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The necrotoxin of Loxosceles reclusa
490
Tenth World Congress
Toxins and other potential virulence factors of Salmonella: recent advances. S.B. BALOD^, A. FARlS, K. KgOVACEK, R. DVAL and I. M/~NSSON (Department of Veterinary Microbiology, Swedish University of Agricultural Sciences, Biomedical Center, Box 583, S-751 23, Uppsala, Sweden).
RESEARCH on Salmonella pathogenicity has become challenging during this century and thus remains an interesting field of research. A number of virulence determinants have been identified in different species of Salmonella, e.g. adhesins, toxins, plasmids and other invasion factors. Salmonella strains of both human and animal origin have been reported to produce a range of toxins such as ¢nterotoxins and cytotoxins which are genetically unrelated to conventional Vibrio cholerae and Escherichia coli toxins. Research on these enteropathogens/(entero) toxins has been further complicated not only by the presence of immunologically different toxins, but also by the lack of a single simple assay system for the detection of activities of these toxins. Current studies in our laboratories have demonstrated the presence of Salmonella strains producing toxins which are active only in in vivo enterotoxin assays such as ligated rabbit ileal loop and rabbit skin permeability tests, while these toxins show no activity in in vitro tissue culture assays (viz., Yl-mouse adrenal cells and Chinese hamster ovary cells). Furthermore, the eventual role in the infection process, of flagella, pili and other adherence proteins present on Salmonella cell surface, remains another interesting aspect of Salmonella pathogenesis. Human and animal isolates of Salmonella have been reported by us to bind to connective tissue proteins, e.g. fibronectin and collagen, which may be exposed in the basement membranes by the cell-damaging activity of Salmonella cytotoxins. Our studies have also demonstrated the presence of fibronectin-binding fimbriae on Salmonella cell surface. Salmonella toxins and other virulence determinants and their potential role in the pathogenesis of Salmonella will be reviewed and discussed in this lecture.
Enterotoxin production by Salmonella in different foods. S. B. BALODA,A. FAres, K. KROVACEK, R. DYAL and I. MJ,NSSON (Department of Veterinary Microbiology, Swedish University of Agricultural Sciences, Biomedical Center, Box 583, S-751 23, Uppsala, Sweden).
FOOD contamination with Salmonella constitutes a major public health problem in both the developed and developing world. This study examines the enterotoxin production by Salmonella inoculated in milk/milk products and meat/meat products at different incubation temperatures. Non-fat milk powder, sweetened condensed milk, flavoured milk, cottage cheese, yoghurt and cream, lamb, chicken, fish, sausage, ham and salami were inoculated with a clinical human diarrhoeal isolate of Salmonella enteritidis and incubated for up to 96 hr at 10, 28, and 37°C. These food samples were later evaluated for enterotoxin production using rabbit ileal loop assay. Syncase medium and nutrient broth were also used as control media for enterotoxin production under similar conditions. The S. enteritidis produced enterotoxin in different types of foods with a varying degree of efficiency at the three incubation temperatures. It produced more enterotoxin in boiled milk than in pasteurized and autoclaved milk. The optimum amount was produced in boiled milk at 28°C. Among milk products, maximum toxin was produced in flavoured milk, followed by cottage cheese, yoghurt, cream and diluted condensed milk. Salmonella enteritidis produced enterotoxin in a variety of meat samples, and the highest amounts were produced in lamb, followed by chicken and fish. Toxin production in the decreasing order was seen in salami, sausage and ham. Maximum enterotoxin production was observed at 37°C, though it was also produced at 28°C and 10°C. A substantial amount of toxin was produced after 24 hr incubation at 37°C, 48 hr incubation at 28°C and 96hr incubation at 10°C.
The necrotoxin of Loxosceles reclusa. J.T. BARRETT, B.J. CAMPBELL and L. J. FORRESTER (Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212, U.S.A.).
TI-~ SUBJECT of this investigation was to determine the biochemical basis of the dermonecrosis that develops following bites of Loxosceles reclusa spiders. Venom collected from surgically removed venom glands was diluted in saline and passed through a Sephadex G75-120 column equilibrated with pH 7.1 HEPES buffer. A dually dermonecrotic and hemolytically active fraction eluted in a single peak. Hemolytic assays were used to further analyze this material as a more quantitative procedure than dermal necrosis assays in vivo. The hemolysin (necrotoxin) is calcium dependent with a pH optimum of 7. I. The toxin degrades sphingomyelin in sheep RBC membranes but releases no water-soluble phosphate nor free fatty acids. Thin layer chromatography of sphingomyelin digestion products after exposure to toxin identified choline and N-acylsphingosine phosphate as the end products. The results indicate that the toxin is a sphingomyelinas¢ D. Further purification of the toxin by isoelectric focusing resulted in the appearance of enzyme activity in four fractions of p l 7.8, 8.2, 8.4, and 8.7. These fractions were immunologically cross-reactive with an antiserum to the pI 8.2 molecule. All four isoforms digested a water-soluble derivative of sphingomyelin, producing the same products as noted above. The p l 8.2 molecule is dermonecrotic and able to aggregate human platelets in a Ca2+-dependent reaction. The ability of L. reclusa sphingomyelinase D to damage cell membranes and to aggregate platelets indicates that this enzyme causes necrosis by direct tissue destruction and thrombosis.
