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 flea, Ctenocephalus canis, by BAsllm (1910), who believed he was dealing with a species of Leishmania. FmTXtAM (1912) placed it in genus Her#etomonas, and provisionally gave it the specific name Ctenocephali. Since, however, crithidia 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 fleas. 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 ! 2 3 ~ f .... 6 7 5 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.