WO2014037103A1 - Vaccin vivant à dérive métabolique atténuée contre la typhose aviaire - Google Patents

Vaccin vivant à dérive métabolique atténuée contre la typhose aviaire Download PDF

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WO2014037103A1
WO2014037103A1 PCT/EP2013/002654 EP2013002654W WO2014037103A1 WO 2014037103 A1 WO2014037103 A1 WO 2014037103A1 EP 2013002654 W EP2013002654 W EP 2013002654W WO 2014037103 A1 WO2014037103 A1 WO 2014037103A1
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antibiotic
vaccine
mutants
strain
salmonella gallinarum
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PCT/EP2013/002654
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Awad SHEHATA
Hesham SULTAN
Monika KRÜGER
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Universität Leipzig
Lohmann Animal Health Gmbh & Co. Kg
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/42—Salmonella

Definitions

  • the present invention relates to the development of a new live fowl typhoid (FT) vaccine candidate against Salmonella Gallinarum, with one or two independent attenuating mutations based on metabolic drift (MD), i.e. spontaneous chromosomal mutations.
  • MD metabolic drift
  • the invention further relates to a method of preparing the new live FT vaccine.
  • FT is a systemic disease in domestic poultry which results in septicaemia. It is caused by Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum. FT is a disease of considerable economic importance in many areas, including Latin America, the Middle East, Africa and Asia. Infection in chickens occurs at all ages and is characterized by severe hepatomegaly and splenomegaly accompanied by liver damage with bronzing aspect, anemia, and septicemia. Infected poultry show poor growth, diarrhea, loss of appetite and somnolence. Lesions may include a swollen, friable and often bile-stained liver. The course of the disease is short but mortality and morbidity rates due to FT may reach up to 80% (32).
  • the object of the present invention is to provide a vaccine against FT which is easy to administer and confers solid immunity against Salmonella Gallinarum.
  • the present invention provides a method for the generation of a live vaccine containing stable bacteria carrying one or two attenuating mutations and a vaccine against FT containing bacteria obtained by said method.
  • the method for the generation of the live vaccine of the present invention is based on live attenuated Salmonella Gallinarum strains having MD spontaneous mutations.
  • MD is a mutation related function or spontaneous mutation. It occurs in all microorganisms as an evolution principle. It occurs in viruses, bacteria and fungi. This means all microorganisms undergo mutation.
  • RNA viruses the incidence of this mutation is about 1 : 10 3 . In bacteria may reach to 1 :10 s . This means the incidence is one chance each 100 million chances in bacteria. Due to its low incidence it is not detectable either in vivo or in vitro. Also the incidence of back mutation can be almost excluded due to its low incidence. As consequences of this mutation, bacteria grow slowly; have longer generation time and show smaller colony size relatively to the wild type strain.
  • the MD mutations are a standard technique which induce clone-specific gradually reduced colony sizes but was so far not used for Salmonella Gallinarum. Linde and co-workers (16) described the MD compartments which can be exploited for vaccination purposes. These MD mutations induce clone-specific gradually reduced colony sizes which are inversely correlated with the degree of the reduction in colony size.
  • Costs and time for the preparation are low and the degree of attenuation via the desired selection of an increased generation time and, thus, reduced colony size, respectively, is, in principle, almost arbitrary.
  • MD mutants represent clones having mutations in metabolic compartments per definitionem resulting in dysfunction (i.e. attenuation is at the expense of the bacterial "fitness"). As a consequence, gradually reduced colony sizes (depending on the clone) compared to the wild strain can be found. Normally, these mutants are eliminated by the immune-competent host or, alternatively, are overgrown by the adapted normal flora.
  • the method for the generation of the Salmonella Gallinarum live vaccine containing stable Salmonella Gallinarum bacteria carrying one or two attenuating mutations comprises the following steps:
  • step (d) optionally growing a clone obtained in step (c) in a medium supplemented with a second antibiotic having a suitable concentration; isolating clones which are MD antibiotic resistant mutants as a second attenuating marker and smaller than clones obtained in step (c);
  • the bacterial clone of step (a) is, preferably, obtained from "wild" virulent strains. These bacteria can be taken from diseased animals (e.g., chicken).
