Evaluation of a protective effect of in ovo delive

Authors: Renata Godlewska Maciej Kuczkowski Agnieszka Wyszyńska Joanna Klim Katarzyna Derlatka Anna WoźniakBiel Elżbieta K JagusztynKrynicka

Publish Date: 2016/07/06

Volume: 100, Issue: 20, Pages: 8855-8864

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Abstract

Campylobacter jejuni is the most prevalent cause of a foodborne gastroenteritis in the developed world with poultry being the main source of infection Campylobacter jejuni like other Gramnegative bacteria constitutively releases outer membrane vesicles OMVs OMVs are highly immunogenic can be taken up by mammalian cells and are easily modifiable by recombinant engineering We have tested their usefulness for an oral in ovo vaccination of chickens Four groups of 18dayold chicken embryos 164 animals underwent injection of wt C jejuni OMVs or modified OMVs or PBS into the amniotic fluid The OMVs modifications relied on overexpression of either a complete wt cjaA gene or the C20A mutant that relocates to the periplasm Fourteen days posthatch chicks were orally challenged with live C jejuni strain Cecum colonization parameters were analyzed by twoway ANOVA with Tukey posthoc test The wtOMVs and OMVs with wtCjaA overexpression were found to confer significant protection of chicken against C jejuni p = 003 and p = 0013 respectively in comparison to PBS controls and are promising candidates for further in ovo vaccine developmentCampylobacter strains are recognized as a major causal agent of bacterial diarrhea both in developing and developed regions of the world Campylobacter infection rates have been increasing steadily over the past decade In EU countries the number of confirmed human campylobacteriosis cases was 236851 in 2014 an increase of 22067 cases 96  compared to 2013 EFSA and ECDC 2015 Mortality associated with Campylobacter infections is relatively low and no specific treatment is required for a vast majority of patients However Campylobacter infections constitute a serious problem due to the high number of cases severity of possible neurological complications as well as high social and economic costs of the disease Kaakoush et al 2015 Kirkpatrick and Tribble 2011The consumption of infected poultry meat is a major source of infection European Food Safety Authority EFSA reported that in 2014 384  of the 6703 samples of fresh broiler meat were found to be Campylobacter positive EFSA and ECDC 2015 Efforts to comply with EU hygiene and biosecurity regulations appear insufficient to control or eliminate Campylobacter from the poultry food chain Havelaar et al 2007 Mangen et al 2007 Elimination of Campylobacter jejuni from chickens would significantly reduce the incidence of campylobacteriosis in humans and seems to be an alternative and more realistic approach for controlling Campylobacter contamination However antiCampylobacter chicken vaccines are not commercially available yetMany Gramnegative bacteria especially pathogenic ones such as Neisseria meningitidis Haemophilus influenzae Helicobacter pylori Borrelia burgdorferi Pseudomonas aeruginosa Shewanella spp Shigella spp Salmonella spp produce outer membrane vesicles OMVs Secretion of these vesicles to a medium is a naturally occurring phenomenon Vesicles contain not only outer membraneassociated proteins but also periplasmic and even cytoplasmlocated molecules virulence factors DNA chains enzymes Kuehn and Kesty 2005Considering their immunogenic and selfadjuvant properties the ability to be taken up by mammalian cells and adjustability of their content by recombinant engineering OMVs are attractive candidates for vaccine delivery vectors Composition of the OMVs LPS glycerophospholipids OM and periplasmic proteins makes them capable of mediating both pro and antiinflammatory activities leading to a clearance of infection or a widespread inflammation MashburnWarren et al 2008 Mashburn and Whiteley 2005 That is why OMVs are useful as vaccines and adjuvants stimulating protective mucosal and humoral immune responses Collins 2011 already constituting a base of several licensed vaccines and gaining an increasing popularity van der Pol et al 2015 For example conventional wildtype outer membrane vesicle wtOMV vaccines are the only formulations that have shown efficacy against serogroup B meningococcal disease Acevedo et al 2014 Asensio et al 2011 Holst et al 2009 Roberts et al 2008 Consequently a novel vaccine 4CMenB against serogroup B meningococcal disease composed of protein antigens identified by reverse vaccinology fHBP fused to GNA2091 GNA2132 fused to GNA1030 and NadA combined with OMVs is now approved in Europe Canada Australia and some Latin American countries Carter 2013 Giuliani et al 2006 Martin and Snape 2013 Serruto et al 2010Campylobacter jejuni OMVs have not yet attracted a comparable interest It has only been shown that C jejuni membrane vesicles contain a toxindubbed cytolethal distending toxin CDT Elmi et al 2012 Lindmark et al 2009 and proteomic analyses of C jejuni OMVs identified 151 proteins including periplasmic outer membrane associated innermembrane and even cytoplasmic ones Among them all three subunits of CDT CdtA CdtB and CdtC and sixteenglycosylated proteins were present Elmi et al 2012 Jang et al 2014One of the latter is CjaA an extracytoplasmic glycosylated and highly immunogenic protein cloned and characterized in our laboratory Pawelec et al 1997 Wyszynska et al 2008 CjaA is conserved between different Campylobacter serotypes Crystallographic analyses of the Escherichia coliproduced rCjaA determined that CjaA binds to the cysteine ligand and is a constituent of the ABC cysteine transport system Muller et al 2005 Expression of CjaA in bacteria cultured on solid media increases under iron deficiency conditions and is higher in clinical Campylobacter isolates than in laboratory strains Cordwell et al 2008 Holmes et al 2005 ShoafSweeney et al 2008 These facts suggest that CjaA participates in a colonization process in vivo

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