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1 Research International Journal of Integrative Biology A journal for biology beyond borders ISSN Infectivity of Leishmania infantum treated with amphotericin B plus Phlebotomus salivary gland in BALB/c mice Carla Maia, Nuno Rolão, Mónica Nunes, Lenea Campino * Unidade de Leishmanioses, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal Submitted: 15 Apr. 2009; Accepted: 28 May 2009 Abstract Amphotericin B (AMB) has been widely used in visceral leishmaniasis treatment in Europe, namely in Leishmania/HIV co-infections and paediatric cases. However, only few studies on resistance to AMB are available so far. The purpose of this work is to anticipate the possibility of emerging AMB-resistant isolates in nature. BALB/c mice were intradermally (ID) inoculated with L. infantum wild-type strain and AMB in vitro treated parasites in presence or absence of Phlebotomus perniciosus salivary gland. The course of infection was followed-up during 12 weeks. Parasite load and cytokine expression were determined. Specific humoral and cellular responses were also evaluated. Our results confirm that ID inoculation of L. infantum in mice causes visceral infection and all infected mice developed a mixed immune response i.e. pro and anti inflamatory responses. No significant difference in the parasite load was observed among infected groups, showing that treatment of parasites with AMB and the presence of saliva did not affect their infectivity on the vertebrate host. However, the presence of AMB treated parasites in the skin of infected mice allow to suggest that AMB resistant strains could arise and be transmitted in nature as a result of the use of AMB on human and canine leishmaniasis therapy. Keywords: Leishmania infantum; Amphotericin B; Phlebotomus perniciosus; saliva; BALB/c. INTRODUCTION Visceral leishmaniasis (VL) is a systemic, vector-borne parasitic disease caused by obligate intracellular protozoa belonging to the Leishmania donovani complex. Parasites are transmitted by phlebotomine sand flies between mammalian hosts. Sand fly saliva has been shown to result in disease exacerbation, probably due to modulation of the immune response to favour parasite survival and replication (Rohousová and Volf, 2006). Treatment options for VL vary among regions but are generally limited to only a few drugs: pentavalent antimonials, amphotericin B (AMB) and recently, miltefosine (Gradoni et al., 2008). AMB has activity against Leishmania due to its higher affinity for ergosterol, the predominant sterol in the parasite membrane. Although this drug has been widely used in * Corresponding author: Lenea Campino, MD, Ph.D. Unidade de Leishmanioses, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa,Portugal Campino@ihmt.unl.pt VL treatment following failure of antimonials treatment and in HIV co-infection cases, there are only few studies on the emergence of AMB resistance (Croft et al., 2006). Secondary non-responsiveness has been reported in immunocompromised patients co-infected with HIV/Leishmania who relapsed after treatment with this drug (Alvar et al., 2008). A further pressure is the insistence to treat L. infantum-infected dogs with AMB. These dogs often remain infected despite repeated treatment and, therefore provide ideal conditions for selection of drug resistance since the dogs are the main reservoir of L. infantum to humans (Al-Mohammed et al., 2005). Although it is widely accepted that resistance to the disease in murine models of L. major infection is correlated with expansion of Th1 cells and production of IFN-γ, and that susceptibility is associated with the development of Th2 cells resulting in production of IL- 4 and IL-10 as well as TGF-β by infected mononuclear cells (Belkaid et al., 2002), in experimental VL a mixed Th1/Th2 immune response was observed (Rolão et al., 2007). The purpose of this work is to anticipate the possibility of emerging AMB-resistant isolates in the field. For that, the course of L. infantum wild-type and AMB treated parasites plus Phlebotomus perniciosus International Journal of Integrative Biology IJIB, 2009, Vol. 6, No. 3, 105

