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The golden hamster (Mesocricetus auratus) is commonly used as a promising model for Leishmania braziliensis infection developing skin-ulcerated lesions. However, different protocols using high concentration of parasites inoculated in the footpad result in severe clinical disease. Here, we further investigate the outcome of the site of infection and concentration of L. braziliensis parasites inoculated on the immunopathogenesis and clinical evolution. Initially, hamsters were infected in the ear dermis or hind footpad with a concentration of 1 × 10

Background Several infectious diseases are associated with hypothalamic-pituitary-adrenal (HPA) axis disorders by elevating circulating glucocorticoids (GCs), which are known to have an immunosuppressive potential. We conducted this study in golden hamsters, a suitable model for human visceral leishmaniasis (VL), to investigate the relationship of Leishmania (L.) infantum infection on cortisol production and VL severity. Methods L. infantum-infected (n = 42) and uninfected hamsters (n = 30) were followed-up at 30, 120, and 180 days post-infection (dpi). Plasma cortisol was analyzed by radioimmunoassay and cytokines, inducible nitric oxide synthase (iNOS), and arginase by RT-qPCR. Results All hamsters showed splenomegaly at 180 dpi. Increased parasite burden was associated with higher arginase expression and lower iNOS induction. Cortisol levels were elevated in infected animals in all-time points evaluated. Except for monocytes, all other leucocytes showed a strong negative correlation with cortisol, while transaminases were positively correlated. Immunological markers as interleukin (IL)-6, IL-1β, IL-10, and transforming growth-factor-β (TGF-β) were positively correlated to cortisol production, while interferon-γ (IFN-γ) presented a negative correlation. A network analysis showed cortisol as an important knot linking clinical status and immunological parameters. Conclusions These results suggest that L. infantum increases the systemic levels of cortisol, which showed to be associated with hematological, biochemical, and immunological parameters associated to VL severity., Author summary Visceral leishmaniasis (VL) is an infectious disease that is common in most tropical countries. VL has high morbidity and leads to death if not properly treated. In Brazil, Leishmania (Leishmania) infantum is the main causative agent of VL. Golden hamsters have proven to be a suitable model for VL. Despite the importance of hypothalamic-pituitary-adrenal (HPA) axis disturbances in infectious disease, few studies have addressed this issue in VL. In this study, we showed that L. infantum-infected hamsters present augmented levels of plasmatic cortisol in association with increased spleen parasite burden. Indeed, a strong positive correlation was observed between cortisol and biochemical parameters (AST/ALT/ALP) related to liver damage, as well as pro-inflammatory cytokines (IL-6 and IL-1β), anti-inflammatory cytokines (IL-10 and TGF-β), and the arginase enzyme that may favor the progression of infection. On the other side, cortisol was negatively correlated with leucocytes, except monocytes, and with IFN-γ and iNOS, which are involved in parasite-killing macrophage function. These results shed light on an unexplored aspect of VL pathogenesis, which is the importance of cortisol production in the disease-associated immune dysfunction.

The golden hamster is a suitable model for studying cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis. Immunopathological mechanismsare wellstablished inthe L. (L.) major-mouse model, in which IL-4 instructs a Th2 response towards progressive infection. In the present study, we evaluatedthe natural history of L. braziliensis infection from its first stagesup to lesion establishment, with the aim ofidentifyingimmunological parameters associated with the disease outcome and parasitismfate. To this end, hamsters infected with 104, 105,or 106 promastigoteswere monitored duringthe first hours (4h, 24h), early (15, 30 days) and late (50 days) post-infection (pi) phases. Cytokines, iNOS and arginasegene expression were quantified in the established lesions by RT-PCR. Compared to the 105 or 106 groups, 104animals presented lower lesions sizes, less tissue damage,and lower IgG levels. Basal gene expression in normal skin was high for TGF-β, and intermediary for TNF, IL-6, and IL-4.At 4hpi, no cytokine induction was observed in the 104 group, while an upregulation of IL-6, IL-10, and IL-4 was observed in the 106 group. At 15dpi, lesion appearance was accompanied byan increasedexpression of all assessed cytokines, markedly in the 105 and 106 groups. Upregulation of all investigated cytokines was observed in the late phase, although less expressive in the 104 group. IFN-γ was the depending variable influencing tissue damage, while IL-6 was associatedto parasite load. The network correlating gene expression and clinical and laboratorial parameters indicated inoculum-independent associations at 15 and 30dpi.A strong positive network correlation was observed in the 104 group, but not in the105 or 106 groups. In conclusion, IL-4, IL-6, IL-10, and TGF-β are linkedto L. braziliensisprogression. However, a balanced cytokine network is the key for an immune response able to reduce the ongoing infection and reduce pathological damage.

