KR20080105099A - Live attenuated salmonella vaccine - Google Patents

Abstract

The present invention is related to double and triple attenuated mutant strains of a bacterium infecting veterinary species such as Salmonella enterica and/or (a pathogenic) Escherichia coli. The mutants of the invention contain at least one first genetic modification and at least one second genetic modification, said first modification in one or more motility genes, and said second modification in one or more genes involved in the survival or the proliferation of the pathogen in the host. The present invention further relates to live attenuated vaccines based on such mutants for preventing amongst others Salmonellosis and/or an infection by an E. coli pathogen in a veterinary species. ® KIPO & WIPO 2009

Description

Attenuated Salmonella Live Vaccine {LIVE ATTENUATED SALMONELLA VACCINE}

The present invention relates to attenuated bacterial mutants, in particular attenuated Salmonella enterica. enterica ) mutants, and attenuated live vaccines comprising them. Double, triple, multiple mutants of the invention advantageously allow serological distinction between vaccinated animals and animals exposed to wild-type fields (not vaccinated), such as wild-type field S. enterica and the like.

The Salmonella are Gram-negative, selective anaerobic, motility, non-lactose fermenting bacteria belonging to Enterobacteriaceae. Salmonella is commonly transmitted to humans by consuming contaminated foods, causing salmonellosis. E. coli is another member of the intestinal bacteria family.

Salmonella have been separated from many animal species, including cattle, chickens, turkeys, sheep, pigs, dogs, cats, horses, donkeys, seals, lizards, and snakes.

95% of the important Salmonella pathogens are the most common forms of S. enterica serotype S. typhimurium and S. enterica serotype enterobacteria (S. enteritis; S. Enteritidis) Together belongs to S. enterica.

Salmonella infections are a serious medical and veterinary problem worldwide and are of interest to the food industry. Contaminated food cannot be easily identified.

Control of salmonellosis is important to avoid the possibility of fatal human infection and significant economic losses to the livestock industry.

Salmonella, which exists everywhere in nature, complicates the control of diseases caused only by the detection and eradication of infected animals.

Several control strategies based on the principles of competitive exclusion and vaccination have been tested, for example to control poultry infection.

Vaccination of farm animals is often considered the most effective way to prevent zoonotic infections caused by, for example, Salmonella.

Whole cell killing vaccines and subunit vaccines are used with variable results in the prevention of Salmonella infection in animals and humans. Inactivated vaccines generally provide poor protection against salmonellosis.

Attenuated Salmonella live vaccines include: (i) their ability to induce cell-mediated immunity in addition to antibody responses; (ii) oral delivery without the risk of needle contamination; (iii) effects after single-dose administration; (iv) induction of an immune response at multiple mucosal sites; (v) low production costs; And (vi) potentially superior to inactivated agents thanks to their possible use as carriers for the delivery of recombinant antigens to the immune system.

The following attenuated mutant strains were tested for their efficiency in inducing a protective immune response in treated animals: (1) Strains carrying mutations in the aro genes ( Alderton et al ., 1991, Avian diseases 35: 435-442; Schiemann and Montgomery, 1991, Veterinary Microbiology 27: 295-308 ), (2) strains with deletions in cya (adenylate cyclase) and / or crp (cyclic AMP receptor) genes ( US 5,389,368; US 5,855,879; US 5,855,880) ; Hassan and Curtiss , 1997, Avian Diseases 41: 783-791; Porter et al . 1993, Avian Diseases 37: 265-273 ) and (3) strains carrying mutations in the genes of the guaBA operon of S. typhoid ( Wang) et al., 2001, Infection and Inmunity 69: 4734-4741; WO99 / 58146 and US Pat. No. 6,190,669 ).

So far, only the vaccine strain Megan ^® Vacl involving the deletion in cya and crp genes were at least partially effective. This strain does not provide full protection (http://www.meganhealth.com/meganvac.html).

McFarland and Stocker (1987, Microbial Pathogenesis 3: 129-141) reported the virulence of the guaA and guaB TnIO insertion mutants of S. murine typhoid and S. dublin in BALB / c mice. At high doses (2.5xlO ⁷ CFU), these authors reported significant mortality in animals resulting from propagation of nutritionally demanding strains.

In addition, the ΔguaBA mutant of S. typhoid bacteria has been proven to provide no suitable candidate for robust and stable protection against typhoid fever. It showed significant residual virulence in mice (Wang et al., 2001).

Thus, there is still a need for improved attenuated live vaccine strains as well as improved attenuated Salmonella live vaccine strains of bacteria that infect common veterinary species.

Vaccines animals produce antibodies against different antigens of the pathogen. The problem is that vaccinated animals can no longer be distinguished from, and possibly infected by, animals in contact with wild strains such as Salmonella strains.

Thus, there is also a need for improved attenuated live strains, such as attenuated Salmonella live vaccine strains that may enable such distinction.

It is an object of the present invention to provide double or triple mutated attenuated Salmonella enterica strains.

Another object of the present invention is to provide live attenuated vaccines against salmonellosis and methods of treatment based thereon.

Another object of the present invention is to provide attenuated Salmonella strains useful as DNA-mediated vaccines expressing foreign body antigens and as live vectors. Thus, such strains are well suited for the development of vaccines including multivalent vaccines.

Another object of the present invention is to provide a method of achieving the S. enterica deletion mutants of the present invention.

Another object of the present invention is to provide an attenuated Salmonella strain that allows serological distinction between vaccinated animals and animals that are likely to be infected without vaccination.

Another object of the present invention is to provide attenuated strains of bacteria that infect common veterinary species, in particular poultry, and methods of making these materials.

The general purpose is to improve food safety and animal health.

Some ΔguaB nutritionally required Salmonella enterica mutants mutated deletions in the guaB gene showed residual virulence. Further modifications (preferably deletions) in one or more genes involved in motility have been found to reduce the remaining virulence without affecting the immunogenic dose of the strain.

Accordingly, a first aspect of the invention relates to attenuated mutant strains of bacteria that infect veterinary species, particularly attenuated S. enterica mutant strains, wherein the mutant strains comprise at least one first genetic modification and Containing at least one second genetic modification, wherein the first modification in one or more (at least one) motility genes, and the second modification in one or more (at least one) genes comprises: Example S. enterica). The term “bacteria infecting veterinary species” in the context of the present invention means bacteria which are particularly pathogenic to veterinary species and which can be attenuated by said genetic modification. The bacteria that infect veterinary species can be Gram-negative. Preferred are Gram-negative bacteria for poultry such as Salmonella, Pest, E. coli, and the like. Most preferred are Salmonella enterica and (pathogenic) Escherichia coli. By "pathogenic to" is meant that bacteria, if not attenuated, can cause infectious diseases in veterinary species.

Genetic modifications of the invention advantageously induce null-function, that is to say, impair or affect gene function. Modifications in the context of the present invention are also referred to as "damage modifications". Modification refers to inactivating the gene in question. Advantageously, the inactivation results in attenuation to the extent that at least the mutant strain is suitable for use in an attenuated live vaccine.

Genetic modification may be the insertion, deletion, and / or substitution of one or more nucleotides in said genes. Mutant strains according to the invention are affected by motility gene function and gene function necessary for the survival or propagation of pathogens by such modifications, and induce null-function (no functional gene product is formed) of the affected genes. .

Deletion mutants are preferred because the insertion mutants can be restored to restore the pathogenicity of the strain.

The first modification is present in one or more (1, 2, 3, ...) motility genes. Examples of genes involved in motility are genes encoding flagellin. Mutants of the invention may have a (damaged) modification in the fliC and / or fljB or fljBA genes (fliC; fljB; fljBA; fliC and fljB; fliC and fljBA; ...). Advantageously, mutants from which all genes encoding flagellin have been detected can be readily distinguished from wild-type motility strains by being able to cluster on LB medium containing 0.4% agar.

The second genetic modification is present in one or more (1, 2, 3, ...) genes involved in the survival or proliferation of the pathogen in its host. Such genes may be house-keeping genes or virulence genes. An example of a house-keeping gene that can induce attenuated strains when gene function is affected is the guaB gene, which encodes the enzyme IMP dehydrogenase. Such mutants are new ( de novo ) may form guanine nucleotides. Also possible are impaired modifications in the guaBA operon that advantageously induce null-function of the gene (s) encoding or regulating appropriate IMP dehydrogenase activity.

Advantageously, the attenuated mutant strains of the invention are immunogenic.

The present invention is particularly aimed at providing attenuated S. enterobacteria and S. rat Typhoid strains.

Preferably, the genetic modifications of the present invention are introduced into parental strain S. enteritis phage phage type 4 strain 76Sa88 or into parental strain S. murine typhoid 1491S96. The 76Sa88 strain is a clinical isolate from turkey obtained from the Veterinary and Agrochemical Research Center (B-1180 Ukkel, Groeselenberg 99, Belgium), harboring the harboring temperature sensitive replication plasmid pKD46 and encoding the bacteriophage lambda red recombinase system. do. The 1491S96 strain is a clinical isolate from chickens.

One of the attenuated S. enterica strains obtained according to the present invention is S. enteritis strain SM73 having accession number LMG P-21642. Another example is the attenuated S. rattypus strain SM89 having accession number LMG P-21643.

Preferred mutants of the invention involve, or include, genetic modifications in the guaB gene and genetic modifications in the fliC gene.

Other preferred mutants of the invention carry or include genetic modifications in the guaB gene and genetic modifications in the fljBA genes.

Another preferred mutant of the invention involves or comprises a genetic modification in the guaB gene, a genetic modification in the fliC gene, and a genetic modification in the fljBA genes.

The attenuated strains of the present invention are well suited for use in attenuated live vaccines. Mutant strains of the invention can encode and express foreign antigens.

Another aspect of the invention

Attenuated mutant strains according to the invention in a pharmaceutically effective or immunological amount; And

-A vaccine for immunizing veterinary species against bacterial infection, comprising a pharmaceutically acceptable carrier or diluent.

Generally about 10 ² cfu to about 10 ¹⁰ cfu, preferably about 10 ⁵ cfu to about 10 ¹⁰ cfu is administered (example of pharmaceutically effective amount or immunity). The immune dose varies with the route of administration. Those skilled in the art will appreciate that an effective dosage for a vaccine administered parenterally may be less than a similar vaccine administered via drinking water and the like.

The attenuated strains of the present invention and pharmaceutical compositions or vaccines comprising the same are very suitable for immunizing veterinary species, livestock, and more particularly animals such as poultry. For example, attenuated Salmonella strains of the present invention, and pharmaceutical compositions or vaccines comprising the same, are directed against veterinary species and in particular poultry, such as salmon, salmonella and possible other diseases (e.g. for multivalent vaccines). It is very suitable for immunization. The attenuated strains of the invention are particularly suitable for protecting the animal / veterinary species in question against the challenge of the pathogen in question (the bacteria that infects the veterinary species).

Thus, a further aspect of the present invention relates to a method of immunizing animals, preferably veterinary species, more preferably poultry, for example chickens, against disease caused by bacteria infecting veterinary species, The method comprises administering an immunological amount of the attenuated mutant strain of the invention and / or a vaccine comprising the same to a veterinary species or animal in need thereof, so that a protective immune response is present in these animals or veterinary species. Is carried out in. The present invention relates in particular to methods for immunizing veterinary species against salmonellosis or against infection by Escherichia coli.

