# Patent

## Title
Prevention of Salmonella recrudescense

## Abstract

The present invention relates to novel Salmonella mutants, to a process for producing the same and to vaccines containing the same, wherein said Salmonella mutants are characterized in that they are not responsive to stress-related recrudescence. It is accordingly an object of the present invention to provide the use of said Salmonella mutants in the vaccination of animals, in particular mammals and birds, more in particular pigs, poultry and cattle.

## Background
FIELD OF THE INVENTION
The present invention relates to novel Salmonella mutants, to a process for producing the same and to vaccines containing the same, wherein said Salmonella mutants are characterized in that they are not responsive to recrudescence.
It is accordingly an object of the present invention to provide the use of said Salmonella mutants in the vaccination of animals, in particular mammals and birds, more in particular pigs, poultry and cattle. 
BACKGROUND TO THE INVENTION
Salmonellae are Gram-negative, facultative anaerobic, motile, non-lactose is fermenting rods belonging to the family Enterobacteriaceae. Salmonellae are usually transmitted to humans by the consumption of contaminated foods and cause salmonellosis.
Salmonellae have been isolated from many animal species including, birds, cattle, sheep, pigs, dogs, cats, horses, donkeys, seals and reptiles. Ninety-five percent or more of the Salmonella serovars (ser.) isolated from food producing animals belong to Salmonella enterica subspecies enterica (S. enterica), with Salmonella ser. Typhimurium (S. Typhimurium), Salmonella ser. Choleraesuis (S. Choleraesuis), Salmonella ser. Derby (S. Derby), Salmonella ser. Infantis (S. Infantis), Salmonella ser. Bredeney (S. Bredeney), Salmonella ser. Rissen (S. Rissen), and Salmonella ser. Anatum (S. Anatum), as the most common serovars in pigs. Salmonella Enteritidis (S. Enteritidis), S. Typhimurium, Salmonella Hadar (S. Hadar), Salmonella Virchow (S. Virchow), S. infantis, Salmonella Kentucky (S. Kentucky), S. Bredeney, Salmonella Agona (S. Agona) and Salmonella paratyphi B (S. paratyphi B) are the most common in poultry.
Salmonella infections are a serious medical and veterinary problem world-wide and cause concern in the food industry. Control of salmonellosis is important to avoid potentially lethal human infections and considerable economic losses for the social security and animal husbandry industry.
There has been a long history of the use of live attenuated Salmonella vaccines as effective vaccines for the prevention of salmonellosis in animals and humans. The live attenuated oral typhoid vaccine, Ty21a (Vivotif®), manufactured by the Swiss Serum Vaccine Institute, has proved to be a successful vaccine for the prevention of typhoid fever and has been licensed in many countries including the US and Europe. However, none of the currently available vaccines confer any protection against recrudescence of infection e.g. triggered by stress (Nakamura et al., 1994; Wallis, 2001; Boyen et al., 2008).
Fasting and transportation of animals is known to cause varying levels of stress, depending on a number of parameters, such as crowding, temperature, social status, and duration of feed deprival/transport. A period of stress results in the release of a variety of neurotransmitters, peptides, cytokines, hormones, and other factors into the circulation or tissues of the stressed organism (Freestone et al., 2008). Besides the fast-acting catecholamines, which are released by the sympathetic nervous system, the hypothalamic-pituitary-adrenal axis becomes activated which results in the release of the slow-acting glucocorticoids by the adrenal gland (Dhabhar F S, 2009). These stress hormones can affect the host immune response via the modulation of various aspects of the immune system. However, the pathogenesis of an infection can also be altered by direct effects of these stress mediators on the bacteria. Bacteria can exploit the neuroendocrine alteration of a host stress reaction as a signal for growth and pathogenic processes (Freestone et al., 2008; Lyte M, 2004). This could partly explain the stress induced recrudescence of Salmonella Typhimurium by pigs (Boyen et al., 2009b).
Pigs secrete cortisol as the predominant glucocorticoid (Worsaae and Schmidt, 1980). Peak levels of cortisol occur immediately after start of transport and remain elevated throughout transport (Bradshaw et al., 1996), and plasma cortisol concentrations are an important measure of stress (Jensen-Waern and Nyberg, 1993).
Stress can increase Salmonella shedding in infected pigs and even cause a recrudescence of Salmonella in carriers (Hurd et al., 2002). Consequently, periods of stress result in increased cross-contamination during transport and lairage and to a higher degree of carcass contamination which could lead to higher numbers of foodborne Salmonella infections in humans (Berends et al., 1996; Wong et al., 2002). Reduction of Salmonella in animal products should thus include monitoring and intervention not only at the farm level, but at all levels of production.
For said reasons, it is desirable to develop a vaccine or vaccine strain that is induces a good immune response, is not responsive to recrudescence and/or that is able to prevent or reduce Salmonella recrudescence in order to keep the Salmonella bacteria and the number of Salmonella contaminated animals from increasing during periods of stress. 
SUMMARY OF THE INVENTION
The present invention is based on the finding that the increased intracellular survival in macrophages under cortisol-induced stress is associated with an upregulation of the genes scsA, scsB, scsC, scsD and/or cbpA of the Salmonella bacteria. These genes are therefore suitable targets in the manufacture of a vaccine to prevent or reduce Salmonella recrudescence in a subject.
It is accordingly a first objective of the present invention to provide a Salmonella mutant strain, having at least one genetic modification within, in particular a deletion of, the scsABCD (or scs) locus or the scsA, scsB, scsC, scsD and/or cbpA gene, more in particular within the scsA gene or the scsABCD locus. As will be apparent to the skilled artisan, said genetic modification includes both a naturally occurring genetic modification within said gene(s), as well as an artificially introduced genetic modification. Preferably the genetic modification is an artificially introduced genetic modification.
With the objective to obtain Salmonella mutant strains, the scs locus, scsA, scsB, scsC, scsD and/or cbpA gene mutations as defined herein, can be applied in wild type Salmonella serovars, including naturally occurring attenuated Salmonella vaccine strains, as well as in artificially attenuated Salmonella vaccine strains. The latter typically comprise one, two, three or more (auxotrophic) mutations. In a particular embodiment the present invention provides the Salmonella mutant strain, having at least one genetic modification within the scs locus or the scsA, scsB, scsC, scsD and/or cbpA genes, in particular the scs locus or the scsA gene, and further comprising one, two, three or more (auxotrophic) mutations.
The Salmonella mutant strain as defined and used herein, includes Salmonella enterica and any serotype of the enterica subspecies, and is typically selected from the group consisting of S. Typhimurium, S. Choleraesuis, S. Derby, S. Infantis, S. Bredeney, S. Rissen, S. Anatum, S. Hadar, S. Virchow, and S. Enteritidis. In a more particular embodiment said strain is Salmonella ser. Typhimurium. 
A further embodiment includes the Salmonella mutant strain for use as a vector to administer a heterologous antigen to a subject for vaccination against an infectious agent.
It is a further objective of the present invention to provide the use of a Salmonella mutant strain as described herein, in the manufacture of a vaccine.
In a further embodiment the present invention provides a composition, in particular a vaccine, comprising the Salmonella strain of the invention, optionally comprising a pharmaceutically acceptable carrier, diluent and/or adjuvant.
A further embodiment provides the Salmonella mutant strain, or the composition of the present invention for use as a medicament. More particular the invention provides the Salmonella mutant strain e.g. as part of a vaccine for use in the prevention or inhibition of recrudescence of said strain in a subject, more specific an animal.
Another embodiment provides the use of the composition of the present invention in the treatment or prevention of Salmonella infection, in particular for immunization of pigs, poultry, and/or cattle, and against recrudescence of Salmonella infection.
It is also an object of the present invention to provide a method for treating or preventing Salmonella recrudescence, comprising administering a Salmonella mutant strain or a composition of the present invention, to a subject.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Recovery of Salmonella Typhimurium bacteria from various organs and gut contents of carrier pigs that were submitted to either feed withdrawal (n=6) or social stress, isolation (n=3) and overcrowding (n=9), 24 hours before euthanasia. Six pigs were not stressed and served as a control group. The log10 value of the ratio of CFU per gram sample is given as the mean+standard deviation. Superscript (*) refers to a significant difference compared to the control group (p≦0.05).
FIG. 2: Comparison of the mean serum cortisol concentrations+standard deviation from pigs that were not stressed (control group, n=6) and pigs that were submitted to either feed withdrawal (n=6) or social stress, isolation (n=3) and overcrowding (n=9), 24 hours before euthanasia. The sera of all pigs were tested in twofold and superscript (*) refers to a significant difference compared to the control group (p≦0.05).
FIG. 3: Recovery of Salmonella Typhimurium bacteria from various organs and gut contents of carrier pigs that were injected with either HBSS (control group, n=9) or 2 mg dexamethasone per kg body weight (dexamethasone group, n=9), 24 hours before euthanasia. The log10 value of the ratio of CFU per gram sample is given as the mean+standard deviation. Superscript (*) refers to a significant difference compared to the control group (p≦0.05).
FIG. 4: Number of intracellular Salmonella Typhimurium bacteria in PAM that were treated with control medium or different concentrations of A) cortisol or B) dexamethasone, for 24 hours after invasion. The log10 values of the number of gentamicin protected bacteria+SD are shown. Results are is presented as a representative experiment conducted in triplicate. Superscript (*) refers to a significant difference compared to the control (p≦0.05).
FIG. 5: Number of intracellular Salmonella Typhimurium WT, ΔcbpA and ΔscsA bacteria in PAM that were treated with control medium or different concentrations of cortisol, for 24 hours after invasion. The log10 values of the number of gentamicin protected bacteria+SD are shown. Results are presented as a representative experiment conducted in triplicate. Superscript (*) refers to a significant difference compared to the control (p≦0.05) group.
FIG. 6: Nucleic acid and amino acid sequences of or encoded by the Salmonella scsA, scsB, scsC, scsD and/or cbpA genes.
FIG. 7: Number of intracellular Salmonella Typhimurium bacteria in PAM that were treated with control medium, 2 μM cytochalasin D, 20 μM nocodazole or the combination of both, for 24 hours after invasion. The white bars represent medium without cortisol and the black bars represent medium with 1 μM cortisol. The log10 values of the number of gentamicin protected bacteria+standard deviation are shown. Results are presented as a representative experiment conducted in triplicate. Superscript (*) refers to a significant difference compared to the condition without cortisol (p≦0.05).
FIG. 8: The invasiveness of Salmonella Typhimurium WT and ΔscsA in PAM is shown. The log10 values of the number of gentamicin protected bacteria+standard deviation are given. Results are presented as a representative experiment conducted in triplicate.
FIG. 9: Recovery of Salmoporc® from various organs of pigs 14 days post vaccination. Black bars represent vaccinated pigs that received an intramuscular injection of dexamethasone (2 mg/kg) and white bars represent vaccinated animals that received an intramuscular injection of HBSS (control group). The mean log10 values of the number of CFU per gram sample with is their standard deviations are given. An asterisk (*) refers to a significant difference (P<0.05) between the control group and the dexamethasone group.
FIG. 10: Recovery of Salmonella Typhimurium 112910aNal20 from various organs from DBA/2J mice 14 days post infection. Black bars represent infected DBA/2J mice that received a subcutaneous injection of dexamethasone (100 mg/kg) 24 h before euthanasia and white bars represent infected mice that received an subcutaneous injection of HBSS (control group). Striped bars represent DBA/2J mice that received 25 mg/kg dexamethasone 24 h and 21 h before euthanasia. The mean log10 values of the number of CFU per gram sample with their standard deviations are given. An asterisk (*) refers to a significant difference (P<0.05) between the control group and the dexamethasone group.
FIG. 11: Recovery of Salmonella Typhimurium 112910aNal20 from various organs from BALB/c mice 7 days post infection. Black bars represent infected BALB/c mice that received a subcutaneous injection of dexamethasone (100 mg/kg) 24 h before euthanasia and white bars represent infected mice that received an subcutaneous injection of HBSS (control group). Striped bars represent BALB/c mice that received 25 mg/kg dexamethasone 24 h and 21 h before euthanasia. The mean log 10 values of the number of CFU per gram sample with their standard deviations are given.
FIG. 12: Recovery of Salmonella Typhimurium 112910a (WT) and its isogenic scs (either scsA, scsB, scsC, scsD or scsABCD) knock-out mutants from various organs from mice 14 days post infection. Black bars represent WT infected DBA/2J mice that received a subcutaneous injection of dexamethasone (100 mg/kg) 24 h before euthanasia. White bars represent WT infected mice that received a subcutaneous injection of HBSS. Gray bars represent either ΔscsA, ΔscsB, ΔscsC, ΔscsD or ΔscsABCD infected DBA/2J is mice that received a subcutaneous injection of dexamethasone (100 mg/kg) 24 h before euthanasia. Striped bars represent either: ΔscsA, ΔscsB, ΔscsC, ΔscsD or ΔscsABCD infected mice that received a subcutaneous injection of HBSS. The mean log10 values of the number of CFU per gram sample with their standard deviations are given. An asterisk (*) refers to a significant difference (P<0.05) with the WT dexamethasone group.