The golden hamster ( Mesocricetus auratus ) is a susceptible model to Leishmania ( Viannia ) spp.; however, available studies employ different infection protocols, which account for clinical and pathological presentation differences. Herein, L . ( V .) braziliensis preparations were standardized to contain 10 4 , 10 5 , or 10 6 parasites to determine an optimal inoculum that ensured cutaneous lesions without causing a disseminated infection in hamsters. Lesion development was followed for 105 days by size measurements, and skin, draining lymph node, spleen, and sera were investigated to check parasite load, spleen visceralization, cytokine expression, histopathological changes, and anti- Leishmania IgG levels. The lesion emergence time was inversely proportional to the parasite concentration in the inocula. Animals infected by 10 4 parasites presented nodular lesions, while those infected with 10 6 parasites often exhibited ulcerated lesions. The differences in the final lesion sizes were observed between 10 4 and 10 5 inocula or 10 4 and 10 6 inocula. High IFNG expression, anti- Leishmania IgG levels, and parasite load occurred independently of the inoculum used. A mild inflammatory skin involvement was observed in animals infected with 10 4 parasites, while extensive tissue damage and parasite spleen visceralization occurred with 10 5 and 10 6 parasites. These results indicate that inocula with different concentrations of parasites generate differences in the time of lesion emergence, clinical presentation, and systemic commitment, despite high and similar IFNG expression and parasite load. This suggests that a modulation in the immune response to different parasite numbers occurs in an early phase of the infection, which could dictate the establishment and magnitude of the chronic phase of the disease.

SUMMARYThe lack of an adequate model forLeishmania(Viannia)braziliensisinfection is a limiting factor for studying American tegumentary leishmaniasis (ATL). The golden hamster (Mesocricetus auratus) is a promising model because besides being highly susceptible to dermotropicLeishmaniainfection, the lesions are very similar to cutaneous leishmaniasis (CL) in humans. However, differentLeishmaniaisolates or species and/or protocols have resulted in different outcomes, whereas no study has evaluated the reproducibility ofL. braziliensisinfection in this model. The natural history ofL. braziliensisinfection in 34 hamsters was evaluated by using a single parasite isolate in 8 independent experiments under similar experimental conditions. Clinical, histological and immunological analyses were performed. The hamsters presented skin ulcers similar to those observed in ATL. The intra-experiment lesion increment tended to show an intermediary variance. Histological analysis of infected skins showed granulomatous reaction, scarce amastigotes, and Schaumann's bodies. Blood lymphocytes proliferated in response to leishmanial antigens. The severity of the infection was positively correlated to spleen weight, and the titres of anti-LeishmaniaIgG antibodies. Our findings indicate that the hamster is an appropriate model for immunopathogenesis studies of CL caused byL. braziliensis, supporting its use in clinical, vaccine and chemotherapy experimental protocols.

