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PAGES: 645-648 DOI: Full paper
Experimental Infection of Canine Peritoneal Macrophages with Visceral and Dermotropic Leishmania Strains

MF Madeira +, EGO Barbosa-Santos, MCA Marzochi

Departamento de Ciências Biológicas, Escola Nacional de Saúde Pública-Fiocruz, Rua Leopoldo Bulhões 1480, 21041-210 Rio de Janeiro, RJ, Brasil


A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell.

Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35oC.

The findings open the possibility of using canine peritoneal cells as a model for the screenning of leishmanicide drugs and to study the pathogenesis of these species.

key words:

Leishmanias are obligate intracellular parasites that multiply inside the cells of the mononuclear phagocytic system of vertebrate hosts. The multiplicity of species associated with transmission conditions by different vector species and host immune conditions generate variable clinical manifestations (Grimaldi Jr 1982, Marzochi & Marzochi 1994).

Changes introduced by humans to natural environments favor the installation of non-sylvan foci, thereby leading to the involvement of various species of domestic animals, mainly canids and equids (Marzochi 1992, Marzochi & Marzochi 1994), thus fostering the maintenance and expansion of peridomiciliary cycles.

The frequent presence of household dogs with high infection rates associated with the human disease in endemic areas for American tegumentary and visceral leishmaniasis, as well as the presence of the same genotypic pattern of the parasite circulating in human and canine populations prove the involvement of dogs in the peri and intradomiciliar transmission cycle of both diseases (Lopes et al. 1984, Pacheco et al. 1986). The same has been observed with regard to equine species in American tegumentary leishmaniasis (ATL) areas (Barbosa-Santos et al. 1994).

Thus, domestic dogs have been the target of several studies and are considered an excellent model for experimental infections (Chapman et al. 1979, Pirmez et al. 1988). However data concerning the treatment of disease in dogs are limited.

In attemps to evaluate experimental treatments various models have been used in studies with these protozoa. However, in vitro cellular models offer the greatest advantages for the objective at hand concerning the in vivo system, including both simplicity and speed in obtaining results (Berman 1985).

Macrophages, described as pioneering cells surviving in axenic conditions (Carrel & Ebeling 1922) have been used under different experimental conditions and obtained from different anatomical sites from various animal species. Such cells represent an ideal target cell population, mainly for studies of parasites from the Leishmania genus.

We have performed an in vitro investigation employing canine macrophages infected separately with three species ofLeishmania in order to monitor the multiplication kinetics and possible standardization for trials using drugs with leish-manicide properties.



Parasites - We used stationary-phase cultured promastigotes of L. (Viannia) braziliensis (MHOM/BR/95DCB-22), amastigotes of L. (L.) amazonesis (MCAN/BR/94DCB-16) and L. (L.) chagasi (MHOM/BR/95/DCB-27).

Macrophages - Macrophages were obtained from healthy adult male and female mongrol dogs kept at the Jorge Waistman Institute of Veterinary Medicine, under the Rio de Janeiro Health Secretariat. The health of the animals was established on the basis of results of a clinical examination and the evaluation for antibodies to Leishmania using the indirect immunofluorescence reaction. The dogs had their peritoneal cavity washed four days after stimulation with Sephadex G50 (Pharmacia) at a concentration of 70mg/kg body weight.

Dogs were anesthetized with an intravenous injection of sodium pentobarbital (Ceme, Brazil) at 25 mg/kg body weight. The peritoneal cavity was washed with a 500 ml volume of 0.85% physiological saline solution containing 0.3% sodium citrate, 100 U penicillin, and 100 µg streptomycin/ml. Approximately 80% of the lavage fluid was recovered, packed at 4oC, and immediately processed.

Isolation and infection of macrophages - The lavage fluid was concentrated by centrifugation at 800 g for 10 min at 4oC and resuspended in RPMI-1640 containing 100 U penicillin and 50 µg streptomycin/ml. Cell viability was determined using 0.2% trypan blue exclusion dye (Phillips 1973) and adjusted to give a cell density of 2x106 per ml, using a Neubauer hemocytometer in RPMI-1640 medium without serum.

Each milliliter of cell suspension was placed in a test tube (Vidrolabor 15cmx10cm) containing a coverslip. The tubes were incubated at 35oC for 2-3 hr. There after, the medium was removed and each tube was washed with RPMI-1640 medium to remove the non-adherent cells. After washing, RPMI-1640 was added, containing antibiotics and 10% sterile inactivated homologous canine serum.

