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The thymus is a lymphoid organ responsible for the development and maturation of T cells, which are part of the Th1, Th2, Th17, and Treg immune responses triggered by visceral leishmaniasis. The maturation and immunological development of T lymphocytes require a bidirectional interaction between the thymic microenvironment of epithelial cells, dendritic cells, and macrophages and the extracellular matrix with differentiating lymphocytes. We evaluated the morphological characteristics and tissue distribution of hematopoietic and stromal cells in the thymuses of hamsters experimentally infected with Leishmania infantum, aiming to gain an insight into the pathophysiology of the disease. Fifteen hamsters were subjected to intraperitoneal experimental infection with 107 L. infantum promastigotes (MHOM/BR/1972/BH46). The animals were divided into three groups, each comprising five infected hamsters, and were then euthanized 15, 60, and 120 days postinfection. The control groups consisted of three groups of five healthy hamsters euthanized simultaneously with the infected ones. Thymic morphology was evaluated through histopathology and the cell composition through immunohistochemistry. We used antibodies to mark mesenchymal cells (anti-vimentin), epithelial cells (anti-cytokeratin), macrophages (anti-MAC387), B lymphocytes (anti-CD79a), and T lymphocytes (anti-CD3). Immunohistochemistry was also used to mark the parasite in the thymus. Infected and control hamsters showed no difference in thymic morphology and degree of atrophy. After 15 days of infection, CD3 + T lymphocytes in the thymus showed an increase that stabilized over time. At 120 days of infection, we detected a significant decrease in CD79a+ B lymphocytes. The parasite was present in the medullary and corticomedullary regions of 9 out of 15 hamsters. These findings confirm that the presence of a parasite can cause changes in a thymus cell population. However, further studies are needed to evaluate these changes' effects on the immune response of infected animals. • Visceral leishmaniasis affects the composition and maturation of TCD3 + lymphocytes in the thymus of infected hamsters. • A decrease in CD79a+ B lymphocytes occurs after 120 days of Leishmania infantum infection in the thymus. • Amastigote forms of the parasite are present in the organ. [ABSTRACT FROM AUTHOR]

Susceptible dogs suffering from canine leishmaniasis (CanL) develop an ineffective humoral immune response that leads to the formation of circulating immune complexes (CIC). These CIC are aggregates of Leishmania proteins and anti- Leishmania immunoglobulins. Their deposition in different tissues is considered the main cause of mortality. For this reason, CIC have been suggested as an excellent CanL biomarker for measuring the progression of the disease and the effectiveness of specific treatments. The present study aims to perform a laboratory validation of a Leishmania -specific method to isolate and quantify CIC in dog serum samples. CIC isolated from serum samples of infected dogs, grouped according to the LeishVet classification, were quantified following a PEG-ELISA procedure. The validation established a cut-off of 0.274 OD. All the parameters analyzed (including linearity, specificity, precision, and robustness) fulfilled the defined criteria, confirmed by statistical analyses. The results also proved the reproducibility and reliability of the method when samples were tested under the same conditions, and the consistency and usefulness of the method for an optimal staging of infected dogs. In conclusion, the laboratory validated method offers a potent tool to clinicians for a proper CanL management and to measure the progression of the disease. [ABSTRACT FROM AUTHOR]

Abstract: Visceral leishmaniasis (VL) caused in the New World by Leishmania (Leishmania) infantum chagasi has dog as important peridomestic reservoir in its transmission cycle. Since VL in infected animals is similar to human VL, its study is interesting for human pathology. During Leishmania infection, insulin-like growth factor-I (IGF-I) plays a role in the host-parasite interaction, favoring parasite growth, particularly acting directly on Leishmania. We evaluated IGF-I mRNA expression in different organs/tissues, which was differently modulated in dogs naturally infected by L. (L.) infantum chagasi. We also evaluated the hepatic IGF-I mRNA and serum IGF-I levels in infected dogs. Hepatic mRNA IGF-I expression was higher in the infected dogs than in control animals. However, the serum levels of IGF-I, which are related to the production of this factor in the liver, were reduced in the infected dogs compared with the non-infected controls. Thus, we suggest interference in post-transcriptional processing in IGF-I production in active visceral leishmaniasis. [Copyright &y& Elsevier]

