BRPI0621489A2 - attenuated mutant strain of veterinary infecting bacteria, vaccine for immunization of veterinary species against a bacterial infection, method for immunizing a veterinary species against a bacterial infection and use of an attenuated mutant strain - Google Patents

Abstract

ATTENUATED MUTANT CEPA OF INFECTING BACTERIA OF VETERINARY SPECIES, VACCINE FOR IMMUNIZATION OF VETERINARY SPECIES AGAINST BACTERIAL INFECTION, METHOD OF IMMUNIZING A VETERINARY SPECIES AGAINST A BUTTERFLY INFECTION. The present invention relates to a mutant with attenuated guaB deletion of a veterinary species infected by bacteria, more particularly, Salmoneila enterica, for use and production. The present invention also relates to a live attenuated vaccine based on said mutant for the prevention of bacterial infection, and more particularly Salmonellosis, in a veterinary species, particularly in poultry.

Description

"Attenuated bacterial mutant CEPA of VETERINARY SPECIES, VETERINARY SPECIES IMMUNIZATION VACCINE AGAINST BACTERIAL INFECTION METHOD FOR IMMUNIZING A VETERINARY SPECIES AGAINST BACTERIAL INFECTION AND A BACTERIAL INFECTION". Field of the invention

The present invention relates to an attenuated bacterial mutant, in particular an attenuated Salmonella enteric mutant, and a live attenuated vaccine comprising the same.

State of the art

Salmonellae is Gram negative, facultative anaerobic, mobile, non-lactose fermentative bacillus belonging to the enterobacteriaceae family. Salmonellae are usually transmitted to humans through the consumption of contaminated food and cause Salmonellosis. Salmonellae have been isolated from many animal species including, cows, chickens, turkey, sheep, pigs, dogs, cats, horses, donkeys, seals, lizards and snakes.

95% of the important Salmonella pathogens belong to Salmonella enterica, with S. enterica serotype Typhimurium (S. typhimurium) and S. enterica serotype Enteritidis (S. enteritidis) as the most common serotype. Salmonella infections are a serious medical and veterinary problem worldwide and a cause related to the food industry. Contaminated foods cannot be readily identified.

Control of Salmonellosis is important to prevent potentially life-threatening infections and significant economic losses to the agricultural industry. The ubiquitous presence of Salmonella in natura complicates disease control precisely by detecting and eradicating infected animals. Strategies for severe control based on principles of exclusion by competition and vaccination have been tested to control infection, for example from poultry.

Vaccination of farmed animals is often considered to be the most effective way to prevent Salmonella zoonoses.

Whole-cell killing and subunit vaccines are used to prevent Salmonella infections in animals and humans with varying results. Inactivated vaccines generally provide little protection against Salmonellosis.

Live attenuated Salmonella vaccines are potentially superior to inactivated preparations due to:

(i) its ability to induce cell-mediated immunity in addition to the antibody response;

(ii) oral release without risk of needle contamination;

(iii) efficacy after single dose administration;

(iv) induction of immune response at multiple mucosal sites;

(v) low cost of production; and

(vi) their possible use as a vehicle for the release of recombinant antigens to the immune system.

Few live attenuated Salmonella vaccines are currently on the market because results with attenuated mutant strains have not always been good. For example, poultry has been vaccinated with mutants -ar or -cAMP, yet many birds die quickly after vaccination. Because Salmonella infection often occurs very easily, vaccination of many young birds is crucial. However, at this stage they are very sensitive to Salmonella, possibly due to the immaturity of their immune system.

In addition to poor protection from vaccinated birds, prolonged excretion of vaccine strains was frequently observed.

Bird vaccination with the Megan Vacl strain, which causes deletion in the cya and crp genes (US Patent 5,389,368; US 5,855,879; US 5,855,880) results in a reduction in the number of birds from which a contaminating virulent Salmonella strain can be isolated. 7 days after contamination. However, it does not provide complete protection (http://www.meganhealth.com/meganvac.html). Thus, there is still a need for improvement of live attenuated Salmonella vaccine strains, and for an improved live attenuated vaccine strain of infectious bacteria of veterinary species in general. Information in the literature on the influence of guaB mutations on the virulence of Salmonella and other bacteria is scarce.

McFarland and Stocker (1987, "Microbial pathogenesis", 3: 129-141) reported reduced virulence of mutants with guaA and guaB Tn10 inserts of S. typhimurium and S. dublin in BALB / c mice. At high dosages (2.5x10 7 cfu for S. typhimurium and 10 4 cfu for S. dublin), however, these authors reported high mortality resulting from the multiplication of the auxotrophic strain. Wang et al., (2001, "Infection and Immunity", 69: 4734-4741) reported a preclinical screening with a typhoid vaccine in humans. The sheath of Wang et al. Comprises a Salmonella typhi AguaBA mutant. This attenuated strain, however, demonstrated significant residual virulence in mice.