Examples of veterinary species to be immunized against salmonellosis: poultry, small livestock or heavy livestock such as chickens, turkeys, ducks, quails, guinea fowls, pigs, sheep, young calves, cows, etc. Immunity is administered to these animals, preferably via an oral, intranasal or parenteral route.

A further aspect of the invention relates to a mutant strain of the invention for use as a medicament (eg for use in a vaccine). Another aspect of the invention is an attenuated mutation of the invention for the manufacture of a medicament, for example a vaccine, or for the prevention (and / or treatment) of diseases caused by pathogens (bacteria that infect veterinary species), such as salmonellosis. And to the use of a sieve strain. Animals or veterinary species to be treated and the recommended dosages are given above.

Another aspect of the invention is the mutants of the invention, in particular flagellin, as serological markers for distinguishing between vaccinated and naturally infected animals, ie wild-type strains, thereby distinguishing between infected animals. It is about mutants.

The present invention relates to a method for serological distinction between, for example, vaccinated animals and animals infected by wild-type strains, wherein the vaccinated animals are mutated by inactivated flagellin genes. Immunized, this method is:

Analyzing the animals in the presence of increased antibodies against flagellin, and

-Distinguishing infected animals from vaccinated animals based on the presence or absence of said antibodies.

The method of the invention is advantageously an in vitro method. Advantageously, animals infected with the Salmonella are distinguished from animals immunized with live attenuated vaccines according to the invention.

Livestock, such as poultry, in particular chickens, are known to generate antibodies against flagellin gene products, in particular FIiC gene product. Thus, the antibodies in question will be detected in an infected animal by a wild type strain (which produces such antibodies), but not in animals vaccinated by a mutant strain in which flagellin gene (s) is inactivated. The latter does not, for example, generate antibodies against FIiC and / or FIjB.

The presence of these antibodies suggests wild type strains and thus infections. Thus, the methods of the invention advantageously allow for the detection or diagnosis of Salmonella infection in animals vaccinated by mutant strains in which flagellin gene (s) have been inactivated. Such mutant strains may be one of the above strains of the invention.

Thus, inactivation of flagellin genes such as fliC can be used, for example, using serological tests based on the detection of FliC protein to diagnose the presence of wild type strains such as wild type S. enterica in (vaccinated) animals. Allow. In the method of the invention, the animals are preferably analyzed for antibodies against FliC.

The method of the invention applies in particular to poultry, more preferably chickens.

Detailed description of the invention

Surprisingly, a combination of flagellin mutations (also called damage modifications, flagellar mutations in gene coding for flagellin) and nutritionally demanded mutations have been shown to induce highly immunogenic attenuated S. enterica strains. lost. Mutant strains according to the present invention are capable of (at least one) modification (s) in kinetic gene (s), and (at least one) modification (s) in gene (s) associated with the survival or proliferation of pathogens in their host. Accompany or include.

Genes involved in survival or proliferation may be house-keeping genes and / or virulence genes. Examples of such house-keeping genes and virulence genes can be found in Mastoeni et al., 2000, The Veterinary Journal 161: 132-164, which is incorporated herein by reference. Preferred examples of house-keeping genes are the guaB gene, but modifications of the aro, pur, dap, pab, sipC, phoP, phoQ, pagC, cya, and / or crp genes can also be predicted.

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

Such inactivation can be obtained through deletions with impaired gene function. One skilled in the art knows how to obtain such mutants, and a simple test can tell whether the gene function is impaired. For example, mutant strains that fail to express a functional guaB gene product cannot grow on at least A medium unless the medium is supplemented with guanine, xanthine, guanosine or xanthosine (eg 0.3 mM). .

Another simple test can tell whether a motility gene function, such as flagellin gene function, is impaired. These mutants do not collect on LB medium containing 0.4% agar.

Modifications in the genes in question should preferably result in attenuation of the mutant strains at least to the extent that they are suitable for use in live vaccines.

The examples below further demonstrate that further mutations in (at least one; one or more) kinetic gene (s) (fliC, flJB and / or fljBA) advantageously relieve the residual pathogenicity of the guaB deletion mutant and provide a lethal dose of wild type S Improved protection of immunized animals against challenge by enterica.

The flagella filaments of all members of the genus Salmonella are flagellin proteins, which are multimers of a single protein ( van Asten et al ., 1995, Journal of Bacteriology 177: 1610-1613 ).

FIiC is a Phase 1 filament subunit protein of flagellin ( Ciacci - Woolwine et al ., 1998, Infection and Immunity 66: 1127-1134 ).

S. mutipus bacteria are located at different sites of the chromosome and have two flagellin genes (fliC and fljB) that show a pace change. The promoter of fljB forms part of a chromosome fragment that can be reversed by site-specific recombination. Depending on the orientation, fljB is expressed with fljA or the latter encodes an inhibitor of the fliC gene; Or fliC is expressed. Escherichia coli, another member of the enterococci, may also possess two flagellin genes that share homology with the fliC and fljB genes of S. enterica , respectively ( Tominaga , 2004, Genes Genet . Syst . 79: 1-8 ).

Inactivation of the fliC gene in S. enteritis (which encodes the major flagella protein) increased both the stability and effectiveness of the vaccine administered to the homocrossed mouse cell line BALB / c. This cell line is very sensitive to systemic salmonellosis.

This demonstrates, among other things, that flagellin is not an essential antigen for inducing a protective immune response against Salmonella in BALB / c mice despite many of the instructions in the literature.

S. enteritis flagellin is immunogenic in chickens and involves H: g, m antigenic determinants ( van Asten et al ., 1995; Wyant et al ., 1999, Infection and Immunity 67: 1338-1346; Ogushi et al ., 2001, The Journal of Biological Chemistry 276: 30521-30526 ).

Flagella from different Gram-negative bacteria (eg, those of S. enterococci and S. typhoid) activate monocytes to produce pre-inflammatory cytokines (eg tumor necrosis factor alpha) There is evidence to mediate the activation of interleukin-1 receptor-associated kinase (IRAK).

Thus, Gram-negative flagellin is believed to play an important and previously unrecognized role in the innate immune response to Gram-negative bacteria. FliC may be particularly important in the process of infection in the gastrointestinal tract ( Ciacci - Woolwine et al ., 1998; Wyant et al., 1999; Moors et al ., 2001, Infection and Immunity 69: 4424-4429 ).

There is much ambiguity in the literature regarding the degree to which flagella contributes to virulence in poultry and / or humans.

Van Asten et al. ( 2000, FEMS Microbiol Lett . 185: 175-9 ) showed that inactivation of the flagellin gene of S. enterobacteria strongly reduced invasion into Caco-2 cells (human colon cancer cell line) (50-fold), while bacterial aggregation was not actually affected. Shows. The report is limited to in vitro results.

On the other hand, Parker and Guard-Petter ( 2002, FEMS Microbiology Letters 204: 287-291 ) found that the fliC :: Tn10 mutant was equally virulent to wild type following the oral challenge of chicks. This suggests that the presence of flagellin is not necessary to at least achieve adequate levels of invasion after oral challenge. When applied subcutaneously, flagella mutants were significantly reduced compared to wild type strains.

Thus, there are many prior art instructions that teach teaching away from constructing S. enterica double (or triple) mutants according to the present invention. Inactivation of one or more motility genes, for example, helps to reduce the remaining virulence of guaB deletion mutants, but other advantages exist for dinner.

For example, inactivation of the fliC gene is serologically based on the detection of antibodies directed against FliC protein to diagnose the presence of wild type S. enterica, eg, S. enteritis, in (vaccinated) animals. The use of the test is advantageously allowed. Immune detection is possible via ELISA, RIA techniques and / or any other known immunological test or format.

IDEXX laboratories use commercially available assays (FlockChek ^® Salmonella enterococcal antibody test kits) to reliably detect antibodies against H-antigenic determinants (H: g, m flagella epitopes) of S. enterobacterial FliC flagellin. Has

The above-mentioned double and / or triple S. enterica mutants of the present invention that result in (damaged) genetic modifications in genes involved in the survival or proliferation of pathogens in host and gene (s) associated with motility are present in the art. It has been shown to have advantages over attenuated S. enterica strains.

Mutant strains of the invention are well suited for use in attenuated live vaccines as live vectors and / or DNA-mediated vaccines. The term "vaccine" is meant to include prophylactic vaccines as well as therapeutic vaccines. Preferably the vaccine is for prophylaxis.

"Live vector" vaccines, also referred to as "carrier vaccines" and "live antigen delivery systems", include an interesting and flexible field of vaccination (Levine et al, 1990, Microecol. Ther. 19: 23-32). . In this approach, a viral or bacterial live vaccine is modified so that it expresses protective foreign body antigens of other microorganisms and delivers those antigens to the immune system, thereby stimulating a protective immune response. The bacterial live vectors that are spreading include, among other things, attenuated Salmonella.

It is an object of the present invention to provide attenuated mutant strains for use in live vaccines, possibly multivalent live vaccines. "Polyvalent or multivalent vaccine" means a vaccine comprising antigenic determinants, in particular from many different disease-causing organisms.

Therefore, one of the objects of the present invention

Attenuated mutant strains of the invention in pharmaceutically effective or immunity amounts; And

Pharmaceutically acceptable carriers or diluents;

To include, for example, to provide a vaccine against salmonellosis.

Another object of the present invention

Attenuated mutant strains of the invention in a pharmaceutically effective or immunological amount, wherein the mutants encode and express foreign antigens; And

Pharmaceutically acceptable carriers or diluents;

To provide a live vector vaccine comprising a.

Certain foreign antigens used in live vectors are not critical to the present invention.

Another object of the present invention

Pharmaceutical attenuated mutant strains of the invention in a pharmaceutically effective or immunological amount, wherein said mutant contains a foreign body antigen which is a plasmid encoded and expressed in eukaryotic cells; And

Pharmaceutically acceptable carriers or diluents;

It is to provide a DNA-mediated vaccine comprising a.

Details regarding the construction and use of DNA-mediated vaccines can be found in US Pat. No. 5,877,159, which is incorporated by reference in its entirety. Again, the particular foreign antigen used in the DNA-mediated vaccine is not important to the present invention.

Determination of whether to express a foreign antigen in a pathogen (using a prokaryotic cell promoter in a live vector vaccine) or in cells invaded by a pathogen (using a progressive cell promoter in a DNA-mediated vaccine) determines the specific antigen. Which vaccine construct provides the best immune response in animal studies or clinical trials, and / or if glycosylation of the antigen is essential for its protective immunogenicity, and / or the correct tertiary form of the antigen Can be based on where is achieved in one form that is better than the other (US Pat. No. 5,783,196).

"Pharmaceutically effective amount" means much greater than universal to overcome (prevent and / or treat) a disease in question, for example salmonellosis. As used herein, “immunity” refers to an amount that in fact can induce a (protective) immune response in an animal that has received a pharmaceutical composition / vaccine. The immune response conducted may be a humoral, mucosal, local and / or cyclic immune response. As is known in the art, the amount required may depend on age, sex, weight and many other factors.

The particular pharmaceutically acceptable carriers or diluents used are not critical to the present invention and are conventional in the art. Examples of diluents are buffers that act against gastric acid in the stomach, such as citrate buffer containing sucrose (pH 7.0), bicarbonate buffer (pH 7.0) alone, or bicarbonate full containing ascorbic acid, lactose and optionally aspartame. Sieve (pH 7.0). Examples of carriers include proteins as found in skim milk; Sugars; Example sucrose; Or polyvinylpyrrolidone.