2 salivary gland infection by intradermal inoculation of BALB/c mice was followed-up. MATERIALS AND METHODS Parasite strains Leishmania infantum MON-1 (MCAN/PT/05/IMT373) wild-type (WT) and AMB treated line R160 were used. R160 was obtained by subculturing WT promastigotes in media containing increasing concentrations of AMB (Fugizone, Bristol-Myers Squibb, Portugal). Every concentration of AMB was maintained until the parasite number was equivalent to the one exhibited in control cultures (without drug). Metacyclic promastigotes were prepared as described by Howard et al. (1987). Phlebotomus perniciosus salivary gland lysate (SGL) Phlebotomus perniciosus colony was maintained in the animal house of the Faculty of Science, Charles University, Prague. SGL was prepared as described by Rohousová et al. (2005). Salivary glands were isolated from non-blood-fed, 5-10 days old females, dissected out in cold Tris buffer (20 mm Tris, ph 7.6) and stored at -70ºC until needed. Mice and infection A total of 96 female BALB/c mice with 6-8 weeks of age were purchased from Harlam Interfauna Ibérica SL (Barcelona, Spain) and housed at the Instituto de Higiene e Medicina Tropical (IHMT) of Lisbon, under stable climatic and dietary conditions. Animal manipulation was approved by the Ethics Committee of the IHMT, Veterinary Authorities and followed the guidelines of the Portuguese Legislation (Lei nº92/95, 12.9). Mice were divided in four groups, each with 24 animals, anaesthetized with 150 mg of ketamin (Imalgene 1000, Rhône Mérieux, Portugal) and 15 mg of xylazin (Rompun, Bayer, Portugal) and inoculated intradermally in the pinna of the right ear with 10 7 metacyclic promastigotes WT (group WT); coinoculated with 10 7 metacyclic promastigotes WT plus SGL (group WSG); co-inoculated with 10 7 metacyclic promastigotes R160 plus SGL (group RSG) and control mice (group C) were co-inoculated with 0.9% physiological solution plus SGL. Sample collection Mice were followed-up for 12 weeks (84 days). At each time-point (D7, D14, D28, D42, D56 and D84), four animals per group were sacrificed and, skin from right and left ears, draining lymph nodes, spleen, liver, femoral bone marrow, peritoneal macrophages and peripheral blood were sampled. Spleen and liver samples were macerated before use in the different assays. Spleen homogenates (after removing 400 μl for DNA and RNA extraction) and peritoneal macrophages were pooled in order to achieve the optimal concentration of mononuclear cells to use in lymphocyte proliferation and nitric oxide measurement assays. All other samples were analysed individually. Sera were processed for anti-leishmania antibodies analysis. Parasite load Parasite load determination was accessed by real-time TaqMan PCR (qrt-pcr), as described by Rolão et al. (2004a). Briefly, skin from ears, lymph node, spleen and liver macerates, bone marrow and peripheral blood from each sacrificed mouse were processed for DNA extraction (PCR-template Preparation kit, Roche Diagnostics GmbH, Germany) and for quantification (GeneQuant, Amersham Biosciences, UK). Mass cultures of L. infantum promastigotes were used to construct the standard curve ranging from 10 5 to 1 parasite. The diluted parasite cultures were processed for DNA extraction, as above and mixed with DNA from healthy mice. Immune response Humoral response Parasite specific antibodies were detected by counterimmunoelectrophoresis (CIE) performed as described previously (Maia et al., 2008). Cellular response The cellular immune response was analyzed by the production of nitric oxide (NO) by peritoneal macrophages and by in vitro lymphocyte proliferation. To determine NO production, peritoneal macrophages were cultured in flat bottom 96 well tissue culture plates (DeltaLab, Spain) at a concentration of 2x10 5 cells/well and incubated for 48 h in the presence of 40 μg/ml of soluble Leishmania antigen (SLA), obtained by freeze-thaw cycles and homogenization, or in the absence of exogenous stimuli, in a humidified atmosphere at 37ºC and 5% CO 2. Supernatants were collected as a source of secreted NO which was quantified by determining the nitrite concentration by the Griess reaction according to the manufacturer s instructions (Griess reagent system, Promega, USA). The nitrite concentration was calculated from a standard curve generated with NaNO 2. A concentration higher than 2 mm was considered indicative of macrophage activation. International Journal of Integrative Biology IJIB, 2009, Vol. 6, No. 3, 106

