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This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sbv) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum, displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum.

Standards of care for human visceral leishmaniasis (VL) are based on drugs used parenterally, and oral treatment options are urgently needed. In the present study, a repurposing strategy was used associating tamoxifen (TMX) with polyethylene glycol-block-polylactide nanocapsules (NC) and its anti-leishmanial efficacy was reported in vivo. Stable surface modified-NC (5 mg/mL of TMX) exhibited 200 nm in size, +42 mV of zeta potential, and 98% encapsulation efficiency. Atomic force microscopy evidenced core-shell-NC. Treatment with TMX-NC reduced parasite-DNA quantified in liver and spleen compared to free-TMX, and provided a similar reduction of parasite burden compared with meglumine antimoniate in mice and hamster models. Image-guided biodistribution showed accumulation of NC in liver and spleen after 30 min post-administration. TMX-NC reduced the number of liver granulomas and restored the aspect of capsules and trabeculae in the spleen of infected animals. TMX-NC was tested for the first time against VL models, indicating a promising formulation for oral treatment.

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP+Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4+IL-4+ cells and increasing CD8+IFN-γ+ cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in almost all countries of Latin America. In Brazil, oral infection is becoming the most important mechanism of transmission of the disease in several regions of the country. The gastrointestinal tract is the gateway for the parasite through this route of infection, however, little is known about the involvement of these organs related to oral route. In this sense, the present study evaluated the impact of oral infection on the digestive tract in mice infected by Berenice-78 (Be-78) T. cruzi strain, in comparison with the intraperitoneal route of infection. In this work, the intraperitoneal route group showed a peak of parasitemia similar to the oral route group, however the mortality rate among the orally infected animals was higher when compared to intraperitoneal route. By analyzing the frequency of blood cell populations, differences were mainly observed in CD4+ T lymphocytes, and not in CD8+, presenting an earlier reduction in the number of CD4+ T cells, which persisted for a longer period, in the animals of the oral group when compared with the intraperitoneal group. Animals infected by oral route presented a higher tissue parasitism and inflammatory infiltrate in stomach, duodenum and colon on the 28th day after infection. Therefore, these data suggest that oral infection has a different profile of parasitological and immune responses compared to intraperitoneal route, being the oral route more virulent and with greater tissue parasitism in organs of the gastrointestinal tract evaluated during the acute phase.

Chagas disease remains neglected, and current chemotherapeutics present severe limitations. Lychnopholide (LYC) at low doses loaded in polymeric poly(d,l-lactide)-block-polyethylene glycol (PLA-PEG) nanocapsules (LYC-PLA-PEG-NC) exhibits anti-Trypanosoma cruzi efficacy in mice infected with a partially drug-resistant strain. This study reports the efficacy of LYC-PLA-PEG-NC at higher doses in mice infected with a T. cruzi strain resistant to benznidazole (BZ) and nifurtimox (NF) treated at both the acute phase (AP) and the chronic phase (CP) of infection by the oral route. Mice infected with the T. cruzi VL-10 strain were treated by the oral route with free LYC (12 mg/kg of body weight/day), LYC-PLA-PEG-NC (8 or 12 mg/kg/day), or BZ at 100 mg/kg/day or were not treated (controls). Treatment efficacy was assessed by hemoculture (HC), PCR, enzyme-linked immunosorbent assay (ELISA), heart tissue quantitative PCR (qPCR), and histopathology. According to classical cure criteria, treatment with LYC-PLA-PEG-NC at 12 mg/kg/day cured 75% (AP) and 88% (CP) of the animals, while at a dose of 8 mg/kg/day, 43% (AP) and 43% (CP) were cured, showing dose-dependent efficacy. The negative qPCR results for heart tissue and the absence of inflammation/fibrosis agreed with the negative results obtained by HC and PCR. Thus, the mice treated with the highest dose could be considered 100% cured, in spite of a low ELISA reactivity in some animals. No cure was observed in animals treated with free LYC or BZ or the controls. These results are exceptional in terms of experimental Chagas disease chemotherapy and provide evidence of the outstanding contribution of nanotechnology in mice infected with a T. cruzi strain totally resistant to BZ and NF at both phases of infection. Therefore, LYC-PLA-PEG-NC has great potential as a new treatment for Chagas disease and deserves further investigations in clinical trials.