Deletion mutants according to the invention are generated through standard homologous recombination techniques, such that the entire gene (s) in question or at least a portion of those genes is replaced with resistance genes and flanking FRT sites.

Preferably, in the second step, the resistance gene is removed by recombination between two FRT sites. One FRT site and priming sites Pl and P2 are left behind by a recombinant molecular mechanism that eliminates the antibiotic resistance gene according to Datsenko and Wanner (200O) (see eg FIG. 4).

The invention will be described in more detail in the following examples and embodiments by reference to the accompanying drawings. Certain embodiments and examples are not intended to limit the scope of the invention as claimed in any way. The principles of the examples given herein for S. enterica are equally applicable to other (gram-negative) strains on poultry, such as (gram-negative) bacteria, particularly pests, (pathogenic) Escherichia coli, etc. that infect veterinary species. It can be applied well.

1 provides a schematic overview of the biosynthetic pathway of guanosine monophosphate. (AICAR: 5′-phosphoribosyl-4-carboxamide-5-aminoimidazole; ATP: Adenosine Triphosphate; G: Guanine; GMP: Guanosine Monophosphate; GR: Guanosine; Hx: Hypoxanthine; HxR : Hypoxanthine riboside (inosine); IMP: inosine monophosphate; X: xanthine, XMP: xanthosine monophosphate; guaA: GMP synthetase, guaB: IMP dehydrogenase; guaC: GMP reductase.)

2 shows contig 1294 (SEQ ID NO: 19) of the S. enteritis genome. The ATG start codon and the TGA stop codon of the guaB gene are bold. N may be A, C, T or G.

Figure 3 shows the sequence of ΔguaB fragment of S. enteritis bacteria cloned at pUC18 (SEQ ID NO: 20). Primers used are indicated by horizontal arrows. The fragment generated by the primers GuaB6-GuaB7 was cloned in pUC18. ATG initiation and TGA termination codons of the guaB gene and the CCCGGG SmaI restriction site are indicated in bold.

Figure 4 shows the nucleotide sequence (SEQ ID NO: 21) of S. enteritis PCR fragment comprising guaB deletion obtained using primer GuaBlO. PCR fragments were amplified by primers GuaB6-GuaB7 using the whole genomic DNA of mutant SM20. The remaining FRT sites are indicated in bold italics and the Pl and P2 primers are indicated by arrows (Datsenko and Wanner, 2000, PNAS 97: 6640-6645). ATG initiation and TGA termination codons of the guaB gene are indicated in bold.

FIG. 5 shows the guaB gene (SEQ ID NO: 22) of S. murine typhoid LT2, section 117 in the complete genome 220. FIG. The ATG start codon and TGA stop codon of the guaB gene are shown in bold.

FIG. 6 shows the nucleotide sequence (SEQ ID NO: 23) obtained after sequencing the PCR fragment amplified by primers FliC1-FliC2 against the entire DNA of mutants SM73 and SM89 using primers FliC1 and FliC2. The remaining FRT sites are indicated in bold italics, the ATG initiation and TAA stop codons are indicated in bold, and Pl and P2 are indicated by arrows.

FIG. 7 shows the nucleotide sequence (SEQ ID NO: 24) obtained after sequencing the PCR fragment amplified by primers FljBA6-FljBA5 against the entire DNA of S. mutiform mutant SM48 using primer F1jBA6. The remaining FRT sites are indicated in bold and Pl and P2 are indicated by arrows.

8-11 show the deposits for SM69, SM73, SM86 and SM89, respectively.

Example  One: guaB  Nutrition that affects genes Demand  mutation

Nutritionally required insertional mutants of wild type S. enterobacteria were obtained through insertional mutagenesis. Only when supplemented with 0.3M guanine, xanthine, guanosine or xanthosine, the mutant strain grows on minimal A medium.

These data strongly suggest that the nutrient mutation of the strain affects the guaB gene encoding the enzyme IMP dehydrogenase (EC 1.1.1.205). This enzyme converts inosine-5'-monophosphate (IMP) into xanthosine monophosphate (XMP) as indicated in FIG.

Insert mutants can be reverted, thereby restoring the pathogenicity of the strain. This may limit its applicability in attenuated live vaccines. In such aspects deletion mutants are preferred. Thus, guaB deletion mutants of S. enterococci and S. mutipus were generated and tested. The guaB genes of all serotypes are given in FIGS. 2 and 5.

Example  2: guaB  Deletion mutants

Construction of guaB deletion mutants

A method for generating deletion mutations in the already disclosed genome of E. coli K12 (Datsenko and Wanner, 2000, PNAS 97: 6640-6645) was applied for this goal. This method relies on homologous recombinants mediated by the bacteriophage λ red recombinase system of linear DNA fragments generated by PCR in which the guaB sequence is replaced with an antibiotic resistance gene. This resistance gene can be deleted from the genome by site-specific recombination surrounded by FRT sites and mediated by FLP recombinase.

Duplicate PCR (Ho et al., 1989, Gene 77: 51-59) was applied to deletion of the 861 bp internal segmenter of the guaB coding sequence. The principle relies on the use of two primer sets, GuaB3-GuaB4 (flanked at the 5 'end of guaB gene) and GuaB5-GuaB2 (flanked at the 3' end of guaB gene). Both sets are partially complementary and contain primers (GuaB4 and GuaB5) to which the SmaI restriction site is added. After annealing and chain stretching of the resulting complementary sequences, PCR generated fragments by the 6 base pair SmaI site replacing the 861 base pair inner segment of the guaB coding sequence with the outer primers GuaB6 and GuaB7. This ΔguaB fragment was cloned in vector pUC18 (see FIG. 3).

The chloramphenicol resistance gene (cat) with its flanking FRT sequences was amplified using plasmid pKD3 DNA as primers Pl and P2 (Datsenko and Wanner, 2000) and template. This PCR fragment was ligated at the SmaI site of the cloned ΔguaB fragment. The desired fragment was generated using nested primers (GuaB6-GuaB7). The resulting PCR fragment was electroporated into S. enteritis 76Sa88, which harbors a temperature sensitive replication plasmid pKD46 and encodes a bacteriophage lambda red recombinase system. Chloramphenicol resistant transformants were tested on minimal A medium and on minimal A medium supplemented with 0.3 mM guanine. ΔguaB :: catFRT mutants were identified by PCR using the following primer combinations: GuaB6-GuaB7, GuaB6-P2, GuaB7-Pl and P1-P2.

S. enteritis ΔguaB :: catFRT mutant (SMl2) was electroporated by a temperature sensitive replication plasmid pCP20 encoding FLP recombinase to remove the cat gene. The resulting strain S. enteritis ΔguaB was named SM20. The PCR fragment in which the deletion was located was obtained using the whole genomic DNA and primer combination GuaB6-GuaB7 of mutant SM20. The ΔguaB mutation was confirmed by sequencing using the primer GuaBlO of this fragment (see FIG. 4).

The sequences of all these primers are given in Table 1.

In order to avoid the presence of possible further mutations caused by the expression of the Red Recombinase system, homologous gene strains were constructed.

ΔguaB :: catFRT mutations of the mutant SM12 is a bacteriophage ^{SM12 P22 HT int -. (Davis} , RW, Botstein D. and Roth, JR (1980) In Advanced Bacterial Genetics, A manual for genetic engineering Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) lysates were transfected with wild type S. enteritis 7676. Cat genes were removed using plasmid pCP20. The resulting strain S. enteritis ΔguaB was called SM69 with accession number LMG P-21641.

The ΔguaB mutant of S. mutipus strain 1491S96 was constructed using the same procedure and the same primers. The resulting strain was named SM19. SM86 (having Accession No. LMG P-21646) is a homologous gene obtained after transfection of ΔguaB :: catFRT to S. mumps strain 1491S96 using bacteriophage P22 HT int ^- lysate of SM9 and restriction of cat genes. Strain.

ΔguaB mutants SMl9, SM20, SM69 and SM86 are sensitive to bacteriophage P22 HT int ⁻ . This demonstrates the presence of intact lipopolysaccharide (LPS).

S. enterococci in mice guaB  Deletion mutant SM20 Virulence and protection test

The virulence of mutant SM20 in mice was tested by oral infection of 6-8 week old female BALB / c mice in two independent experiments (Pattery et al., 1999, MoI. Microbiol. 33 (4): 791-805). These tests were performed as described above. Wild-type strain S. enteritis 76Sa88 was performed in parallel with the positive control. S. enteritis 76 Sa88 ΔaroA mutant SM50 was included in the experiment as a vaccine control. This mutant involved the correct deletion of the complete aroA coding sequence and was constructed by the method of Datsenko and Wanner (2000).

Complete data are given in Tables 2 and 3. These results indicate that ΔguaB mutant SM20 is strongly attenuated in mice but still shows some residual pathogenicity when administered at such large doses. Oral immunization with such mutants induces protective immunity against infection with large doses of the corresponding pathogenic wild type S. enterococci strain 76Sa88. This protection is at least the same as that provided by the S. enterobacterial AaroA mutant SM50.

Homogeneous Genes in Mice guaB  Deletion mutants SM69  And SM86 Virulence and protection test by

The virulence of the mutants SM69 and SM86 in mice was tested by oral infection of 6-8 week old female BALB / c mice. These tests were performed as described above. Wild-type strains S. enteritis 76Sa88 and S. rattypus 1491S96 were tested in parallel with positive controls.

Complete data are given in Tables 6, 7, and 10-13. These results indicate that ΔguaB mutants SM69 and SM86 are strongly attenuated in mice but still show some residual pathogenicity when administered at such large doses. Oral immunization with mutants induces protective immunity against infection by large doses of corresponding pathogenic wild type strains.

Example  3: S. enterobacteria and S. Bacteriostatic Flagellin  Mutants

It was next tested whether additional (more) modifications in the motility gene (eg the flagellin gene) could further reduce the residual pathogenicity left in single mutants such as SM20 accompanied by deletion mutations in the guaB gene. .

The only one gene coding for flagellin, S. enterobacterial strains containing fliC, was used in preliminary experiments. Double mutants were constructed when the guaB and fliC genes of S. enteritis were inactivated. For S. mutipus, double (ΔguaBΔfliC; AguaBAfljBA) and triple (ΔguaBΔfliCΔfIjBA) mutants were constructed.

Δ fliC  Mutants ( SM24 , SM30 )of Construction

PCR using the FliCP1-FliCP2 primer combination on template plasmid pKD3 (catFRT) or pKD4 (kanFRT) amplifies the recombinant fragment containing the antibiotic resistance gene along with the FRT sites and priming sites P1 and P2, and the fliC coding sequence Has an extension homologous to the early 50 (1-50) and terminal (1468-1518) 50 nucleotides of In this region, S. mutipus 1491S96 and S. enteritis 76Sa88 have 100% and 98% sequence identity with the primers, respectively. Primer FliCPl contains an additional G at position 37 compared to SEQ ID NO 22. Thus, the ΔfliC mutant allele encodes 16 amino acid peptides, of which the first 12 amino acids correspond to the amino terminus of FliC. The internal segment of 1416 bp (51-1467) of the fliC coding sequence (1-1518) will be substituted.

The resulting PCR product (1 μg) was previously induced by 0.2% arabinose and electroporated with S. mutipus 1491S96 (pKD46) and S. enteritis 76sa88 (pKD46) encoding the lambda recombinase system.

