RESUMO
The emergence of livestock-associated methicillin-resistant Staphylococcus aureus strains (LA-MRSA) and the potential role of pigs in the evolution of these strains has led to increased interest in research of these microorganisms. However, this has contributed to a lack of research in the isolation and characterization of methicillin-susceptible S. aureus strains (MSSA). In this study, the prevalence of S. aureus in pigs in the nursery and finishing stages were analyzed. The susceptibility profiles to antibiotics, tolerance to heavy metals, and biofilm production of the isolates were evaluated using phenotypic and genotypic techniques. A total of 1,250 colonies suggestive of Staphylococcus spp. were isolated from 128 pigs, of which 63.6% (n = 795) belonged to this microbial genus. Sixty-seven colonies isolated from 34 animals (26.5%) were confirmed as S. aureus (8.4%). No strains resistant to copper, zinc, or methicillin were detected; however, all strains presented a resistance profile to at least three different classes of antimicrobials and 21 produced biofilms. These data are of concern, as they indicate the need for increased surveillance in the use of antimicrobials as well as reinforce the importance of studies on MSSA strains.(AU)
A emergência de cepas de Staphylococcus aureus resistentes à meticilina associadas à pecuária (LA-MRSA) e o papel potencial dos suínos na evolução dessas cepas têm levado ao aumento do interesse na pesquisa desses microrganismos. No entanto, isso tem contribuído para a falta de estudos sobre o isolamento e a caracterização de cepas de S. aureus sensíveis à meticilina (MSSA). Neste estudo, foi analisada a prevalência de S. aureus em suínos nas fases de creche e terminação. Os perfis de suscetibilidade aos antibióticos, a tolerância a metais pesados e a produção de biofilme dos isolados foram avaliados por meio de técnicas fenotípicas e genotípicas. Um total de 1.250 colônias sugestivas de Staphylococcus spp. foi isolado de 128 suínos, das quais 63,6% (n = 795) pertenciam a esse gênero microbiano. Sessenta e sete colônias isoladas de 34 animais (26,5%) foram confirmadas como S. aureus (8,4%). Nenhuma cepa resistente ao cobre, ao zinco ou à meticilina foi detectada; entretanto, todas as cepas apresentaram perfil de resistência a pelo menos três classes diferentes de antimicrobianos e 21 produziam biofilme. Esses dados são preocupantes, pois indicam a necessidade de maior vigilância no uso de antimicrobianos, bem como reforçam a importância de estudos com cepas de MSSA.(AU)
Assuntos
Animais , Staphylococcus aureus/isolamento & purificação , Suínos , Fatores de Virulência/análise , Staphylococcus aureus Resistente à Meticilina , Resistência Microbiana a Medicamentos , BiofilmesRESUMO
IgG and IgM deposits in kidneys of dogs with visceral leishmaniasis (VL) were studied in 25 symptomatic dogs (case) and 15 asymptomatic dogs (control) by an immunohistochemical method. All tested dogs were positive for VL by polymerase chain reaction, enzyme-linked immunosorbent assay, and indirect immunofluorescence test. Kidney fragments were submitted to immunohistochemical reaction. Many morphological patterns of distribution of subendothelial granules were identified for IgG and IgM in glomerular capillaries: global, segmental, diffuse, or focal. Intensity of immunohistochemical reaction to IgG was not significantly different when comparing the symptomatic and the asymptomatic animal groups by Fisher's exact test. IgM reactions were significantly different between groups (P<0.01). Deposits of IgM on mesangial cells and in inflammatory interstitial infiltrate were rarely seen, although IgG reactions were frequent at these sites. This study concluded that immunohistochemical reactions for IgM were more intense than those observed for IgG in canine VL, and these reactions were characterized by distribution of subendothelial granules in glomerular capillaries.(AU)
Caracterizou-se a deposição de IgG e IgM em rins de cães com leishmaniose visceral (LV) pelo uso da técnica imunoistoquímica. Foram estudados rins de 25 cães sintomáticos (caso) e de 15 cães assintomáticos (controle). Todos os animais foram positivos para leishmaniose pela reação em cadeia da polimerase, pelo ELISA e pela imunofluorescência indireta. Fragmentos renais foram submetidos à reação imunoistoquímica. Diversos padrões morfológicos de distribuição de grânulos subendoteliais de IgG e IgM foram identificados nos capilares glomerulares: global, segmentar, difuso ou focal. A intensidade da reação imunoistoquímica da IgG, medida pelo teste exato de Fisher não diferiu entre os grupos sintomáticos e assintomáticos e a intensidade de reação da IgM foi diferente entre os grupos (P<0,01). Depósitos de IgM nas células mesangiais e no infiltrado inflamatório raramente foram visualizados, no entanto as reações IgG foram freqüentemente visualizadas nesses locais. Concluiu-se que as reações de IgM foram mais intensas que as reações de IgG na LV canina e caracterizam-se pela distribuição de grânulos subendoteliais nos capilares glomerulares.(AU)
Assuntos
Animais , Imunoglobulina G , Imunoglobulina M , Leishmaniose Visceral , Imuno-Histoquímica/métodos , Rim/anatomia & histologia , Cães/imunologiaRESUMO
