Avaliação hematológica de cães naturalmente infectados por Leishmania (Leishmania) chagasi submetidos a tratamento com antimoniato de meglumina

The present research was carried out aiming to assess the hematological response of dogs with visceral leishmaniasis submitted to treatment. For this, seven animals naturally infected by Leishmania sp. were submitted to a treatment with 75 mg/kg meglumine antimoniate subcutaneously, 12-12h/3 weeks. In all animals, a complete blood count and bone marrow aspiration biopsy were carried out for a descriptive evaluation at up to seven moments: before the treatment, 30, 60, 90, 120, 150 and 180 days after the start of the treatment. Before the beginning of the experiment hematological alterations were observed in four of the seven dogs (57.1%), among them, nonregenerative anemia, lymphopenia, lymphocytosis and monocytosis. During the course of the experiment the occurrence of leukocytoses, such as left shift neutrophilia and eosinophilia, were observed in some of the animals. Before the beginning of the treatment (M1), the occurrence of erythrocytic hypoplasia was detected by bone marrow cytology in two of the dogs (28.6%). This was reversed through an increase in the amount of erythroid progenitor cells after the administration of meglumine antimoniate. Thus, it can be concluded that the treatment led to normalization of the hematological alterations and recovery of the bone marrow.

Avaliação da reatividade cruzada entre antígenos de Leishmania spp e Trypanosoma cruzi na resposta sorológica de cães pela técnica de imunofluorescência indireta (RIFI)

Visceral Leishmaniasis (VL) is a zoonosis caused by Leishmania spp. protozoa. Dog is the main parasite's reservoir, especially in urban areas. Indirect Immunofluorescent Antibody Test (IFAT) is commonly used for canine visceral leishmaniasis (CVL) diagnosis, by detection of antibodies against the parasite. However, Trypanosoma cruzi (T.cruzi) cross reactions can occur in serological diagnosis. In order to evaluate the occurrence of cross reactions between antigens of Leishmania spp. and T.cruzi, 150 blood samples of CVL epidemiological inquiry positive dogs were collected and sera were tested by IFAT. Dogs were culled at Zoonosis Control Center of Bauru (SP), an endemic area for VL. Another 150 dogs' blood samples were collected in Botucatu (SP), a non endemic area for LV, and sera were also tested by IFAT. Serum samples of dogs from Bauru were positive, both for Leishmania spp. and for T. cruzi, showing high cross-reactions incidence, reinforcing the need of diagnosis confirmation by other tests. Serum samples of dogs from Botucatu demonstrated low prevalence of positive results by IFAT, both for Leishmania spp. and T.cruzi. However, despite the low number of positive serological results for these parasites, the epidemiological and serological investigation for LV and Chagas disease in dogs from Botucatu must be kept, considering the importance of dog as domestic reservoir for both parasites.

Vascularização arterial e venosa do linfonodo poplíteo em cães

Poplíteo lympho nodes in the dogs is placed, to the right and the left in popliteo an appointed space fossa, in distal part of the muscles femoral biceps, laterally and semi-tendinous, medially, projecting in the height of the face volume of the joint to femoro tibial e femoro patellar (joint of the knee). In this study twenty and five dogs, males and females adult, originating the Araçatuba municipal kennel had been used whose captures had been effected by the animal sanitary defense of this city. The arterial vessels destined to this structure always derive, of both the sides, the femoral artery distal volume and vary of 10 the 1, more frequently of 2 (7 times, 28%) to the right and of 6 the 2, equally more frequently of 2 (8 times, 32%). So soon as one has still to right 5 and 6 branches (3 times, 12%), 1, 7 and 10 (1 time, 4%). Relatively to the veins derived from this lympho nodes, always converges to the lateral safena vein, these vessels oscillates between 9 and 2, more frequently of 3 (6 times, 24%), to the right and of 12 the 2, more frequently of 3 (10 times, 40%) to the left. Thus, others deriving branches of popliteo lympho nodes right and that if they insert in the above-mentioned vein are in number of 2 and 5 (5 times, 20%), 4 and 6 (2 times, 8%) and 8 and 9 (1 time, 4%). In spite of, to the left side it is examined in number de 2 branches (6 times, 24%), 4 (4 times, 16%), 6 (3 times, 12%) and finishing 9 and 12 branches (1 time, 4%). The size of popliteo lympho node in seropositive dogs for Visceral Leishmaniasis can meets enters 7,8 × 3,8 × 6,1 a 50,0 × 20,7 × 28,5, in mm, being average 26,18 × 10,5 × 15,97 mm for right and 26,98 × 11,14 × 15,25 mm for left (concerning the measures dorsoventral, latero-lateral and cranial-caudal, respectively).