Antibiotic resistance candidate substitution mutants were identified by PCR using primers FliC1 and FliC2 and whole DNA of mutant strains and wild type strains. Restriction analysis was performed to distinguish between PCR fragments of approximately the same size. The enzyme EcoRV was used to limit the wild type S. murine typhoid PCR fragment amplified by FliC1-FliC2. Two fragments (470 bp and 1021 bp) were obtained. Fragments amplified for the fliC substitution mutant do not contain EcoRV restriction sites. In the case of S. enterobacteria, the enzyme ApoI was used. This enzyme cleaves two wild-type fliC fragments of S. enteritis into two pieces (345bp and 1147bp). The fragment obtained for the fliC substitution mutant does not contain an ApoI restriction site.

The motility of the mutants was tested on LB medium with 0.4% agar (Miller, 1992, A short course in bacterial Genetics, a laboratory manual and handbook for Escherichia coli and related bacteria.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Wild type S. mutipus bacteria and wild type S. enteritis bacteria are collected on this medium. When the S. mutipus fliC substitution mutant is fully assembled, the fljB gene is still present in the mutant. S. enterobacterial fliC substitution mutants no longer collect. These results were confirmed by microscopic observation.

Electrically competitive cells of different mutants were prepared and extracted from plasmid pCP20 (extracted from S. pneumoniae χ3730, R. Curtiss, III, SM Kelly, PA Gulig, CR. Gentry-Weeks and JE to remove antibiotic resistance genes). Galan.Avirulent Salmonella expressing virulence antigens from other pathogens for use as oraly-administered vaccines.In: J. Roth, Editor, Virulence Mechanisms, American Society for Microbiology, Washington DC (1988), p. 311). By transformation. The transformants were incubated at 43 ° C. This will remove the temperature sensitive pCP20 plasmid and should remove the antibiotic resistance gene. Loss of the antibiotic resistance gene was confirmed in S. mutipus and S. enteritis ΔfliC :: catFRT mutants.

Deletion mutants originating from chloramphenicol resistance substitution mutants were identified by PCR using the primer combination FliC1 / FliC2. For both S. mucinus ΔfliC and S. enteritis ΔfliC, a fragment of 185 bp was amplified.

Deletion was confirmed by sequencing fragments amplified using primers FliC3.

The resulting mutants were tested on LB medium containing 0,4% agar: wild type S. murine typhoid and wild type S. enterobacteria were clustered on this medium, and also S. rat typhoid ΔfliC was collected (fljB flagella). The gene still exists). As expected, S. enterococci ΔfliC do not collect.

Δ fljBA  Mutants ( SM48 )of Construction

S. mutipus contains the second flagellin gene, fljB. This gene is expressed with fljA, which encodes for an inhibitor of fliC. In this case, both fljA and fljB were deleted. FljB is 15Obp long and is encoded for the protein flagellin. FljA is 539 bp in length and encoded for an inhibitor of fliC. Total length of deleted fragments (fljBA): 2127 bp.

Primers were designed that showed homology with the sequences of the fljBA gene and 51 nucleotide homology and the sequences of the template plasmid, and flanked the antibiotic resistance gene and the FRT sites. Primer FljBAP1 shows homology with the sequence starting from the start codon of fljB up to 51 bp downstream (1-51), and primer F1JBAP2 shows homology with the sequence starting from the stop codon of fljA and up to 51 bp upstream (2076-2127). see. Primers FljBAP1 and F1JBAP2 show homology at their 3 ′ ends with priming sites Pl and P2 in the template plasmid flanking the resistance gene by FRT sites.

PCR using primers FljBAP1 and F1JBAP2 (sequences in Table 1) and template DNA pKD3 (catFRT) or pKD4 (kanFRT) amplified fragments to the desired length.

1 μg of PCR product was electroporated with S. murine typhoid transformed with pKD46 or pKD20. Selected karamycin and chloramphenicol resistant transformants were identified by PCR.

Mutants were tested on containing LB medium containing 0,4% agar. Wild-type S. mutipus, S. mutipus ΔfljBA :: kanFRT and S. mutipus ΔfljBA :: catFRT were assembled (fliC is still present). The motility of the three strains was confirmed by microscopic observation.

The electrocompetitive cells of the different mutants were electroporated by pCP20 plasmid (derived from S. pneumoniae LT2 restriction mutant χ3730) to remove the antibiotic resistance gene. After incubation at 28 ° C. for 2 hours, the cultures were plated on LB medium with carbenicillin. After incubation of the transformants at 43 ° C. on LB, they were tested for loss of plasmid and antibiotic resistance genes. Deleted mutations were identified by PCR and sequencing of the fragments.

PCR using the primer combination FljBA6 / FljBA5 (sequences in Table 1) amplified a fragment of 2112 bp for wild type S. murine typhoid and a fragment of 185 bp for S. mutipus ΔfljBA mutant SM48.

Deletion in mutant SM48 was confirmed by sequencing using primer FljBA6 on PCR fragments obtained using primers FljBA6-F1jBA5 (FIG. 7).

S. Fungus  1491 S96  Δ fljBA Δ fliC  Double mutants ( SM23 )of Construction

Strain S. mutipus ΔfljBA :: kanFRT (pKD46) was used to construct the double mutants. The electrocompetitive cells were prepared at a temperature of 28 ° C. (temperature sensitive plasmid pKD46). The electrocompetitive cells were electroporated by recombinant fliC fragments, where the fliC gene was replaced with a chloramphenicol resistance gene (see early examples). To select and identify candidate mutants, the procedure used for the construction of fliC mutants was followed. Desired genotype: S. bacterium ΔfljBA :: kanFRT ΔfliC :: catFRT. To remove the antibiotic resistance gene, the protocol already described was followed. Deletion in S. mutipus ΔfljBA ΔfliC (SM23) was confirmed by PCR.

The double mutant S. mutipus ΔfljBA ΔfliC (SM23) did not move on LB medium with 0,4% agar as expected. Non-mobility of the strain was confirmed by microscopic observation.

nutrition Demand  And combination of flagella mutations

P22-trait introduction (Davis, RW, Botstein D. and Roth, JR (1980) In Advanced Bacterial Genetics, A manual for genetic engineering.Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) was used to combine the mutations. . P22-lysates of substitutional mutants were used to construct combined deletion mutants. Transduction was confirmed by PCR (same protocol and primers were used as in previous confirmations). In order to eliminate antibiotic resistance genes, an already disclosed protocol using the pCP20 helper plasmid was used. Deletion was confirmed by PCR. The mutants constructed in this manner are as follows: S. rat Typhoid bacterium AfliC ΔguaB (SM32), S. rattypus ΔfljBA ΔguaB (SM35), S. rat typhoid ΔfliC ΔfljBA ΔguaB (SM27) and S. enteritis ΔfliC ΔguaB (SM21).

Of homologous gene deletion mutants Construction

The possibility that additional unknown mutations (which may affect attenuation of strains) are present in candidate vaccine strains, dedicated to the use of the method disclosed by Datsenko and Wanner (2000) for the construction of deletion mutants. To rule out, homologous gene deletion mutants were constructed. Mutations were transduced against wild-type backgrounds by P22 phage transduction (Davis, RW, Botstein D. and Roth, JR (1980) In Advanced Bacterial Genetics, A manual for genetic engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Antibiotic resistance substitution mutants were used as donor strains. For the confirmation of removal and deletion of antibiotic resistance genes, the same protocols were used as in previous experiments.

Constructed Mutants: S. Enterococci ΔguaB (SM69, with Accession No. LMG P-21641); S. enterococci ΔfliC (SM71); S. mucinus ΔguaB (SM86, has Accession No. LMG P-21646); S. bacillus ΔfliC (SM91); S. murine bacterium ΔfljBA (SM90), S. enterococci ΔguaB ΔfliC (SM73, with Accession No. LMG P-21642); S. strain Typhoid ΔguaB ΔfliC (SM10); S. bacterium ΔguaB ΔfljBA (SM87); S. mutiform ΔfljBA ΔfliC (SM83); S. murine bacterium ΔguaB ΔfljBA ΔfliC (SM89, has Accession No. LMG P-21643).

Example  4: Virulence and Protective Experiments with S. Enterococcal Vaccine Strains

About the virulence of S. enterovirus vaccine strains fliC  Effect of Gene Inactivation

To study the effect of inactivation of the fliC gene on immunogenicity of S. enterovirus vaccine strains, two independent virulence and protection tests were performed at 7 week old females with both mutants SM20 (ΔguaB) and SM21 (ΔguaB ΔfliC). It was performed in BALB / c mice (Tables 4 and 5).

For virulence analysis, mice were orally infected at a dose of about 10 ⁸ CFU, which corresponds to 10 ⁵ times LD ₅₀ of wild type strains (Pattery et al., 1999, Molecular Microbiology 33: 791-805). Mice were observed for 21 days. All mice inoculated with wild type S. enterococci strain 76Sa88 died within 9 days of infection, while uninfected control mice survived healthy for a 21 day observation period. In the first experiments, mice infected with S. enterococci ΔguaB mutant SM20 showed typical disease symptoms (reduced activity, disturbed hair and bent, etc.) and one in 10 died. No disease symptoms were observed by SM20 in the second experiment. The S. enterococci ΔguaB ΔfliC mutant SM21 was asymptomatic in both experiments.

Mutants to be protected SM20  And SM21 Efficacy: Protective Trials

The efficacy of the mutants SM20 and SM21 to be protected was tested 3 weeks after initial immunization by oral challenge with wild type S. enteritis strain 76Sa88 of about 10 ⁵ LD ₅₀ (LD50 = 10 ³ CFU). Mice were observed for 21 days. All mice not immunized died after challenge. In the second experiment, one of three mice vaccinated with SM20 died. All other vaccinated mice survived the challenge with no observable disease symptoms. These data show that all mutants are attenuated and applied to protection against the challenge by the corresponding wild type strain.

There are no additional unknown mutations, and mutations containing selective resistance genes have been transferred to the wild-type background by P22 transduction to confirm that they may contribute to attenuation of candidate vaccine strains (Davis , RW, Botstein D. and Roth, JR (1980) In Advanced Bacterial Genetics, A manual for genetic engineering.Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

S. On virulence of enterococci fliC  Efficacy of gene inactivation: virulence and protection tests by isogene strains

Virulence and protection tests in BALB / c mice were repeated by homogeneous strains SM69 (ΔguaB), SM71 (ΔfliC) and SM73 (ΔguaB ΔfliC) after confirmation by PCR and phenotypic characterization as previously described. In this virulence assay, Δflic mutants were included to study the effect of inactivation of the fliC gene on virulence of S. enteritis. Conditions similar to those described above were used for virulence and challenge experiments. Data obtained for the transfectants SM69 and SM73 (Tables 6 and 7) confirmed the observations made in the initial experiments.

The comparison between all guaB deletion mutants showed that the S. enterococci ΔguaB ΔfliC double mutant SM73 was more attenuated and better protected against the challenge by larger doses of wild-type strain than the S. enterococci ΔguaB single mutant SM69. Suggests providing. Virulence analysis performed by S. enteritis ΔfliC mutant SM71 shows that such mutants remain virulent as wild-type strains under the conditions applied.

Immunological Responses and Antibody Production

54 days after the initial immunization in the first experiment, blood samples were collected from the tail arteries of mice. Anti-lipopolysaccharide (LPS) IgG titers were determined by enzyme-linked immunosorbent assay (ELISA) using S. enteritis LPS (Sigma) for coating. Comparison between the sera of mice immunized with SM20 and SM21 showed that in both cases anti-LPS serum IgG responses were derived and no significant difference in titer was measured. Oral immunization with the second and third doses did not increase anti-LPS IgG levels in serum at 66 and 95 days after initial immunization (data not shown).

