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Journal of Experimental Medicine

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C O M P A R A T I V E STUDIES OF HERPETOMONADS AND
LEISHMANIAS.
I. CULTIVATION OF H E R P E T O • O N A D S

FROM" INSECTS A N D PLANTS.

BY H I D E Y O NOGUCHI, M.D., AICD E V E L Y N B. TILDEN.

(From the Laboratories of The Rockefeller Institute for Medical Research.)

P~TES I0 TO 15.
(Received for publication, March 16, 1926.)
Our interest in the herpetomonads parasitic in insects and plants invarious parts of the world lies in the possible relationship of some ofthem to the parasites of kala-azar, of India, China, and Mediterranean countries, oriental sore, of Palestine, and-espundia, of SouthAmerica. The mode of infection of man by the leishmanias is notunderstood, and experimental work on the problem of transmission
has, until recently# been rendered extremely difficult by the lack ofsusceptible laboratory animals. The geographic distribution andepidemlology of the various forms of leishmaniasis have indicatedthat the parasites may be carried by some insect, and many attempts
have been made to find evidence of this mode of transmission.
That the leishmanias may be taken up by insects and are capable of survivingin the insect gut was first shown in the case of Leishmania donovanl by Patton, 2
who fed bedbugs on kala-azar patients and observed the development of theparasites to the flagellate stage in the bugs. Wenyon3 later reported the ingestion of Leishmania tropica from open lesions of cutaneous leishmaniasis by flies,
mosquitoes, and bedbugs, and the development of the parasites in some of these
insects. The sand fly (Phlebotomus) is looked upon at present4as the most probable vector of leishmaniasis, but the experimental evidence is fragmentary.
1Young, C. W., and Liu, Pao-Yung, Proc. Soc. Exp. Biol. and Med., 1926,
xxiii, 392.
Patton, W. S., Scient. Mere. Off. Med. and San. Dept. Gov. India, No. 27, 1907.
a Wenyon, C. M., Parasitology, 1911, iv, 273.
4 Sinton, J. A., Indian J. Met. Research, 1924-25, xii, 701.
307

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HERPETOMONADS AND LEISHMANIAS.

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The search for pathogenic varieties of herpetomonads among the flagellates
so commonly found inhabiting the intestinal tract of insects was begun in 1913b~¢ Laveran and Franchini, who reported having induced in rats and mice ad~'sease not unlike kah-azar or oriental sore by inoculation of flagellates obtained
from the dog flea, mosquito, sheep ked, and house fly, and even of flagellates ofplant origin. 5 Pure cultures were used for inoculation in most instances. Laterthey obtained somewhat similar results with cultures of a flagellate obtained
from the sand fly.6 Fantham and Porter 7 induced symptoms of kala-azar byinoculation of various insect flagellates and believed that the leishmaniases
would prove to be insect-borne herpetomoniases, but Hoare8 repeated their experiments and obtained only negative results. Becker 9 and Drbohlav I° also
failed to infect animals with flagellates of insect origin. Strong, Is who foundflagellate infections in the hemipteran, Chariesterus cuspidatus, of Panama, in its
plant hosts, and in a lizard presumed to prey upon the insect, was able to cause
ulceration of the skin in a monkey by inoculation of the flagellate material fromthe intestinal tract of the lizard and to demonstrate leishmaniform parasites
in the skin lesion; but he found the plant and insect flagellates to be noninfective.
While a very large number of the plant and insect flagellates have been described, the greater part have been studied only under natural conditions and on
the basis of morphological features, and they have usually been given specificnames designating their hosts. It has been recognized, however, that the same
flagellate may live in several hosts, and Becker, 9 having demonstrated by crossinfection experiments that the flagellates of Musca domestica could thrive equallywell in the intestinal tract of several other species of flies, and that the flagellates
inhabiting each of the fly species which he studied showed a similar adaptability,
concluded that the herpetomonads of flies all represent the same species. Drbohlay x° confirmed Becker's experiments with fly flagellates, using a cultivatedstrain of Herpetomonas muscarum, but was unable to infect the dog flea or water
strider with any flagellate save the one naturally occurring in these insects. He
was unsuccessful in establishing Leishmania infantum in any flagellate hoststudied.
A number of the flagellates of invertebrates and plants have been cultivated,among them tlerpetomonas culicis from Culex pipiens (Novy, MacNeal,
Laveran, A., and Franchini, G., Compt. rend. Acad., 1913, civil, 744; Bull.
Soc. path. exot., 1914, vii, 665; 1919, xii, 379; 1920, xiii, 796.
6 Laveran, A., and Franchini, G., Bull. Soc. path. exot., 1920, xiii, 569.
Fantham, H. B., and Porter, A., Proc. Cambridge Phil. Soc., 1915, xviii~ 39.
s Hoare, C. A., Parasitology, 1921, xiii, 67.
9 Becker, E. R., J. Parasitol., 1923, x, 25.
10 Drbohlav, J. J., Am. J. Hyg., 1925, v, 580, 599.
u Strong, R. P., Am. J. Trop. Med., 1924, iv, 1.

