Marcos José Marques

Prevalence and Factors Associated with Leishmania infantum Infection of Dogs from an Urban Area of Brazil as Identified by Molecular Methods

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<twice the Brazilian minimum salary (OR 2.3; 95%CI 1.4–3.8), knowledge of the owner regarding the vector (OR 1.9; 95%CI 1.1–3.4), the dog staying predominantly in the backyard (OR 2.2; 95%CI 1.1–4.1), and a lack of previous serological examination for VL (OR 1.5; 95%CI 1.1–2.3). Conclusions PCR detected a high prevalence of L. infantum infection in dogs in an area under the Control Program of VL intervention. Socioeconomic variables, dog behavior and the knowledge of the owner regarding the vector were factors associated with canine visceral leishmaniasis (CVL). The absence of previous serological examination conducted by the control program was also associated with L. infantum infection. It is necessary to identify the risk factors associated with CVL to understand the expansion and urbanization of VL.

Performance of LBSap Vaccine after Intradermal Challenge with L. infantum and Saliva of Lu. longipalpis: Immunogenicity and Parasitological Evaluation

In the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21+) and both CD4+ and CD8+ T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4+ T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-γ and low IL-10 and TGF-β1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions.

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.

Molecular diagnosis of canine visceral leishmaniasis : a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantum infection.

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.

Risk Factors for Seroconversion by Leishmania infantum in a Cohort of Dogs from an Endemic Area of Brazil

Visceral leishmaniasis (VL) has recently emerged in various urban and peri-urban areas of Brazil and other countries. Understanding the urbanization of VL requires identification of risk factors associated with human and canine infection. To determine the predictors of risk for canine VL, a survey was conducted of 1,443 dogs, from which a cohort was selected (n = 455) and evaluated for approximately 26 months. Serology was conducted with two enzyme-linked immunosorbent assays (ELISA): one conducted in the Laboratory of Zoonosis of the Belo Horizonte Health Department (LZOON) and the other in the Laboratory of Immunopathology of the Federal University of Ouro Preto (LIMP). A molecular diagnostic method (PCR–restriction fragment length polymorphism) and a structured questionnaire were also used. To identify the factors associated with seroconversion, two time-dependent Cox regression models were performed with different sensitivities (model 1, seroconversion by ELISA/LZOON; model 2, seroconversion by ELISA/LIMP). The overall incidences of seroconversion were 6.5/1000 dogs-months and 11.2/1000 dogs-months for ELISA/LZOON and ELISA/LIMP, respectively. Increased risk of seroconversion was associated with short fur (model 1: hazard ratio [HR] 1.9), the presence of dry leaves (model 1: HR 2.8) or manure (model 1: HR 3.5) in the backyard, dogs sleeping predominantly in the backyard (model 2: HR 2.1), the presence of symptoms (model 2: HR 2.0), and positive molecular results during follow-up (model 2: HR 1.5). Decreased risk was associated with insecticide spraying in the house (model 2: HR 0.5). These results indicate that more-vulnerable domiciles, certain dog behaviors, lack of vector control measures, and positive molecular results were associated with the occurrence of canine VL. Furthermore, it is important to emphasize that PCR-positive dogs should be monitored, owing to the possibility of seroconversion. Identifying risk factors for seroconversion in dogs is crucial for developing adequate strategies for VL prevention and control.

Canine visceral leishmaniasis: Incidence and risk factors for infection in a cohort study in Brazil

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 reservoirs 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 baseline 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 infection 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 Surveillance 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.

Natural Products from Garcinia brasiliensis as Leishmania Protease Inhibitors

The infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which cause renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro Leishmania protease inhibition activity of extracts (hexanic, ethyl-acetate, and ethanolic) and fukugetin, a bioflavonoid purified from the ethyl-acetate extract of the pericarp of the fruit of Garcinia brasiliensis, a tree native to Brazilian forests. The isolated compound was characterized by using spectral analyses with nuclear magnetic resonance, mass spectroscopy, ultraviolet, and infrared techniques. The ethyl-acetate extract and the compound fukugetin showed significant activity as inhibitors of Leishmanias proteases, with mean (±SD) IC(50) (50% inhibition concentration of protease activity) values of 15.0±1.3 μg/mL and 3.2±0.5 μM/mL, respectively, characterizing a bioguided assay. In addition, this isolated compound showed no activity against promastigote and amastigote forms of L. (L.) amazonensis and mammalian cells. These results suggest that fukugetin is a potent protease inhibitor of L. (L.) amazonensis and does not cause toxicity in mammalian or Leishmania cells in vitro. This study provides new perspectives on the development of novel drugs that have leishmanicidal activity obtained from natural products and that target the parasites proteases.

Occurrence of a conserved domain in ATP diphosphohydrolases from pathogenic organisms associated to antigenicity in human parasitic diseases

A polypeptide (r78-117) belonging to the potato apyrase was identified as a conserved domain shared with apyrase-like proteins from distinct pathogenic organisms, and was obtained as a 6xHis tag polypeptide (r-Domain B). By ELISA, high IgG, and IgG1 and IgG2a subtypes levels were detected in BALB/c mice pre-inoculated with r-Domain B. In Schistosoma mansoni adult worm or Leishmania (V.) braziliensis promastigote preparation, anti-r-Domain B antibodies inhibit 22-72% of the phosphohydrolytic activities and when immobilized on Protein A-Sepharose immunoprecipitate 42-91% of them. Western blots of the immunoprecipitated resin-antibody-antigen complexes identified bands of mw similar to those predicted for parasite proteins. Total IgG and subclasses of patients with leishmaniasis or schistosomiasis exhibited cross-immunoreactivity with r-Domain B. Therefore, the domain B within both S. mansoni SmATPDase 2 (r156-195) and L. (V.) braziliensis NDPase (r83-122) are potentially involved in the host immune response, and also seem to be conserved during host and parasites co-evolution.

Autochthonous canine visceral leishmaniasis in a non-endemic area: Bom Sucesso, Minas Gerais State, Brazil

The article begins by describing a dog with characteristic symptoms of visceral leishmaniasis. A serum sample from this animal was positive by indirect immunofluorescence (IIF) performed in anti-Leishmania total IgG in 1999. Tissues from the same dog were also positive by polymerase chain reaction (PCR) in 2004, identifying Leishmania DNA in the cerebellum, liver, kidney, and intestine. This is the first report of a dog with autochthonous visceral leishmaniasis in the county of Bom Sucesso, Minas Gerais State, Brazil. The finding of this IIF-positive dog led to a canine visceral leishmaniasis epidemiological investigation in the county. The investigation was conducted from March 1999 to December 2005. IIF was positive for Leishmania in 22 (3%) of 734 examined dogs. Among the 22 IIF-positive dogs, six presented characteristic symptoms of canine visceral leishmaniasis. The results of this epidemiological investigation were sent to local and State public health authorities, requesting visceral leishmaniasis control and preventive measures to interrupt transmission of the disease and avoid the occurrence of human cases.

Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies

Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control.