Example  5: Fungus  characteristics In the introduction  Virulence and protection experiments

Virulence and protection experiments were performed by transfectants in 7 week old female BALB / c mice. Mice were orally inoculated with about 10 ⁸ cells. Mice were observed daily for a period of 21 days. After this period, mice were challenged with 10 ⁸ cells of wild type pathogenic strains and mice were observed over a 21 day period.

Disease symptoms and survival rates are noted in Tables 10-13. Single and double flagella mutants were left with great virulence when administered orally, and all mice died. Mice inoculated with the guaB mutant showed mild symptoms of the disease (the mice fought and only two survived). The combined ΔguaB ΔfljBA, ΔguaB ΔfliC and ΔguaB ΔfliC ΔfljBA mutants were greatly attenuated. No disease symptoms were observed for 21 days after vaccination. These mutants provided good protection after challenge with large doses of wild type strains. Only reduced activity of mice can be observed after challenge.

These results show that flagellar mutations do not affect the immunogenic dose of the strain when administered to BALB / c mice. Flagellar mutations may be useful as serological markers to distinguish between vaccine strains and wild type strains. The combination of nutritionally demanded mutations and flagellar mutation (s) provides the best results with regard to reduced virulence of mutants in mice and protection against the corresponding wild type strain.

Attenuation of the S. murine ΔguaB ΔfliC ΔfljBA triple deletion mutant and the S. murine bacteria double deletion mutants, ΔguaB ΔfljBA and ΔguaB ΔfliC, was similar.

Example  6: Evaluation of stability of S. enterovirus vaccine strains

Intratracheal  Or in chicks inoculated at day 1 by the oral gavage route SM69 Stability evaluation

The purpose of this study was to evaluate the stability of S. enteritis ΔguaB mutant strain SM69 master seed in 1 day old chickens. Mortality was used as the main parameter for determining stability.

One day old chicks were bent with legs and placed randomly in each of the four treatment groups (Group 1: SM69-IT, Group 2: SM69-OG, Group 3: PBS-IT and Group 4: PBS-OG). After master seed inoculation, chickens in groups 1 and 2 were placed in one isolator and chickens in groups 3 and 4 were placed in another isolator.

Chickens in groups 1 and 2 were inoculated with SM69 master seed by actual titer of 1.3 × 10 ⁸ CFU / 0.2 ml per bird, either by the endotracheal (IT) route or the oral gavage (OG) route, respectively. Chickens in groups 3 and 4 received 0.2 ml of PBS (phosphate buffered saline) per bird by intratracheal or oral gavage, respectively.

After inoculation with SM69 or PBS, chick mortality was observed daily up to 38 days post inoculation. Table 8 summarizes the results of mortality rates for all four groups. In group 1, one died during the inoculation due to inoculation trauma. Two died on day 2 after inoculation (DPI). Three died on days 3 to 13 (at 3, 5 and 13 DPI, respectively). Thus, all six died in group 1. In group 2, two died overall. One died from inoculation trauma and one died five days after inoculation. No chickens died in the PBS treated groups by the tracheal or oral gavage route.

This study suggests that the S. enterococci ΔguaB mutant strain SM69 is not safe when administered at 1.3 × 10 ⁸ CFU per day of chick by the intratracheal or oral gavage route.

Intratracheal  Or in chicks inoculated at 2 weeks of age by the oral gavage route SM69 Stability evaluation

Subsequently, the stability of the S. enterococci ΔguaB mutant strain SM69 was evaluated in 2 week-old SPF chickens by intratracheal and oral gavage route. Mortality was the main criterion for determining stability, and body weight was used as the second criterion.

Two week old chicks were bent with legs and placed randomly in each of the four treatment groups: SM69-IT, SM69-OG, Poulvac ST-IT and PBS-IT. Ten chickens in group 1 were inoculated with SM69 by the intratracheal route; Ten chickens in group 2 were inoculated with SM69 by oral gavage; Ten chickens in group 3 were inoculated with Salmonella bactypus AroA ^- vaccine (Poulvac® ST) by the endotracheal route; Five chickens in group 4 were inoculated with PBS by the intratracheal route.

Chickens in groups 1 and 2 were inoculated by SM69 master seed with an actual titer of 2.3 × 10 ⁸ CFU / 0.2 ml per animal, either by the intratracheal or oral gavage route. Chickens in group 3 received 2.2 × 10 ⁸ CFU / 0.2 ml of Poulvac® ST per animal by the intratracheal route. Chickens in group 4 received 0.2 ml of PBS per animal by the intratracheal route.

After inoculation, chickens in groups 1 and 2 were placed in one isolator and chickens in groups 3 and 4 were placed in another isolator.

After inoculation, mortality was observed daily up to 21 days after inoculation. Body weights of all chickens were also recorded at the end of the study period (21 days). Poulvac® ST and PBS were used as endotracheal procedure controls.

During the 21 day observation period, one of the SM69 endotracheal treatment groups (Group 1) died of infected yolk sac. No mortality was associated with SM69 inoculation and suggests stability at titers tested at 2.3 × 10 ⁸ CFU per animal by intratracheal and oral gavage route. As expected, no deaths were observed in Poulvac® ST treated chickens or PBS treated chickens with titers of 2.2 × 10 ⁸ CFU per animal, suggesting that this study is valid (Table 9).

Body weights were compared among the groups under analysis of the ANOVA model of body weights as treatment and dependent variables included as independent variables. Group comparisons were made using the Tukey test for multiple comparisons. The effective level was set at p <0.05. This study was considered valid because control chickens (PBS control group) remained healthy and there were no clinical signs or mortality of the disease throughout the study.

There were no significant differences in final body weights in chickens receiving SM69, Poulvac® ST, or PBS (Table 9) by intratracheal or oral gavage vaccination. Although no baseline for 1 day old chickens in each group has been established, there is little chance that there will be a significant difference in initial weight among the 4 groups because the chickens were randomly placed in each of the 4 treatment groups.

From this experiment it can be concluded that SM69 is safe when administered at tested titers of 2.3 × 10 ⁸ CFU per 2 weeks old chick by the tracheal or oral gavage route.

Intratracheal  Or in chicks inoculated at day 1 by the oral gavage route SM73 Stability evaluation

The stability of the S. enterobacterial deletion mutant strain SM73 (ΔguaB ΔfliC) was evaluated in chickens by intratracheal and oral gavage route. Mortality was the main criterion for determining stability, and body weight was used as the second criterion.

All chickens were curved and randomly assigned to one of four groups of chickens included in this study (Group 1: SM73-IT, Group 2: SM73-OG, Group 3: Poulvac ST-IT and Group). 4: PBS-IT). Ten of group 1 were inoculated by the endotracheal route with SM73; Ten of group 2 were inoculated by oral gavage with SM73; Ten of group 3 were inoculated by the intratracheal route with S. mutiform AroA ^- vaccine (Poulvac® ST); Five of group 4 were inoculated by the endotracheal route with PBS. A total of four isolators were used (one in each group), where chickens were housed during the study.

Chickens were inoculated at 1 day of age. Chickens in groups 1 and 2 were inoculated with SM73 master seeds by an intratracheal or oral gavage route with an actual titer of 2.5 × 10 ⁷ CFU / 0.2 ml per animal. Chickens in Group 3 received Poulvac® ST by the endotracheal route at 2.1 x 10 ⁷ CFU / 0.2 ml per animal. Chickens in group 4 received 0.2 ml of PBS per animal by the intratracheal route.

Mortality was observed and body weight was recorded for SM69 as described above. Poulvac® ST and PBS were once again used as endotracheal procedure controls.

During the 21 day observation period, no mortality was recorded for any chicken. There was no further difference in the final body weight of chickens inoculated with PBS-IT, Poulvac ST-IT and SM73-OG. The mean weight of the SM73-IT groups was significantly lower compared to the SM73-OG group (p = 0.009), but this is most likely due to experimental error.

From this experiment it can be concluded that SM73 is safe when administered at tested titers of 2.5 × 10 ⁷ CFU per day of chick by the tracheal or oral gavage route.

Deposits were made at BCCM / LMG Culture Collection, Laboratorium voor Microbiologie, KL Ledeganekstraat 35, B-9000 Gent (Belgium) for the following microorganisms in accordance with the Budapest Treaty: Salmonella enteritis B. SM69, accession no. LMG P-21641 (deposit date: 9 August 2002); S. Enterococci SM73, Accession No. LMG P-21642 (Deposit: August 9, 2002), S. Rattypus SM86, Accession No. LMG P-21646 (Deposit: August 28, 2002) and S. Rat Typhoid SM89, Accession No. LMG P-21643 (Deposit: August 9, 2002). Deposits are given by J.-P. Professor Hernalsteens (formerly Vrije Universiteit Brussel, Laboratorium Genetische Virologie, Paardenstraat 65, B-1640 Sint-Geneius-Rhode, current address: Vrije Universiteit Brussel, Onderzoeksgroep Genetische Virologie, Pleinlaan 2, B-1050 Brussels, Belgium) lost.

Table 1: Primer Sequences

SEQ ID number primer order One GuaB2 5 'CGTTCAGGCG CAACAGGCCG TTGT 3' 2 GuaB3 5 'GGCTGCGATT GGCGAGGTAG TA 3' 3 GuaB4 5 'GGTGATCCCG GGCGTCAAAC GTCAGGGCTT CTTTA 3' 4 GuaB5 5 'TTGACGCCCG GGATCACCAA AGAGTCCCCG AACTA 3' 5 GuaB6 5 'GCAACAACTC CTGCTGGTTA 3' 6 GuaB7 5 'AGACCGAGGA TCACTTTATC 3' 7 GuaB10 5 'AGGAAGTTTG AGAGGATAA 3' 8 P1 5 'GTGTAGGCTG GAGCTGCTTC 3' 9 P2 5 'CATATGAATA TCCTCCTTAG 3' 10 FIiCPl 5 'ATGGCACAAG TCATTAATAC AAACAGCCTG TCGCTGGTTG ACCCAGAATA ATGTGTAGGC TGGAGCTGCT TC 3' 11 FIiCP2 5 'CGCATTAACG CAGTAAAGAG AGGACGTTTT GCGGAACCTG GTTMGCCTGC GCCACATATG AATATCCTCC TTAG 3' 12 FliCl 5 'ATGGCACAAG TCATTAATAC AAACAG 3' 13 FliC2 5 'CGCATTAACG CAGTAAAGAG AGGAC 3' 14 FliC3 5 'TATCGGCAAT CTGGAGGCAA 3' 15 FIjBAPl 5 'ATGGCACAAG TAATCAACAC TAACAGTCTG TCGCTGCTGA CCCAGAATAA CTGTGTAGGC TGGAGCTGCT TC 3' 16 F1JBAP2 5 'TTATTCAGCG TAGTCCGAAG ACGTGATCCT GCTCACCCAG TCAAACATAA CCATATGAAT ATCCTCCTTA G 3' 17 FIjBA5 5 'CAGCGTAGTC CGAAGACGTG ATC 3' 18 FLjBA6 5 'ACACTAACAG TCTGTCGCTG CT 3'