Dogs are important hosts and reservoirs of leishmaniasis, a disease caused by protozoan parasites from the genus Leishmania, affecting ~12 million people worldwide. The detection of visceral leishmaniasis (VL) in dogs by real-time PCR (qPCR) may improve on diagnosis, but the different qPCR methods available for Leishmania DNA detection have not been established as routine in diagnostic tools and/or epidemiologic studies for canine VL. Here, we compared three qPCR assays (DNApol, Linj31, and LDON) in the detection of VL by Leishmania infantum in spleen (n = 48; 7), skin (n = 48; 7), and whole blood (n = 44; 7) samples from serologically positive and negative dogs, respectively. Overall, the DNApol performed better than the Linj31 and LDON assays in the detection of positive samples in all tissues tested, yielding from 66.7 to 100.0% of positivity for both skin and spleen samples. For spleen samples, we observed no statistically significant differences between positive detection by the LDON and DNApol assays. Whole blood samples yielded the lowest rates of positive detection, regardless of the qPCR assay used. In contrast, positive detection of Leishmania DNA was as efficient from skin samples using the DNApol assay as from spleen samples using either the DNApol or the LDON assay. Although qPCR assays from skin samples may not be practical for use in the field, our study suggests that the DNApol and LDON assays from skin samples could be used in future to evaluate canine VL treatment in veterinary clinics.

Chagas disease (CD) is endemic in Latin America. Drugs available for its treatment are benznidazole (BZ)/nifurtimox (NF), both with low efficacy in the late infection and responsible for several side effects. Studies of new drugs for CD among natural products, and using drug combinations with BZ/NF are recommended. Silibinin (SLB) is a natural compound that inhibits the efflux pump (Pgp) of drugs in host cell membranes, causes death of trypanosomatids, has anti-inflammatory activity, and was never assayed against T. cruzi. Here, in vitro and in vivo activities of SLB, SLB+BZ, and BZ against T. cruzi Y strain were evaluated. Cytotoxicity of SLB in VERO cells by the MTT method revealed IC50 of 250.22 μM. The trypanocidal activity evaluated by resazurin method in epimastigotes showed that SLB 25 μM inhibited parasite growth. SLB IC50 and selectivity index (SI) for amastigote were 79.81 μM and 3.13, respectively. SLB100+BZ10 showed higher parasite inhibition (91.44%) than SLB or BZ. Swiss mice infected with Y strain were treated with SLB, SLB+BZ, and BZ. Parasitemia was evaluated daily and 90, 180, and 240 days after treatment in surviving animals by hemoculture, blood qPCR, and after euthanasia, by qPCR in heart tissue. SLB monotherapy was not able to control the parasitemia/mortality of the animals. Parasitological negativation of 85.7–100% was observed in the experimental groups treated with SLB+BZ. Although SLB had shown activity against T. cruzi in vitro, it was not active in mice. Thus, the results of the therapeutic effect observed with SLB+BZ may be interpreted as a result from BZ action.

Pentavalent antimonial has been the first choice treatment for visceral leishmaniasis; however, it has several side effects that leads to low adherence to treatment. Liposome-encapsulated meglumine antimoniate (MA) arises as an important strategy for chemotherapy enhancement. We evaluated the immunopathological changes using the mixture of conventional and pegylated liposomes with MA. The mice were infected with Leishmania infantum and a single-dose treatment regimen. Comparison was made with groups treated with saline, empty liposomes, free MA, and a liposomal formulation of MA (Lipo MA). Histopathological analyses demonstrated that animals treated with Lipo MA showed a significant decrease in the inflammatory process and the absence of granulomas. The in vitro stimulation of splenocytes showed a significant increase of gamma interferon (IFN-γ) produced by CD8 + T cells and a decrease in interleukin-10 (IL-10) produced by CD4 + and CD8 + T cells in the Lipo MA. Furthermore, the Lipo MA group showed an increase in the IFN-γ/IL-10 ratio in both CD4 + and CD8 + T cell subsets. According to the parasite load evaluation using quantitative PCR, the Lipo MA group showed no L. infantum DNA in the spleen (0.0%) and 41.4% in the liver. In addition, we detected a low positive correlation between parasitism and histopathology findings (inflammatory process and granuloma formation). Thus, our results confirmed that Lipo MA is a promising antileishmanial formulation able to reduce the inflammatory response and induce a type 1 immune response, accompanied by a significant reduction of the parasite burden into hepatic and splenic compartments in treated animals.