International patent application WO 99/58146 and U.S. Patent 6,190,669 describes Salmonella typhi vaccine strains harboring a AguaBa mutation, which are used as a living vector to release exogenous antigens. Objectives of the invention

An object of the present invention is to provide attenuated enteric Salmonella strains.

Another object of the present invention is to provide a live attenuated Salmonellosis vaccine and prevention methods based thereon.

It is yet another object of the present invention to provide live attenuated strains which are useful as a live vector and as DNA-mediated vaccines expressing exogenous antigens. Said strains are highly suitable for vaccine development including polyvalent vaccines.

Still another object of this invention is to provide a method for achieving an S. enteral deletion mutation for use in a live attenuated vaccine.

Another further object of this invention is to provide the same material and method for the preparation of attenuated strains of veterinary infecting bacteria, particularly poultry.

The overall objective is to improve food safety and animal health. Summary of the Invention

A first aspect of the invention relates to an attenuated enteric mutant Salmonella strain which is unable to reshape guanine nucleotides, and wherein said mutation contains a deletion mutation in the guaB gene.

The principle as shown herein for S. enteral may be applied to any organism that may use the guanine nucleotide as an intermediate. One aspect of the invention therefore relates to an attenuated mutant strain of a veterinary species infecting bacterium that contains a deletion mutation in the guaB gene. The term "veterinary species infecting bacterium" in the context of the invention refers in particular to bacteria which are pathogenic to veterinary species, and which may be attenuated by a deletion mutation in the guaB gene. The infectious bacterium of veterinary species may be a gram-negative bacterium. Preferred are Gram-negative poultry bacteria such as Salmonella, Pasteurella, Escherichia coli, etc. The most preferred strains are those of enteric (pathogenic) Salmonella and E. coli. By "pathogenic a" it should be understood that the bacterium, if not attenuated, is capable of causing an infectious disease in veterinary species.

Mutants of the invention fail to express a functional guaB gene product. In other words, the function of the guaB gene is impaired / defective, whereby an attenuated auxotrophic strain is obtained.

The mutant strain of the invention, which carries a deletion mutation in the guaB gene, cannot grow in minimal medium A unless supplemented with 0.3 mM guanine, xanthine, guanosine or xanthosine.

The present invention in particular aims to provide attenuated strains of S. enteritidis and S. typhimurium. Preferably, the deletion mutation in the guaB gene is introduced into the strain of the S. enteritidis fact type 76Sa88 or into the parent strain of S typhimurium 149IS96.

One of the attenuated S. enterica strains obtained according to the invention is a S. enteritidis SM69 strain having deposit number LMG P-21641. Another example is the S. typhimurium SM86 strain having deposit number LMG P-21646.

The attenuated mutant strains of the invention are highly suitable for use in a live attenuated vaccine including a multivalent or multivalent vaccine. The attenuated mutant strain of the invention may encode and express an exogenous antigen and may as such be used as a live vector and / or as a DNA-mediated vaccine.

A second aspect of the invention relates to a pharmaceutical composition or vaccine for immunization of a veterinary species against a bacterial infection (e.g. Salmonella caused by Salmonella) comprising:

a pharmaceutically effective amount or an immunizing amount of a mutant strain according to the invention which is unable to re-form guanine nucleotides due to deletion mutation in the guaB gene; and a pharmaceutically acceptable carrier or diluent. Preferred compositions are those comprising a mutant Salmonella enterica strain according to the invention.

The attenuated strain of the invention can transfer DNA encoding an exogenous antigen into a eukaryotic cell. This exogenous antigen may be encoded by and expressed from a plasmid comprising the attenuated mutant strain of the invention.

In general about 10 cfu to about 1010 cfu, preferably about 105 cfu to about 1010 cfu is administered (examples of a pharmaceutically effective amount or an immunizing amount). An immunizing dose varies according to the route of administration. Those skilled in the art may appreciate that the effective dose for a parenterally administered vaccine may be lower than a similar vaccine that is administered via drinking water, and the like. The attenuated strains of the invention, and pharmaceutical compositions or vaccines comprising them, are highly suitable for immunizing an animal or veterinary species against a bacterial infection (e.g. Salmonellosis) and possibly other diseases (in the case of a multivalent vaccine). .

