[show abstract][hide abstract] ABSTRACT: The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8+ T cells, and high NO production.
We investigated the immunogenicity and protective effect of LBSapSal vaccination after intradermal challenge with 1 x 107 late-log-phase L. infantum promastigotes in the presence of sand fly saliva of Lutzomyia longipalpis. The dogs were followed for up to 885 days after challenge.
The LBSapSal vaccine presents extensive antigenic diversity with persistent humoral and cellular immune responses, indicating resistance against CVL is triggered by high levels of total IgG and its subtypes (IgG1 and IgG2); expansion of circulating CD5+, CD4+, and CD8+ T lymphocytes and is Leishmania-specific; and reduction of splenic parasite load.
These results encourage further study of vaccine strategies addressing Leishmania antigens in combination with proteins present in the saliva of the vector.
[show abstract][hide abstract] ABSTRACT: The techniques used for diagnosis of canine visceral leishmaniasis (CVL) in Brazil ELISA and IFAT have been extensively questioned because of the accuracy of these tests. A recent change in the diagnosis protocol excluded IFAT and included the Dual-Path Platform (DPP). We evaluated the prevalence and incidence rates of Leishmania spp. before and after the change in the protocol. In addition, based on our results, we propose a new alternative that is less expensive for the screening and confirmation of CVL. Plasma samples were obtained from a serobank from dogs evaluated in a cross-sectional study (1,226 dogs) and in a cohort study of susceptible animals (n = 447), followed for 26 months. Serology testing was performed using ELISA, IFAT, and DPP. The incidence and prevalence of CVL were determined by using the protocol of the Visceral Leishmaniasis Control and Surveillance Program until 2012 (ELISA and IFAT using filter paper) and the protocol used after 2012 (DPP and ELISA using plasma). The prevalence was 6.2% and the incidence was 2.8 per 1,000 dog-months for the protocol used until 2012. For the new diagnosis protocol for CVL resulted in an incidence of 5.4 per 1,000 dog-months and a prevalence of 8.1%. Our results showed that the prevalence and incidence of infection were far greater than suggested by the previously used protocol and that the magnitude of infection in endemic areas has been underestimated. As tests are performed sequentially and euthanasia of dogs is carried out when the serological results are positive in both tests, the sequence does not affect the number of animals to be eliminated by the Control Program. Then we suggest to municipalities with a large demand of exams to use ELISA for screening and DPP for confirmation, since this allows easier performance and reduced cost.
PLoS ONE 01/2014; 9(3):e91009. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test in a broad range of serum samples using antigens and fluorescent antibodies that had been stored for 1 year at 4°C. Non-infected control dogs and Leishmania infantum-infected dogs were tested and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs' clinical status, the prototype test had an outstanding accuracy in all groups with positive serology (asymptomatic-II, oligosymptomatic, symptomatic). However, in dogs which present positive results by PCR-RFLP but negative by conventional serology (asymptomatic-I), it was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune®, Leish-Tec®, LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi, Leishmania braziliensis, Ehrlichia canis, and Babesia canis) were used to determine cross-reactivity and specificity, and the prototype presented high performance particularly in dogs with B. canis and E. canis (100% and 93.3% specificity, respectively). In conclusion, our data reinforce the prototype's potential for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis, with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens.
Clinical and vaccine Immunology: CVI 10/2013; · 2.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the studies presented here, dogs were vaccinated against Leishmania (Leishmania) chagasi challenge infection using a preparation of Leishmania braziliensis promastigote proteins and saponin as adjuvant (LBSap). Vaccination with LBSap induced a prominent type 1 immune response that was characterized by increased levels of interleukin (IL-) 12 and interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) upon stimulation with soluble vaccine antigen. Importantly, results showed that this type of responsiveness was sustained after challenge infection; at day 90 and 885 after L. chagasi challenge infection, PBMCs from LBSap vaccinated dogs produced more IL-12, IFN-γ and concomitant nitric oxide (NO) when stimulated with Leishmania antigens as compared to PBMCs from respective control groups (saponin, LB- treated, or non-treated control dogs). Moreover, transforming growth factor (TGF)-β decreased in the supernatant of SLcA-stimulated PBMCs in the LBSap group at 90 days. Bone marrow parasitological analysis revealed decreased frequency of parasitism in the presence of vaccine antigen. It is concluded that vaccination of dogs with LBSap vaccine induced a long-lasting type 1 immune response against L. chagasi challenge infection.