IgG and IgM deposits in kidneys of dogs with visceral leishmaniasis (VL) were studied in 25 symptomatic dogs (case) and 15 asymptomatic dogs (control) by an immunohistochemical method. All tested dogs were positive for VL by polymerase chain reaction, enzyme-linked immunosorbent assay, and indirect immunofluorescence test. Kidney fragments were submitted to immunohistochemical reaction. Many morphological patterns of distribution of subendothelial granules were identified for IgG and IgM in glomerular capillaries: global, segmental, diffuse, or focal. Intensity of immunohistochemical reaction to IgG was not significantly different when comparing the symptomatic and the asymptomatic animal groups by Fisher's exact test. IgM reactions were significantly different between groups (P<0.01). Deposits of IgM on mesangial cells and in inflammatory interstitial infiltrate were rarely seen, although IgG reactions were frequent at these sites. This study concluded that immunohistochemical reactions for IgM were more intense than those observed for IgG in canine VL, and these reactions were characterized by distribution of subendothelial granules in glomerular capillaries.
Caracterizou-se a deposição de IgG e IgM em rins de cães com leishmaniose visceral (LV) pelo uso da técnica imunoistoquímica. Foram estudados rins de 25 cães sintomáticos (caso) e de 15 cães assintomáticos (controle). Todos os animais foram positivos para leishmaniose pela reação em cadeia da polimerase, pelo ELISA e pela imunofluorescência indireta. Fragmentos renais foram submetidos à reação imunoistoquímica. Diversos padrões morfológicos de distribuição de grânulos subendoteliais de IgG e IgM foram identificados nos capilares glomerulares: global, segmentar, difuso ou focal. A intensidade da reação imunoistoquímica da IgG, medida pelo teste exato de Fisher não diferiu entre os grupos sintomáticos e assintomáticos e a intensidade de reação da IgM foi diferente entre os grupos (P<0,01). Depósitos de IgM nas células mesangiais e no infiltrado inflamatório raramente foram visualizados, no entanto as reações IgG foram freqüentemente visualizadas nesses locais. Concluiu-se que as reações de IgM foram mais intensas que as reações de IgG na LV canina e caracterizam-se pela distribuição de grânulos subendoteliais nos capilares glomerulares.
RESUMO
SUMMARY: In a screening of 65 derivatives of natural quinones using bloodstream trypomastigotes of Trypanosoma cruzi, the 3 naphthoimidazoles derived from beta-lapachone - N1, N2 and N3--were selected as the most active. Investigation of their mode of action led to the characterization of mitochondrion, reservosomes and DNA as their main targets, and stimulated further studies on death pathways. Ultrastructural analysis revealed both autophagic (autophagosomes) and apoptotic-like (membrane blebbing) phenotypes. Flow cytometry analysis showed, in N2-treated trypomastigotes, a small increase of phosphatidylserine exposure, and a large increase in the percentage of necrosis, caused by N1 or N2. These death phenotypes were not detected in treated epimastigotes. The strong increase in labelling of monodansyl cadaverine, the inhibition of the death process by wortmannin or 3-methyladenine, the overexpression of ATG genes in treated epimastigotes, together with ultrastructural evidence point to autophagy as the predominant phenotype induced by the naphthoimidazoles. However, there are other pathways occurring concomitantly with variable intensities, justifying the need to detail the molecular features involved.
Assuntos
Autofagia/efeitos dos fármacos , Imidazóis/farmacologia , Naftoquinonas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Citometria de Fluxo , Imidazóis/síntese química , Imidazóis/química , Microscopia Eletrônica , Naftoquinonas/síntese química , Naftoquinonas/química , Testes de Sensibilidade Parasitária , Fenótipo , Tripanossomicidas/síntese química , Tripanossomicidas/química , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestruturaRESUMO
Mast cells (MC) secrete diverse pre-stored chemical mediators that are pivotal in inflammatory and fibrotic etiologies, such as Trypanosoma cruzi-induced myocardiopathy. However, due to reduced number of cardiac MC, in situ and in vitro identification, and difficult tissue isolation, these cells are rarely addressed. In this work we optimized the identification of cardiac and peritoneal MC and developed an enzymatic method for MC isolation using control and T. cruzi-infected mice. MC were identified by: toluidine blue (TB); alcian blue (AB)/safranin (S); AB or a mixed solution composed by AB/S/TB. Previous evaluations of cardiac MC in T. cruzi infection were based on TB staining and our results using AB/S/TB solution showed an increase in, at least, five times the detection of MC. This mixed solution may improve the identification of MC populations also from skin, mucosa and tissues that are infected by other pathogens or under the influence of chronic inflammation, leading to more precise results. Furthermore, the appropriate combination of samples (frozen/unfixed/thick slices) and staining protocols can assure the best evaluation of MC. We have also isolated cardiac MC using collagenase and developed a highly efficient 60%/70% Percoll-graded protocol that enriched in, at least, 95% the population of cardiac MC.