HIDEYO NOGUCHI AND EVELYN B. TILDEN

309

and Torreyt2), H. ctenocephali of the dog flea (Tyzzer and Walker, 13 Wenyon,14
Laveran and FranchiniS), H. pulicis from Pulex irritans (WenyontS), H. ctenopsyll~e5of the mouse flea, H. phlebotomi6 of the sand fly, II.jaculum ~of the scorpion,
Crithidia melophagi5 of the sheep ked, and II. tarantol~r16 of the gecko (Laveranand Franchini). Laveran and Franchini obtained pure cultures also fromplants, tr N6ller ts cultivated a number of insect herpetomonads and several
trypanosomes. Drbohlav1° obtained pure line strains of H. muscarum. The
medium used has usually been the N. N. N. medium or some modification of it,
though Franchini and Laveran made use also of blood bouillon, 16and Franchinit*
later found that a number of flagellates would grow in a mixture of bouillon andplant latex.
The purpose of the present investigation was to cultivate a numberof herpetomonads from different sources and compare their culturaland biological properties. Serological comparison of the leishmanias ~° had already indicated the value of immunological properties
for differentiation and had suggested t h a t these and other biologicalproperties might be used for comparative studies of the leishmanias
with other herpetomonads. We have been able to supplement thisstudy by comparison of our strains with other cultivated flagellates,
H. clenocephali, isolated by Tyzzer and Walker in 1919, is and T.
rotatorium, isolated by Drbohlav, 1° cultures of which were furnished
us through the courtesy of Dr. E. E. Tyzzer, of Harvard University
Medical School.
Pure cultures of herpetomonads have been isolated from the following insects and plants: Oncopeltus fasciatus (Fig. 1), Oncopellus sp.? 21(Fig. 2), Lygaeus kalmii (Fig. 3) (two strains), Anopheles quadrimacula12Novy, F. G., MacNeal, W. J., and Torrey, H. N., Y. Infect. Dis., 1907, iv, 223.
13Tyzzer, E. E., and Walker, E. L., J. Med. Research., 1919, xl, 129.
14Wenyon, C. M., Tr. Soc. Trop. Med. and Hyg., 1914, vii, 104.
tSWenyon, C. M., Arch. Protistenk., 1913, xxxi, 1.
16 Laveran, A., and Franchini, G., Bull. Soc. path. exot., 1921, xiv, 323.
17Laveran, A., and Franchini, G., Bull. Soc. path. exot., 1920, xiii, 796.
as N611er, W., Arch. Schiffs- u. Tropen-Hyg., 1917, xxi, 53.
19 Franchini, G., Bull. Soc. path. exot., 1923, xvi, 41.
2o Noguchi, H., Proc. Internat. Conf. Health Prob. Trop. America, Kingston,
Jamaica, British West Indies, july 22 to August 1, 1924, published in Boston,
1924, p. 455.
21Specimens of this insect have recently been submitted for identification to
Dr. H. G. Barber of the American Museum of Natural History, who was able
at once to classify it as to genus. The species has not yet been determined.

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HERPETOMONADS AND LEISHMANIAS.

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tus, Culex pipiens, Musca domestica, bluebottle flies (two strains),
Asclepias syriaca (two strains), and Asclepias nivea. I n addition, two
impure cultures were obtained, one from Oncopeltus cingulifer (Fig. 4),
and one from the plant on which it feeds, Asclepias curassavica.
Attempts to purify these two strains were unsuccessful, and they soon
died out.
The specimens of O. cingulifer were collected in Tela, Honduras, ~° those of
Oncopeltus sp.? in the vicinity of Lima, Peru, 2~those of O. fasciatus and L. kalmii
on Long Island and in Shandaken, N. Y., those of Anopheles quadrimaculatus in
Leesburg, Georgia,~s and those of Culex pipiens near Jersey City. The houseand bluebottle flies were collected on the grounds of The Rockefeller Institute.
The specimens of A. curassavica were brought from Tela, 2° Honduras, and thoseof A. syriaca from Baltimore, Long Island, and Shandaken. For the Haitianmilkweeds, A. nivea, we are indebted to Dr. Francis O. Holmes, of the Boyce
Thompson Institute for Plant Research, Yonkers, N. Y., who infected them byallowing infected specimens of Oncopeltus fasciatus to feed upon the seed pods.
For identification of the latex-feeding insects we are indebted to Dr. H. G.
Barber, of the American Museum of Natural History. The bluebottle flies
were no doubt Calliphora, but identification of the species was not possible inthese instances.

