WO2012092226A1 - Veterinary vaccine composition against infections caused by salmonella - Google Patents

Abstract

The invention relates to attenuated Salmonella strains, and to vaccine compositions useful for preventing and/or controlling Salmonella infections or diseases in animals. The invention also provides a safe and effective method of protecting and vaccinating animals against Salmonella infections.

Description

VETERINARY VACCINE COMPOSITION AGAINST

INFECTIONS CAUSED BY SALMONELLA

FIELD OF THE INVENTION

The present invention relates to vaccines, in particular to novel double or triple deleted attenuated Salmonella strains which can provide live avirulent vaccines for protecting animal against Salmonella infections. The present invention also pertains to vaccine compositions and methods for treating and preventing a Salmonella infection in animals.

BACKGROUND OF THE INVENTION

Salmonella is a gram-negative, rod-shaped, bacterium that is non-spore forming and more precisely a genus of the family Enterobacteriaceae. Salmonella has been associated with a wide spectrum of infectious diseases, which cause public health problems worldwide. Environmental sources of these microorganisms include water, soil, insects, factory surfaces, animal feces, raw meats, and raw poultry. Particularly, Salmonella infections have widespread occurrences in animals, especially in poultry, cattle and swine, and can cause fatal conditions like septicemia that affect food producing animals with serious economic consequences. The ubiquitous presence of Salmonella in nature complicates the control of the disease just by detection and eradication of infected animals.

Based on the serologic classification determined using an array of specific antisera, many serotypes or serovars of Salmonella have been isolated from animals. Salmonella have been classified according to a nomenclature system of the WHO and Center for Disease Control and Prevention (CDC). According to the CDC nomenclature system, Salmonella enterica is the type species of Salmonella. Most medically important Salmonella serovars of Salmonella enterica includes for example Salmonella typhimurium, Salmonella enteritidis, Salmonella dublin, Salmonella choleraesuis, Salmonella lyphisius, Salmonella brandenburg, Salmonella heidelberg and Salmonella abortusovis . These strains are most frequently associated with clinical salmonellosis and are a common cause of infection in many animals including bovines, ovines, swines and avians. More particularly, Salmonella typhimurium can cause bird and mammalian salmonellosis, Salmonella dublin can cause cattle samonellosis, Salmonella lyphisius can cause hog salmonellosis; Salmonella heidelberg can cause poultry salmonellosis, and Salmonella brandenburg can cause sheep and cattle salmonellosis. Prevention of salmonellosis in farm animals is thus an important task in zootechny due to both the economic consequences of infectious diseases outbreaks in animals in intensive rearing, and the implications of such diseases on health of the consumers.

Numerous attempts have been made to protect animals by immunization with a variety of vaccines, Many of the vaccines provide only poor to moderate protection and often require large doses to be completely efficacious. Previously used vaccines against salmonellae have generally fallen into four categories: (i) subunit vaccines, including cell fractions or lysates, intact antigens, fragments thereof, or synthetic analogs of naturally occurring antigens or epitopes; (ii) antiidiotypic antibodies; (iii) killed vaccines which corresponds to the whole killed etiologic agent; or (iv) an avirulent or attenuated derivative of the etiologic agent used as a live vaccine. Among these subunit vaccines and whole-cell killed vaccines are in use and give variable results in the prevention of Salmonella infections in animals. Inactivated vaccines in general provide poor protection against Salmonellosis.

Live attenuated Salmonella vaccines are potentially superior to inactivated preparations owing to: (i) their ability to induce cell-mediated immunity in addition to antibody responses; (ii) effectiveness after single-dose administration; (iii) induction of immune responses at multiple mucosal sites; (iv) low production cost; and (v) their possible use as carriers for the delivery of recombinant antigens to the immune system. The ability of live attenuated Salmonella to colonize the gut-associated lymphoid tissue (GALT; Fever's patches) and the deep tissues following oral administration is the factor that aids stimulation of all arms of the immune response, including mucosal, humoral and cellular immunities. To this regard, Salmune®. an attenuated live Salmonella typhimurium sold by CEVA, has been widely and efficiently used as a live bacterial vaccine for chickens for the prevention of disease and colonization of the internal organs and the intestine, including the ceca, by multiple types of salmonellae including group B Salmonella heidelberg, Salmonella typhimurium, group C Salmonella hadar and Salmonella kentucky, and group D Salmonella enteritidis. Salmune® vaccine contains an attenuated Salmonella typhimurium strain which has been chemically mutated to render the vaccine strain permanently attenuated by limiting its invasiveness and colonization time within the chicken's internal organs and intestine.

It is clear that the selection of appropriate targets for attenuation which will result in a suitable vaccine candidate is not straightforward and cannot easily be predicted. Many factors may influence the suitability of the attenuated strain as an appropriate vaccine, and there is much research being carried out to identify suitable strains. Safe and rationally designed attenuated Salmonella strains can be produced by introducing defined non-reverting mutations into the bacterial genome. Several approaches to obtain Salmonella attenuated strains were used inter alia by Cardenas et al., Clin Microbial Rev. 5:328-342 ( 1992); Chatfield et al., Vaccine 7:495-498 ( 1989). These approaches include temperature sensitive mutants (Germanier et al., Infect Immun. 4:663-673 (1971)), mutants defective for biochemical factors, such as -aroA, -asd, -cys, or -thy (Galan et al., Gene 94:29- 35 (1990); Hoiseth et al., Nature 291 :238-239 (1981); Robertsson et al., Infect Immun. 41 :742-750 (1983)), Apur and Adap (Clarke et al., J Vet Res. 51 :32-38 (1987); McFarland et al., Microb Pathog. 3: 129- 141 (1987)]), carbohydrates synthesis defective strains, such as AgalE (Hone et al, J Infect Dis. 156: 167-174 (1987)]), and lipopolysaccharide synthesis defective strains (e.g., galE, pmi, rfa). However, the effectiveness of these approaches is doubtful and currently no such optimum vaccine able to confer long term resistance against Salmonella infections is available, further many attenuated strains tested as vaccine candidates lead to vaccinemia or abscesses in the human patient. The international publication WO 2007/112518 describes the vaccine strain MeganVac, which carries deletions in the cya (adenylate cyclase) and crp genes (cyclic AMP receptor), but still does not provide full protection. There is thus still a need for improved live attenuated Salmonella vaccine strains, as well as for improved live attenuated vaccine strains of bacteria infecting animals in general.