A further aspect of the invention therefore relates to a method for immunizing animals, preferably veterinary species, more preferably poultry such as chickens, against infection by a bacterium (e.g. Salmonellosis caused by Salmonellae), said A method comprising the step of: administering to the animal or veterinary species, in need thereof, an immunizing amount of an attenuated mutant strain of the invention and / or a vaccine comprising the same, whereby a protective immune response is then commanded in the animal. animal or veterinary species.

Examples of veterinary species to be immunized against Salmonellosis: poultry, small and large farm animals such as chickens, turkey, ducks, quail, pheasant (Numididae), pigs, sheep, young calves, cattle raising, etc. ..

In general, an appropriate dose is administered to these animals, preferably via the oral, nasal or parenteral route. A last aspect of the invention relates to a mutant strain of the invention for use as a medicament (for example for use in a vaccine). Still another aspect of the invention relates to the use of an attenuated mutant strain of the invention for the preparation of a medicament, such as a vaccine, for the prevention (and / or treatment) of a disease caused by a pathogen (the infecting bacterium). ) such as Salmonellosis. Examples of animals and veterinary species to be treated and recommended doses are given below.

Detailed Description of the Invention

It has been surprisingly found that a deletion mutation in the guaB gene can lead to attenuated Salmonella enterica strains with a significantly reduced virulence and can induce an immune response in a farm animal. Said deletion would be attenuated in any organism that could use the guanine nucleotide as an intermediate.

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

The deletion mutation according to the invention is one in which the purine metabolic pathway enzyme IMP dehydrogenase (encoded by guaB) is inactivated. Said inactivation may be achieved via a deletion in which the guaB gene function is defective, leading to null functioning (without a functional gene product formed) of the affected gene (s). It is well known to one skilled in the art that obtaining such mutants and a simple test can tell whether the guaB gene function is defective. The mutant strain that fails to express a functional guaB gene product cannot grow in minimal A medium unless this medium is supplemented with (e.g. 0.3mM) guanine, xanthine, guanosine or xanthosine.

The invention aims to provide, among others, attenuated S. enteritidis and S. typhimurium strains since these strains are the most common S. enterica serotypes.

The present invention provides attenuated strains. The invention provides among other strains of attenuated Salmonella enterica for use, inter alia, as a live attenuated vaccine against Salmonellosis, as a live vehicle and / or as DNA-mediated vaccines expressing exogenous antigens. As used herein, an "exogenous antigen" means an antigen external to Salmonella.

Live vector vaccines also termed "vehicle vaccines" and "live antigen delivery systems" comprise an area of excitation and a versatile area of vaccinology (Levine et al., 1990, Microecol. Ther., 19: 23- 32). In this research, a live virus or bacterial vaccine is modified so that it expresses protection from the exogenous antigen of another microorganism, and releases those antigens into the immune system, thereby stimulating a protective immune response. The living bacterial vectors being declared include, among others, attenuated Salmonella.

An object of the invention is to provide attenuated strains, such as s strains. attenuated enteric acid for use in a live vaccine, possibly a multivalent or multivalent live vaccine.

It is therefore an object of the invention to provide a vaccine against, for example, Salmonellosis comprising:

a pharmaceutically effective amount or an immunizing amount of a mutant of the invention (e.g., a Salmonella enterica mutant) that is unable to form a new guanine nucleotide, said mutant strain containing a deletion mutation in the guaB gene;

a pharmaceutically acceptable carrier or diluent. Another object of the invention is to provide a live vector vaccine comprising:

a pharmaceutically effective amount or an immunizing amount of a mutant of the invention (e.g., a Salmonella enterica mutant) that is unable to form a new guanine nucleotide, said mutant strain encoding and expressing an exogenous antigen; and

a pharmaceutically acceptable carrier or diluent.

The particular exogenous antigen employed in the live vector (S. enterica) is not critical to the present invention.

Still another object of the invention is to provide a DNA mediated vaccine comprising:

a pharmaceutically effective amount or an immunizing amount of a mutant of the invention (e.g., a Salmonella enteric mutant) that is unable to form a new guanine nucleotide, said mutant strain containing a mutation in the guaB gene; wherein said mutant strain contains a plasmid encoding and expressing in a eukaryotic cell, an exogenous antigen; and

a pharmaceutically acceptable carrier or diluent. Details such as the construction and use of DNA-mediated vaccines can be found in US Patent 5,877,159, which is incorporated herein by reference in its entirety. Again, the particular exogenous antigen employed in DNA-mediated vaccine is not critical to the present invention.

The decision expressing the exogenous antigen in, for example, S. enteral (using a prokaryotic promoter in a live vector vaccine) can be based on the vaccine's construction so that the particular antigen gives an improved immune response in animal studies or screening. and / or, if glycosylation of an antigen is essential for its protective immunogenicity, and / or if the correct tertiary conformation of an antigen is best achieved with

one form of expression than with the other (U.S. Patent No. 5,783,196).

