Guarantee the safety of live Salmonella vaccines by introducing deletions in genes crucial for egg contamination

01 January 2013 → 31 December 2016
Regional and community funding: IWT/VLAIO
Research disciplines
  • Natural sciences
    • Animal biology
  • Agricultural and food sciences
    • Veterinary medicine
    • Other veterinary sciences
    • Other agricultural, veterinary and food sciences
salmonella vaccincs commercial vaccins control program
Project description

Salmonella enterica subspecies enterica serovar Enteritidis is a pandemic pathogen, present in
countries with industrial poultry production since the 1990s. Ingestion of this foodborne
pathogen by humans results in gastroenteritis and is linked to contaminated eggs and egg
products. Salmonellosis caused by Salmonella Enteritidis in chickens however does not lead to
clinical symptoms but causes enormous economic losses. Consequently, there is continuing
interest in finding ways of preventing flock infection of laying hens with Salmonella. Control of
Salmonella infections in poultry farms begins with good farming practices and appropriate
management. In laying hens vaccination is an important tool to protect against Salmonella
colonization. Vaccination against Salmonella Enteritidis is vastly undertaken in many countries
around the world. Studies have reported that cases of human salmonellosis due to food
poisoning decreased significantly after the implementation of a widespread vaccination
program in commercial layers. Although great efforts have been made, recently, atypical
pathogenic Salmonella strains emerged. At the start of this doctoral thesis, studies in numerous
countries worldwide confirm the rapid emergence and dissemination of a monophasic variant of
Salmonella Typhimurium, i.e. 1,4,[5],12:i:-. Cases of human infection caused by the emerging
monophasic variant have been linked to a number of sources, predominantly pigs. Strains from
this serotype have also been found in chicken meat, broilers and recently in laying hens. This
shows that the monophasic variant 1,4,[5],12:i:- represents a significant and potential emerging
threat to humans, not only through porcine meat, but also through chicken product
consumption. Consequently it has been included in actions implementing the legislation of the
EU to detect and control Salmonella serovars of public health significance in laying hens,
broilers, breeders and turkeys.
While the efficacy of the commercial live vaccines AviPro® Salmonella VacE and VacT to protect
laying hens from oviduct colonization and egg contamination by Salmonella Enteritidis has been
proven, no data have been published yet on potential effects of this vaccine on caecal, spleen
and liver colonization by the emerging monophasic serotype 1,4,[5],12:i:-. Therefore, in the first
study of this thesis (chapter 3.1), two short-term (two weeks) trials, either using a high or a low
infection dose, and 1 longer term study (6 weeks) were carried out to evaluate the protective
effect against gut and internal organ colonization after vaccination with Salmonella
Typhimurium strain Nal2/Rif9/Rtt, a strain contained in the commercially available live vaccines
AviPro® Salmonella Duo and AviPro® Salmonella VacT, at day of hatch. Oral administration of
the vaccine strain at day of hatch, reduced colonization with a strain of the monophasic variant
of Salmonella Typhimurium, 1,4,[5],12:i:-, after challenge at day 2. The Salmonella 1,4,[5],12:i:-
serotype is Typhimurium-like and can thus, as shown in this study, also be controlled in the early
immune deprived stage by using live Salmonella Typhimurium vaccines. This is of value for
layers as well as for broilers and can be part of a control program for the new emerging
serotype 1,4,[5],12:i:-. These monophasic Salmonella Typhimurium 1,4,5,12:i:- variants are lacking the fljB-encoded second phase antigen. It has been suggested that the lack of flagella changes virulence characteristics of Salmonella but the exact role of flagella in the pathogenesis of Salmonella
infections in chickens was not yet completely clear. Little was known yet about the role of
flagellin in oviduct colonization. The glandular epithelial cells of the oviduct in laying hens
express Toll-like receptors (TLRs). These interact with MAMPs, like LPS and flagellin. Binding of
MAMPs to TLRs should normally initiate the innate immune response, leading to inflammation
and tissue damage. Salmonella Enteritidis however is able to colonize the oviduct without
causing an inflammatory reaction. Indeed we found that expression of flagella by Salmonella
Enteritidis is downregulated following colonization of the chicken oviduct and in chicken OEC
(chapter 3.2). The result of these studies indicate that Salmonella Enteritidis is capable of
avoiding an effective inflammatory response when colonizing the chicken oviduct and when
invading in chicken OEC through downregulation of flagellar gene expression and in this way
suppressing the flagellin-TLR5 activation pathway. Further studies are needed to identify the
signaling and sensing mechanisms involved in the downregulation of flagella expression by
Salmonella Enteritidis in the environment of the chicken oviduct. This information could be
important for future vaccine development.
Current commercial live vaccines contain strains harboring undefined mutations in one or more
genes on the chromosome. Strains harboring point mutations might, however, revert to a
virulent phenotype and are thus considered to be unsafe. Future live vaccines should therefore
contain fully defined strains carrying (multiple) gene deletions for purposes of safety. Most
experimental vaccines contain strains deleted for genes important for metabolism or virulence.
Numerous experimental vaccines were already tested in various animal hosts, including
chickens, but data on the protection of these live vaccines against egg contamination are scarce.
A vaccine strain used for the prevention of (vertical) egg contamination of Salmonella Enteritidis
ideally induces local immunity in the reproductive tract. From a public health point of view, it
may not persist here and preferably does not survive in egg white. A logical approach is to
eliminate genes playing a role in egg white survival. In the third chapter (chapter 3.3), defined
mutants in MDR transporters and the TolC outer membrane channel were used as vaccine
strains. The TolC outer membrane channel is used by MDR transporters (eg acrAB, acrEF,
mdtABC) to export host antibacterial compounds and bacterial molecules such as siderophores,
and is involved in survival in harmful environments, including egg white. The ΔtolC and
∆acrABacrEFmdtABC vaccine strains can no longer survive in egg white, thereby eliminating the
risk of human exposure through eggs. These genes were never associated with protective
immunity in chickens, allowing wild type-like antigen presentation. Data from this chapter
indicate that Salmonella Enteritidis ΔtolC and ΔacrABacrEFmdtABC strains are safe vaccines that
can induce protection against internal organ colonization after intravenous inoculation of a
Salmonella Enteritidis challenge strain. The vaccine strains were able to completely prevent egg
contamination with Salmonella Enteritidis in a 6 months in vivo challenge trial.
In conclusion, a number of control measures were being used to avoid Salmonella infections in
the poultry industry. In spite of these measures, new (monophasic) variants arise. Current
commercial available vaccines are able to protect against these upcoming variants. It is
important however to keep evaluating the protection offered by current commercial vaccines
against new upcoming variants in order to respond as quickly as possible to epidemiological
changes. At the same time it is important to guarantee the safety of vaccine strains by deletion
of whole gene(s). The Salmonella Enteritidis ΔtolC and ΔacrABacrEFmdtABC vaccine strains are
safe and could be used to prevent egg contamination.