Influence of apoptosis on the cutaneous and peripheral lymph node inflammatory response in dogs with visceral leishmaniasis

In canine visceral leishmaniasis (CVL), the abnormalities most commonly observed in clinical examination on the animals are lymphadenomegaly and skin lesions. Dogs are the main domestic reservoir for the protozoon Leishmania (L.) chagasi and the skin is the main site of contamination by the vector insect. Some protozoa use apoptosis as an immunological escape mechanism. The aim of this study was to correlate the presence of apoptosis with the parasite load and with the inflammatory response in the skin and lymph nodes of dogs naturally infected with Leishmania (L.) chagasi. Thirty-three dogs from the municipality of Araçatuba (São Paulo, Brazil) were used, an endemic area for CVL. Muzzle, ear and abdominal skin and the popliteal, subscapular, iliac and mesenteric lymph nodes of symptomatic (S), oligosymptomatic (O) and asymptomatic (A) dogs were analyzed histologically. The parasite load and percentage apoptosis were evaluated using an immunohistochemical technique. Microscopically, the lymph nodes presented chronic lymphadenitis and the skin presented plasmacytic infiltrate and granulomatous foci in the superficial dermis, especially in the ear and muzzle regions. The inflammation was most severe in group S. The parasite load and apoptotic cell density were also greatest in this group. The cause of the lymphoid atrophy in these dogs was correlated with T lymphocyte apoptosis, thus leaving the dogs more susceptible to CVL. The peripheral lymph nodes presented the greatest inflammatory response. Independent of the clinical picture, the predominant inflammatory response was granulomatous and plasmacytic, both in the skin and in the peripheral lymph nodes. The ear skin presented the greatest intensity of inflammation and parasite load, followed by the muzzle skin, in group S. The ear skin area presented a non-significant difference in cell profile, with predominance of macrophages, and a significant difference from group A to groups O and S. It was seen that in these areas, there were high densities of parasites and cells undergoing apoptosis, in group S. The association between apoptosis and parasite load was not significant in the lymph nodes, but in the muzzle regions and at the ear tips, a positive correlation was seen between the parasite load and the density of cells undergoing apoptosis. The dogs in group S had the highest parasite load and the greatest number of apoptotic cells, thus suggesting that the parasite had an immune evasion mechanism, which could be proven statistically in the skin. © 2012 Elsevier B.V.

Pro-inflammatory cytokines predominate in the brains of dogs with visceral leishmaniasis: A natural model of neuroinflammation during systemic parasitic infection

Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. To investigate the pathogenesis of brain alterations occurring during visceral leishmaniasis infection, the expression of the cytokines IL-1β, IL-6, IL-10, IL-12p40, IFN-γ, TGF-β and TNF-α and their correlations with peripheral parasite load were evaluated in the brains of dogs naturally infected with Leishmania infantum. IL-1β, IFN-γ and TNF-α were noticeably up-regulated, and IL-10, TGF-β and IL-12p40 were down-regulated in the brains of infected dogs. Expression levels did not correlate with parasite load suggestive that the brain alterations are due to the host's immune response regardless of the phase of the disease. These data indicate the presence of a pro-inflammatory status in the nervous milieu of dogs with visceral leishmaniasis especially because IL-1β and TNF-α are considered key factors for the initiation, maintenance and persistence of inflammation. © 2012 Elsevier B.V.