In vaccines of the present invention, the pharmaceutically effective amount or immunizing amount of the mutants of the present invention to be administered will vary depending upon the age, weight and sex of the subject. By an "immunizing amount" as used herein should be understood in fact an amount that is capable of inducing an immune response in the animal receiving the pharmaceutical composition / vaccine. The invoked immune response may be a humoral, mucosal, local immune response and / or a cellular immune response.

The particular pharmaceutically acceptable carrier or diluent employed is not critical to the present invention, and is conventional in the art. Examples of diluents include, stomach gastric acid buffering buffer, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone, or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame. Examples of carriers include: proteins, for example as found in skim milk; sugars; for example sucrose; or polyvinylpyrrolidone.

The deletion mutation according to the invention was created via standard homologous recombination technique, thereby departing from the guaB gene, for example, part of the guaB coding sequence, in a first step is repositioned by a resistance gene and flanked FRT sites.

Preferably, in a second step, said resistance gene is removed by recombination between the two FRT sites. An FRT site and the starting sites Pl and P2 remain by the molecular mechanism of recombination by removing the antibiotic resistance gene according to Datsenko and Wanner (2000) (see, for example, Figure 4).

A particular example of the invention relates, for example, to the S. enteritidis guaB deletion mutation comprising a mutated guaB gene or the coding sequence comprising SEQ.ID.NO:12.

Brief Description of Drawings

Figure 1 is a schematic representation of a biosynthetic guanosine monophosphate pathway (adapted from Zalkin and Nygaard, 1996, in Escherichia coli and Salmonella, Cellular and Molecular Biology, Second Edition, 1996 - FC, Neidhardt ed., ASM Press. , Washington DC, Vol. 1, Ch., 34: 561-579). AICAR: 5'-phosphoribosyl-4-carboxamide-5-aminoimidazole; ATP: adenonsine 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; Figure 2 represents the genome of S. enteritidis contig 1294 (SEQ. ID. NO; 10). The initiation codon ATG and the terminus codon TGA of the guaB gene are in bold; Figure 3 depicts the sequence of the S. enteritidis AguaB fragment cloned into pUC18 (SEQ ID NO: 11). Primers that have been used are indicated by the horizontal arrows. The fragment generated with GuaB6-GuaB7 primers was cloned into pUC18. The start codon ATG and the end codon TGA of the guaB gene and the SmaI CCCGGG restriction site are shown in bold;

Figure 4 is the nucleotide sequence of the S. enteritidis PCR fragment, which includes the guaB deletion obtained after sequencing using the GuaBlO primer (SEQ ID NO: 12). The PCR fragment was amplified with GuaB6-GuaB7 primers using the total genomic DNA of the SM20 mutant. The remaining FRT site is indicated in bold italics and the P1 and P2 primers by arrows (Datsenko and Wanner, 2000, PNAS, 97: 6640-6645). The guaG gene ATG start codon and TGA stop codon are shown in bold; and

Figure 5 represents the S. typhimurium LT2 guaB gene, section 117 of 220 of the complete genome (SEQ ID NO: 13). The ATG start codon and the guaB gene end codon TGA are in bold.

The invention will be described in more detail with reference to the following examples and configurations with respect to the accompanying drawings. The particular embodiments and examples are in no way intended to be limiting to the scope of protection of the invention as claimed.

Examples

Example 1: Auxotrophic mutation affects the guaB people. An auxotrophic insertion mutation of a wild type of S. enteritidis was obtained via mutagenic insertion. Only when supplemented with 0.3 mM guanine, xanthine, guanosine or xanthosine could the mutant strain grow in minimal medium A.

These data strongly suggest that auxotrophic mutation of the strain affects the guaB gene encoding the enzyme dehydrogenase IMP (EC 1.1.1.205). This enzyme converts inosine-5'-monophosphate (IMP) to xanthosine monophosphate (XMP) as shown in Figure 1.

An insertion mutation can reverse, thereby restoring the pathogenicity of the strain. This limits its applicability in a live attenuated vaccine. In this regard, deletion mutation is preferred. The guaB deletion mutants of S. enteritidis and S. typhimurium were therefore created and tested. The guaB genes of both serotypes are given in figures 2 and 5.

Example 2: guaB deletion mutants.

Construction of guaB deletion mutants

The guaB deletion mutants were created according to the method for producing deletion mutations in the Escherichia coli K12 genome (Datsenko and Wanner, 2000, PNAS, 97: 6640-5, incorporated herein by reference in their entirety).