Assuntos
Doença de Chagas , Mastócitos/citologia , Miócitos Cardíacos/citologia , Trypanosoma cruzi , Animais , Separação Celular/métodos , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos CBA , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coloração e Rotulagem , Fatores de TempoRESUMO
This study analyses the anti-proliferative effect of lemongrass essential oil and its main constituent (citral) on all 3 evolutive forms of Trypanosoma cruzi. Steam distillation was used to obtain lemongrass essential oil, with chemical composition determined by gas chromatography (GC) and GC coupled to mass spectrometry (GC-MS). The IC50/24 h (concentration that reduced the parasite population by 50%) of the oil and of citral upon T. cruzi was determined by cell counting in a Neubauer chamber, while morphological alterations were visualized by scanning and transmission electron microscopy. Treatment with the essential oil resulted in epimastigote growth inhibition with IC50=126.5 microg/ml, while the IC50 for trypomastigote lysis was 15.5 microg/ml. The IC50/48 h for the Association Index (% macrophage infection x number of amastigotes per cell) was 5.1 microg/ml, with a strong inhibition of intracellular amastigote proliferation. Ultrastructural analysis demonstrated cytoplasmic and nuclear extraction, while the plasma membrane remained morphologically preserved. Our data show that lemongrass essential oil is effective against T. cruzi trypomastigotes and amastigotes, and that its main component, citral, is responsible for the trypanocidal activity. These results indicate that essential oils can be promising anti-parasitic agents, opening perspectives to the discovery of more effective drugs of vegetal origin for treatment of parasitic diseases. However, additional cytotoxicity experiments on different cell lines and tests in a T. cruzi-mouse model are needed to support these data.
Assuntos
Cymbopogon/química , Óleos Voláteis/farmacologia , Extratos Vegetais/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Cymbopogon/toxicidade , Cromatografia Gasosa-Espectrometria de Massas , Concentração Inibidora 50 , Estágios do Ciclo de Vida/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Óleos Voláteis/química , Óleos Voláteis/toxicidade , Extratos Vegetais/química , Extratos Vegetais/toxicidade , Testes de Toxicidade , Tripanossomicidas/química , Tripanossomicidas/toxicidade , Trypanosoma cruzi/ultraestruturaRESUMO
Three naphthoimidazoles presenting aromatic groups attached to the imidazole ring were the most active against trypomastigotes of Trypanosoma cruzi between 45 derivatives from beta-lapachone. N1 is active against the three forms of the parasite. In this work, we investigated N2 and N3 and analyzed the effect of the three derivatives on metacyclogenesis, endocytosis, and cell cycle. In epimastigotes, N2 and N3 blocked the cell cycle, inhibited succinate cytochrome c reductase, metacyclogenesis, and induced damage to mitochondrion, Golgi, and reservosomes. In treated trypomastigotes, there were alterations in the mitochondrion, nucleus and kinetoplast, and DNA fragmentation. Preincubation with cysteine protease inhibitors reversed the effect of N1, N2, and N3. Such reversion and ultrastructural alterations suggest the involvement of autophagy in parasite death. Ultrastructural, flow cytometry, and biochemical studies suggest that naphthoimidazoles interferes with the energetic metabolism and induces DNA fragmentation.