3 Parasites/μg DNA Parasites/μg DNA Parasites/μg DNA Infectivity of L. infantum treated with amphotericin B plus Phlebotomus saliva in BALB/c mice The lymphocyte proliferation assay was done according to Rolão et al. (2007). Proliferation responses to SLA or to the mitogen concanavalin A (Con A) were expressed as the cell proliferation index (SI), calculated as the coefficient between the scintillations per min(cpm) of stimulated cells and the cpm of nonstimulated cells.. A SI 1.5 was considered indicative of a positive cellular response WT Days post infection To determine cytokine and inducible nitric oxide synthase (inos) relative expression, total RNA was extracted from each ear, lymph node, spleen and liver macerates, peripheral blood and bone marrow by using the RNA extraction kit, (Roche Diagnostics GmbH). Total RNA was reverse transcribed (RT) into cdna using 200U M-MLVRT (Gibco, USA), at 37 º C for 60 min in the presence of 3 mm of 5x M-MLV RT buffer (250 mm Tris HCl, ph 8.3, 375 mm KCl, and 15 mm MgCl2) (Gibco), 10 mm BSA (Boehringer, Germany), 1 mm dntps (Gibco), 40 U rrnasin ribonuclease inhibitor and 10X oligo (dt) (Promega). The samples were then heated 10 min at 95 º C for RT inactivation and cooled to 4 º C. qrt-pcr was performed using commercialised primers from Applied Biosystems (USA) for IFN-γ (Mm ), IL-4 (Mm ), IL-10 (Mm ), TNF-α (Mm ), TGF-β (Mm ) and inos (Mm ). Relative quantification of the target was analyzed using the comparative threshold cycle and the value for each sample was normalized using glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (Mm ) as housekeeping gene. After this normalization, the expression of each target from each infected group was related to the values of control group. Statistical analysis Statistical analysis was performed using the SPSS 16.0 software (SPSS Inc., USA). Nonparametric Friedman test was used to compare: (i) the parasite load in the different tissues from the different infected groups; (ii) lymphocyte proliferation and NO production in the four groups. The correlation between the kinetics of parasite load and cytokine expression was determined by Spearman s rank correlation analysis. Differences were considered statistically significant when p values < RESULTS Parasite load The course of infection was followed for 12 weeks. Leishmania DNA was detected in the three infected groups at D7 (Fig. 1). At this time-point, only in mice from WSG group parasite DNA was observed in the internal organs (liver, spleen and bone marrow). In the other two infected groups there was only local Left ear Lymph node Liver Spleen Blood Bone marrow (a) WSG Days post infection Left ear Lymph node Liver Spleen Blood Bone marrow (b) RSG Days post infection Left ear Lymph node Liver Spleen Blood Bone marrow (c) Figure 1: Course of infection in WT (a.), WSG (b.) and RSG (c.) groups during experimental period. WT: mice group inoculated with Leishmania infantum wild-type parasites; WSG: mice group co-inoculated with L. infantum wildtype parasites plus one salivary gland lysate; RSG: mice group coinoculated with L. infantum amphotericin B treated parasites plus one salivary gland lysate. dissemination (left ear and regional lymph node). All groups presented a sterile phase (e.g. without detection of parasites) at D14 and D28. Visceralization of infection in RSG and WT was observed at D42 and D56, respectively. At D56 only on animals from RSG group Leishmania DNA was detected in the skin. After visceralization bone marrow presented the highest parasite load in the three infected groups. No significant difference in the parasite load of internal organs was observed between infected groups throughout the observation period. At D84 no parasites were detected in all animal groups. Parasite DNA was not detected on samples from Group C during the 12 weeks. International Journal of Integrative Biology IJIB, 2009, Vol. 6, No. 3, 107

4 Immune response Humoral response No anti-leishmania antibodies were detected by CIE during the whole study. Cellular response Throughout the 12 week course of infection, spleen cells from control and infected animal groups were tested for the capacity to respond to in vitro stimulation with SLA or ConA. All groups proliferated in the presence of the mitogen (data not showed). At D14 only splenocytes from groups co-inoculated with SGL had a positive proliferation to SLA, while 28 days after infection an antigen-specific response was observed in the WT group (Fig. 2). Spleen cells from mice of group C showed no detectable antigen-specific lymphoproliferative responses throughout the whole study. Figure 2: Proliferative response of spleen cells from WT, WSG, RSG and C groups to soluble Leishmania antigen. C: mice from control group co-inoculated with 0.9% physiological solution