  • any antibiotic capable of inducing MD mutations can be used for the purposes of the present invention, e.g., streptomycin, rifampicin, fosfomycin, fusidic acid or nalidixic acid.
  • streptomycin is known to lead rapidly to the development of resistant and dependent strains among the micro-organisms.
  • the antibiotic of step is streptomycin (Sm) or rifampicin (Rif). If Sm is used as the first antibiotic, then it is preferred to use Rif as the second antibiotic or vice versa.
  • the genetic modification may affect a chromosome of the bacterium.
  • the chromosomic modification is a rare event which, once carried out, ensures the stability of the acquired properties.
  • the bacterial strains according to the invention characterized by one or two attenuating mutations are in the first place non-virulent strains selected from natural virulent strains, for their growth capacity on a medium with a high content of an antibiotic such as streptomycin, and in addition, which can be developed satisfactorily in the presence of the antibiotic (step (b)).
  • the strains of step (c) are mutants selected from the MD antibiotic resistant strains and provide stable MD mutants containing single attenuating marker after at least 30 passages, preferably at least 50 passages, with colony size of > 50% of the colony size of the wild type strain.
  • step (c) is carried out by passaging a clone of step (b) once in the presence of the antibiotic which is used in the induction of the metabolic drift mutation and then passaging colonies which show reduced size relative to wild type Salmonella Gallinarum at least 30 times on media free from antibiotic.
  • Step (d) allows the isolation of second MD antibiotic resistance mutants.
  • concentration of the antibiotic in step (d) can be determined by the person skilled in the art according to routine procedures.
  • step (e) the strains of the previous steps (c) or (d) characterized by a reduced colony size are isolated and serially passaged once on media containing the same antibiotic which is used for the induction of the first and/or second metabolic drift mutation in order to check stability. At least 30 serial passages, preferably 50 serial passages, are carried out.
  • step (f) the clones isolated from step (e) having the graduated reduction of the colony size as stable property are provided.
  • single antibiotic-resistant mutants are generated by spatulating approximately 10 8 -10 12 , preferably about 10 10 , cfu of fresh Salmonella Gallinarum on an agar plate, preferably Caso agar, supplemented with a suitable amount of a first antibiotic (e.g. 300-700 pg, preferably about 500 pg, streptomycin per ml, or 100-500 pg, preferably about 300 pg, rifampicin per ml). Plates are incubated aerobically at about 37 °C for 1-3 days; preferably about 48 h.
  • a first antibiotic e.g. 300-700 pg, preferably about 500 pg, streptomycin per ml, or 100-500 pg, preferably about 300 pg, rifampicin per ml.
  • approximately 10 8 -10 12 , preferably about 10 10 , cfu fresh cultures of single mutants (SG-Sm or SG-Rif) were used for isolation of second MD markers.
  • the SG-Sm or SG-Rif were spatulated on an agar plate, preferably Caso agar, containing either 100-500 pg, preferably about 300 pg, rifampicin per ml, or 300-700 pg, preferably about 500 pg, Sm per ml, respectively. Plates were incubated aerobically at about 37 °C for 1-3 days, preferably about 48 h. The same procedures were carried out for resistant colonies to obtain stable mutants containing two MD attenuating markers as mentioned above. The resulting MD mutants containing two attenuating markers were designated SG-Sm-Rif or SG-Rif-Sm.
  • the present invention provides a method for the generation of a Salmonella Gallinarum live vaccine containing stable Salmonella Gallinarum bacteria carrying an attenuating mutation, wherein said method comprises the following steps: (a) providing a wild type Salmonella Gallinarum strain and plating and growing that isolate in the presence of a first antibiotic;
  • step (c) passaging the colonies isolated in step (b) once on antibiotic-supplemented agar and then on antibiotic-free agar;
  • step (d) the mutants of step (d) are grown in the presence of a second antibiotic and steps (b) to (d) are repeated.