Abstract: In this study, we summarized the major phenotypic/functional aspects of circulating leukocytes following canine immunization with Leishvaccine and Leishmune®. Our findings showed that Leishvaccine triggered early changes in the innate immunity (neutrophils and eosinophils) with late alterations on monocytes. Conversely, Leishmune® induced early phenotypic changes in both, neutrophils and monocytes. Moreover, Leishvaccine triggered mixed activation-related phenotypic changes on T-cells (CD4+ and CD8+) and B-lymphocytes, whereas Leishmune® promoted a selective response, mainly associated with CD8+ T-cell activation. Mixed cytokine profile (IFN-γ/IL-4) was observed in Leishvaccine immunized dogs whereas a selective pro-inflammatory pattern (IFN-γ/NO) was induced by Leishmune® vaccination. The distinct immunological profile triggered by Leishvaccine and Leishmune® may be a direct consequence of the distinct biochemical composition of these immunobiological, i.e. complex versus purified Leishmania antigen along with Bacillus Calmette-Guérin (BCG) versus saponin adjuvant. Both immunobiologicals are able to activate phagocytes and CD8+ T-cells and therefore could be considered as a putative vaccines against canine visceral leishmaniasis (CVL). [Copyright &y& Elsevier]

Abstract: Canine visceral leishmaniasis is an important emerging disease with a multitude of clinical symptoms, including neurological alterations. Matrix metalloproteinases (MMP) are proteases implicated with the extracellular matrix remodelling and, within the central nervous system, these enzymes are involved with blood–brain-barrier disruption and inflammation. To establish the involvement of MMP-2 and -9 within the nervous tissue of dogs with spontaneous visceral leishmaniasis, fragments of nervous tissue from oligosymptomatic (n =9), symptomatic (n =8), neurological (n =12) and normal dogs (n =8) were subjected to zymographic and to immunohistochemical analysis. Immunohistochemistry evidenced MMP-2 staining in inflammatory cells inside and outside blood vessels. MMP-9 was found in endothelial cells and in the ependyma. Zymographic evaluation revealed only the latent forms of MMP-2 and -9 within the nervous tissue. ProMMP-9 activity in the infected animals was found higher than the normal dogs, but with no difference among the infected dogs. Oligosymptomatic dogs presented the highest proMMP-2 activity, followed by the symptomatic and then, by the neurological and the normal dogs. In summary, the nervous tissue compartment seems to be preserved in dogs with VL, due to the absence of active MMPs, even though the elevated levels of proMMP-2 and -9 would indicate a pro-inflammatory state in the brain. [Copyright &y& Elsevier]

Abstract: The Leishmune® vaccine has been used in endemic areas to prevent canine visceral leishmaniasis in Brazil, but cytokine production induced by vaccination has rarely been investigated in dogs. This study aimed to evaluate the immune response of dogs vaccinated with Leishmune FML vaccine (Fort Dodge) against total antigen of Leishmania (Leishmania) chagasi (TAg) and FML. Twenty healthy dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniasis area, received three consecutive subcutaneous injection of Leishmune vaccine at 21-day intervals. PBMC were isolated before and 10 days after completing vaccination and lymphoproliferative response and antibody production against FML or total promastigote antigen were tested. Cytokines IFN-γ, IL-4 and TNF-α were measured in culture supernatant and CD4+/CD25+ and CD8+/CD25+ T cell presence was determined. Analysis of the data indicated that the vaccine conferred humoral responses (100%) against both antigens and cellular immunity to FML (85%) and total antigen (80%), the supernatant of cultured cells stimulated with TAg and FML showed an increase in IFN-γ (P <0.05), and the vaccine reduced CD4+/CD25+ T cell presence compared to that observed before vaccination. These responses may constitute part of the immune mechanism induced by Leishmune. [Copyright &y& Elsevier]

