LaSap vaccine: Immunotherapy and immunochemotherapy associated with allopurinol in dogs naturally infected with Leishmania infantum.

Canine visceral leishmaniasis is a parasitic zoonosis that has a profound impact on public health in countries where it is endemic. Chemotherapeutic treatments cannot keep dogs stable for long periods, and the risk of generating parasitic resistance must be considered. Forty-four symptomatic and naturally infected dogs with Leishmania infantum were tested with two treatment protocols (i) immunotherapy with LaSap vaccine and (ii) immunochemotherapy with LaSap vaccine plus allopurinol. At 90 days after the end of the treatment, it was verified that, although both protocols had generated significant clinical improvements with a greater production of IFN-γ/IL-10, in relation to the parasite load, mainly in the skin, the dogs treated only with immunotherapy maintained the same profile. These results indicate that LaSap is a good strategy to control dog parasitism.

Dogs with canine visceral leishmaniasis have a boost of extracellular vesicles and miR-21-5p up-expression.

This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra-centrifuged to recover EVs (Can-EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-poli-acrylammide gel eletroforesis (SDS-PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum-EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can-EVs from dogs with CanVL (7.78 × 1010 Can-EVs/mL) were higher (p < .0001) than the non-infected dogs (mean: 1.47 × 1010 Can-EVs/mL). These results suggested that concentrations of Can-EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR-21-5p, miR-146a-5p, miR-125b-5p, miR-144-3p, miR-194-5p, miR-346, miR-29c-3p, miR-155-5p, miR-24-3p, miR-181a-5p, and miR-9-5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up-expressed miR-21-5p and miR-146a-5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can-EVs, as well as, the up-expression of miR-21-5p and miR-146a-5p in infected dogs.

Down regulation of IL-10 and TGF-ß1 mRNA expression associated with reduced inflammatory process correlates with control of parasitism in the liver after treatingL. infantuminfected dogs with the LBMPL vaccine therapy.

The liver plays an important role in human and canine visceral leishmaniasis, then it is considered as target to understand the mechanisms involved in the parasite control and a parameter to assess therapeutic responses. In this sense, our study focuses on evaluating the major alterations in the liver by histological (morphometric parenchyma inflammation/semi-quantitative portal inflammation), immunohistochemical assays (parasitism), and qPCR (parasitism and cytokine gene expression) in Leishmania infantum naturally infected dogs and treated with LBMPL vaccine. Animals were divided in four groups: NI group (n = 5): uninfected and untreated dogs; INT group (n = 7): L. infantum-infected dogs and not treated; MPL group (n = 6): L. infantum-infected dogs that received only monophosphoryl lipid A adjuvant, and LBMPL group (n = 10): L. infantum-infected dogs that received treatment with the vaccine composed by L. braziliensis disrupted promastigotes associated with MPL adjuvant. Ninety days after the end of treatments, the dogs were euthanized, and the liver was collected for the proposed evaluations. Significantly lower portal inflammatory reactions, and lower parenchyma inflammation were observed in the LBMPL group compared to INT and MPL groups. iNOS mRNA expression was higher in LBMPL group and in contrast, IL-10 and TGF-ß1 mRNA expression was lower in this group when compared to INT group. Immunohistochemical and qPCR analysis showed significant parasite load reduction in LBMPL group compared to INT and MPL animals. Our data suggest that in naturally Leishmania-infected dogs, LBMPL vaccine reduces the damage in the hepatic tissue, being able to attenuate the type 2 immune response. It could be associated with a marked reduction in the parasitism decreasing liver inflammation in treated dogs. Along with previously obtained data, our results suggest that LBMPL vaccine can significantly contribute to the therapy strategy for L. infantum infected dogs.

Diagnostic Classification of Cases of Canine Leishmaniasis Using Machine Learning.

Proposal techniques that reduce financial costs in the diagnosis and treatment of animal diseases are welcome. This work uses some machine learning techniques to classify whether or not cases of canine visceral leishmaniasis are present by physical examinations. For validation of the method, four machine learning models were chosen: K-nearest neighbor, Naïve Bayes, support vector machine and logistic regression models. The tests were performed on three hundred and forty dogs, using eighteen characteristics of the animal and the ELISA (enzyme-linked immunosorbent assay) serological test as validation. Logistic regression achieved the best metrics: Accuracy of 75%, sensitivity of 84%, specificity of 67%, a positive likelihood ratio of 2.53 and a negative likelihood ratio of 0.23, showing a positive relationship in the evaluation between the true positives and rejecting the cases of false negatives.