The original aim of the work that led to the invention was the identification of ideal genes that are involved in the virulence pathways of pathogenic bacteria, the identification and deletion of which would render Salmonella avirulent while retaining immunogenicity and suitability for use as vaccines. The present invention thus relates to an improved attenuated live vaccine, containing double and/or triple gene mutations in Salmonella strains, which have a moderate level of attenuation and which may be used in an immunologically effective amount to penetrate and briefly colonize the internal organs as well as cecum of the vaccinated animals. Attenuated live vaccines according to the invention also provide efficient protection against homologous challenge and mostly afford cross protection. In order to guarantee high efficiency and reliable safety of a live attenuated vaccine, it is essential that virulence and immunity are well balanced. The inventors of the present invention have aimed to achieve this balance.

Additionally the attenuated Salmonella strains of the present invention are advantageous because the said mutations can be easily transduced into all Salmonella strains, further the strains of the invention lack genes conferring resistance to antibiotics or other genetic markers foreign to the strain and still further the strains are traceable and distinguishable from wild type Salmonella strains by microbiological or molecular biology assays.

SUMMARY OF THE INVENTION

The present invention is based on the finding that at least two specific mutations introduced into a Salmonella strain can produce a vaccine having a high degree of immunogenicity and a low risk of reverting to a virulent form.

The present invention further relates to attenuated Salmonella strain, wherein said strain contains a first mutation in one or more invasion genes and at least another mutation in one or more genes involved in the survival and/or proliferation of Salmonella in the host and/or one or more genes involved in the motility of Salmonella.

An object of the present invention is to provide attenuated Salmonella with a double or a triple mutation.

Another object of the present invention is to provide a live vaccine composition comprising immunogenically effective amounts of attenuated Salmonella strains with a double or a triple mutation, optionally with pharmaceutically acceptable carriers and/or diluents and/or excipients, and/or adjuvants.

Yet another object of the present invention is to provide a method for preventing and/or treating Salmonella infections caused by Salmonella species, including but not limited to Salmonella typhimurium, Salmonella enteritidis and Salmonella dublin, Salmonella choleraesuis, Salmonella iyphisius, Salmonella brandenburg, Salmonella Heidelberg and Salmonella abortusovis in bovines, ovines, swines and avians.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1: corresponds to a gel electrophoresis of PCR products from each of live consecutive passages of the master seed at passages +1 through +5, using primers to amplify the invA gene, and showing that the invA deletion is missing 1.8 kb of DNA compared to the parent strain and remains stable (i.e., unchanged in size) through the fifth passage. P* means positive control, which is the parent strain Salmonella typhimurium 076-94. N** means negative control. FIGURE 2: is a gel electrophoresis of the PGR product from the MS and each passage +1 through +5, using primers to amplify the rfall gene, shows that the rfall deletion is missing 0.3 kb of DNA compared to the parent strain and remains stable (i.e., unchanged in size) through the fifth passage. P* means positive control, which is the parent strain Salmonella typhimurium 076-94. N** means negative control.

FIGURE 3: shows nucleic acid sequence for fliC gene.

FIGURES 4A-4B: A. Backbone Biological Agent, Salmonella enterica serovar typhimurium 076-94 strain. B. double mutant, Salmonella AinvAArfaH typhimurium (Regulated Biological Agent or RBA).

FIGURES 5A-5D: show the procedure for deletion of invA from Salmonella typhimurium 076-94 Strain. A. PGR generation of the invA construct, i.e., the Cm^R- containing construct for homologous recombination with the chromosomal invA. B. Insertion of helper plasmid pKD46 into Salmonella typhimurium 076-94 Strain. Upon activation of the araC activator, pKD46 expresses λ Red recombinase, which enhances the recombination of linear DNA into a chromosome by promoting efficient repair/recombination of a double- strand break. C. Chromosomal integration of the invA construct. D. Elimination of the chloramphenicol genetic resistance marker Cm^R. The pCP20 plasmid is an ampicilin and chloramphenicol resistant plasmid that shows thermal induction of flippase recombination enzyme (FLP) synthesis and temperature-sensitive replication. Abbreviations used denote the following: IH ^,nvA: 50-bp homologue of 5' end of invA, H2^mvA: 50-bp homologue of 3' end of invA, Cm^R: gene that provides chloramphenicol resistance, pKD3: template plasmid that contains the Cm^R gene, PI : priming site 1 of plasmid pKD3, P2: priming site 2 of plasmid pKD3, FRT: flippase recognition target site.

FIGURES 6A-6D: show deletion of rfciH from Salmonella typhimurium AinvA. A. PCR generation of the rfaH construct, i.e. the Cm^R-containing construct that will replace rfali. B. Insertion of helper plasmid pKD46 into Salmonella typhimurium AinvA. Upon activation of the araC activator, pKD46 expresses λ Red recombinase, which enhances the recombination of linear DNA into a chromosome by promoting efficient repair/recombination of a double-strand break. C. Chromosomal integration of the rfall construct. D. Elimination of the chloramphenicol genetic resistance marker Cm^R.

The pCP20 plasmid is an ampicillin and chloramphenicol resistant plasmid that shows thermal induction of flippase recombination enzyme (FLP) synthesis and temperature- sensitive replication. Abbreviations used denote the following Hl^rfaH: 55 -bp homologue of 5' end of rfaH, H2^rfaH: 62-bp homologue of 3 ' end of rfaH, Cm¹*: gene that provides chloramphenicol resistance, pKD3 : template plasmid that contains the Cm^R gene, PI : pri ing site 1 of plasmid pKD3, P2: priming site 2 of plasmid pKD3, FRT: flippase recognition target site.

FIGURE 7: gives rfaH gene and protein.

DETAILED DESCRIPTION OF THE INVENTION

The following discussion is intended to be illustrative of the present invention and is not intended to limit the scope of the present invention to specific embodiments and specific examples.

Definitions

The term "gene" used herein refers to the coding sequence and to its regulatory sequences such as the promoter and termination signals.

The term "mutation" according to the invention may relate to an insertion, a deletion, and/or a substitution of one or more nucleotides in said genes, deletion mutants being the most preferred. The terms alteration or deletion of the gene used herein refers to modification or a mutation of the gene as to inhibit or abolish expression and/or biological activity of the encoded gene product. The modification or mutation may act through affecting transcription or translation of the gene or its mRNA, or the mutation may affect the polypeptide product itself in such a way as to render it inactive.

In preferred embodiments, inactivation is carried by deletion of a portion of the coding region of the gene because a deletion mutation reduces the risk that the mutant will revert to a virulent state. For example, some, most (e.g., half or more) or virtually all of the coding region may be deleted (partial or complete deletion). Alternatively, the mutation may be an insertion or deletion of even a single nucleotide that causes a frame shift in the open reading frame, which in turn may cause premature termination of the encoded polypeptide or expression of a completely inactive polypeptide.