Abstract: In murine models of leishmaniasis, IgG subclass expression is a proxy measure for Th1/Th2 cellular immune response bias. However, in dogs, the reservoir of zoonotic visceral leishmaniasis, no consistent association has been described between IgG subclass ratios and disease resistance. Inconsistent results may reflect lack of specificity of commonly used commercial antibodies. Our aim was to measure IgG1 and IgG2 responses to crude Leishmania antigen using commercial polyclonal antibodies for comparison with a panel of commercially unavailable monoclonal antibodies, in a cohort of 60 naturally infected dogs, and to compare associations between subclass responses and clinical or parasitological outcomes. IgG1 and IgG2, measured by both antibodies, were higher in clinically symptomatic than in asymptomatic dogs (P ≤0.03), reflecting general upregulation of IgG in infected dogs. Unlike the murine model, canine IgG2:IgG1 ratios were not predictive of clinical or parasitological outcomes of infection. Associations between subclass levels and positivity by bone marrow culture and PCR were not consistent when measured with different antibodies. Further research is needed to re-evaluate the specificity of commercially available IgG subclass antibodies. [Copyright &y& Elsevier]

Abstract: Vaccine candidates, including live and/or killed parasites, Leishmania-purified fractions, defined recombinant antigens and antigen-encoding DNA-plasmids have been proposed to use as vaccine anti-Leishmania. More recently, the hamsters have been used to pre-selection of antigens candidate to apply in further experiments using canine model. In this report we evaluated the kinetics of cell migration in dermal inflammatory infiltrate, circulating leukocytes and the presence of nitric oxide (NO)/induced nitric oxide synthase during the early (1–24h) and late (48–168h) periods following inoculation of hamsters with antigenic components of anti-canine visceral leishmaniasis vaccines Leishmune® and Leishmania braziliensis antigen (LB) with and without saponin (Sap) adjuvant. Our results show that LB caused an early reduction of lymphocytes in the dermis while Sap and LBSap triggered a late recruitment, suggesting the role of the adjuvant in the traffic of antigen-presenting cells and the induction of lymphocyte migration. In that manner our results suggest that the kinetics of cell migration on hamster model may be of value in the selection of vaccine antigens prior the tests in dogs particularly in respect of the toxicity of the preparations. [Copyright &y& Elsevier]

Abstract: Human visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL) are the most important emerging diseases with high prevalence in Latin American countries and are mainly caused by Leishmania (L.) chagasi (Syn= L. infantum). CVL has a great impact on Brazilian public health because domestic dogs are the most important VL peri-domicile reservoirs in both urban and peri-urban areas. Our findings highlight the complexity of cellular immunological events related to the natural infection from dogs by L. chagasi, additionally correlating major peripheral blood phenotypic markers with clinical status and tissues parasite density. Our main results demonstrated that lower frequency of circulating B cells and monocytes are important markers of severe CVL, whereas increased levels of CD8+ lymphocytes appear to be the major phenotypic feature of asymptomatic disease. Determination of the isotypes patterns during CVL demonstrated that asymptomatic dogs and those with low parasitism are associated with an increase of IgG1, while the symptomatic dogs and those with high parasitism are associated with an increase of IgG, IgG2, IgM, IgA and IgE immunoglobulins. Pioneer findings obtained by our group showed a correlation between clinical status of CVL with degree of tissue parasite density. This data demonstrated that asymptomatic dogs presented low parasitism while symptomatic dogs are associated with high parasite load in various tissues such as skin, bone marrow and spleen. We have also investigated the association between tissue parasitism and CVL clinical forms. Regardless of clinical status, skin and spleen are the major sites of high parasite density during ongoing CVL. Furthermore, we demonstrated that bone marrow and spleen parasite density are the most reliable parasitological markers to decode the clinical status of CVL. In this article, we have reviewed some aspects of the histopathological and immunological events occurring in natural and experimental L. chagasi/L. infantum infection, pointing out the main L. chagasi-parasitized tissue. We have discussed the importance of the association between parasite density, immunological/histopathological aspects and clinical status of the CVL, their current applications, challenges for the future and potential opportunities in CVL research. [Copyright &y& Elsevier]