Method of Cultivation.
For the initial cultivation of the flagellates from insects, solid blood
agar plates were employed which contained various c a r b o h y d r a t e s
and were adjusted to different hydrogen ion concentrations, p H 5,
5.5, 6, 6.5, 7, and 7.5. The proportions of the constituents were as
follows:
Defibrinated horse or rabbit blood . . . . . . . . . . . . . . . . . . . . . . . . . . .
300 cc.
2 per cent nutrient agar, melted . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
500 cc.
Mixed sugar solution, sterilized by filtration through Berkefeld
filters: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25 cc.
Glucose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20 gm.
Saccharose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10 "Inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 "~ These specimens were collected by Dr~ T. S. Battistini, of Lima.
28Dr. Mark F. Boyd, of the International Health Board, was kind enough t oinform us of the presence of flagellates in these mosquitoes and also to dissect
insects and inoculate culture media.

HIDEYO NOGUCHI AND EVELYN B. TILDEN
Levulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 gm.
Raffmose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 "Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . .
100 cc.
0.9 per cent sodium chloride solution . . . . . . . . . . . . . . . . . . . . . . . .

311

175 cc.

Various bacteria, molds, and yeasts are usually associated with the flagellates
in the insect gut and by their more rapid growth render the isolation of thelatter extremely difficult. In several instances impure cultures were obtamec~
by inoculating plates with ascending dilutions of the suspension of teased viscera,
and these were purified by transfers to new plates repeatedly until some purecolonies of flagellates appeared. Later, however, in the work with flies, it was
found necessary to resort to Barber's technique 24 of picking out one flagellate ora group of them for inoculation of the plates. Theoretically this procedure
ought to give fairly constant results, but actually many plates implanted witha single flagellate failed to show any growth, though plates spread gently withseveral organisms usually yielded some isolated colonies. Probably not every
individual was capable of adapting itself to the cultural conditions provided.
I t was noticed that whenever an impure culture was obtained by the plate
method, the contaminating organisms were usually yeasts, which had apparently
suppressed the growth of bacteria to some extent by the production of acidity.
The observation suggested the use of culture media adjusted to concentrationsof acid which were not sufficient to interfere with the growth of the flagellates,
but in which the growth of bacteria was reduced so far as possible. The neutral
and slightly alkaline media were not omitted in routine work, however. As 'arule, four or five plates of a given hydrogen ion concentration were used; a small
drop of the suspension of teased viscera in 0.5 per cent saline was spread on thefirst plate with a sterile bent pipette, and the other plates were brushed successively with the same pipette. The first plate was usually overgrown bybacteria, but one of the others might yield fairly discrete colonies of bacteria andan occasional pure colony of flagellates.
The cultivation of plant flagellates was much more difficult, not because of thecontaminants--for in this instance the only contaminants present are the yeasts
and molds from the stem or leaves--but because of the difficulty of adaptationof the flagellates to the media, as shown by the fact that when large numbersof them were introduced they usually lost their motility within a few days andfinally degenerated. Pure cultures have thus far been obtained only on leptospira medium, ~ and only three times among many attempts. In one other in24 Barber, M. A., Philippine Y. Sc., Section 13, 1914, ix, 307.
26 The formula is as follows: 0.9 per cent NaC1, 800 parts; fresh rabbit serum,
100 parts; 2 per cent nutrient agar (pH 6.5 to 7.0), 100 parts; rabbit hemoglobin(made by laking 1 part of defibrinated blood with 3 parts of distiUed water), 10
parts.

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HERPETOMONADS AND LEISIIMANIAS.

I

stance an impure culture of flagellates from A. curassa~icawas obtained on a
medium composed of the usual leptospira medium plus glucose, inulin, and tapioca.
The isolation experiments were carried out at room temperature, which varied
from 18°C. to 28°C. during the day and was probably somewhat lower at night.
All the plates were sealed with adhesive tape and kept in a moist chamber.
Examinations were not made until after 7 to 10 days, experience having soon
show_-, that there was no advantage in earlier examination. The colonies ofO.dgellates develop very slowly, and they are so minute that very close examination is necessary to find them. In initial cultures l0 to 14 days were required inmost instances for the colonies to become visible as tiny dew-like spots. Whenassociated with bacteria, the flagellates can only be detected by microscopic
examination, and for this purpose the dark-field microscope offers the mostconvenient and rapid means of detection.
From the pure colonies of flagellates found on plates with well separated
colonies, subcultures were readily obtained on similar media (plates or slants),
and still more certainly on leptospira medium, with or without carbohydrates.
On this medium growth is remarkably rapid and prolific, in marked contrast tothat of the first generation. All the strains isolated during the present experiments grow much more rapidly and profusely than any of the leishmanias or thestrains of flagellates from dog flea or frog. They grow well at 37°C., as well as
25°C., while for the other flagellates the lower temperature is more suitable.