As used herein, the term "attenuated" refers to a microorganisms and/or bacteria strains and/or viruses that have been genetically modified so as to not cause illness in an animal model. The terms "attenuated" and "avirulent" are used interchangeably herein. By "immunizing amount" as used herein is in fact meant an amount that is able to induce an immune response in the animal that receives the pharmaceutical composition/vaccine. The immune response invoked may be a humoral, mucosal, local and/or a cellular immune response.

As used herein "animals" refers to non human species including but not limited to cattle, young calves, swine, chickens, turkeys, geese, ducks, pheasants, bantam, quail, pigeons, sheep, etc.

The term "multivalent," as used herein may be bivalent, trivalcnt, quadravalent, etc., and refers to a vaccine which has other antigenic components from related and/or unrelated microorganism and/or viruses, or their components and fragments along with the attenuated Salmonella strain. For example, the "multivalent" vaccine could be a combined vaccine or an attenuated Salmonella strain comprising the antigenic components.

The present invention provides vaccines prepared from an attenuated Salmonella strains, for the immunization of an animal susceptible to Salmonella infections, such as bovines. ovines, swines and avians. Pathogenic Salmonella, according to this invention, is made avirulent as a result of non-reverting mutations that are created in at least two coding sequences, the products of which typically act in concert to produce non reverting attenuated Salmonella.

Salmonella strains as used in the present invention are intended to mean all medically important Salmonella serovars of Samonella enterica, such as for examples Salmonella lyphimurhim, Salmonella enteritidis, Salmonella dublin, Salmonella choleraesitis, Salmonella typhisius, Salmonella brandenburg, Salmonella heidelberg, and Salmonella abortusovis, which are frequently associated with clinical salmonellosis in bovines, ovines, swines and avians.

Advantageously, the attenuated Salmonella strains of the invention are immunogenic and comprise a first mutation which modifies a gene responsible for invasion of salmonella in host cells. The attenuated Salmonella strains also comprise a second mutation that modifies a gene that is responsible for biosynthesis of somatic lipopolysaccharide and/or a gene which is involved in the motility of Salmonella and/or a gene that is responsible for production of heme, Genes of particular interests include:

- the InvA and/or InvF genes which when present act to trigger internalization of Salmonella into epithelial cells; - the rfaH gene which when present acts to regulate the production of amphipathic LPS (lipopolysaccharide) required to survive stressful environments, the growth in epithelial and macrophage cells, and the resistance to intracellular antimicrobial peptides;

- the fliC gene which when present encodes the flagellar protein (flagellin) required in the mobility and invasion of certain host cells. Advantageously mutants in which genes encoding flagellin are deleted, are incapable of swarming and can thereby easily be distinguished from wild-type motile strains. For example mutant failing to express the fliC gene such as the flagellin gene function may be detected by the absence of swarming on LB medium containing 0.4% agar; and

- the Hem A gene which when present converts 5-amonolcvulinic acid (ALA) to heme which serves in Salmonella both for respiration and oxygen defense {i.e., Iron binding protein).

InvA , rfaH and fliC genes are well known to one skilled in the art. In particular, these genes have described and characterized by Galan, J. E., and R. Curtiss III. (Proc. Natl. Acad. Sci. USA 86:6383-6387. 1989). FliC gene has been described and characterized by Ciacci- Woolwine et al., 1998 (Infection and Immunity 66:1127-1134).

A preferred attenuated Salmonella strain of the invention carries or comprises a mutation in the invA or in the invF gene and a mutation in the rfaH gene. Most preferably, the attenuated Salmonella strains comprise a mutation in the invA gene and a mutation in the rfaH gene.

Another preferred attenuated Salmonella strain of the invention carries or comprises a mutation in the invA gene or in the invF and a mutation in the fliC gene. Most preferred attenuated Salmonella strains comprise a mutation in the invA gene and a mutation in the fliC gene. These attenuated Salmonella strains may also comprise a further mutation in the HemA gene.

Yet another preferred attenuated Salmonella strain carries or comprises a mutation in the invA gene or the invF gene, a mutation in the rfaH gene, and a mutation in the fliC gene. Most preferred attenuated Salmonella strains comprise a mutation in the invA gene, a mutation in the rfaH gene, and a mutation in the fliC gene. These attenuated Salmonella strains are thus designated hereinafter as AinvAArfaH or AinvAAfliC double-deleted attenuated Salmonella strain, or Aim A AtfaHAfliC triple-deleted attenuated Salmonella strain.

It has been surprisingly found that AinvAArfaH and AinvAAfliC double-deleted, or AinvAArfaHAfliC triple-deleted attenuated Salmonella strains are more efficacious than currently used live attenuated vaccine, particularly for the protection of cecum of vaccinated animals, such as bovine, ovine, swine and avian. Also, these attenuated Salmonella strains advantageously have limited invasiveness and colonization time within the animal's internal organs and intestine.

Genetic alterations, deletions or mutations according to the invention may be obtained via insertion, deletion, site-specific mutation(s), and/or substitution(s) of one or more nucleotides within said genes so as to inactivate the genes. Preferably, attenuated Salmonella strains are double- or triple-deleted mutants. The mutations introduced into the bacterial vaccine generally knock out the function of the gene completely. This may be achieved either by abolishing synthesis of any polypeptide at all from the gene or by making a mutation that results in synthesis on non-functional polypeptide. In order to abolish synthesis of any polypeptide, either the entire gene or its 5'-end may be deleted. A deletion or insertion within the coding sequence of a gene may be used to create a gene that synthesizes only nonfunctional polypeptide, such as for example, a polypeptide that contains only the N-terminal sequence of the wild-type protein. The mutations are non-reverting mutations. These are mutations that show essentially no reversion back to the wild-type when the bacterium is used as a vaccine.