Avaliação clínica e parasitológica de cães naturalmente infectados por Leishmania (Leishmania) chagasi submetidos a tratamento com antimoniato de meglumina e alopurinol

Aiming to assess the efficacy of the treatment, to verify the occurrence of possible disease relapses and to search for the presence of parasites after the treatment, seven dogs naturally infected by Leishmania sp., were submitted to a treatment with meglumine antimoniate and allopurinol. For this, lymph node and bone marrow aspiration biopsies were carried out at seven moments. After the end of the six-month observation period all dogs were submitted to euthanasia. Then, spleen and liver imprints and in vitro cultures were carried out to search for amastigote forms of the parasite. All animals presented remission of the symptoms and during all the observation period no dog presented relapse of the disease, although amastigote forms of the parasite were observed in two of the animals at the end of the experiment. Thus, it was possible to conclude that the treatment promotes clinical healing but it does not eliminate the parasites completely.

Detecção de anticorpos anti-leishmania infantum syn chagasi em cães de São José do Rio Preto, São Paulo

Visceral leishmaniasis (VL) has been a widespread zoonosis in São Paulo since 1998, when the first autochthonous canine case was identified in Araçatuba. The aim of this study was to determine the occurrence of anti-Leishmania infantum syn chagasi antibodies in serum samples of 584 dogs from São José do Rio Preto, São Paulo, a non endemic area for the disease. Five dogs (0.86%) seroconverted by ELISA and one (0.17%) by immunochromatography. The indirect immunofluorescent reaction, carried out in 138 animals whose optical densities were above or close to ELISA's cutt-off point, evidenced two dogs (1.45%) with titers above 1:40. Only one dog was serum-reactive on the three techniques. Although there was not a history of displacing this animal to endemic areas, the dog had been acquired in a region with canine and human cases of VL. These results suggests that there were no autochthonous cases of canine VL in this population.

Soroprevalência de anticorpos contra Leishmania infantum e outras espécies de protozoários e rickettsia em cães

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

Subclinical muscle injuries in dogs infected with Leishmania (Leishmania) infantum chagasi

Although canine visceral leishmaniasis (CVL) has been extensively studied, muscular damage due to Leishmania (Leishmania) infantum chagasi infection remains to be fully established. The aim of this study was to describe the electromyographic and histological changes, as well as search for the presence of amastigote forms of Leishmania spp, CD3+ T-lymphocytes, macrophages and IgG in skeletal muscles of dogs with visceral leishmaniasis (VL). Four muscles (triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius) from a total of 17 naturally infected and six healthy dogs were used in this study. Electromyographic alterations such as fibrillation potentials, positive sharp waves and complex repetitive discharges were observed in, at least, three muscles from all infected dogs. Myocyte necrosis and degeneration were the most frequent muscular injury seen, followed by inflammatory reaction, fibrosis and variation in muscle fibers size. Immunohistochemistry in muscle samples revealed amastigote forms in 4/17 (23. 53%), IgG in 12/17 (70. 58%), CD3+ T-lymphocytes in 16/17 (94. 12%) and macrophages in 17/17 (100%) dogs. Statistically positive correlation was observed between: inflammatory infiltrate (p=0. 0305) and CD3+ immunoreaction (p=0. 0307) in relation to the number of amastigote forms; inflammatory infiltrate (p=0. 0101) and macrophage immunoreaction (p=0. 0127) in relation to the amount of CD3+; and inflammatory infiltrate (p=0. 0044) and degeneration/necrosis (p<0. 0001) in relation to the presence of macrophages. Our results suggest that different mechanisms contribute to the development of myocytotoxicity, including celular and humoral immune responses and direct muscular injury by the parasite. Nevertheless, the catabolic nature of the disease can probably interact with other factors, but cannot be incriminated as the only responsible for myositis.

