ABSTRACT
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.
Subject(s)
Antibodies, Bacterial/physiology , Chickens/immunology , Egg Yolk/immunology , Immunoglobulins/physiology , Shiga Toxin 2/immunology , Animals , Antibodies, Bacterial/isolation & purification , Antibody Affinity , Immunoglobulins/isolation & purification , Mice , Mice, Inbred Strains , Neutralization Tests , RabbitsABSTRACT
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.
Subject(s)
Bothrops , Crotalid Venoms/enzymology , Muscle, Skeletal/drug effects , Phospholipases A/isolation & purification , Phospholipases A/pharmacology , Amino Acid Sequence , Animals , Argentina , Chromatography, Ion Exchange , Edema/chemically induced , Electrophoresis, Polyacrylamide Gel , Hemorrhage/chemically induced , Immunodiffusion , In Vitro Techniques , Mice , Molecular Sequence Data , Muscular Diseases/chemically induced , Phospholipases A/analysis , Phospholipases A2 , Survival RateABSTRACT
N:NIH mice were vaccinated according to the WHO recommendations for the potency test with the Second International Standard for Pertussis Vaccine (ISPV). Blood for serological investigation was taken from the animals on day 14 post immunization before intracerebral challenge with Bordetella pertussis 18323 was done. The relationship between anti-pertussis toxin, anti-filamentous hemagglutinin and anti-adenylate cyclase antibody levels as measured by ELISA and protection from intracerebral challenge was studied. The proportion of surviving mice increased in correlation with increasing anti-PT titres; a protective level of 4 ELISA units/ml was found. Such relationship between protection against intracerebral challenge and antibody titres was not found for anti-FHA nor for anti-AC antibodies, thus suggesting that these antibodies do not play an important role in protection in this model. The excellent correlation between anti-PT antibody titres and protection suggests that the measure of anti-PT response could be a useful tool for estimating the potency of whole-cell vaccines. The development of an alternative method for testing the potency of pertussis whole-cell vaccines based on the anti-PT response should be considered.