Herein, we evaluated the treatment strategy employing a therapeutic heterologous vaccine composed of antigens of Leishmania braziliensis associated with MPL adjuvant (LBMPL vaccine) for visceral leishmaniasis (VL) in symptomatic dogs naturally infected by Leishmania infantum. Sixteen dogs received immunotherapy with MPL adjuvant (n = 6) or with a vaccine composed of antigens of L. braziliensis associated with MPL (LBMPL vaccine therapy, n = 10). Dogs were submitted to an immunotherapeutic scheme consisting of 3 series composed of 10 subcutaneous doses with 10-day interval between each series. The animals were evaluated before (T0) and 90 days after treatment (T90) for their biochemical/hematological, immunological, clinical, and parasitological variables. Our major results showed that the vaccine therapy with LBMPL was able to restore and normalize main biochemical (urea, AST, ALP, and bilirubin) and hematological (erythrocytes, hemoglobin, hematocrit, and platelets) parameters. In addition, in an ex vivo analysis using flow cytometry, dogs treated with LBMPL vaccine showed increased CD3+ T lymphocytes and their subpopulations (TCD4+ and TCD8+), reduction of CD21+ B lymphocytes, increased NK cells (CD5−CD16+) and CD14+ monocytes. Under in vitro conditions, the animals developed a strong antigen- specific lymphoproliferation mainly by TCD4+ and TCD8+ cells; increasing in both TCD4+IFN-γ+ and TCD8+IFN-γ+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF-α and reduced levels of IL-10. Concerning the clinical signs of canine visceral leishmaniasis, the animals showed an important reduction in the number and intensity of the disease signs; increase body weight as well as reduction of splenomegaly. In addition, the LBMPL immunotherapy also promoted a reduction in parasite burden assessed by real-time PCR. In the bone marrow, we observed seven times less parasites in LBMPL animals compared with MPL group. The skin tissue showed a reduction in parasite burden in LBMPL dogs 127.5 times higher than MPL. As expected, with skin parasite reduction promoted by immunotherapy, we observed a blocking transmission to sand flies in LBMPL dogs with only three positive dogs after xenodiagnosis. The results obtained in this study highlighted the strong potential for the use of this heterologous vaccine therapy as an important strategy for VL treatment.

Canine visceral leishmaniosis (CVL) is a zoonosis of major public health impact caused by organisms of the genus Leishmania which is transmitted to human and animals by phlebotomine sand flies. The skin is the first point of contact with Leishmania parasites for sandy fly vectors and it is considered an important reservoir compartment in infected dogs. The aim of this study was to determine the main histophatologic alterations in ear skin of dogs naturally infected by Leishmania infantum with different clinical status and different degrees of parasitism. Therefore, thirty-four dogs naturally infected with L. infantum were grouped according to their clinical status in asymptomatic (AD, n = 11), oligosymptomatic (OD, n = 11) and symptomatic dogs (SD, n = 12) as well as their degrees of parasite load in the skin as low (LP, n = 11), median (MP, n = 11) and high (HP, n = 12) parasitism. Additionally, ten dogs were used as control (CD, n = 10). At necropsy, skin samples were collected for further histological and parasitological analysis. The OD and SD groups presented higher parasite burden than AD group. The inflammation was higher in SD group when compared to OD and AD. The LP, MP and HP groups showed an increasing inflammatory process, indicating that a great parasite load is accompanied by a major inflammatory process in the skin. The number of mast cells was higher in the OD and LP groups than CD group, suggesting that these cells may be involved in tissue remodeling, since that an increase of type III collagen fibers and decrease type I collagen fibers were observed in these groups. Taken together, our results enable a better understanding of the alterations in skin of CVL dogs and consequently new insights about the pathogenesis of CVL.