  • the present invention also provides live bacterial strains obtainable by the methods of the invention as well as a vaccine comprising live bacterial strains of the invention and a biologically acceptable carrier.
  • the vaccinating compositions may of course be constituted by means of freshly cultivated bacteria which are then freeze-dried for the ease of conservation. To administer the vaccinating bacteria, the medium in which they are suspended is not critical. Of course, this medium must not interfere with the good viability of the bacteria that they contain.
  • the vaccine of the present invention is administered in an amount suitable for immunization of chickens and may additionally contain one or more common auxiliary agents.
  • the employed term "amount suitable for immunization” comprises any amount of bacteria with which the chickens can be immunized.
  • the “amount suitable for immunization” may be determined using methods known to one skilled in the art.
  • the preferred administration of the vaccine is the oral route but also injection may be used at various sites of the chickens intramuscularly, subcutaneously, intradermally or in any other form of application. It may also be favourable to carry out one or more "booster injections" having about equal amounts.
  • biologically acceptable carrier comprises any auxiliary agents suitable for a vaccine.
  • auxiliary agents are, e.g., buffered common salt solutions, water, emulsions, such as oil/water emulsions, wetting agents, adjuvants, sterile solutions, etc.. It is favourable for the carriers to be non-immunogenic. Carriers, such as serum albumin, fibrinogen or transferrin or a fragment thereof are preferred. In a preferred embodiment, however, no adjuvants are necessary at all.
  • the vaccine of the present invention may be prophylactic, that is, the compounds are administered to prevent or delay the development of an infection or colonisation, e.g. an infection, caused by Salmonella Gallinarum to avoid the onset of FT.
  • Mutants strains of the present invention in particular those with two attenuating markers, would meet the security demands of the WHO.
  • the incidence of back mutation per one attenuating marker may reach 10 ⁇ 9 , in relation to the observed frequency of back mutations of spontaneously attenuated Shigella-tested in Volunteers- which was about 10 ⁇ 9 (10).
  • Two markers Salmonella Gallinarum mutants assure safety and the probability of a back mutation can almost be excluded (the incidence of back mutation is 10 ⁇ 18 ).
  • MD mutant vaccine is the possibility to differentiate vaccine strain from pathogenic wild strains using differential culture media, which are easily achieved by the addition of the antibiotics involved in the genetic changes.
  • MD mutations were performed using Sm and Rif as attenuating and epidemiological markers.
  • the Sm attenuation is based on a natural ribosome misreading, which can heightened by certain mutations (3, 12, 4).
  • five MD mutant strains could be isolated and proved to be stable by unabatedly reduced colony sizes over 30 passages on Caso agar medium. It has been demonstrated that the MD Salmonella Gallinarum mutants (10 8 cfu) did not cause death of any infected birds, compared with Salmonella Gallinarum wild type strain which caused high mortality rates (40-60%).
  • Salmonella Gallinarum mutants did not cause death of any infected birds while Salmonella Gallinarum wild type bacteria caused high mortality in commercial broiler chickens (Table 1 ). MD mutants have mutations in gene codifying essential enzymes and metabolic regulatory centers. As a consequence the generation time is increased and the virulence is reduced (16). The Salmonella Gallinarum mutants could be recovered from internal organs as liver and spleen but significantly lower than wild type strain (Table 2), similar results were reported for Salmonella Dublin MD mutants (24, 22).
  • Salmonella Gallinarum invades the mononuclear phagocyte system within macrophages of internal organs as liver and spleen (2), and unlimited proliferation of virulent strains in the host results in bacteremia, acute illness and death (5).
  • the presence of bacterial antigens in the host tissue is essential for inducing protective immunity.
  • high numbers of residential live vaccine strains in the internal organs may cause significant lesions, functional organ impairment and appearance of clinical symptoms (5).
  • Viable counts from the spleen and liver the development of hematological changes in the form of anemia, and pathological lesions were shown to be correlated, 10 4 CFU in spleen and liver were required for the development of significant pathological- and hematological changes (5).