Description of Flagellates Studied.
Seven of the twelve strains isolated were derived from milkweedfeeding insects and their plant hosts. When the cultures came to bestudied with respect to their serological and carbohydrate-fermenting
properties, it was found t h a t the seven strains represented in reality
only two species of flagellates. Similarly, the two strains from mosquitoes behaved as one serologically. If we were to classify the strainsaccording to host species, we should have to create several names fororganisms which b y morphology and biological reactions show identical characteristics, while if we were to depend on morphology alone,a n u m b e r of flagellates which behave differently would have to beclassed as a single species. T h e s t u d y of biological relationships,
however, made it possible to identify a given flagellate in different
environments and to separate different flagellates occurring in thesame host. T h e classification of the strains was accordingly v e r ymuch simplified. In order to avoid confusion it seems desirable toemploy throughout this paper the nomenclature finally adopted,

313

HIDEYO NOGUCHI AND EVELYN B. TILDEN

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314

HERPETOMONADSAND

LEISIIMANIAS.

I

although the manner in which it was arrived at is chiefly discussed in
Par t I I of our report. 26
Table I is a r6sum6 of the names, source, and morphology of all
the flagellates grown in pure culture. Identical strains from two or
more sources have been named in accordance with the source fromwhich they were first cultivated (H. oncopelti, H. lygxorum). 27 The
mosquito flagellates, which proved identical, have been called Herpetomonas culicidarum, since the Same strain was isolated from twogenera of the family Culicid2. As will be clear from the experiments
described in Part II of this report, it was not possible to identify thisorganism with others previously described as occurring in mosquitoes.
The naming of the fly strains was a more difficult problem, inasmuch
as each of the three proved to be distinct from the others. The preparations of the flagellates found in the gut of Musca domestica show
chiefly large forms with double flagellum which conform with .thedescriptions of H. muscx domesticx, but the organisms seen in young
cultures are not unlike H. oncopdti and H. lyg~eorum. As the cultures
grow older much larger forms appear, but these do not correspondexactly with the naturally occurring individuals, and it is difficult to
exclude the possibility that some other strain than H. muscle domesticae
was simultaneously present in the insect and grew more readily inculture. It was likewise impossible to identify two distinct strainsfrom Calliphora as H. calliphorx. The strains were finally given the
new species designations, Herpetomonas muscidarum, H. media, andH. parva.
All the flagellates cultivated have a single anterior flagellum, arisingnear the parabasal body, which is situated beside, or in front of, thenucleus. No undulating membrane, such as characterizes Crithidia
and Trypanosoma, has been demonstrated in any of the strains, either
under natural conditions or in culture. The term Leptomonas, while
it has priority over Herpetomonas, is less well defined, and it seems
reasonable, as suggested by Wenyon, 15 to restrict the name to thespecies for which it was created until it can be proved that Herpetomonas is actually a synonym of it. Werner's objection to Herpeto~6'Noguchi, I-I., J. Exp. Med., 1926, xliv, 327.
~7The name H. lyg~eihas been used by Patton (Arch. Protistenk., 1909, xiii, 1)
to designate a flagellate species found in Lyg~us militaris of India.

HIDEYO NOGUCHI AND EVELYN B. TILDEN

313

monas, 2s on the ground that it was created for a biflagellate genus,
has been invalidated by Wenyon's cytological study 16 of H. muscle
domestic~e, which demonstrates that the biflagellated forms are actuallyindividuals in a very early stage of division.
Herpetomonas oncopelti, n. sp. (Figs. 5, 6, 9, 10, 13, 14, 17, 18).
Strain/.--This strain (Fig. 5) was o~tained from the intestinal tract of Oncopeltus fasciatus (Fig. 1) cat~,ht in $kandaken, N.Y. The insect feeds on thelatex of the common milkweed, Asclef~as syriaca, which is of widespread occurrence in the temperate regions of the United States. According to Holmes,29
the insect is rarely found north of New Jersey, being replaced in the northeasternstates by the species Lyg~us kalmii, which also feeds on the latex of A. syriaca.
Only three specimens of O. fasciatus have been obtained in the course of thepresent study (one on Long Island, and two in Shandaken), and only one of thesewas infected with herpetomonads. Farther south, however, in New Jersey and
Maryland, Holmes found that the majority of specimens harbored flagellates.