[show abstract][hide abstract] ABSTRACT: Canine visceral leishmaniasis (CVL) is a parasitic disease endemic in many countries, and dogs present as the major natural reservoir of the parasite, Leishmania chagasi (syn. L. infantum). Biomarkers in the canine immune system is an important technique in the course of developing vaccines and treatment strategies against CVL. New methodologies for studying the immune response of dogs during Leishmania infection and after receiving vaccines and treatments against CVL would be useful. In this context, we used peripheral blood mononuclear cells (PBMCs) from healthy dogs to evaluate procedures related to (i) establishment of in vitro conditions of monocytes differentiated into macrophages infected with L. chagasi and (ii) purification procedures of T-cell subsets (CD4(+) and CD8(+)) using microbeads. Our data demonstrated that after 5 days of differentiation, macrophages were able to induce significant phagocytic and microbicidal activity after L. chagasi infection and also showed increased frequency of parasitism and a higher parasite load. Although N-acetyl-β-d-glucosaminidase (NAG) levels presented similar levels of macrophage culture and L. chagasi infection, a progressive decrease in myeloperoxidase (MPO) levels was a hallmark over 5 days of culture. High purity levels (>90%) of CD4 and CD8 T cells were obtained on a magnetic separation column. We concluded that monocytes differentiated into macrophages at 5 days and displayed an intermediate frequency of parasitism and parasite load 72h after L. chagasi infection. Furthermore, the purification system using canine T-lymphocyte subsets obtained after 5 days of monocyte differentiation proved efficient for CD4 or CD8 T-cell purification (≥90%). The in vitro analysis using L. chagasi-infected macrophages and purified T cells presented a prospective methodology that could be incorporated in CVL vaccine and treatment studies that aim to analyze the microbicidal potential induced by specific CD4(+) and/or CD8(+) T cells.
[show abstract][hide abstract] ABSTRACT: The complex interplay between cytokines and chemokines regulates innate and adaptive immune responses against pathogens; specifically, cytokine and chemokine expression drives activation of immune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs with Leishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components, and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12, IFN-γ, TNF-α, IL-4, IL-13, TGF-β and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24 and 48h after inoculation. We also evaluated the correlation between cytokine and chemokine expression and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and 24h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression, IFN-γ and IL-13 expression, and IL-13 and TGF-β expression, suggesting that a mixed cytokine microenvironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant alone induced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4 and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initially highest in the LBSap group, but at 48h, expression was highest in the LB group. Information about the kinetics of the immune response to this vaccine gained using this dog model will help to elucidate the mechanisms of and factors involved in a protective response against Leishmania infection and will aid in establishing rational approaches for the development of vaccines against canine visceral leishmaniasis.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The control of canine visceral leishmaniasis (CVL) is imperative, but euthanasia of seropositive dogs has been highly criticized. Commonly used, immunodiagnostic tests, including Dual-Path Platform®, enzyme-linked immunosorbent assay, and immunofluorescent antibody test, have failed at detecting asymptomatic dogs in endemic areas. In this context, new serological methods are needed. Flow cytometry serology has demonstrated potential as a test with excellent performance for CVL. In this study, we proposed to establish the best conditions for preserving Leishmania infantum promastigote antigens employed in this serology test. During 12 months of follow-up, promastigotes were maintained in different preservatives (phosphate-buffered saline with 3% fetal bovine serum, phenol 0.35%, thimerosal 0.01%, and formaldehyde 0.5%) and stored at 3 distinct temperatures (25 °C, 4 °C, and -20 °C). During the study period, the morphological characteristics of the promastigotes were assessed by flow cytometry according to the forward and side scatter parameters and also under optical microscopic analysis. Reactivity performance was evaluated as the percentage of positive fluorescent parasites in the sera of naturally infected and noninfected dogs. Microbiological analysis was performed at 2 time points, the first and sixth months, to rule out contamination of stored promastigotes. Taken together, our results indicated that the best conditions to preserve fixed L. infantum antigens were storage in formaldehyde at 4 °C. Promastigotes presented the best morphological profile, with appropriate antigenic stability even at 4 °C, in an inexpensive preservative for a long period of conservation.
Diagnostic microbiology and infectious disease 05/2013; · 2.45 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(12):e82947. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2012; 7(11):e49780. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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).
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.
[show abstract][hide abstract] ABSTRACT: A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was evaluated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE and Leishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45 kDa, and were related to the resistance pattern against Leishmania infection. Immunophenotypic analysis revealed increased circulating CD21+ B-cells and CD5+ T-cells, reflected by higher counts of CD4+ and CD8+ T-cells. The observed interaction between potential antigen-presenting cells (evaluated as CD14+ monocytes) and lymphocyte activation status indicated a relationship between innate and adaptive immune responses. The higher frequency in L. chagasi antigen-specific CD8+ T-lymphocytes, and their positive association with intense cell proliferation, in addition to the progressively higher production of serum nitric oxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies on immunological response after challenge with L. chagasi may provide important information that will lead to a better understanding on vaccine trial and efficacy.
[show abstract][hide abstract] ABSTRACT: Cellular and humoral immune responses of dogs to a candidate vaccine, composed of Leishmania braziliensis promastigote protein plus saponin as adjuvant, have been investigated as a pre-requisite to understanding the mechanisms of immunogenicity against canine visceral leishmaniasis (CVL). The candidate vaccine elicited strong antigenicity related to the increases of anti-Leishmania IgG isotypes, together with higher levels of lymphocytes, particularly of circulating CD8(+) T-lymphocytes and Leishmania chagasi antigen-specific CD8(+) T-lymphocytes. As indicated by the intense cell proliferation and increased nitric oxide production during in vitro stimulation by L. chagasi soluble antigens, the candidate vaccine elicited an immune activation status potentially compatible with effective control of the etiological agent of CVL.