Epidemiology of human immunodeficiency virus-visceral leishmaniasis-co-infection

In Brazil, the rates of mother-to-child-transmission (MTCT) of human immunodeficiency virus (HIV) decreased from 20% to 1-2% in some regions. However, the country contains 90% of individuals infected with visceral leishmaniasis (VL) in Latin America, and the west region of São Paulo state faces an alarming expansion of the disease. We describe the epidemiological aspects of the expanding infection of VL and a case report of an HIV-VL-co-infected child from the west region of São Paulo state. The patient was an AIDS-C3 with low levels of CD4, high viral load, severe diarrhea, oral and perineal candidiasis, severe thrombocytopenia, and protein-caloric malnourishment. She evolved with sepsis, renal and cardiac failure. An rK rapid diagnosis test, indirect fluorescent antibody test (IFAT), and bone marrow aspirate were performed for VL. Her symptoms improved significantly after liposomal amphotericin B administration. From the 45 municipalities that compose the Regional Health Department of Presidente Prudente, Lutzomyia longipalpis vectors were found in 58% of them. VL infected dogs were found in 33% of those municipalities, infected dogs and humans were found in 29%, 20% are starting and 33% of the municipalities are preparing VL investigation. It is likely, in this patient, that VL advanced the clinical progression of the HIV disease and the development of AIDS severity. Supported by favorable conditions, the region becomes a new frontier of VL in Brazil. © 2013.

Improvement in clinical signs and cellular immunity of dogs with visceral leishmaniasis using the immunomodulator P-MAPA

This study investigated the immunotherapeutic potential of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-γ in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4+ and CD8+ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-γ. An increase in CD8+ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs. © 2013 Elsevier B.V.

Leishmaniasis causes oxidative stress and alteration of oxidative metabolism and viability of neutrophils in dogs

The aim of the present study was to test the hypothesis that oxidative stress and alteration of oxidative metabolism and apoptosis of neutrophils in dogs vary with the stage of leishmaniasis and to determine the contribution of uremia to such alterations. Dogs with leishmaniasis were classified into two stages: moderate (Leish II, n = 20) or very severe (i.e. with concurrent uremia; Leish IV, n = 20) according to the LeishVet Consensus. The two leishmaniasis groups were compared with uremic dogs without leishmaniasis (Uremic, n = 10) and to healthy dogs (Control, n = 30). To determine oxidative stress, total antioxidant/oxidant capacity, lipid peroxidation, total glutathione and the plasma antioxidants albumin, uric acid and bilirubin were quantified. Superoxide production was determined using the hydroethidine probe and viability and apoptosis were measured using annexin V-PE by capillary flow cytometry. Oxidative stress was present in both uremia and leishmaniasis with reduced total antioxidant capacity and was associated with increased induced production of superoxide and apoptosis. The greatest amount of oxidants was observed in animals with moderate disease only. Neutrophils from uremic dogs with and without leishmaniasis had decreased viability and an increased apoptosis rate in addition to increased lipid peroxidation. In conclusion, oxidative stress occurs in both stages of leishmaniasis with differences in intensity and levels of plasma markers; however, uremia does contribute to the decreased spontaneous viability of neutrophils in dogs in the final stage of the disease. © 2013 Elsevier Ltd. All rights reserved.

Longitudinal analysis of serological tests officially adopted by the Brazilian Ministry of Health for the diagnosis of canine visceral leishmaniasis in dogs vaccinated with Leishmune®

Development of vaccines against canine visceral leishmaniasis (CVL) may provide a prophylactic barrier, but antibody response detected by standard diagnostic techniques may not separate vaccinated from naturally infected dogs. Moreover, anti-Leishmania antibody levels in vaccinated dogs may be detectable for months. Accordingly, the aim of the present study was to comparatively evaluate an in-house ELISA with three serological tests officially adopted by the Brazilian Ministry of Health for the diagnosis of CVL in dogs vaccinated with Leishmune®. A total of 18 mongrel dogs were submitted to a complete protocol of the vaccine, monitored and evaluated in 5 times (T0-T4) up to 180 days after T0. Twenty-one days after the first dose (T1), 50% of the dogs were seropositive by the in-house ELISA and 5.5% by IFAT, while by the official ELISA and DPP® CVL rapid test all dogs tested negative. At time T2, 42 days after of the first dose, 100%, 83.3%, 11.1%, and 5.5% of the dogs were seropositive by the in-house ELISA, IFAT, official ELISA kit and the DPP® CVL rapid test, respectively. Ninety days after the first dose (T3), 100%, 83.3%, 72.2% and 33.3% of the dogs were seropositive by the in-house ELISA, official ELISA kit, IFAT, and the DPP® CVL rapid test, respectively. Finally, at time T4, 88.8%, 33.3%, 11.1% and 5.5% of the dogs were seropositive by the in-house ELISA, official ELISA kit, DPP® CVL rapid test and IFAT, respectively. In conclusion, dogs vaccinated with Leishmune® cross-react by an in-house ELISA and by the three official Brazilian serological tests for the diagnosis of canine visceral leishmaniasis up to six months after the first vaccine dose, and may be mistakenly diagnosed and removed. © 2013 Elsevier B.V.

