Experimental transmission and biological comparison of strains of Trypanosoma rangeli

EXPERIMENTAL

PARASITOLOGY

11,

Experimental of

1-9 (1961)

Transmission Strains

of

and

Biological

Trypanosoma

Comparison

rangeli

Eleanor Johnson Tohie’ U.S. Departnzent of Health, Education, and Welfare, Public Health Service, Institutes of Health, National Institute of Allergy and Infectious Diseases,’ Maryland (Submitted

for publication,

2 September

National Bethesda,

1960)

The infectivity of strains of Trypanosonza rangeli is compared under experimental conditions. The various strains react differently in the white rat, a vertebrate hcst, as well as in Rhodnius prolixus, an invertebrate host. The survival of T. rangeli in the bloodstream of the rat and infections in the intestine, hemolymph, and salivary glands of R. prolixus are discussed. Evidence suggests that R. prolixus may not be the natural invertebrate host for all strains of T. rangeli or that trypanosomes designated as T. rangeli actually represent more than one species. Passage through the insect host enhanced ability of one strain to invade the salivary glands following inoculation into hemocoele. Maintenance of the strain was thus possible by cyclical transmission.

Trypanosoma rangeli has been reported to be nonpathogenic to various vertebrate hosts including man (Groot, Renjifo and Uribe, 1951; Pifano, 1954; Zeledon, 1954; and Herbig-Sandreuter, 1957). Groot and his collaborators studied four human volunteers and observed no clinical manifestations except a slight local reaction at the site of inoculation in one. Herbig-Sandreuter investigated the possibility of the existence of tissue forms and after extensive histological analysis of serial sections of numerous white mice as well as of organs and tissues of mice and monkeys she concluded that T. rangeli can be of little importance as a pathogenic agent to man. She found no signs of tissue forms or histopathological changes. These findings are in agreement with those of Groot et aZ., who did not find parasites in the viscera or tissues of mice. On the other hand Grewal ( 1957) reports that T. rangeli is pathogenic to its invertebrate host, Rho&&s prolixus. The entire insect apparently is “blocked” by parasites interfering with the molting process, thus causing death prior to or during molting.

1 With the technical 2 Laboratory

assistance of Flora of Parasitic Diseases.

the the

Noticeable differences in the infectivity of several strains of T. rangeli for laboratory rats interested us in comparing these strains, principally in one of the natural invertebrate hosts, R. prolixus. It was thought that if T. rangeli was pathogenic to R. prolixus the virulence of a strain might be increased by passage through this host. MATERIALS

AND METHODS

Five strains originating in endemic areas and nine substrains, eight isolated in our were used. Colombian strains: laboratory,, Pereira, Molano, and Cancerosa, were received from Dr. J. Boshell in October, 1948. Pereira from the city of Villavicencio and Molano from Bogota were original strains isolated from humans. Cancerosa was produced in Colombia by inoculating a cancer patient with material from the Pereira strain. When received by us Molano and Cancerosa had been transferred two times in vitro and Pereira four times, making them one to two months old. Venezuelan strains: San Juan de 10s Morros (Strain 1) and El Tocuyo (Strain 2), isolated from humans, were received from Dr. Felix Pifano in July, 19.58. Cultures of both strains were from transfer sixty, making

C. Gilliam.

1

2

TOBIE

the strains approximately two years and four months old when received. The Panamanian strain, Caescende de Paula from Santa Rica was sent to this country by Dr. Carl Johnson. It was isolated from a patient in November, 1958, and a fifth transfer culture was received in February, 1959. The strains produced in our laboratory will be referred to by the letter and number given the experimental animal from which it was isolated

Infectivity

(for example W410) in addition to the strain from which it originated. Young nursling rats were used as the vertebrate host. In addition some weanling rats, a few adult rats, a few mice, and one dog were inoculated. Rhodnius prolixus nymphs and adults, usually females, raised in our laboratory were used as the insect host. All strains were maintained in the medium used in our laboratory for the cultivation of