DESCRIPTION OF THE INVENTION
The present invention provides mutant strains of Salmonella, in particular Salmonella enterica, that are useful as live or attenuated vaccines for inducing immunological protection against Salmonella, and characterized in that they prevent or reduce intracellular proliferation in macrophages of the Salmonella bacteria triggered by specific circumstances. The mutant strains of the present invention are characterized in that they contain at least one genetic modification within the scsA, scsB, scsC, scsD and/or cbpA gene. The present invention thus relates to a Salmonella strain in which at least one genetic modification within the scsA, scsB, scsC, scsD and/or cbpA gene was introduced.
Salmonella Typhimurium is able to penetrate the mucosal barrier, interact with cells of the immune system and reside in these cells as an intracellular pathogen (Finlay B & Brumell, 2000). Stress induced recrudescence is associated with increased proliferation of the bacterium inside the macrophage. It has now been demonstrated that Salmonella strains comprising a genetic modification in at least one of the scsA, scsB, scsC, scsD and cbpA genes, and in particular in the scs locus or in the scsA gene, yield mutants that no longer exhibit increased proliferation in macrophages in response to stress factors, characterized by elevated cortisol levels. As such the Salmonella mutant strains of the present invention are particularly useful in preparing a vaccine strain that is not responsive to stress-related is recrudescence in a subject. As used herein, the term “recrudescence” refers to reappearance of an infection after it has been quiescent i.e. after a period of latency or relative inactivity; i.e. a new outbreak or the return (become active again) of an infection. “Salmonella recrudescence” or “recrudescence of a Salmonella infection” is characterized by re-excretion of the Salmonella vaccine strain or the Salmonella infection strain by the subject. This results in an increased risk of carrier subjects to begin shedding again, persistent shedders to excrete Salmonella at higher number, and Salmonella-free subjects of becoming infected. In a specific embodiment the recrudescence is stress induced recrudescence. “Stress” or “stress factors” include but are not limited to feed withdrawal, social stress, crowding, temperature changes, social status, rehousing, transport and starvation before slaughter, and is characterized by an increased cortisol blood level.
A “subject” as used herein includes a human or animal, in particular birds (poultry; chicken, turkey, etc.), pigs, or cattle.
In a particular embodiment, the Salmonella mutant strains of the present invention are used as attenuated live vaccines. It is well established that live attenuated micro-organisms are highly effective vaccines; immune responses elicited by such vaccines are often of greater magnitude and of longer duration than those produced by non-replicating immunogens. One explanation for this may be that live attenuated strains establish limited infections in the host and mimic the early stages of natural infection. In addition, unlike killed preparations, live vaccines are often more potent in inducing mucosal immune responses and cell-mediated responses, which may be connected with their ability to replicate in epithelial cells and antigen-presenting cells, such as macrophages, respectively. However, concerns remain over the safety of using live-attenuated vaccines. There may also be a risk of the attenuated strain reverting to virulence, thus having the potential to cause disease and abortion in the vaccinated animal. Surprisingly, it has been demonstrated by the present invention that the vaccine strains and methods as described herein is overcome the risk of stress induced recrudescence of the Salmonella vaccine strain in a vaccinated subject, thereby significantly improving the safety of live vaccines.
The “genetic modification” may be an insertion, a deletion, and/or a substitution of one or more nucleotides in said genes. Deletion mutants (of the complete gene or part thereof) are preferred. The genetic modifications or mutations may be introduced into the microorganism using any known technique. Preferably, the mutation is a deletion mutation, where disruption of the gene is caused by the excision of nucleic acids. Alternatively, mutations may be introduced by the insertion of nucleic acids or by point mutations. Methods for introducing the mutations into the specific regions will be apparent to the skilled person and are preferably created using the one step inactivation method described by Wanner and Datsenko (2000). Other methods can be applied to achieve a site directed mutagenesis (eg. using suicide plasmids), however the one-step inactivation method is generally accepted as the best and fastest way to achieve a knock-out deletion mutant.
The Salmonella mutant strain of the present invention can optionally further comprise one or more additional mutations. Suitable genes for said mutations include but are not limited to genes such as aroA, purA, dam, his, cya/crp, htrA, Lon, phoP/phoQ, guaBA, nuoG, rpoS, rpoE, surA, thyA, aceA and the like. Other genes that may be affected to improve the safety of the vaccine include virulence factors, such as for example SPI-1, SPI-2, SPI-3, SPI-4, SPI-5 and/or related effectors, flagellum-associated genes, fimbria-associated genes, LPS-associted genes and adhesines; quorum sensing and/or biofilm associated genes; genes involved in outer membrane proteins; and regulators of anyone of the aforementioned genes.
Although any serotype of S. enterica may be used, in preferred embodiments, the modifications are inserted into S. enterica serovars, such as for example Salmonella ser. Typhimurium (S. Typhimurium), Salmonella ser. Choleraesuis (S. Choleraesuis), Salmonella ser. Derby (S. Derby), Salmonella ser. Infantis (S. Infantis), Salmonella ser. Bredeney (S. Bredeney), Salmonella ser. Rissen (S. Rissen), and Salmonella ser. Anatum (S. Anatum). In a particular embodiment said modification(s) are inserted a Salmonella ser. Typhimurium background.
The scs locus (scsABCD) consists of two operons, one operon consisting of the single scsA gene and another operon (scsBCD) containing the scsB, scsC and scsD genes encoding proteins that may mediate copper tolerance indirectly by catalyzing the correct folding of periplasmic copper-binding target proteins via a disulfide isomerise-like activity (Gupta et al., 1997). It is demonstrated in the present invention that scsA and scsABCD are vital for dexamethasone induced recrudescence of Salmonella in a DBA/2J mice model, without increasing the virulence of the Salmonella Typhimurium strain used. Deletion of scsA or the entire scs locus in Salmonella Typhimurium live vaccines reduces stress related recrudescence of live vaccine strains.
Preferably, the mutants of the present invention contain at least one genetic modification within the scsA gene, or within the complete scs locus (scsABCD). As used herein, the scsA, scsB, scsC, scsD or cbpA gene is meant to include any homolog or artificial sequence that is substantially identical, i.e. at least 70%, 75%, 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and preferably 100% identical to the corresponding scs locus, scsA, scsB, scsC, scsD or cbpA gene as found in Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 chromosome with NCBI reference sequence NC—003197.1 GI:16763390, and as provided in FIG. 6 (SEQ ID NO 1-5). In said reference sequence the scsA gene corresponds to CDS 1200154.1200516 having GeneID 1252631 and encoding the “suppressor for copper-sensitivity protein A” (NP—460086.1). The scsB gene corresponds to CDS 1200565.1202451 having GeneID 1252632 and encoding the protein “suppressor for copper-sensitivity B” (NP—460087.1). The scsC gene corresponds to CDS 1202448.120307 having GeneID 1252633 and encoding the protein “suppressor for copper-sensitivity C” (NP—460088.1). The scsD gene corresponds to CDS 1203061.1203567, is having GeneID 1252634 and encoding the protein “suppressor for copper-sensitivity D” (NP—460089.1). The cbpA gene is characterized by GeneID 1252630 and encodes the protein “curved DNA-binding protein CbpA” (NP—460085.1). In a specific embodiment, the present invention encompasses a Salmonella mutant strain comprising a genetic modification in, and in particular a deletion of, the scsA gene or the scsABCD locus, as compared to the corresponding wild type sequence as found in Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 chromosome with NCBI reference sequence NC—003197.1 GI:16763390.
The percentage identity of nucleic acid and polypeptide sequences can be calculated using commercially available algorithms which compare a reference sequence with a query sequence. The following programs (provided by the National Center for Biotechnology Information) may be used to determine homologies/identities: BLAST, gapped BLAST, BLASTN and PSI BLAST, which may be used with default parameters.
As already mentioned, safety of (existing) attenuated vaccine strains can be highly improved by applying the methods of the present invention. It is thus also an object of the invention to convert Salmonella vaccine strains, including naturally occurring attenuated Salmonella enterica vaccine strains, as well as artificially attenuated Salmonella enterica vaccine strains, into mutant strains by introducing one or more genetic modifications in the scsA gene, or in the complete scs locus, into said (existing) vaccine strains.
Hence, a particular embodiment of the present invention relates to a method to convert a Salmonella vaccine strain into a safer vaccine strain, said method comprising the following steps:

    
    
        obtaining a Salmonella enterica (vaccine) strain, and
        substituting or deleting part or all of the scsA gene, or the scs locus, in particular deleting the complete scsA gene or the scs locus.
    
    


The method optionally further comprises one or more of the following steps:

    
    
        creating a PCR adjusted antibiotic resistance cassette,
        inserting a helper plasmid in the Salmonella enterica (vaccine) strain,
        substituting part or all of the scsA gene, or the scs locus, with the PCR adjusted antibiotic resistance cassette,
        controlling the substitution with PCR and sequencing,
        inserting the helper plasmid in the substituted target strain,
        deleting the antibiotic resistance cassette and the helper plasmids, and
        controlling the deletion with PCR and sequencing.
    
    


More specific, construction of deletion mutants in genes according to the one-step inactivation method is e.g. described by Datsenko and Wanner (2000) optionally with some modifications (Donné et al., 2005).
In said embodiment wherein a Salmonella enterica vaccine strain is converted into a mutant strain as provided herein, the vaccine strain according to the invention can optionally include additional mutations (also referred to as auxotrophic mutations). Suitable genes for the auxotrophic mutation include but are not limited to genes such as aroA, purA, dam, his, cya/crp, htrA, Lon, phoP/phoQ, guaBA, nuoG, rpoS, rpoE, surA, thyA, aceA and the like. Other genes that may be affected to improve the safety of the vaccine include virulence factors, such as for example SPI-1, SPI-2, SPI-3, SPI-4, SPI-5 and/or related effectors, flagellum-associated genes, fimbria-associated genes, LPS-associted genes and adhesines; quorum sensing and/or biofilm associated genes; genes involved in outer membrane proteins; and regulators of anyone of the aforementioned genes. When modifying existing vaccines according to the method of the current invention, it is important that the further modification does not affect the already weakened strain in its immunogenic and protective effect.
In a further embodiment, the Salmonella mutant is very suitable as a delivery vector. Any of the Salmonella mutant strains described herein can be used as a vector to administer an antigen, DNA or RNA, to a subject for vaccination against an infectious agent, e.g. bacteria, viruses or parasites. Antigen delivery is can be accomplished by introducing into the Salmonella mutant strain a heterologous nucleic molecule encoding the antigen. The antigen-encoding nucleic acid molecule to be introduced into the attenuated Salmonella strain can be present, for example, in a plasmid vector that includes a regulatory sequence, such as a promoter, and, optionally, a sequence encoding a secretion signal.
The promoter can be a prokaryotic promoter, for example, a Salmonella promoter, which directs expression of the antigen in the Salmonella vector. Examples of such promoters are well known to the person skilled in the art. Alternatively, the promoter can be an eukaryotic promoter. Use of such promoters allows for expression of target antigen in a eukaryotic cell, with Salmonella acting as the delivery vehicle for this DNA immunization approach. The construction of such vectors is known in the art. Of course, numerous eukaryotic promoters are known in the art and can be used. Introduction of a plasmid into the Salmonella mutant strain can be accomplished using any of a number of standard methods, such as electroporation or bacteriophage transduction (e.g. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994).
In a further embodiment, the mutant Salmonella strains are used to manufacture a (pharmaceutical) composition or a vaccine composition, which may be administered to the subject via the parenteral, mucosal or oral route. Inactivated or live vaccines can be produced using art known procedures and typically include a pharmaceutically acceptable carrier or diluent, and optionally an adjuvant.
It is accordingly an object of the present invention to provide a pharmaceutical composition or a vaccine against Salmonella recrudescence comprising:

    
    
        a mutant strain according to the invention; and
        a pharmaceutically acceptable carrier or diluent.
    