Trypanosoma cruzi infection may be caused by different strains with distinct discrete typing units (DTUs) that can result in variable clinical forms of chronic Chagas disease. The present study evaluates the immune response and cardiac lesions in dogs experimentally infected with different T. cruzi strains with distinct DTUs, namely, the Colombian (Col) and Y strains of TcI and TcII DTU, respectively. During infection with the Col strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of interleukin (IL)-4. The latter suggests that during the acute phase, infection with the Col strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium. Conversely, infection with the Y strain led to leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. The Y strain stimulated the production of interferon-γ by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed, demonstrating that strains of different DTUs interact differently with the host.

The recent increase in immigration of people from areas endemic for Chagas disease ( Trypanosoma cruzi ) to the United States and Europe has raised concerns about the transmission via blood transfusion and organ transplants in these countries. Infection by these pathways occurs through blood trypomastigotes (BT), and these forms of T. cruzi are completely distinct of metacyclic trypomastigotes (MT), released by triatomine vector, in relation to parasite–host interaction. Thus, research comparing infection with these different infective forms is important for explaining the potential impacts on the disease course. Here, we investigated tissue parasitism and relative mRNA expression of cytokines, chemokines, and chemokine receptors in the heart during acute infection by MT or BT forms in dogs. BT-infected dogs presented a higher cardiac parasitism, increased relative mRNA expression of pro-inflammatory and immunomodulatory cytokines and of the chemokines CCL3/MIP-1α, CCL5/RANTES, and the chemokine receptor CCR5 during the acute phase of infection, as compared to MT-infected dogs. These results suggest that infection with BT forms may lead to an increased immune response, as revealed by the cytokines ratio, but this kind of immune response was not able to control the cardiac parasitism. Infection with the MT form presented an increase in the relative mRNA expression of IL-12p40 as compared to that of IL-10 or TGF-β1. Correlation analysis showed increased relative mRNA expression of IFN-γ as well as IL-10, which may be an immunomodulatory response, as well as an increase in the correlation of CCL5/RANTES and its CCR5 receptor. Our findings revealed a difference between inoculum sources of T. cruzi , as vectorial or transfusional routes of T. cruzi infection may trigger distinct parasite–host interactions during the acute phase, which may influence immunopathological aspects of Chagas disease.

Reverse vaccinology or immunoinformatics is a computational methodology which integrates data from in silico epitope prediction, associated to other important information as, for example, the predicted subcellular location of the proteins used in the design of the context of vaccine development. This approach has the potential to search for new targets for vaccine development in the predicted proteome of pathogenic organisms. To date, there is no effective vaccine employed in vaccination campaigns against visceral leishmaniasis (VL). For the first time, herein, an in silico, in vitro, and in vivo peptide screening was performed, and immunogenic peptides were selected to constitute VL peptide-based vaccines. Firstly, the screening of in silico potential peptides using dogs naturally infected by L. infantum was conducted and the peptides with the best performance were selected. The mentioned peptides were used to compose Cockt-1 (cocktail 1) and Cockt-2 (cocktail 2) in combination with saponin as the adjuvant. Therefore, tests for immunogenicity, polyfunctional T-cells, and the ability to induce central and effector memory in T-lymphocytes capacity in reducing the parasite load on the spleen for Cockt-1 and Cockt-2 were performed. Among the vaccines under study, Cockt-1 showed the best results, eliciting CD4+ and CD8+ polyfunctional T-cells, with a reduction in spleen parasitism that correlates to the generation of T CD4+ central memory and T CD8+ effector memory cells. In this way, our findings corroborate the use of immunoinformatics as a tool for the development of future vaccines against VL.