TABLE I of Culture Strains of Trypanosoma

rangeli to Animals Inoculated

IP Number

infected

Age of culture years

Animal and its age

Pereira


3-g-Day rats Weanling mice Weanling rats S-Day rats J-Day rats

9/23 o/3 O/3 l/8 O/6

Pereira Ala


3-Day g-Day

2/5

l-2
Weanling mice S-11-Day rats 3-Day rats

214 3/14 O/3

l-2 1-2

Young dog 5-Day rats

2/2

<1 <1 l-2 2-3 > 10

3-Day mice Weanling mice S-Day rats d-Day rats g-Day rats

017 O/4 O/6 O/6 O/6

4-5 3-4

6-S-Day rats Adult rats

012

Venezuela Str. 1 D2b

<1

J-Day

O/7

Venezuela Str. 2

2-3 3-4 4-S

New-born mice Adult rats 6-&Day rats

<1
Adult rats S-Day rats

3/5

<1

6-g-Day

6/10

<1
Adult rats Weanling mice 6-Day rats

5/s 4/4

l-2

6-7-Day

9/g

Strain

Molano

Molano

J2a

Cancerosa

Venezuela Str. 1

Venezuela Str. 2 IV Venezuela Str. 2 W410c Panama


Panama ~42~

Rats rats

rats

6-1 l-Day

<1 5 Isolated from animals inoculated IP with culture material. b Isolated from animal inoculated IP with positive hemolymph. C Isolated from animals infected by bite of R. prolixus.

rats

rats rats

Number

inoculated

7/9

l/l

O/8

o/13

l/2 8/8 2/2

S/6

s/5

INFECTIVITY

OF VARIOUS TRYPANOSOMA

the lewisi group of trypanosomes (Johnson, 1947) except that the medium was enriched with 30 ml of blood per 100 ml of base. Transfers were made every 10 to 14 days. This same medium was used to determine infection in animals by culture methods. Animals were infected either by intraperitoneal inoculation (IP) or by allowing an infected insect to feed on an animal. It was often possible to detect an infection by examining a fresh drop of blood microscopically. However, when parasites were not found, 0.2 ml of blood from the animal was cultured on the medium mentioned above. Flagellates could be found as early as 5 days after inoculation of the media but 8 to 10 days was set for the first examination of these isolation cultures to allow time for some multiplication and thus avoid missing a positive culture. An animal was considered negative when 2 weeks after its blood had been introduced into media, flagellates had not developed. The insects, Rhodnius prolixus, were infected in two ways. To produce what we consider a natural infection they were fed on infected animals. To produce an infection by inoculation a drop of infected material was injected with a short 27-gauge needle into the hemocoele (HC) through the lateral margin of the abdominal segments (the connexivum). This technique was suggested to us by Dr. Felix Pifano. Adult triatomas were given a chance to feed at least every two weeks. Females were preferred because they engorged rapidly and defecated almost immediately. Nymphs usually fed only once between molts. Insects were fed and kept individually except when fresh first or second instar nymphs were fed on an infected rat. However, they usually were separated before there was a chance of infecting a rat so that each one could be followed individually. Salivary gland infections were proved by showing that the vertebrate host became infected or by microscopic examination of the glands. Intestinal infections were proved by examining fecal material microscopically. Before discarding triatomas as negative they were dissected and hemolymph, salivary glands, and intestinal contents examined microscopically. Legs were cut off to obtain hemolymph. The head was pulled away gently from the body bringing

3

STRAINS

the glands out from the thorax. The abdominal body-wall was cut away to reveal the intestine which was removed either intact or in sections. RESULTS

Infectivity

of Culture Strains of T. rangeli Animals Inoculated IP

to

Table I summarizes data on the infectivity of T. rangeli for animals inoculated IP with culture material. Differences are evident in the infectivity of the strains. For example the Panama strain infected all but one animal regardless of the age of the animal while the Cancerosa strain did not infect a single animal. When the original isolation culture of Venezuela Strain 2 W410 was used at 22 days the first four rats inoculated (four of the ten, Table I) were not infected. Parasites from the bloodstream when introduced into media rounded up. During the first days in vitro clusters of leishmania-like forms were seen (Fig. 1). Flagellate forms became evident later. These early forms may not have been infective and this may explain the four negative W”10 rats. Material from the first transfer later infected the remaining six rats. The Venezuela Strain 1 D2 culture was isolated from a rat infected by IP inoculation of hemolymph from a triatoma inoculated HC with Strain 1 culture material. This was also an original isolation culture, used at 24 days, and this may account for its lack of infectivity to rats. TABLE

II

Znfectivity of a Culture Strain of Trypanosoma rangeli to Rats during Successive Isolations in Vitro Culture strain Molano Molano Molano Molano

Age of culture* years

No. infected Litter

No. inoculated

l-2 J 2/6 J2 l-2 L 2/2 Ll l-2 s 9/9 S9 2-3 U 4/4 Molano U2 2-3 Y 3/9 Molano Y2 3-4 2 4/4 Molano 24 3-4 AA 4/4 Molano AA1 4-s BB 2/2 Molano AA1 4-5 cc 515 * See Table I. In this series the age of the original strain determines the age of the subculture strain.