Abstract: Cytokines play a decisive role in the regulation of the immune response directed against Leishmania infantum, determining the prevention or progression of the infection. This review focuses on advances in the canine immune response knowledge over the recent years and examines the principal role of the cytokine pattern elicited after infection with L. infantum. [Copyright &y& Elsevier]

Abstract: We have previously reported the applicability of flow cytometry anti-fixed Leishmania infantum chagasi promastigotes IgG (FC-AFPA-IgG) as a novel serological device for laboratorial diagnosis of CVL. Herein, we validate throughout a blind study applied into a broader range of coded sera samples that FC-AFPA-IgG at serum dilution 1:8192 have an outstanding performance to discriminate the serological reactivity of canine visceral leishmaniasis (CVL, n =64) and Leishmune® vaccines (VAC, n =62) and non-infected controls (NI, n =25). Moreover, we have evaluated the performance of indirect immunofluorescence antibody test (IFAT) and the crude-antigen enzyme-linked immunosorbent assay (ELISA) in parallel with FC-AFPA-IgG, to discriminate the seroreactivity of NI, CVL and VAC. Our data demonstrated that both ELISA and FC-AFPA-IgG showed similar performance to detect the seronegativity in 100% of NI, whereas FC-AFPA-IgG displayed better performance to exclude seropositivity in 100% of VAC. The high kappa agreement indexes observed suggested similar performance between these two serological testes when distinct clinical forms of CVL become a challenge. Furthermore, the FC-AFPA-IgG applied at sera dilution 1:8192 showed a remarkable performance to discriminate CVL from other co-endemic canine infections with high co-negativity in dogs infected with Trypanosoma cruzi and Leishmania braziliensis (86% and 84%, respectively). In conclusion, the data presented here re-emphasize the applicability of FC-AFPA-IgG as an innovative methodology able to discriminate post-infection imunomediated seroreactivity from that triggered by prophylactic immunization with minor cross-reactivity with other relevant canine pathogens, which may contribute as a supplementary assay for the CVL immunodiagnosis. [Copyright &y& Elsevier]

Abstract: Nramp1 (Slc11a1) is linked to resistance to Leishmania in mice, but its role in canine leishmaniasis is not clear. In this study we sequenced the Nramp1 cDNA from dogs whose macrophages allowed or restricted intracellular growth of Leishmania chagasi. Peripheral blood monocyte-derived macrophages were isolated from 29 dogs, cultured and inoculated with L. chagasi. This approach resulted in the identification of dogs whose macrophages were resistant or susceptible to L. chagasi. Nramp1 cDNA sequences of these dogs were identical. mRNA levels of Nramp1, IFNγ, IL-4 and the subunit p35 of IL-12 were assessed in the spleen of naturally infected symptomatic and asymptomatic dogs in comparison to uninfected controls. Although not statistically significant, asymptomatic dogs had a tendency for higher levels of Nramp1 mRNA (p =0.11). Expression of Nramp1 was then compared between phenotypically resistant and susceptible dogs, without any significant difference between these groups. [Copyright &y& Elsevier]

Abstract: Associations among parameters commonly used as markers of infection by Leishmania sp., or of susceptibility to visceral leishmaniasis, were investigated in 325 stray dogs from an area where this disease is endemic. Evidence of infection (presence of Leishmania in splenic cultures, positive leishmanin skin test (LST) or detection of anti-Leishmania antibody activity in the serum) was found in 57% of the animals. Both evidence of weight loss (χ 2-test, P =0.0005) and presence of specific antibody activity in the serum (χ 2-test, P <0.0001) were directly associated with positive splenic culture. The frequencies of animals with positive splenic culture were directly correlated with the intensities of antibody activity in the serum as measured by ELISA (relative risk of 3.4 for animals with moderate antibody levels and relative risk of 8.43 for animals with high-antibody levels). A negative association was observed between positive leishmanin skin test results and emaciation (χ 2, P =0.0089). Furthermore, animals with positive splenic cultures and negative leishmanin skin test results had higher levels of total serum IgG (Kruskal–Wallis test, P =0.001) and IgG2 (Kruskal–Wallis test, P =0.05) than animals with negative splenic cultures, and were more emaciated than animals with negative LST results and positive splenic cultures. The data presented herein suggest that associating these common parameters may improve their performance in predicting susceptibility to canine visceral leishmaniasis. [Copyright &y& Elsevier]

