Comparative analysis of biological aspects of Leishmania infantum isolates

Leishmania infantum infantum (LII) is one of the species that causes visceral leishmaniasis (VL) in the Old World, while L. infantum chagasi (LIC), and is present in the New World. Few studies address the biological differences, as well as the behaviour of these strains during infection. These parasites live inside the cells of their hosts, continuously evading the microbicidal mechanisms and modulating the immune response of these cells. One of the mechanisms used by these protozoa involves the L-arginine metabolism. Given the importance of the understanding of differences between Leishmania species, as well as establishing a better murine model to study leishmaniases, the objectives of this work were to analyse the biological and molecular differences between two Leishmania infantum strains (LII and LIC) and the degree of susceptibility of mice with different genetic backgrounds to infection, as well as to understand the role of arginase (ARG)/nitric oxide synthase (NOS) in the parasite-host relationship. The infectivity in vivo and in vitro of LII and LIC was performed in BALB/c and Swiss Webster mice, as well the NOS and ARG activities. The LII strain showed more infective than the LIC strain both in vivo and in vitro. In animals infected by both strains, a difference in NOS and ARG activities occurred. In vitro, promastigotes of LII isolated from BALB/c and Swiss Webster mice showed higher ARG activity than the LIC during the growth curve, however, no difference was observed in intracellular NO production by promastigotes between these strains. A comparison of the sequences of the ARG gene was made and both strains were identical. However, despite the similarity, the strains showed different expression of this gene. It can be concluded that although L. chagasi strains are considered identical to L. infantum strains, both have different biological behaviour.

lineages are used aiming to mimic human leishmaniasis manifestations [16] . 72 Resistance/susceptibility to Leishmania major infection has been associated with the 73 balance of Th1 and Th2 immune responses [17][18][19] ; however, for other Leishmania 74 species, such profile is not so clear-cut, hampering the study of the pathophysiology 75 of the disease and the development of novel drugs. BALB/c mice (inbred) are highly  Corraliza and collaborators (1994) [23] . Briefly, spleen and liver cells were previously 132 washed in sucrose and KCL solution and an anti-proteolytic buffer was added. After 133 cell lysis, L-arginine (0.5M) at pH 9.7 was added.  96h. The amount of urea produced by them was measured as previously described. mice infected by LIC (Fig 1B). Comparing LII and LIC, the former was always more 238 infective in both mice lineages, such difference being of about 3-fold in BALB/c mice 239 at 60 dpi. In the liver, although with lower amounts of parasite than in the spleen, 240 also higher parasite was detected in the case of LII infection (Fig. S1A). LIC when compared to LII, being the differences for the two parasite strains 262 statistically significant (p ≤ 0.05) at 60 dpi (Fig 1C) to LII one (Fig S1B). ARG was observed in both mice lineages infected by LIC or LII (Fig S1C). with significant differences from the 2 nd to 6 th days of culture (Fig 2). Although the four isolates presented the same growth profile, their 308 proliferation rates were different. So, the next step was to evaluate whether these 309 parasites present differences in infectivity, using the complement lysis test in  To confirm the hypothesis that LII is more infective than LIC, the four parasite 329 isolates from BALB/c and Swiss mice, were used to infect peritoneal macrophages 330 obtained from the same mice they were isolated from (Fig 3) and after 24 and 72 h, 331 the infection index was calculated.    Interestingly, the intracellular NO production in the four isolates showed no 367 significant differences, possibly because LII and LIC produce similar basal levels of 368 NO (Fig 4A). higher urea levels compared to LIC, demonstrating highest ARG activity (Fig 4B).

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Higher enzyme levels occurred in LII parasites with higher percentages of metacyclic 389 forms, suggesting that ARG activity may be related to the parasite infectivity. Since Swiss Webster) (Fig 4C), indicating that such differences may be associated with directly influence the course of the infection [29,30] . It is known that T-helper cells- Marques and collaborators (2015) [34] have reported that LII parasites were able to 445 survive high amounts of NO added to the culture.

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Regarding ARG activity, lower urea production was observed in both spleen 447 and liver of infected animals when compared to uninfected controls. However, spleen 448 cells from group BALB/c infected by LII showed higher urea production than of the 449 other infected groups. This higher ARG production may be correlated to higher host's immune response, which may be related to parasite virulence factors [27] .

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There are studies indicating that differences in infectivity between L. infantum strains 459 could be related to modulation by the parasites of the expression of L-arginine 460 metabolic enzymes during mouse infection [34] . Studies about differences between 461 parasites from different geographic regions are needed. Therefore, the present study  The data presented in this study indicate that the strains of L. infantum from 516 the Old and New World present different biological behaviours, although they are 517 considered identical by some authors. This demonstrates the importance of 518 conducting further studies on these differences, as this may assist in the search for 519 better alternatives to combat these parasites.

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It was also concluded that mice with different genetic backgrounds present

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The author(s) declare(s) that they have no conflict of interest to disclose 529 530