Angel Alberto Justiz Vaillant

Purification of Immunoglobulin Y (IgY) from the Ostrich (Struthio camelus) by Staphylococcal Protein a (Spa) Affinity Chromatography

Immunoglobulin Y (IgY) is the major protein present in the avian egg yolk. This antibody fulfils important functions in the protection of Ostrich birds against infections. The aim of this study was to demonstrate the binding capacity of Staphylococcal proteins A (SpA) to Ostrich IgY and assess purification of the IgY by SpA affinity chromatography. Chloroform polyethylene glycol (Polson), affinity chromatography, Enzyme-Linked Immunosorbent Assay (ELISA)and Western blotting methods were used in the process. Results obtained revealed that Ostrich IgY has heavy chain of 70 kDa and light chain of 30 kDa confirming results by Western blot. In addition livetins (egg yolk proteins)were shown in the protein electrophoresis that preceded the Western blot. The binding capacity between SpA and Ostrich IgY is important because SpA can be used as a reagent in immunoassays for antibody detection against microbial agents that usually infect livestocks. This is the first time the use of SpA for purification of Ostrich IgY is being reported in literature.

In vitro Inhibition of Staphylococcus aureus Isolates by Anti-Anti-Idiotypic Antibodies to Staphylococcal Protein (SpA)

This study investigates the ability of antibodies to SpA (a protein produced by the bacterium Staphylococcus aureus) to inhibit the growth of the bacteria. Chickens immunised with SpA produced anti -SpA antibodies in their eggs (primarily the yolk). These anti-SpA antibodies were used to feed chicks, which produce antibodies that recognise the original antigen. When these antibodies were purified from their sera and included in the growth medium of the Staphylococcus aureus, there was inhibition of the bacterial growth. This indicates that these antibodies had specifically bound to the cell surface of the bacteria and prevented growth of the bacteria, i.e. the antibodies could protect against bacterial infection. The potential of such antibody in the hyper-immune egg to act as oral therapeutic agents is discussed.

Prevalence of Salmonella organisms in poultry and poultry environments in Jamaica.

Aim: This study was undertaken to determine the prevalence of Salmonella spp contamination in the Jamaican poultry industry and its environments. Materials and Methods: A total of 45 farms across 6 Jamaican parishes were selected for this study. A total of 6693 specimens from animals and the environment were investigated for the presence of Salmonella spp. All specimens were placed in an igloo with ice packs and transported to the laboratory for analysis. Bacteriological media obtained from Difco Laboratories Detroit MI U.S.A were used for the isolation and identification of Salmonella spp. Salmonella serological typing was performed to determine the Salmonella serovar by standard procedures. Results: This study revealed a low prevalence of Salmonella contamination/infection in both small and large entities in the poultry industry in Jamaica. The overall prevalence was 1 % (79 positive out of 6693 specimens). However, a higher prevalence of Salmonella was observed in the case of those operations which practiced “organic” poultry farming. It was shown that two Salmonella serovars including Augustenborg and Kentucky, identified Research Article British Microbiology Research Journal, 3(4): 461-469, 2013 462 during the study, are newly reported serovars in Jamaica. The sources of Salmonella infection varied from poultry itself to other species, such as rodents, pigs and insects. Improper disposal of broken eggs, wet bedding and other fomites contributed to Salmonella contamination. Conclusions: The results of the study indicate possibility of salmonellosis (zoonosis) in Jamaica, although the prevalence of Salmonella spp was low, and the need for improved quality of the food industry, animal care and human health to prevent salmonellosis.

The Chicken and Egg System for the Development of Anti-Idiotypic Vaccines

This study investigates the use of the chicken and egg system for the development of an oral HIV vaccine. Brown leghorn chickens were immunized with keyhole limpet hemocynin conjugated with a HIV-gp120 peptide (fragment 254-274). An indirect ELISA for antibodies to HIV-gp120 was used to measure anti-HIV antibody titres in the watery soluble fraction of eggs up to 14 weeks after the second week post-immunization. Over a period of 10 weeks, 3 cats were fed with the eggs from the immunized chickens and 2 cats with eggs from non-immunized chickens. An indirect enzyme linked-immunosorbent assay (ELISA) and a binding inhibition assay were used to assess the antibody response to HIV-gp120 peptide in the cat serum. The most important finding was the development of serum antiHIV antibodies in cats fed with eggs from chickens that were positive for anti-HIV antibodies. These feline anti-HIV antibodies bound to the original HIV-gp120 peptide and also inhibited the binding of egg yolk anti-HIV antibodies to the HIV gp120 peptide, showing that the anti-HIV antibody raised in cats after feeding, was an anti-anti-idiotypic antibody. The results of this study suggest that eggs from immunized hens could be considered in the management of HIV infections.

Bacterial Proteins and Their Proposed Interactions with Fc or Fab Fragments of Immunoglobulins

The reactivity of Immunoglobulin Binding Proteins (IBP) to Fc and/or Fab fragments of immunoglobulins was summarized in this review. Staphylococcal protein A (SpA), Streptococcal protein G and Peptostreptococcal protein L (SpL) were the IBP reported. SpA reacted with IgG from skunk, coyote, raccoon, mule and donkey. SpG reacted almost with the entire panel of immunoglobulins and SpL binding was restricted to some immunoglobulins including raccoon, ostrich and duck. The various immunological techniques that have been used to test the binding capacity of IBP to Igs were double immunodiffusion, Enzyme-Linked Immunosorbent Assay (ELISA), SpA-affinity chromatography and immunoblot analysis. These protein-protein interactions are important because they can be used in the immunodiagnosis and in the purification of intact Igs or their fragments.