Abstract: While enlargement of popliteal lymph nodes (LN) is frequently described in canine visceral leishmaniasis (CVL), there are few histopathologic studies of lymph nodes during this chronic immunopathological condition. Besides a detailed histopathologic analysis, we have characterized the parasite load and major immunophenotypic features of the LN in Leishmania (Leishmania) chagasi-infected dogs. Our major histopathological findings highlight that hypertrophy/hyperplasia of LN cortical and medullary zones was the principal characteristic observed in asymptomatic dogs (AD), whereas atrophy of LN cortical zone was predominant in symptomatic animals (SD). The LN parasite density detected by anti-Leishmania immunohistochemical assay or expressed as Leishman Donovan Units was also highly correlated with the skin parasitism, the most reliable parameter to decode the clinical status of CVL. The major LN immunophenotypic changes during ongoing CVL were an increased frequency of T-lymphocytes, particularly CD8+ T-cells, up-regulation of MHC-II expression by lymphocytes and decreased levels of CD21+ B-cells. Our findings further demonstrated that changes in the LN B-lymphocyte compartment exhibited a negative correlation with the skin parasite load. Conversely, we also showed evidence for a positive association between skin parasitism and LN T-cell-mediated immunity, suggesting that T-cells, especially CD8+ lymphocytes, may have a Type-2 immunological profile in this lymphoid tissue in response to CVL. [Copyright &y& Elsevier]

Abstract: Leishmania promastigotes interact with macrophages through the association of multiple membrane surface receptors. Macrophage complement receptor CR3 (CD11b/CD18 or Mac-1) has been implicated in the interaction of both human and murine macrophages with serum-opsonized promastigotes. The aim of this study was to determine CR3 expression in the livers and spleens of dogs naturally infected with Leishmania (Leishmania) chagasi. CR3 expression in liver was higher in asymptomatic than in symptomatic animals. Moreover, the hepatic parasitism load determined by immunocytochemical analysis was lower in parallel with higher numbers of granulomas. In contrast, in spleens, CR3 expression was higher in symptomatic animals than in asymptomatic ones. However, the tissue parasite load was greater in spleens of symptomatic dogs. There was a strict correlation between the parasite load and cellular CR3 expression in the spleens of dogs naturally infected with L. chagasi. CR3 macrophage integrins could be essential receptors for Leishmania survival. Considering that the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their spleens, we can conclude that these splenic cells (monocyte-macrophages) might serve to perpetuate intracellular infection. [Copyright &y& Elsevier]

Abstract: We investigated the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) during canine visceral leishmaniasis (VL) to gain a better understanding of the role of such multi-functional cytokines in parasite resistance. IL-6 and TNF-α levels were measured by capture ELISA in sera from 8 healthy dogs from a non-endemic area (control group) and in sera from 16 dogs from Araçatuba, SP, Brazil, an area endemic for leishmaniosis. The dogs from the endemic area were selected by positive ELISA serology against total Leishmania chagasi antigen, positive spleen imprints for Leishmania, and the presence of at least three clinical signs associated with active visceral leishmaniasis (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotory difficulty, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). Enhanced systemic IL-6 production was found in sera from dogs with the active disease compared to healthy dogs (t-test, P <0.05). In contrast, TNF-α did not differ between the two groups studied. There was no correlation between IL-6 production and anti-leishmanial antibody titers in the sera. Our findings suggest that IL-6 is a good marker of active disease during leishmaniasis, and that other cytokines may be involved in the hypergammaglobulinemia characteristic of canine visceral leishmaniasis. [Copyright &y& Elsevier]

Abstract: 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-γ and TNF-α 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-γ increase in parasitism (IL-10: r =0.3428; p =0.0438 and INF-γ: 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-γ was related with the increase in parasitic load. [Copyright &y& Elsevier]

