Prevalence of Endoparasites in Urban Stray Dogs from Brazil Diagnosed with Leishmania, with Potential for Human Zoonoses.

BACKGROUND: In tropical environments, abandoned animals can be an important source for human zoonotic infections, such as human visceral leishmaniasis and other vector-borne diseases. Here, we report the frequency of protozoan and helminth intestinal parasites in stray dogs, which might have an implication for human health in urban Brazilian settings. MATERIAL AND METHODS: We performed necropsies on 93 animals, euthanized due to canine visceral leishmaniasis control program, and examined their intestines looking for the presence of helminths; we determined the parasite load, and the elimination of eggs and cysts of protozoan parasites in fecal samples. Further, we performed serology tests for the detection of specific antibodies against Toxoplasma gondii. RESULTS: Overall, a high prevalence of intestinal parasites with potential for human zoonoses resulted and only 8.6% of examined intestines remained negative. The most prevalent helminths were Ancylostoma caninum and Dipylidium caninum. For nematodes, high worm burdens were detected for A. caninum and Trichuris vulpis. Additionally, we analyzed worm burdens and quantitative stool examinations, but found no significant association between positive serology for Leishmania infection and intestinal parasite burden. Interestingly, serology for T. gondii infection revealed a prevalence of 33.3% and a positive result was significantly associated with a higher A. caninum adult worm recovery (p = 0.0087). CONCLUSION: Our results showed stray dogs living in urban areas are heavily parasitized, which presents a potential risk for humans. Beyond the control of canine visceral leishmaniasis, we propose an improvement of the control program to reduce the risk for other parasitic diseases in dogs and humans.

Fcγ-RI, Fcγ-RII and IL-10 as predictive biomarkers for post-therapeutic cicatrization time in monocytes from cutaneous leishmaniasis patients.

Cutaneous leishmaniasis (CL) treatment is based on therapy with Glucantime® , yet, there are few laboratory methods to monitor its success. In this study, ex vivo and in vitro evaluations of peripheral blood monocytes were performed in a longitudinal study to characterize the impact of Glucantime® on overall phenotypic/functional features of these cells from CL patients to identify predictive biomarkers for post-therapeutic monitoring by flow cytometry. The ex vivo evaluation from CL patients demonstrated a modulatory profile before treatment, with a decrease in TLR-2, FcγRII, HLA-DR, CD86, IFN-γR, TNF, IL-12, NO, and an increase in FcγRIII and IL-10R. Conversely, treatment changes some of these biomarker expressions by decreasing FcγRIII and IL-10R and increasing IFN-γR, IL-12 and NO. Moreover, an in vitro analysis of these patients showed a reduced phagocytic capacity of Leishmania braziliensis and higher levels of IL-10 and TGF-ß modulating functional profile. Regardless of the compromised L. braziliensis phagocytic capacity, treatment re-established the production of IL-12, IL-10, TGF-ß and NO at the basal level. Notably, monocytes from patients with early cicatrization showed enhanced FcγRI and FcγRII expressions and reduced IL-10, which was further corroborated by a baseline fold change analysis. Finally, the logistic regression model emphasized the performance of FcγRI, FcγRII and IL-10 as robust predictive biomarkers for post-therapeutic cicatrization during cutaneous leishmaniasis.

Distinct pattern of immunophenotypic features of innate and adaptive immunity as a putative signature of clinical and laboratorial status of patients with localized cutaneous leishmaniasis.