Polymerase chain reaction (PCR) and its variations represent highly sensitive and specific methods for Leishmania DNA detection and subsequent canine visceral leishmaniasis (CVL) diagnosis. The aim of this work was to compare three different molecular diagnosis techniques (conventional PCR [cPCR], seminested PCR [snPCR], and quantitative PCR [qPCR]) in samples of skin and spleen from 60 seropositive dogs by immunofluorescence antibody test and enzyme-linked immunosorbent assay. Parasitological analysis was conducted by culture of bone marrow aspirate and optical microscopic assessment of ear skin and spleen samples stained with Giemsa, the standard tests for CVL diagnosis. The primers L150/L152 and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNA minicircle in the cPCR, and snPCR and qPCR were performed using the DNA polymerase gene (DNA pol α) primers from Leishmania infantum. The parasitological analysis revealed parasites in 61.7% of the samples. Sensitivities were 89.2%, 86.5%, and 97.3% in the skin and 81.1%, 94.6%, and 100.0% in spleen samples used for cPCR, snPCR, and qPCR, respectively. We demonstrated that the qPCR method was the best technique to detect L. infantum in both skin and spleen samples. However, we recommend the use of skin due to the high sensitivity and sampling being less invasive.

Specific chemotherapy using benznidazole (BNZ) for Chagas disease during the chronic stage is controversial due to its limited efficacy and toxic effects. Although BNZ has been used to treat Chagas disease since the 1970s, few studies about the biodistribution of this drug exist. In this study, BNZ tissue biodistribution in a murine model and its pharmacokinetic profile in plasma were monitored. A bioanalytical high-performance liquid chromatography method with a UV detector (HPLC-UV) was developed and validated according to the European Medicines Agency for quantification of BNZ in organs and plasma samples prepared by liquid-liquid extraction using ethyl acetate. The developed method was linear in the BNZ concentration, which ranged from 0.1 to 100.0 μg/ml for plasma, spleen, brain, colon, heart, lung, and kidney and from 0.2 to 100.0 μg/ml for liver. Validation assays demonstrated good stability for BNZ under all conditions evaluated. Pharmacokinetic parameters confirmed rapid, but low, absorption of BNZ after oral administration. Biodistribution assays demonstrated different maximum concentrations in organs and similar times to maximum concentration and mean residence times, with means of 40 min and 2.5 h, respectively. Therefore, the biodistribution of BNZ is extensive, reaching organs such as the heart and colon, which are the most relevant organs affected by Trypanosoma cruzi infection, and also the spleen, brain, liver, lungs, and kidneys. Simultaneous analyses of tissues and plasma indicated high BNZ metabolism in the liver. Our results suggest that low bioavailability, instead of inadequate biodistribution, could be responsible for therapeutic failure during the chronic phase of Chagas disease.

Hematological analysis has limited applications for disease diagnosis in Leishmania infantum–infected dogs, but it can be very important in evaluating the clinical forms of the disease and in understanding the evolution of canine visceral leishmaniasis (CVL) pathogenesis. Recently, we demonstrated that alterations in leucopoiesis and erythropoiesis are related to clinical status and bone marrow parasite density in dogs naturally infected by L. infantum. To further characterize these alterations, we evaluated the association between the hematological parameters in bone marrow and peripheral blood alterations in groups of L. infantum–infected dogs: asymptomatic I (AD-I: serum negative/PCR+), asymptomatic II (AD-II: serum positive), oligosymptomatic (OD), and symptomatic (SD). Results were compared with those from noninfected dogs (NID). The SD group was found to present a decrease in erythropoietic lineage with concomitant reductions in erythrocytes, hemoglobin, and hematocrit parameters, resulting in anemia. The SD group also had increased neutrophils and precursors and decreased band eosinophils and eosinophils, leading to peripheral blood leucopenia. In the AD-II group, lymphocytosis occurred in both the peripheral blood and the bone marrow compartments. The SD group exhibited lymphocytosis in the bone marrow, with lymphopenia in the peripheral blood. In contrast, the AD-I group, showed no significant changes suggestive of CVL, presenting normal counts in bone marrow and peripheral blood. Our results showed for the first time that important changes in hematopoiesis and hematological parameters occur during ongoing CVL in naturally infected dogs, mainly in symptomatic disease. Taken together, our results based on myelogram and hemogram parameters enable better understanding of the pathogenesis of the anemia, lymphocytosis, and lymphopenia, as well as the leucopenia (eosinopenia and monocytopenia), that contribute to CVL prognosis.

