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J L Brunton - One of the best experts on this subject based on the ideXlab platform.
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development of Verotoxin 2 and Verotoxin 2 variant vt2v specific oligonucleotide probes on the basis of the nucleotide sequence of the b cistron of vt2v from escherichia coli e32511 and b2f1
Journal of Clinical Microbiology, 1991Co-Authors: T Morooka, M. A. Karmali, Robert Clarke, S De Grandis, J L BruntonAbstract:We and others have noted that there are serological differences between Verotoxin 2 (VT2) (also known as Shiga-like toxin II) produced by Escherichia coli C600(933W) and the VT2 variant (VT2v) produced by strain E32511. Recent reports have described nucleotide sequence differences between the VT2v B subunit cistron of E32511 and B2F1 and that of VT2. We have confirmed the sequence differences and have used them to design oligonucleotide probes which differentiate the B subunit cistron of VT2v from that of VT2. Isolates of VT-producing E. coli obtained from human as well as food and veterinary sources were classified according to the toxin phenotype by using a toxin neutralization assay with VT2-specific monoclonal antibody and VT2v-specific polyclonal antisera. Using the oligonucleotide probes in colony hybridization, we detected 35 of 35 VT2 producers and 16 of 16 VT2v producers. One VT2 producer was falsely identified as containing the VT2v gene. The E32511 strain in our collection hybridized only with the VT2-specific probe. Southern hybridization of radiolabeled oligonucleotide probes showed that strains carried zero to one copy of the VT2 gene and zero to two copies of the VT2v gene. We conclude that colony hybridization with the VT2- and VT2-specific probes is highly predictive of the toxin phenotypes in the clinical isolates described in this study. Images
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development of Verotoxin 2 and Verotoxin 2 variant vt2v specific oligonucleotide probes on the basis of the nucleotide sequence of the b cistron of vt2v from escherichia coli e32511 and b2f1
Journal of Clinical Microbiology, 1991Co-Authors: J Hii, Carlton L Gyles, M. A. Karmali, T Morooka, Robert Clarke, S De Grandis, J L BruntonAbstract:We and others have noted that there are serological differences between Verotoxin 2 (VT2) (also known as Shiga-like toxin II) produced by Escherichia coli C600(933W) and the VT2 variant (VT2v) produced by strain E32511. Recent reports have described nucleotide sequence differences between the VT2v B subunit cistron of E32511 and B2F1 and that of VT2. We have confirmed the sequence differences and have used them to design oligonucleotide probes which differentiate the B subunit cistron of VT2v from that of VT2. Isolates of VT-producing E. coli obtained from human as well as food and veterinary sources were classified according to the toxin phenotype by using a toxin neutralization assay with VT2-specific monoclonal antibody and VT2v-specific polyclonal antisera. Using the oligonucleotide probes in colony hybridization, we detected 35 of 35 VT2 producers and 16 of 16 VT2v producers. One VT2 producer was falsely identified as containing the VT2v gene. The E32511 strain in our collection hybridized only with the VT2-specific probe. Southern hybridization of radiolabeled oligonucleotide probes showed that strains carried zero to one copy of the VT2 gene and zero to two copies of the VT2v gene. We conclude that colony hybridization with the VT2- and VT2-specific probes is highly predictive of the toxin phenotypes in the clinical isolates described in this study.
Shinichi Yoshida - One of the best experts on this subject based on the ideXlab platform.
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brain lesions in rabbits given an intravenous injection of Verotoxin 2 and protection by anti vt2 antibody
Nihon rinsho. Japanese journal of clinical medicine, 1997Co-Authors: J Fujii, Shinichi YoshidaAbstract:Magnetic resonance imaging was obtained to determine the effects of VT2 toxemia on the rabbit's CNS. The first lesion was noted at 24 h in the hypothalamic area of all experimental animals. The rabbits accompanied with the brain stem lesion were dead within 6 days. We examined the integrity of cerebrospinal fluid-brain barrier (CBB) using a tracer. The tracer was detected throughout the cytoplasm of the ependymal cell layer covering the third ventricle after intrathecal injection of the tracer, which means a deterioration of CBB. Furthermore, we examined whether anti-VT2 antibody injected intrathecally protects rabbits from brain damage. All the rabbits survived when they were given an intrathecal injection of rabbit anti-VT2 antibody 2h before the intravenous injection of VT2.