Serum antibodies blocked by glycan antigens in canine visceral leishmaniasis serology are mostly IgA immune complexes.

Immune complexes (ICs) are found in canine visceral leishmaniasis (CVL) and interfere with the serum detection of antibodies. Dissociation of these monovalent complexes by dissociative enzyme-linked immunosorbent assay (ELISA) removes false-negative results and allows some characterization of antibodies and antigens. We studied the serology of dogs with suspected CVL in an endemic area, testing two Leishmania (Leishmania) [L. (L.)] infantum antigens. We analysed the presence of immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) antibodies specific to promastigote soluble extract (PSE) and low-molecular weight glycans (glycan­bovine serum albumin (BSA) complex ­ GBC) by conventional and dissociative ELISA. Our results showed a significant fraction of IgA ICs (46.5% for PSE and 47.6% for GBC), followed by IgG ICs (10% for PSE and 23.5% for GBC). IgM ICs were more frequent for PSE (22.7%). Hypergammaglobulinaemia in CVL would be related to the presence of IgA and IgG ICs, resulting in deficient elimination of these antibodies. Our data confirmed the presence of ICs that can generate false-negative results in conventional serology. The production of IgA antibodies and the high frequency of blockade by glycan antigens suggest the active participation of this immunoglobulin and its ICs in the immunopathology of CVL, indicating a new path for further research.

Immunohistochemical study of renal fibropoiesis associated with dogs naturally and experimentally infected with two different strains of Leishmania (L.) infantum.

The objectives of this work were to study some pathological aspects of kidneys obtained from dogs naturally infected with Leishmania infantum and from dogs experimentally infected with two different strains of L infantum with special emphasis on fibrotic process. Seventy eight specimens of paraffin-embedded kidney fragments were collected as follows: (a) CNI group composed by 62 kidney samples of adult mongrel dogs, naturally infected with L infantum; (b) BH401 group composed by five kidney samples of adult Beagles experimentally infected with L infantum strain MCAN BR/2002/BH401; (c) BH400 group composed by eleven kidney samples of adult Beagles experimentally infected with L infantum strain MCAN/BR/2000/BH400, at the same dose and same route of the previous group, denominated group BH400; Control group (CC) composed by four kidney samples of adult Beagles. All animals revealed glomerular and interstitial fibropoiesis associated with different types of glomerulonephritis and chronic interstitial nephritis. Fibrosis was markedly more intense in the BH401 group, followed by animals in the CNI group. Markers for myofibroblasts (mesenchymal markers) such as alpha-actin (α-SMA), vimentin and the cytokine transforming growth factor beta (TGF-ß) were done by immunohistochemistry. BH401 group showed higher expression of all these markers than others. Intracellular amastigotes forms of Leishmania was mainly found in BH401. These results could be indicating that the MCAN/BR/2002/BH401 strain is a good choice for the study of renal LVC experimental model.

Canine visceral leishmaniasis: Detection of Leishmania spp. genome in peripheral blood of seropositive dogs by real-time polymerase chain reaction (rt-PCR).

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoa of the genus Leishmania. Among the species, L. infantum and/or L. infantum (chagasi) are the most important species affecting the Americas. Domestic dogs are the main reservoir of the parasite and participate effectively in the parasite' transmission cycle. The Canine Visceral Leishmaniasis Control Program (PCLV) adopted in Brazil present as strategies the vector control, health education and serological diagnosis of CVL in dogs followed by culling of the seropositive ones. The resolution to eliminate seropositive dogs by euthanasia, when necessary, are the most controversial and least accepted by society. The diagnostic methods for canine visceral leishmaniasis, currently indicated and approved in Brazil by the Ministry of Health from Brazil are the Dual Path Platform (DPP)® as a screening test and the Enzyme immunoassay test (ELISA®). This study aimed to verify the presence of Leishmania spp. DNA in peripheral blood samples of dogs presenting positive serological results byDPP® and ELISA® tests,throughreal-time polymerase chain reaction (rt-PCR), using the pair of primers 150-152 already described. For this purpose, were collected blood samples from 185 seropositive dogs among them, 41 (22%) exhibited some clinical signal of disease, whereas 144 (78%) was asymptomatic. The animals were also analyzed according to gender, race and hair size. According to the results of rt-PCR, it was observed that among the185 seropositive dogs analyzed, only 132 (71%) presented positive results for CVL and 53 (29%) presented negative results. From this, 41/41 symptomatic dogs were positive (100%), while among the asymptomatic dogs, 91/144 were positive (63, 2%) and 53/144 were negative (36, 8%). Concerning the hair size of seropositive dogs, we found that 41 (22%) had long hair, while 144 (78%) had short hair. No statistical significance occurred between the results of rt-PCR, ELISA and DPP tests and the profile of the animals (gender, size of the dogs and hair size), probably due to the small number of samples and the sampling differences of each profile. But statistical significance occurred between the results of rt-PCR and the clinical evaluation, since the rt-PCR was positive in all symptomatic dogs. Thus, through these results, we reached at the following question, which may contribute to an important current debate: the dogs presenting CVL seropositive diagnosis confirmed by tests distributed by the Ministry of Health were in reality ill or were they seropositive by living in an endemic area of the disease? Would these asymptomatic seropositive dogs spread the disease to the inhabitants even presenting a low parasite charge circulating in the blood.