Mutation methods that may be employed to practice the invention may include cloning the DNA sequence of the wild-type gene into a vector, e.g., a plasmid or cosmid, and inserting a selectable marker into the cloned DNA sequence or deleting a part of the DNA sequence, resulting in its inactivation. A deletion may be introduced by, for example, cutting the DNA sequence using restriction enzymes that cut at two points in the coding sequence and ligating together the two ends in the remaining sequence. A plasmid carrying the inactivated DNA sequence can be transformed into the bacterium by known techniques. It is then possible by suitable selection to identify a mutant wherein the inactivated DNA sequence has recombined into the chromosome of the bacterium and the wild-type DNA sequence has been rendered non-functional in a process known as homologous recombination. In one example, a strategy using counter selectable markers can be employed which has commonly been utilized to delete genes in many bacteria. For a review, see for example, Reyrat, et ah, Infection and Immunity 66:4011-4017 (1998). In this technique, a double selection strategy is often employed wherein a plasmid is constructed encoding both a selectable and counter selectable marker, with flanking DNA sequences derived from both sides of the desired deletion. The selectable marker is used to select bacteria in which the plasmid has integrated into the genome in the appropriate location and manner. The counter selectable marker is used to select for the very small percentage of bacteria that have spontaneously eliminated the integrated plasmid. A fraction of these bacteria will then contain only the desired deletion with no other foreign DNA present. The key to the use of this technique is the availability of a suitable counter selectable marker. In another technique, the ere -lox system is used for site specific recombination of DNA. The system consists of 34 base pair lox sequences that are recognized by the bacterial ere recombinase. If the lox sites are present in the DNA in an appropriate orientation, DNA flanked by the lox sites will be excised by the ere recombinase, thereby resulting in the deletion of all sequences except for one remaining copy of the lox sequence. Using standard recombination techniques, it is possible to delete the targeted genes of interest in the Salmonella genome and to replace it with a selectable marker, such as for example the kanamycin resistance gene, that is flanked by the lox sites. Transient expression by electroporation of a suicide plasmid containing the ere gene under control of a promoter that functions in Salmonella of the ere recombinase should result in efficient elimination of the lox flanked marker. This process would result in a mutant containing the desired deletion mutation and one copy of the lox sequences. These techniques of gene inactivation and other non limiting equivalents are well known to a person of ordinary skill in the art.

Vaccines according to the present invention may be prepared from an attenuated double or triple mutants of serovars of Samonella enterica, such as for examples Salmonella typhimurium, Salmonella enteritidis, Salmonella dublin, Salmonella choleraesuis, Salmonella typhis ius, Salmonella brandenburg, Salmonella Heidelberg, and Salmonella abortusovis, wherein said mutant contains a first mutation in one or more invasion genes and at least another mutation in one or more genes involved in the survival and/or proliferation of Salmonella in the host and/or one or more genes involved in the motility of Salmonella.

Preferably, double or triple attenuated Salmonella strains, specifically Salmonella typhimurium are used as vaccines for treating Salmonellosis in bovine, ovine, swine or avian. Most preferably the genetic modifications of the invention are introduced into parent strain 076-94 (isolated by Hy-line International, Dallas Center, IA). Serotyping was done by National Veterinary Services Laboratory Bacterial Typing Section. Other preferred strains may be Salmonella enterica subsp. enterica serovar Typhimurium (strain: LT2; SGSC 1412; ATCC 700720. NCBI GeneBank Gene ID: 1255503, Locus tag STM3977, protein accession no. P_462862.1.). Another aspect of the invention relates to a medicament or a vaccine for administering to an animal, comprising an immunogcnically effective amount of one or more attenuated Salmonella strains as described above, and optionally pharmaceutically acceptable carriers and/or diluents and/or excipients, and/or adjuvants.

Yet another aspect of the invention relates to the use of the medicament or a vaccine containing one or more attenuated Salmonella strains as described above for the prevention and/or the treatment of Salmonella infections or Salmonellosis.

In a preferred aspect the present invention provides a safe and effective live attenuated vaccine composition which comprises an immunogcnically effective amount of at least one double or triple attenuated Salmonella strain as described above and optionally pharmacologically acceptable carriers and/or diluents and/or excipients, and/or adjuvants.

The vaccine according to this aspect may thus comprise one or more double or triple attenuated Salmonella strains of the serovars of Samonella enterica. Preferably, the vaccine comprises one or more double or triple attenuated Salmonella strains chosen from among Salmonella typhimurium, Salmonella enteritidis, Salmonella dublin, Salmonella choleraesuis, Salmonella (yphisius, Salmonella brandenburg, Salmonella heidelberg, and Salmonella abortusovis.

In yet another preferred embodiment, the attenuated live vaccine composition according to the invention is useful for immunizing a animal against infection and disease caused by Salmonella species or serovars, including for example Salmonella typhimurium.

The vaccine composition of the invention effectively infects animals without causing serious disease and stimulates humoral (antibody-based) immunity and cell-mediated immunity sufficient to provide resistance to any future infection by virulent Salmonella. In other words, the vaccines of the present invention effectively induces protective immunity in vaccinated animals. In particular, as shown in the Examples below, the vaccine composition allows for colonization of the internal organs and the intestine, including the ceca, and allows for protection against multiple types of salmonellae including group B Salmonella heidelberg and Salmonella typhimurium, group C Salmonella hadar and Salmonella kentucky, and group D Salmonella enteritidis.

The attenuated Salmonella strains of the invention and pharmaceutical compositions or vaccines comprising the same are highly suitable for immunizing animals such as bovine, ovine, swine and avian and more specifically poultry, cattle and swine against Salmonellosis and possibly other diseases (e.g., in the case of a multivalent vaccine). The attenuated strains of the invention are particularly suited to protect animal species against Salmonella.

Preferably the vaccine of the present invention may be administered to various animals such as cattle, young calves, swine, chickens, turkeys, geese, ducks, pheasants, bantam, quail, pigeons or sheep.

The vaccine may be advantageously combined with additional commercialized attenuated vaccines, such as Enterisol Ileitis FF, Enterisol SC-54 FF and Ingelvac® ERY- ALC (commercialized by Boehringer Ingelheim Vetmedica, Inc.) for the prevention of swine ileitis, and/or with Argus® (commercialized by Intervet Inc) for the prevention of pneumonia, diarrhea, septicemia and mortality caused by Salmonella; and/or with Paracox ® (com mc rc i al i zed by Intervet), Livacox® (commercialized by Merial), Coxabic® (commercialized by Novartis), and/or with Immunocox II (commercialized by Vetech).

When the vaccine according to the present invention is administered to swine/ pig, the vaccine may be administered in association with further attenuated microorganisms and/or viruses which in their virulent form are known to be pathogenic in pigs. These microorganisms may include, but are not restricted to pseudorabies virus, porcine influenza virus, porcine circovirus, porcine parvovirus, rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Haemophilus parasuis, Pasleurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae, Brachyspira hyodysenleriae and/or Actinohacillus pleuropneumonias

When the vaccine according to the present invention is administered to poultry, it may be advantageously associated or combined with further attenuated microorganisms and/or viruses which in their virulent forms are known to be infectious to poultry. Such microorganisms may include, but are not restricted to infectious Bronchitis virus, Newcastle Disease virus, Turkey Rhinotracheitis virus, Marek's virus, Avian Rcovirus, Infectious Bursal Disease (Gumboro), Chicken Anaemia agent, Mycoplasma gallisepticum, Haemophilus paragaUinarum (Coryza), Chicken Poxvirus, Avian Encephalomyelitisvirus, Duck Plague virus, Egg Drop syndrome virus, Infectious Laryngotracheitis virus, Herpes Virus of Turkeys, Eimeria species, Ornithobacterium rhinotracheale, Pasteurella multocida, and/or Mycoplasma synoviae.