4

TOBIE

It is apparently possible to extend the infectivity of a culture strain for vertebrates by repeatedly isolating a strain and using the newest in vitro strain for each succeeding inoculation into vertebrates. Using Molano as a parent strain we isolated and passed the infection seven times in succession (Table II). After three years of maintenance in this

way the strain infected seven out of seven rats while at the beginning it had infected only two of six. Survival of T. rangeli in the Bloodstream of the Rat When rats were infected by inoculation of culture material IP or by insect bite the

FIG. 1. Leishmania forms in ?-day isolation culture from rat infected by cyclical transmission. (Giemsa stain, 1000 X) FIG. 2. Dividing flagellate from bloodstream of rat inoculated with Tvypanosoma rangeli, Molano strain culture material; I-day infection. (Giemsa stain, 1000 x) FIG. 3. Adult trypanosome from bloodstream of rat inoculated with Trypanosonza range& Molano strain culture material; 3-day infection. (Giemra stain, IO00 X) FIG. 4. Trypanosoma rangeli from salivary glands of Rho&ins prolixus inoculated HC with Venezuela Strain 2 15 culture material. (Giemsa stain, 1000 X)

INFECTIVITY

OF VARIOUS TRYPANOSOMA

flagellates transformed into adult trypanosomes. During the first days of the infection a few trypanosomes showing evidence of division were found (Fig. 2). These were probably crithidial or metacyclic forms ready to divide when injected. After the process of transformation to adult trypanosomes (Fig. 3) is completed parasitemia remains at a very low level and eventually dies out.

Strains

of Trypanosoma

Of the first triatomas inoculated HC with the two strains from Venezuela, but not used for other studies, one inoculated with Strain 2 developed a salivary gland infection and passed the infection to a young rat while feeding on it. The culture substrain 15 from this rat gave us a strain which when inoculated HC into triatomas invaded the salivary glands. The infection was transmitted to rats by bugs infected in this manner as early as 11 days after inoculation and they continued to infect rats until they were sacrificed or died. One bug which died at 194 days had probed and tried to feed 6 days earlier, infecting the rat. Table III gives data on R. prolixus inoculated HC with nine strains of T. rangeli. Strain 1 from Venezuela and the strain from Panama were not capable of progressing to the salivary glands even though the Panama strain was a relatively “young” III

rangeli

to Rhodnius

prolixus

When

Survival .4ge of culture

Glands

invaded

Strain

years

> 10

O/7

lo/IO

2-3 3-4 4-5

O/5

6/6”

O/4 O/4

2/3 4/4

l/SC

7/7”

O/4 O/4

4/4 l/4 0

Venezuela

Str.

Str.

1

2

2-3 3-4 P5

No. checked

No. checked (Heavy)

When examined in days 7-133 4

(Rare)

8-64 10-58 4-14 44-77 11

Venezuela

Str. 2 ISa

<1

19/19

Venezuela

Str. 2 Lgn

<1

9/9

F

Venezuela

Str.

<1

4/4

e

3-20

O/7

o/24

l-55

2 W-‘lOn

l-2

Panama Panama

M4lb

<1

Panama

Y.‘2 b

<1

O/2

a Isolated from animals infected by bite. b Isolated from animals inoculated with culture material. c The one infected I5 referred to in text. (6 Hemolymph infected rats. e Hemolymph was not inoculated into rats since salivary gland infections

HC

Inoculated

in hemolymph

Glands not invaded

Cancerosa Venezuela

of the

Infectivity of Culture Strains of T. rangeli to R. prolixus when Inoculated HC

TABLE of Culture

5

total of 91 days in the bloodstream rat without significant multiplication.

Eight rats, infected with Venezuela Str. 2 15 by bite, and known to be positive, were sacrificed at intervals from 22 to 71 days. Two were negative at 43 and 50 days but the others, including three at 57, 64, and 71 days were positive. In another group of six infected in the same way and with the same strain all were negative when sacrificed at 59 days. In rats inoculated IP with culture material of the Pereira and Molano strains infections were demonstrated at 70, 79, and 86 days. Three weanling rats inoculated with infected blood were found positive on days 7, 36, and 54 respectively. The donor rat inoculated with culture material had been infected for 37 days. The parasites had thus persisted for a

Infectivity

STRAINS

O/l

41

O/7

16 Hours

developed.