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.

Autochthonous Outbreak and Expansion of Canine Visceral Leishmaniasis, Uruguay.

We report an outbreak of canine visceral leishmaniasis in Uruguay. Blood specimens from 11/45 dogs tested positive for Leishmania spp. Specimens of Lutzomyia longipalpis sand flies were captured; typing revealed Leishmania infantum. Our findings document an expansion of visceral leishmaniasis to southern South America and risk for vectorborne transmission to humans.

Immunohistochemical study of hepatic fibropoiesis associated with canine visceral leishmaniasis.

Hepatic fibropoiesis has been confirmed in canine visceral leishmaniasis. In fibrotic disease, hepatic stellate cells (HSC) play an important role in fibropoiesis, undergoing activation by TGF-ß to acquire characteristics of myofibroblasts. These cells show extensive capacity for proliferation, motility, contractility, collagen synthesis and extracellular matrix component synthesis. The aim of this work was to identify markers of HSC activation in 10 symptomatic and 10 asymptomatic dogs naturally infected with Leishmania (Leishmania) infantum. Eight uninfected dogs were used as controls. Alpha-actin (α-SMA), vimentin and cytokeratin were investigated by immunohistochemistry as HSC markers. The cytokine TGF-ß in tissue was also evaluated by immunohistochemistry. All infected dogs showed higher numbers of reticular fibres than controls. Fibropoiesis found in infected dogs was always associated with the presence of parasites and chronic granulomatous hepatitis. Positive correlation was found among fibropoiesis, parasite tissue load and expression of α-SMA. There was no correlation between fibropoiesis, vimentin and cytokeratin markers. The expression of cytokine TGF-ß was higher in infected dogs than in controls, but not significantly different between symptomatic and asymptomatic dogs. These results confirm previous work describing the intense hepatic fibropoiesis in dogs naturally infected with Leishmania infantum, but now associated them with overexpression of TGF-ß, where α-SMA may be a superior marker for activated HSC cells in CVL.

A new Leishmania-specific hypothetical protein and its non-described specific B cell conformational epitope applied in the serodiagnosis of canine visceral leishmaniasis.

The serodiagnosis of canine visceral leishmaniasis (CVL) presents problems related to its sensitivity and/or specificity. In the present study, a new Leishmania-specific hypothetical protein, LiHyD, was produced as a recombinant protein (rLiHyD) and evaluated in ELISA experiments for the CVL serodiagnosis. LiHyD was characterized as antigenic in a recent immunoproteomic search performed with Leishmania infantum proteins and the sera of dogs developing visceral leishmaniasis (VL). Aiming to compare the efficacy between whole proteins and synthetic peptides, two linear and one conformational B cell epitopes of LiHyD were synthesized and also evaluated as diagnostic markers. The four antigens were recognized by the sera of dogs suffering VL. On the contrary, low reactivity was observed when they were assayed with sera from non-infected healthy dogs living in endemic or non-endemic areas of leishmaniasis. In addition, no reactivity was found against them using sera from dogs experimentally infected by Trypanosoma cruzi, Babesia canis, or Ehrlichia canis, or sera from animals vaccinated with the Leish-Tec® vaccine, a prophylactic preparation commercially available for CVL prevention in Brazil. As comparative diagnostic tools, a recombinant version of the amastigote-specific A2 protein and a soluble crude Leishmania extract were studied. Both antigens presented lower sensitivity and/or specificity values than the LiHyD-based products. The rLiHyD presented better results for the CVL serodiagnosis than its linear epitopes, although the peptide recreating the conformational epitope resulted also appropriate as a diagnostic marker of CVL. To the best of our knowledge, this is the first study showing the use of a conformational epitope derived from a Leishmania protein for serodiagnosis of CVL.

Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples.

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.

A potential link among antioxidant enzymes, histopathology and trace elements in canine visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is a severe and fatal systemic chronic inflammatory disease. We investigated the alterations in, and potential associations among, antioxidant enzymes, trace elements and histopathology in CVL. Blood and tissue levels of Cu-Zn superoxide dismutase, catalase and glutathione peroxidase were measured in mixed-breed dogs naturally infected with Leishmania infantum chagasi, symptomatic (n = 19) and asymptomatic (n = 11). Serum levels of copper, iron, zinc, selenium and nitric oxide, and plasma lipid peroxidation were measured. Histological and morphometric analyses were conducted of lesions in liver, spleen and lymph nodes. We found lower blood catalase and glutathione peroxidase activity to be correlated with lower iron and selenium respectively. However, higher activity of Cu-Zn superoxide dismutase was not correlated with the increase in copper and decreased in zinc observed in infected animals compared to controls. Organ tissue was characterized by lower enzyme activity in infected dogs than in controls, but this was not correlated with trace elements. Lipid peroxidation was higher in symptomatic than in asymptomatic and control dogs and was associated with lesions such as chronic inflammatory reaction, congestion, haemosiderin and fibrosis. Systemic iron deposition was observed primarily in the symptomatic dogs showing a higher tissue parasite load. Dogs with symptomatic CVL displayed enhanced LPO and Fe tissue deposition associated with decreased levels of antioxidant enzymes. These results showed new points in the pathology of CVL and might open new treatment perspectives associated with antioxidants and the role of iron in the pathogenesis of CVL.

Global Distribution of Canine Visceral Leishmaniasis and the Role of the Dog in the Epidemiology of the Disease.

Visceral leishmaniasis is a disease caused by protozoa of the species Leishmania (Leishmania) infantum (syn = Leishmania chagasi) and Leishmania (Leishmania) donovani, which are transmitted by hematophagous insects of the genera Lutzomyia and Phlebotomus. The domestic dog (Canis familiaris) is considered the main urban reservoir of the parasite due to the high parasite load on its skin, serving as a source of infection for sandfly vectors and, consequently, perpetuating the disease in the urban environment. Some factors are considered important in the perpetuation and spread of canine visceral leishmaniasis (CVL) in urban areas, such as stray dogs, with their errant behavior, and houses that have backyards with trees, shade, and organic materials, creating an attractive environment for sandfly vectors. CVL is found in approximately 50 countries, with the number of infected dogs reaching millions. However, due to the difficulty of controlling and diagnosing the disease, the number of infected animals could be even greater. In the four continents endemic for CVL, there are reports of disease expansion in endemic countries such as Brazil, Italy, Morocco, and Tunisia, as well as in areas where CVL is not endemic, for example, Uruguay. Socio-environmental factors, such as migration, drought, deforestation, and global warming, have been pointed out as reasons for the expansion into areas where it had been absent. Thus, the objective of this review is to address (i) the distribution of CVL in endemic areas, (ii) the role of the dog in the visceral leishmaniasis epidemiology and the factors that influence dog infection and the spread of the disease, and (iii) the challenges faced in the control of CVL.

Synthetic Peptides Selected by Immunoinformatics as Potential Tools for the Specific Diagnosis of Canine Visceral Leishmaniasis.

Diagnosing canine visceral leishmaniasis (CVL) in Brazil faces challenges due to the limitations regarding the sensitivity and specificity of the current diagnostic protocol. Therefore, it is urgent to map new antigens or enhance the existing ones for future diagnostic techniques. Immunoinformatic tools are promising in the identification of new potential epitopes or antigen candidates. In this study, we evaluated peptides selected by epitope prediction for CVL serodiagnosis in ELISA assays. Ten B-cell epitopes were immunogenic in silico, but two peptides (peptides No. 45 and No. 48) showed the best performance in vitro. The selected peptides, both individually and in combination, were highly diagnostically accurate, with sensitivities ranging from 86.4% to 100% and with a specificity of approximately 90%. We observed that the combination of peptides showed better performance when compared to peptide alone, by detecting all asymptomatic dogs, showing lower cross-reactivity in sera from dogs with other canine infections, and did not detect vaccinated animals. Moreover, our data indicate the potential use of immunoinformatic tools associated with ELISA assays for the selection and evaluation of potential new targets, such as peptides, applied to the diagnosis of CVL.