Table 2: Virulence Tests of S. Enterococci ΔguaB Mutant SM20 in BALB / c Mice

infection Survival rate Date of death The condition of mice Strain dosage 1st experiment Negative Control: Milk Of 11/11 Of No disease symptoms Positive control: S. enteritis 76Sa88 2.1 x ^{10 8} 0/5 7,7,8, 8,9 Vaccine Control: S. Enterococci ΔaroA SM50 2.5 x 10 ⁸ 10/10 Of No disease symptoms S. enterococci ΔguaB SM20 5.1 x ^{10 8} 9/10 13 Disease symptoms between 7 and 14 days after infection 2nd experiment Negative Control: Milk Of 4/4 Of No disease symptoms Positive control: S. enteritis 76Sa88 1.4 x ^{10 8} 0/3 8,9,9 Vaccine Control: S. Enterococci ΔaroA SM50 2.1 x ^{10 8} 3/3 Of No disease symptoms S. enterococci ΔguaB SM20 1.9 x ^{10 8} 3/3 Of No disease symptoms

 Table 3: Challenges of mice vaccinated with S. enterococci guaB mutant SM20

Vaccination challenge Survival rate Date of death Mouse Status Strain dosage Strain dosage 1st experiment Negative Control: Milk Of Negative Control: Milk Of 6/6 Of No disease symptoms Negative Control: Milk Of S. Enterococci 76Sa88 1.5 x ^{10 8} 0/5 7,8,8, 8,9 Disease symptoms starting 5 days after challenge Vaccine Control: S. Enterococci ΔaroA SM50 2.5 x 10 ⁸ S. Enterococci 76Sa88 1.5 x ^{10 8} 3/5 9,13 Disease symptoms between 7 and 14 days after challenge S. enterococci ΔguaB SM20 5.1 x ^{10 8} S. Enterococci 76Sa88 1.5 x ^{10 8} 5/5 Of Mice rarely become active between 11 and 14 days after challenge

Table 3 continued:

Vaccination challenge Survival rate Date of death Mouse Status Strain dosage Strain dosage 2nd experiment Negative Control: Milk Of Negative Control: Milk Of 2/2 Of No disease symptoms Negative Control: Milk Of S. Enterococci 76Sa88 1.5 x ^{10 8} 0/2 9,18 Vaccine Control: S. Enterococci ΔaroA SM50 2.1 x ^{10 8} S. Enterococci 76Sa88 1.5 x ^{10 8} 1/3 9,21 Disease symptoms between 7 and 21 days after challenge S. enterococci ΔguaB SM20 1.9 x ^{10 8} S. Enterococci 76Sa88 1.5 x ^{10 8} 2/3 10 Disease symptoms starting 9 days after challenge

Table 4: Virulence Tests of S. Enteritis Mutants SM20 and SM21 in BALB / c Mice

infection Survival rate Date of death Mouse Status Strain dosage 1st experiment Negative Control: Not Infected Of 11/11 Of Asymptomatic Positive control: S. enteritis 76Sa88 2.1 x ^{10 8} 0/5 7,7,8, 8,9 Serious symptoms from day 5 S. enterococci ΔguaB SM20 5.1 x ^{10 8} 9/10 13 Mild to severe symptoms from day 7 to day 17 S. Enterococci ΔguaB ΔfliC SM21 4.3 x ^{10 8} 10/10 Of Asymptomatic 2nd experiment Negative Control: Not Infected Of 4/4 Of Asymptomatic Positive control: S. enteritis 76Sa88 1.4 x ^{10 8} 0/3 8,9,9 Serious symptoms from day 6 S. enterococci ΔguaB SM20 1.9 x ^{10 8} 3/3 Of Asymptomatic S. Enterococci ΔguaB ΔfliC SM21 3.2 x ^{10 8} 3/3 Of Asymptomatic

Table 5: Challenges of mice vaccinated with S. enterococci mutants SM20 and SM21

Vaccination challenge Survival rate Date of death Mouse Status Strain dosage Strain dosage 1st experiment Negative Control: Milk Of S. enteritis wild type strain 76Sa88 1.6 x ^{10 8} 0/5 7,8,8, 8,9 Severe symptoms starting on day 6 S. enteritis wild type strain 76Sa88 2.1 x ^{10 8} S. enteritis wild type strain 76Sa88 1.6 x ^{10 8} Of Of Of S. enterococci ΔguaB SM20 5.1 x ^{10 8} S. enteritis wild type strain 76Sa88 1.6 x ^{10 8} 4/4 Of Asymptomatic S. Enterococci ΔguaB ΔfliC SM21 4.3 x ^{10 8} S. enteritis wild type strain 76Sa88 1.6 x ^{10 8} 5/5 Of Asymptomatic

Table 5: continued

Vaccination challenge Survival rate Date of death Mouse Status Strain dosage Strain dosage 2nd experiment Negative Control: Milk Of S. enteritis wild type strain 76Sa88 1.5 x ^{10 8} 0/2 9,18 Severe symptoms starting on day 6 S. enteritis wild type strain 76Sa88 1.4 x ^{10 8} S. enteritis wild type strain 76Sa88 1.5 x ^{10 8} Of Of Of S. enterococci ΔguaB SM20 1.9 x ^{10 8} S. enteritis wild type strain 76Sa88 1.5 x ^{10 8} 2/3 10 No symptoms except one day with severe symptoms S. Enterococci ΔguaB ΔfliC SM21 3.2 x ^{10 8} S. enteritis wild type strain 76Sa88 1.5 x ^{10 8} 3/3 Of Asymptomatic

Table 6: Virulence Testing of S. Enterococci Mutants SM71, SM73 and SM69 in BALB / c Mice

infection Survival rate death rate Mouse Status Strain dosage Negative Control: Milk Of 4/4 Of Asymptomatic Positive control: S. enteritis 76Sa88 3.7 x ^{10 8} 0/3 7,8,9 Serious symptoms that started on day 5 S. enteritis ΔfliC SM71 1.4 x ^{10 8} 0/3 6,8,8 Serious symptoms that started on day 4 S. enterococci ΔguaB SM69 7.6 x ^{10 8} 5/5 Of Mild symptoms from 11 to 18 days S. Enterococci ΔguaB ΔfliC SM73 1.2 x ^{10 8} 5/5 Of Reduced activity from day 11 to day 13

Table 7: Challenges of mice vaccinated with S. enterococci mutants SM71, SM73 and SM69

Vaccination challenge Survival rate Date of death Mouse Status Strain dosage Strain dosage Negative Control: Milk Of S. enteritis wild type strain 76Sa88 3.1 x ^{10 8} 0/4 8,8,8, 9 Severe symptoms starting on day 5 S. enteritis wild type strain 76Sa88 3.7 x ^{10 8} S. enteritis wild type strain 76Sa88 3.1 x ^{10 8} Of Of Of S. enteritis ΔfliC SM71 1.4 x ^{10 8} S. enteritis wild type strain 76Sa88 3.1 x ^{10 8} Of Of Of S. enterococci ΔguaB SM69 7.6 x ^{10 8} S. enteritis wild type strain 76Sa88 3.1 x ^{10 8} 2/5 8,8,19 Severe symptoms starting on day 5 S. Enterococci ΔguaB ΔfliC SM73 1.2 x ^{10 8} 5/5 Of Asymptomatic

Table 8: Stability assessment of Salmonella mutant SM69 in 1 day old chickens

infection group N Titer Route Survival rate Date of Death (DPI) Strain S. Enterococci SM69 1: SM69-IT 10 1.3xlO ⁸ CFU / 0.2 ml Intratracheal 4/10 * 0,2,3 5,13 S. Enterococci SM69 2: SM69-OG 10 1.3xlO ⁸ CFU / 0.2 ml Oral Gastrointestinal Nutrition 8/10 ** 0,5 Negative Control: PBS 3: PBS-IT 10 PBS-0.2 ml Intratracheal 10/10 - Negative Control: PBS 4: PBS-OG 10 PBS-0.2 ml Oral Gastrointestinal Nutrition 10/10 -

In IT

OG Oral Gastrointestinal Nutrition

Days after DPI inoculation

In group 1, one died during the inoculation; 1 died on DPI 3, 5 and 13 and 2 died on DPI 2 respectively

** In group 2, one died during the inoculation; 1 died on DPI 5

Table 9: Stability Assessment of Salmonella Mutant SM69 in Two-Week Chickens

infection group N Titer Route Survival rate Day of Death Average (DPI) Weight Standard weight Strain S. Enterococci SM69 1: SM69-IT 10 2.3xlO ⁸ CFU / 0.2 ml IT 9/10 * 13 0.429 0.064 S. Enterococci SM69 2: SM69-OG 10 2.3xlO ⁸ CFU / 0.2 ml OG 10/10 - 0.420 0.044 Vaccine Control: Poulvac® ST * 3: Poulvac-IT 10 2.2xlO ⁸ CFU / 0.2 ml IT 10/10 - 0.423 0.046 Negative Control: PBS 4: PBS-IT 5 PBS-0.2 mL OG 5/5 - 0.388 0.019

In IT

OG Oral Gastrointestinal Nutrition

Days after DPI inoculation

Death due to yolk sac infection

** S. rat Typhoid AroA ^against live S. typhoid ^- live vaccine

Table 10: Virulence experiments with S. murine typhoid mutant strains in BALB / c mice. Oral inoculation with approximately 10 ⁸ cells of S. mutiform strain 1491S96

infection Strains (S. bacterus 1491S96) Survival Rate (Day Date) Mouse Status Wild type SM2 0/3 (9,9,10) Disease symptoms starting on day 4 ΔfliC SM91 0/5 (9,10,11,15,15) Disease symptoms starting on day 5 ΔfljBA SM90 0/5 (8,10,10,11,34) Serious illness symptoms starting on day 7 ΔflJBA ΔfliC SM83 0/5 (8,9,12,14,15) Serious illness symptoms starting on day 7 ΔguaB SM86 2/5 * (2,2,2) Minor disease symptoms from 9th to challenge date ΔguaB ΔfljBA SM87 5/5 No symptoms ΔguaB ΔfliC ΔfljBA SM89 5/5 No symptoms Control (not infected) 4/4 No symptoms

* Died after fighting

Table 11: Challenge experiment with S. murine typhoid mutant strain in BALB / c mice. Oral Vaccination with Approximately 10 ⁸ Cells of S. mutiform Strain 1491S96

Vaccination challenge Strains (S. bacterus 1491S96) Strains (S. bacterus 1491S96) Dose (CFU) Survival Rate (Day Date) Mouse Status ΔguaB SM86 S. bacillus 1491S96 1.3 x 10 ⁷ 2/2 Severe symptoms up to 14 days later, one mouse showed obvious symptoms and the other became healthy again ΔguaB ΔfljBA SM87 S. bacillus 1491S96 1.3 x 10 ⁷ 5/5 Reduced activity between 8 and 10 days ΔguaB ΔfliC ΔfljBA SM89 S. bacillus 1491S96 1.3 x 10 ⁷ 5/5 Reduced activity between 8 and 10 days Control (not infected) S. bacillus 1491S96 1.3 x 10 ⁷ 0/4 Severe symptoms beginning on day 6

Table 12: Virulence Experiments with S. mutiform 1491S96 Mutant Strains in BALB / c Mice

infection Strains (S. bacterus 1491S96) dosage Survival Rate (Day Date) Mouse Status Wild type SM2 0.8 x ^{10 8} 1/4 (11,13,14) Disease symptoms starting on day 6 ΔguaB SM86 0.8 x ^{10 8} 5/5 Mild symptoms at 13 and 14 days ΔguaB ΔfliC SM91 2.5 x 10 ⁸ 5/5 No symptoms ΔguaB ΔfljBA SM87 1.5 x ^{10 8} 5/5 No symptoms ΔguaB ΔfliC ΔfljBA SM89 1.7 x ^{10 8} 5/5 No symptoms Control (not infected) - 5/5 No symptoms