After 24 hr in dishes, the cell monolayers were infected at a ratio of 5-10 parasites per host cell with incubation at 35oC when the infection was with L. (L.) amazonensis and L. (L.) chagasi and 34oC when the infection was by L. (V.) braziliensis for a period of 2-3 hr. After this period, the cell monolayers were washed, complete RPMI was added, and the respective temperatures were maintained. At 24 hr intervals the coverslips were washed in PBS, fixed in methanol, and stained with Giemsa. The percentage of infected cells was determined by individual counts on the slide covers under light microscopy, making a total of 100 cells.



The results of this investigation as shown in Fig. 1 indicate that 24 hr after infection the percentage of infected cells were 81% for L. (V.) braziliensis, 67% for L. (L.) amazonensis, and 90% for L. (L.) chagasi. These values incresead over the course of the study and by the sixth day post-infection they stood at 92%, 95%, and 98% of infected cells, respectively.

Fig. 2 shows peritoneal macrophages infected with L. (V.) braziliensisL. (L.) amazonensis and L. (L.) chagasi at 3, 5 and 6 day post-infection respectively. At least two different patterns of infection were observed during the experiment on macrophages infected by each Leishmania specie. The aspect of cells parasited with L. (L.) amazonensis is typical provoving the appearance of many vacuoles. By the way on the two others epecies the parasite multiplication is closer.



The use of canine peritoneal macrophages for in vitro studies has been sparsely documented, although different cell lines have been used experimentally in various investigations, displaying advantages and disadvantages (Mattock & Peters 1975, Chang 1980, Berman & Wyler 1980).

To our knowledge this is the first report concerning the utilization of canine peritoneal macrophages experimentally infected with three species of Leishmania, since the literature only includes utilization of these cells in association with infection by Ehrlichia canis (Nyindo et al. 1971, Stephenson & Osterman 1977). Lack of utilization of this in vitro cellular model may be the resulted the inherent difficulties associated with keeping larger animals as compared to smaller rodents.

The specific purposes of this investigation led us to develop a relatively simple technique for washing the peritoneal cavity of dogs, providing up to 109 cells on total volume, while utilization of special cannulae in this procedure (Zeman & Neri 1970) eliminates the need to sacrifice the animals.

In our study, the characteristics displayed by the cells cultured in vitro were similar to those described elsewhere, presenting single spherical and kidney-shaped nuclei located excentrically in the cell (Stephenson & Osterman 1977). However, in some cases there was not a good spreading of the cells, which may be linked to biological factors includind state of cell maturation and age and sex of the donor animals, factors which cannot be controlled by our methodology.

Numerous reports on the evaluation of the in vitro multiplication of different Leishmania species suggest a correlation with the in vivo system, e.g., where it is possible to observe the limited capacity of L. (V.) braziliensis to grow in murine macrophages, while L. amazonensis displays exuberant multiplication in this cell type (Zeledón et al. 1969, Grimaldi Jr et al. 1983).

Actually the utilization of two forms, amastigotes and viable promastigotes could be start and to follow up the macrophage infection by Leishmania (Berman & Lee 1984). In this study it was used to infect the canine cells a pool of metacyclic and non-metacyclic promastigotes harvested from stationary phase culture of L. (V.) braziliensis and amastigotes of L. (L.) amazonensis and L. (L.) chagasi and the three species clearly behaved differently with regard to their multiplication rate. The L. (L.) chagasi displayed greater capacity for multiplication than the two others (data not show).

So in order to make sure if the macrophage infection by L. (L.) chagasi really display greater affinity for canine cells as seen in this study a specific experiment is being realized with only Leishmania metaciclic promastigotes forms.

Our results support the hypothesis that these three organisms behave differently, both in their morphology and location in the host cell, showing characteristic patterns and the formation of large vacuoles as observed in the murine cells infected by L. (L.amazonensis (Grimaldi Jr et al. 1983).

In addition the standartization of this efficacious system open perspectives to study the role of this animal as an important reservoir for both visceral and mucocutaneous leishmaniasis by L. (V.) braziliensis, besides the avaliation of new efficient drugs and/or natural products with potential leishmanicide activity.



To Roger Magno Macedo Silva for technical assistance.



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This work was supported by grants from the Oswaldo Cruz Foundation (Papes/Fiocruz 0250250378).