Canine Visceral Leishmaniasis: A Histological and Immunohistochemical Study of Fibropoiesis in Chronic Interstitial Pneumonitis.

We studied some fibrotic aspects of chronic interstitial pneumonitis in the lungs of dogs infected with Leishmania infantum. The lungs of eleven naturally infected dogs, twelve experimentally infected with two distinct strains of L. infantum (BH401 and BH46), and six uninfected (controls) dogs, were analyzed by histological, parasitological, and immunohistochemical studies. Conventional histology (HE), collagen deposition (Gomori's silver staining for reticulin collagen fibers), and immunohistochemistry for myofibroblast characterization were carried out based on the cellular expression of alpha-smooth muscle actin, vimentin, cytokeratin, E-cadherin, snail antigen homologue 1 (SNAI1) (Snail), and the cytokine expression of transforming growth factor-beta (TGF-ß). Parasitological screening was carried out using conventional polymerase chain reaction (PCR) and the immunohistochemical reaction of streptavidin-peroxidase for visualizing Leishmania amastigotes. Dogs naturally infected with L. infantum and experimentally infected with L. infantum BH401 strains showed intense interstitial pneumonitis characterized by thickening of the alveolar septa as a consequence of an intense diffuse and focal (plaques) chronic exudate of mononuclear cells associated with fibrogenesis. The expression of alpha-actin, vimentin, and TGF-ß was higher in the lung interstitium of all infected dogs than in the other two groups (BH46 strain and controls). Moreover, in both the naturally and experimentally infected dog (BH401 strain) groups, the expression of Snail was moderate to intense in contrast to the other groups. Based on these immunohistochemical results, we concluded that mesenchymal cells are active in promoting changes in the extracellular matrix in the lungs of dogs naturally and experimentally infected with L. infantum, but it depends on the virulence of the parasite.

Comparative Study of Colorimetric In Situ Hybridization and Quantitative Real-Time Polymerase Chain Reaction for Diagnosis of Infection by Leishmania infantum in Dogs in Formalin-Fixed and Paraffin-Embedded Skin.

The zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and dogs are reservoirs for this parasite. For the diagnosis of Leishmania at the species level in dogs in formalin-fixed, paraffin-embedded skin (FFPES) samples, colorimetric in situ hybridization (CISH) and quantitative real-time polymerase chain reaction (qPCR) are options, but their sensitivities are not well established. Therefore, the aim of this study was to determine the sensitivity of these two techniques in FFPES for the diagnosis of the L. infantum infection in dogs using culture as the reference standard. The FFPES of 48 dogs with cutaneous infection by L. infantum confirmed by culture and by multilocus enzyme electrophoresis were examined by CISH and qPCR using specific probes for L. infantum. The sensitivities of qPCR, CISH and their combination were, respectively, 77.0%, 58.0% and 83.3%. The sensitivities of qPCR in dogs with and without clinical signs were, respectively, 74.2% and 82.4%. The sensitivities of CISH in dogs with and without clinical signs were, respectively, 61.3% and 52.9%. The CISH and qPCR showed satisfactory sensitivities for the diagnosis of L. infantum in the FFPES of dogs, even in dogs without clinical signs, and their combination increases the sensitivity for this diagnosis.

Pharmacokinetics, Dose-Proportionality, and Tolerability of Intravenous Tanespimycin (17-AAG) in Single and Multiple Doses in Dogs: A Potential Novel Treatment for Canine Visceral Leishmaniasis.