Abstract: The role of anti-leishmanial immune response underlying the susceptibility/resistance during canine visceral leishmaniasis (CVL) has been recognized throughout ex vivo and in vitro investigations. Recently, we demonstrated that immunoglobulin levels (Igs), as well as the parasite load are relevant hallmarks of distinct clinical status of CVL. To further characterize and upgrade the background on this issue, herein, we have evaluated, in Leishmania (Leishmania) chagasi naturally infected dogs, the relationship between tissue parasitism (skin, bone marrow, spleen, liver and lymph node), the CVL clinical status (asymptomatic (AD), with no suggestive signs of the disease; oligosymptomatic (OD), with maximum three clinical signs—opaque bristles; localized alopecia and moderate loss of weight; symptomatic (SD), serologically positive with severe clinical signs of visceral leishmaniasis), and the humoral immunological profile of anti-Leishmania immunoglobulins (IgG, IgG1, IgG2, IgM, IgA and IgE). Our major statistically significant findings revealed distinct patterns of tissue parasite density within L. chagasi-infected dogs despite their clinical status, pointing out the spleen and skin as the most relevant sites of high parasitism during ongoing CVL. Parasite density of bone marrow and spleen were the most reliable parasitological markers to decode the clinical status of CVL. Moreover, the parasite density of bone marrow better correlates with most anti-Leishmania Igs reactivity. Additionally, a prognostic hallmark for canine visceral leishmaniasis was found, highlighting strong correlation between IgG1 and asymptomatic disease, but with IgA, IgE and IgG2 displaying better association with symptomatic disease. The new aspects of this study highlighted pioneer findings that correlated the degree of tissue parasite density (low (LP), medium (MP) and high (HP) parasitism) with distinct patterns of anti-Leishmania Igs reactivity. In this scope, our data re-enforce the anti-Leishmania IgG but with IgA reactivity as the better marker for overall tissue parasitism. The association between clinical status, Ig profile and the tissue parasitism support a novel investigation on the impact of humoral immune response and susceptibility/resistance mechanism during ongoing CVL. [Copyright &y& Elsevier]

Abstract: Visceral leishmaniasis (VL) presents vigorous Th2 immune response, which is mainly characterized in human by augmented expression of Il-4, polyclonal B cell activation, intense hypergammaglobulinemia and production of antileishmanial IgE antibodies. However, few aspects of this type of immune response have been demonstrated in studies of canine visceral leishmaniasis (CVL). This work investigated by ELISA and western immunoblotting the production of antileishmanial IgE antibodies (IgE Ab) in symptomatic and asymptomatic dogs naturally infected by Leishmania chagasi, and also compared this IgE immune response with those of IgG, IgG1 and IgG2 antibodies. Three groups of dogs were evaluated: 12 VL dogs with positive Leishmania biopsies (GI), 44 dogs with a positive leishmanial indirect fluorescent antibody test (IFAT), 30 of them presenting clinical signs of VL and 14 asymptomatic (GII) and 21 healthy dogs living in kennels located in leishmaniasis endemic areas (GIII), which were seronegative in the IFAT. Eighteen dogs from an area free of CVL were used as controls (GIV). Antileishmanial IgE antibodies were detected in 4 of 12 VL dogs from group I (33%) and 14 of 30 symptomatic dogs from group II (47%). While all asymptomatic dogs from group II (100%) were seronegative for antileishmanial IgE Ab, 7 of 21 healthy animals from group III (33%) had these immunoglobulins. A strong correlation was verified between antileishmanial IgG and IgG2 antibody titers in all symptomatic dogs, but only 15 of these 42 animals (36%) produced simultaneously IgE, IgG, IgG1 and IgG2 antibodies to Leishmania. IgE antibodies recognized leishmanial antigens of 12, 36, 61, 81 and 118 KDD, while a more complex pattern of immunoblotting was verified mainly for IgG and IgG2 antibodies from symptomatic animals. IgG1 and IgG2 antibodies shared the recognition of L. chagasi polypeptides of 118, 81, 61, 36, 18, 14 and 12 KDD, being more intense the immune reactions between IgG1 Ab and the leishmanial polypeptides of 61 and 36 KDD, and also between IgG2 antibodies and the antigens of 26, 21, 18, 14 and 12 KDD. Our results suggest that the polyclonal production of antileishmanial antibodies that includes IgE Ab could characterize a Th2 immune response in CVL and can help the laboratory diagnosis of this disease. [Copyright &y& Elsevier]

