Multiplex flow cytometry serology to diagnosis of canine visceral leishmaniasis.

An accurate diagnosis of visceral leishmaniasis is an essential tool for control of the disease. While serologic methods are very useful, these conventional methodologies still present limitations in terms of sensitivity and specificity. The use of flow cytometry is a worldwide trend in the development of high-performance diagnostic methods. Herein, we describe a new flow cytometry serology test, characterized by the employment of the Cytometric Bead Array microspheres A4 and E4 coated with the recombinant antigens rLci1A and rLci2B respectively, to improve the serodiagnosis of canine visceral leishmaniasis. The tests were conducted in a wide variety of sera groups (n = 140), where the diagnostics development would be optimized accounting not just the ability to identify infected dogs with different clinical status, but also to exclude cross-reaction and differentiate vaccinated dogs from dogs infected. Serological testing of the antigenic system A4-rLci1A showed a sensitivity of 90.0% and specificity of 75%, while the E4-rLci2B testing demonstrated a sensitivity of 95.0% and specificity of 82.5%. The use of a multiplex assay of A4-rLci1A and E4-rLci2B, resulted in a diagnostic improvement, with a sensitivity of 95.0% and specificity of 91.2%. Our results show that this novel flow cytometry serology test is a viable tool for sensitive and specific serodiagnosis. Notably, the combination of distinct antigenic systems allows us to test for antibodies to multiple recombinant antigens from a single serum sample. This benefit emphasizes the importance of this methodology as an alternative in the serological diagnosis.

Mixed Formulation of Conventional and Pegylated Meglumine Antimoniate-Containing Liposomes Reduces Inflammatory Process and Parasite Burden in Leishmania infantum-Infected BALB/c Mice.

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.

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs.

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.

Canine visceral leishmaniasis biomarkers and their employment in vaccines.

The natural history of canine visceral leishmaniasis (CVL) has been well described, particularly with respect to the parasite load in different tissues and immunopathological changes according to the progression of clinical forms. The biomarkers evaluated in these studies provide support for the improvement of the tools used in developing vaccines against CVL. Thus, we describe the major studies using the dog model that supplies the rationale for including different biomarkers (tissue parasitism, histopathology, hematological changes, leucocytes immunophenotyping, cytokines patterns, and in vitroco-culture systems using purified T-cells subsets and macrophages infected with L. infantum) for immunogenicity and protection evaluations in phases I and II applied to pre-clinical and clinical vaccine trials against CVL. The search for biomarkers related to resistance or susceptibility has revealed a mixed cytokine profile with a prominent proinflammatory immune response as relevant for Leishmania replication at low levels as observed in asymptomatic dogs (highlighted by high levels of IFN-γ and TNF-α and decreased levels in IL-4, TGF-ß and IL-10). Furthermore, increased levels in CD4+ and CD8+ T-cell subsets, presenting intracytoplasmic proinflammatory cytokine balance, have been associated with a resistance profile against CVL. In contrast, a polyclonal B-cell expansion towards plasma cell differentiation contributes to high antibody production, which is the hallmark of symptomatic dogs associated with high susceptibility in CVL. Finally, the different studies used to analyze biomarkers have been incorporated into vaccine immunogenicity and protection evaluations. Those biomarkers identified as resistance or susceptibility markers in CVL have been used to evaluate the vaccine performance against L. infantum in a kennel trial conducted before the field trial in an area known to be endemic for visceral leishmaniasis. This rationale has been a guiding force in the testing and selection of the best vaccine candidates against CVL and provides a way for the veterinary industry to register commercial immunobiological products.

Setting the proportion of CD4+ and CD8+ T-cells co-cultured with canine macrophages infected with Leishmania chagasi.

New methods for evaluating the canine immune system are necessary, not only to monitor immunological disorders, but also to provide insights for vaccine evaluations and therapeutic interventions, reducing the costs of assays using dog models, and provide a more rational way for analyzing the canine immune response. The present study intended to establish an in vitro toll to assess the parasitological/immunological status of dogs, applicable in pre-clinical trials of vaccinology, prognosis follow-up and therapeutics analysis of canine visceral leishmaniasis. We have evaluated the performance of co-culture systems of canine Leishmania chagasi-infected macrophages with different cell ratios of total lymphocytes or purified CD4(+) and CD8(+) T-cells. Peripheral blood mononuclear cells from uninfected dogs were used for the system set up. Employing the co-culture systems of L. chagasi-infected macrophages and purified CD4(+) or CD8(+) T-cell subsets we observed a microenvironment compatible with the expected status of the analyzed dogs. In this context, it was clearly demonstrated that, at this selected T-cell:target ratio, the adaptive immune response of uninfected dogs, composed by L. chagasi-unprimed T-cells was not able to perform the in vitro killing of L. chagasi-infected macrophages. Our data demonstrated that the co-culture system with T-cells from uninfected dogs at 1:5 and 1:2 ratio did not control the infection, yielding to patent in vitro parasitism (≥ 80%), low NO production (≤ 5 µM) and IL-10 modulated (IFN-γ/IL-10 ≤ 2) immunological profile in vitro. CD4(+) or CD8(+) T-cells at 1:5 or 1:2 ratio to L. chagasi-infected macrophages seems to be ideal for in vitro assays. This co-culture system may have great potential as a canine immunological analysis method, as well as in vaccine evaluations, prognosis follow-up and therapeutic interventions.

Evaluation of change in canine diagnosis protocol adopted by the visceral leishmaniasis control program in Brazil and a new proposal for diagnosis.

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.

Analysis using canine peripheral blood for establishing in vitro conditions for monocyte differentiation into macrophages for Leishmania chagasi infection and T-cell subset purification.

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-ß-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 72 h 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.

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs

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.