Case Reports on the Use of Intravenous Immunoglobulins (IVIG) in theTreatment of Systemic Lupus Erythematosus (SLE)

Introduction: This work represents a preliminary study of the treatment of systemic lupus erythematosus with a commercially available intravenous immunoglobulin. The aim of this study was to assess the efficacy of this product in three patients aged 16, 34 and 49 diagnosed with systemic lupus erythematosus at the Internal Medicine Service of “Freire de Andrade” Hospital, Cuba. Case report summary: The patients had a history of treatment with several drugs, including immunesuppressants. However recurrent respiratory tract infections, skin rash as well as several immunological abnormalities were present. Intravenous immunoglobulin (5-10 g/day) was given intravenously during five consecutive days, in the absence of other types of immunotherapy. An immunological profile before and after the intravenous immunoglobulin therapy was performed. In addition the patients were clinically evaluated using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score. The intravenous immunoglobulin therapy was highly effective in all patients due to its immunosuppressive, anti-inflammatory, immunomodulating and antimicrobial properties. It also prevented some of the frequent complications associated with the traditional immunotherapy in systemic lupus erythematosus. Conclusion: We recommend new clinical studies on large group of patients to establish the efficacy and side effects of intravenous immunoglobulin as a first line therapy in systemic lupus erythematosus.

Use of Dot Blots Analysis in the Separation of Anti-HIV Antibodies in Animals

In this study antibodies against the fragment 254-274 from gp120 of human immunodeficiency virus (HIV) were produced in cats, rats and chicken that can be used as reagents in the preparation of diagnostic kits. Enzyme linked immunosorbent assay showed the presence of anti-HIVgp120 antibodies. Dot blot analysis was used as a separation technique and confirmed the results, proving its efficiency in the detection of proteins. This study also demonstrated that orally given antibodies to an animal as oral vaccine, initiated immune responses of the antibodies in the animals which may be useful for protection, antibody purification and development of reagents for immunodiagnostic procedures.

Preliminary Study of the Identification of Proteins in Tissue Fluids of theSea Mussel Isognomon alatus

The aim of this preliminary study was to investigate the presence of proteins in the tissue fluids of the sea mussel Isognomon alatus. Protein extraction was done by the chloroform-cold ethanol technique. Immunization for production of antibody to be used as reagents in Western blotting, assessment of the protein concentration by the Bradford method, protein characterization by native polyacrylamide gel electrophoresis (PAGE) were also performed as a part of the methodology of this study. The results showed a protein content of 65 mg/ml in tissue fluids and a protein band approximately of 220 kDa in PAGE that was further confirmed by the Western blotting. Future work should investigate the structure and function of the proteins separated from the tissue fluids and we considered it as a limitation of this investigation. The sea bivalve literature is scanty. However the limitation of this work we still can conclude that there are high molecular weight proteins in large concentrations in tissue fluids of the sea mussel Isognomom alatus.

Laying Hen's Primary Immune Response to Staphylococcal Protein A (SpA)

Our aim was to provide information about the production of Egg White Immunoglobulin (EWIg) with specificity to Staphylococcal protein-A, a surface antigen of Staphylococcus aureus and to study the inhibition of this bacterium growth in pre- and post-immunized hens. A sandwich Enzyme-Immunosorbent Assay (ELISA) showed a large concentration of anti-SpA antibodies in the eggs from hens immunized with protein A. The titer of these antibodies was at least 5 to 6-folds of that of the eggs from pre-immunized hens 10 days post-immunization. Inhibition of the growth of S. aureus by anti-SpA antibodies purified by SpA-affinity chromatography (PURE-1A) was observed in laying hens vaccinated. Growth of the bacteria in blood agar plates occurred in antibody samples from preimmunized laying hens only. Inhibition of the agglutination of SpA-bearing Staphylococcus aureus cells by purified anti-SpA antibodies was observed in vitro. The authors are not aware of previous studies of the primary immune system response developed in eggs from laying hens, so this research could set a precedent in the field of egg white immunoglobulin technology. The use of hyper-immune eggs as alternative to the use of antibiotics could be advantageous for the large amount of antibodies produced, low cost, the reduction of antigenic variation and very low toxicity.

Separation and Reactivity of Avian Immunoglobulin Y

Immunoglobulin Y (IgY) is the mayor protein present in the avian egg yolk. This antibody fulfils important functions in the protection of the embryo against several challenging stimuli. Separation of IgY from the egg yolk of several birds was carried out by the Polson method. Their capacity to react with immunoglobulin-binding bacterial protein: protein A, L or LA was investigated. The cross-reactivity of an anti-chicken-IgY-HRP conjugate with different avian IgY was tested by ELISA. The results showed that protein L reacts with bantam hem IgY; and proteins A and LA react with ostrich, bantam hen or duck IgYs. These findings are important for the development of methods of detection and purification of avian IgY proteins.