Assuntos
Antiprotozoários/farmacologia , Bignoniaceae/química , Fragmentação do DNA/efeitos dos fármacos , Imidazóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Naftoquinonas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Ciclo Celular/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Imidazóis/síntese química , Imidazóis/química , Concentração Inibidora 50 , Camundongos , Microscopia Eletrônica de Varredura , Naftoquinonas/síntese química , Naftoquinonas/química , Testes de Sensibilidade Parasitária , Trypanosoma cruzi/citologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestruturaRESUMO
Podisus nigrispinus (Dallas) is a common predator in agricultural and natural systems in Neotropical America. Its feeding strategy involves extra-oral digestion and to better understand this process its salivary glands were extracted and subjected to morphological and preliminary enzyme characterization. The salivary glands of P. nigrispinus are formed by a pair of main and accessory gland complexes. The main salivary glands are further divided into an anterior and a posterior lobe. The compartmentalization of the salivary gland complex is likely to be important for the production, activation and release of the digestive enzymes used in the extra-oral digestion of prey items. Proteases and lipase, important digestive enzymes involved in zoophagy, were detected in the salivary glands of P. nigrispinus. The prevailing trypsin-like protease activity was characterized by using the serine-protease substrate N-alpha-benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and the trypsin inhibitors tosyl-L-lysine chloromethyl ketone (TLCK) and benzamidine. The KM value obtained for trypsin-like activity was 1.57 mm and the different peaks of optimum pH and temperature activity suggest the presence of multiple forms of this enzyme in P. nigrispinus. Detection of amylase activity in the salivary glands of this predator suggests its ability to digest starch and obtain nutrients from plants, which may have adaptative value under prey scarcity.
Assuntos
Heterópteros/enzimologia , Heterópteros/ultraestrutura , Amilases/análise , Amilases/metabolismo , Animais , Benzamidinas/farmacologia , Benzoilarginina Nitroanilida/metabolismo , Digestão/fisiologia , Feminino , Heterópteros/fisiologia , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/análise , Lipase/metabolismo , Masculino , Microscopia Eletrônica de Varredura/veterinária , Glândulas Salivares/enzimologia , Glândulas Salivares/ultraestrutura , Serina Endopeptidases/análise , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Temperatura , Tosilina Clorometil Cetona/farmacologiaRESUMO
In human and canine renal histological studies of visceral leishmaniasis (VL), the etiological agent is rarely found in situ. The objective of this study was to evaluate PCR in identifying the etiological agent in spleen, liver, lymph node, and kidneys of VL-seropositive dogs. Twenty-five symptomatic (case group) and 15 asymptomatic (control group) VL-seropositive dogs of different breeds, sexes, and ages from Teresina, Piauí State, Brazil, were used. Serologic diagnosis was made by enzyme-linked immunosorbent assay and indirect immunofluorescence test. Animals were subjected to euthanasia and necropsy. Renal fragments were immersed in buffered formaldehyde solution. Spleen, liver, lymph node, and kidney samples were collected and frozen at -70ºC until DNA extraction. After dehydration and diaphanization, renal fragments were infiltrated and embedded in paraffin, cut at 3 mm, and stained with hematoxylin-eosin (HE). DNA amplification used an automatic thermocycler with specific Leishmania primers. All case-group dogs and 2 controls showed positive results in spleen, liver, or lymph node PCRs. There was a significant difference by Fisher exact test. In symptomatic seropositive dogs, renal histopathological evaluation showed one animal (4%) with amastigote forms of Leishmania in inflammatory infiltrate, and kidney PCRs detected Leishmania DNA in eight animals (32%). The conclusion was that PCR is more precise than the conventional histopathology in detecting the Leishmania parasite in kidney
Assuntos
Animais , Masculino , Feminino , Cães , Cães , Leishmania , Reação em Cadeia da Polimerase/métodos , RimRESUMO
Visceral leishmaniasis (VL) is a zoonosis that affects both animals and man. Dogs are the etiological agents main reservoir. The aim of this study was to evaluate the clinical laboratory aspects and renal histopathology of VL dogs. Thirty-four symptomatic (case) and 17 asymptomatic (control) VL seropositive dogs of different breeds, sexes, and ages from Teresina, Piauí State, Brazil, were used. Diagnosis was confirmed by enzyme-linked immunosorbent assay and indirect immunofluorescence test. Clinical and laboratory tests included blood cell count and renal function analysis (urea and creatinine). Animals were subjected to euthanasia and necropsy. Renal fragments were prepared by the usual histological techniques and stained with hematoxylin-eosin and periodic acid-Schiff. Physical examination showed that lymph node hypertrophy (85.29%) and skin lesions (35.29%) were frequent in the case group. Anemia was found in 55.88% of the case and in 11.76% of the control group. There was a significant difference between groups by Fishers exact test. Two case-group dogs showed azotemia. Renal histopathological evaluation showed that 61.76% case and 17.65% control-group dogs had membranoproliferative glomerulonephritis. Mesangial proliferative glomerulonephritis was seen in 32.35% case and 64.70% control-group animals. There was a significant difference for both types of glomerulonephritis between groups. Amastigote forms of Leishmania were found in the renal parenchyma, in the inflammatory infiltrate of one case-group dog. We concluded that, in canine VL, regardless of the clinical signs at physical examination, the kidneys are frequently compromised