  • the degree of attenuation depends on the number of attenuating marker. For highly pathogenic bacteria more than one MD attenuating marker may be required to ensure safety.
  • the incidence of back mutation per one attenuating marker may reach 10 "9 , in relation to the observed frequency of back mutations of spontaneously attenuated Shigella-tested in Volunteers- which was about 10 "9 (10).
  • Salmonella Gallinarum mutants which assure safety and the probability of a back mutation can almost be excluded (The incidence of back mutation is 10 "18 ).
  • the reduced colony sizes were stable after at least 50 passages on culture media.
  • An additional advantage of MD mutant vaccine is the possibility to differentiate vaccine strain from pathogenic wild strains using differential culture media, which are easily achieved by the addition of the antibiotics involved in the genetic changes.
  • high challenge strain number could be recovered from liver and spleen at 7 and 14 dpch.
  • the challenge strain still remains in some vaccinated chickens in the liver and/or spleen on the 7th dpch but most of vaccinated chickens were negative at 14th dpch. This attributed to that the challenge strain is cleared by the acquired immunity (31, 22).
  • the present invention demonstrates that the live FT vaccine of the present invention fulfills the security demand of the World Health Organization (WHO).
  • the vaccine proved to be genetically stable, safe and effective in experimental studies.
  • This attenuated vaccine strain is designed sufficiently invasive to induce immunity in poultry, in particular chickens, and the vaccinated animals were protected against experimental Salmonella Gallinarum challenge.
  • the vaccine can be used for poultry and can be administered orally which is an easy and cost effective alternative to injection.
  • Fig. (1 ) Colony morphology of Salmonella Gallinarum wild and MD-mutants strains showing the gradual diminish size.
  • Fig. (2) Determination of the pathogenicity of Salmonella Gallinarum wild type strain by measuring the survival rate of infected chicken
  • Fig. (3) Chickens infected with wild type Salmonella Gallinarum showing enlarged liver , presence of necrotic foci (A), nephritis and accumulation of urates in ureters (B).
  • Fig. (5) Pathological changes in liver and spleen. Liver of chickens infected with Salmonella Gallinarum wild type strain showed perivascular aggregation of granulocytes (A) and degragation of hepatocytes (B) (H&E X400). Spleen showed depletion of lymphocytes with hemorrages (C) (H&E X400).
  • Liver of chickens vaccinated with SG-Rif1 mutant showed mild congestion of blood vessels (D) (H&E X100) with thrombus formation (E) (H&E X250). Spleen showed mild congestion.
  • Fig. (6) Isolation of metabolic drift mutants, spontaneous mutants; for details, see Example 4.
  • Fig. (7) Safety (A) and immunogenicity (B) of MD mutants; for details, see Example 4.
  • Fig. (8) Colony morphology of Salmonella Gallinarum wild and MD-mutant strains; for details, see Example 4.
  • Example 1 General materials and methods 1.1 Salmonella Gallinarum wild strain
  • a Salmonella Gallinarum wild strain (Z34/11 ) was obtained from one week old chickens kept in backyard which showed high mortality, white diarrhoea with pasting feathers around the vent ("pasty vent”). Dead birds were submitted for diagnosis at Institute of Poultry Diseases, Free University, Berlin, Germany. At necropsy the liver was enlarged and showed multiple small white focal necroses. Necrotic foci and greyish white nodules were also observed in the caeca. The spleen was enlarged and the ureters were distended with urates. The initial isolation was carried out through culturing of internal organs (liver and spleen) on Blood and Gassnar agar (BioMerieux, Nurtingen, Germany).
  • the preliminary identification was carried out biochemically as well as serologically and finally analysed using Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF).
  • the isolated Salmonella Gallinarum was passaged on Caso agar (3.5% Caso, 0.3% yeast extract, 0.1 glucose, 0.5% Agar Agar; SIFIN, Berlin, Germany) and used for the development of MD mutant vaccine.
  • the minimal inhibitory concentration (MIC) of streptomycin (Sm) (Roth, Germany) and rifampicin (Rif), (Infecto Pharm, Germany) was determined.