a b s t r a c t Zoonotic visceral leishmaniasis in Brazil is caused by Leishmania infantum parasites and is transmitted by sand flies of the Phlebotominae family. Dogs are the main urban reser- voirs and represent the major source of contagion for the vectors. Studies have shown that most infected dogs are polymerase chain reaction-positive months before seroconversion. Herein, we describe a cohort study designed to identify the incidence of and risk factors for L. infantum infection as detected by polymerase chain reaction-restriction fragment length polymorphism. To determine the risk factors for infection, we conducted a base- line canine survey (n = 1443) from which dogs were selected for the cohort study (n = 282) involving three evaluations over the course of a 26-month follow-up period. Serology, molecular tests, and a structured questionnaire were used. The risk factors for infection were identified by means of the Cox regression model. The overall infection incidence was 5.8 per 100 dog-months (95% confidence interval 5.1-6.5). Increased risk of infec- tion was associated with the presence of previous cases of canine visceral leishmaniasis in the domiciles (hazard ratio (HR) 1.4; 95% confidence interval (CI) 1.1-1.8) and unplastered house walls (HR 3.6; 95% CI 1.6-8.1). These risk factors suggest that insecticide spraying in cracks and crevices in unplastered walls can reduce biting rates within and around homes. Furthermore, our results demonstrate that the Visceral Leishmaniasis Control and Surveil- lance Program should adopt environmental management measures in homes with previous cases of canine visceral leishmaniasis, because these homes are more likely to maintain the transmission cycle.

Background Various factors contribute to the urbanization of the visceral leishmaniasis (VL), including the difficulties of implementing control measures relating to the domestic reservoir. The aim of this study was to determine the prevalence of canine visceral leishmaniasis in an urban endemic area in Brazil and the factors associated with Leishmania infantum infection among seronegative and PCR-positive dogs. Methodology A cross-sectional study was conducted in Belo Horizonte, Minas Gerais, Brazil. Blood samples were collected from 1,443 dogs. Serology was carried out by using two enzyme-linked immunosorbent assays (Biomanguinhos/FIOCRUZ/RJ and “in house”), and molecular methods were developed, including PCR-RFLP. To identify the factors associated with early stages of infection, only seronegative (n = 1,213) animals were evaluated. These animals were divided into two groups: PCR-positive (n = 296) and PCR-negative (n = 917) for L. infantum DNA. A comparison of these two groups of dogs taking into consideration the characteristics of the animals and their owners was performed. A mixed logistic regression model was used to identify factors associated with L. infantum infection. Principal Findings Of the 1,443 dogs examined, 230 (15.9%) were seropositive in at least one ELISA, whereas PCR-RFLP revealed that 356 animals (24.7%) were positive for L. infantum DNA. Results indicated that the associated factors with infection were family income, Author Summary Visceral leishmaniasis (VL) is a disease caused by the parasite Leishmania infantum, and dogs are the most important domestic reservoirs of the agent. During recent decades, VL has expanded to large Brazilian urban centers. In the present work, we have demonstrated by using molecular techniques that the rate of canine infection as detected by serology has been considerably underestimated. Two groups of seronegative dogs (infected and non-infected according to molecular methods) were further evaluated from data obtained through interviews with owners of the animals. The factors associated with Leishmania infection in dogs were a family income of less than two minimum salaries, the knowledge of the owner regarding the vector, the dog spending most of its time in the backyard and the dog never having had a previous serological examination. Awareness regarding the factors associated with canine infection will improve health services and the understanding of the disease's expansion in urban areas.