In this study, we have analysed the phenotypic features of innate/adaptive immunity of patients with localized cutaneous leishmaniasis (LCL), categorized according to their clinical/laboratorial status, including number of lesion (L1; L2­4), days of illness duration (≤60;>60) and positivity in the Montenegro skin test (MT−;MT+). Our findings highlighted a range of phenotypic features observed in patients with LCL (↑%HLA-DR+ neutrophils; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio; ↑HLA-DR in B lymphocytes, ↑%CD23+ neutrophils, monocytes and B cells; ↑α-Leishmania IgG and ↑serum NO2⁻ + NO3⁻). Selective changes were observed in L1 (↑%HLA-DR+ neutrophils, ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑serum NO2⁻ + NO3⁻) as compared to L2­4 (↑%CD5− B cells; ↑CD23+ B cells and ↑α-Leishmania IgG). Whilst ≤60 presented a mixed profile of innate/adaptive immunity (↓%CD28+ neutrophils and ↑%CD4+ T cells), >60 showed a well-known leishmanicidal events (↑CD8+ T cells; ↑serum NO2⁻ + NO3⁻ and ↑α-Leishmania IgG). MT+ patients showed increased putative leishmanicidal capacity (↑%HLA-DR+ neutrophils; ↑%CD23+ monocytes; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑ serum NO2⁻ + NO3⁻). Overall, a range of immunological biomarkers illustrates the complex immunological network associated with distinct clinical/laboratorial features of LCL with applicability in clinical studies.

Humoral and cellular immune responses in dogs with inapparent natural Leishmania infantum infection.

Molecular analysis, serology and immunophenotyping for T lymphocytes and their subsets, B lymphocytes and monocytes were performed on dogs naturally infected with Leishmania infantum. Animals were categorised as asymptomatic dogs I (AD-I), with negative serology and positive molecular results, and asymptomatic dogs II (AD-II), with positive serology and positive molecular results, and these were compared to symptomatic dogs (SD) and control dogs (CD). AD-I exhibited immunophenotypic features similar to those of CD, including isotype profiles and concentrations of monocytes. Similar biomarkers were found in AD-II and SD, such as, higher levels of immunoglobulins IgG, IgG2, IgM and IgA and higher concentrations of eosinophils. High frequencies of T lymphocytes and CD4(+) T cells were observed in both AD-I and AD-II compared to SD, whereas CD8(+) T cells were higher only in AD-II compared with SD. Analysis of B lymphocytes revealed an increased frequency of this cell type only in AD-II animals compared with SD. Asymptomatic dogs appear to have a dichotomous infection spectrum that can influence the humoral and cellular immunological status during canine visceral leishmaniasis.

Evaluation of the influence of tissue parasite density on hematological and phenotypic cellular parameters of circulating leukocytes and splenocytes during ongoing canine visceral leishmaniasis.

During Leishmania infection, tissue parasitism at different sites may differ and imply distinct immunopathological patterns during canine visceral leishmaniasis (CVL). For this reason, we have assessed by flow cytometry the impact of spleen and skin parasite density on the phenotypic profile of splenocytes and circulating leukocytes of 40 Brazilian dogs naturally infected by Leishmania chagasi categorized according to splenic and cutaneous parasite load. Our major statistically significant findings demonstrated that dogs with splenic high parasitism presented a significant decrease in absolute counts of CD5+ T lymphocytes in comparison with dogs presenting splenic medium parasitism. Moreover, a decrease in the absolute number of circulating monocytes was observed as a hallmark of high parasitism. The increased frequency of CD8+ T cells is associated with low splenic parasitism during CVL. Although we did not found any significant differences between the immunophenotypic analysis performed in circulating lymphocytes according to cutaneous parasite load, there were negative correlations between CD5+ and CD8+ T cells and cutaneous parasite density reemphasizes the role of T cell-mediated immune response in resistance mechanisms during ongoing CVL. These results add new insights about the pathogenesis of CVL and may help in the establishment of additional tools for future studies on drugs and vaccine approaches.

Analysis of the cytokine profile in spleen cells from dogs naturally infected by Leishmania chagasi.