plus one salivary gland lysate; WT: mice group inoculated with Leishmania infantum wild-type parasites; WSG: mice group coinoculated with L. infantum wild-type parasites plus one salivary gland lysate; RSG: mice group co-inoculated with L. infantum amphotericin B treated parasites plus one salivary gland lysate. Figure 3: Nitric oxide production by macrophages from WT, WSG, RSG and C groups stimulated with soluble Leishmania antigen during experimental period. C: mice from control group co-inoculated with 0.9% physiological solution plus one salivary gland lysate; WT: mice group inoculated with Leishmania infantum wild-type parasites; WSG: mice group coinoculated with L. infantum wild-type parasites plus one salivary gland lysate; RSG: mice group co-inoculated with L. infantum amphotericin B treated parasites plus one salivary gland lysate. To detect if macrophages had been activated, the production of nitrites was measured with the highest amount (4 mm) being observed at D14 for all infected groups (Fig. 3). The cells of these groups also produced NO at D28 and D56. Macrophages from group C showed NO 2 mm throughout the study. In all infected groups there was a mixed immune response with concomitant expression of proinflamatory, anti-inflamatory and regulatory cytokines (Fig. 4 [Supplementary data]). During observation period, IFN-γ, TGF-β, IL-4, IL-10 and TNF-α expressions by bone marrow cells from WT mice group was statistically different among them (p<0.05). TNF-α was the most expressed cytokine. Moreover, IFN-γ expression was positively correlated with IL-4 and IL-10 (rs=1) (Table 1 [Supplementary data]). IL-4 expression was positively correlated with IL-10 (rs=1). With the exception of IL-4 and IL-10, the expression of cytokines by hepatocytes was statistically different (p<0.05). There was a positive correlation between INF-γ and IL-10 and between TNF-α and IL-4. IL-10 expression by spleen cells was positively correlated with TGF-β (rs=1). IL-4, TNF-α and inos presented identical expression kinetics throughout the study. TNF-α expression by blood cells presented a positive correlation with IL-4 (rs=1). These two cytokines presented similar kinetics to TGF-β (p=0.051). There were no statistical differences or correlation between the expression of the different cytokines and inos in both ear and regional lymph node. In WSG group there were no statistical differences between the expression of the different cytokines and inos in the studied organs. However, in liver cells there was a positive correlation between IL-10 and both IL-4 and TGF-β (rs=1). The positive correlation between IL-10 and IL-4 and between TNFα and inos was also observed in splenocytes (rs=1). IFN-γ expression by blood cells was positively correlated with IL-4 (rs=1). In RSG group there were no statistical differences between the expression of the different cytokines and inos in the studied organs. TNF-α expression by liver and spleen cells presented a negative correlation with TGF-β and IL-10 (rs=1). In hepatocytes IL-10 expression was positively correlated with IL-4 and IFN-γ (rs=1). In blood cells, the kinetics of IFN-γ was positively correlated with IL-4 kinetics (rs=1). TNF-α was the most expressed cytokine in all the organs analyzed at the end of the study. There were no statistical differences in cytokines and inos expression among infected mice groups neither significant correlation between cytokines and parasite load. International Journal of Integrative Biology IJIB, 2009, Vol. 6, No. 3, 108

5 DISCUSSION Intraperitoneal and endovenous inoculation routes are the most used in experimental VL infections (Rolão et al., 2004b; 2007), however, subcutaneous (SC) and intradermal (ID) inoculation are the ones that most mimic natural infection in which parasite is transmitted by the bite of a sand fly. Melby et al. (1998) infected BALB/c mice with 5x10 6 L. donovani amastigotes inoculated SC in the hind footpad. Parasites were detected in the draining popliteal lymph node over an 8-week observation period, but there were no detectable parasites in internal organs. A similar result was obtained by Streit et al. (2001) after the SC inoculation of 10 7 L. chagasi promastigotes in the back of the mice. Spleen cells from these infected animals had a positive proliferation response to SLA with the development of a Th1 response with concomitant IFN-γ, IL-12 and inos expression and with supression of TGF-β production. Those authors suggested that the inoculation