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magnetic resonance imaging and histopathological study of brain lesions in rabbits given intravenous Verotoxin 2
Infection and Immunity, 1996Co-Authors: J Fujii, Yoshimasa Kinoshita, Toshiro Kita, A Higure, T Takeda, N Tanaka, Shinichi YoshidaAbstract:When rabbits were given intravenously purified Verotoxin 2 (VT2) at 5 microg/kg of body weight, they developed hemorrhagic diarrhea, flaccid paresis, an ataxic gait, an opisthotonic posture, and convulsions. To examine the effects of VT2 toxemia on the rabbit central nervous system, magnetic resonance imaging and ultrastructural studies were performed. At 24, 57, and 80 h after injection of VT2 into 12 rabbits, T2-weighted images of the central nervous system were obtained. The initial lesion was noted at 24 h in the hypothalamic areas of all experimental animals. At 57 h, the T2 value increased in the medulla of the cerebral hemisphere or the hippocampus, with a brain stem lesion in six rabbits (50%). The rabbits with the brain stem lesions, in which neurological signs were very severe, died within 6 days. Lesions in the cerebellar hemisphere and/or vermis were noted in four rabbits (33%) that survived more than 1 month. To better understand the pathogenesis of VT2 in these brain lesions, we examined the deterioration of the blood-brain barrier and cerebrospinal fluid-brain barrier by using horseradish peroxidase as a tracer. The tracer was detected by electron microscopy both in the subendothelial layer, including the basal lamina, and throughout the cytoplasm of the ependymal cell layer covering the ventricle after intravenous or intrathecal treatment with horseradish peroxidase. We also determined the localization of VT2 by immunoelectron microscopy and found that it was localized on edematous endothelial cells of capillaries, ependymal cells, and myelin sheaths. The present study suggests that VT2 was conveyed from the endothelial and ependymal cell layers and caused edematous changes in the rabbit brain.
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direct evidence of neuron impairment by oral infection with Verotoxin producing escherichia coli o157 h in mitomycin treated mice
Infection and Immunity, 1994Co-Authors: J Fujii, Toshiro Kita, T Takeda, N Tanaka, Shinichi Yoshida, H Kobayashi, K Ohsato, Yasuo MizuguchiAbstract:We developed a mouse model of acute encephalopathy induced by Verotoxin 2 variant (VT2v)-producing Escherichia coli. Three-week-old mice were inoculated intragastrically with approximately 10(10) CFU of E. coli O157:H- strain E32511/HSC and simultaneously given an intraperitoneal injection of mitomycin (MMC; 2.5 mg/kg). Drinking water containing 5 g of streptomycin sulfate per liter was given ad libitum from 3 days before the infection. From 1 to 2 days after bacterial inoculation, clinical features including weight loss, weakness, and flaccid paralysis of the extremities developed, usually culminating in death within 4 days. Diarrhea was not observed during the course of disease. No mice died in the absence of streptomycin or MMC treatment for 2 weeks after the oral bacterial infection. Judging from the clinical course and the biochemical and histological examination, the cause of death was not likely to be attributable to renal failure or to a side effect of MMC. To better understand the cause of death, we examined the brain cortex and spinal cord of the moribund mice by electron microscopy. Mice showing mortal symptoms were given horseradish peroxidase intravenously. The tracer was present in the endothelial basal lamina, in the surrounding extracellular spaces, and even in the neuron fibers of the brain cortex. Furthermore, immunoreactivity of VT2v, proved by the use of rabbit anti-VT2 serum, was localized selectively in the damaged myelin sheaths of neuron fibers which were accompanied by edematous axons in the brain cortex and spinal cord. These findings strongly suggest that VT2v is toxic to both endothelial cells and neurons in the central nervous system and subsequently causes fatal acute encephalopathy.
J Fujii - One of the best experts on this subject based on the ideXlab platform.
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brain lesions in rabbits given an intravenous injection of Verotoxin 2 and protection by anti vt2 antibody
Nihon rinsho. Japanese journal of clinical medicine, 1997Co-Authors: J Fujii, Shinichi YoshidaAbstract:Magnetic resonance imaging was obtained to determine the effects of VT2 toxemia on the rabbit's CNS. The first lesion was noted at 24 h in the hypothalamic area of all experimental animals. The rabbits accompanied with the brain stem lesion were dead within 6 days. We examined the integrity of cerebrospinal fluid-brain barrier (CBB) using a tracer. The tracer was detected throughout the cytoplasm of the ependymal cell layer covering the third ventricle after intrathecal injection of the tracer, which means a deterioration of CBB. Furthermore, we examined whether anti-VT2 antibody injected intrathecally protects rabbits from brain damage. All the rabbits survived when they were given an intrathecal injection of rabbit anti-VT2 antibody 2h before the intravenous injection of VT2.
