Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.

Salmonella Enteritidis Flagella may Enhance Attachment and Invasion of Hen Ovarian Granulosa Cells and Induce Protective Immune Response in Egg-laying Hens.

Aims: To study the effect of flagellin on bacterial attachment and invasion of avian ovary cells in vitro by comparing the attachment and invasion of wild-type S. Enteritidis with nonmotile mutants. To assess the immunogenic properties of extracted flagellin against Salmonella Enteritidis experimental infection in laying hens. Methodology: Non-flagellated mutants for wild-type S. Enteritidis (phage type 8, 13A and 28) were produced by using a strain of S. Enteritidis, SA4502, which carried an fliC::Tn 10 to transfer fliC::Tn 10 insertion into the wild type strains using phage 22 (P22)-mediated transduction with selection for antibiotic resistance encoded within the mutant alleles. Granulosa cells were harvested from Single Comb White Leghorn hens between 18-45 weeks of age. Flagellin was purified from the studied bacterial cultures of Salmonella Enteritidis following reported methods. Laying hens were immunized with the flagellin with adjuvant Results: Non-motile mutants of S. Enteritidis phage wild types were analyzed to confirm the elimination of H1 flagellin synthesis. Wild-type and fliC mutant strains were assessed for their ability to adhere to hen's ovarian granulosa cells. The adherence of the mutant strain was reduced nearly ten-fold compared with that of the wild-type phage 8. Similarly, light microscopic observation of fixed cover slips from wild-type phage types and its mutant strain revealed fewer numbers of the bacterial mutants adhered to the cultured granulosa cell monolayer. Light microscopy revealed similar findings for mutant phage types 28 and 13 A when compared to the wild-type control. There was five folds rise in the egg yolk antibody during the 2-3 weeks post-immunization. No rise was detected in the egg yolk samples from the control hens injected with the placebo mixture without flagellin. Conclusion: It was concluded that Flagellin has an important role in the attachment and invasion of Salmonella Enteritidis to avian ovary cells and that it can be used as immunogenic components to induce a protective immune response in vaccinated hens against challenge infection with the wild type strains.

TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.

Identification of putative new Escherichia coli flagellar antigens from human origin using serology, PCR-RFlP and DNA sequencing methods

Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO) of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein) by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.

Identificación de antígenos inmunorreactivos de Leptospira interrogans / Identification of immunoreactive antigens of Leptospira interrogans

Se estudió un lote de 28 sueros de llama (Lama gama) de la provincia de Jujuy, Argentina, a fin de identificar antígenos inmunorreactivos contra Leptospira interrogans. Se utilizaron distintas preparaciones antigénicas de la bacteria para estudiar la inmunorreactividad mediante microaglutinación (MAT), ELISA y Western inmunoblot. Un pool de sueros bovinos positivos a la MAT fue empleado como control. Todos los sueros de llama fueron negativos mediante MAT e igual resultado se observó mediante ELISA. Dos de los 28 sueros de llama y el pool de sueros bovinos positivos, al ser evaluados por Western inmunoblot, arrojaron resultados positivos y permitieron identificar proteínas inmunorreactivas. Por MALDI-TOF se logró establecer que la proteína asociada a los dos sueros de llama inmunorreactivos era una flagelina periplásmica de Leptospira interrogans serovar Lai STR, mientras que la asociada al pool de sueros bovinos positivos a Leptospira sp. se trataba de una lipoproteína de la membrana externa de Leptospira interrogans serovar Ballum, LipL21. Estas proteínas podrían ser utilizadas en el diseño de un nuevo ELISA aplicado al diagnóstico temprano de leptospirosis, ya sea en distintos tipos de ganado como así también en reservorios silvestres.

A batch of 28 llama (Lama gama) sera from Jujuy province in Argentina was studied in order to identify immune reactive antigens to Leptospira interrogans. Different antigenic preparations from the bacterium were used to study the immune reactivity by the microagglutinattion (MAT), ELISA and Western immunoblot tests. A control pool of positive bovine sera was used. All the llama sera were negative to MAT as well as to ELISA. Two of the llama sera and the positive bovine sera pool rendered positive results when evaluated by Western immunoblot, allowing the identification of immune reactive proteins. These proteins were identified by MALDI-TOF. A periplasmic flagellin of Leptospira interrogans serovar Lai STR called FlaB1 was identified from the reactive llama sera, and an external membrane lipoprotein of Leptospira interrogans serovar Ballum called LipL21 was identified from the pool of bovine positive sera. These proteins could be used in a new ELISA applied to the early diagnosis of leptospirosis in different kind of cattle or wild reservoirs.

Anti-flagellin antibody responses elicited in mice orally immunized with attenuated Salmonella enterica serovar Typhimurium vaccine strains

In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.

Intervención del flagelo de campylobacter jejuni subsp. jejuni en la adherencia a cultivos celulares: evidencias bacteriológicas e inmunológicas / Intervention of campylobacter campylobacter jejuni subsp. jejuni flagella in the adhesion to cellular cultures: bacteriological and immunological evidences

The participation of the flagella of a virulent strain (O52) of campylobacter jejuni subsp. jejuni in the adhesion to HEp-2 cells and their inhibition by means of homologous polyclonal antibodies, moniclonal antiflagella antibodies and colostral natural antibodies (IgA) was studied. An aflagellated strain (T1) was used as negative control. Adhesion was observed in higher rates with O52 strain (72 percent) than with T1 strain (27,5 percent). Polyclonal, monoclonal and colostral antibodies inhibited O52 strain adhesion in more than 70 percent (p<0,001). T1 strain adhesion was inhibited only by polyclonal and colostral natural antibodies. Our results suggest that the flagella of C. jejuni subsp. jejuni could participate effectively in the adhesion process. However, the inhibition of T1 strain by polyclonal and colostral antibodies suggest the existence of other kinds of adhesins in the bacterial surface

Monoclonal antibodies against protein antigens of salmonellae causing paratyphoid fever and their diagnostic application.

Hybrid clones producing monoclonal antibodies (MAbs) specific for Salmonella paratyphi A (72 clones), S. paratyphi B (9 clones) and S. paratyphi C (8 clones) were produced by using the affinity purified Salmonella protein (Bp) as immunogens. MAbs to S. paratyphi A and S. paratyphi B reacted specifically with the 52 kDa homologous flagellin protein components while those to S. paratyphi C reacted with a 61 kDa flagellin protein component. The MAbs against S. paratyphi A and S. paratyphi B were used to establish a double antibody sandwich ELISA for detection of the 52 kDa flagellin antigens in serum samples from patients with acute paratyphoid A and paratyphoid B fever. With this assay system, 6.25 ng per ml of flagellin antigens of S. paratyphi A and S. paratyphi B could be detected. However, the assay system could not detect the flagellin antigens in patients' sera. The presence of IgM antibodies to the 52 kDa antigens of S. paratyphi A and S. paratyphi B in the acute sera from paratyphoid A or paratyphoid B patients suggested that the 52 kDa protein components of both salmonellae are good immunogens for human and might be used as antigens for early diagnosis of paratyphoid A and paratyphoid B fever.