    


The particular pharmaceutically acceptable carriers or diluents employed are not critical to the present invention, and are conventional in the art. Examples is of diluents include: buffer for buffering against gastric acid in the stomach, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone, or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame. Examples of carriers include: proteins, e.g., as found in skimmed milk; sugars, e.g. sucrose; or polyvinylpyrrolidone.
The particular adjuvants employed are not critical to the present invention, and are conventional in the art. Examples of adjuvants include, but are not limited to, tensoactive compounds (such as Quil A), mineral salts (such as aluminium hydroxide), micro-organism derived adjuvants (such as muramyl dipeptide), oil-in-water and water-in-oil emulsions (such as Freund's incomplete adjuvant), particulate antigen delivery systems (such as liposomes, polymeric atmospheres, nanobeads, ISCOMs and ISCOMATRIX), polysaccharides (such as micro-particulate inulin), nucleic acid based adjuvants (such as CpG motivs), cytokines (such as interleukins and interferons), activators of Toll-like receptors and eurocine L3 en N3 adjuvantia.
As is known to the skilled artisan, the dose or amount varies according to the route of administration. Those skilled in the art may find that the effective dose for a vaccine administered parenterally may be smaller than a similar vaccine which is administered via drinking water, and the like. The number of microorganisms that are required to be present in the formulations can be determined and optimised by the skilled person. However, in general, a patient may be administered approximately 107-1010 colony-forming units (CFUs), preferably approximately 104-109 CFUs in a single dosage unit.
The composition or vaccine comprising are highly suitable for protecting animals against Salmonella recrudescence. The mutant Salmonella strains of the invention, and composition or vaccine comprising the same, are highly suitable for immunizing veterinary species, in particular pigs, cattle and poultry, and even more in particular pigs.
It is thus an object of the present invention to provide the use of mutant strains is of Salmonella enterica of the present invention for preparing a medicament which is employed for the prophylactic and/or therapeutic treatment of Salmonella infection in animals, in particular in pigs. The present invention thus also encompasses the mutant strains of Salmonella enterica as described herein for treating or preventing salmonellosis.
As already mentioned hereinbefore, the mutant microorganisms and vaccine compositions of the present invention may be prepared by known techniques.
The choice of particular Salmonella enterica microorganism, can be made by the skilled person without undue experimentation. A preferred microorganism is selected from the group consisting of S. Typhimurium, S. Choleraesuis, S. Derby, S. Infantis, S. Bredeney, S. Rissen, S. Anatum, S. Hadar, S. Virchow, and S. Enteritidis. 
In one embodiment the microorganism is Salmonella Typhimurium; more in particular the Salmonella Typhimurium strain MB2486, also known as the Salmonella Typhimirium strain 112910a (Boyen F. et al., 2005; Boyen F. et al., 2006). The latter strain has been deposited on Mar. 5, 2010 with BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie—Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium and has accession number LMG P-25625.
In a particular embodiment of the present invention, the mutant strains are scsA, scsB, scsC, scsD and/or cbpA deletion mutants, and preferably scs or scsA deletion mutants, in Salmonella Typhimurium strain MB2486. This strain is a well-characterized porcine field strain that is able to cause persistent infections in pigs, both in field and experimental conditions and is responsive to stress (cortisol) induced recrudescence. This strain does not harbour the virulence plasmid. The contribution of virulence plasmids to the systemic phase of Salmonella infections is well described (Barth and Bauerfeind, 2005; Rychlik et al., 2006). At least six serotypes of Salmonella (serotypes Abortusovis, Choleraesuis, Dublin, Enteritidis, Gallinarum/Pullorum, and Typhimurium) are known to harbour a virulence plasmid. This does not mean is that all isolates of these serotypes carry the virulence plasmid. Pigs generally carry more Salmonella Typhimurium strains lacking the virulence plasmid, compared to for example cattle or horses (Bauerfeind et al., 2001). The virulence plasmid has also been reported to be often absent from strains isolated from clinically healthy pigs or pigs showing only diarrhoea (Namimatsu et al., 2006). In contrast, the virulence plasmid was frequently observed in the isolates from systemically infected pigs (Namimatsu et al., 2006). It can therefore be assumed that strains lacking the virulence plasmid are still capable of colonizing the gut of pigs, but will less frequently lead to systemic infections, both in pigs, other animals and humans.
The invention will be described in further details in the following examples and embodiments by reference to the enclosed drawings. Particular embodiments and examples are not in any way intended to limit the scope of the invention as claimed. The rationale of the examples given here for the serotype S. Typhimurium are equally well applicable to other Salmonella enterica serotypes infecting veterinary species, such as for example Salmonella ser. Choleraesuis (S. Choleraesuis), Salmonella ser. Derby (S. Derby), Salmonella ser. Infantis (S. Infantis), Salmonella ser. Bredeney (S. Bredeney), Salmonella ser. Rissen (S. Rissen), and Salmonella ser. Anatum (S. Anatum).
The following Examples illustrate the invention
Example 1
Materials and Methods
1. Chemicals
Cortisol and dexamethasone (Sigma-Aldrich, Steinheim, Germany) stock solutions of 10 mM were prepared in water and stored at −20° C. Serial dilutions of cortisol were, depending on the experiment, prepared in Luria-Bertani broth (LB, Sigma-Aldric) or in the corresponding cell medium.
2. Bacterial Strains and Growth Conditions
Salmonella Typhimurium strain 112910a, isolated from a pig stool sample and characterized previously by Boyen et al. (2008), was used as the wild type strain (WT) in which the spontaneous nalidixic acid resistant derivative strain (WTnal) was constructed (Boyen et al., 2008). For fluorescence microscopy, Salmonella Typhimurium strain 112910a carrying the pFPV25.1 plasmid expressing green fluorescent protein (gfp) under the constitutive promoter of rpsM was used (Boyen et al., 2008; Van Immerseel et al., 2004).
Unless otherwise stated, the bacteria were generally grown overnight (16 to 20 hours) as a stationary phase culture with aeration at 37° C. in 5 ml of LB broth. To obtain highly invasive late logarithmic cultures for invasion assays, 2 μl of a stationary phase culture were inoculated in 5 ml LB broth and grown for 5 hours at 37° C. without aeration (Lundberg et al., 1999).
For the oral inoculation of pigs, the WTnal was used to minimize irrelevant bacterial growth when plating tonsillar, lymphoid, intestinal and faecal samples. The bacteria were grown for 16 hours at 37° C. in 5 ml LB broth on a shaker, washed twice in Hank's buffered salt solution (HBSS, Gibco, Life Technologies, Paisley, Scotland) by centrifugation at 2300×g for 10 min at 4° C. and finally diluted in HBSS to the appropriate concentration of 107 colony forming units (CFU) per ml. The number of viable Salmonella bacteria per ml inoculum was determined by plating 10-fold dilutions on Brilliant Green agar (BGA, international medical products, Brussels, Belgium) supplemented with 20 μg per ml nalidixic acid (BGANAL, Sigma-Aldrich) for selective growth of the mutant strains.
3. Cell Cultures
Porcine pulmonary alveolar macrophages (PAM) were isolated by broncho-alveolar washes from lungs of euthanized 3 to 4 week old piglets, obtained from a Salmonella-negative farm, as described previously (Dom et al., 1992). The isolated cells were pooled and frozen in liquid nitrogen until further use. Prior to seeding the cells, frozen aliquots of approximately 108 cells per ml were thawed and washed 3 times in Hank's buffered salt solution with Ca2+ and Mg2+ (HBSS+, Gibco) with 10% (v/v) fetal calf serum (FKS, Hyclone, Cramlington, England) at 4° C. Finally, these cells were cultured in Roswell Park Memorial Institute medium (RPMI, Gibco) containing 10% (v/v) FKS, 1% (v/v) L-glutamine (Gibco), 1% (v/v) sodium pyruvate (Gibco), 1% (v/v) MEM non essential amino acids (NEAA, Gibco) and 1% (v/v) penicillin-streptomycin (Gibco).
The polarized intestinal porcine epithelial (IPEC-J2) cell line is derived from jejunal epithelia isolated from a neonatal piglet (Rhoads et al., 1994; Schierack et al., 2006).
4. In vivo Trials
4.1 Experimental Inoculation of Piglets
A standardized infection model was used to create Salmonella carrier pigs (Boyen et al., 2009a). For this purpose 4 four-week-old piglets (commercial closed line based on Landrace) were obtained from a serologically negative breeding herd (according to the Belgian Salmonella monitoring program). The Salmonella-free status of the piglets was tested serologically using a commercially available Salmonella antibody test kit (IDEXX, Hoofddorp, The Netherlands), and bacteriologically via faecal sampling. The piglets were housed in pairs in separate isolation units at 25° C. under natural day-night rhythm with ad libitum access to feed and water. Seven days after they arrived, the piglets were orally inoculated with 2 ml of a suspension containing 107 CFU of WTnal per ml HBSS.
In a first in vivo trial, we investigated the effect of different types of stress on the recrudescence of Salmonella Typhimurium by pigs. In a second in vivo trial, we injected pigs intramuscularly with 2 mg dexamethasone per kg body weight to test our hypothesis that cortisol plays a role in the recrudescence of Salmonella Typhimurium in pigs.
4.2 Effect of Different Types of Stress on the Salmonella Typhimurium Load in Carrier Pigs
The animal experiment was approved by the ethical committee of the Faculty of Veterinary Medicine, Ghent University (EC 2007/101).
At day 23 post inoculation, pigs were submitted to either social stress (n=12) or feed withdrawal stress (n=6), mimicking the transport and starvation period before slaughter. The remaining six pigs were not stressed and served as a negative control group. To induce social stress, the piglets were mixed for 24 hours. One piglet was removed from its pen and transferred to another pen, which already contained 2 piglets. This was done in triplicate, so finally there were three groups of 3 piglets per pen and three groups of 1 piglet per pen. To mimic feed withdrawal stress, three groups of 2 piglets per pen were starved for 24 hours. After the stress period, the animals were humanely euthanized. Blood samples were taken and the serum cortisol concentrations were determined via a commercially available enzyme-linked immunosorbent assay (ELISA, Neogen, Lansing, USA) for the quantitative analysis of cortisol levels in biological fluids. This was conducted according to the manufacterer's instructions. Furthermore, samples of tonsils, ileocaecal lymph nodes, ileum, caecum, colon and contents of ileum, caecum and colon were collected for bacteriological analysis to determine the number of Salmonella bacteria, with a detection limit of 50 CFU per gram tissue or contents.
4.3 Effect of Dexamethasone on the Salmonella Typhimurium Load in Carrier Pigs
This in vivo experiment was approved by the ethical committee of the Faculty of Veterinary Medicine, Ghent University (EC 2010/108). The animals (n=18) were housed and inoculated as described above to create Salmonella carrier pigs (Boyen et al., 2008). At day 42 post inoculation, pigs were intramuscularly injected with either dexamethasone (Kela laboratoria, Hoogstraten, Belgium) (n=9) or HBSS (n=9). Dexamethasone is a long-acting glucocorticoid with a half-life of 36 to 72 hours (Shefrin et al., 2009), which was used at a concentration of 2 mg dexamethasone per kg body weight. It has been described that this concentration does not cause immunosuppression of the pig (Flaming et al., 1994). At 24 hours after dexamethasone injection, the animals were humanely euthanized and samples of tonsils, ileocaecal lymph nodes, ileum, caecum, colon and contents of ileum, caecum and colon were collected for bacteriological analysis, with a detection limit of 83 CFU per gram tissue or contents.
4.4 Bacteriological Analysis
All tissues and samples were weighed and 10% (w/v) suspensions were prepared in buffered peptone water (BPW, Oxoid, Basingstoke, UK). The samples were homogenized with a Colworth stomacher 400 (Seward and House, London, UK) and the number of Salmonella bacteria was determined by plating 10-fold dilutions on BGANAL plates. These were incubated for 16 hours at 37° C. The samples were pre-enriched for 16 hours in BPW at 37° C. and, if negative at direct plating, enriched for 16 hours at 37° C. in tetrathionate broth (Merck, Darmstadt, Germany) and plated again on BGANAL. Samples that were negative after direct plating but positive after enrichment were presumed to contain 50 or 83 CFU per gram tissue or contents (detection limit for direct plating). Samples that remained negative after enrichment were presumed to contain less than 50 or 83 CFU per gram tissue or contents and were assigned value ‘1’ prior to log transformation. Subsequently the number of CFU for all samples derived from all piglets was converted logarithmically prior to calculation of the average differences between the log10 values of the different groups and prior to statistical analysis.
5. Cytotoxicity Assays
The cytotoxic effect of cortisol from 0.001 to 100 μM on PAM and IPEC-J2 cells was determined using the lactate dehydrogenase cytotoxicity detection kit (LDH, Roche Applied Science, Bazel, Switzerland). Therefore PAM were seeded in a 96-well microplate at a density of approximately 2×105 cells per well and were allowed to attach for at least 2 hours. The IPEC-J2 cells were seeded and allowed to grow for at least 24 hours in a 96-well microplate at a density of approximately 2×104 cells per well in Dulbecco's modified Eagle's is medium (DMEM, Gibco) supplemented with 46.5% (v/v) Ham's F12 medium (Gibco), 5% (v/v) FKS and 1% insulin-transferrin-selenium-A supplement (ITS, Gibco).
The LDH test was used in accordance to the manufacturer's instructions and the absorbance was measured at 492 nm using a microplate ELISA reader (Multiscan M S, Thermo Labsystems, Helsinki, Finland). The percentages of cortisol induced cytotoxicity were calculated using the following formula:

% cytotoxicity=100×((a−c)/(b−c))

In this formula a=OD492 derived from the wells incubated with cortisol, b=OD492 derived from the wells incubated with 1% (v/v) Triton X-100 (Sigma-Aldrich), c=OD492 derived from untreated control wells.
6. Effect of Cortisol on the Growth and Gene Expression of Salmonella Typhimurium 
6.1 Effect of Cortisol on the Growth of Salmonella Typhimurium 
The effect of cortisol concentrations ranging from 0.001 to 100 μM on the growth of Salmonella Typhimurium WT was examined during 24 hours. Therefore, Salmonella Typhimurium was grown in LB broth whether or not supplemented with cortisol. The number of CFU per ml was determined at different time points by titration of 10-fold dilutions of the bacterial suspensions on BGA. After incubation for 24 hours at 37° C., the number of colonies was counted.
6.2 Effect of Cortisol on the Gene Expression of Salmonella Typhimurium 
RNA was isolated from Salmonella Typhimurium WT at logarithmic and stationary growth phase in presence or absence of 1 μM cortisol (Lundberg et al., 1999). Two OD600 nm units of each culture were incubated in one-fifth culture volume 5% (v/v) phenol pH 4.3/95% (v/v) ethanol solution for 30 min on ice to stabilize the RNA. Subsequently, the RNA was extracted using the SV Total RNA purification kit (Promega, Leiden, the Netherlands).
Each condition was tested in triplicate and transcriptomic techniques involved Salmonella Typhimurium microarrays constructed at the Institute of Food Research, Norwich, UK, as described previously (Clements et al., 2002; Yberg et al., 2006). The microarray used in this study was the whole-genome SALSA cDNA microarray covering 5080 genes and data analysis was performed as described by Eriksson-Ygberg et al., 2006.
7. Invasion and Intracellular Survival Assays
To examine whether the ability of Salmonella Typhimurium to invade and proliferate in PAM and IPEC-J2 cells was altered after exposure of these cells to cortisol, invasion and intracellular survival assays were performed.
For the invasion assays, PAM and IPEC-J2 cells were seeded in 24-well plates at a density of approximately 5×105 cells and 105 cells per well, respectively. PAM were allowed to attach for at least 2 hours and IPEC-J2 cells were allowed to grow for at least 24 hours. Subsequently, the cells were exposed to different concentrations of cortisol ranging from 0.001 to 100 μM. After 24 hours the invasion assay was performed as described by Boyen et al., 2009a. Finally the PAM and IPEC-J2 cells were washed 3 times and lysed for 10 min with 1% (v/v) Triton X-100 or 0.2% (w/v) sodium deocxycholate (Sigma-Aldrich), respectively and 10-fold dilutions were plated on BGA plates.
To assess intracellular growth, cells were seeded and inoculated as described in the invasion assay, but the medium containing 100 μg per ml gentamicin was replaced after 1 hour incubation with fresh medium containing 20 μg per ml gentamicin whether or not supplemented with different concentrations of cortisol or dexamethasone ranging from 0.001 to 100 μM. The number of viable bacteria was assessed 24 hours after infection.
To determine whether the observed effect was cortisol specific, invasion and proliferation assays were performed also after exposure of the cells to epinephrine, norepinephrine and dopamine at a concentration of 1 μM (Rupprecht et al. 1997; Rupprecht and Holsboer 1999).
To visualize the effect of cortisol on the intracellular proliferation of Salmonella bacteria, PAM were seeded in sterile Lab-tek® chambered coverglasses (VWR, Leuven, Belgium), inoculated with gfp-producing Salmonella at a is multiplicity of infection of 2:1 as described by Boyen et al., 2009a and exposed to cortisol at a high physiological stress concentration of 1 μM (Wei et al., 2010) in cell medium or to cell medium only. After 24 hours at 37° C., cells were washed three times to remove unbound bacteria and cellTraceTM calcein red-orange (Molecular Probes Europe, Leiden, The Netherlands) was added for 30 min at 37° C. Afterwards, cells were washed three times and fluorescence microscopy was carried out. In 100 macrophages, the number of macrophages containing gfp-Salmonella was counted and the average number of cell associated bacteria was calculated.
8. Statistical Analysis
All in vitro experiments were conducted in triplicate with 3 repeats per experiment, unless otherwise stated. All statistical analyses were performed using SPSS version 17 (SPSS Inc., Chicago, Ill., USA). Normally distributed data were analyzed using unpaired Student's t-test or one-way ANOVA to address the significance of difference between mean values with significance set at p≦0.05. Bonferroni as post hoc test was used when equal variances were assessed. If equal variances were not assessed, the data were analyzed using Dunnett's T3 test. Not normally distributed data were analyzed using the non parametric Kruskal-Wallis analysis, followed by Mann-Whitney U test.
Results
1. Feed Withdrawal Results in Increased Numbers of Salmonella Typhimurium Bacteria in the Gut of Pigs and Elevated Cortisol Blood Levels
Carrier pigs subjected to feed withdrawal, 24 hours before euthanasia, showed elevated numbers of Salmonella Typhimurium in their bowel contents and organs in comparison to the control group. This increase was significant in the ileum (p 0.001), ileum contents (p=0.022) and colon (p=0.014). As illustrated in FIG. 1, the number of Salmonella Typhimurium bacteria was also increased in the caecum (p=0.136), caecum contents (p=0.156) and is colon contents (p=0.074). The social stress groups (overcrowding and isolation) showed no significant difference in comparison to the control group.
Pigs that were subjected to feed withdrawal (p=0.004) and overcrowding (p=0.001) showed significantly elevated serum cortisol levels compared to the control group that had a mean cortisol concentration±standard deviation of 48.65±4.67 nM. Pigs that were starved 24 hours before euthanasia had the highest mean serum cortisol level±standard deviation of 66.88±6.72 nM. Pigs that were housed per 3 and housed separately, 24 hours before euthanasia had a mean cortisol concentration±standard deviation of 59.26±3.47 nM and 53.66±2.06 nM respectively. The sera of all pigs were tested in twofold and the results are shown in FIG. 2.
2. Dexamethasone Increases the Number of Salmonella Typhimurium Bacteria in the Gut of Carrier Pigs
Carrier pigs that were intramuscularly injected with 2 mg dexamethasone per kg body weight, 24 hours before euthanasia, showed elevated numbers of Salmonella Typhimurium in their gut tissues and contents in comparison to the control group that was intramuscularly injected with HBSS. This increase was significant in the ileum (p=0.018), colon (p=0.003) and caecum (p=0.014). As illustrated in FIG. 3, the number of Salmonella Typhimurium bacteria was also increased in the ileum contents (p=0.067), caecum contents (p=0.157) and colon contents (p=0.229).
3. Cortisol does not Affect Salmonella Growth and Gene Expression
Cortisol concentrations ranging from 0.001 to 100 μM did not affect the growth of Salmonella (data not shown). The exposure of both a stationary and logarithmic phase culture of Salmonella Typhimurium to cortisol at a high physiological stress concentration of 1 μM did not significantly affect gene expression levels as assessed by microarray analysis (data not shown).
4. Cortisol and Dexamethasone Promote the Intracellular Proliferation of Salmonella Typhimurium in Porcine Macrophages but not in Porcine Enterocytes
The results of the intracellular survival assay of Salmonella Typhimurium in PAM with or without prior exposure to cortisol or dexamethasone are summarized in FIG. 4. The intracellular proliferation of Salmonella Typhimurium was higher in PAM that were treated with cortisol or dexamethasone, for 24 hours, in comparison to non-treated cells. Exposure to concentrations of cortisol 100 nM led to a significant dose-dependent increase of the number of intracellular Salmonella Typhimurium bacteria. The same tendency was seen in PAM that were exposed to dexamethasone.
Cortisol and dexamethasone concentrations from 0.001 to 100 μM did neither affect the intracellular proliferation of Salmonella Typhimurium in IPEC-J2 cells, nor the invasion in PAM and IPEC-J2 cells (data not shown).
The enhanced intracellular proliferation of Salmonella Typhimurium in PAM exposed to a high physiological stress concentration of 1 μM cortisol was confirmed in a proliferation assay with gfp-Salmonella. The proliferation rate of intracellular bacteria that were exposed to 1 μM cortisol for 24 hours was increased in comparison with the control PAM, resulting in a higher mean bacterial count±standard deviation (3.1±2.72 versus 2.0±1.48 bacteria per macrophage, respectively).
Epinephrine, norepinephrine and dopamine at a concentration of 1 μM did neither affect the intracellular proliferation, nor the invasion of Salmonella Typhimurium in PAM and IPEC-J2 cells (data not shown).
Discussion
Our findings showed that a natural stress stimulus like feed withdrawal causes recrudescence of a Salmonella Typhimurium infection in carrier pigs, which could have a serious economic impact. Until now, the mechanism of stress related recrudescence of Salmonella in pigs is not well known.
We showed that social stress and starvation result in elevated serum cortisol levels. To verify whether an increase in corticosteroids could induce recrudescence of Salmonella Typhimurium in pigs, we conducted an in vivo trial in which carrier pigs were intramuscularly injected with dexamethasone. The in vivo trial showed that dexamethasone treatment causes recrudescence of Salmonella Typhimurium in carrier pigs. This implies that the release of corticosteroids in the bloodstream could alter the outcome of a Salmonella Typhimurium infection in pigs resulting in recrudescence of the infection.
We showed that this cortisol mediated effect was not the result of a direct effect on the bacterium, such as increased growth or altered pathogenicity of the bacterium. Earlier research has shown that norepinephrine in vitro promotes the growth and the motility of Salmonella enterica (Bearson B L & Bearson S M, 2008; Methner et al., 2008) and that in vitro pretreatment of Salmonella Typhimurium with norepinephrine is associated with an increased replication of this microorganism in various tissues of experimentally infected pigs (Toscano et al., 2007). However, we provide evidence that cortisol does not cause an increase in growth or any significant changes in the gene expression of Salmonella Typhimurium, at a physiological stress concentration of 1 μM.
In contrast to the absence of a direct effect on the bacterium, we showed that cortisol and dexamethasone promote intracellular proliferation of Salmonella Typhimurium in porcine macrophages, in a dose-dependent manner at concentrations (0.001 to 100 μM) that did not exert a notable effect on cell viability. Salmonella Typhimurium is able to survive and even multiply intracellularly after bacterial entry into host cells (Finlay B & Brumell, 2000). Therefore, we examined whether this increase of serum cortisol levels could result in altered host-pathogen interactions of Salmonella Typhimurium with PAM and IPEC-J2 cells.
Example 2
In Vivo Expression Technology (IVET) Screening for Intracellularly Cortisol Induced Genes of Salmonella Typhimurium 
All the colonies showing low-level lacZY expression were analysed to identify is genes that are intracellularly expressed in PAM that might be essential for Salmonella survival in PAM. In total, we purified and sequenced 287 and 69 colonies from PAM whether or not treated with 1 μM cortisol, respectively. An overview of the identified genes is given in table 1. The represented data are the result of 3 independent experiments for PAM whether or not treated with 1 μM cortisol. Of all genes, only STM4067 was found in all 3 independent experiments and in both conditions. STM4067 encodes the putative ADP-ribosylglycohydrolase, which was identified by Van Parys et al. (2011) as a factor for intestinal Salmonella Typhimurium persistence in pigs.
CbpA, pflC, pflD and scsA were identified in all 3 independent experiments, however only in PAM that were treated with 1 μM cortisol. This implies that these genes might be intracellularly cortisol induced genes of the bacterium. PfIC and pflD encode the pyruvate formate lyase activase II and the formate acetyltransferase 2, respectively. These genes both play a role in the anaerobic glucose metabolism (Nollet et al., 2005). CpbA encodes the curved DNA binding protein which is a molecular hsp40 chaperone that is involved in bacterial responses to environmental stress and which is homologous to DnaJ (Van Parys et al., 2011). ScsA encodes the suppressor of copper sensitivity protein and according to Gupta et al. (1997), it possibly functions as a peroxidase by preventing the formation of free hydroxyl radicals resulting from the reaction of copper with hydrogen peroxide (Williams & Newell, 1970).







TABLE 1







List of genes of Salmonella Typhimurium


induced intracellularly in PAM.