route was probably responsible for the polarization of the immune response and subsequently control of infection. In the present study, the selection of 10 7 promastigotes per mouse was due to the use of this concentration in most of the VL experiments (Streit et al., 2001; Ahmed et al., 2003; Rolão et al., 2007) so the application of a different parasite number would have limited the comparison of results. Animals from the group coinoculated with the wild-type strain plus salivary gland lysate (WSG) visceralized one week after inoculation. This early parasite spreading to visceral organs could be related with the presence of sand fly saliva since in the animals inoculated with the same strain without saliva (WT) parasites were only detected in internal organs later. Other authors (Ahmed et al., 2003) observed parasites in the liver and spleen of infected animals one week after ID co-inoculation of saliva plus L. infantum not treated with AMB. The co-inoculation of saliva with parasites has been shown to result in disease exacerbation probably due to the modulation of immune response to favour parasite survival and replication (Titus and Ribeiro, 1988; Rohousová and Volf, 2006). Our results showed that although dissemination to internal organs happened earlier in coinoculated animals, there were no significant differences in parasite load on the infected mice between the three groups. The same result was obtained in the only two similar studies done so far by Ahmed et al. (2003) and Paranhos-Silva et al. (2003) on mice and dogs co-inoculated with L. infantum and L. chagasi ( syn. L. infantum) plus P. perniciosus or Lutzomyia longipalpis saliva, respectively. The observation of a lack of a significant effect of salivary components on infection may reflect population variation in the ability of specific sand fly colonies to cause enhancement of infectivity as suggested by Warburg et al. (1994). Infection of different animal models with natural infected sand flies, or with different concentrations of saliva and/or secretory promastigote gel may be required to determine if sand fly saliva components can exarcebate the visceralization or establishment of Leishmania infection (Ahmed et al., 2003; Rogers et al., 2002). As far as we are aware, the present paper is the first work that followed-up a murine infection with L. infantum species treated with drug (AMB) plus P. perniciosus saliva. Mbongo et al. (1998) used a non common inoculation route (retroorbital sinus route) to infect BALB/C mice with 10 8 AMB resistant L. donovani (without saliva) but parasites were only observed in the liver and spleen of mice infected with the wild-type strain (control group). In the present study, the presence of saliva seemed to have facilitated the dissemination of parasites to internal organs, but the infection with AMB treated strain plus saliva visceralized later than the infection with the wild typestrain plus saliva. This delay in visceralization was also observed by Al-Mohammed et al. (2005). These authors concluded about the slower multiplication and dissemination of AMB-resistant parasites in comparison with control group. The negative results of PCR on liver and spleen samples from mice sacrificed at D14, D28 and D84 were not due to the heterogeneous distribution of the parasites since whole organs were macerated and homogenized prior to DNA extraction. A possible reason could be the persistence of parasites in other tissues not analysed. Throughout the experimental infection there was no detection of specific anti- Leishmania antibodies in mice sera from the four groups. Paranhos-Silva et al. (2003) had similar results after inoculating ID Beagle dogs with L. chagasi. Although those authors did not observed specific antibodies in animal sera during whole study, Leishmania DNA in internal organs was detected. These findings are consistent with observations made in human and canine natural infection where anti- Leishmania antibodies were undetectable. Our results related with spleen cells from infected mice showed a transient positive Leishmania-specific lymphoproliferative response. This observation differs from those where suppression of proliferative response to SLA has been reported in L. infantum experimentaly infected rodents (Riça-Capela et al., 2003; Rolão et al., 2007). It was verified a positive correlation between IL-4 and IL-10, in hepatic cells from WSG and RSG mice, spleen cells of WSG and bone marrow cells of WT mice. The association of the expression or production of these cytokines has been described by others (Miralles et al., 1994; Wilson et al., 2002) in murine International Journal of Integrative Biology IJIB, 2009, Vol. 6, No. 3, 109

