RESUMO
The exotoxin SLT-IIeB from the Escherichia coli Ee strain was expressed in E. coli, and the recombinant protein was purified, mixed with the Ee strain, then emulsified with oil-emulsion adjuvants to obtain a mixed subunit bacterin. Groups of Kunming mice were immunized at weeks 0 and 2, and challenged intraperitoneally with the Ee strain at week 4. Antibodies were detected by ELISA and an agglutination test. After the second immunization, the antibody level increased and the rate of immune protection against the Ee strain was 70 and 91.7% in the subunit bacterin and bacterin groups, respectively. Therefore, the mixed subunit bacterin provided good protection against the homologous Ee strain, which provides a basis for further research, into high-efficacy vaccines against porcine edema disease.
Assuntos
Vacinas Bacterianas/administração & dosagem , Edematose Suína/genética , Infecções por Escherichia coli/genética , Toxina Shiga II/genética , Animais , Vacinas Bacterianas/genética , Edematose Suína/tratamento farmacológico , Edematose Suína/patologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/patologia , Imunização , Camundongos , Subunidades Proteicas/genética , Toxina Shiga II/administração & dosagem , Suínos/microbiologiaRESUMO
Shiga toxin type 2 (Stx2), a toxin secreted by Shiga toxin-producing Escherichia coli (STEC), could be one of the causes of maternal and fetal morbimortality not yet investigated. In this study, we examined the effects of Stx2 in rats in the early stage of pregnancy. Sprague-Dawley pregnant rats were intraperitoneally (i.p.) injected with sublethal doses of Stx2, 0.25 and 0.5 ng Stx2/g of body weight (bwt), at day 8 of gestation (early postimplantation period of gestation). Maternal weight loss and food and water intake were analyzed after Stx2 injection. Another group of rats were euthanized and uteri were collected at different times to evaluate fetal status. Immunolocalization of Stx2 in uterus and maternal kidneys was analyzed by immunohistochemistry. The presence of Stx2 receptor (globotriaosylceramide, Gb3) in the uteroplacental unit was observed by thin layer chromatography (TLC). Sublethal doses of Stx2 in rats caused maternal weight loss and pregnancy loss. Stx2 and Gb3 receptor were localized in decidual tissues. Stx2 was also immunolocalized in renal tissues. Our results demonstrate that Stx2 leads to pregnancy loss and maternal morbidity in rats in the early stage of pregnancy. This study highlights the possibility of human pregnancy loss and maternal morbidity mediated by Stx2.
Assuntos
Feto/efeitos dos fármacos , Toxina Shiga II/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Creatinina/sangue , Decídua/patologia , Comportamento de Ingestão de Líquido/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Masculino , Gravidez , Ratos Sprague-Dawley , Toxina Shiga II/administração & dosagem , Útero/efeitos dos fármacos , Útero/patologiaRESUMO
Infection by Shiga toxin-producing Escherichia coli causes hemorrhagic colitis, hemolytic uremic syndrome (HUS), acute renal failure, and also central nervous system complications in around 30% of the children affected. Besides, neurological deficits are one of the most unrepairable and untreatable outcomes of HUS. Study of the striatum is relevant because basal ganglia are one of the brain areas most commonly affected in patients that have suffered from HUS and since the deleterious effects of a sub-lethal dose of Shiga toxin have never been studied in the striatum, the purpose of this study was to attempt to simulate an infection by Shiga toxin-producing E. coli in a murine model. To this end, intravenous administration of a sub-lethal dose of Shiga toxin 2 (0.5 ηg per mouse) was used and the correlation between neurological manifestations and ultrastructural changes in striatal brain cells was studied in detail. Neurological manifestations included significant motor behavior abnormalities in spontaneous motor activity, gait, pelvic elevation and hind limb activity eight days after administration of the toxin. Transmission electron microscopy revealed that the toxin caused early perivascular edema two days after administration, as well as significant damage in astrocytes four days after administration and significant damage in neurons and oligodendrocytes eight days after administration. Interrupted synapses and mast cell extravasation were also found eight days after administration of the toxin. We thus conclude that the chronological order of events observed in the striatum could explain the neurological disorders found eight days after administration of the toxin.