Freq. + 
% + 
Freq. − 
% − 



Gene product 
1 μM
1 μM
1 μM
1 μM


Gene
description*
cortisol
cortisol
cortisol
cortisol















cbpA
curved DNA-binding 
3/3
9.5





protein CbpA






cmk
cytidylate kinase
2/3
1.4




dnaC
DNA replication protein 
1/3
0.7





DnaC






dnaK
molecular chaperone 


1/3
 4.3



DnaK






dnaT
primosomal protein Dnal
1/3
0.7




efP
elongation factor P


1/3
 2.9


entF
enterobactin synthase 
1/3
0.7





subunit F






eutA
reactivating factor for


1/3
 1.4



ethanolamine ammonia 







lyase






folA
dihydrofolate reductase
1/3
0.7




gppA
guanosine pentaphosphate
1/3
1.4





phosphohydrolase






gyrB
DNA gyrase, subunit B
1/3
0.3




lysS
lysyl-tRNA synthetase
2/3
1.0




marC
multiple drug resistance
1/3
0.3





protein MarC






menA
1,4-dihydroxy-2-naphtoate
2/3
6.0





octaprenyltransferase






menG
ribonuclease activity 
2/3
4.6





regulator protein RraA






nlpB
lipoprotein


2/3
16.0


parE
DNA topoisomerase IV


1/3
17.5



subunit B






pflC
pyruvate formate lyase II
3/3
3.6





activase






pflD
formate 
3/3
3.6





acetyltransferase 2






prfC
peptide chain release 
1/3
0.3





factor 3






proP
proline/glycine betaine
1/3
0.7





transporter






prpD
2-methylcitrate 
2/3
0.7





dehydratase






prpE
propionyl-CoA synthetase
2/3
0.7




ratB
outer membrane protein


1/3
 1.4


rfaD
ADP-L-glycero-D-


1/3
 2.9



mannoheptose-6-







epimerase






rnT
ribonuclease T
1/3
2.9




rpoE
RNA polymerase sigma 
1/3
0.3





factor RpoE






rpoN
RNA polymerase factor


1/3
 1.4



sigma-54






rpoZ
DNA-directed RNA
1/3
0.3





polymerase subunit 







omega






scsA
suppression of copper
3/3
8.1





sensitivity protein A






STM0014
putative transcriptional
1/3
0.3





regulator






STM0266
putative cytoplasmic 
1/3
0.3





protein






STM0272
putative chaperone 
1/3
0.3





ATPase






STM0409
putative hypothetical 
1/3
0.7





protein






STM2314
putative chemotaxis 
1/3
0.7





signal transduction 







protein






STM2840
putative anaerobic 
1/3
0.3





nitric oxide reductase 







flavorubredoxin






STM4067
putative ADP-
3/3
36.3 
3/3
21.7



ribosylglycohydrolase






tolC
outer membrane channel
1/3
0.7





protein






torA
trimethylamine N-oxide
1/3
1.0





reductase subunit






trpS
tryptophanyl-tRNA 
1/3
0.7





synthetase






yabN
transcriptional regulator 
1/3
0.7





SgrR






ybdZ
cytoplasmic protein
1/3
0.7




ycgB
SpoVR family protein
1/3
0.3




yfeA
hypothetical protein
1/3
0.3




yfeC
negative regulator
1/3
0.3




ygdH
nucleotide binding
1/3
0.7




ygfA
ligase
1/3
0.3




yggE
periplasmic immunogenic
2/3
3.9





protein






yhbG
ABC transporter 


1/3
 1.4



ATP-binding







protein YhbG






yjbB
transport protein
1/3
0.3
1/3
 2.9


yjjk
RNA polymerase factor


1/3
26.2



sigma-54






yqjE
inner membrane protein
1/3
1.0




yqjg
glutathione S-transferase
1/3
1.7











The represented data are the result of 3 independent experiments for PAM whether (+1 μM cortisol) or not (−1 μM cortisol) treated with cortisol. The frequency (Freq.) shows the fraction of positive samples in relation to the total number of independent experiments. If an expressed gene was found more than once, then the contribution of the gene in relation to the total number of tested colonies is expressed as percentage (%). Superscript (*) refers to gene product description according to the National Center for Biotechnology Information (NCBI)
Example 3
Deletion of scsA Results in the Inhibition of the Cortisol Induced Increased Intracellular Proliferation of Salmonella Typhimurium in PAM and in an Upregulation of the scsBCD Operon
Salmonella Typhimurium deletion mutants ΔscsA and ΔcbpA, were constructed according to the one-step inactivation method described by Datsenko and Wanner (2000) and slightly modified for use in Salmonella Typhimurium as described by Boyen et al. (2006c). Primers used to create the gene-specific linear PCR fragments (cbpA and scsA forward and reverse) are given in table 2. The targeted genes were completely deleted from the start codon through the stop codon, as confirmed by sequencing.







TABLE 2







Primers used in this study.










Primers
Sequences







cbpA
5′-GAAACCTTTTGGGGTCCCT



forward
TCTGTATGTATTGATTTAGCGAGATGAT




GCTTGTGTAGGCTGGAGCTGCTTC-3′ 




(SEQ ID NO: 11)



 



cbpA
5′-GTGTGCAAACAAAATTCGGTG



reverse
ATGGTAAAGGTGACAGTGATGTTAG




CCATCATATGAATATCCTCCTTAG-3′ 




(SEQ ID NO: 12)



 



scsA
5′-CAAAACCGCGCCAGTGGCTAAGAT



forward
AACTCGCGTTAAACAGTGAGGG




CGCATGTGTAGGCTGGAGCTGCTTC-3′ 




(SEQ ID NO: 13)



 



scsA
5′-ATTTTTTCTCCGTGAATGAGTAA



reverse
TTAACCGTTAGCAATAACCGGTCT




GCATATGAATATCCTCCTTAG-3′ 




(SEQ ID NO: 14)










Following the IVET screening, the intracellular survival assay was repeated with Salmonella Typhimurium ΔscsA and ΔcbpA and compared to the WT. These results are shown in FIG. 5. The intracellular proliferation of Salmonella Typhimurium WT and ΔcbpA was higher in cortisol treated PAM, for 24 hours, in comparison to non-treated cells. Exposure to cortisol concentrations of respectively ≧10 nM and 500 nM led to a significant dose-dependent increase of the number of intracellular Salmonella Typhimurium WT or ΔcbpA bacteria. Cortisol concentrations from 0.001 to 100 μM did not affect the intracellular proliferation of Salmonella Typhimurium ΔscsA in PAM. This implies that the scsA gene is at least partly responsible for the increased intracellular survival of Salmonella WT in cortisol exposed PAM.
Microarray analysis of Salmonella Typhimurium ΔscsA in comparison to the WT, results in 57 and 19 genes that are differentially regulated, by ≧1.5 fold with p≦0.05, in the logarithmic and stationary phase culture, respectively. This analysis established that the deletion of scsA results in the upregulation of the scsBCD operon. ScsB, scsC and scsD were upregulated with a fold change of respectively 34.16, 19.63 and 6.50 in a stationary phase culture and 32.09, 19.90 and 6.33 in a logarithmic phase culture. The results are provided in Table 3 and 4.
In the stationary phase culture, an increased expression of Salmonella pathogenicity island (SPI-1) Type III Secretion system (T3SS) Needle Complex Protein Prgl (1.81) and the SPI-1 T3SS effector protein SipA (1.71) was observed. Furthermore, Salmonella Typhimurium ΔscsA grown to a logarithmic phase culture showed an increased expression of the T3SS effector protein SipC (2.19). However, the invasion capacity of Salmonella Typhimurium ΔscsA was not altered in comparison to the WT strain (FIG. 8).







TABLE 3







Microarray data of the stationary phase culture, showing genes


differentially regulated, by ≧1.5 fold with p ≦ 0.05, between the wild type and the


ΔscsA derivative Salmonella Typhimurium strain.










Fold




change




compared 



Gene
to 1
Gene product description





scsB
34.16 
suppressor for copper-sensitivity B (gi|2327004); suppression of




copper sensitivity: lipoprotein modification in Igt mutants of E. coli




[Salmonella typhimurium LT2].


scsC
19.63 
S. typhimurium suppressor for copper-sensitivity C (gi|2327005)


scsD
6.5 
S. typhimurium suppressor for copper-sensitivity D (gi|2327006)


prgK
2.649
lipoprotein; may link inner and outer membranes; PRGK protein




precursor. (SW:PRGK_SALTY); cell invasion protein [Salmonella




typhimurium LT2].


corA
2.444
Mg transport system I; magnesium and cobalt transport protein




CORA. (SW:CORA_SALTY); MIT family Mg2+/Ni2+/Co2+




transport protein [Salmonella typhimurium LT2].


PSLT092
2.089
conjugal transfer pilus assembly protein TraU


PSLT015
1.947
putative outer membrane protein


prgl
1.806
PRGI protein. (SW:PRGI_SALTY); cytoplasmic cell invasion




protein [Salmonella typhimurium LT2].


rpsG
1.777
initiates assembly; (SW:RS7_SALTY); 30S ribosomal subunit




protein S7 [Salmonella typhimurium LT2].


sipA
1.713
SipA (gi|1172128); cell invasion protein [Salmonella typhimurium




LT2].


ptrB
1.57 
similar to E. coli protease II (AAC74915.1); Blastp hit to




AAC74915.1 (686 aa), 89% identity in aa 1-680; protease II




[Salmonella typhimurium LT2].


PSLT070
0.618
psiA—plasmid SOS inhibition protein A


yfgJ
0.591
similar to E. coli orf, hypothetical protein (AAC75563.1); Blastp hit




to AAC75563.1 (83 aa), 63% identity in aa 13-83; putative




cytoplasmic protein [Salmonella typhimurium LT2].


yjbG
0.57 
similar to E. coli orf, hypothetical protein (AAC76998.1); Blastp hit




to AAC76998.1 (245 aa), 74% identity in aa 1-245; putative




periplasmic protein [Salmonella typhimurium LT2].


yceO
0.554
similar to E. coli orf, hypothetical protein (AAC74142.1); Blastp hit




to AAC74142.1 (46 aa), 64% identity in aa 10-46; putative inner




membrane protein [Salmonella typhimurium LT2].


phnW
0.543
2-aminoethylphosphonate-pyruvate aminotransferase phnW




(gi|11354251); 2-aminoethylphosphonate transport [Salmonella




typhimurium LT2].


prpD
0.535
S. typhimurium PRPD protein. (SW:PRPD_SALTY)


STM4218
0.525
hypothetical protein; putative inner membrane protein [Salmonella




typhimurium LT2].


STM1698A
0.344
















TABLE 4







Microarray data of the logarithmic phase culture, showing genes


differentially regulated, by ≧1.5 fold with p ≦ 0.05, between the wild type and the


ΔscsA derivative Salmonella Typhimurium strain.










Fold




change




compared 



Gene
to 1
Gene product description












scsB
32.09
suppressor for copper-sensitivity B (gi|2327004); suppression of




copper sensitivity: lipoprotein modification in Igt mutants of E. coli




[Salmonella typhimurium LT2].


scsC
19.9
S. typhimurium suppressor for copper-sensitivity C (gi|2327005)


scsD
6.325
S. typhimurium suppressor for copper-sensitivity D (gi|2327006)


yjiY
4.529
similar to E. coli putative carbon starvation protein (AAC77310.1);




Blastp hit to AAC77310.1 (721 aa), 96% identity in aa 6-721;




putative carbon starvation protein [Salmonella typhimurium LT2].


yjiA
2.929
similar to E. coli orf, hypothetical protein (AAC77308.1); Blastp hit




to AAC77308.1 (284 aa), 90% identity in aa 1-284; putative




cobalamin synthesis protein [Salmonella typhimurium LT2].


yjiX
2.358
similar to E. coli orf, hypothetical protein (AAC77309.1); Blastp hit




to AAC77309.1 (67 aa), 94% identity in aa 1-67; putative




cytoplasmic protein [Salmonella typhimurium LT2].


sipC
2.191
sspC protein (gi|7443298); cell invasion protein [Salmonella




typhimurium LT2].


STM1785
2.061
putative cytoplasmic protein [Salmonella typhimurium LT2].


STM4596
1.987
similar to E. coli orf, hypothetical protein (AAC73478.1); Blastp hit




to AAC73478.1 (222 aa), 31% identity in aa 55-222; putative




inner membrane protein [Salmonella typhimurium LT2].


ygaE
1.984
similar to E. coli putative transcriptional regulator (AAC75711.1);




Blastp hit to AAC75711.1 (226 aa), 87% identity in aa 10-225;




putative GntR family transcriptional repressor [Salmonella




typhimurium LT2].


phoQ
1.97
ligand is Mg+; virulence sensor protein PHOQ




(SW:PHOQ_SALTY); sensory kinase protein in two-component




regulatory system with PhoP [Salmonella typhimurium LT2].


pduT
1.838
polyhedral bodies; similar to E. coli detox protein (AAC75510.1);




Blastp hit to AAC75510.1 (111 aa), 41% identity in aa 16-93,




31% identity in aa 16-90; propanediol utilization protein




[Salmonella typhimurium LT2].


orgA
1.765
Putative RBS for orgA; RegulonDB:STMS1H002934


STM3355
1.756
similar to E. coli L-tartrate dehydratase, subunit A (AAC76097.1);




Blastp hit to AAC76097.1 (303 aa), 54% identity in aa 6-299;




putative tartrate dehydratase alpha subunit [Salmonella




typhimurium LT2].


rph
1.721
ribonuclease PH. (SW:RNPH_SALTY); RNase PH [Salmonella




typhimurium LT2].


pykF
1.702
formerly F; fructose stimulated; pyruvate kinase I.