Biochemical and nutritional evaluation of patients with visceral leishmaniasis before and after treatment with leishmanicidal drugs

Introduction Visceral leishmaniasis (VL) is caused by the intracellular protozoan Leishmania donovani complex. VL may be asymptomatic or progressive and is characterized by fever, anemia, weight loss and the enlargement of the spleen and liver. The nutritional status of the patients with VL is a major determinant of the progression, severity and mortality of the disease, as it affects the clinical progression of the disease. Changes in lipoproteins and plasma proteins may have major impacts in the host during infection. Thus, our goal was evaluate the serum total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, glucose, albumin, globulin and total protein levels, as well as the body composition, of VL patients before and after treatment. Methods Nutritional evaluation was performed using the bioelectrical impedance analysis (BIA) to assess body composition. Biochemical data on the serum total cholesterol, HDL, LDL, triglycerides, glucose, albumin, globulin and total protein were collected from the medical charts of the patients. Results BIA indicated that both pre-treatment and post-treatment patients exhibited decreased phase angles compared to the controls, which is indicative of disease. Prior to treatment, the patients exhibited lower levels of total body water compared to the controls. Regarding the biochemical evaluation, patients with active VL exhibited lower levels of total cholesterol, HDL, LDL and albumin and higher triglyceride levels compared to patients after treatment and the controls. Treatment increased the levels of albumin and lipoproteins and decreased the triglyceride levels. Conclusions Our results suggest that patients with active VL present biochemical and nutritional changes that are reversed by treatment.

Evaluation of canine and feline leishmaniasis by the association of blood culture, immunofluorescent antibody test and polymerase chain reaction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of P-MAPA immunomodulator on Toll-like receptor 2, ROS, nitric oxide, MAPKp38 and IKK in PBMC and macrophages from dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CD4+FOXP3+cells produce IL-10 in the spleens of dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological changes and parasite load of the adrenal from dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serological and infection statuses of dogs from a visceral leishmaniasis-endemic area

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Temporal, spatial and spatiotemporal analysis of the occurrence of visceral leishmaniasis in humans in the City of Birigui, State of São Paulo, from 1999 to 2012

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of PCR with stained slides of bone marrow and lymph nodes aspirates with suspect diagnosis for leishmaniasis

Visceral leishmaniasis (VL), also known as kala-azar, is a disseminated protozoan infection caused by Leishmania donovani complex. Traditionally the definite diagnosis is made by amastigote detection in the tissue. The aim this study was to evaluate the PCR technique in stained slides of bone marrow and lymph nodes aspirates with suspect diagnosis for leishmaniasis. Slides were selected totaling 62 suspect cases (33 bone marrow samples and 29 lymph node samples) and 17 positive cases (8 bone marrow and 9 lymph node). From 62 suspect cases, 39 (62.90%) were confirmed to be positive being 17 (n = 29) lymph node aspirates and 22 (n = 33) bone marrow. This finding is in agreement with the higher sensitivity of the PCR assay compared to direct microscopic observation. In conclusion, the findings of this study supports the use of PCR on archive cytological preparation stained slides for the diagnosis of canine visceral leishmaniasis, emphasizing the higher sensitivity of this technique when compared to direct microscopic examination and mostly the use of the suspect status for the cytology samples that presents the previously mentioned particularities with focus on detecting the oligosymptomatic or assymptomatic dogs in endemic areas functioning as potential reservoirs for this disease. (C) 2014 Elsevier B.V. All rights reserved.