  • Nutrient agar, glucose and yeast extract were purchased from SIFIN (Berlin, Germany). Streptomycin (Sm) was obtained from Roth and rifampicin (Rif) from infecto Pharm.
  • Single antibiotic-resistant mutants were generated by spatulating approximately 10 10 cfu of fresh Salmonella Gallinarum isolate on Caso agar supplemented with 500 pg Sm or 300 pg Rif per ml. Plates were incubated aerobically at 37 °C for 48 h. Small resistant colonies were passaged once on the antibiotic supplemented Caso ager then on antibiotic-free Caso agar. Stable diminished colony sizes after at least 50 passages on Caso agar served as a criterion of stability and considered single marker MD mutants.
  • the Sm and Rif attenuating marker clones were designated as SG-Sm and SG-Rif, respectively.
  • liver and spleen were collected from euthanized birds at 7 and 14 dpv, and the viable bacterial cells were then determined quantitatively on Caso-agar. The bacterial count was expressed in log-m CFU/g. A sample was assumed as negative when it still negative after enrichment in Selenite broth (Roth, Germany) for 16 h.
  • the Salmonella Gallinarum re-isolation was confirmed by MALDI-TOF. To distinguish the mutants from the wild type strain, specimens collected from vaccinated birds were cultured on Caso-agar supplemented with antibiotic which involved in the genetic changes.
  • Tissue samples from liver and spleen were fixed in 10% neutral formalin and processed by conventional methods for histopathology. Sections were stained with the haematoxylin-eosin (HE) stain and examined for histopathological changes. Histopathological scores (Hp score) were determined as described by Matsuda and coworkers (22).
  • the Salmonella Gallinarum whole cells (10 8 cfu /ml) were inactivated with 1 % formalin in PBS and used as antigen for ELISA.
  • ELISA 96-well microlon high binding microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with 50 ⁇ antigen solution diluted in coating buffer (200 mM sodium bicarbonate, pH 9.6) and incubated overnight at 4 °C. Plates were washed (30 seconds) five times with washing buffer (PBS, 0.05% Tween20) and blocked for 1 h at 37 °C with 300 ⁇ /well blocking buffer (PBS, 0.05% Tween20, 3% bovine serum albumin, BSA).
  • HRP Horseradish Peroxidase-labelled
  • the Salmonella Gallinarum wild type strain showed MIC values of 16 and ⁇ 1.0 for Sm and Rif, respectively.
  • the isolation of MD mutant depends on the unequivocal aspects of differences in colony size between the wild type strain and their MD mutants with different marker (single or double marker).
  • Five MD mutant strains could be isolated and designated, SG-Rifl , SG-Sm6, SG-Rif1-Sm4, SG-Sm6-Rif10 and SG-Rif1-Sm10.
  • the colony sizes in relation to wild type strain were reduced to 50%, 40%, 30%, 30% and 20%, respectively, ( Figure 1 ).
  • the probability of a back mutation can almost be excluded as the reduced colony sizes were stable after at least 50 passages on culture media.
  • the Salmonella Gallinarum mutant strains behaved as attenuated strains in comparison with wild type strain in commercial layer chickens (Table 1 ).
  • Chickens received SG-Rifl or SG-Sm6 (one marker vaccine) showed residual gross lesions in the liver and spleen but significantly lower (P ⁇ 0.001 ) than positive control chickens (infected with SG wild type strain).
  • vaccinated groups with double marker vaccine demonstrated no significant differences in the mean gross lesion values compared to the negative control group, which resulted in 0 (Table 1 ).
  • Table (2) Bacterial recovery from internal organs of chicken vaccinated with different MD mutants in comparison with wild type strain
  • IgG reaction was analyzed by determining the Salmonella Gallinarum-specific IgG at 15 dpv in serum using indirect ELISA.
  • An increase in the OD of specific serum IgG in chickens vaccinated with mutant SG strains was observed 15 dpv compared with non-treated group ( Figure 4).