Morphology in Insect Host.--The forms found in the insect (Figs.
5, 9, 17) have relatively narrow bodies, the anterior end is somewhat
rounded, and the body gradually narrows to a point at the posterior
end. The flagellum originates near the parabasal body, which isround or oval and is situated closer to the anterior end than to the
nucleus. In most specimens the flagella are about the length of thebody, perhaps longer. When the organisms are moving swiftly inforward motion, the flagellum vibrates rapidly; in less active specimens
it has an undulating, serpentine motion and may sometimes be alternately coiled into loops and stretched out. None of the flattened or
twisted ribbon-like bodies so characteristic of plant herpetomonads
has been encountered. Occasionally short, broad, pear-shaped
forms have been observed, some of which have long and others very
short flagella.
Cultural Properties.--Once obtained in culture, the organism iseasily grown on the semisolid leptospira medium or on any of the blood
agar slants, such as the N.N.N. or N6ller's medium, which have been
widely employed for the cultivation of leishmanias and insect flagellates. On leptospira medium the growth becomes visible within 48~8 Werner, H., Arch. Protistenk., 1909, xiii, 19.
29 Holmes, F. O., Biol. Bull., 1925, xlix, 323.

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HERPETOMONADS AND LEISHMANIAS.

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hours at room temperature as a grayish white, usually homogeneous
mass, which gradually extends deeper into the medium. At 37°C.
growth can be recognized macroscopically after 24 hours. On blood
agar slants grayish, dew-like, moist and shining colonies become
visible within 72 hours at room temperature and 48 hours at 37°C.
They gradually spread and become confluent, and the layer of growth.assumes a light grayish color. The colonies are of a sticky consistency, and a thin grayish pellicle may form on the surface of the condensation water.
The morphological features vary greatly according to the medium
on which the organism is grown. In young colonies on the surfaceof blood agar slants (Figs. 6,a, 10,a) most of the individuals are pearshaped, oval, or truncated at the anterior end, with a short flagellum
which moves constantly from side to side. The flagellum arises far
in the interior of the cell, the parabasal body lying at one side of thenucleus. After some days the organisms become aggregated into
rosettes of varying size, the flagella being directed inward, the bodies
becoming uniformly narrower, pyriform, or spindle-shaped, and theflagella longer. In the condensation water much longer, slenderer
forms are found, with flagella as long as the body.
The flagellates grown on leptospira medium are of elongate form,
with flagella usually the length of the body (Figs. 6,b, 10,b). Unusually long forms are found in cultures which have been kept at room
temperature more than 2 or 3 weeks, and these resemble the specimensof the organisms in the insect host. The parabasal body occupies aposition anterior to the nucleus, the anterior portion of the organism iswide and rounded, and the body narrows down to a slender, pointed
posterior portion. These long forms with well developed flagella travel
about freely and swiftly in all directions, in contrast to the short truncated forms, with vibrating flagella, which rotate swiftly but do nottravel out of the microscopic field.
Numerous bizarre forms appear in media having a pH near 5. In
some the cytoplasm is divided except at the anterior portion, where asingle flagellum arises, the division beginning near the anterior portion
or at any point farther toward the posterior end. There may be one,
two, or several clefts in a single organism, and the lobes are usuallyunequal in size. It is difficult to interpret this phenomenon, which

HIDEYO NOGUCHI AND EVELYN B. TILDEN

317

has also been observed u n d e r n a t u r a l conditions in the insect host, b u t
it a p p e a r s to be an a b n o r m a l multiple division.

Strain 2 (from. Oncopeltus sp.t).--Figs. 9, 10. Another species of Oncopeltus(Fig. 2) was obtained by Dr. Tel~maco S. Battistini in the vicinity of Lima. Its
plant host is also a species of Asclepias. (The specimens of milkweeds which
Dr. Battistini collected, however, proved to be free from flagellates.) The initial
impure culture of this flagellate was purified by the Barber method. No morphological differences could be detected between this strain and the one derived
from Oncopeltus fasciatus.
Strain 3 (from Lyg~eus kalmii No. /).--Figs. 17, 18. Lygseus kalmii (Fig. 3)
resembles O. fasciatus in appearance, and its geographic distribution is somewhat
similar. In the Catskill Mountains (Shandaken), at an elevation of 1000 feetabove sea level, Lyg~eus kalmii far exceeded O. fasciatus in number. Four of thetwenty-four specimens collected harbored flagellates, and in two instances cultivation was successful. While the two strains were morphologically indistinguishable in culture, they proved subsequently to be distinct species, one of which
is identical with H. oncopelti.
Strain 4 (from Asclepias syriaca No. 213).--The occurrence of herpetomonads
in Asclepias syriaca (Fig. 30) was first reported by Holmes,3° who regarded theorganisms found by him as identical with H. elmassiani of Migone, describedby Franfa. 31 Of two strains of flagellates isolated from two infected milkweeds
collected in Shandaken, N. Y., one proved to be H. oncopelti. The initial growth
was obtained on leptospira medium and was not detected until several weeks
after inoculation. The culture was purified by plating.
Strain 5 (from Asclepias nivea).--Figs. 13, 14. Through the kindness of Dr.
Francis O. Holmes, of the Boyce Thompson Institute for Plant Research, a plantof the species Asdepias nivea, a Haitian milkweed, was placed at our disposal.
It had been infected by allowing infected specimens of Oncopeltus fasciatus tofeed on the seed pods. The morphology of these flagellates in the latex (Fig. 13)
is similar to that of H. oncopelti in Ascleplas syriaca (Fig. 30). They are characteristically ribbon-like, with twisted bodies, dear, almost hyaline protoplasm,and short flagellum. In culture (Fig. 14), however, they cannot be distinguishedfrom the culture forms derived from hemipterans. The initial culture was pure,and, as in the case of all the plant strains, was obtained on leptospira medium.
Only one tube of twelve inoculated yielded growth.
Herpetomonas lyg~eorum, n. sp. (Figs. 11, 12, 19, 20).
Strain 1 (from Lyg~us kalmii No. 2).--As has already been mentioned, cultures of herpetomonads were obtained from two insects of the species LyMeus
S°Holmes, F. 0., Phytopathology, 1924, x.lv, 146.
81Fran~a, C., Ann. Soc. belge reed. trop., 1920-21, i, 245.

