Construction and Evaluation of a Dcya Dcrp Salmonella typhimurium Strain Expressing Avian Pathogenic Escherichia coli O78 LPS as a Vaccine to Prevent Airsacculitis in Chickens

Abstract

Avian pathogenic strains of Escherichia coli cause a number of extraintestinal diseases in poultry, including airsacculitis and colisepticemia. Expression of O78 lipopolysaccharide (LPS) is frequently associated with pathogenic isolates. Salmonella, a common poultry contaminant, is a major public health concern. The purpose of this work was to develop an E. coli vaccine for poultry with the use of an attenuated Salmonella typhimurium carrier that would benefit both the bird and the consumer. Orally administered attenuated S. typhimurium delta cya delta crp strains have been shown to provide excellent protection against wild-type Salmonella challenge in chickens. This work describes the construction of a delta cya delta crp derivative of an avian pathogenic S. typhimurium that expresses both the homologous group B determinants (O1,4,5,12) and the heterologous E. coli O78 LPS O antigens. This was accomplished by inserting the E. coli rfb region, which encodes the genes required for O78 expression, into the chromosomal cya gene of S. typhimurium, creating a defined deletion/insertion mutation. A delta crp mutation was introduced in a subsequent step. Expression of both O antigens was stable in vitro and in vivo. Vaccination of white leghorn chicks at day of hatch and 14 days with the recombinant vaccine strain induced serum immune responses against both S. typhimurium and E. coli LPS and protected the birds against subsequent challenge with an avian pathogenic E. coli O78 strain. Introduction of a mutation in rfc, which encodes the O antigen polymerase, reduced the chain length of the S. typhimurium LPS without affecting the expression of O78. The rfc mutation further enhanced the ability of the vaccine strain to protect chickens against E. coli challenge.

Mechanisms of intrinsic resistance to antimicrobial peptides of Edwardsiella ictaluri and its influence on fish gut inflammation and virulence

“…The flanking regions were ligated and cloned into pMEG-375 digested with SphI and XbaI. To construct E. ictaluri mutants, the suicide plasmid was conjugationally transferred from Escherichia coli x7213 (Roland et al, 1999) to E. ictaluri strains. Strains containing single-crossover plasmid insertions were isolated on BHI agar plates containing Col and Amp.…”

Salmonella entericaSerovar Typhimurium Strains with Regulated Delayed Attenuation In Vivo

“…PCR amplification with primers designed for specific modifications was used to alter promoter, ribosome binding/Shine-Dalgarno (SD), and start codon sequences. Conjugational transfer of suicide vectors for generation of unmarked deletion and deletion-insertion mutations was performed by standard methods (52,63) using the suicide vector donor strain 7213 (Table 1). Since live vaccine strains cannot display resistance to antibiotics, we used means to generate defined deletion mutations using suicide vector technologies that did not use drug resistance markers or leave molecular scars.…”

SalmonellaVaccine Vectors Displaying Delayed Antigen SynthesisInVivoTo Enhance Immunogenicity

“…Suicide vectors and P22-mediated transduction were used to generate defined deletion/deletion-insertion mutations (19,35,64). Transfer of recombinant suicide plasmids to Salmonella was accomplished by conjugation using Escherichia coli 7213 (Asd Ϫ ) as the plasmid donor (61). Bacteriophage P22HTint-mediated general transduction was performed by standard methods (69).…”