Antibodies anti-Shiga toxin 2 B subunit from chicken egg yolk: isolation, purification and neutralization efficacy.

Shiga toxins (Stx1 and Stx2) are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC), a foodborne pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The aim of this study was to evaluate the antibodies against Stx2 obtained from egg yolks of laying hens immunized with a recombinant Stx2B subunit. A high specific response in serum was observed 25 days after the first immunization and IgY antibodies were extracted from day 47th and purified from egg yolk. A concentration of 0.84 mg of total IgY/ml of egg yolk was obtained, of which 8% were antigen specific. The ability of anti-Stx2B IgY to recognize Stx2B and Stx2 either in solid-phase or in solution were evaluated and compared with anti-Stx2B rabbit antibodies by Western blotting and ELISA. The protective efficacy of IgY against Stx2 was determined by in vitro and in vivo experiments. The results show that IgY was able to recognize Stx2B and Stx2 in denatured conditions, attached to a solid-phase and free in solution. The anti-Stx2B IgY could effectively block the biological activity of Stx2 on Vero cells and protect mice from Stx2 challenge. The data suggest that immunization of hens with Stx2B could be a strategy to obtain at low cost a relatively high concentration of anti-Stx2 egg yolk IgY, able to neutralize Stx2 lethal activity. IgY technology could be an useful tool for research, diagnosis and therapy of EHEC infection.

Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms.

Species of the genus Bothrops induce the vast majority of snakebite envenomings in Latin America. A preclinical study was performed in the context of a regional network of public laboratories involved in the production, quality control and development of antivenoms in Latin America. The ability of seven polyspecific antivenoms, produced in Argentina, Brazil, Peru, Bolivia, Colombia and Costa Rica, to neutralize lethal, hemorrhagic, coagulant, defibrinogenating and myotoxic activities of the venoms of Bothrops neuwiedi (diporus) (Argentina), Bothrops jararaca (Brazil), B. neuwiedi (mattogrossensis) (Bolivia), Bothrops atrox (Peru and Colombia) and Bothrops asper (Costa Rica) was assessed using standard laboratory tests. Despite differences in the venom mixtures used in the immunization of animals for the production of these antivenoms, a pattern of extensive cross-neutralization was observed between these antivenoms and all the venoms tested, with quantitative differences in the values of effective doses. This study reveals the capacity of these antivenoms to neutralize, in preclinical tests, homologous and heterologous Bothrops venoms in Central and South America, and also highlight quantitative differences in the values of Median Effective Doses (ED50s) between the various antivenoms.

Characterization of a basic phospholipase A2-homologeu myotoxin isolated from the venom of the snake Bothrops neuwiedii (yarará chica) from Argentina.

A basic protein was isolated by CM-Sephadex C-25 chromatography from the venom of Bothrops neuwiedii from Argentina, and named B. neuwiedii myotoxin I. This protein exerted local myotoxic and edema-forming effects in mice, with potencies comparable to other myotoxins isolated from Bothrops spp. venoms. When injected by i.v. route at doses up to 4.7 mg/kg of body weight, the toxin was not lethal. In vitro, the toxin had no detectable phospholipase A2 activity on egg yolk phospholipids. B. neuwiedii myotoxin I appeared as a homodimer in sodium dodecylsulphate-polyacrylamide gel electrophoresis, with a subunit molecular weight of 15 kD. Gel immunodiffusion revealed a pattern of partial antigenic identity between the newly isolated myotoxin and myotoxin II from Bothrops asper venom. The sequence of B. neuwiedii myotoxin I was determined for the first 40 amino acid residues, showing high homology to several class II phospholipase A2 myotoxins of the Lys-49 family from crotalids. Altogether, results suggest that this toxin is a new member of the Lys-49 phospholipase A2-homologues with myotoxic, cytolytic, and edema-inducing activities.

Comparative study of the cytolytic activity of myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro.

A rapid in vitro cytolytic effect of some myotoxic phospholipases A2 (PLA2s) isolated from the venoms of Viperidae snakes has been previously described. This study was undertaken to investigate if cytolytic activity is a common property of the myotoxic proteins from this group. Murine endothelial cells (tEnd) and skeletal muscle myotubes (C2C12) were utilized as targets. The release of lactic dehydrogenase was quantified as a measure of cell damage, 3 h after exposure of cells to the different PLA2s, including representatives from the genera Bothrops, Agkistrodon, Trimeresurus, Crotalus (family Viperidae), and Notechis (family Elapidae). All of the group II myotoxic PLA2s tested displayed rapid cytolytic activity when tested in the micromolar range of concentrations (8-32 microM). In contrast, the group I myotoxic PLA2 notexin was devoid of this activity. Aspartate-49 and lysine-49 PLA2 group II variants showed a comparable cytolytic effect. Skeletal muscle myotubes, obtained after fusion and differentiation of C2C12 myoblasts, were significantly more susceptible to the cytolytic action of myotoxins than endothelial cells, previously reported to be more susceptible than undifferentiated myoblasts under the same assay conditions. Cytolytic activity appears to be a common characteristic of group II myotoxic PLA2s of the Viperidae. Bee venom PLA2, a group III enzyme of known myotoxicity, also displayed cytotoxic activity on C2C12 myotubes, being devoid of activity on endothelial cells. These results suggest that in vitro differentiated skeletal muscle myotubes may represent a suitable model target for the study of myotoxic PLA2s of the structural group II found in snake venoms.