(SW:KPY1_SALTY); pyruvate kinase I [Salmonella typhimurium




LT2].


yhjS
1.642
similar to E. coli putative protease (AAC76561.1); Blastp hit to




AAC76561.1 (523 aa), 80% identity in aa 1-523; putative




cytoplasmic protein [Salmonella typhimurium LT2].


sprB
1.641
transcriptional regulator SprB (gi|5007028); transcriptional




regulator [Salmonella typhimurium LT2].


pduQ
1.637
similar to E. coli CoA-linked acetaldehyde dehydrogenase and




iron-dependent alcohol dehydrogenase; pyruvate-formate-lyase




deactivase (AAC74323.1); Blastp hit to AAC74323.1 (891 aa),




40% identity in aa 574-860, 35% identity in aa 456-556;




propanediol utilization propanol dehydrogenase [Salmonella




typhimurium LT2].


yajG
1.626
similar to E. coli putative polymerase/proteinase (AAC73537.1);




Blastp hit to AAC73537.1 (226 aa), 85% identity in aa 20-226;




putative lipoprotein [Salmonella typhimurium LT2].


STM4519
1.599
Paralog of E. coli putative aldehyde dehydrogenase




(AAC74598.1); Blastp hit to AAC74598.1 (470 aa), 42% identity in




aa 19-465


hepA
1.591
Ortholog of E. coli probable ATP-dependent RNA helicase




(AAC73170.1); Blastp hit to AAC73170.1 (968 aa), 93% identity in




aa 1-968


talA
1.587
similar to E. coli transaldolase A (AAC75517.1); Blastp hit to




AAC75517.1 (316 aa), 89% identity in aa 1-316; transaldolase A




[Salmonella typhimurium LT2].


nhaB
1.568
Na+/H+ antiporter; regulator of intracellular pH; similar to E. coli




Na+/H+ antiporter, pH independent (AAC74270.1); Blastp hit to




AAC74270.1 (513 aa), 92% identity in aa 1-513; NhaB family of




transport protein [Salmonella typhimurium LT2].


yaeJ
1.535
Ortholog of E. coli orf, hypothetical protein (AAC73302.1); Blastp




hit to AAC73302.1 (140 aa), 85% identity in aa 1-136


STM1560
1.531
similar to E. coli 1,4-alpha-glucan branching enzyme




(AAC76457.1); Blastp hit to AAC76457.1 (728 aa), 30% identity in




aa 235-407, 28% identity in aa 524-576; putative alpha




amylase [Salmonella typhimurium LT2].


tolC
1.528
specific tolerance to colicin E1; segregation of daughter




chromosomes; role in organic solvent tolerance; similar to E. coli




outer membrane channel; specific tolerance to colicin E1;




segregation of daughter chromosomes (AAC76071.1); Blastp hit




to AAC76071.1 (495 aa), 89% identity in aa 1-495; outer




membrane channel [Salmonella typhimurium LT2].


ydcR
1.502
similar to E. coli multi modular; putative transcriptional regulator;




also putative ATP-binding component of a transport system




(AAC74521.1); Blastp hit to AAC74521.1 (468 aa), 87% identity in




aa 1-468; putative gntR family regulatory protein [Salmonella




typhimurium LT2].


fliN
0.66
component of motor switch and energizing; flagellar motor switch




protein FLIM. (SW:FLIN_SALTY); flagellar biosynthesis protein




[Salmonella typhimurium LT2].


ratB
0.658
RatB (gi|5107806); putative outer membrane protein [Salmonella




typhimurium LT2].


melB
0.655
melibiose carrier protein (thiomethylgalactoside




permease)(melibiose permease) (Na+ (Li+)/melibiose symporter)




(melibiosetransporter). (SW:MELB_SALTY); GPH family




melibiose permease II [Salmonella typhimurium LT2].


STM1808
0.654
similar to E. coli orf, hypothetical protein (AAC74867.1); Blastp hit




to AAC74867.1 (119 aa), 42% identity in aa 2-113; putative




cytoplasmic protein [Salmonella typhimurium LT2].


STM2694
0.644
Putative RBS for STM2694; RegulonDB:STMS1H002780


STM2745
0.624
Putative inner membrane protein


amyA
0.619
cytoplasmic alpha-amylase. (SW:AMY2_SALTY); cytoplasmic




alpha-amylase [Salmonella typhimurium LT2].


STM2718
0.599
Putative RBS for STM2718; RegulonDB:STMS1H002803


STM2239
0.593
Putative RBS for STM2239; RegulonDB:STMS1H002376


STM2708
0.588
Putative RBS for STM2708; RegulonDB:STMS1H002793


STM2789
0.568
similar to E. coli orf, hypothetical protein (AAC75706.1); Blastp hit




to AAC75706.1 (360 aa), 86% identity in aa 34-360; putative




cytoplasmic protein [Salmonella typhimurium LT2].


yoaG
0.568
similar to E. coli orf, hypothetical protein (AAC74866.1); Blastp hit




to AAC74866.1 (60 aa), 96% identity in aa 1-60; putative




cytoplasmic protein [Salmonella typhimurium LT2].


ygdK
0.561
Ortholog of E. coli orf, hypothetical protein (AAC75853.1); Blastp




hit to AAC75853.1 (147 aa), 88% identity in aa 1-144


pyrL
0.524
pyrbi operon leader peptide (attenuator).(SW:LPPY_SALTY);




pyrBI operon leader peptide [Salmonella typhimurium LT2].


yigF
0.521
hypothetical 14.6 Kda protein in corA-rarD intergenic region.




(SW:YIGF_SALTY); putative inner membrane protein [Salmonella




typhimurium LT2].


STM0763
0.509
similar to E. coli cyn operon positive regulator (AAC73441.1);




Blastp hit to AAC73441.1 (299 aa), 25% identity in aa 16-288;




transcriptional regulator, lysR family [Salmonella typhimurium




LT2].


STM4219
0.509
putative cytoplasmic protein [Salmonella typhimurium LT2].


PSLT069
0.508
Plasmid SOS inhibition protein B


STM1012
0.498
probable regulatory protein (gi|7467281); Gifsy-2 prophage




putative regulatory protein [Salmonella typhimurium LT2].


STM0298
0.493
similar to E. coli orf, hypothetical protein (AAC73370.1); Blastp hit




to AAC73370.1 (384 aa), 28% identity in aa 221-295; putative




integrase core domain [Salmonella typhimurium LT2].


STM1033
0.475
similar to E. coli ATP-dependent proteolytic subunit of clpA-clpP




serine protease, heat shock protein F21.5 (AAC73540.1); Blastp




hit to AAC73540.1 (207 aa), 29% identity in aa 73-203; Gifsy-2




prophage Clp protease-like protein [phage Gifsy-2].


STM0348
0.459
hypothetical protein; putative inner membrane protein [Salmonella




typhimurium LT2].


STM0283
0.447
putative inner membrane protein [Salmonella typhimurium LT2].


STM4316
0.441
hypothetical protein; putative cytoplasmic protein [Salmonella




typhimurium LT2].


ssaL
0.41
secretion system apparatus protein SSAL. (SW:SSAL_SALTY);




secretion system apparatus protein [Salmonella typhimurium




LT2].


STM1016
0.392
S. typhimurium hypothetical protein (gi|7467246)


pldB
0.38
similar to E. coli lysophospholipase L(2) (AAC76828.1); Blastp hit




to AAC76828.1 (340 aa), 81% identity in aa 1-336;




lysophospholipase L(2) [Salmonella typhimurium LT2].


PSLT083
0.309
conjugal transfer protein TrbD


wzzE
0.305
Ortholog of E. coli putative transport protein (AAC76790.1); Blastp




hit to AAC76790.1 (349 aa), 91% identity in aa 2-346









Example 4
Effect of Cortisol on the Protein Expression of Salmonella Typhimurium Infected Primary Porcine Macrophages
A comparative proteome study was conducted to reveal the effects of cortisol on the protein expression of Salmonella Typhimurium infected primary porcine alveolar macrophages (PAM). We used a gel-free approach called isobaric tags for relative and absolute quantification (iTRAQ) in which four different isobaric labels are used to tag N-termini and lysine side chains of four different samples with four different isobaric reagents. Upon collision-induced dissociation during MS/MS, the isobaric tags are released, which results in four unique reporter ions that are used to quantify the proteins in the four different samples (Ross et al., 2004).
Sample preparation: PAM were isolated and cultured as described in Verbrugghe et al. (2011), they were seeded in 175 cm2 cell culture flasks at a density of approximately 5×107 cells per flask and were allowed to attach for 2 hours. Subsequently, PAM were washed 3 times with Hank's buffered salt solution with Ca2+ and Mg2+ (HBSS+, Gibco) and a gentamicin protection invasion assay was performed as described by Boyen et al. (2009a). Briefly, Salmonella was inoculated into the cell culture flasks at a multiplicity of infection (MOI) of 10:1. To synchronize the infection, the inoculated flasks were centrifuged at 365×g for 10 min and incubated for 30 min at 37° C. under 5% CO2. Subsequently, the cells were washed 3 times with HBSS+ and fresh medium supplemented with 100 μg/ml gentamicin (Gibco) was added. After 1 hour, the medium was replaced by fresh medium containing 20 μg/ml gentamicin, with or without 1 μM cortisol (Sigma-Aldrich). Twenty-four hours after infection, the cells were washed 3 times with HBSS+ and treated with lysis buffer containing 1% (v/v) Triton X-100 (Sigma-Aldrich), 40 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris, Sigma-Aldrich), a cocktail of protease inhibitors (PIs; Sigma-Aldrich) and phosphatase inhibitors (PPI, Sigma-Aldrich), 172.6 U/ml deoxyribonuclease I (DNase I, Invitrogen, USA) and 100 mg/ml ribonuclease A (RNase A, Qiagen, Venlo, The Netherlands). Subsequently, cell debris and bacteria were removed by centrifugation at 2300×g for 10 min at 4° C. Two % (v/v) tributylphosphine (TBP, Sigma-Aldrich) was added to the supernatant followed by centrifugation at 17 968×g for 10 min. The supernatant was held on ice until further use and the pellet was dissolved and sonicated (6 times 30 sec), using an ultrasonic processor XL 2015 (Misonix, Farmingdale, N.Y., USA), in reagent 3 of the Ready Prep Sequential extraction kit (Bio-Rad, Hercules, Calif., USA). This was centrifugated at 17 968×g for 10 min. Both supernatants were combined and a buffer switch to 0.01% (w/v) SDS in H2O was performed using a Vivaspin column (5000 molecular weight cut off Hydrosarts, Sartorius, Germany). Protein concentration was determined using the Bradford Protein Assay (Thermo Fisher Scientific, Rockford, USA) according to the manufacterer's instructions.
Trypsin digest and iTRAQ labeling: Digest and labeling of the samples (100 μg proteins per sample) with iTRAQ reagents was performed according to the manufacturer's guidelines (AB Sciex, Foster City, Calif., USA). Individual samples of cortisol treated or untreated PAM were analyzed in the same run, making paired comparisons possible and minimizing technical variation. Each condition was run in duplicate using different labels of the four-plex labeling kit. The experiment was conducted in twofold and the labeling of the samples was as follows: run 1 (untreated PAM sample 1: 114—untreated PAM sample 2: 115—treated PAM sample 1: 116—treated PAM sample 2: 117)—run 2 (untreated PAM sample 3: 114—untreated PAM sample 4: 115—treated PAM sample 3: 116—treated PAM sample 4: 117). After labeling, 6 μl of a 5% (v/v) hydroxylamine solution was added to hydrolyze unreacted label and after is incubation at room temperature for 5 min, the samples were pooled, dried and resuspended in 5 mM KH2PO4 (15% (v/v) acetonitrile) (pH 2.7). The combined set of samples was first purified on ICAT SCX cartridges, desalted on a C18 trap column and finally fractionated using SCX chromatography. Each fraction was analyzed by nano LC-MSMS as described by Bijttebier et al. (2009).
Data analysis: With no full pig protein database available, different search parameters and databases, both EST and protein, were validated to obtain maximum spectrum annotation. Best results (39% of spectra annotated above homology threshold with a 3.71% false discovery rate in the decoy database) were obtained when searching NCBI Mammalia. For quantification, data quality was validated using ROVER (Colaert et al., 2011). Based on this validation a combined approach was used to define recurrently different expression patterns. In a first approach, the four ratios that can be derived from each run (114/116, 115/117, 114/117 and 115/116) were log-transformed and a t-test was used to isolate protein ratios significantly different from 0 in each run. In a second approach, the two runs were merged into one file and the 114/116 and 115/117 ratios of each run were log-transformed and these ratios were multiplied (log*log). Proteins with recurrent up- or downregulation result in positive log*log protein ratios and those >0.01 were retained and listed. Proteins that were present in both lists were considered unequivocally differentially expressed. This combined approach allows defining proteins with relatively low, but recurrent expressional differences.
The Contribution of the Cytoskeleton to Cortisol Induced Intracellular Proliferation of Salmonella Typhimurium in Primary Porcine Macrophages
The contribution of the cytoskeleton during the cortisol induced increased proliferation of Salmonella Typhimurium in PAM was investigated using cytochalasin D (Sigma) for the inhibition of F-actin polymerization, and nocodazole (Sigma) as an inhibitor for microtubule formation. Therefore, PAM is were seeded in 24-well plates at a density of approximately 5×105 cells per well, allowed to attach for 2 hours and infected with Salmonella, as described in the iTRAQ analysis. To assess the intracellular proliferation, the medium containing 100 μg/ml gentamicin was replaced after 1 hour incubation with fresh medium containing 20 μg/ml gentamicin, with or without 1 μM cortisol, 2 μM cytochalasin D and/or 20 μM nocodazole. Twenty-four hours after infection, the number of viable bacteria was determined by plating 10-fold dilutions on Brilliant Green Agar (BGA, international medical products, Brussels, Belgium).
Results
Differential Protein Expression of Salmonella Typhimurium Infected Primary Porcine Macrophages after Exposure to Cortisol
Peptides from trypsin digested proteins were labeled with isobaric mass tag labels and analyzed by 2-D LC MSMS. Collision-induced dissociation results in the release of these isobaric tags, which allows relative quantification of the peptides. A broad comparison between cortisol treated and untreated Salmonella Typhimurium infected PAM, resulted in the identification of 23 proteins with relatively low, but recurrent expressional differences, as shown in Table 5. Two of these proteins showed higher levels in untreated PAM, whereas 21 of them were more abundant in cortisol treated PAM. Proteomic analysis revealed a cortisol increased expression of beta tubulin, capping protein beta 3 subunit, thymosin beta-4, actin-related protein 3B, tropomyosin 5, and elongation factor 1-alpha 1 isoform 4, which are 6 proteins that are involved in reorganizations of the cytoskeleton. Furthermore, cortisol caused an increased expression of transketolase, Cu—Zn superoxide dismutase, glutaredoxin and prostaglandin reductase 1 (15-oxoprostaglandin 13-reductase) which play a role in the macrophage defense mechanisms.







