== Growth inhibition activity assays of anti EspBrec IgY antibodies extracted from yolk of ostriches onE

== Growth inhibition activity assays of anti EspBrec IgY antibodies extracted from yolk of ostriches onE. were purified by precipitation with 19% sodium sulfate and 20% ammonium sulphate methodologies. Purified IgY 50L aliquots were incubated in 850L BHI broth made up of 50L inoculums of five strains ofS. aureusand five strains ofE.coliduring four hours at 37C. Growth inhibition was evaluated followed by photometry reading (DO550nm). Egg yolk IgY preparation from hiperimmunized birds contained antibodies that inhibited significantly (p<0,05) growth of strains tested. Potential use of ostrich IgY polyclonal antibodies as a diagnostic and therapeutic tool is usually proposed for diseased animals. Keywords:Ostrich IgY antibodies, inhibition, recombinant proteins,E. coli,S. aureus == INTRODUCTION == Ostrich (Struthio GGACK Dihydrochloride camelus) raising need experience and information from farmers and successful ostrich farming is largely dependent on the ability of farmers to rear sufficient numbers of viable and healthy chicks. However, high mortality of ostrich chicks particularly during the first months of life is a problem (8). Mortality can be reduced by correct housing, feeding and health management of chicks at hatch and during the brooding period, and some microorganisms were isolated and involved in ostrich chicks mortality (19,28). Among microorganisms isolated from ostrichesE. coliand staphylococci may colonize and eventually cause diseases in the host (1,17,19).Staphylococcus aureuscan pose as an infectious agent due to virulence factors secreted by some clone and multidrug resistance is one of the main features (20). The bacteria can also secrete toxins with superantigenic properties (10), with enterotoxins secretion commanded by theagrloci after activation of RNA III Activating Protein -RAP (7). Anti-RAP antibodies inhibited in vivo contamination caused byS. aureusin mouse (6,11) GGACK Dihydrochloride and in vitro growth and secretion of enterotoxins by different strains ofS. aureuswere inhibited by specific antibodies developed against synthetic peptide RIP involved inagractivation (27,28).S. aureusis also considered one of the main agents causing mastitis in cattle (1,29). Diseases caused byEscherichia colialso pose as important losses for human and animals. Pathogenic strains enterotoxigenicE.coli(ETEC), enteropathogenicE.coli(EPEC), enteroinvasiveE. coli(EIEC), enterohemorragicE. coli(EHEC), Rabbit Polyclonal to ZNF420 enteroagregativeE. coli(EAEC) and extraintestinal patogenicE. coli(EXPEC) are among the causes of diarrhea in human and animal leading to economic loss for farmers, including birds and mammals (18). Antibodies developed against several microorganisms in chickens, also known as IgY, has been proved to be an alternative for immunological diagnostic tool, as well as suggested as immunotherapic purposes (4,12,13,15,17,21,31). The aim of this work was to demonstrate for the first time the inhibitory activity of polyclonal antibodies developed in ostriches against recombinant proteins recRAP and recSEC fromS. aureusand recBFPA and recEspB fromE.coli, towards clinical and standard strains ofS. aureusandE. coliin vitro. == MATERIALS AND METHODS == == Recombinant proteins/antigens == Four ostriches in breeding season were immunized three times with recombinant proteins (200g/animal) at intervals of 21 days in the brachial muscle of birds. The selected recombinant proteins were recSEC and recRAP derived fromS. aureusand recEspB and recBFPA fromE. coli, previously used by others (2,26). From each female blood and non-fertilized eggs were collected before and GGACK Dihydrochloride after each immunization step, in order to obtain unfavorable controls. == Immunization scheme == Four ostriches were primed on day 0 by injecting 200g of antigens previously incorporated in complete Freund’s adjuvant into the wing muscle. The birds were subsequently injected two times with the antigens in physiological saline at 21 days intervals. Blood samples and eggs were collected immediately before and during the course of immunization. The blood was allowed to clot at room temperature and the centrifuged sera stored at -20 C. The collected eggs were stored at 4 C before being used to prepare IgY. == Purification of chicken IgY == The method used by others (3,27) was carried out by diluting one part of egg yolk in nine equal volumes of distilled water, pH 5.0 to 6.0. The mixture was allowed to stand overnight at 4C. Non-immunoglobulin proteins precipitated after centrifugation at 10,000 xgfor 30 minutes at 4C were discarded, and the supernatant was collected. Following the material rich in IgY antibodies was submitted to second precipitation step by two methodologies. After the mixtures were allowed to stand at room temperature, part was precipitated with 19% sodium sulphate solution (w/v) (Merck, Germany) and other part was precipitated with 20% ammonium sulphate.