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Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis.

Cunha J, Carrillo E, Sánchez C, Cruz I, Moreno J, Cordeiro-da-Silva A - Parasit Vectors (2013)

Bottom Line: In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development.The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection.This work is supported by other studies that present the parasite's inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.

ABSTRACT

Background: Leishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infective Leishmania species and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity of Leishmania strains in the outcome of visceral leishmaniasis.

Methods: In this study we have characterized four strains of L. infantum in terms of molecular typing, in vitro cultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence by in vitro and in vivo infectivity in a murine model of visceral leishmaniasis.

Results: Molecular typing unraveled a new k26 sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain. In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loads in vitro that were corroborated with the in vivo assays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection.

Conclusion: Differences in in vitro and in vivo infectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite's inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis.

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Leishmania-specific humoral response.Leishmania-specific sera reactivity of naive and infected animals 2 and 6 weeks post-infection was analyzed by ELISA. Specific (A) IgG2a and (B) IgG1 were quantified and are depicted as means ± SD of one representative experiment of 2 independents. Statistically significant differences are pointed out as given by one-way ANOVA followed by Dunnett’s multiple comparison test.
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Figure 7: Leishmania-specific humoral response.Leishmania-specific sera reactivity of naive and infected animals 2 and 6 weeks post-infection was analyzed by ELISA. Specific (A) IgG2a and (B) IgG1 were quantified and are depicted as means ± SD of one representative experiment of 2 independents. Statistically significant differences are pointed out as given by one-way ANOVA followed by Dunnett’s multiple comparison test.

Mentions: The enlargement in the B cell population explains the elevated titers of anti-Leishmania antibodies quantified in the chronic infection by HL (Figure 7). Both IgG2a and IgG1 were generated in high levels, leading to the exacerbation of the pathology, as described by others [46-48]. However, in E390M infections we did not detect any B cell expansion, as measured at 2 weeks post infection and later at 6 weeks, though IgG2a and IgG1 were significantly increased compared to age-matched naïve mice. We speculate that this antibody production could be in part related to the k26 gene. Leishmania k26 (also known as HASPB) protein, as well as SHERP that share the same locus on chromosome 23 [49], are stage-regulated proteins, expressed only in the mammalian host infective forms (metacyclic promastigotes and amastigotes) [50]. k26 has a central core composed of a 10–11 amino acid repeats (PKEDGHTQKND/PKEDGRTQKN in Additional file 3: Figure S2) which present high inter and intra-specific variability [49,51,52], a feature that makes it an interesting tool for molecular typing of different Leishmania species and strains [21]. Other than this, k26 is a highly immunogenic antigen [49] with proven efficacy as a vaccine in murine models of visceral leishmaniasis by L. donovani[51,53] and partial efficacy in canine leishmaniasis by L. infantum[54]. Its immunogenic properties make k26 an interesting antigen that has been studied for diagnostic purposes [55-57]. As we reported in this work, E390M k26 protein has more repeats than MON-1 strains, which may influence the type and the strength of the humoral response as those amino acid repeats were determined to be B cell epitopes [52]. We point towards this argument since Bibiano and ST strains, that proved to be more infective than E390M, were not able to produce specific antibodies nor increased splenic cellularity in the acute or the chronic phases of murine VL.


Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis.

Cunha J, Carrillo E, Sánchez C, Cruz I, Moreno J, Cordeiro-da-Silva A - Parasit Vectors (2013)

Leishmania-specific humoral response.Leishmania-specific sera reactivity of naive and infected animals 2 and 6 weeks post-infection was analyzed by ELISA. Specific (A) IgG2a and (B) IgG1 were quantified and are depicted as means ± SD of one representative experiment of 2 independents. Statistically significant differences are pointed out as given by one-way ANOVA followed by Dunnett’s multiple comparison test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3649922&req=5

Figure 7: Leishmania-specific humoral response.Leishmania-specific sera reactivity of naive and infected animals 2 and 6 weeks post-infection was analyzed by ELISA. Specific (A) IgG2a and (B) IgG1 were quantified and are depicted as means ± SD of one representative experiment of 2 independents. Statistically significant differences are pointed out as given by one-way ANOVA followed by Dunnett’s multiple comparison test.
Mentions: The enlargement in the B cell population explains the elevated titers of anti-Leishmania antibodies quantified in the chronic infection by HL (Figure 7). Both IgG2a and IgG1 were generated in high levels, leading to the exacerbation of the pathology, as described by others [46-48]. However, in E390M infections we did not detect any B cell expansion, as measured at 2 weeks post infection and later at 6 weeks, though IgG2a and IgG1 were significantly increased compared to age-matched naïve mice. We speculate that this antibody production could be in part related to the k26 gene. Leishmania k26 (also known as HASPB) protein, as well as SHERP that share the same locus on chromosome 23 [49], are stage-regulated proteins, expressed only in the mammalian host infective forms (metacyclic promastigotes and amastigotes) [50]. k26 has a central core composed of a 10–11 amino acid repeats (PKEDGHTQKND/PKEDGRTQKN in Additional file 3: Figure S2) which present high inter and intra-specific variability [49,51,52], a feature that makes it an interesting tool for molecular typing of different Leishmania species and strains [21]. Other than this, k26 is a highly immunogenic antigen [49] with proven efficacy as a vaccine in murine models of visceral leishmaniasis by L. donovani[51,53] and partial efficacy in canine leishmaniasis by L. infantum[54]. Its immunogenic properties make k26 an interesting antigen that has been studied for diagnostic purposes [55-57]. As we reported in this work, E390M k26 protein has more repeats than MON-1 strains, which may influence the type and the strength of the humoral response as those amino acid repeats were determined to be B cell epitopes [52]. We point towards this argument since Bibiano and ST strains, that proved to be more infective than E390M, were not able to produce specific antibodies nor increased splenic cellularity in the acute or the chronic phases of murine VL.

Bottom Line: In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development.The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection.This work is supported by other studies that present the parasite's inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.

ABSTRACT

Background: Leishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infective Leishmania species and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity of Leishmania strains in the outcome of visceral leishmaniasis.

Methods: In this study we have characterized four strains of L. infantum in terms of molecular typing, in vitro cultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence by in vitro and in vivo infectivity in a murine model of visceral leishmaniasis.

Results: Molecular typing unraveled a new k26 sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain. In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loads in vitro that were corroborated with the in vivo assays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection.

Conclusion: Differences in in vitro and in vivo infectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite's inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis.

Show MeSH
Related in: MedlinePlus