The dose of vaccine may vary according to the age and size of the host, the severity of the infection, the mode of administration and the like. In general, suitable effective amounts per dosage unit may be about 10² to 10⁴ colony forming units (cfu), about 10² to 10¹⁴ cfu. preferably about 5.0x l 0² to 5.0* 10¹⁰ cfu, more preferably about 2.0 10⁶ to 6.0 < 10⁶ cfu of the attenuated Salmonella strain sufficient to provide about 10 to 10 cfu, preferably about 5.0x l 0² to 5.0x l 0¹⁰ cfu, more preferably about 2.0* 10⁶ cfu to 6.0* 10⁶ cfu per dosage unit. Those skilled in the art may find that these effective doses can be varied according to route, animal, age, sex, weight etc., for example for a parenteral vaccine dose may be smaller than a similar vaccine which is administered via drinking water, and the like.

Further the number of doses may be determined by a person of skill in art depending on the immune status of animal. For example for animals like poultry, the vaccine may be administered in an amount effective to induce protection of the vaccinated subject, in one or more doses, each dose containing for example 10⁵ to 1 0^s E1D₅₀.

Particular pharmaceutically acceptable carriers or diluents or excipients or adjuvants employed are routine and used conventionally in the art. For example pharmacologically acceptable carriers suitable for use in the vaccine composition of the invention may be any conventional liquid carriers suitable for veterinary pharmaceutical compositions, preferably a balanced salt solution such as sterile phosphate buffered saline, more preferably distilled water. Other suitable media can include emulsions. Examples of diluents may include, but are not limited to: buffer agents against gastric acid in the stomach, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone, or bicarbonate buffer (pll 7.0) containing ascorbic acid, lactose, and optionally aspartame. Examples of carriers include, but are not limited to: proteins, such as proteins found in skimmed milk, sugars, such as sucrose, or polyvinylpyrrolidon etc. Other preferred excipients may be selected from preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents, stabilizers, and the like.

The vaccine according to the present invention may further comprise an effective amount of adjuvant. Adjuvants are well known in the art and may include oily emulsions, oil in water emulsions, chitosan, aluminum salts or gels, such as aluminum hydroxide or aluminium phosphate, saponins, vitamins, extracts from bacterial cell walls, polymers based on polyacrylic acid, such as carbopols, non ionic block polymers, fatty acid amines, such as avridin and DDA, polymers based on dextran, such as dextran sulphate and DEAE dextran, biodegradable microcapsules, liposomes, viral immune stimulators, such as MDP, LPS, glucans and the like.

According to another embodiment of the invention, the vaccine may further comprise other additional microorganisms and/or antigens, such as viruses, bacteria, any other parasites etc. These may be live attenuated microorganisms or killed inactivated microorganisms, either whole microorganisms or subunits. chimeric or recombinant microorganisms, disrupted microorganisms, mutant microorganisms, defective microorganisms, or combinations thereof. The vaccine may also be an antigen including epitopes or antigenic parts of the microorganism's structure, such as preparations of antigenic proteins from pathogens, recombinant proteins, preferably viral antigen, such as viral capsid proteins, cell wall proteins, peptides, polysaccharides, lipopolysaccharides and glycoproteins etc. Alternatively, the vaccine may be formulated as a multivalent vaccine along with other components as specified above.

The vaccine composition according to the present invention may be administered by any conventional means, such as for examples via oral, nasal, ocular, mucosal, parenteral vaccination routes or via in ovo vaccination. It is also suitable for mass application, such as via drinking water or spraying via eye-drops, sprays or aerosols.

The vaccine composition according to the invention may be formulated for administration as liquids or dry powders, aerosols, sprays, eye-drops, or in drinking water. For example, in the case of poultry, the vaccine compositions may be administered to poultr by spraying directly onto the heads of the birds. Alternatively, one or more drops of the vaccine composition may be placed directly into the eyes of each individual bird. Preferred compositions according to the invention are formulated as liquid eye-drops.

Vaccine compositions for administration as aerosols, eye-drops, sprays, or drinking water may optionally contain one or more excipients of the type usually included in such conventional compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, lachrymal blocker agents, buffering agents, stabilizers, and the like. In actual practice, the attenuated Salmonella strains according to the invention may be admixed with a liquid carrier and administered as a spray or drinking water additive.

Surprisingly, it has now been found that a vaccine composition which comprises an immunogenically effective amount of attenuated Salmonella strain according to the present invention and optionally a pharmacologically acceptable carrier, diluent, excipient or adjuvant, is safe and effective for the protection of animals against disease and infection caused by Salmonella infections. Advantageously, the composition of the invention comprises attenuated Salmonella strain as immunogens capable of inducing both cellular and humoral immunity responses in animals, while demonstrating safety.

In the case of poultry, the vaccine composition may for example be used for the vaccination of healthy chickens at day one of age using coarse spray or drinking water. A single spray application of this vaccine to broilers at the hatchery, aids in the prevention of Salmonella colonization throughout the production cycle. If water vaccination is used for day-old chicks, a second vaccination is required at seven days of age. If chickens are maintained past seven weeks of age, a repeat vaccination is recommended. Dosage units may be contemplated b the skilled artisan. If two dosage units are selected, then vaccination is typically effected at about day 1 post-hatch and again at about one to two weeks of age. A dosage unit is desirably about 0.5 to 1 ml of vaccine per bird, but that quantity may be optimized to deliver an immunogcnically effective amount of the attenuated Salmonella strain hereinabove described.

A further aspect of the invention relates to a method of immunizing and/or preventing and/or protecting animals against Salmonella infection or disease which comprises the step of administering to said animals, such as bovine, ovine, swine and avian, an immunogcnically effective amount of an attenuated Salmonella strain and/or of the vaccine composition as described above, whereby a protective immune response is induced in the immunized subjects. It is another object of this invention to provide a method of preventing and/or treating infections or diseases caused by Salmonella species including Salmonella typhimurium, Salmonella enterilidis, Salmonella dub! in, Salmonella choleraesuis, Salmonella typhis ins, Salmonella brandenburg, Salmonella heidelberg, and Salmonella abortusovis, in bovines, ovines, swines and avians.

Moreover, there is provided a use of a vaccine composition as defined above for protecting, preventing or immunizing animals against Salmonella infections.