In the New World, dogs are considered the main reservoir of visceral leishmaniasis (VL). Due to inefficacies in existing treatments and the lack of an efficient vaccine, dog culling is one of the main strategies used to control disease, making the development of new therapeutic interventions mandatory. We previously showed that Tanespimycin (17-AAG), a Hsp90 inhibitor, demonstrated potential for use in leishmaniasis treatment. The present study aimed to test the safety of 17-AAG in dogs by evaluating plasma pharmacokinetics, dose-proportionality, and the tolerability of 17-AAG in response to a dose-escalation protocol and multiple administrations at a single dose in healthy dogs. Two protocols were used: Study A: four dogs received variable intravenous (IV) doses (50, 100, 150, 200, or 250 mg/m2) of 17-AAG or a placebo (n = 4/dose level), using a cross-over design with a 7-day "wash-out" period; Study B: nine dogs received three IV doses of 150 mg/m2 of 17-AAG administered at 48 h intervals. 17-AAG concentrations were determined by a validated high-performance liquid chromatographic (HPLC) method: linearity (R2 = 0.9964), intra-day precision with a coefficient of variation (CV) ≤ 8%, inter-day precision (CV ≤ 20%), and detection and quantification limits of 12.5 and 25 ng/mL, respectively. In Study A, 17-AAG was generally well tolerated. However, increased levels of liver enzymes-alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT)-and bloody diarrhea were observed in all four dogs receiving the highest dosage of 250 mg/m2. After single doses of 17-AAG (50-250 mg/m2), maximum plasma concentrations (Cmax) ranged between 1405 ± 686 and 9439 ± 991 ng/mL, and the area under the curve (AUC) plotting plasma concentration against time ranged between 1483 ± 694 and 11,902 ± 1962 AUC 0-8 h µg/mL × h, respectively. Cmax and AUC parameters were dose-proportionate between the 50 and 200 mg/m2 doses. Regarding Study B, 17-AAG was found to be well tolerated at multiple doses of 150 mg/m2. Increased levels of liver enzymes-ALT (28.57 ± 4.29 to 173.33 ± 49.56 U/L), AST (27.85 ± 3.80 to 248.20 ± 85.80 U/L), and GGT (1.60 ± 0.06 to 12.70 ± 0.50 U/L)-and bloody diarrhea were observed in only 3/9 of these dogs. After the administration of multiple doses, Cmax and AUC 0-48 h were 5254 ± 2784 µg/mL and 6850 ± 469 µg/mL × h in plasma and 736 ± 294 µg/mL and 7382 ± 1357 µg/mL × h in tissue transudate, respectively. In conclusion, our results demonstrate the potential of 17-AAG in the treatment of CVL, using a regimen of three doses at 150 mg/m2, since it presents the maintenance of high concentrations in subcutaneous interstitial fluid, low toxicity, and reversible hepatotoxicity.

Diagnosis of Canine Visceral Leishmaniasis by Flow Cytometry Serology using the rMELEISH Multiepitope Antigen Coupled in a Functional Bead.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease, with dogs being the main reservoir of the Leishmania infantum parasite. OBJECTIVE: To develop a new flow cytometry test to diagnosis canine VL (CVL) diagnosis. METHODS: The current study addresses a new flow cytometry test using beads coupled to the multiepitope antigen rMELEISH. RESULTS: In the study set of samples a sensitivity (87.1%) and specificity (89.9%) was observed. Considering the dogs' clinical status, 20/20 (100.0%) of the symptomatic sera tested positive, while 19/22 (86.4%) of the oligosymptomatic and 16/20 (80.0%) of asymptomatic were positive. In the non-infected control, all samples (0/30) tested as negative. In the cross-reaction control, the test was more efficient in dogs infected with L. braziliensis (2/10) and Trypanosoma cruzi (0/10), than those with Babesia canis (4/10) and Ehrlichia canis (4/10). Dogs immunized with different vaccines (Leishmune, Leish-Tec®, or LBSap) did not present serological reactivity. CONCLUSION: The flow cytometry serology through coupling the antigen rMELEISH in functional beads showed high accuracy in diagnosing CVL.

Canine visceral leishmaniasis as a systemic fibrotic disease.

We propose that canine visceral leishmaniasis (CVL) is a systemic fibrotic disease, as evidenced by the wide distribution of fibrosis that we have found in the dogs suffering from chronic condition. The inflammatory cells apparently direct fibrosis formation. Twenty-four cases (symptomatic dogs) were identified from a total of one hundred and five cases that had been naturally infected with Leishmania chagasi and had been documented during an epidemiological survey of CVL carried out by the metropolitan area of the municipality of Belo Horizonte, MG, Brazil. The histological criterion was intralobular liver fibrosis, as has been described previously in dogs with visceral leishmaniasis. In addition to the findings in the liver, here we describe and quantify conspicuous and systemic deposition of collagen in other organs, including spleen, cervical lymph nodes, lung and kidney of all the infected symptomatic dogs. Thus we report that there is a systematic fibrotic picture in these animals, where inflammatory cells appear to direct fibrosis in all organs that have been studied. Therefore we propose that CVL is a systemic fibrotic disease.