In Brazil, programs based on elimination of infected dogs have not curtailed the spread of visceral leishmaniasis (VL), suggesting that other reservoirs of infection exist. Persons with active VL can infect the sand fly vector, but in endemic areas, persons with asymptomatic infections, whose infectivity to sand flies is unknown, are far more numerous. In this study, a polymerase chain reaction-based assay detected kinetoplast DNA of Leishmania chagasi in the blood of eight of 108 asymptomatic persons living with patients with recently diagnosed VL. These eight persons had low or unmeasurable levels of IgG antibodies to Leishmania, demonstrating the insensitivity of serology for subclinical infection. All eight persons had positive leishmanin skin test results, as did 70% of persons living in households of persons with active VL. Even if a small proportion of such asymptomatic persons are infective to sand flies, they represent a formidable reservoir of infection in endemic areas.

The sensitivity and specificity of PCR, serology (ELISA) and lymphoproliferative response to Leishmania antigen for the detection of Leishmania infantum infection were evaluated in a cohort of 126 dogs exposed to natural infection in Brazil. For PCR, Leishmania DNA from bone-marrow was amplified with both minicircle and ribosomal primers. The infection status and time of infection of each dog were estimated from longitudinal data. The sensitivity of PCR in parasite-positive samples was 98%. However, the overall sensitivity of PCR in post-infection samples, from dogs with confirmed infection, was only 68%. The sensitivity of PCR varied during the course of infection, being highest (78–88%) 0–135 days post-infection and declining to around 50% after 300 days. The sensitivity of PCR also varied between dogs, and was highest in sick dogs. The sensitivity of serology was similar in parasite-positive (84%), PCR-positive (86%) and post-infection (88%) samples. The sensitivity of serology varied during the course of infection, being lowest at the time of infection and high (93–100%) thereafter. Problems in determining the specificity of serology are discussed. The sensitivity and specificity of cellular responsiveness were low. These data suggest that PCR is most useful in detecting active or symptomatic infection, and that serology can be a more sensitive technique for the detection of all infected dogs.

The failure of control programs for visceral leishmaniasis (VL) that depend on elimination of infected dogs suggests that other reservoir hosts may p

Leishmania (V.) braziliensis and Leishmania(L.) amazonensis are the most pathogenic agents of American Cutaneous Leishmaniasis in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including: localized cutaneous leishmaniasis (LCL^DTH+/++), borderline disseminated cutaneous leishmaniasis (BDCL^DTH±), anergic diffuse cutaneous leishmaniasis (ADCL^DTH-), and mucosal leishmaniasis (ML^DTH++++). It has recently been demonstrated, however, that while L. (V.) braziliensis shows a clear potential to advance the infection from central LCL (a moderate T-cell hypersensitivity form) towards ML (the highest T-cell hypersensitivity pole), L. (L.) amazonensis drives the infection in the opposite direction to ADCL (the lowest T-cell hypersensitivity pole). This study evaluated by immunohistochemistry the expression of Toll-like receptors (TLRs) 2, 4, and 9 and their relationships with CD4 and CD8 T-cells, and TNF-α, IL-10, and TGF-β cytokines in that disease spectrum. Biopsies of skin and mucosal lesions from 43 patients were examined: 6 cases of ADCL, 5 of BDCL, and 11 of LCL caused byL. (L.) amazonensis; as well as 10 cases of LCL, 4 of BDCL, and 6 of ML caused byL. (V.) braziliensis. CD4⁺ T-cells demonstrated their highest expression in ML and, in contrast, their lowest in ADCL. CD8⁺ T-cells also showed their lowest expression in ADCL as compared to the other forms of the disease. TNF-α⁺showed increased expression from ADCL to ML, while IL-10⁺and TGF-β⁺ showed increased expression in the opposite direction, from ML to ADCL. With regards to TLR2, 4, and 9 expressions, strong interactions of TLR2 and 4 with clinical forms associated with L. (V.) braziliensis were observed, while TLR9, in contrast, showed a strong interaction with clinical forms linked to L. (L.) amazonensis. These findings strongly suggest the ability of L. (V.) braziliensis and L. (L.) amazonensis to interact with those TLRs to promote a dichotomous T-cell immune response in ACL.