The present invention also provides a product or a vaccination kit comprising a means for dispensing the vaccine compositions comprising an immunologically effective amount of the attenuated Salmonella strain as described above. More precisely, the vaccination kit comprises a dispensing device adapted for dispensing the vaccine composition to bovine, ovine, swine and avian. Such dispensing device may, for example, be an aerosol, a spray or eye-drop delivery system, in ovo delivery system and may be arranged to dispense only a single dose, or a multiplicity of doses. The vaccination kit according to the present invention may further comprise technical instructions with information on the administration and dosage of the vaccine composition.

For a more clear understanding of the invention, the following examples are set forth below. These examples are merely illustrative and are not to be understood to limit the scope or underlying principles of the invention in any way. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the examples set forth herein below and the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

EXAMPLES

Example 1: Construction of Salmonella deleted mutants: AinvAArfaH

A schematic representation of the Salmonella typhimurium parental strain ST 076-94 strain Backbone Biological Agent (BBA) is provided in Figure 4A. Two genes, invA and rfaH, were deleted from the genome of ST 076-94 strain to yield the double mutant AinvAArfaH Salmonella Typhimurium (RBA) (Figure 4B). The invA gene is located between the invB and invE genes. The rfaH gene is located between the yigW and yigC genes. DNA sequence data indicated that the length of invA is 2058 bp and the length of rfaH is 489 bp. The chloramphenicol resistance (Cm^R) gene was used as a reporter gene to replace both the invA gene and the rfaH gene. The Cm^R gene was then removed from the organism through the process described below.

Flow diagrams for the construction of the RBA are provided in Figures 5A to 5D and 6A to 6D, and described below.

The relevant region of the ST 076-94 genome, the BBA, is shown in Figure 4A.

The Cm^R gene contained in the pKD3 plasmid (Figures 5A and 6A) was used as a reporter gene to replace the invA gene and the rfaH gene (Figures 5C and 6C). The Cm^R gene was then removed from the organism (Figures 5D and 6D).

Shuttle vectors and detailed processes to construct the S. typhimurium AinvAArfaH are provided herein below. The general procedures are also reported by Datsenko, K. A., and B. L. Wanner. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA. 97:6640-6645. 2000; Ellis, H. M D. Yu, T. DiTizio, and D. L. Court. High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. USA. 98:6742-6. 2001 et Yu, D., H. M. Ellis., E. C. Lee, N. A. Jenkins., N. G. Copeland, and D. L. Court. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA. 97:5978-5983. 2000.

(i) The methods used for invA gene deletion are shown in Figures 5A-D and described in sections ii-v below, and those used for rfaH gene deletion are shown in Figures 6A-D and described in sections vi-x below. Plasmids pKD3, pKD46 and pCP20 used in these processes were purchased from E. coli Genetic Resource Center Yale University as a Gene Disruption set.

(ii) Generation of an invA construct used to delete the invA gene was described in Figure 5A. The invA construct is a PGR product that contained a Cm^R gene flanked by sequences homologous to the invA gene. Briefly, two primers for PCR were designed. The forward primer (Primer 1) included the priming site 1 (PI) of the template pKD3 plasmid and a 50-base-pair (bp) sequence homologous to the 5' end of the invA gene (Hl^invA). The reverse primer (Primer 2) included the priming site 2 (P2) of the pKD3 plasmid and a 50-bp sequence homologous to the 3' end of the invA gene (H2^invA). The length of the invA construct was 1016 bp, which included two Flippase Recognition Target sites (FRT) and the Cm^R gene, and was flanked by HI and H2.

(iii) Before the invA construct is introduced into the ST 076-94 genome replacing the invA gene, the pKD46 helper plasmid must be electroporated into the bacteria (Figure 5B). Under the arabinose-induced control of the araC activator, this plasmid expresses λ Red recombinase, which enhances the recombination of linear DNA into a chromosome by promoting efficient repair/recombination of a double-strand break. The genetic map of the p D46 plasmid and its insertion into ST 076-94 were shown in Figure 5B.

(iv) The invA construct was introduced by electroporation into the ST 076-94 strain harboring the pKD46 plasmid expressing λ Red recombinase (Figure 5C). After electroporation and recombination events, organisms with the Cm^R gene, and therefore without the invA gene, were isolated as chloramphenicol-resistant colonies and are referred to as Salmonella typhimurium AinvACm^K.

(v) The Cm^R gene was then removed by using helper plasmid pCP20, which encodes the flippase recombination enzyme (FLP), by following procedure outlined in Cherepanov, P. P., and W. Wackernagel. Gene disruption in Escherichia coli: TcR and raR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene. 158:9-14. 1995 (Figure 5D). This enzyme facilitated a recombination event between the two FRT sites flanking the Cm^R gene, consequentially removing the Cm^R gene. The resultant organism was called Salmonella typhimurium AinvA, and was used as the starting material from which the rfali gene was deleted as described in the following sections.

(vi) A rfali construct was made using PCR methods and strategies similar to those used to make the invA construct (Figure 6 A). The length of the rfali construct was 1 135 bp, included two FRT sites and the Cm^R gene, and was flanked by 56 and 63 -bp sequences, which were homologous to the 5' and 3' ends, respectively, of the rfaH gene and were referred to as H 1 ^rJaH and H2^rfaH, respectively (Figure 6A).

(vii) The pKD46 helper plasmid was electroporated into Salmonella typhimurium AinvA (Figure 6B).

(viii) The rfaH construct was electroporated into Salmonella typhimurium AinvA harboring the pKD46 plasmid. After electroporation and recombination events, organisms with the Cm^R gene, and therefore without the rfaH gene (Salmonella typhimurium AinvAArfaHCm^R) were isolated as chloramphenicol-resistant colonies (Figure 6C).

(ix) The Cm^R gene was eliminated from Salmonella typhimurium AinvA ArfaHCm¹¹ by- exploiting the pCP20 helper plasmid (Figure 6D). The pCP20 plasmid was removed by raising the temperature to 43 °C.

(x) The resultant strain, Salmonella typhimurium AinvAArfaH did not contain either the invA gene or the rfaH gene and was the RBA (Figure 4B) in the attenuated Salmonella typhimurium vaccine of the current invention.

E. coli BW25 141/pKD3 (B. L. Wanner strain) was used to transform and amplify the pKD3 plasmid; E. coli BW251 13/p D46 (B. L. Warmer strain) was used to transform and amplify the pKD46 plasmid; and DH5a/pCP20 (W. Wackernagel strain) was used to transform and amplify the pCP20 plasmid.