Recent studies suggest that asymptomatic dogs infected with canine visceral leishmaniasis (CVL) develop a Th1 immunological profile whilst oligosymptomatic and symptomatic CVL-infected animals present a Th2 profile. In the present study, an RT-PCR method has been standardised and employed to evaluate the frequency and the semi-quantitative level of expression of the cytokines IL-4, IL-10, IL-12, INF-gamma and TNF-alpha in splenocytes of 30 dogs naturally infected with Leishmania chagasi and of 7 non-infected dogs (NID). An increase in the level of expression of IL-12 (p=0.059) was detected in all CVL-infected dogs compared with NID. In dogs exhibiting high parasitism, the frequency of expression of IL-10 was higher (p=0.011) than in animals presenting low parasitism or medium parasitism (MP) and in NID animals, whilst the level of expression of IL-10 was higher (p=0.0094) than in animals exhibiting MP and in the NID group. Positive correlations between the levels of expression of IL-10 with respect to the progression of the disease (IL-10: r=0.3510; p=0.0337) and the levels of expression of IL-10 and INF-gamma increase in parasitism (IL-10: r=0.3428; p=0.0438 and INF-gamma: r=0.4690; p=0.0045) were observed. Such data suggest that CVL is marked by a balanced production of Th1 and Th2 cytokines, with a predominant accumulation of IL-10 as a consequence of an increase in parasitic load and progression of the disease, and INF-gamma was related with the increase in parasitic load.

Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi.

Canine visceral leishmaniasis (CVL) manifests itself as a broad clinical spectrum ranging from asymptomatic infection to patent severe disease. Despite relevant findings suggesting changes on lymphocytes subsets regarding the CVL clinical forms, it still remains to be elucidated whether a distinct phenotypic profile would be correlated with degree of tissue parasite density. Herein, we have assessed the correlation between the clinical status as well as the impact of bone marrow parasite density on the phenotypic profile of peripheral blood leucocytes in 40 Brazilian dogs naturally infected by Leishmania chagasi. Our major findings describe the lower frequency of B cells and monocytes as the most important markers of severe CVL. Our main statistically significant findings reveal that the CD8(+) T cell subset reflects most accurately both the clinical status and the overall bone marrow parasite density, as increased levels of CD8(+) lymphocytes appeared as the major phenotypic feature of asymptomatic disease and dogs bearing a low parasite load. Moreover, enhanced major histocompatibility complex (MHC)-II density as well as a higher CD45RB/CD45RA expression index seems to represent a key element to control disease morbidity. The association between clinical status, bone marrow parasitism and CD8(+) T cells re-emphasizes the role of the T cell-mediated immune response in the resistance mechanisms during ongoing CVL. Higher levels of circulating T lymphocytes (both CD4(+) and CD8(+) T cells) and lower MHC-II expression by peripheral blood lymphocytes seem to be the key for the effective immunological response, a hallmark of asymptomatic CVL.

Relationship between canine visceral leishmaniosis and the Leishmania (Leishmania) chagasi burden in dermal inflammatory foci.

The skin is the first point of contact with organisms of the genus Leishmania from sand fly vectors, and apparently normal skin of sick dogs harbours amastigote forms of Leishmania chagasi. In relation to canine visceral leishmaniosis (CVL), the ear skin was examined in 10 uninfected dogs (UDs) and in 31 dogs dogs naturally infected with L. chagasi. The infected animals consisted of 10 symptomless dogs (SLDs), 12 mildly affected dogs (MADs) and nine affected dogs (ADs). A higher parasite burden was demonstrated in ADs than in SLDs by anti-Leishmania immunohistochemistry (P<0.01), and by Leishman Donivan Unit (LDU) indices (P=0.0024) obtained from Giemsa-stained impression smears. Sections stained with haematoxylin and eosin demonstrated a higher intensity of inflammatory changes in ADs than in SLDs (P<0.05), and in the latter group flow cytometry demonstrated a correlation (P=0.05/r=0.7454) between the percentage of CD14(+) monocytes in peripheral blood and chronic dermal inflammation. Extracellular matrix assessment for reticular fibres by staining of sections with Masson trichrome and Gomori ammoniacal silver demonstrated a decrease in collagen type I and an increase in collagen type III as the clinical signs increased. The data on correlation between cellular phenotypes and histological changes seemed to reflect cellular activation and migration from peripheral blood to the skin, mediated by antigenic stimulation. The results suggested that chronic dermal inflammation and cutaneous parasitism were directly related to the severity of clinical disease.