This method relies on a homologous recombination, mediated by the bacteriophage λ di recombinase system Red, from a linear DNA fragment generated by PCR.

The guaB sequence is replaced here by an antibiotic resistance gene. This resistance gene is flanked by FRT sites (FLP recognition target sites) and can be extracted from the genome by site-specific recombination mediated by FLP recombinase. Overlap PCR (Ho et al., 1989, "Gene", 77: 51-59) was applied to construct a linear fragment. The principle relies on the use of two sets of primers, one downstream pair (GuaB3-GuaB4; GuaB3: 5'-GGCTGCGATT GGCGAGGTAG TA 3 ', SEQ ID NO: 2; GuaB4: 5'-GGTGATCCCG GGCGTCAAAC GTCAGGGCTT , SEQ ID NO: 3) and an upstream primer pair (GuaB5-GuaB2; GuaB5: 5 'TTGACGCCCG GGATCACCAA AGAGTCCCCG AACTA 3', SEQ ID NO: 4; GuaB2: 5 '- CGTTCAGGCG CAACAGGCCG IDGT 3', SEQ : 1) the guaB gene.

Both sets contain primers (GuaB4, GuaB5) that are partially complementary and to which an SmaI restriction site has been added.

Following annealing of the resulting complementary sequences and chain elongation, PCR with the exteriorized primers (GuaB6-GuaB7; GuaB6: 5 'GCAACAACTC CTGCTGGTTA 3'; SEQ ID NO: 5; GuaB7: 5 'AGACCGAGGA TCACTTTATC 3'; SEQ ID NO: 6) producing a 6 base pair fragment at the SmaI site replacing an internal segment of base pair 861 of the guaB coding sequence. This AguaB fragment was cloned into the pUC18 vector (see figure 3).

The chloramphenicol (cat) resistance gene with its flanking FRT sequences was amplified using the P1 (5 'GTGTAGGCTG GAGCTGCTTC 3', SEQ ID NO: 8) and P2 (5 'CATATGAATA TCCTCCTTAG 3', SEQ ID NO: 9 primers ) (Datsenko and Wanner, 2000) and plasmid pKD3 DNA (Datsenko and Wanner, 2000) as a standard.

This PCR fragment was ligated at the SmaI site of the cloned AguaB fragment. The desired fragment was produced using the sheltered primers (GuaB6-GuaB7). The resulting PCR fragment was electroporated into S. enteritidis type 4 strain 76Sa88 (a clinical isolate from a turkey obtained from the Veterinary and Agrochemical Research Center, Groeselenber, 99, B-1180 Ukkel, Belgium) and of a temperature sensitive replication plasmid pKD46 encoding the bacteriophage lambda Red recombinase system.

Chloramphenicol resistant transformants were tested in minimal A medium and minimal A supplemented with 0.3 mM guanine. AguaB :: catFRT mutants were confirmed by PCR using the following primer combinations: GuaB6-GuaB7; GuaB6-P2; GuaB7-Pl and Pl-P2.

The S. enteritidis AguaB :: catFRT mutant (SM12) was transformed with the temperature sensitive replication plasmid pCP20 by electroporation. Plasmid pCP20 encodes the FRP site-recognizing FLP recombinase to remove the cat gene. The resulting strain was named SM20.

The PCR fragment in which the deletion is located was obtained using the total mutant SM20 genomic DNA and the GuaB6-GuaB7 primer combination (see Figure 4).

The AguaB mutation was confirmed by sequencing this fragment using the GuaB10 primer (5 'AGGAAGTTTG AGAGGATAA 3', SEQ ID NO: 7).

The sequences of all the above mentioned primers are given in Table 1.

To avoid the presence of possible additional mutations caused by the expression of the Red recombinase system, an isogenic strain was constructed.

The DguaB :: catFRT mutation of the SM12 mutant was transduced by the bacteriophage P22 HT int- (Davis, RW, Botstein D., and Roth, JR (1980) "In Advanced Bacterial Genetics, A Manual for Genetic Engineering", Cold Spring Harbor Cold Spring Harbor, NY), for S.

Wild allele enteritidis 76Sa88. The cat gene was removed using plasmid pCP20. The resulting strain was named SM69 (deposit number LMG P-21641). A AguaB mutant of the S. typhimurium 1491S96 strain was constructed using the same procedures and primers. The resulting S. typhimurium AguaB :: catFRT and S. typhimurium AguaB strains were named Sm9 and SM19, respectively. SM86 (having deposit number LMG P-21646) is the isogenic strain obtained from it after transduction by bacteriophage P22 HT int- lysate from strain SM9 and excision of cat gene using plasmid pCP20.