The final product of the methods as described in the earlier sections, referred to as Salmonella typhimurium AinvAArfaH, was selected by its inability to grow on ampicillin and chloramphenicol, which indicated that the pCP20 plasmid and Cm^R gene were not present, and its inability to swarm on LB plates when compared to the wild-type, parent strain ST 076-94, which indicated successful deletion of the invA and rfaH genes.

Absence of the invA and rfaH genes was confirmed by PGR and sequencing of the loci, by the method reported in Sambrook, J., and D. W. Russell. Molecular Cloning: A Laboratory Manual 3 ^rd edition. Cold Spring Harbor Laboratory Press. 2001.

Physical Characterization of Salmonella typhimurium AinvAArfaH

Physical map: shown in Figure 4B, was characterized by PCR and subsequent sequencing was carried out using primers that bind to sequences outside of the region of the deletion site. Phenotypic Characteristics: The rfaH gene deletion yields colonies that had lost swarming capability in the presence or absence of surfactin and this can distinguish the S. typhimurium AinvAArfaH from wild type strains.

Genotypic Characteristics: Comparison of the sequence of Salmonella typhimurium 436-09 AinvAArfaH in the region of the invA gene to that of Salmonella typhimurium STL2 (GeneBank No. AE006468) confirmed the deletion of the invA gene in the former genome. Comparison of the sequence of Salmonella typhimurium 436-09 AinvAArfaH in the region of the invA gene to the linear invA construct confirmed the absence of the cat gene in Salmonella typhimurium 436-09 AinvAArfall (Figure 4B). Comparison of the sequence of Salmonella typhimurium 436-09 AinvAArfall in the region of the rfaH gene to that of Salmonella typhimurium STL2 (GeneBank No. AE006468) confirmed the deletion of the rfaH gene in the former genome. Comparison of the sequence of Salmonella typhimurium 436-09 AinvAArfaH in the region of the rfaH gene to the linear rfaH construct confirmed the absence of the cat gene in Salmonella typhimurium 436-09 AinvAArfaH.

Virulence Characteristics : The purpose was to determine the identity, purity, absence of extraneous agents, and genetic stability of the master seed (MS) bacterium, used for production of Salmonella typhimurium Vaccine, which was defined as Salmonella typhimurium 436-09 AinvA (invA gene deletion) ArfaH (rfaH gene deletion). Identity of Salmonella typhimurium 436-09 AinvAArfaH was done by confirmation of gene deletions using PCR, DNA sequencing, serology, biochemical reactions, a swarm test and gram staining. Genetic stability was confirmed by use of PCR on five consecutive passes of the master seed (MS+5). All test results on this master seed, including identity, purity, extraneous agents, and genetic stability, were satisfactory.

Genetic stability: this was confirmed by PCR and gel electrophoresis of the product from each of five consecutive passes of the MS, and by sequencing of the MS+5. Gel electrophoresis (Figure 1) of the PCR product from the MS and each passage +1 through +5, using primers to amplify the invA gene, shows that the invA deletion is missing 1.8 kb of DNA compared to the parent strain and remains stable (i.e., unchanged in size) through the fifth passage. Gel electrophoresis (Figure 2) of the PCR product from the MS and each passage +1 through +5, using primers to amplify the rfaH gene, shows that the rfaH deletion is missing 0.3 kb of DNA compared to the parent strain and remains stable (i.e., unchanged in size) through the fifth passage. The two sets of primers to test genetic stability (the absence of the invA and rfali genes) designed for these PGR experiments are provided in Table 1 :

Example 2: Biological Properties of the Salmonella typhimurium AinvAArfaH Virulence Characteristics

Infectious dose₅o testing of the double, gene-deleted vaccine in chicks showed that virulence was reduced by at least 4 logs (> 5.2 x 10⁷ colony forming units, CFU) as compared to the parental strain (< 4.8 x 10^'' CFU). Additionally, an in vitro invasion assay demonstrated invasion of epithelial cells by the RBA was reduced by 81% compared to the parental strain. Therefore, it was not expected to be virulent for chicks.

Attenuated Salmonella typhimurium based vaccines were tested for 10X overdose safety in chickens. Five hundred (500) one-day-old specific pathogen free (SPF) chicks were randomly divided into groups, such that 125 birds were vaccinated with a 10X dose of the attenuated Salmonella typhimurium vaccine, (Group 1), 125 birds were used as the corresponding contact controls (Group 2), 125 birds are vaccinated with a 10X dose of the parental strain (Group 3) and 125 birds were used as the corresponding contact controls (Group 4). At 7, 14, 21 , 28 and 49 days post vaccination, 25 chickens from each of Groups 1 -4 were evaluated for recovery of salmonella from the liver/spleen and the cecum.

Attenuated Salmonella typhimuriu vaccines were tested for safety in turkeys, pigeons and quail. Forty one-day-old SPF turkeys, 40 adult quails and 40 adult pigeons were divided into three groups, such that 20 birds were vaccinated with a 10X dose of attenuated Salmonella typhimurium vaccine and 20 birds were vaccinated with a 10X dose of the parental strain. Mortality and clinical disease were recorded. At 7 and 21 days post vaccination, ten birds per vaccine group were evaluated for colonization and persistence of each strain in the heart, liver, lung, kidney, spleen, ileo-cecal junction with cecal pouches, midgut, and duodenum with pancreas. Virulence, shed spread, and tissue tropism were tested in adult mice. Ten adult mice were vaccinated orally with a 10X dose of attenuated Salmonella typhimurium vaccine and ten adult mice were used as the corresponding contact controls. Also, ten adult mice were vaccinated orally with a 10X dose of the parental strain and ten adult mice were used as the corresponding contact controls. Mortality and clinical disease were then recorded. At 7 and 21 days post vaccination, five vaccinates per group and five contact controls per group were necropsied. The lung, spleen, kidney, liver, duodenum, jejunum, ileum, and ileo-cecal junction of each animal were tested for the presence of Salmonella.

Virulence was further evaluated by testing attenuated Salmonella typhimurium Vaccine and the parental strain for their ability to invade porcine, bovine, primate and canine cell lines in a comparative in vitro invasion assay at 100X (100:1, bacteria to cell) infection density.

Reversion to virulence by attenuated Salmonella typhimurium vaccine master seed in chickens was tested in a back passage study. Attenuated Salmonella typhimurium vaccine and the parent strain are passed at least five times in birds, and tested for virulence, as measured by infection and persistence in the internal organs and intestines, and genetic stability, as determined by PGR.