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magnetic resonance imaging and histopathological study of brain lesions in rabbits given intravenous Verotoxin 2
Infection and Immunity, 1996Co-Authors: J Fujii, Yoshimasa Kinoshita, Toshiro Kita, A Higure, T Takeda, N Tanaka, Shinichi YoshidaAbstract:When rabbits were given intravenously purified Verotoxin 2 (VT2) at 5 microg/kg of body weight, they developed hemorrhagic diarrhea, flaccid paresis, an ataxic gait, an opisthotonic posture, and convulsions. To examine the effects of VT2 toxemia on the rabbit central nervous system, magnetic resonance imaging and ultrastructural studies were performed. At 24, 57, and 80 h after injection of VT2 into 12 rabbits, T2-weighted images of the central nervous system were obtained. The initial lesion was noted at 24 h in the hypothalamic areas of all experimental animals. At 57 h, the T2 value increased in the medulla of the cerebral hemisphere or the hippocampus, with a brain stem lesion in six rabbits (50%). The rabbits with the brain stem lesions, in which neurological signs were very severe, died within 6 days. Lesions in the cerebellar hemisphere and/or vermis were noted in four rabbits (33%) that survived more than 1 month. To better understand the pathogenesis of VT2 in these brain lesions, we examined the deterioration of the blood-brain barrier and cerebrospinal fluid-brain barrier by using horseradish peroxidase as a tracer. The tracer was detected by electron microscopy both in the subendothelial layer, including the basal lamina, and throughout the cytoplasm of the ependymal cell layer covering the ventricle after intravenous or intrathecal treatment with horseradish peroxidase. We also determined the localization of VT2 by immunoelectron microscopy and found that it was localized on edematous endothelial cells of capillaries, ependymal cells, and myelin sheaths. The present study suggests that VT2 was conveyed from the endothelial and ependymal cell layers and caused edematous changes in the rabbit brain.
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direct evidence of neuron impairment by oral infection with Verotoxin producing escherichia coli o157 h in mitomycin treated mice
Infection and Immunity, 1994Co-Authors: J Fujii, Toshiro Kita, T Takeda, N Tanaka, Shinichi Yoshida, H Kobayashi, K Ohsato, Yasuo MizuguchiAbstract:We developed a mouse model of acute encephalopathy induced by Verotoxin 2 variant (VT2v)-producing Escherichia coli. Three-week-old mice were inoculated intragastrically with approximately 10(10) CFU of E. coli O157:H- strain E32511/HSC and simultaneously given an intraperitoneal injection of mitomycin (MMC; 2.5 mg/kg). Drinking water containing 5 g of streptomycin sulfate per liter was given ad libitum from 3 days before the infection. From 1 to 2 days after bacterial inoculation, clinical features including weight loss, weakness, and flaccid paralysis of the extremities developed, usually culminating in death within 4 days. Diarrhea was not observed during the course of disease. No mice died in the absence of streptomycin or MMC treatment for 2 weeks after the oral bacterial infection. Judging from the clinical course and the biochemical and histological examination, the cause of death was not likely to be attributable to renal failure or to a side effect of MMC. To better understand the cause of death, we examined the brain cortex and spinal cord of the moribund mice by electron microscopy. Mice showing mortal symptoms were given horseradish peroxidase intravenously. The tracer was present in the endothelial basal lamina, in the surrounding extracellular spaces, and even in the neuron fibers of the brain cortex. Furthermore, immunoreactivity of VT2v, proved by the use of rabbit anti-VT2 serum, was localized selectively in the damaged myelin sheaths of neuron fibers which were accompanied by edematous axons in the brain cortex and spinal cord. These findings strongly suggest that VT2v is toxic to both endothelial cells and neurons in the central nervous system and subsequently causes fatal acute encephalopathy.