Assuntos
Corpo Estriado/efeitos dos fármacos , Corpo Estriado/ultraestrutura , Toxina Shiga II/toxicidade , Administração Intravenosa , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/ultraestrutura , Corpo Estriado/patologia , Modelos Animais de Doenças , Edema , Masculino , Mastócitos/patologia , Camundongos , Atividade Motora/efeitos dos fármacos , Necrose , Neurônios/efeitos dos fármacos , Neurônios/patologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Oligodendroglia/ultraestrutura , Toxina Shiga II/administração & dosagem , Sinapses/efeitos dos fármacos , Sinapses/patologia , Sinapses/ultraestruturaRESUMO
Shiga toxin-producing Escherichia coli produces watery and hemorrhagic diarrhea, and hemolytic uremic syndrome (HUS) characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. Central nervous system (CNS) complications are observed in around 30% of infant population with HUS. Common signs of severe CNS involvement leading to death include seizures, alteration of consciousness, hemiparesis, visual disturbances, and brain stem symptoms. The purpose of the present work was to study the effects of Shiga toxin 2 (Stx2) in the brain of rats intraperitoneally (i.p.) injected with a supernatant from recombinant E. coli expressing Stx2 (sStx2). Neurological alterations such as postural and motor abnormalities including lethargy, abnormal walking, and paralysis of hind legs, were observed in this experimental model of HUS in rats. Neuronal damage, as well as significant decrease in aquaporin 1 (AQP1) and aquaporin 4 (AQP4) expression levels were observed in the brain of rats, 2 days after sStx2 injection, compared to controls. Downregulation of aquaporin protein levels, and neuronal alterations, observed in brain of rats injected with sStx2, may be involved in edema formation and in neurological manifestations characteristic of HUS.
Assuntos
Aquaporina 1/genética , Aquaporina 4/genética , Encéfalo/metabolismo , Infecções por Escherichia coli/genética , Escherichia coli/metabolismo , Síndrome Hemolítico-Urêmica/genética , Neurônios/metabolismo , Toxina Shiga II/metabolismo , Animais , Aquaporina 1/metabolismo , Aquaporina 4/metabolismo , Encéfalo/efeitos dos fármacos , Escherichia coli/genética , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Masculino , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Toxina Shiga II/administração & dosagem , Toxina Shiga II/genética , Toxina Shiga II/toxicidadeRESUMO
Infection associated with Shiga toxin-producing Escherichia coli (STEC) and subsequent Hemolytic-Uremic Syndrome (HUS) have become relevant in public health since STEC is considered as one of the most important emergent pathogens. STEC infection may either be asymptomatic or begin with watery diarrhea associated with hemorrhagic colitis and HUS. The major virulence factor of STEC is Shiga toxin type 1 or 2 (Stx1, Stx2) although strains that express only Stx2 are highly prevalent. Up to now, it has not been established whether STEC infection affect pregnant women. In this study, we evaluated the effect of Stx2 on maternal lethality, fetal status and delivery time by injecting Stx2 in rats in the late stage of pregnancy. Stx2 induced fetal resorption, placental abruption, intrauterine hemorrhage and fetal death at 1-2 days post-injection in a dose-dependent manner. With 2ng Stx2/g body weight, placentas and fetuses presented extensive necrotic areas, while uteri and kidneys showed normal histology. Immunolocalization of Stx2 was observed in placentas and fetuses. With 4 and 6ng Stx2/g body weight maternal death was also observed. Those rats that survived after Stx2-treatment were able to become pregnant and deliver normal pups at term. Our results show, for the first time, that the preterm labor with fetal death observed in treated rats may be a consequence of the action of Stx2 on the feto-maternal unit. Although there are no reports of Stx2 effects in human pregnancy, we speculate that STEC infections could be one of the causes not yet determined of fetal morbimortality.
