RESUMEN
The objectives of this study were to determine the prevalence of enteric verocytotoxigenic E. coli (VTEC) infection in a population of cats in Ontario, and to determine whether an association exists between the presence of VTEC and feline diarrhea. Fecal samples from 179 cats, representing 113 cats with diarrhea and 66 cats with normal feces, were cultured for E. coli. The fecal cultures were screened for verocytotoxin activity with a Vero cell assay. Confirmation of the presence of verocytotoxin (VT) genes was done with polymerase chain reaction (PCR) amplification; the frequency of occurrence of the genes for generic VT, VT1, and VT2 was determined. VTEC-positive samples were defined as those that demonstrated cytotoxicity on the Vero cell assay and yielded E. coli possessing one or more of the VT genes. All VTEC-positive isolates were serotyped. The overall prevalence of enteric VTEC infection in the cats was 12.3% (22/179). Statistical analysis of the case-control data showed no significant association between VTEC infection and diarrheal illness. The majority of the cats with VT-positive E. coli were positive for the presence of the generic VT, rather than for VT1 or VT2; it is therefore possible that a novel verocytotoxin gene may exist in E. coli isolated from cats. Eight VTEC strains were identified by serotyping; 4 of these serotypes have previously been isolated from humans, and 2 from cattle, suggesting that cats may be capable of acting as reservoirs for human and bovine VTEC serotypes.
Asunto(s)
Toxinas Bacterianas/biosíntesis , Enfermedades de los Gatos , Diarrea/veterinaria , Enterotoxinas/biosíntesis , Infecciones por Escherichia coli/veterinaria , Escherichia coli/patogenicidad , Animales , Toxinas Bacterianas/genética , Estudios de Casos y Controles , Gatos , Bovinos , Chlorocebus aethiops , Diarrea/microbiología , Enterotoxinas/genética , Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/fisiopatología , Heces/microbiología , Humanos , Reacción en Cadena de la Polimerasa , Células VeroRESUMEN
The etiology of acute, nonviral diarrhea in dogs is poorly understood. Enterotoxigenic and verotoxigenic Escherichia coli are causal agents of diarrhea in humans, pigs, and cattle, but the association of these toxigenic E. coli with diarrhea in dogs has not been explored to a significant extent. In this study, DNA hybridization and PCR amplification were used to identify the frequency with which the genes for E. coli enterotoxins (STap, STb, and LTI) and verotoxins (VT1 and VT2) occur in association with diarrhea in dogs. Genes for VT1 (8.9%), VT2 (22.2%), STa (26.7%), and STb (4.4%) were identified in E. coli cultured from feces of 20 of 45 dogs (44.4%) with diarrhea. Genes for VT2, STa, and STb were not identified in feces from normal dogs. Genes for VT1 were observed in similar proportions in fecal samples from diarrheic (8.9%) and normal (12.3%) dogs. Heat labile enterotoxin (LTI) was not detected in fecal samples from either diarrheic or normal dogs. Our results suggest that heat stable enterotoxins and VT2 may be causally associated with diarrhea in dogs. Dogs appear to be able to carry VT1-producing E. coli without showing overt signs of disease.
Asunto(s)
Toxinas Bacterianas/genética , Diarrea/veterinaria , Enfermedades de los Perros/microbiología , Enterotoxinas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Enfermedad Aguda , Animales , Toxinas Bacterianas/química , Secuencia de Bases , Diarrea/microbiología , Perros , Enterotoxinas/química , Escherichia coli/química , Heces/microbiología , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/veterinaria , Toxina Shiga IRESUMEN
A simplified dot-blot procedure is described for the detection of fowlpox virus (FPV) in infected monolayers of chicken embryo fibroblasts (CEF) cultured in 96-well microtiter plates. The relative resistance of DNA to hot NaOH, which hydrolyzes other macromolecules including RNA and protein, was exploited to solubilize virus infected cells and denature intracellular DNA in a simple, quick manner. Moreover, there was no need to purify virus or isolate viral DNA from cellular DNA prior to dot blotting. After incubation of CEF with FPV, the extracellular fluid from infected cells was collected for storage in 96-well microtiter plates. The remaining cell monolayers in each well were then solubilized with hot NaOH. The solubilized and denatured DNA was transferred to a nylon membrane using a dot-blot vacuum filtration manifold. Hybridization was carried out with a 32P-labeled FPV DNA probe. With this methodology it was possible to detect specific viral DNA sequences following the infection of cell monolayers with as little as 1 infectious unit per well. The ability to detect specific viral DNA sequences in infected cells, without the need to isolate pure viral DNA, made it possible to analyze large numbers of samples in a single experiment. Moreover, sufficient fowlpox virus was present in the extracellular media from each well for further amplification and analysis of selected samples.
Asunto(s)
ADN Viral/análisis , Fibroblastos/microbiología , Virus de la Viruela de las Aves de Corral/aislamiento & purificación , Animales , Células Cultivadas , Embrión de Pollo , Virus de la Viruela de las Aves de Corral/genética , Hidrólisis , Métodos , Hibridación de Ácido Nucleico , Sensibilidad y Especificidad , Hidróxido de SodioRESUMEN
Both dietary zinc and copper deficiencies can cause lipid peroxidation in microsomes in rats. The cytochrome P-450 enzyme system can generate active oxygens by uncoupling of the P-450-oxy complex in the catalytic cycle and/or the electron transfer mediated by the NADPH-cytochrome P-450 reductase. The effects of dietary zinc and copper deficiencies on NADPH-dependent H2O2 generation, the catalytic activity of the cytochrome P-450 enzyme with aminopyrine as the substrate and the activity of NADPH-cytochrome P-450 reductase were determined. Zinc deficiency caused increased H2O2 production, increased NADPH-cytochrome P-450 reductase activity, decreased aminopyrine demethylation and two- and fivefold increases in iron concentration in lung and liver microsomes, respectively, compared to Zn-adequate, ad libitum--fed controls. Active oxygen generation by uncoupling of the cytochrome P-450 enzyme system and accumulation of iron are thus possible mechanisms by which zinc deficiency causes microsomal lipid peroxidation. Copper deficiency did not affect H2O2 production; however, it caused two- and fourfold increases in iron concentration in lung and liver microsomes, respectively, compared to Cu-adequate, ad libitum--fed controls. The mechanism by which cooper deficiency causes microsomal lipid peroxidation is still unknown but could be related to the observed accumulation of iron.