Background Cutaneous leishmaniasis (CL) is a neglected disease with a broad spectrum of clinical manifestations, ranging from small cutaneous nodules to severe mucosal tissue destruction. Leishmania (Viannia) braziliensis is the main species attributed to CL in the Americas. However, studies of experimental infection are limited in the murine model due to the self-resolutive pattern of the disease. Previously, our group demonstrated that the hamster model reproduces many of the clinical and histopathological features observed in humans. Herein, we standardized a RT-qPCR gene expression assay to evaluate a panel of immunological markers and a qPCR assay in order to quantify with high sensitivity and reproducibility the parasite load in skin lesions. Methods Hamsters were intradermally infected in the footpad with 105 promastigotes of L. (V.) braziliensis and 110 days post-infection skin lesions and popliteal lymph nodes were removed for RNA and DNA extraction, both from the same tissue fragment. Gene expression of IFN-ɣ, IL-10, TGF-β TNF, IL-4, IL-6, iNOS and arginase were measured using non-infected animal tissue as a calibrator. Parasite load was quantified from DNA extracted from lesions by qPCR targeting Leishmania kDNA and normalized by hamster GAPDH, using a SYBR Green-based absolute quantification methodology. Results A relative quantification RT-qPCR assay was standardized for the evaluation of mRNA levels from skin and lymph node samples of golden hamsters, with PCR efficiencies ranging from 92.3 to 116.4 %. In uninfected animals, higher basal mRNA levels in lymph nodes were observed for IFN-ɣ, TGF-β, TNF and IL-4 (111.4 ± 92.2; 5.6 ± 1.2; 5.3 ± 1.7; and 60.3 ± 26.8, respectively) in comparison to skin. In golden hamsters infected with L. (V.) braziliensis, an increase in the expression of all immunological markers evaluated was observed, ranging from 2.7 ± 0.2 for TGF-β to 1018.5 ± 809.0 for iNOS in skin lesions, and 2.4 ± 1.6 for TGF-β to 600.2 ± 666.4 for iNOS in popliteal lymph nodes. Interestingly, significantly higher levels of IFN-ɣ, TNF and IL-10 mRNA were observed in skin in comparison to lymph nodes, while a lower significant level of arginase mRNA was observed in skin. In parallel, parasite loads were quantified by qPCR from the skin lesions of infected animals, ranging from 27.0 to 6647.0, with a median of 553.4 (416.7–1504.0) parasites/mg skin equivalents, whereas lesion size varied from 0.3 to 3.1 mm. Despite the tendency of larger lesions to present higher parasite load, the correlation observed was not statistically significant. Conclusions In this study, we describe for the first time a sensitive, reproducible and cheaper molecular assay to quantify from the same tissue fragment the gene expression of immunological markers and the parasite load in skin lesions, observing a mixed profile of immune response in the hamster model infected by L. (V.) braziliensis. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1647-6) contains supplementary material, which is available to authorized users.

Background Previous results have shown that oral and intranasal administration of particulate Leishmania (Leishmania) amazonensis antigens (LaAg) partially protects mice against L. amazonensis infection. However, vaccination studies on species of the subgenus Viannia, the main causative agent of cutaneous and mucosal leishmaniasis in the Americas, have been hampered by the lack of easy-to-handle bio-models that accurately mimic the human disease. Recently, we demonstrated that the golden hamster is an appropriate model for studying the immunopathogenesis of cutaneous leishmaniasis caused by L. (Viannia) braziliensis. Using the golden hamster model, our current study investigated whether the protective effect of intranasal immunisation with LaAg can be extended to L. braziliensis infection. Methodology/Principal Findings Golden hamsters vaccinated with either two intranasal (IN) doses of LaAg (10 µg) or two intramuscular doses of LaAg (20 µg) were challenged 2 weeks post-vaccination with L. braziliensis. The results showed that IN immunisation with LaAg significantly reduced lesion growth and parasitic load as well as serum IgG and IgG2 levels. At the experimental endpoint on day 114 post-infection, IN-immunised hamsters that were considered protected expressed IFN-γ and IL10 mRNA levels that returned to uninfected skin levels. In contrast to the nasal route, intramuscular (IM) immunisation failed to provide protection. Conclusions/Significance These results demonstrate for the first time that the nasal route of immunisation can induce cross protection against L. braziliensis infection., Author Summary Leishmaniasis is a disease that is common in most tropical countries. In Brazil, the cutaneous form of the disease is highly prevalent, with approximately 28,000 new cases reported annually. L. (Viannia) braziliensis is the main causative agent of cutaneous leishmaniasis; however, vaccine studies against protozoans of the subgenus Viannia have been largely neglected, mainly due to the high resistance of most mouse strains to the infection. Here, the authors used the golden hamster, which is highly susceptible to dermotropic Leishmania spp infection. It was previously shown that oral and intranasal vaccination with whole L. (Leishmania) amazonensis antigens (LaAg) protected mice against L. amazonensis infection. In the present study, the authors investigated whether the protective effect of intranasal immunisation with LaAg can be extended to L. braziliensis infection using the golden hamster model. The results showed that intranasal immunisation with LaAg significantly reduced lesion growth and parasitic load as well as IgG and IgG2 serum levels. At the endpoint of the experiment, intranasally immunised hamsters that were considered protected expressed IFN-γ and IL10 mRNA at levels similar to those in uninfected skin. These data show that the use of a proper animal model and/or different vaccination strategies may facilitate the development of an effective vaccine against L. braziliensis.