Assuntos
Morte Fetal/induzido quimicamente , Nascimento Prematuro/induzido quimicamente , Toxina Shiga II/farmacologia , Feto Abortado/efeitos dos fármacos , Animais , Feminino , Idade Gestacional , Injeções Intraperitoneais , Masculino , Gravidez , Nascimento Prematuro/mortalidade , Ratos , Ratos Sprague-Dawley , Toxina Shiga II/administração & dosagem , Toxina Shiga II/metabolismo , Análise de Sobrevida , Útero/metabolismo , Útero/patologiaRESUMO
Shiga toxin (Stx) from enterohemorrhagic Escherichia coli (STEC) is the main cause of hemorrhagic colitis which may derive into Hemolytic Uremic Syndrome (HUS) and acute encephalopathy, one of the major risk factors for infant death caused by the toxin. We have previously demonstrated that intracerebroventricular administration of Stx2 causes neuronal death and glial cell damage in rat brains. In the present work, we observed that the intracerebroventricular administration of Stx2 increased the expression of glial fibrillary acidic protein (GFAP) leading to astrogliosis. Confocal microscopy showed reactive astrocytes in contact with Stx2-containing neurons. Immunocolocalization of increased GFAP and Stx2 in astrocytes was also observed. This insult in the brain was correlated with changes in the expression and activity of neuronal nitric oxide synthase (nNOS) by using the NADPH-diaphorase histochemical technique (NADPH-d HT). A significant decrease in NOS/NADPH-d-positive neurons and NOS/NADPH-d activity was observed in cerebral cortex and striatum, whereas an opposite effect was found in the hypothalamic paraventricular nucleus. We concluded that the i.c.v. administration of Stx2 promotes a typical pattern of brain injury showing reactive astrocytes and an alteration in the number and activity of nNOS/NADPH-d. According to the functional state of nNOS/NADPH-d and to brain cell morphology data, it could be inferred that the i.c.v. administration of Stx2 leads to either a neurodegenerative or a neuroprotective mechanism in the affected brain areas. The present animal model resembles the encephalopathy developed in Hemolytic Uremic Syndrome (HUS) patients by STEC intoxication.
Assuntos
Química Encefálica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/biossíntese , Óxido Nítrico Sintase Tipo I/biossíntese , Toxina Shiga II/toxicidade , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/genética , Imuno-Histoquímica , Injeções Intraventriculares , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , NADPH Desidrogenase/metabolismo , Neostriado/fisiologia , Óxido Nítrico Sintase Tipo I/genética , Células Piramidais/efeitos dos fármacos , Células Piramidais/enzimologia , Células Piramidais/metabolismo , Ratos , Ratos Sprague-Dawley , Toxina Shiga II/administração & dosagem , Toxina Shiga II/isolamento & purificaçãoRESUMO
Shiga toxin (Stx) from enterohemorrhagic Escherichia coli (STEC) is the main cause of hemorrhagic colitis which may derive to hemolytic-uremic syndrome (HUS). HUS is characterized by acute renal failure, thrombocytopenia and microangiopathic hemolytic anemia. Mortality in the acute stage has been lower than 5% of total affected argentine children with endemic HUS. Common signs of severe CNS involvement leading to death included seizures, alteration of consciousness, hemiparesis, visual disturbances, and brainstem symptoms. The main purpose of the present work was to study the direct involvement of Stx2 in brain cells by intracerebroventricular (i.c.v.) administration of Stx2. Immunodetection of Stx2 was confirmed by immunoelectron cytochemistry in different subsets and compartments of affected caudate putamen cells of corpus striatum. Transmission electron microscopy (TEM) studies revealed apoptotic neurons, glial ultrastructural alterations and demyelinated fibers. The i.c.v. microinfusion was applied for the first time in rats to demonstrate the direct action of Stx2 in neurons and glial cells. The toxin may affect brain neuroglial cells without the involvement of proinflammatory or systemic neurotoxic elements.