Shizuo Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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new application of indirect fluorescent antibody ifa technique using latex particles coupled with Verotoxin 2 from escherichia coli o157 h7 in order to determine colostral antibody titers in immunized dairy cows
Journal of Immunoassay & Immunochemistry, 2014Co-Authors: Tetsuro Seita, Takashi Kuribayashi, Katsunori Furuhata, Seiji Yamaguchi, Shizuo YamamotoAbstract:A simple and novel assay method for determining colostral and serum against soluble verotxin 2 (VT2) titers by indirect fluorescent antibody (IFA) assay using latex sensitized with VT2 was devised. The latex particles did not auto-fluoresce, and non specific reactions disappeared after washing with phosphate buffered saline containing 3 M Nacl. The highest titer measured by neutralizing test was observed at 1 day after delivery. The highest titer for each immunoglobulin class measured by enzyme-linked immunosorbent assay (ELISA) or IFA using latex sensitized with VT2 was also observed at 1 day after delivery. The changes in titer measured by each method showed similar patterns. Furthermore, the titers for IgG antibody were higher than those for IgM or IgA antibodies. Thus, the titers of bovine immune colostral antibody and each immunoglobulin class could be measured by IFA using latex sensitized with VT2.
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comparison of efficacies of bovine immune colostral antibody and each immunoglobulin class against Verotoxin 2 flagellum and somatic cells of escherichia coli o157 h7 in mice
Journal of Microbiology Immunology and Infection, 2013Co-Authors: Tetsuro Seita, Takashi Kuribayashi, Toshio Honjo, Shizuo YamamotoAbstract:Purpose The efficacy of bovine immune colostral (colostral) antibodies against Verotoxin (VT) 2, flagellum and somatic cells of Escherichia coli ( E. coli ) O157:H7 in mice was determined. Methods Three major immunoglobulin (Ig) classes were isolated from the colostral antibody against VT2 by affinity chromatography and were used for estimation. Mice inoculated with VT2 were administered each Ig class from the colostral antibody, colostral antibody (colostral whey containing antibody) or serum antibody against VT2 at 1 hour after VT2 inoculation. Results All control mice (20/20) died after administration of sterilized saline instead of the colostral antibody. The survival rate was 93.3% (14/15) after administration of S-IgA or IgM antibody, or colostral antibody. Survival rates for IgG antibody and serum antibody administration were 80% (12/15) and 60% (9/15), respectively. Serum concentrations of VT2, which was absorbed from the small intestine in mice after administration of VT2 and colostral antibody, were measured by fluorescence enzyme immunoassay (FEIA). Serum concentrations of VT2 after administration of colostral antibody were lower than those after administration of sterilized saline. Mice inoculated with VT2-producing E. coli 157:H7 were administered anti-flagellum or anti-somatic colostral antibodies. Survival rates for E. coli O157:H7-infected mice administered the anti-flagellum and anti-somatic colostral antibodies were 52.4% (11/21) and 22.2% (4/18), respectively. Furthermore, survival rates increased to 89.5% (17/19) with combined administration of anti-flagellum and anti-VT2 colostral antibodies. Conclusion These results suggest that colostral antibodies against VT2, flagellum and somatic cells are effective against E. coli O157:H7 infection.
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bovine colostral antibody against Verotoxin 2 derived from escherichia coli o157 h7 resistance to proteases and effects in beagle dogs
Comparative Medicine, 2009Co-Authors: Takashi Kuribayashi, Tetsuro Seita, Mariko Matsumoto, Katsunori Furuhata, Kazutoshi Tagata, Shizuo YamamotoAbstract:In July 1996, a widespread outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection occurred among schoolchildren in Sakai, Japan, followed by numerous other similar outbreaks of food poisoning throughout the country.4,19 Escherichia coli O157:H7 infection is monitored in Japan, in accordance with the Infection Diseases Control Law, and in 2005, 3589 cases were reported.10 Enterohemorrhagic Escherichia coli infection occurs in many industrialized nations21 and is an emergent infectious disease of significant clinical importance.12,13,23 Therapeutic approaches for EHEC infection are the subject of widespread discussion.9,25,31 Generally, the treatment for bacterial food poisoning is antibiotic administration. However, antibiotic therapy is not recommended for food poisoning caused by EHEC infection, because it increases the risk of serious complications, such as hemolytic uremic syndrome, due to the release of Verotoxin (VT) from killed bacteria. Therefore, alternative therapeutic approaches, such as inhibiting VT activity or absorption from the intestine, are required. We previously obtained a colostral antibody against VT2 from cows immunized with the toxin and confirmed the neutralization efficacy of this reagent against VT2 in mice.15 However, before this bovine colostral antibody can be administered to patients infected with E. coli O157, its resistance to decomposition by intestinal proteases must be investigated. Each immunoglobulin class reportedly differs in its resistance to protease degradation in vitro,1,3,18, 22,26,28 but such resistance has not been confirmed in vivo. Furthermore, few animal models are available for evaluating for E. coli O157:H7 infection. The weaned immature mouse model has been used to study E. coli O157:H7 infection and VT,15 and beagle dogs pretreated with fradiomycin before inoculation with E. coli O157:H7 developed diarrhea. We chose to use this canine model in the current study. In this study, we investigated the resistances of bovine colostral antibody and individual immunoglobulin classes to proteases in the small intestine of beagle dogs. We also evaluated the efficacy of this colostral antibody against VT2 in beagle dogs.