SUMMARY: In the present study, we assessed morphological changes and cytokine production afterin vitrointeraction with causative agents of American cutaneous leishmaniasis and compared the microglia and macrophage immune responses. Cultures of microglia and macrophages infected with stationary-phase promastigotes ofLeishmania(Viannia)shawi, Leishmania(Viannia)braziliensisorLeishmania(Leishmania)amazonensiswere evaluated 24, 48 and 72 h after interaction. Macrophages only presented the classical phagocytic process while microglia also displayed large cytoplasmic projections similar to the ruffles described in macropinocytosis. In the macrophage cultures, the percentage of infected cells increased over time, in a fashion that was dependent on the parasite species. In contrast, in microglial cells as the culture time progressed, there was a significant reduction in the percentage of infected cells independent of parasite species. Measurements of cytokines in macrophage cultures 48 h after interactions revealed distinct expression patterns for different parasites, whereas in microglial cultures they were similar for allLeishmaniatested species. Taken together, our results suggest that microglia may have a higher phagocytic ability and cytotoxic potential than macrophages for all investigated species. The robust response of microglia against all parasite species may suggest microglia have an important role in the defence against cerebral leishmaniasis.

We present here a cross-sectional study analyzing the IgG1 and IgG2 immune responses to natural canine Leishmania (L.) infantum chagasi-infection and their relationships with delayed-type hypersensitivity (DTH) in 50 mongrel dogs with previous positive serodiagnoses (IFAT-IgG) (56% with subclinical status [= apparently healthy] and 44% clinically sick), living in endemic areas for visceral leishmaniasis in the Brazilian Amazon. IgG1 and IgG2 responses were measured using commercial polyclonal antibodies in ELISA, while DTH was elicited by intradermal skin test using cultured promastigotes L. (L.) i. chagasi-antigen. Data analyses used Chi-square and Pearson's r coefficient (95% confidence interval). Regarding DTH and the clinical statuses of dogs, it was noted that 100% of the animals showing positive DTH (n = 8) were from the subclinical group, while 100% showing negative DTH were from the clinically sick group; higher IgG2 than IgG1 responses were observed in both clinical groups. However, when this comparison was made between the subclinical and sick groups, higher IgG1 responses were noted in the dogs from the sick rather than the subclinical group, while no differences were noted between the IgG2 responses in the dogs from both clinical groups. Additionally, we found lower IgG1 responses in dogs from the subclinical group showing positive DTH than in the dogs from the subclinical or sick groups with negative DTH; no differences were found between the IgG2 responses of these two clinical groups. These findings suggest that the IgG1, but not the IgG2, response is associated with susceptibility to canine visceral leishmaniasis (CVL). Nous présentons ici une étude transversale analysant les réponses immunitaires IgG1 et IgG2 à l'infection canine naturelle par Leishmania (L.) infantum chagasi et leurs relations avec une hypersensibilité retardée (HR) chez 50 chiens errants avec des sérologies positives antérieures (IFAT-IgG) (56 % avec statut sous-clinique [= apparemment en bonne santé] et 44 % malades cliniquement) vivant dans une zone endémique de leishmaniose viscérale dans l'Amazonie brésilienne. Les réponses IgG1 et IgG2 ont été mesurées en utilisant des anticorps polyclonaux commerciaux en ELISA, tandis que l'HR a été provoquée par un test cutané intradermique utilisant un antigène de promastigotes cultivés de L. (L.) i. chagasi. Les analyses de données ont utilisé le coefficient de Chi-carré et Pearson (intervalle de confiance de 95 %). En ce qui concerne l'HR et les états cliniques des chiens, on a noté que 100 % (n = 8) des animaux présentant une HR positive provenaient du groupe sous-clinique, tandis que 100 % montrant une HR négative provenaient du groupe cliniquement malade. Des réponses IgG2 supérieures aux IgG1 ont été observées dans les deux groupes cliniques. Cependant, lorsque cette comparaison a été faite entre les groupes sous-clinique et malade, des réponses IgG1 plus élevées ont été observées chez les chiens du groupe malade que chez le groupe sous-clinique, alors qu'aucune différence n'a été notée entre les réponses IgG2 chez les chiens des deux groupes cliniques. En outre, nous avons trouvé des réponses IgG1 plus faibles chez les chiens du groupe sous-clinique présentant une HR positive que chez les chiens des groupes sous-cliniques ou malades avec HR négative. Aucune différence n'a été trouvée entre les réponses IgG2 de ces deux groupes cliniques. Ces résultats suggèrent fortement que la réponse IgG1, mais pas IgG2, est associée à une susceptibilité à la leishmaniose viscérale canine.