6

TOBIE

strain. However, Venezuela Strain 2 substrains 1.5, L8, and W410 isolated from rats infected by bite, produced salivary gland infections in all of the triatomas inoculated. Some bugs reached a point where they were no longer able to feed. This undoubtedly was due to the heavy gland infection. Glands which were removed were filled with flagellates. They could be seen microscopically moving within the intact glands, which had lost some of their bright red color. A smear from an infected gland is shown in Fig. 4. Though Strain 1 and 2 from Venezuela and the Cancerosa strain were not able to progress to the salivary glands (except in one bug inoculated with Venezuela Strain 2) flagellates remained alive and in some cases multiplied in the hemolymph, while those of the Panama strain were destroyed within 24 hours (Table III). Substrains M41 and Y42 isolated from rats inoculated with the original Panama strain did not survive any better. The old strain, Cancerosa, developed heavy hemolymph infections, but the flagellates did not infect young rats. Three of the Venezuela Stain 2 W410 triatomas oozed hemolymph at the site of inoculation while feeding at 5, 13, and 20 days respectively. Flagellates were numerous and active. In a few insects, which did not develop demonstrable salivary gland infections, low grade gut infections were noticed. We can only speculate that perhaps a few flagellates got into a salivary gland and were picked up

Infectivity

of Trypanosoma

rangeli

to Rhodnius

while feeding. It is of course possible, in the case of these low grade intestinal infections, that a few parasites could have entered the digestive tube from the hemolymph by penetrating the gut wall. However, in the triatomas inoculated HC, where salivary gland infections developed, intestinal infections were never found until after the first time the infection was transmitted, indicating that the insect ingested its own parasites as it fed. To prove that this could happen a fresh insect and an infected insect were fed on a fresh rat at the same time. Intestinal infection in the originally non-infected triatoma was evident when the insect defecated following the next feeding, one to two weeks later. Infectivity of the Bloodstream Form of T. rangeli to R. pro&us when Ingested while Feeding Even though T. rangeli is presumably more virulent for R. prolixus, one of its invertebrate hosts, than for its known vertebrate hosts, the insects did not always develop intestinal infections when fed on an infected animal. When triatomas were inoculated HC with Venezuela Strain 2 substrains salivary gland infections developed readily (Table III). These insects transmitted the infection to young rats by bite and fresh insects in turn picked up the infection from rats thus infected, while feeding on them. In sixteen out of twenty female triatomas fed on rats infected in this way intestinal infections de-

TABLE IV prolixus When Ingested Rhodnius

Age of strain years

Strain Venezuela Str. Venezuela Str. Venezuela Str. Venezuela Str. Venezuela Str. Venezuela Str. Panama Panama Y2

2 2 IS

2-3
1

1 D2 2 2 W410

a Heavy parasitemia. b Rare parasites dying out.

Rats

fed on rats Inoculated IP with cultures No. inf./ No. inoc.

Days survival in intestine

< 74 134a

o/2 O/7

<1
firolixus

Infected by bite Days survival No. inf./No. fed in intestine 111 X/20

While Feeding on Infected

4-5

2/2 2/2

l-2

o/g

0

o/2

0

21 35b

INFECTIVITY

OF VARIOUS TRYPANOSOMA

veloped and were maintained in the gut for 134 days with no indication that they might die out (Table IV). In contrast to this it was noted that in triatomas fed on rats inoculated IP with Panama culture material no intestinal infection developed (Table IV). The response to a recently isolated subculture Y2 was the same. Triatomas were fed on animals inoculated IP with Venezuela Strain 1 and substrain D2 even though infection in the rats was not detected by microscopic examination of the blood, since occasionally xenodiagnosis will demonstrate an infection when other diagnostic methods fail. No infection resulted. When insects were fed on rats inoculated with culture material of Venezuela Strain 2 and substrain W*lO, intestinal infections of varying degrees developed but in those infected with the natural Strain 2 the parasites died out by 35 days. At the same time, in bugs infected with the substrain W*lO, parasites were present but extremely rare. In one bug only rounded-up, non-motile, disintegrating forms were seen. To see whether the forms in the intestine were really capable of developing further, ten of those with intestinal infections (15 strain, Table IV) and thirteen fresh triatomas were inoculated HC with positive fecal material from the 15 group. Salivary gland infections developed in all of them. The intestinal forms, however, were never found to be infective for rats when inoculated IP. Development