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ITERPETOMONADS AND LEISI:IMANIAS.

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kalmii. The preparations made from suspensions of the intestinal tract show thatthe forms in Lygaeus No. 2 were a trifle narrower and longer than the majorityof those in Lygaeus No. 1, and had somewhat longer flagella. The morphologicalcharacteristics of the two cultures, however, are very much the same (Figs. 18,
20). The two species can be distinguished only by serological and fermentation
reactions.
Strain 2 (from Asclepias syriaca No. 46).--Figs. 11, 12. The impure cultureof this strain was discovered several weeks after inoculation of leptospira medium
with the plant latex. Purification was accomplished by the Barber technique.
In the plant (Fig. 11) and also in culture (Fig. 12), this organism is indistinguishable from the strain from A. syriaca No. 213, except for slight difference in size,
but serologically and in fermenting properties it is identical with H. lygaeorum.

Herpetomonas sp.? from Oncopeltus cingulifer (Figs. 7, 8, 33, 34).
Oncopeltus cingulifer (Fig. 4) feeds on the latex of Asclepias curassavica, a
Honduran milkweed, in which flagellates were first found by Hegner31 The
relation between the insect strain and that of its plant host could not be ascertainedin this instance, since pure cultures were not obtained. The impure growth was
detected 75 days after inoculation on one of several dozen plates which had been
similarly inoculated and kept under identical conditions. The flagellates of
O. cingulifer differed in appearance from H. oncopelti in having a considerably
longer flagellum.
Herpetomonas sp.? from Asclepias curassavica (Figs. 15, 16, 31, 32).
An impure culture of this organism was obtained on a semisolid (leptospira)medium containing glucose, inulin, and tapioca. The flagellates in the culture(Figs. 16, 32) bore rather slight resemblance to the forms found in the latex(Figs. 15, 31) and were much longer than those of the pure cultures isolated subsequenfly from American and Haitian milkweeds (Figs. 12, 14). Their morphological features do not correspond with those of the cultural forms (impure) of
H. elmassiani obtained by Migone from Aranjia angustifolia in Paraguay, as
described by Franga. sl

Herpelomonas culicidarum, n. sp. (Figs. 21, 22).
Through the cooperation of Dr. Mark F. Boyd, of Leesburg, Georgia, 23 wewere able to secure an impure culture of a Herpetomonas from Anopheles quadrimaculatus on a blood sugar slant (pH 5). The strain was purified by plating
on similar medium. The original film preparation made by Dr. Boyd (Fig. 21)
contained numerous flagellates of truncated and spindle shape, with short flagella.
The flagellates in culture (Fig. 22) were considerably longer, in this instance, thanthose in the original material. In all other instances the reverse was true.

ttlDE¥O NOGUCHI AND EVELYN B. TILDEN

319

Strain from Culex pipiens.--Small numbers of herpetomonads were found in
the intestines of two larvm of Culex pipiens, collected in New Jersey, and fromone of them a culture was obtained on acid blood agar plates. The immunological and fermenting properties of this strain are identical with those of the flagellate from A. quadrimaculatus.
Herpetomonas muscidarum, n. sp. (from Musca domestica) (Figs.
23, 24).
Many of the house flies collected about the grounds of The Rockefeller Institute harbored herpetomonads, some of them very large forms with long active
flagellum, in many instances double, as described by yon Prowazek. 32 A sectionof intestinal tract from one of the infected flies, in which the flagellates wereswarming as in culture, was picked out intact with a fine pipette, washed, and thecontents suspended in 0.5 per cent saline. Inoculation of acid blood agar withascending dilutions of this suspension yielded some pure colonies.
While the cultural properties of this organism are very much the same as thoseof the other cultivated strains of the series, in morphology it is slightly different.
In young cultures on leptospira medium (Fig. 24, a) the individuals are no larger
and the flagella no longer than in the case of H. oncopeltiand H. lyg~eorum,but after
several weeks in this medium extremely long forms appear, with flagella notunlike those of the organisms in the original material (Fig. 24, b).