Potential for Horizontal Gene Transfer

It is well known that Salmonella typhimurium bacteria can acquire genes through horizontal gene transfer (HGT). Attenuated Salmonella typhimurium vaccine contained no new genes as a source of DNA and a loss of function in two genes due to deletions in the chromosome. Intracellular conjugation frequency was dependent upon the probability of coinfection of the vaccine and wild type. Since our studies showed that invasion of epithelial cells by the invA^' vaccine strain was reduced by over 80%, the probability of coinfection was greatly reduced. It is feasible that a HGT event could occur to restore the function of one gene. However, the vaccine had two deletions. The potentially transferred DNA segment that would contain both invA and rfaH genes is 1,145,012 bp in length. Since DNA that comprised a plasmid is only about 44,000 bp in length, the 1,145,012-bp piece of DNA was too long to be included in a single DNA fragment. Therefore, it was highly unlikely that these rare events would occur simultaneously.

Salmonella invA mutants were significantly impeded in their ability to enter cultured epithelial cells. Tissue tropism of Salmonella typhimurium Vaccine

Tissue tropism of attenuated Salmonella typhimurium vaccine was determined compared to that of the parental strain.

About one hundred (100) one-day-old SPF chicks was divided and randomly assigned to five groups. Four groups were vaccinated with a 10X dose of various attenuated Salmonella typhimurium vaccines (ArfaH, AinvAArfall, AinvAAInvF, AinvAAfliC) and the last with the parental strain. At 7 days post vaccination, the liver, spleen, cecum, were collected and evaluated for the presence of salmonella.

The Salmonella typhimurium AinvAArfaH had two deletions, one in invasion gene (invA) and one in the LPS formation (rfaH) gene, attenuating the wild-type virulence phenotype. The Salmonella typhimurium AinvAArfaH was likely to colonize the cecum. Some chicks may show invasion of intestinal epithelium with penetration to the liver and spleen. The vaccine should then clear from the internal organs and cecum within 21-28 days post vaccination.

Claims

1. An attenuated Salmonella strain, comprising a first mutation in one or more invasion genes and at least another mutation in one or more genes involved in the survival and/or proliferation of Salmonella in the host and/or one or more genes involved in the motility of Salmonella.

2. The attenuated Salmonella strain according to claim 1 , wherein the first mutated invasion gene is the invA gene and/or the invF gene.

3. The attenuated Salmonella strain according to claim 1 or 2, wherein the second mutated gene is chosen from among the rfaH gene, fliC and HemA.

4. The attenuated Salmonella strain according to any one of claims 1 to 3, wherein the mutation comprises deletion of the gene.

5. The attenuated Salmonella strain according to claim 4, comprising a genetic deletion of invA and rfaH.

6. The attenuated Salmonella strain according to claim 5, further comprising a genetic deletion of the fliC gene.

7. The attenuated Salmonella strain according to claim 4, comprising deletion of invA and fliC genes.

8. The attenuated Salmonella strain of claim 7, further comprising deletion of rfaH gene.

9. The attenuated Salmonella strain according to any one of claims 1 to 8, wherein mutations of said genes are partial or complete.

10. The attenuated Salmonella strain according to any one of claims 1 to 9, wherein said Salmonella belongs to Salmonella serovars of Samonella enterica.

1 1. The attenuated Salmonella strain according to claim 10, wherein the Salmonella is chosen from among Salmonella lyphimurium. Salmonella enteritidis, Salmonella dublin, Salmonella choleraesuis, Salmonella typhisius, Salmonella brandenburg. Salmonella heidelberg, and Salmonella abortusovis.

12. The attenuated Salmonella strain according to claim 1 1 , wherein said Salmonella strain is Salmonella lyphimurium.

13. An aviiiilent live culture which comprises the attenuated Salmonella strain as described in any one of claims 1 to 12.

14. A vaccine composition comprising an immunologically effective amount of the attenuated Salmonella strain of any one of claims 1-13 and optionally a pharmacologically acceptable carrier, diluent or excipient.

15. The vaccine composition of claim 14, further comprising an effective amount of adjuvant.

16. A multivalent vaccine which comprises attenuated Salmonella strain according to any one of claims 1 to 13.

17. The vaccine composition according to claim 14 or 15, which is associated with another vaccine chosen among Enterisol Ileitis FF, Enterisol SC-54 FF, Ingelvac® ERY- ALC, Argus®, Paracox®, Livacox®, Coxabic®, and/or with Immunocox II®.

18. The vaccine composition according to claim 14 or 15, which is administered to swine in association with an attenuated microorganisms and/or viruses pathogenic in pigs, chosen among pseudorabies virus, porcine influenza virus, porcine parvovirus, porcine circovirus, rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchisepiica, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae, Brachyspira hyodysenteriae and/or Actinobacillus pleuropneumoniae.

19. The vaccine composition according to claim 14 or 15, which is administered to avian in association with an attenuated microorganisms pathogenic in poultry, chosen among infectious Bronchitis virus, Newcastle Disease virus. Turkey Rhinotrachcitis virus, Marek's virus, Avian Reovirus, Infectious Bursal Disease (Gumboro), Chicken Anaemia agent. Mycoplasma gallisepticum, Haemophilus paragallinarum (Coryza), Chicken Poxvirus, Avian Encephalomyclitisvirus, Duck Plague virus. Egg Drop syndrome virus. Infectious Laryngotracheitis virus, Herpes Virus of Turkeys, Eimeria species, Ornithobacterium rhinotr ache ale, Pasteurella multocida, and/or Mycoplasma synoviae.

20. The vaccine composition of any one of claims 14 to 19, wherein the vaccine composition is formulated for administration as liquids or dry powders, aerosols, sprays, eyedrops, in drinking water.

21. A method of protecting and/or treating animals against Salmonella infections, which comprises administering to said animal an immunologically effective amount of attenuated Salmonella strain of any one of claims 1 to 13 or an effective amount of the vaccine composition of any one of claims 14 to 20.

22. The method according to claim 21 wherein the animal is selected from the group comprising of bovine, ovine, swine and avian.

23. The method according to claims 21 or 22 wherein attenuated Salmonella strain is administered via oral, nasal, ocular, mucosal, parenteral vaccination routes or via in ovo vaccination.

24. Use of the attenuated Salmonella strain according any one of claims 1 to 13 for the preparation of a vaccine composition for protecting and/or treating animals against Salmonella infections.

25. A vaccination kit comprising a dispensing device and an immunologically effective amount of the vaccine of any one of claims 14 to 20.

26. The vaccination kit of claim 25, wherein the dispensing device is adapted for ocular eye-drops, spray, aerosol delivery or in ovo delivery.

27. The vaccination kit of any one of claims 25 to 26 further comprising technical instructions with information on the administration and dosage of the vaccine composition.