A Live Salmonella enterica Serovar Enteritidis Vaccine Allows Serological Differentiation between Vaccinated and Infected Animals

Abstract

Three precisely defined deletion mutants of Salmonella enterica serovar Enteritidis were constructed, a guanine auxotrophic ⌬guaB mutant, a nonflagellated ⌬fliC mutant, and an auxotrophic and nonflagellated ⌬guaB ⌬fliC double mutant. All three mutants were less invasive than the wild-type strain in primary chicken cecal epithelial cells and the human epithelial cell line T84 and less efficiently internalized in the chicken macrophage cell line HD11. The ⌬fliC mutant was pathogenic in orally infected BALB/c mice, while the ⌬guaB mutant was attenuated and conferred protection against a challenge with the pathogenic parent strain. The ⌬guaB ⌬fliC double mutant was totally asymptomatic and conferred better protection than the ⌬guaB mutant. This indicates that the major flagellar protein flagellin is not required for efficient vaccination of BALB/c mice against Salmonella infection. The ⌬guaB ⌬fliC mutant was also safe for vaccination of 1-day-old chickens. After two immunizations, it induced statistically significant protection against infection of the internal organs of the birds by a virulent S. enterica serovar Enteritidis challenge strain but not against intestinal colonization. These data demonstrate that nonflagellated attenuated Salmonella mutants can be used as marker vaccines.

Vaccine potential of a nonflagellated, virulence-plasmid-cured (fliD–, pSEVΔ) mutant of Salmonella Enteritidis for chickens

“…Our results are in harmony with the in vitro invasion results of previous publications comparing wild-type strains and nonflagellated (fliC-deleted) mutants of different Salmonella serovars, indicating that flagellae are necessary for the proper invasion of S. Enteritidis into human Caco-2 and Hep-2 tumour cells (Van Asten et al, 2000;La Ragione et al, 2003), but differ somewhat from those obtained on fliC-deleted mutants of different strains of S. Enteritidis (Adriaensen et al, 2007;Methner et al, 2011b). Adriaensen et al (2007) found that the fliCΔ mutant of their wild-type S. Enteritidis 76Sa88 showed less reduced invasion of human (T84) or chicken intestinal epithelial cells. Unfortunately, these authors did not compare their double-deletion mutant (ΔguaB ΔfliC) in day-old chicks to either the ΔfliC single mutant or to the wild parent strain.…”

“…The most straightforward solution to provide a negative serological marker for a Salmonella strain is the elimination of flagellae. A nonflagellated S. Enteritidis vaccine candidate strain (ΔguaB, ΔfliC) has been produced and proven to be promising in the protection of chicks against homologous challenge (Adriaensen et al, 2007). Further fliC-S. Enteritidis vaccine candidates have been reported by Methner et al (2011a) and Matulova et al…”

“…At the same time, here we confirm that the flagellar protein FliD itself is not required for the efficient vaccination of chickens. Although there are several data supporting the role of flagellae in cellular and humoral immune responses to Salmonella (Salazar-Gonzalez and McSorley, 2005), the results of Kodama and Matsui (2004) and Adriaensen et al (2007) also suggest that Salmonella flagellin is not a major protective antigen in mice. As stated above, our results confirm and extend these observations on the lack of a major protective role of S. Enteritidis flagellin against Salmonella infection in chicks.…”

“…Live attenuated Salmonella vaccines produced by targeted mutagenesis have also been extensively tested in several animal species, and it is known that such strains more readily promote a long-lasting cellmediated immunity than the killed vaccines (Zhang-Barber et al, 1999;Van Immerseel et al, 2005;Barrow, 2007;Pasquali et al, 2008). Recently a nonflagellated (ΔguaB, ΔfliC) mutant produced by Adriaensen et al (2007), and the nonflagellated (ΔphoP, ΔfliC) mutant of S. Enteritidis produced by Methner et al (2011b) have been demonstrated to confer sufficient protection against organ invasion by a virulent S. Enteritidis challenge strain, but either no reduction or only a weak reduction of colonisation was proven, respectively. Besides, neither of these two papers reported about cloacal shedding of the challenge S. Enteritidis strain.…”

“…One of them is the need for serological markers, so that naturally infected and vaccinated flocks could be distinguished by simple serological assays. For that purpose, three recent studies have reported the use of nonflagellated fliC (H1 flagellin) deletion mutants of S. Enteritidis (Adriaensen et al, 2007;Methner et al, 2011b;Matulova et al, 2013), produced by the method reported by Datsenko and Wanner (2000). None of these fliC-, nonmotile vaccine candidates were reported to be devoid of the S. Enteritidis virulence plasmid.…”