Herpetomonas media, n. sp. (from Calliphora No. 1) (Figs. 25, 26).
This culture, which was obtained by the Barber method, is a pure line strain.
Morphologically this organism resembles H. muscidarum, but it never attains
the size of that strain, even in old cultures, and it is immunologically distinct.

Herpelomonas parva, n. sp. (from Calliphora No. 2) (Figs. 27, 28).
This culture also represents a pure line strain. I t is decidedly smaller thanthe other two strains from flies. On blood agar slants the colonies of media
and parva are light gray, heavier than those of muscidarum, and show less tendency to coalesce.

Herpelomonas ctenocephali; Trypanosoma rotatorium; the Leishmanias.
Morphologically, H. ctenocephali is quite unlike any of our strains. In its
leaf-like form it resembles rather the flagellates found in the plant latices, though
the flagellum is much longer. The frog trypanosome is again entirely different
from any of the other flagellates studied. On the other hand, the morphologicalfeatures of the leishmanias, while distinctive, approach those of the culture
a2 von Prowazek, S., Arb. k. Gsndhtsamte, 1904, xx, 440.

320

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LEISHM.ANIAS.

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forms of our insect and plant flagellates; they are pyriform and stocky whengrown on the surface of blood agar slants, but characteristically longer andslenderer on leptospira medium, and have longer flagella.
The flagellates of the dog flea and frog grow very slowly, 1 to 2 weeks being
required for the growth on blood agar slants to become visible. The leishmanias
grow somewhat more rapidly, but much more slowly than the plant and insect
strains. All the strains studied show more rapid and prolific growth on leptospira medium than on plates or slants.
Table II is a comparison of certain morphological features of theflagellates studied. Preparations of cultures grown on leptospiramedium were used in making the measurements of culture forms,
except in the case of Leishmania brasiliensis; in this instance the stocky
forms obtained from a blood slant culture were measured.
The preparations used as a basis of comparison were all made by
fixing dried films in methyl alcohol and staining with Giemsa's solution. The early part of the work was not done under laboratory conditions, and this method was the only one which could be conveniently
carried out. Later the cultures were studied by cytological methods(wet fixation in Schaudinn's sublimate alcohol, followed by staining
with iron-hematoxylin or Giemsa's solution, with subsequent differentiation), but since these preparations furnished no additional information regarding the structure of the organisms and were notavailable in all instances, they have not been especially mentioned
in the present study.
SUMMARY.

Nine strains of herpetomonads have been isolated in pure culture
from eight varieties of insects, and three strains from two species ofplants. Four of the cultures were derived from latex-feeding insects(Oncopeltus fasciatus, Oncopeltus sp. ?, Lyg~eus kalmii) and three fromlatex plants (A sclepias syriaca, A sclepias nivea) ; two from mosquitoes(Culex pipiens and Anopheles quadrimaculatus), one from the house fly(Musca domestica), and two from bluebottle flies. In addition impure
cultures have been obtained from Oncopeltus cingulifer and from its
plant host, Asclepias curassavica.
The flagellates cultivated, all of which belong to the genus tlerpetomonas, have been identified chiefly by their biological relationships,

HIDEYO NOGUCHI AND EVELYN B. TILDEN

323

which will be described in detail in Part I I of this report. The sevenstrains from latex-feeding insects and latex plants represent two distinct species, which have been designated H. oncopelti and H. lyg~eorum.
The two strains from mosquitoes proved to be the same organism andhave been called Herpetomonas culicidarum. The culture obtained
from Musca domestica contained larger individuals than those of any
other strain, and the organism is morphologically distinct from eitherof the Calliphora strains. None of the fly flagellates cultivated could
be identified with t h e species II. muscae domestic~e or H. calliphor~e,and hence they have been given new names, Herpetomonas muscidarum, H. media, and H. parva.
Blood agar plates were used for initial cultivation of the strainsfrom insects and the semisolid leptospira medium for isolation of theplant flagellates. A number of the strains were purified by plating onacid blood agar, a procedure which reduces considerably the growth ofbacterial contaminants. The Barber technique was utilized for
isolation of the flagellates from flies, because of the very large numberof bacteria found with them in these insects, and, in one or two instances, for the purification of impure cultures. Once they have beenobtained in culture, all the strains grow well on leptospira medium,
as well as on blood slants. Growth takes place both at 26°C. andat 37 °.
The morphology of the organisms is considerably modified by cultivation. This is especially true of the plant flagellates. In the latex
they have ribbon-like bodies, often twisted, and comparatively shortflagella; the protoplasm is clear, almost hyaline. The flagellates seenin the gut and feces of insects are usually large, slender organisms, withflagella as long as or even longer than the body, which contains numerous volutin granules in the cytoplasm. In cultures under parallel
conditions the flagellates from both these sources become shorter andthicker, the plant forms no longer appear flat and ribbon-like, and ingeneral the organisms approach one another in morphological features.
Even in the case of the least modified insect flagellates, i.e. those fromflies, there is never exact correspondence between the natural and thecultivated forms.
The morphological features of the cultivated flagellates vary according to the medium on which the organisms are grown and the age

324

HERPETOMONADSAND

LEISHMANIAS.

