The Haemoculture of Trypanosoma minasense Chagas, 1908

Trypanosoma minasense was isolated for the first time in blood axenic culture from a naturally infected marmoset, Callithrix penicillata, from Brazil. The parasite grew profusely in an overlay of Roswell Park Memorial Institute medium plus 20% foetal bovine serum, on Novy, McNeal and Nicolle medium (NNN), at 27°C, with a peak around 168 hr. The morphometry of cultural forms of T. minasense , estimates of cell population size and comparative growth in four different media overlays always with NNN, were studied. The infectivity of cultural forms to marmosets (C. penicillata and C. jacchus) and transformation of epimastigotes into metacycliclike forms in axenic culture in the presence of chitin derivates (chitosan) were evaluated.

Infections with T. minasense are usually scanty, and division stages have never been detected in the simian host. It does not infect triatomine bugs and nothing is known about the vectors in nature. Attempts to infect laboratory rodents and dogs have also failed (Hoare 1972). Rodhain (1937), Deane and Damasceno (1961) and Marinkelle (1966) were unable to cultivate this trypanosome in axenic culture media, such as Novy, McNeal and Nicolle (NNN), and previous attempts to cultivate this parasite from blood samples of marmosets and monkeys from several localities in southeastern and northern Brazil have also been unsuccessful (Lourenço-de-Oliviera, unpublished data).
This paper records the axenic culture of T. minasense in a modified "Roswell Park Memorial Institute -Novy, McNeal and Nicolle" (RPMI-NNN) medium.

MATERIALS AND METHODS
A specimen of the marmoset C. penicillata (No. 63), caught in Felixlândia (MG) in May 1991, was shown to be naturally infected with T. minasense following the examination of thick and thin blood smears. No other blood parasites were detected, and xenodiagnosis made with six nymphs of 3rd and 4th instar Rhodnius prolixus was negative. The parasite did not grow in blood-agar culture medium NNN/LIT .
The absence of T. cruzi and T. rangeli in the cultures was tested by the inoculation of 0.1 ml of the culture (10 6 parasites/ml) into ten 10 days-old albino mice. Their blood were examined (fresh and Giemsa-stained smears) from 7 to 20 days after inoculation. Then, all the inoculated mice were sacrificed and haemoculture (NNN + LIT) was made and the cultures microscopically examined during one month.
In addition, we tried to infect 30 nymphs of 3rd and 4th instar R. prolixus by feeding them through a membrane (Garcia et al. 1984) on sterile defibrinated sheep blood mixed with the same culture. The search for parasites in the bugs was conducted 5, 13 and 25 days after the blood meal.
A one month old C. penicillata marmoset (No. 71), born and reared in the laboratory and negative for trypanosomes, was inoculated intraperitoneally (IP) and subcutaneously (SC) with 0.5 ml of a 13 days-old sample (4th passage) of the original culture of T. minasense. The search for parasites in the marmoset was made by thin and thick Giemsa-stained blood smears from day 5 to day 30 post inoculation (p.i).
Two adult C. jacchus marmosets (No. 74 and 76) were injected (IP/SC) with 0.5 ml of the 2nd passage of the original culture, previously cryopreserved for one month in liquid nitrogen. The animals were submitted to blood examination (thin and thick smears), xenodiagnosis (six nymphs of 3rd instar of R. prolixus) and haemoculture (NNN + LIT) for 30 days.
A third adult C. jacchus marmoset (No. 77), was inoculated intravenously (IV) with 0.5 ml of the 2nd subculture of the original isolation of the trypanosome, previously cryopreserved for eleven months, and diluted after quick centrifugation with sterile saline. The animal was examined (thick blood smears) from day 7 to day 120 p.i. Another attempt to infect the same animal was made by injecting it (IV) with material from subcultures, diluted in PBS pH 7.2. The marmoset was then examined from the day 7 to day 15 p.i.
We have tried to stimulate the transformation of T. minasense epimastigotes into metacyclic-like trypomastigotes in axenic culture, using chitin derivates (chitosan) according to the method of Wallbanks et al. (1989), using three concentrations: 150, 300, 600 mg of chitosan/5 ml of RPMI. Parasites were sampled at roughly 24 hr periods, when fresh medium (RPMI) was replaced.
The estimates of cell population size were made by a direct count in a haemocytometer (Neubauer).