Table 13: Protection experiments with S. murine typhi 1491S96 mutant strains in BALB / c mice

Vaccination challenge Strains (S. bacterus 1491S96) dosage Strains (S. bacterus 1491S96) dosage Survival Rate (Day Date) Mouse Status ΔguaB SM86 0.8 x ^{10 8} S. bacillus 1491S96 2.7 x ^{10 8} 5/5 Reduced activity from 6 to 16 days ΔguaB ΔfliC SM91 2.5 x 10 ⁸ S. bacillus 1491S96 2.7 x ^{10 8} 5/5 Reduced activity starting at day 7; Weak symptoms from 8 to 14 days ΔguaB ΔfljBA SM87 1.5 x ^{10 8} S. bacillus 1491S96 2.7 x ^{10 8} 3/5 (10,28) Weak symptoms between 6 and 16 days ΔguaB ΔfliC ΔfljBA SM89 1.7 x ^{10 8} S. bacillus 1491S96 2.7 x ^{10 8} 4/5 (19) Weak symptoms between 8 and 16 days Control (not infected) - S. bacillus 1491S96 2.7 x ^{10 8} 0/5 (8,9,10,11,16) Severe symptoms starting on day 6

                         SEQUENCE LISTING <110> Fort Dodge Animal Health   <120> LIVE ATTENUATED SALMONELLA VACCINE <130> BP.FDAH.002A / EP <160> 24 <170> PatentIn version 3.3 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GuaB2 primer <400> 1 cgttcaggcg caacaggccg ttgt 24 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GuaB3 primer <400> 2 ggctgcgatt ggcgaggtag ta 22 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> GuaB4 primer <400> 3 ggtgatcccg ggcgtcaaac gtcagggctt cttta 35 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> GuaB5 primer <400> 4 ttgacgcccg ggatcaccaa agagtccccg aacta 35 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GuaB6 primer <400> 5 gcaacaactc ctgctggtta 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GuaB7 primer <400> 6 agaccgagga tcactttatc 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GuaB10 primer <400> 7 aggaagtttg agaggataa 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> P223 primer <400> 8 gtgtaggctg gagctgcttc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> P223 primer <400> 9 catatgaata tcctccttag 20 <210> 10 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> FliCP1 primer <400> 10 atggcacaag tcattaatac aaacagcctg tcgctggttg acccagaata atgtgtaggc 60 tggagctgct tc 72 <210> 11 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> FliCP2 primer <400> 11 cgcattaacg cagtaaagag aggacgtttt gcggaacctg gttmgcctgc gccacatatg 60 aatatcctcc ttag 74 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> FliC1 primer <400> 12 atggcacaag tcattaatac aaacag 26 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> FliC2 primer <400> 13 cgcattaacg cagtaaagag aggac 25 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FliC3 primer <400> 14 tatcggcaat ctggaggcaa 20 <210> 15 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> FljBAP1 primer <400> 15 atggcacaag taatcaacac taacagtctg tcgctgctga cccagaataa ctgtgtaggc 60 tggagctgct tc 72 <210> 16 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> FljBAP2 primer <400> 16 ttattcagcg tagtccgaag acgtgatcct gctcacccag tcaaacataa ccatatgaat 60 atcctcctta g 71 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> FljBA5 primer <400> 17 cagcgtagtc cgaagacgtg atc 23 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FLjBA6 primer <400> 18 acactaacag tctgtcgctg ct 22 <210> 19 <211> 2903 <212> DNA <213> Salmonella Enteritidis <220> <221> misc_feature <223> Contig 1294 of the S. Enteritidis genome <220> <221> misc_feature (222) (1581) .. (1583) N is a, c, g, or t <220> <221> misc_feature (222) (1657) .. (1657) N is a, c, g, or t <400> 19 ctgatcgaac aagccttcgg cctgcagttt ggcttttagc tgctcatatt tctgctgcaa 60 caagccttcg cccgcgggct gcatactttc ggcgatgatt tgataatcgc cgcgcggctc 120 gtacagcgta atgttggcgc gtaccagcac ctgctgcccg tgctgcgggc ggaacgtcac 180 ccggcgattg ctgttacgga acatcgcaca gcgcacctga gcggtatcgt ctttgagcgt 240 aaagtaccag tggcccgacg caggctgcgt gaaattagaa atctcgccgc tgatccatac 300 ctgtcccatc tcctgttcta acagcagacg aaccgtctgg ttaaggcggc ttacggtaaa 360 aattgaggaa gtttgagagg ataacatgtg agcgggatca aattctaaat cagcaggtta 420 ttcaatcgat agtaacctgc tcacggggga tcgcaagcac tatttgcaaa aaaatgtaga 480 tgcaaccgat tacgttctgt ataatgccgc gggaatattt attaacctcc caggcgagat 540 attgcccatg ctacgtatcg ctaaagaagc tctgacgttt gacgacgtcc tccttgttcc 600 cgctcactcc accgttttgc cgaatactgc cgatctcagc acgcagttga cgaaaactat 660 tcgtctgaat attcctatgc tttctgcggc gatggacacc gtgacggaag cgcgcctggc 720 aattgccctg gcccaggaag gcggcattgg ttttatccac aaaaacatgt ctattgagcg 780 ccaggcggaa gaagttcgcc gcgtgaagaa acacgagtcc ggcgtagtga ccgacccgca 840 gaccgtcctg ccaaccacca cgttgcatga agtgaaagcc ctgaccgagc gtaacggttt 900 tgcgggctat ccggtggtga ctgaagataa cgagctggtg ggtatcatca ccggtcgtga 960 cgtgcgtttt gtgactgacc tgaaccagcc ggtgagtgtc tacatgacac cgaaagagcg 1020 tctggtgacc gttcgtgaag gcgaagcccg tgaagtcgtg ctggcaaaaa tgcacgaaaa 1080 acgcgtagaa aaagcgctgg tcgttgatga taacttccat ctgcttggca tgattaccgt 1140 aaaagatttc cagaaagcgg aacgtaaacc aaactcctgt aaagatgagc agggccgttt 1200 acgtgtcggc gcggcggtcg gcgcaggcgc gggcaacgaa gagcgcgttg acgcgctggt 1260 ggcggcaggc gttgacgtac tgctgatcga ctcctctcac ggtcactctg aaggcgtgtt 1320 gcaacgtatc cgtgagacgc gtgctaaata tcctgacctg caaatcatcg gcgggaacgt 1380 tgcgacgggc gcaggcgctc gcgcactggc ggaagccggt tgcaacgcgg tgaaagtggg 1440 tatcggcccg ggttccatct gtacgactcg tatcgggact ggtgtgggcg ttccgcagat 1500 caccgctgtt tctgacgcgg tggaagcgct ggaaggcacc gggattccgg ttatcgctga 1560 cggcggtatc cgtttctccg nnnacatcgc caaagccatc gccgcaggcg cgagcgcggt 1620 aatggtgggt tctatgctgg ccggtaccga agaatcnccg ggcgaaatcg aactctacca 1680 gggccgttct tacaaatctt atcgcggtat gggttctctg ggcgcgatgt ccaaaggttc 1740 ctccgaccgt tacttccaga gcgacaacgc cgccgacaaa ctggtgccgg aaggtatcga 1800 aggccgcgta gcctataaag gtcgcctgaa agagatcatt caccagcaga tgggcggcct 1860 gcgctcctgt atggggctga ccggttgtgc taccatcgac gaactgcgta ctaaagcgga 1920 gtttgtgcgt atcagcggtg cgggtatcca ggaaagccac gttcacgacg tgaccatcac 1980 caaagagtcc ccgaactacc gtctgggctc ctgattttct tcgcccgacc ttcgcgtcgg 2040 gcgatttatt taatctgttt cacttgcctc ggaataagcg tcaatgacgg aaaacattca 2100 taagcatcgc atcctcattc tggacttcgg ttctcagtac actcaactgg ttgcgcgccg 2160 cgtgcgtgag ctgggtgttt actgtgaact gtgggcgtgg gatgtgacag aagcacaaat 2220 tcgtgacttc aacccaagcg gcattattct ttccggcggc ccggaaagca ccaccgaaga 2280 aaacagcccg cgcgcgccgc agtatgtctt tgaagcaggc gtgccggtat ttggcgtttg 2340 ctatggtatg cagaccatgg cgatgcagct tggcggtcat gtagaaggtt ctaatgagcg 2400 tgaatttggt tatgcgcagg tcgaagtgtt gaccgacagc gcgctggttc gcggtattga 2460 agattccctg accgcagacg gcaaaccgct gctggacgtg tggatgagcc acggcgataa 2520 agtgacggcg attccgtccg acttcgtgac cgtcgccagc accgagagct gcccgttcgc 2580 cattatggcc aacgaagaaa aacgcttcta cggcgtacag ttccacccgg aagtgactca 2640 cacccgccag ggtatgcgca tgctggagcg ttttgtacgc gatatctgcc agtgtgaagc 2700 gttgtggacg ccggcgaaga tcatcgatga cgccgtggcg cgcattcgcg agcaggtagg 2760 cgacgataaa gtgatcctcg gtctctccgg cggcgtggat tcttccgtca ccgccatgct 2820 gctgcaccgc gcgatcggta aaaatctgac ctgtgttttc gtcgacaacg gcctgttgcg 2880 cctgaacgaa gccgagcagg tga 2903 <210> 20 <211> 1995 <212> DNA <213> Artificial Sequence <220> <223> Sequence of the delta-guaB fragment of S. Enteritidis cloned in        pUC18 <220> <221> misc_feature <222> (55) .. (55) N is a, c, g, or t <400> 20 ggctgcgatt ggcgaggtag tagtccatcg ccatgcccag acgctgctgg cgganctgga 60 tctgacgcaa caactcctgc tggttacggc tgacaatttc tgccgctgct gatggcgtag 120 cgcgcgcagg tcggcgacaa aatcagctat cgtgacgtcc gtttcgtgac cgacggcgct 180 gaccaccgga atgcggctgg caaaaatcgc ccgcgccacg cgttcgtcgt taaaactcca 240 caaatcttcc agcgaaccgc cgccgcgccc gacgatcagt acatcacatt cgccgcgcgc 300 gttcgccagt tcgatagcac gaacgatctg ccccggcgcg tcgtcgccct ggactgcggt 360 tggatagata ataacgggca gggatgggtc acgacgcttt agcacgtgga gaatatcgtg 420 cagcgccgcg ccggttttcg aagtgatcac cccaacgcag tgggccgggg agggcaacgg 480 ctgtttatgc tgctgatcga acaagccttc ggcctgcagt ttggctttta gctgctcata 540 tttctgctgc aacaagcctt cgcccgcggg ctgcatactt tcggcgatga tttgataatc 600 gccgcgcggc tcgtacagcg taatgttggc gcgtaccagc acctgctgcc cgtgctgcgg 660 gcggaacgtc acccggcgat tgctgttacg gaacatcgca cagcgcacct gagcggtatc 720 gtctttgagc gtaaagtacc agtggcccga cgcaggctgc gtgaaattag aaatctcgcc 780 gctgatccat acctgtccca tctcctgttc taacagcaga cgaaccgtct ggttaaggcg 840 gcttacggta aaaattgagg aagtttgaga ggataacatg tgagcgggat caaattctaa 900 atcagcaggt tattcaatcg atagtaacct gctcacgggg gatcgcaagc actatttgca 960 aaaaaatgta gatgcaaccg attacgttct gtataatgcc gcggcaatat ttattaacct 1020 cccaggtgag atattgccca tgctacgtat cgctaaagaa gctctgacgt ttgacgcccg 1080 ggatcaccaa agagtccccg aactaccgtc tgggctcctg attttcttcg cccgaccttc 1140 gcgtcgggcg atttatttaa tctgtttcac ttgcctcgga ataagcgtca atgacggaaa 1200 acattcataa gcatcgcatc ctcattctgg acttcggttc tcagtacact caactggttg 1260 cgcgccgcgt gcgtgagctg ggtgtttact gtgaactgtg ggcgtgggat gtgacagaag 1320 cacaaattcg tgacttcaac ccaagcggca ttattctttc cggcggcccg gaaagcacca 1380 ccgaagaaaa cagcccgcgc gcgccgcagt atgtctttga agcaggcgtg ccggtatttg 1440 gcgtttgcta tggtatgcag accatggcga tgcagcttgg cggtcatgta gaaggttcta 1500 atgagcgtga atttggttat gcgcaggtcg aagtgttgac cgacagcgcg ctggttcgcg 1560 gtattgaaga ttccctgacc gcagacggca aaccgctgct ggacgtgtgg atgagccacg 1620 gcgataaagt gacggcgatt ccgtccgact tcgtgaccgt cgccagcacc gagagctgcc 1680 cgttcgccat tatggccaac gaagaaaaac gcttctacgg cgtacagttc cacccggaag 1740 tgactcacac ccgccagggt atgcgcatgc tggagcgttt tgtacgcgat atctgccagt 1800 gtgaagcgtt gtggacgccg gcgaagatca tcgatgacgc cgtggcgcgc attcgcgagc 1860 aggtaggcga cgataaagtg atcctcggtc tctccggcgg cgtggattct tccgtcaccg 1920 ccatgctgct gcaccgcgcg atcggtaaaa atctgacctg tgttttcgtc gacaacggcc 1980 tgttgcgcct gaacg 1995 <210> 21 <211> 349 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the S. Enteritidis PCR fragment, which        includes the guaB deletion, obtained using primer GuaB10 <400> 21 catgtgtgag cgggatcaaa ttctaaatca gcaggttatt caatcgatag taacctgctc 60 acgggggatc gcaagcacta tttgcaaaaa aaatgtagat gcaaccgatt acgttctgta 120 taatgcccgg caatatttat taacctccca ggtgagatat tcgccatgta cgtatcgcta 180 aagaagccct gacgtttgac gcccgtgtag gctggagctg cttcgaagtt cctatacttt 240 ctagagaata ggaactccgg aataggaact aaggaggata ttcatatggg atcaccaaag 300 atccccgaac taccgtctgg gctctgattt tcttcgcccg accttcgcg 349 <210> 22 <211> 1553 <212> DNA <213> Salmonella Typhimurium <220> <221> misc_feature <223> guaB gene of S. Typhimurium LT2, section 117 of 220 of the        complete genome <400> 22 atttgcaaaa aaatgtagat gcaaccgatt acgttctgta taatgccgcg gcaatattta 60 ttaacctccc aggtgagata ttgcccatgc tacgtatcgc taaagaagcc ctgacgtttg 120 acgacgtcct ccttgttccc gctcactcca ccgttttgcc gaatactgcc gatctcagca 180 cgcagttgac gaaaactatt cgtctgaata ttcctatgct ttctgcggcg atggacaccg 240 tgacggaagc gcgcctggca attgccctgg cccaggaagg cggcattggt tttatccaca 300 aaaacatgtc cattgagcgc caggcggaag aagttcgccg cgtgaagaaa cacgagtccg 360 gcgtagtgac cgacccgcag accgtcctgc caaccaccac gttgcatgaa gtgaaagccc 420 tgaccgagcg taacggtttt gcgggctatc cggtggtgac tgaagataac gagctggtgg 480 gtatcatcac cggtcgtgac gtgcgttttg tgactgacct gaaccagccg gttagcgttt 540 acatgacgcc gaaagagcgt ctggtgaccg ttcgtgaagg cgaagcccgt gaagtcgtgc 600 tggcaaaaat gcacgaaaaa cgcgtagaaa aagcgctggt cgttgatgat aacttccatc 660 tgcttggcat gattaccgta aaagatttcc agaaagcgga acgtaaacca aactcctgca 720 aagatgagca gggccgttta cgtgtcggcg cggcggtcgg cgcaggcgcg ggcaacgaag 780 agcgcgttga cgcgctggtg gcggcaggcg ttgacgtact gctgatcgac tcctctcacg 840 gtcattcaga aggcgtgttg caacgtatcc gtgaaacccg tgctaaatat cctgacctgc 900 aaatcatcgg cggcaacgtc gcgacaggcg caggcgctcg cgcactggcg gaagccggtt 960 gcagcgcggt gaaagtcggt attggcccgg gttccatctg taccactcgt atcgtgactg 1020 gcgtgggcgt tccgcagatc accgctgttt ctgacgcagt tgaagcgctg gaaggtaccg 1080 gtattccggt tatcgctgac ggcggtatcc gtttctccgg cgacatagcc aaagcgattg 1140 ccgcaggtgc aagcgcggta atggtgggtt ccatgctggc gggtacggaa gaatccccgg 1200 gcgaaatcga actctaccag ggccgttctt acaaatctta ccgcggcatg ggctcgctgg 1260 gtgcgatgtc caaaggttcc tccgaccgtt acttccagag cgacaacgcc gctgacaaac 1320 tggtgccgga aggtatcgaa ggtcgcgtag cctataaagg tcgcctgaaa gagatcattc 1380 accagcagat gggcggcctg cgctcctgta tggggctgac cggttgtgct accatcgacg 1440 aactgcgtac taaagcggag tttgtgcgta tcagcggtgc gggcattcag gaaagccacg 1500 ttcacgacgt gaccatcacc aaagagtccc cgaactaccg tctgggctcc tga 1553 <210> 23 <211> 193 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence obtained after sequencing the PCR fragment        amplified with primers FliC1-FliC2 on total DNA of the mutants        SM73 and SM89, using primers FliC1 and FliC2 <400> 23 ctatggcaca agtcattaat acaaacagcc tgtcgctggt tgacccagaa taatgtgtag 60 gctggagctg cttcgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact 120 aaggaggata ttcatatgtg gcgcaggcga accaggttcc gcaaaacgtc ctctctttac 180 tggcgttaat gcg 193 <210> 24 <211> 140 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence obtained after sequencing the PCR fragment        amplified with primers FljBA6-FljBA5 on total DNA of the S.        Typhimurium mutant SM48, using primer FljBA6 <400> 24 agaataactg tgtaggctgg agctgcttcg aagttcctat actttctaga gaataggaac 60 ttcggaatag gaactaagga ggatattcat atggttatgt ttgactgggt gagcaggatc 120 acgtcttcgg actacgctgt 140  