TABLE 5







Differential protein expression of Salmonella infected macrophages


after exposure to cortisol.












T-
log*


Protein name*
Function*
test
log 





Cytochrome c 
This protein is one of the nuclear-coded
0.7
0.8


oxidase
polypeptide chains of cytochrome c oxidase,




subunit 5B,
the terminal oxidase in mitochondrial electron




mitochondrial
transport.




Pulmonary 
Pulmonary surfactant-associated proteins
0.7
0.8


surfactant-
promote alveolar stability by lowering the




associated 
surface tension at the air-liquid interface in the




protein B
peripheral air spaces.




Tropomyosin 5
Is an actin-binding protein that regulates actin
1.2
1.2



mechanics.




Cathepsin B 
Thiol protease which is believed to participate
1.2
1.2


precursor
in intracellular degradation and turnover of





proteins.




Peptidyl-prolyl 
Peptidyl-prolyl cis-trans isomerase B
1.2
1.2


cis-trans 
accelerates the folding of proteins. It catalyzes




isomerase B
the cis-trans isomerization of proline imidic





peptide bonds in oligopeptides.




Transketolase
Is an enzyme of the pentose phasphate
1.2
1.3



pathway and the calvin cycle that catalysis the





conversion of Sedoheptulose 7-phosphate +





D-glyceraldehyde 3-phosphate to D-ribose 5-





phosphate + D-xylulose 5-phosphate in both





directions.




Translation 
This protein promotes the GTP-dependent
1.3
1.3


elongation 
binding of aminoacyl-tRNA to the A-site of




factor 1
ribosomes during protein biosynthesis.




alpha 2 





isoform 1





L-lactate
Is an enzyme that catalyses the conversion
1.2
1.2


dehydrogenase 
from (S)-lactate + NAD+ to pyruvate + NADH




A chain
in the final step of anaerobic glycolysis.




Cu-Zn-
Is an enzyme that catalysis the dismutation of
1.2
1.2


superoxide
superoxide into oxygen and hydrogen




dismutase
peroxide.




Cytochrome c 
This protein is one of the nuclear-coded
1.2
1.3


oxidase
polypeptide chains of cytochrome c oxidase,




subunit IV
the terminal oxidase in mitochondrial electron





transport.




Malate
Is an enzyme in the citric acid cycle that
1.2
1.3


dehydrogenase,
catalyzes the conversion of (S)-malate +




mitochondrial
NAD+ into oxaloacetate + NADH and vice





versa




Elongation 
This protein promotes the GTP-dependent
1.2
1.3


factor
binding of aminoacyl-tRNA to the A-site of




1-alpha 1 
ribosomes during protein biosynthesis.




isoform 4





Thymosin 
Plays an important role in the organization of
1.2
1.3


beta-4
the cytoskeleton. Binds to and sequesters





actin monomers (G actin) and therefore





inhibits actin polymerization.




Capping 
Cellular component of the F-actin capping
1.2
1.3


protein
protein complex that binds to and caps the




beta 3
barbed ends of actin filaments, thereby




subunit
regulating the polymerization of actin





monomers but not severing actin filaments.




Annexin A1
Calcium/phospholipid-binding protein which
1.3
1.3



promotes membrane fusion and is involved in





exocytosis. This protein regulates





phospholipase A2 activity.




Neutral alpha-
Cleaves sequentially the 2 innermost alpha-
1.3
1.3


glucosidase AB
1,3-linked glucose residues from the





Glc2Man9GlcNAc2 oligosaccharide precursor





of immature glycoproteins.




CD14 antigen
The protein is a surface antigen that is
1.3
1.3



preferentially expressed on





monocytes/macrophages. It cooperates with





other proteins to mediate the innate immune





response to bacterial lipopolysaccharide.




Beta tubulin
Tubulin is the major constituent of
1.3
1.4



microtubules. It binds two moles of GTP, one





at an exchangeable site on the beta chain and





one at a non-exchangeable site on the alpha-





chain.




Actin-related 
May function as ATP-binding component of
1.4
1.4


protein 3B
the Arp2/3 complex which is involved in





regulation of actin polymerization and together





with an activating nucleation-promoting factor





(NPF) mediates the formation of branched





actin networks.




Granulins
Granulins have possible cytokine-like activity.
1.4
1.5



They may play a role in inflammation, wound





repair, and tissue remodeling.




Glutaredoxin
Is a redox enzyme that uses glutathione as a
1.4
1.4



cofactor and which plays a role in cell redox





homeostasis.




Vat1 protein
This protein belongs to the oxidoreductases
1.7
1.7



that play a role in oxidation-reduction





processes.




Prostaglandin
Catalyzes the conversion of leukotriene B4
1.8
1.8


reductase 1
into 12-oxo-leukotriene B4. This is an initial





and key step of metabolic inactivation of





leukotriene B4.





Differentially expressed proteins identified in cortisol treated PAM in comparison to untreated PAM, by use of iTRAQ analysis coupled to 2-D LC MSMS. Superscript (*) refers to protein description according to the UniProtKB/Swiss-Prot protein sequence database.


T-test: Protein ratio treated/untreated PAM of the T-test approach


Log*log: Protein ratio treated/untreated PAM of the log*log approach






Cortisol Induced Increased Survival of Salmonella Typhimurium is Both Microfilament and Microtubule Dependent
As earlier described, exposure to 1 μM cortisol for 24 hours led to a significant increase of the number of intracellular Salmonella Typhimurium bacteria compared to untreated PAM (Verbrugghe et al., 2011). In the present study, we showed that this cortisol induced increased intracellular proliferation of Salmonella Typhimurium is microfilament and microtubule dependent. The is treatment of Salmonella Typhimurium infected PAM with cytochalasin D and/or nocodazole resulted in the inhibition of the cortisol induced increased survival of the bacterium. Results are summarized in FIG. 7.
Example 5
Reducing Stress Induced Recrudescence of Live Salmonella Vaccine Strains
5.1. Materials and Methods
Animal experiments were carried out in strict accordance with the recommendations in the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The experimental protocols and care of the animals were approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University (EC 2011/099 and EC 2011/116).
Salmonella Strains
For oral vaccination of pigs, a commercially available, live, attenuated, Salmonella Typhimurium vaccine (Salmoporc® IDT, Rodleben) was used. This strain is formulated based on a double attenuated Salmonella Typhimurium mutant strain (phage type DT 9), unable to synthesize both adenine and histidine (Lindner et al., 2007). The strain is distinguishable from field isolates of the same serotype on the basis of its auxotrophy using a rapid test (IDT Salmonella Diagnostic Kit) within 24-48 hours (Eddicks et al., 2009).
Knock-out Mutants
Salmonella Typhimurium strain 112910a, phage type 120/ad, isolated from a pig stool sample and characterized previously (Boyen et al., 2008b), and several isogenic knock-out mutants, were used in this study. The knock-out mutants where constructed as described before (Boyen et al, 2006), primers is used in this study are shown in table 6.
Briefly, the genes of interest were first substituted by a PCR adjusted antibiotic resistance cassette (kanamycin) using the helper plasmid pKD46. This plasmid encodes the phage λ Red system, which promotes recombination between the native gene and the PCR adjusted antibiotic resistance cassette. Recombinant clones were selected by plating on Luria-Bertani agar (LB; Sigma Aldrich Chemie Gmbh, Steinheim, Germany) containing 100 μg/ml kanamycin. The substitution was confirmed by PCR. In the last step, the antibiotic resistance cassettes were eliminated using the helper plasmid pCP20. The targeted genes were completely deleted from the start codon through the stop codon, as confirmed by sequencing.







TABLE 6







Primers used in this study to create the deletion mutants 


ΔscsA, ΔscsB, ΔscsC, ΔscsD, ΔscsABCD and ΔcbpA.








Primers
Sequences 5′-3′





scsA 
CAAAACCGCGCCAGTGGCTAAGATAACTCGCGTTAAACAGTGAGGGCGCAT


forward
GTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 13)


 


scsA 
ATTTTTTCTCCGTGAATGAGTAATTAACCGTTAGCAATAACCGGTCTGCATAT


reverse
GAATATCCTCCTTAG (SEQ ID NO: 14)


 


scsB 
CGGTTATTGCTAACGGTTAATTACTCATTCACGGAGAAAAAATTGTGTAGGC


forward
TGGAGCTGCTTC (SEQ ID NO: 15)


 


scsB 
CGCGATGCTCAGCGTCGAAAACAGCGCCAGCAGTAAAACAATCATGTATTC


reverse
ATATGAATATCCTCCTTAG (SEQ ID NO: 16)


 


scsC 
GCGATGCGGTATTACAAACGTTGAAAAAAGCGAAAGGAATAACCCAATGAT


forward
GTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 17)


 


scsC 
GCTTCACGCAGCCAACGCCGCAGTTTACCCGCCATTCATATGAATATCCTC


reverse
CTTAG (SEQ ID NO: 18)


 


scsD 
GCCCTGGGATACGCtGGAAGCGGTGGTGAAAGAAAAACTGGCGTCTGCCAT


forward
GTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 19)


 


scsD 
GATTTCGCAAAACGGGGGTTTTTCTTACAGTAAACGCGTTAGCGCCGGGAC


reverse
ATATGAATATCCTCCTTAG (SEQ ID NO: 20)


 


scsABCD
CAAAACCGCGCCAGTGGCTAAGATAACTCGCGTTAAACAGTGAGGGCGCAT


forward
GTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 21)


 


scsABCD
GATTTCGCAAAACGGGGGTTTTTCTTACAGTAAACGCGTTAGCGCCGGGAC


reverse
ATATGAATATCCTCCTTAG (SEQ ID NO: 22)


 


cbpA 
GAAACCTTTTGGGGTCCCTTCTGTATGTATTGATTTAGCGAGATGATGCTTG


forward
TGTAGGCTGGAGCTGCTTC (SEQ ID NO: 11)


 


cbpA 
GTGTGCAAACAAAATTCGGTGATGGTAAAGGTGACAGTGATGTTAGCCATC


reverse
ATATGAATATCCTCCTTAG (SEQ ID NO: 12)