Canine visceral leishmaniasis poses important concerns for public health and veterinary medicine in many areas of the world. Resistance to it seems to be associated with cellular specific immune responses of the so-called Th1 type. Interleukin-12 (IL-12) is one of the most potent inducers of Th1 type of immune responses to co-administered antigens. Herein, the cloning of canine IL-12, as a single-chain fusion protein (sccaIL-12), and its expression in biologically active form in COS-7 cells is reported. Supernatants from these cells stimulated the expression of comparable amounts of interferon gamma mRNA in peripheral blood mononuclear cells from dogs with natural visceral leishmaniasis. In addition, after stimulation with sccaIL-12, there was no difference between interferon gamma mRNA expressions in peripheral blood mononuclear cells of dogs with visceral leishmaniasis and from normal healthy control animals. [Copyright &y& Elsevier]

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an important disease of domestic dogs. Here, we present data on the IgG subclass antibody response to crude L. infantum antigen in a cohort of naturally infected Brazilian dogs. Specific IgG1–IgG4 responses could be detected in 98, 58, 70 and 82%, respectively of 57 dogs that were seropositive for specific IgG. Levels of all IgG subclasses were strongly inter-correlated. Levels of all IgG subclasses increased at the time of seroconversion, before reaching a plateau after several months. Levels of all IgG subclasses were higher in sick dogs than healthy dogs, and levels of all except IgG2 were higher in parasite-positive (by PCR) than parasite-negative dogs. However, levels of IgG2 relative to IgG1 were lower in sick or parasite-positive dogs compared to healthy or parasite-negative infected dogs. In contrast to previous studies, the results suggest that canine VL is associated with upregulation of specific antibody of all IgG subclasses, particularly IgG1, IgG3 and IgG4. [Copyright &y& Elsevier]

Visceral leishmaniasis (VL) is an important zoonotic vector-borne disease and domestic dogs are considered the main domiciliary and peri-domiciliary reservoir of Leishmania (Leishmania) infantum in South America. Distinct eco-epidemiological scenarios associated to the prevalence of the disease, clusters of parasite genotypes and chemotypes of vectors population are described in Brazil, especially in the state of São Paulo (SP). In this context, the purpose of the present study is to evaluate the clinical signs, histopathological lesions, parasite load and cytokine profile by immunohistochemistry (IHC) in popliteal lymph nodes of canines naturally infected with L. infantum , from different municipalities of the state of SP. Eighty-three dogs with VL, 61 from northwest SP (NWSP) and 22 from southeast SP (SESP), were clinically classified in stage II, with no babesiosis and ehrlichiosis. Subcapsular inflammatory infiltration and histiocytosis were significantly higher in the SESP group (p = 0.0128; 0.0077, respectively). On the other hand, dogs from NWSP revealed 4.6-fold significantly higher parasite burden (p = 0.0004) and higher IHC scores of IL-1β (p = 0.0275) and IL-4 (p = 0.0327) in the popliteal lymph node tissues, which may be associated with the susceptibility and progression of the disease in these dogs. Differences in immune response profile associated with higher parasite load in dogs can also contribute to explain the distinct eco-epidemiological patterns of VL in specific geographic regions. • We evaluated clinical signs, parasite load, histopathological lesion and cytokine profile in canine visceral leishmaniasis cases from different eco-epidemiological scenarios. • Higher parasitic load and lower score of IL-4 and IL-1β in popliteal lymph node samples can contribute to explain the different eco-epidemiological scenarios of VL in São Paulo state. • These data improve understanding the host factors in dogs naturally infected with L. infantum. [ABSTRACT FROM AUTHOR]