Iof the culture. The flagellates grown on the surface of blood slants
are pyriform, with truncated anterior portion, and short flagellum;in the condensation water, however, the individuals are elongatedand have long active flagella. On the leptospira medium the slenderactive forms with long flagella predominate. In the presence offermentable carbohydrate, or in medium containing considerable acid,peculiar bifurcated or multifurcated individuals are seen. Similar
forms have been seen under natural conditions. Cultures of Leishmania behave in the same way under the conditions described.
There is a striking difference in rapidity of growth between theorganisms isolated by us and the leishmanias, H. ctenocephaH, andT. rotatorium. While the stock cultures of the group first mentioned
multiply rapidly at 37°C., growth becoming visible within 24 hours,
the latter group grow scarcely at all at 37 ° and only slowly at 25 ° ,1 to 2 weeks being required for growth to become macroscopicallydemonstrable.
While the flagellum of the leishmanias, as also of H. ctenocephali,is long, serpentine in its movements, and heavy, having the appearance
of being enveloped by a sheath throughout its entire length, that of therecently isolated strains is thin, less flexible, and without the sheathlike appearance. The only exceptions to this rule are the flagellates
from Musca domestica and Calliphora No. 1, which have a long flagellum not unlike that of the leishmanias.
As the foregoing observations indicate, morphological differentiation of the flagellates studied, while not impossible, is subject to error
by reason of the variations due to age and cultural conditions. The
flagellates of the latex-feeding insects, the plants, the flies, and themosquitoes can readily be distinguished from Leishmania by theirrapid growth at 37°C., but their differentiation from one another ispossible only by serological and fermentation reactions.
EXPLANATION OF PLATES.
PLATE 10.
FIGS. i to 4. Latex-feeding insects, natural size. Fig. 1, specimen of Oncopeltus fasciatus caught on Long Island. Fig. 2, Oncopeltus sp. from Lima, Pera.
Fig. 3, Lyg~us kalmii, specimen from Long Island. Fig. 4, Oncopeltus cingulifer
from Honduras.

HIDEYO NOGUCHI AND

EVELYNB. T I L D E N

325

PLATES 11 TO 14.
FIGS. 5 to 28. Herpetomonads of insects and plants, as they appear undernatural conditions and in culture. Drawn from preparations stained with
Giemsa's solution after fixation by methyl alcohol. Magnification X 1200.
The species name and source are indicated in each instance, a, forms from~lant cultures, b, forms from cultures on leptospira medium.
PLATE 15.
Magnification × 1200.
FIG. 29. Herpetomonas davidi, original preparation made by Lafont, 83 presented by Professor Emile Brumpt. Giemsa's stain.
FIGs. 30 to 34. Drawings from preparations fixed in methyl alcohol andstained with Giemsa's solution. Fig. 30, flagellates from a specimen of A.
syriaca, a plant of the group found by Holmes3° in Maryland and studied byhim. Fig. 31, flagellates from the latex of Asclepias curassavica (Honduras).
Fig. 32, impure culture of flagellates from Asdepias curassavica. Fig. 33, flagellates from the gut of Oncopeltus cingulifer, which feeds on Asclepias curassavica.
Fig. 34, impure culture of flagellates of Oncopeltus cingulifer.

33Lafont, A., Compt. rend. Soc. biol., 1909, lxvi, 1011.

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIV.

PLATE 10.

(Noguchi and Tilden: Herpetomonads and leishmanias. I.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIV.

PLATE 11.

(Noguchl and Tilden: Herpetomonads and leishmanias, I.)

THE JOURNAL OF E X P E R I M E N T A L M E D I C I N E VOL. X L I V ,PLATE 12.

(Noguchi and Tilden; Herpetomonads and leishmanias. I.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIV.

PLATE 13.

(Noguchl and Tilden: I-Ierpetomonads and leishmanias. I.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIV.

PLATE 14.

(Noguchi and Tilden: Herpetomonads and leishmanias. I.)

THE JOURNAL OF EXPER M E N T A L M E D I C I N E VOL. X L I V .

PLATE 15.

(Noguchi and Tilden: He,'petomonads and leishmanias. I.)