Claims (34)

The attenuated mutant contains at least one first genetic modification and at least one second genetic modification, wherein the first modification of one or more kinetic genes and the second modification of the one or more genes are associated with the survival or Attenuated mutant strains of bacteria that infect veterinary species, which are associated with proliferation. The mutant strain of claim 1, wherein said veterinary species are poultry. The mutant strain of any one of the preceding claims, which is a Salmonella enterica or E. coli strain. The mutant strain of claim 1, wherein the motility gene is a gene encoding flagellin. The mutant strain of claim 4, wherein the mutant strain is by mutation in the fliC and / or fljB or fljBA genes. The mutant strain according to claim 4 or 5, which can be collected on LB medium containing 0.4% agar. The mutant strain of claim 1, wherein the gene involved in survival is a house-keeping gene or a virulence gene. 8. The mutant strain of claim 7, wherein said house-keeping gene inactivated is a guaB gene. The mutant strain of claim 8, wherein said mutant strain contains deletion mutations that impair guaB gene function. The method according to claim 8 or 9, wherein the new ( de novo) a mutant strain that is unable to form guanine nucleotides. The mutant strain of claim 1, wherein the mutant strain encodes and expresses a foreign body antigen. The mutant strain according to any one of the preceding claims, which is an attenuated S. enterobacteria or S. rat Typhoid strain. The mutant strain of any one of the preceding claims, wherein said genetic modifications are introduced into a parent strain S. enteritis bacterium phage type 4 strain 76Sa88. The mutant strain according to claim 13, which is an attenuated S. enteritis strain SM73 having accession number LMG P-21642. The mutant strain according to any one of claims 1 to 12, wherein the genetic modifications are introduced into the parent strain S. murine typhi 1491S96. 16. The mutant strain according to claim 15, which is attenuated S. mutipus strain SM89 having accession number LMG P-21643. The mutant strain of claim 1, which contains a genetic modification in the guaB gene and a genetic modification in the fliC gene. The mutant strain of claim 1, which contains a genetic modification in the guaB gene and a genetic modification in the fljBA genes. The mutant strain of claim 1, which contains a genetic modification in the guaB gene, a genetic modification in the fliC gene, and a genetic modification in the fljBA genes. The mutant strain of claim 1, wherein the modification is selected from the group consisting of insertion, deletion, and / or substitution of one or more nucleotides in the genes. A mutant strain of any one of the above terms in a pharmaceutically effective or immunological amount; And a pharmaceutically acceptable carrier or diluent. A vaccine for immunizing veterinary species against bacterial infection. The vaccine of claim 21, wherein the mutant strain encodes and expresses a foreign body antigen. 23. The vaccine of claim 21 or 22 wherein the mutant strain comprises a plasmid that encodes and expresses foreign body genes in eukaryotic cells. 23. A bacterial infection comprising administering to a veterinary species in need of treatment a mutant strain according to any one of claims 1 to 20 and / or a vaccine according to claims 21 to 23. How to immunize veterinary species against. The method of claim 24, wherein the veterinary species are poultry. The method of claim 24 or 25, wherein the mutant strain is an attenuated strain of S. enterica or E. coli. 27. The method of any one of claims 24 to 26, wherein the mutant strain and / or vaccine is administered via oral, intranasal or parenteral routes. The mutant strain according to any one of claims 1 to 20, which is used for the manufacture of a vaccine for the prevention and / or treatment of veterinary species, preferably poultry infection. As a serological distinction between vaccinated animals and animals infected by wild type strains, the vaccinated animals are immunized with mutant strains in which flagellin genes have been inactivated. Analyzing the animals in the presence of increased antibodies against flagellin; And Distinguishing infected animals from vaccinated animals based on the presence or absence of said antibodies; Comprising a serological distinction. 30. The mutant strain of claim 29, wherein said antibodies are raised by an animal infected by a wild type strain, but, such as the mutant according to any one of claims 4-20, And not caused by the animal vaccinated. 31. The method of any one of claims 29-30, wherein the presence of the antibodies indicates the presence of wild-type strains and the resulting infection. 32. The method of any one of claims 29-31, wherein the animals infected by the Salmonella family are distinguished from animals immunized with the live attenuated vaccine according to any one of claims 21-23. 33. The method of any one of claims 29-32, wherein said animals have been analyzed for increased antibodies against FliC. 33. The method according to any one of claims 29 to 32, wherein said animal is a veterinary species, preferably poultry, more preferably chicken.

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