The AguaB Sm19, SM20, SM86 and SM69 mutant are sensitive to bacteriophage P22. This proves the presence of intact lipopolysaccharide (LPS).

Virulence and Protection Tests with the guaB SM20 deletion mutant in mice.

The virulence of the SM20 mutant in mice was tested by oral infection of 6-8 week old female BALB / c mice in two independent experiments. These were performed as described above. The wild type strain S. enteritidis 76Sa88 was tested in parallel as a positive control. The S. enteritidis SM50 AaroA mutant was included in the experiment as a control vaccine. This mutant carries an accurate deletion of the complete aroA coding sequence and was constructed by the method of Datsenko and Wanner (2000).

Complete data are listed in Tables 2 and 3. These results demonstrate that the AguaB SM20 mutant is strongly attenuated in mice but still demonstrate some pathogenicity residues when administered at high doses. Oral immunization with the mutant induces immune protection against high-dose infection of a pathogenic corresponding wild-type S. enteritidis 76Sa88 strain. The protection is at least equal to the protection conferred by the S. enteritidis AaroA SM50 mutant.

Virulence and protection tests with guaB SM69 and SM86 isogenic deletion mutants in mice.

The virulence of SM69 and SM86 mutants in mice was tested by oral infection of 6-8 week-old female BALB / c mice. These were performed as described above. The wild allele strain S. enteritidis 76Sa88 and S. typhimurium 1491S96 were tested in parallel as a positive control.

Complete data are illustrated in tables 4-7. These results demonstrate that the AguaB SM69 and Sm86 mutants are strongly attenuated in mice but still show some residual pathogenicity when administered at high doses. Oral immunization with the mutants induces protective immunity against infection by a high dose of the corresponding pathogenicity of the wild allele strain.

Evaluation of the safety of the S. enteritidis AguaB deletion mutation in day-old chickens with intratracheal and oral feeding.

The aim of this study was to evaluate the safety of the S. enteritidis SM69 germ master mutant AguaB strain in one day old chickens. Mortality was used as a primary parameter for determining safety.

One-day-old chickens had their legs tied and randomly placed 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 inoculation with the master germ, the birds in groups 1 and 2 were placed in one isolation and those in group 3 and 4 in another isolation.

Group 1 and 2 hens were inoculated with the SM69 master via intratracheal (IT) or oral feeding (OG), respectively, with a current titer of 1.28 108 cfu / 0.2 ml per bird. Group 3 and 4 chickens were administered at a dose of 0.2 ml PBS (phosphate buffered saline) per bird by intratracheal or oral feeding, respectively.

Following inoculation of SM69 or PBS, chicken mortality was observed daily until the 38th day after inoculation. Table 8 summarizes the mortality results in all 4 groups. In the If group a bird died during inoculation due to the trauma of inoculation. Two birds died two days after inoculation (DPI). Three birds died from the third to the thirteenth day (on the 3rd, 5th and 13th DPI, respectively). A total of 6 birds died in group 1. In group 2, two birds died in total. One death during inoculation due to inoculation trauma and one death on the 5th day after inoculation.

No birds died in the PBS-treated group, either intratracheally or via the oral feeding route. This study indicates that the S. enteritidis AguaB mutant SM69 strain is unsafe when administered at 1.28 x 108 cfu per day-old bird via intratracheal or oral feeding.

Evaluation of the safety of S. enteritidis SM69 deletion mutation in two-week-old chickens

intratracheal route and oral feeding route.

The safety of the S. enteritidis SM69 mutant AguaB strain was then evaluated with two-week-old pathogen-free chickens (SPF) via the intratracheal route and oral feeding route. Mortality was used as a primary criterion and body weight as a secondary criterion for determining safety.

The 2-week-old birds had their legs tied and were randomly placed in each of the four treatment groups: SM69-IT, SM69-OG, Poulvac ST-IT and PBS-IT. Ten birds in group 1 were inoculated with SM69 by intratracheal route; ten birds in group 2 were inoculated with SM69 by oral feeding; ten birds in group 3 were inoculated with an S. typhimurium AroA ~ vaccine (Poulvac® ST) by the intratracheal route; and five birds from group 4 were inoculated with PBS by intratracheal route. The chickens in group 1 and 2 were inoculated with SM69 main spread by intratracheal route or by oral route, respectively, with a current titer of 2.304 χ 108 cfu / 0.2 ml per bird. The chickens in group 3 were administered with Poulvac® ST by the intratracheal route at 2.19 χ 108 cfu / 0.2 ml per bird. The chickens in group 4 were administered intratracheally with 0.2 ml PBS per bird. After inoculation, the birds from the treatment of groups 1 and 2 were placed in one isolation and those of groups 3 and 4 in another isolation.