For experimental infection of mice, an invasive, spontaneous nalidixic acid resistant Salmonella Typhimurium strain 112910aNal20, resistant to 20 μg/ml nalidixic acid, was used.
Effect of Dexamethasone on Recrudescence of a Live Salmonella Typhimurium Vaccine Strain in Pigs
In this in vivo experiment we investigated whether a subcutaneous injection of dexamethasone is able to induce recrudescence of a live commercial Salmonella Typhimurium vaccine in pigs. For that purpose, twenty, three-week-old, piglets were used. The Salmonella-free status of the piglets was is tested serologically using a commercially available enzyme-linked immunosorbent assay (ELISA) (IDEXX Laboratories) according to the manufacturer's instructions. All animals were housed together at 25° C. under natural day-night rhythm with ad libitum access to feed and water and were orally vaccinated with 1 ml of the live Salmonella Typhimurium vaccine, Salmoporc®. Two weeks later, ten animals received an intramuscular injection of 2 mg dexamethasone (Kela laboratoria, Hoogstraten, Belgium) per kg body weight, to mimic pre-slaughter stress conditions. This dose was shown to cause recrudescence of Salmonella Typhimurium in pigs (Verbrugghe et al., 2011). Ten pigs served as a control group and were intramuscularly injected with 2 ml of Hank's buffered salt solution (HBSS; Gibco Life Technologies, Paisley, Scotland). Twenty-four hours later, all animals were humanely euthanized and organ samples were taken for bacteriological analysis.
Developing a Mice Model that Mimics Stress Related Recrudescence of Salmonella Typhimurium 
In this in vivo experiment we evaluated whether dexamethasone increases the number of Salmonella Typhimurium bacteria in the gut of Salmonella Typhimurium infected mice in order to create a mice model that allows screening of bacterial genes that might be involved in dexamethasone induced recrudescence of Salmonella. For that purpose, eighteen, four week old DBA/2J mice, intermediately sensitive to Salmonella Typhimurium infections (Sebastiani et al., 2002) and eighteen, four week old BALB/c mice, highly susceptible to Salmonella Typhimurium infections (Sebastiani et al., 2002), were housed in filter-topped cages at 25° C. under natural day-night rhythm with ad libitum access to feed and water and enriched with mouse houses and play tunnels. Five days after arrival, all mice were infected with a total of 1·106 CFU of Salmonella Typhimurium strain 112910aNal2° by the orogastric route. At day 7 post inoculation (p.i.) six BALB/c mice were subcutaneously injected once with 100 mg/kg dexamethasone. Simultaneously, six BALB/c mice received a subcutaneous injection of 25 mg/kg dexamethasone, which was repeated after three hours. Fourteen days p.i. six DBA/2J mice were subcutaneously (SC) injected once with 100 mg/kg dexamethasone and contemporary six DBA/2J mice received a SC injection of 25 mg/kg dexamethasone (repeated after three hours). Six mice of each strain received a SC injection of 200 μl HBSS (24 h before euthanasia) and were used as a control group. Twenty-four hours after the SC injection of dexamethasone, all animals were humanely euthanized and samples of spleen, liver and cecum were collected for bacteriological analysis.
The Role of scs Genes in a Mice Model Mimicking Stress Related Recrudescence of Salmonella Typhimurium 
A mice model was used to verify whether scsA, scsB, scsC, scsD or the entire scs locus is important in dexamethasone related recrudescence in vivo. Therefore, three to four week old DBA/2J mice were used and randomly allocated in six groups of sixteen mice. The animals were housed in filter-topped cages at 25° C. under natural day-night rhythm with ad libitum access to feed and water and enriched with mouse houses and play tunnels. Mice were inoculated with a total of 1·106 CFU of Salmonella Typhimurium or its isogenic scsA, scsB, scsC, scsD or scsABCD knock-out mutants. At day 14 p.i., eight animals of each group were SC injected with 100 mg/kg dexamethasone and eight mice were SC injected with 200 μl HBSS and served as a control group. Twenty-four hours later, all mice were humanely euthanized. Spleen, liver and cecum samples were examined for the number of Salmonella Typhimurium bacteria.
Bacteriological Analysis
All organ samples were weighed and 10% (w/v) suspensions were prepared in buffered peptone water (BPW, Oxoid, Basingstoke, United Kingdom). The samples were homogenized with a Colworth stomacher 400 (Seward and House, London, United Kingdom) and the number of Salmonella bacteria was determined by plating 10-fold dilutions on XLD plates (for porcine organ samples and organ samples of the last mice in vivo trial) or on BGANal20 plates (for samples collected to optimize the mice model). All plates were incubated for 16 hours at 37° C. The samples were pre-enriched for 16 hours in BPW at 37° C. and, if negative at direct plating, enriched for 16 hours at 37° C. in tetrathionate broth (Merck KGaA, Darmstadt, Germany) and plated again on BGANal20 or XLD plates.
Samples that were negative after direct plating but positive after enrichment were presumed to contain 83 CFU/gram tissue or contents (detection limit for direct plating). Samples that remained negative after enrichment were presumed to contain less than 83 CFU/gram tissue or contents and were assigned value ‘1’ prior to log transformation. Subsequently the number of CFU for all samples derived from all animals was converted logarithmically prior to calculation of the average differences between the log10 values of the is different groups and prior to statistical analysis.
Statistical Analysis
In all experiments, statistical analysis was performed using a one-way ANOVA test (in case of homogeneity of variances), with posthoc Bonferroni corrections or a nonparametric Mann-Whitney-U-test (in case of non-homogeneity of variances), using the SPSS Statistics 19.0 software (SPSS Inc., Chicago, USA). A P-value of <0.05 was considered significant.
5.2. Results
Dexamethasone Promotes Recrudescence of a Salmonella Typhimurium Live Vaccine Strain in Pigs
In this experiment we determined to which extent the commercially available, Salmonella Typhimurium live vaccine, Salmoporc®, is subject to recrudescence when vaccinated pigs are treated with 2 mg/kg dexamethasone, 24 hours before euthanasia. FIG. 9 illustrates that recovery of Salmoporc® was higher in organ samples and contents of vaccinated pigs treated with dexamethasone compared to vaccinated pigs that received a saline solution. This elevation was significantly different (P<0.05) for ileocecal lymph nodes, colon contents and cecum contents.
A Subcutaneous Injection of Dexamethasone Results in Recrudescence of Salmonella Typhimurium 112910Nal20 in DBA/2J Mice but not in BALB/c Mice
A mice model was optimized to demonstrate that a subcutaneous injection of 100 mg/kg dexamethasone (or two injections of 25 mg/kg with an interval of three hours) is capable to induce recrudescence of Salmonella Typhimurium is strain 112910aNal20 in DBA/2J or BALB/c mice. Salmonella infected DBA/2J mice, subsequently injected with dexamethasone had a significantly (P<0.05) higher number of Salmonella Typhimurium bacteria in the spleen, the liver and the cecum, compared to DBA/2J mice that were injected with a saline solution. Results are shown in FIG. 10. Our study proved that Salmonella Typhimurium 112910a infections in DBA/2J mice are non-lethal. Bacterial growth in DBA/2J mice is controlled after several days and we assume that it reaches a plateau phase and subsequently declines, while in BALB/c mice the bacterial load in organ samples increases gradually (unpublished results). Therefore, DBA/2J mice and not BALB/c mice allowed us to investigate stress related recrudescence in animals that carry Salmonella Typhimurium asymptomatically.
FIG. 11 shows that the number of Salmonella Typhimurium bacteria in organs of infected BALB/c mice, subsequently injected with dexamethasone, was not significantly different (P>0.05) from Salmonella numbers isolated from the spleen, the liver and the cecum of infected BALB/c mice that were injected with HBSS 24 h before euthanasia.
scsA and scsABCD Determine Dexamethasone Induced Recrudescence of Salmonella Typhimurium 112910a
Bacterial counts in the spleen (P>0.05), liver (P>0.05) and caecum (P<0.05) of DBA/2J mice infected with ΔscsA and subsequently injected with 100 mg/kg dexamethasone, were reduced compared to bacterial numbers in organs of mice infected with its isogenic wild type strain, subsequently injected with dexamethasone. The Salmonella Typhimurium load in organ samples of mice, infected with ΔscsB or ΔscsC and subsequently injected with 100 mg/kg dexamethasone, was not significantly different from that in organs of DBA/2J mice infected with the wild type strain and subsequently injected with dexamethasone. The Salmonella Typhimurium load in the liver of mice is infected with ΔscsD or ΔscsABCD, subsequently injected with 100 mg/kg dexamethasone, was significantly different from that in the liver of DBA/2J mice infected with the wild type strain and subsequently injected with dexamethasone. Results are shown in FIGS. 12A-E.
None of the DBA/2J mice infected with ΔscsA, ΔscsABCD or their isogenic wild type strain died as a result of the infection, whereas eleven mice infected with either ΔscsB (n=3), ΔscsC (n=4) or ΔscsD (n=4) died as a consequence of challenge.
The DBA/2J mice model allowed us to investigate whether scs genes are able to reduce dexamethasone induced recrudescence of Salmonella in vivo. Our results indicated that mice infected with ΔscsA, ΔscsD or ΔscsABCD did not show recrudescence of Salmonella Typhimurium after a subcutaneous injection with dexamethasone. This was not the case for mice infected with either ΔscsB or ΔscsC. Furthermore, deletion of ΔscsB, ΔscsC or ΔscsD increased virulence of Salmonella Typhimurium 112910a in DBA/2J mice.
In conclusion, we showed that scsA and scsABCD are able to abolish dexamethasone induced recrudescence of Salmonella in a DBA/2J mice model, without increasing the virulence of the Salmonella Typhimurium strain used. Therefore, deletion of scsA or the entire scs locus in Salmonella Typhimurium live vaccines might help to reduce stress related recrudescence of live vaccine strains.
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## Claims

1. A Salmonella mutant strain having at least one genetic modification within the cbpA gene or having at least one genetic modification within the scs locus.

2. The Salmonella mutant strain of claim 1, wherein the genetic modification in the scs locus is located within the scsA, scsB, scsC, or scsD gene.

3. The Salmonella mutant strain of claim 1, wherein the genetic modification is a deletion of at least a portion of the cbpA gene or the scs locus.

4. The Salmonella mutant strain according to claim 1, wherein the Salmonella strain consists of Salmonella enterica subspecies enterica.

5. The Salmonella mutant strain according to claim 4, wherein the Salmonella strain consists of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium).

6. The Salmonella mutant strain according to claim 1, further comprising one or more additional mutations in a gene other than the cbpA gene or scs locus.

7. A composition comprising the Salmonella mutant strain of claim 1, and a pharmaceutically acceptable carrier or diluent.

8. A vaccine comprising the Salmonella mutant strain according to claim 1, and a pharmaceutically acceptable carrier or diluent.

9. The vaccine of claim 8 wherein the vaccine is a live attenuated vaccine.

10. A method for converting a Salmonella vaccine strain into a Salmonella mutant vaccine strain by introducing at least one genetic modification within the cbpA gene or the scs locus into said strain; said method comprising:
obtaining a Salmonella vaccine strain;
introducing a genetic modification within the cbpA gene or the scs locus of said vaccine strain; thereby obtaining said Salmonella mutant vaccine strain.

11. The method according to claim 10 where the genetic modification in the scs locus is located within the scsA, scsB, scsC, or scsD gene.

12. The method according to claim 10 wherein the genetic modification is a deletion of at least a portion of the cbpA gene or the scs locus.

13. The method of claim 10 wherein the Salmonella vaccine strain consists of Salmonella enterica strain.

14. The method of claim 13 wherein the Salmonella enterica strain consists of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium).

15. The method according to claim 10, wherein the Salmonella vaccine strain is an attenuated strain.

16. The method according to claim 10, further comprising:
creating a PCR adjusted antibiotic resistance cassette,
inserting a first helper plasmid into the Salmonella vaccine strain,
substituting part or all of the cbpA gene or the scs locus with the PCR adjusted antibiotic resistance cassette,
controlling the substitution with PCR and sequencing,
inserting a second helper plasmid into the substituted target strain,
deleting the antibiotic resistance cassette and the helper plasmids, and
controlling the deletion with PCR and sequencing.

17. A method for preventing, inhibiting or reducing recrudescence of a Salmonella infection comprising administering a Salmonella mutant vaccine strain as defined in claim 10 to a subject in need thereof.

18. The method according to claim 17, wherein the recrudescence is stress-induced.

19. The method according to claim 17, wherein the subject is selected from the group consisting of a pig, poultry and cattle.

20. A method for immunization of pigs, poultry and cattle against Salmonella infection comprising administering a Salmonella mutant strain vaccine as defined in claim 10 to a subject in need thereof.

21. A method for administering an antigen heterologous to Salmonella to a subject for vaccination against an infectious agent comprising:
providing a Salmonella mutant strain according to claim 1;
introducing a heterologous nucleic molecule encoding the antigen into the Salmonella mutant strain; and
administering said Salmonella mutant strain to a subject in need thereof.