6 experimental infections. Although for some authors L. major infection triggered a Th2 response associated with the inhibition of Th1, in our work there was a positive correlation between IL-4 (Th2) and INF-γ (Th1) in the peripheral blood from WSG and RSG and in bone marrow of WT mice. The co-existence of these cytokines has been associated with the regression of symptoms in human VL (Liew and O Donnell, 1993). In blood and hepatic cells from WT mice there was also an association between IL-4 and TNF-α. Both cytokines are crucial for the resolution of hepatic infection through the formation of granulomas: TNF-α is essential for leucocyte recruitment to the liver and IL-4 is responsible for the priming of CD8+ T cells that are critical for long term protection (Stanley and Engwerda, 2007). The simultaneous production of IFNγ and IL-10 by hepatic cells of RSG and WT as well as by bone marrow cells from WT observed here, has already been described in human and canine VL (Sundar et al., 1997; Lage et al., 2007) and probably indicates a possible regulation of the immune response in controlling the potential damage from the increased expression of pro-inflammatory cytokines (Belkaid et al., 2002). In hepatic and spleen cells from RSG it was verified a negative correlation between TNF-α and TGF-β. This regulatory cytokine could have been the responsible for the highest parasite load observed in the spleen of the RSG mice. As showed in other studies (Wilson et al., 2002; Melby et al., 1998; Rolão et al., 2007) the highest expression of TGF-β was generally observed in the days with higher parasitism suggesting that this cytokine favors parasite multiplication, probably through deactivation of macrophage leishmanicidal activity and inhibition of the development of a Th1- type immune response. Experimental murine infections with L. donovani and L. infantum have been showed the co-existence of IFN-γ expression and production with high parasitism (Miralles et al., 1994; Wilson et al., 1996; Ahmed et al., 2003; Rolão et al., 2007). IFN-γ is responsible for macrophage activation and consecutive parasite destruction through inos expression and NO production (Farrell, 2002). inos expression by spleen cells from the infected groups was higher when Leishmania DNA was detected. The highest inos expression in the RSG group could be related to the highest parasite load observed in this group. The increased expression of inos by the spleen and lymph node of mice infected with L. donovani was also obtained by Melby et al. (1998). Carvalho et al. (1992) associated the absence of IFN-γ in blood from L. infantum infected children with progression of infection to active disease. In our study, INF-γ was expressed in the blood suggesting that the presence of INF-γ in blood can be used as a prognostic value of the course of infection. Since TNF-α is one of the cytokines responsible for macrophage activation, the reason for the absence of parasite DNA in the different tissues at D84 was probably due to the fact that this cytokine was the most expressed among the organs analysed. As observed by others (Melby et al., 1998; Rolão et al., 2007; Stanley and Engwerda, 2007) in this study a Th1/Treg or Th2/Treg or Th1/Th2 responses were developed in the different tissues /organs from infected mice groups. Taking into account that cytokines expression was organ-specific, the evaluation of immune response to Leishmania infection should be determined locally. The detection of Leishmania in the skin of the mice inoculated with AMB treated parasites associated with the increase use of this drug on human and canine leishmaniasis therapy allow us to hypothesize that AMB resistant strains could arise and be transmitted in nature. Acknowledgement We thank P. Volf, V. Volfova and J. Hostomska from Charles University, Prague and J. Cristóvão, M. Marques, S. Henriques, E. Cabrita from IHMT, for technical support. This work was supported by EU/FEDER, POCI/CVT/56357/2004 from Fundação para a Ciência e a Tecnologia (FCT), Portugal. Carla Maia (SFRH/BPD/44082/2008) is a fellowship from FCT. References Ahmed S, et al. (2003) Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis. Infect. Immun., 71: Al-Mohammed H, et al. 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