Alterações eletromiográficas e histopatológicas da musculatura de cães naturalmente infectados por Leishmania infantum

The aim of this study was to evaluate the electromyographic and histopathological changes in skeletal muscles of dogs naturally infected by L. infantum. Twenty five mixed breed adult dogs with parasitological, molecular and serological diagnosis were selected. The evaluated muscles were: triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius. One dog had locomotor clinical signs with hind limbs paresis associated with severe muscle atrophy. Twenty-three (92%) had some type of muscular change, and in 22 (88%) such changes were directly identified by electromyography. Even without any clinical signs of the disease, 10 (40%) dogs had electromyographic and histopathological changes. Leishmania antigens were detected in muscles of four (16%) dogs. The electromyographic evaluation indicated the occurrence of chronic polymyositis in 13 (52%) dogs, the presence of both acute and chronic muscle inflammation four (16%), acute myopathy in two (8%) and absence of electromyographic abnormalities in three (12%) dogs. The most frequently observed histopathological changes were degeneration and necrosis of myofibers and inflammatory infiltration observed in 12 (48%) dogs. Other changes were decreased diameter of muscle fibers in 15 (60%) and peri or endomysial fibrosis in 14 (56%) animals. The changes observed in the present study showed that even in the absence of clinical signs, most dogs infected by Leishmania infantum have chronic polymyositis.

T and B lymphocytes in the brains of dogs with concomitant seropositivity to three pathogenic protozoans: Leishmania chagasi, Toxoplasma gondii and Neospora caninum

Background: Visceral leishmaniasis is a disease with great variability regarding the clinical manifestations in humans and dogs. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterize the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis. Concomitant with Leishmania chagasi, dogs may be infected by opportunistic pathogens, such as Toxoplasma gondii and Neospora caninum, which may contribute to the occurrence of lesions in the central nervous system. Hence, we aimed to compare the T and B lymphocytes population in the brains of infected dogs with seropositivity to L. chagasi, T. gondii and N. caninum concurrently (n = 24), seropositivity only to L. chagasi (n = 31), and seropositivity to T. gondii and N. caninum (n = 16). Uninfected dogs were used as control (n = 10). Results: Inflammatory lesions, characterised by mononuclear cell accumulation, composed mainly of CD3+ T lymphocytes predominated in several encephalic regions of the dogs from all the three infected groups, with no difference among them (P = 0.0004), whereas CD79α+ B lymphocytes were detected in very small intensity and presented no difference among groups (P = 0.5313). Furthermore, no association among diseases was detected at the serological enquire. Conclusions: We demonstrate that the peripheral infection by L. chagasi per se can promote the influx of lymphocytes within the nervous milieu as occurs during Toxoplasma and Neospora infections, and the concomitant seropositivity against these pathogens does not exacerbate the inflammatory brain lesions. Therefore, these findings give additional support that the brain should be included in the list of organs affected by visceral leishmaniasis and that even asymptomatic infected dogs may develop brain lesions. © 2013 Sakamoto et al.; licensee BioMed Central Ltd.

Genetic Structure of Lutzomyia longipalpis Populations in Mato Grosso Do Sul, Brazil, Based on Microsatellite Markers