After inoculations, mortality was observed daily up to 21 days after inoculation. The body weight of all birds was also related at the end of the study period (21 days). Poulvac® ST and PBS were used as intratracheal procedure control.

During the 21-day observation period, a bird in the intratracheal treatment group SM69 (group 1) died from the infected egg yolk sac. No mortality was associated with SM69 inoculation, indicating that the SM69 strain is safe in the titration tested, 2.304 x 108 cfu per bird by intratracheal route or oral route. As expected, no deaths were observed either in Poulvac® ST-treated birds at a titration of 2.19 x 108 cfu per bird or in PBS-treated birds, indicating that the study was valid (Table 9).

Body weight was compared between groups in a variant model analysis (ANOVA) with body weight as the dependent variable and treatment included as an independent variable. Group comparisons were made using the Turkey test for multiple comparisons. The significance level was represented as ρ x 0.05. The study was considered valid because of the control of the remaining healthy chickens (PBS control group) free of clinical signs of disease or mortality during the study.

There were no significant differences in end-of-body weight in chickens that were administered SM69 intratracheally or orally, Poulvac® ST, or PBS (Table 5). Although no baseline was established in each group for day-old birds, it was reasonable that there was no significant difference in initial body weight between the four groups as birds were randomly placed within each of the two groups. four treatment groups.

Since no mortality has been attributed to the SM69 inoculum, it can be concluded that the S. enteritidis, SM69, mutant strain AguaB is safe when administered at the tested titration of 2.304 cfu / 0.2 ml per dose for birds with 2 weeks of age by both intratracheal and oral inoculation. SM69 inoculations had no effect on the final body weight of the birds, bird weight being the second safety parameter evaluated here.

A deposit was made in accordance with the Budapest Treaty on the BCCM / LMG Culture Collection, "Laboratorium voor Microbiologie", K.L. Ledeganckstraat 35, B-9000 Gent (Belgium) for the following micro-organisms: Salmonella enteriditis SM69 under deposit number LMG P-21641 (filing date: August 9, 2002) and S. typhimurium SM86 under deposit number LMG P-21646 (filing date: August 28, 2002). The deposits were made in the name of Prof. J. -P. Hernalsteen, previous address: Vrije Universiteit Brussel, "Laboratorium Genetische Virologie", Paardenstraat 65, B-1640 Sint- Genesius-Rhode, current address: Vrije Universiteit Brussel, Onderzoeksgroep Genetische Virologie, Pleinlaan 2, B-1050 Brussels, Belgium.

TABLE 1

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Evaluation of mutation safety by deletion of S. enteritidis guaB SM69 strain in one day old chickens.

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<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 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence

<22O>

<223> GuaB7 primer <400> 6

agaccgagga tcactttatc 20

<210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GuaBlO primer <400> 7 aggaagtttg agaggataa 19

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> P1 primer

<400> 8

gtgtaggctg gagctgcttc ??? 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> P2 primer

<400> 9

catatgaata tcctccttag 20

<210> 10

<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)

<223> is not a, c, g, or t

<220>

<221> misc_feature

<222> (1657). . (1657)

<223> is not a, c, g, or t

<400> 10 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 3 00

ctgtcccatc tcctgttcta acagcagacg aaccgtctgg ttaaggcggc ttacggtaaa 360

aattgaggaa gtttgagagg ataacatgtg agcgggatca aattctaaat cagcaggtta 42 0

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 84 0

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 13 8 0

tgcgacgggc gcaggcgctc gcgcactggc ggaagccggt tgcaacgcgg tgaaagtggg 1440

tatcggcccg ggttccatct gtacgactcg tatcggggact 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

tttgtgcgt 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 2 903 <210> 11

<211> 1995

<212> DNA

<213> Artificial Sequence <220>

<223> Sequence of S. Enteritidis delta-guaB fragment cloned into pUC18

<220>

<221> misc_feature

<222> (55). . (55)

<223> is not a, c, g, or t

<400> 11

ggctgcgatt ggcgaggtag tagtccatcg ccatgcccag acgctgctgg cgganctgga 60

tctgacgcaa caactcctgc tggttacggc tgacaatttc tgccgctgct gatggcgtag 120

cgcgcgcagg tcggcgacaa aatcagctat cgtgacgtcc gtttcgtgac cgacggcgct 18 0

gaccaccgga atgcggctgg caaaaatcgc ccgcgccacg cgttcgtcgt taaaactcca 240

caaatcttcc agcgaaccgc cgccgcgccc gacgatcagt acatcacatt cgccgcgcgc 3 00

gttcgccagt tcgatagcac gaacgatctg ccccggcgcg tcgtcgccct ggactgcggt 360

tggatagata ataacgggca gggatgggtc acgacgcttt agcacgtgga gaatatcgtg 42 0

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 72 0

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> 12 <211> 349 <212> DNA

<213> Artificial Sequence <220>

<223> Nucleotide sequence of the S.Enteritidis PCR fragment, which includes the guaB deletion, obtained using the GuaBlO primer