Tenth World Congress
Toxins and other potential virulence factors of Salmonella: recent advances. S.B. BALOD^, A. FARlS, K. KgOVACEK, R. DVAL and I. M/~NSSON (Department of Veterinary Microbiology, Swedish University of Agricultural Sciences, Biomedical Center, Box 583, S-751 23, Uppsala, Sweden).
RESEARCH on Salmonella pathogenicity has become challenging during this century and thus remains an interesting field of research. A number of virulence determinants have been identified in different species of Salmonella, e.g. adhesins, toxins, plasmids and other invasion factors. Salmonella strains of both human and animal origin have been reported to produce a range of toxins such as ¢nterotoxins and cytotoxins which are genetically unrelated to conventional Vibrio cholerae and Escherichia coli toxins. Research on these enteropathogens/(entero) toxins has been further complicated not only by the presence of immunologically different toxins, but also by the lack of a single simple assay system for the detection of activities of these toxins. Current studies in our laboratories have demonstrated the presence of Salmonella strains producing toxins which are active only in in vivo enterotoxin assays such as ligated rabbit ileal loop and rabbit skin permeability tests, while these toxins show no activity in in vitro tissue culture assays (viz., Yl-mouse adrenal cells and Chinese hamster ovary cells). Furthermore, the eventual role in the infection process, of flagella, pili and other adherence proteins present on Salmonella cell surface, remains another interesting aspect of Salmonella pathogenesis. Human and animal isolates of Salmonella have been reported by us to bind to connective tissue proteins, e.g. fibronectin and collagen, which may be exposed in the basement membranes by the cell-damaging activity of Salmonella cytotoxins. Our studies have also demonstrated the presence of fibronectin-binding fimbriae on Salmonella cell surface. Salmonella toxins and other virulence determinants and their potential role in the pathogenesis of Salmonella will be reviewed and discussed in this lecture.
Enterotoxin production by Salmonella in different foods. S. B. BALODA,A. FAres, K. KROVACEK, R. DYAL and I. MJ,NSSON (Department of Veterinary Microbiology, Swedish University of Agricultural Sciences, Biomedical Center, Box 583, S-751 23, Uppsala, Sweden).
FOOD contamination with Salmonella constitutes a major public health problem in both the developed and developing world. This study examines the enterotoxin production by Salmonella inoculated in milk/milk products and meat/meat products at different incubation temperatures. Non-fat milk powder, sweetened condensed milk, flavoured milk, cottage cheese, yoghurt and cream, lamb, chicken, fish, sausage, ham and salami were inoculated with a clinical human diarrhoeal isolate of Salmonella enteritidis and incubated for up to 96 hr at 10, 28, and 37°C. These food samples were later evaluated for enterotoxin production using rabbit ileal loop assay. Syncase medium and nutrient broth were also used as control media for enterotoxin production under similar conditions. The S. enteritidis produced enterotoxin in different types of foods with a varying degree of efficiency at the three incubation temperatures. It produced more enterotoxin in boiled milk than in pasteurized and autoclaved milk. The optimum amount was produced in boiled milk at 28°C. Among milk products, maximum toxin was produced in flavoured milk, followed by cottage cheese, yoghurt, cream and diluted condensed milk. Salmonella enteritidis produced enterotoxin in a variety of meat samples, and the highest amounts were produced in lamb, followed by chicken and fish. Toxin production in the decreasing order was seen in salami, sausage and ham. Maximum enterotoxin production was observed at 37°C, though it was also produced at 28°C and 10°C. A substantial amount of toxin was produced after 24 hr incubation at 37°C, 48 hr incubation at 28°C and 96hr incubation at 10°C.
The necrotoxin of Loxosceles reclusa. J.T. BARRETT, B.J. CAMPBELL and L. J. FORRESTER (Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212, U.S.A.).
TI-~ SUBJECT of this investigation was to determine the biochemical basis of the dermonecrosis that develops following bites of Loxosceles reclusa spiders. Venom collected from surgically removed venom glands was diluted in saline and passed through a Sephadex G75-120 column equilibrated with pH 7.1 HEPES buffer. A dually dermonecrotic and hemolytically active fraction eluted in a single peak. Hemolytic assays were used to further analyze this material as a more quantitative procedure than dermal necrosis assays in vivo. The hemolysin (necrotoxin) is calcium dependent with a pH optimum of 7. I. The toxin degrades sphingomyelin in sheep RBC membranes but releases no water-soluble phosphate nor free fatty acids. Thin layer chromatography of sphingomyelin digestion products after exposure to toxin identified choline and N-acylsphingosine phosphate as the end products. The results indicate that the toxin is a sphingomyelinas¢ D. Further purification of the toxin by isoelectric focusing resulted in the appearance of enzyme activity in four fractions of p l 7.8, 8.2, 8.4, and 8.7. These fractions were immunologically cross-reactive with an antiserum to the pI 8.2 molecule. All four isoforms digested a water-soluble derivative of sphingomyelin, producing the same products as noted above. The p l 8.2 molecule is dermonecrotic and able to aggregate human platelets in a Ca2+-dependent reaction. The ability of L. reclusa sphingomyelinase D to damage cell membranes and to aggregate platelets indicates that this enzyme causes necrosis by direct tissue destruction and thrombosis.