  • Liver of chickens infected with Salmonella Gallinarum wild type strain showed perivascular aggregation of granulocytes and degregation of hepatocytes with a mean Hp score of 5 (Figure 5).
  • spleen showed depletion of lymphocytes with hemorrages.
  • liver of chickens vaccinated with Salmonella Gallinarum mutants showed mild congestion of blood vessels with thrombus formation with mean scores of ⁇ 1. Spleen showed mild congestion (Figure 5).
  • chickens from vaccinated group demonstrated no significant differences in the mean gross lesions values compared to the negative control at 7 and 15 dpch.
  • Gallinarum strain did not grow on Caso agar containing antibiotics (Sm or Rif), confirming that the isolated strain is wild type.
  • Example 4 Further approach of Isolating MD single and double marker mutants
  • Single antibiotic-resistant mutants were generated by spatulating approximately 10 10 colony-forming units (CFU) of fresh Salmonella Gallinarum wild type on Caso agar supplemented with 300 mg Rifampicin per milliliter. Plates were incubated aerobically at 37 C for 48 hr. Many antibiotic resistant colonies are arise (Figure 6, (a) big colonies, (b) intermediate colonies, (c) very small colonies).
  • CFU colony-forming units
  • Stable diminished colony sizes after at least 50 passages on Caso agar served as a criterion of stability and considered single-marker MD mutants.
  • one marker is enough to be used for vaccination purposes because the incidence of back mutation per one attenuating marker could reach 10 9 . This means one chance to 1.000.000.000 bacteria.
  • FIG. 7B shows the invasiveness of Salmonella Gallinarum wild type in chickens vaccinated with different MD mutants. The invasiveness of wild type was higher in chickens vaccinated with the smallest clone (Rif1- Sm10).
  • Fig. 8 The colony morphology of the Salmonella Gallinarum wild and MD-mutant strains is shown in Fig. 8.
  • the antibiotic resistant metabolic drift mutants are characterized by (a) slow growth, (b) extended generation time, (c) reduced colony size, (d) attenuation and (e) being suitable as a vaccine candidate. They are highly immunogenic because all genes are expressed (no gene deletion).
  • Mizuno, T., Ploeg, R., Trott, D. A new concept to stimulate mucosal as well as systemic immunity by parenteral vaccination as applied to the development of a live attenuated Salmonella enterica serovar Dublin vaccine. Vet. Res. 38: 773-794. 2007.

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Abstract

La présente invention concerne le développement d'un nouveau vaccin candidat vivant contre la typhose aviaire (FT) dirigé contre Salmonella Gallinarum (SG), présentant deux mutations d'atténuation indépendantes basées sur la dérive métabolique (MD), à savoir des mutations chromosomiques spontanées. Ce vaccin candidat répond aux exigences de sécurité de l'Organisation Mondiale de la Santé (OMS). Le vaccin s'est avéré génétiquement stable, sûr et efficace dans les études expérimentales. Cette souche de vaccin atténuée est destinée à être suffisamment invasive pour induire une immunité chez la volaille, en particulier les poulets, et les animaux vaccinés ont été protégé contre la provocation expérimentale par SG. Le vaccin peut être utilisé pour le poulet et peut être administré par voie orale, ce qui constitue une alternative simple et rentable à l'injection.
PCT/EP2013/002654 2012-09-05 2013-09-04 Vaccin vivant à dérive métabolique atténuée contre la typhose aviaire WO2014037103A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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KR102077701B1 (ko) * 2018-12-18 2020-02-14 전북대학교산학협력단 살모넬라 갈리나룸 사균체를 포함하는 가금 티푸스의 예방 또는 치료용 백신 조성물

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US5792452A (en) 1993-10-04 1998-08-11 Linde; Klaus Live salmonella vaccine having an increased stability
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KR102077701B1 (ko) * 2018-12-18 2020-02-14 전북대학교산학협력단 살모넬라 갈리나룸 사균체를 포함하는 가금 티푸스의 예방 또는 치료용 백신 조성물

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