATroN, W. S. & RAO, S. (1921). ?ndiany. reed. Res., 8, (~21 S~lou'rT, II. E. (1923). Ibid., 10, 721. W~,'qVON, C.M. (1914). Trans. Soc. trop. ivied. Hy2, , 7, 9). -- ~ - (192~). Protozoology. Londo:t : B~.i!liltre, Tindali & Cox.