R. prolixus”

of a Strain of Trypanosoma Strain inoculated (date)

Development of a Strain of T. rangeli Capable of Being Maintained by Cyclical Transmission Table V summarizes data on the development of a strain which we have maintained by natural transmission. Triatoma 41F inoculated HC with 15 culture material infected rat Ul by feeding on it. Following this the strain has completed the natural cycle through four generations, alternating between R. prolixus and the rat. Young nymphs were used in the development of this strain since previous attempts to pass the infection through adults in a natural manner had been disappointing. In only one adult, 75F (Tables IV and V), was the cycle completed. Of forty young nymphs fed on rats infected by the four naturally infected triatomas used in maintaining the strain, only five were known to develop salivary gland infections. Four of the five were first instar nymphs and were infected during their first blood meal after hatching. One was a second instar nymph. Two died during molting before they could be used to continue the strain. Of the forty fed, others also died during molting, and at least some may have been infected. Almost all developed intestinal infections which they maintained as long as we observed them. Rats were infected by these nymphs for the first time between sixteen and twenty-four days. The day depended to some extent on when the nymphs molted and were fed. Bugs in which salivary gland

TABLE V rangeli Capable of Being Maintained HC

4F

Venezuela Str. 2 culture

41F

Venezuela Str. 2 I5 culture (1-9-59)

75F”

Pos. fecal material from 41F

Infected by feeding on rat number (date)

by Cyclical

Transmission

Passed infection to rat number (date) 15 (11-19-S)

(9-22-58)

(7-8-59)

7

STRAINS

Ul Ul

(3-4-59)

121N

K43

139N

S41 (2-8-60)

(12-11-59)

201N

L53

(3-2-59)

F31 (7-1-59) K43 (12-9-59) S*l

(l-27-60)

L53

(S-18-60)

(s-23-60)

0 F = adult female; N = nymph. b f5F was inoculated HC with fecal material from 41F before it was known that it had passed the infection to Fa1. It is doubtful that this had anything to do with the ability to pass the infection later.

TOBIE

8

infections were not demonstrated two to three weeks after the infective feeding never showed gland invasion later. They remained negative into the adult stage, which was as long as they were observed. I)lSCUSSION

The mode of transmission of T. rangeli by the bite of triatomas was suggested by Pifano and Mayer (1949) when they found flagellates in the fluid from the proboscis of R. prolixus which had been infected in what they term the “natural way.” The cycle of development of T. rangeli was studied and described by Rey-Matiz ( 1941)) Groot (1952; 1954), Pifano et al. (1948), Pifano, (1954), and Herbig-Sandreuter (1955). For a critical review of the literature see Zeledon (1954). The trypanosomes in the bloodstream of a vertebrate are ingested by the intermediate host. There is a period of development and multiplication in the intestinal canal before they pass into the hemocoele. After a few days of growth in the hemolymph they invade the salivary glands where they become metacyclic trypanosomes. In this report it is shown that various strains identified as T. rangeli react differently in the vertebrate host as well as in the invertebrate host. One strain survived in the bloodstream of the rat yet was killed off rapidly by the insect when inoculated HC and did not develop in the intestine. Other strains survived in the hemolymph of the insect yet were never detectable in the bloodstream of the rat. One strain multiplied in the hemolymph yet was not capable of invading the salivary glands or infecting the rat. In view of these observations it appears possible that either R. prolixus is not the natural invertebrate host for all strains of T. range2i or that trypanosomes designated as T. rangeli actually represent more than one species. We have compared intestinal infections, salivary gland infections, and hemolymph infections of T. rangeli in R. prolixus. The cycle of development of T. rangeli in the insect is not complete when the parasites are found only in the fecal material or in the hemolymph, but only when salivary gland invasion has occurred. An intestinal infection is sufficient for xenodiagnosis, but there is evi-