<400> 12

catgtgtgag cgggatcaaa ttctaaatca gcaggttatt caatcgatag taacctgctc 60

acgggggatc gcaagcacta tttgcaaaaa aaatgtagat gcaaccgatt acgttctgta 12 0

taatgcccgg caatatttat taacctccca ggtgagatat tcgccatgta cgtatcgcta 180

aagaagccct gacgtttgac gcccgtgtag gctggagctg cttcgaagtt cctatacttt 240

ctagagaata ggaactccgg aataggaact aaggaggata ttcatatggg atcaccaaag 3 00

atccccgaac taccgtctgg gctctgattt tcttcgcccg accttcgcg 349

<210> 13

<211> 1553

<212> DNA

<213> Salmonella Typhimurium

<220>

<221> misc_feature

<223> S. Typhimurium LT2 Gene guaB, Complete Genome Section 117 to 220

<400> 13

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 3 00

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 60 0

A tggcaaaaat gcacgaaaaa cgcgtagaaa aagcgctggt cgttgatgat aacttccatc 660

f tgcttggcat gattaccgta aaagatttcc agaaagcgga acgtaaacca aactcctgca 72 0

aagatgagca gggccgttta cgtgtcggcg cggcggtcgg cgcaggcgcg ggcaacgaag 78 0

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 gtgcgatgtc caaaggttcc tccgaccgtt tggtgccgga aggtatcgaa ggtcgcgtag accagcagat gggcggcctg cgctcctgta aactgcgtac taaagcggag tttgtgcgta ttcacgacgt gaccatcacc aaagagtccc

cggggggggggggggggggggg

Claims (17)

1. Attenuated mutant strain of veterinary infecting bacteria, characterized in that it is unable to re-form the guanine nucleotide, said mutant strain containing a deletion mutation in the guaB gene. Mutant strain according to claim 1, characterized in that the veterinary species is a domestic bird. Mutant strain according to any one of claims 1 to 2, characterized in that it is a strain of Salmonella enterica or a strain of Escherichia coli. Mutant strain according to any one of claims 1 to 3, characterized in that said mutant strain encodes and expresses an exogenous antigen. Mutant strain according to any one of claims 1 to 4, characterized in that the strain is an Enteritidis or a Typhimurium strain. Mutant strain according to any one of claims 1 to 5, characterized in that said mutation is introduced into the parent strain S. Enteritidis type 4, strain 76Sa88. Mutant strain according to claim 6, characterized in that it is S. Enteritidis strain SM69 having deposit number LMG P-21641. Mutant strain according to any one of claims 1 to 7, characterized in that said mutation is introduced into the S. typhimurium 1491S96 source strain. Mutant strain according to claim 8, characterized in that it is the strain s. typhimurium SM86 having deposit number LMG P-21646. Vaccine for the immunization of veterinary species against a bacterial infection, comprising: a pharmaceutically effective amount or an immunizing amount of a mutant strain as defined in any one of claims 1 to 9 which is unable to form. new guanine nucleotides due to a mutation deletion in the guaB gene; and a pharmaceutically acceptable carrier or diluent. Vaccine according to claim 10, characterized in that said mutant strain encodes and expresses an exogenous antigen. Vaccine according to either of claims 10 and 11, characterized in that said mutant strain comprises a plasmid encoding and expressing in an eukaryotic cell an exogenous gene. A method for immunizing a veterinary species against a bacterial infection, comprising the steps of: administering to a veterinary species, in need thereof, an immunizing amount of a mutant strain as defined in any one of claims 1 to 4. And / or the vaccine defined in any one of claims 10 to 12. Method according to claim 13, characterized in that the veterinary species is a domestic bird. Method according to either of claims 13 or 14, characterized in that the mutant strain and / or vaccine are administered via an oral, nasal or parenteral route. Attenuated mutant strain according to any one of claims 1 to 9, characterized in that it is used as a medicament. Use of an attenuated mutant strain as defined in any one of claims 1 to 9, characterized in that it is for the preparation of a medicament or a vaccine for the prevention and / or treatment of a bacterial infection in a veterinary species, preferably a domestic bird.