Background:Lutzomyia longipalpis (Diptera: Psychodidae) is the major vector of Leishmania (Leishmania) infantum and thus plays a crucial role in the epidemiology of American visceral leishmaniasis (AVL). This vector is the best studied species of sand fly in the Neotropical region. Many studies claim that this vector is in fact a species complex; however there is still no consensus regarding the number of species that belong into this complex or the geographical distribution of sibling species. The aim of the present study was to analyze the genetic relationships within Lu. longipalpis populations in the state of Mato Grosso do Sul (MS), Brazil.Methodology/Principal Findings:We collected 30 Lu. longipalpis (15 females and 15 males) from five localities (Campo Grande, Três Lagoas, Aquidauana, Miranda and Bonito) and 30 Lu. Cruzi from Corumbá, totaling 180 sandflies from MS, and 30 Lu. longipalpis from Estrela de Alagoas, state of Alagoas (AL), Northeast Brazil. We show that eight previously described microsatellite loci were sufficient in distinguishing Lu. longipalpis from Lu. Cruzi, which is a closely related species, and in differentiating between Lu. longipalpis collected in MS versus Estrela de Alagoas. Analyses of the genotypes revealed introgression between sympatric Lu. longipalpis and Lu. Cruzi.Conclusions/Significance:Our findings support the hypothesis of cryptic species within the Lu. longipalpis complex. Furthermore, our data revealed introgression between Lu. longipalpis and Lu. cruzi. This phenomenon should be further investigated to determine the level and incidence of hybridization between these two species. We also demonstrated that microsatellite markers are a powerful tool for differentiating sand fly populations and species. The present study has elucidated the population structure of Lu. longipalpis in MS and, by extension, the Neotropical Lu. longipalpis complex itself. © 2013 Santos et al.

Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector

We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n= 24) and asymptomatic (n= 14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p= 0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p= 0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p= 0.027) and was directly correlated with antibody levels (p= 0.0379). Age and gender did not influence the transmissibility. Our data show that asymptomatic dogs are highly infective and competent for establishing sand fly infection, indicating their role in maintaining L. (L.) infantum chagasi cycle as well as their involvement in VL spreading in endemic areas. © 2013 Elsevier B.V.

CD95 (FAS) and CD178 (FASL) induce the apoptosis of CD4+ and CD8+ cells isolated from the peripheral blood and spleen of dogs naturally infected with Leishmania spp

Infected dogs are urban reservoirs of Leishmania chagasi, which is a causative agent of visceral leishmaniasis (VL). Dogs exhibit immune suppression during the course of this disease, and lymphocyte apoptosis is involved in this process. To investigate apoptosis and the expression levels of FAS-FAS-associated death domain protein (CD95 or APO-1), FASL-FAS ligand protein (CD178), and TRAIL-TNF-related apoptosis-inducing ligand (CD253) receptors in peripheral blood mononuclear cells and spleen leukocytes from 38 symptomatic dogs with moderate VL and 25 healthy dogs were evaluated by flow cytometry. The apoptosis rate of blood and splenic CD4+ and CD8+ cells was higher in infected dogs than in healthy dogs. The expression levels of FAS and FASL in blood and splenic CD4+ cells were lower in infected dogs than in healthy dogs. FAS expression in CD8+ cells was higher in infected dogs than in healthy dogs; in contrast, FASL expression was lower in infected dogs. The expression of the TRAIL receptor increased only in splenic CD8+ cells from infected dogs. The FAS and FAS-L blocking antibodies confirmed the importance of these receptors in apoptosis. Our results enhance the current understanding of the immune response in dogs infected with L. chagasi, facilitating the future development of therapeutic interventions to reduce lymphocyte depletion. © 2013 Elsevier B.V.

Prevalence of Toxoplasma gondii and Leishmania spp. infection in cats from Brazil

A total of 386 feline blood samples from Brazil were collected and analyzed by the indirect immunofluorescence antibody test (IFAT) for the presence of Toxoplasma gondii and Leishmania spp. antibodies. Specific antitoxoplasma IgG were found in 63 of 386 (16.3%) cats and immunoglobulin G against Leishmania spp. was detected in two serum samples. The overall prevalence was significantly higher in adult cats than in juvenile cats for T. gondii infection. There were no significant differences between positivity and gender or breed. The frequency of T. gondii antibodies found in domestic cats of Brazil suggests active transmission within an urban environment. This study proved the occurrence of two important protozoan zoonosis in felines from Brazilian endemic area for visceral leishmaniasis. © 2013.

Estrutura populacional de Lutzomya longipalpis através da amplificação e sequenciamento do segmento ribossomal 12S de DNA mitocondrial

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

Identificação e análise de polimorfismos no gene que codifica a Lectina ligante de Manose (MBL) na espécie Canis lúpus familiaris

Pós-graduação em Biologia Geral e Aplicada - IBB