dence that all strains do not develop in the gut of R. prolixus. We do not know how the parasites pass from the intestine to the hemocoele. It has been suggested that there are tissue forms which break out of the intestinal wall. Rey-Matiz (1941) described intestinal forms in R. prolixus which he considered intracellular. Grewal (1957) did not find evidence of this. Passage through the insect host enhanced the infectivity of one strain so that salivary gland invasion regularly followed inoculation HC and thereafter maintenance of the strain was possible by cyclical passage. Even though the infectivity of this strain was enhanced, it still was difficult to infect R. prolixus nymphs and adults in the natural manner i.e. by feeding them on infected rats. T. rangeli does not complete its cycle of development in more than a small percentage of insects, partly because it increases the insect mortality. Grewal (1957) attributes the high mortality of infected R. prolixus entirely to the pathogenicity of T. rangeli. However, we find that regardless of rearing conditions there is considerable loss of uninfected insects in each stage due to inability to feed and to molt. Since so few insects become infected by ingesting infected blood under controlled experimental conditions, one is inclined to think there must be some other means by which so many (Groot, 1952; Pifano, 1954) become infected in nature. Reichenow ( 1957) considers T. rangeli a parasite of triatomid bugs with the vertebrate an accidental host and he speculated that the cyst-like forms found in the fecal material are those which transmit the infection from insect to insect, Pifano and his collaborators (1949) were not able to pass the infection from insect to insect by inheritance, coprophagy, or cannibalism. Thus it seems that the possibility of transmission from insect to insect needs clarification. REFERENCES M. S. 1957. Pathogenicity of Trypanosoma rangeli Tejera, 1920 in the invertebrate host. Experimental Parasitology 6, 123-130. GROOT, H. 1952. Further observations on TrypanoSoma ariarii of Colombia, South America. Ameri-

GREWAL,

can Journal 1, 585-592.

of Tropical

Medicine

and Hygiene

INFECTIVITY

GROOT, H. 1954. Estudios manos

clasificados

OF VARIOUS

sobre 10s trypanosomas coma

T. rangeli

hu-

con especial

referencia a su evolution en Rhodnius prolirus p a su comparacion con T. ariarii. Anales de la So&dad de biologia de Bogotd 6, 109-126. GROOT, H., RENJIFO, S., AND URIBE, C. 1951. TrypaMosonta ariarii, n. sp., from man, found in Colombia. American Journal of Tropical Medicine 31, 673-691. HERBIC-SA~TDREUTER,A. 1955. Experimentelle Untersuchungen iiber den Cyclus von Trypanosoma rangeli Tejera 1920 im Warmbliiter und in Rhodnius prolixus. Acta Tropica 12, 261.264. HERBIG-SAKDREUTER, .4. 1957. Further studies on Trypanosoma rangeli Tejera 1920. Acfa Tropica 14, 193-207. JOHKSON, E. M. 1947. The cultivation of Trypanosoma conorhini. Journal of Parasitology 33, 85. PIFANO C., F. 1954. Nueva trypanosomiasis humana de la region neo-tropica producida por el Trypanosoma rangeli, con especial referencia a Venezuela. Archives venezolanos de patologia tropical y parasitologia mldica 2( 2)) 89-l 20. PIFAXO C., F., AND MAYER, M. 1949. Hallazgo de formas el jugo

evolutivas de1 Trypanosoma rangeli de la trompa de Rhodnius proliws

en de

TRYPANOSOMA

STRAINS

9

Venezuela. drchivos venezolanos de patologia tropical y parasitologia mtdica l(2), 153-158. PIFANO C., F., MAYER, M., MEDINA, R., AND BENAIM PINTO, H. 1948. Primera comprobacion de Trypanosoma rangeli en el organism0 human0 por cultivo de sangre periferica. Archives venezolanos de patologia tropica! y parasitologia mtdica l(l),

1-31.

PIFANO C., F., MAYER, M., R~MER, M., DOMIPSGUEZ, E., MEDINA, R., AND BENAIM PINTO, E. 1949. Estado actual de las investigaciones en Venezuela sobre una nueva trypanosomiasis humana de la region neotropica producida por el Trypanosoma rang&. Archives Venezolanos de patologia tropical y parasitologia mCdica l(2), 135-152. REY-MAXZ, H. 1941. Observaciones sobre trypanosomas en Colombia. Revista de la facultad mCdicina, L’niversidad narional (Bogotci) 10, 23-49. REICHEP~OW, E. 1957. t;‘ber Trypanosoma rangeli und die Entwicklung des Blutparasitismus der SPugetiertrypanosomen. Zeitschrift fiir Tropenmedizin und Parasitologie 8, 219-224. ZELEDON, R. 1954. Tripanosomiasis rangeli. Rev&a de biologia tropical, Universidad de Costa Rica 2, 231-268.