RESULTS
NNN medium overlaid with RPMI gave the best growth-rate of T. minasense (Table I).
The blood of all mice inoculated with T. minasense culture, their respective haemocultures and xenodiagnoses (gut contents and faeces) were negative. Based on these experiments we concluded that the culture was free from both T. rangeli and T. cruzi, and composed purely of T. minasense.
The epimastigotes and trypomastigotes found in the culture were quite distinct. Fig. 1  II show, respectively, camera-lucida drawings and the measurements of the most commonly detected cultural forms. We observed a great variety of forms in the culture of T. minasense, as follows: round epimastigotes with a long, free flagellum; short, long and slender epimastigotes; and large epimastigotes reaching up to 70.0 µm in length and 4.5 µm in width. Stout epimastigotes, sometimes provided with a free flagellum of up to 42.0 µm long were also seen; epimastigotes in binary fission; and long and short trypomastigotes, some of which appeared to be metacyclic forms.
The injected C. penicillata (No. 71) marmoset became infected, presenting the following parasitaemia (parasites/thick blood smear of 5 µl, according to Earle & Perez 1932): 5th day, 2 forms; 7th day, 18; 12th day, 28; 19th day, 18 and 21st day, 7. The blood stream parasites found in the 5th day were little thinner than the typical T. minasense trypomastigotes seen later on the 7th day. The peak of parasitaemia was on the 12th day after inoculation. No division stages were seen.
In NNN + RPMI, the isolate of T. minasense presented a maximum growth at 168 hr, with about 8.9 x 10 6 parasites/ml (Fig. 2).
The percentage of metacyclic-like forms detected in the cultures with added chitosan were periodically evaluated. The highest number of trypomastigotes was recorded after the first change of media, with 300 mg of chitosan/5 ml of RPMI (Table III).

DISCUSSION
Hoare (1972)  T. lambretchi is easily cultured in Tobie's blood-agar diphasic medium, with either human or rabbit blood, although development is slow (Lambrecht 1965, Marinkelle 1968). Pro-   Recently, we have failed to isolate T. minasense from two adult C. penicillata marmosets from Minas Gerais, using blood culture in four tubes with NNN + RPMI supplemented with 20% FBS. Its seems that the isolation and maintenance of T. minasense in these media is somewhat inconsistent and may depend on the phase of the infection at the time the culture is made. The haemoculture of T. minasense from the C. penicillata marmoset No. 63, for instance, was positive only in a second attempt made 17 months after the first negative one.
Actually, we have made several previous attempts to cultivate T. minasense from blood samples of infected marmosets from Minas Gerais (C. penicillata) and Rio de Janeiro (C. jacchus), in NNN blood-agar (Miles et al. 1980) and in NNN with overlay of LIT, brain heart infusion (BHI, Difco), RPMI and Schneider's. These either gave negative results or the culture was not successfully maintained. Other attempts with VERO cells with nutrient media such as LIT, RPMI or FBS have also been made. The longest maintenance of T. minasense in all these media (76 days) was obtained when the haemoculture was started in NNN + LIT and the flagellates later seeded in BHI, RPMI or Schneider's medium (Lourenço-de-Oliveira, unpublished data).   mastigotes, usually in clusters, were the stages most commonly found, but short, stumpy, slowly-moving epimastigotes and occasional trypomastigotes were also detected in T. lambretchi cultures (Marinkelle 1976). According to Marinkelle (1976), there have been no previous reports on the multiplication in vivo or in vitro of any other trypanosome belonging to the subgenus Megatrypanum of neotropical non-human primates. However, a strain of T. devei, from the tamarin Saguinus midas niger, was cultivated by Lanham et al. (1984) in blood-agar medium. The trypanosome grew profusely as long slender epimastigotes. This is the first description of T. minasense multiplying in axenic culture, and the description and illustration of the cultural forms presented here now allows a better characterization of this parasite outside the vertebrate host, as well as a comparison with, and differentiation from other Megatrypanum species, such as T. devei and T. lambretchi, as described by Marinkelle (1976) and Lanham et al. (1984), and other New World trypanosomes such as T. cruzi and T. rangeli (viz. Hoare 1972).
The culture of T. minasense also now permits biochemical analysis using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on a discontinuous SDS buffer system, in which the parasite has a quite distinct peptideme from T. rangeli, T. saimrii-like, T. cruzi and T. conorhini (Ziccardi 1995).