T Morooka - One of the best experts on this subject based on the ideXlab platform.
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development of Verotoxin 2 and Verotoxin 2 variant vt2v specific oligonucleotide probes on the basis of the nucleotide sequence of the b cistron of vt2v from escherichia coli e32511 and b2f1
Journal of Clinical Microbiology, 1991Co-Authors: T Morooka, M. A. Karmali, Robert Clarke, S De Grandis, J L BruntonAbstract:We and others have noted that there are serological differences between Verotoxin 2 (VT2) (also known as Shiga-like toxin II) produced by Escherichia coli C600(933W) and the VT2 variant (VT2v) produced by strain E32511. Recent reports have described nucleotide sequence differences between the VT2v B subunit cistron of E32511 and B2F1 and that of VT2. We have confirmed the sequence differences and have used them to design oligonucleotide probes which differentiate the B subunit cistron of VT2v from that of VT2. Isolates of VT-producing E. coli obtained from human as well as food and veterinary sources were classified according to the toxin phenotype by using a toxin neutralization assay with VT2-specific monoclonal antibody and VT2v-specific polyclonal antisera. Using the oligonucleotide probes in colony hybridization, we detected 35 of 35 VT2 producers and 16 of 16 VT2v producers. One VT2 producer was falsely identified as containing the VT2v gene. The E32511 strain in our collection hybridized only with the VT2-specific probe. Southern hybridization of radiolabeled oligonucleotide probes showed that strains carried zero to one copy of the VT2 gene and zero to two copies of the VT2v gene. We conclude that colony hybridization with the VT2- and VT2-specific probes is highly predictive of the toxin phenotypes in the clinical isolates described in this study. Images
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development of Verotoxin 2 and Verotoxin 2 variant vt2v specific oligonucleotide probes on the basis of the nucleotide sequence of the b cistron of vt2v from escherichia coli e32511 and b2f1
Journal of Clinical Microbiology, 1991Co-Authors: J Hii, Carlton L Gyles, M. A. Karmali, T Morooka, Robert Clarke, S De Grandis, J L BruntonAbstract:We and others have noted that there are serological differences between Verotoxin 2 (VT2) (also known as Shiga-like toxin II) produced by Escherichia coli C600(933W) and the VT2 variant (VT2v) produced by strain E32511. Recent reports have described nucleotide sequence differences between the VT2v B subunit cistron of E32511 and B2F1 and that of VT2. We have confirmed the sequence differences and have used them to design oligonucleotide probes which differentiate the B subunit cistron of VT2v from that of VT2. Isolates of VT-producing E. coli obtained from human as well as food and veterinary sources were classified according to the toxin phenotype by using a toxin neutralization assay with VT2-specific monoclonal antibody and VT2v-specific polyclonal antisera. Using the oligonucleotide probes in colony hybridization, we detected 35 of 35 VT2 producers and 16 of 16 VT2v producers. One VT2 producer was falsely identified as containing the VT2v gene. The E32511 strain in our collection hybridized only with the VT2-specific probe. Southern hybridization of radiolabeled oligonucleotide probes showed that strains carried zero to one copy of the VT2 gene and zero to two copies of the VT2v gene. We conclude that colony hybridization with the VT2- and VT2-specific probes is highly predictive of the toxin phenotypes in the clinical isolates described in this study.
