PREPARATION OF LIVE VACCINES

Abstract

Described is a method for the generation of a live vaccine containing stable bacteria carrying at least three attenuating mutations and a vaccine containing bacteria obtained by said method.

Claims

1. A method for the generation of a stable attenuated bacterial strain carrying three to seven attenuating mutations, wherein said method comprises the following steps: (a) obtaining a starting natural bacterial strain and growing said strain in the presence of a first antibiotic; isolating from the strain of (a) colonies that are dependent on the first antibiotic and have a size of ≤10% as compared to a colony of a corresponding starting natural bacterial strain provided in step (a), wherein mini colony is characterized by colonies having a size of ≤10% as compared to a colony of a corresponding starting natural bacterial strain; (b) growing a clone of (b) in the absence of the first antibiotic and isolating attenuated revertants characterized by a colony size which is ≥50% as compared to a colony of a corresponding starting natural bacterial strain provided in step (a); (c) growing a clone obtained in step (c) in a medium supplemented with a second antibiotic; (d) isolating and serially passaging colonies to identify clones stably being of reduced size but greater than 25% in size as compared to a colony of a corresponding starting natural bacterial strain provided in step (a); and (e) isolating clones of step (e) having reduced colony size as a stable property; wherein the first antibiotic is selected from streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, and nalidixic acid; and wherein the second antibiotic is selected from streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, nalidixic acid, and fosfomycin; to provide a stable attenuated bacterial strain carrying three to seven attenuating mutations; and wherein colony size is characterized by the diameter of the colony.

2. The method of claim 1, wherein the first and second antibiotic is selected from streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, and nalidixic acid.

3. The method of claim 1, wherein the first antibiotic is streptomycin, and wherein the second antibiotic is rifampicin.

4. The method of claim 1, wherein the bacterial strain is selected from the group consisting of Aeromonas species (sp.), Bacillus cereus, Bordetella sp., Campylobacter sp., Escherichia coli, Klebsiella sp., Listeria sp., Ornithobacterium rhinotracheale, Pasteurella/Avibacterium sp., Pseudomonas sp., Riemerella sp., Salmonella sp., Shigella sp., Staphylococcus aureus, and Yersinia sp.

5. The method of claim 1, wherein the bacterial strain is Salmonella or Campylobacter.

6. The method of claim 5, wherein Salmonella mutants selected in steps (a) and (b) are isolated from log phase cultures and as mini-colonies that start appearing after at least or more than 48 h at 37° C. incubation.

7. The method of claim 5, wherein Campylobacter mutants selected in steps (a) and (b) are isolated as mini-colonies that start appearing after at least or more than 72 h at 39° C. incubation.

8. The method of claim 1, wherein the bacterial strain carries at least four attenuating mutations, and steps (a) to (c) are repeated at least once.

9. The method of claim 1, wherein serially passaging comprises at least 30 serial passages.

10. The method of claim 1, wherein the second antibiotic of step (d) comprises a concentration of at least fourfold MIC and up to tenfold MIC.

11. The method of claim 1, wherein the second antibiotic is rifampicin, streptomycin, or fosfomycin and comprises a concentration of tenfold MIC.

12. The method of claim 1, wherein the second antibiotic is fusidic acid and comprises a concentration of fourfold MIC.

13. The method of claim 1, wherein the bacterial strain is Salmonella, the first antibiotic is streptomycin, and the second antibiotic is rifampicin.

14. The method of claim 1, wherein the stable attenuated bacterial strain comprises a degree of attenuation that does not exceed the degree of attenuation of vaccine strains having two metabolic drift attenuating mutations.

15. The Salmonella enterica strain deposited with the German Type Culture Collection (Deutsche Sammlung von Mikrorganismen und Zellkulturen (DSMZ)) on Nov. 27, 2012 under accession number DSM 26682.

16. A vaccine comprising the Salmonella strain of claim 15.

17. The Campylobacter strains deposited with the German Type Culture Collection (Deutsche Sammlung von Mikrorganismen und Zellkulturen (DSMZ)) on Nov. 27, 2012 under accession numbers DSM 26683 or DSM 26684.

18. A vaccine comprising one or more strains according to claim 17.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIGS. 1A-1D show examples of Salmonella vaccine strains characterized by three attenuating mutations.

Incubation of the plates was at 37° C. for about 20 hours and resulted in the following:

[0037] FIG. 1A Salmonella infantis (wild strain/Sm-id 4.22/Rif 2 (and Iet 4));

[0038] FIG. 1B Salmonella virchow (wild strain/Sm-id 9.3/Rif 2);

[0039] FIG. 1C Salmonella hadar (wild strain/Sm-id 4.1/Sm 2 (and Rif 1);

[0040] FIG. 1D Salmonella paratyphi B, variant Java 3.2 (wild strain/Sm-id.1.1/Rif 3 (and Rif 2)).

[0041] FIGS. 2A-2D show examples of Campylobacter vaccine strains characterized by three attenuating mutations Incubation of the plates was at 39° C. for about 48 and 72 hours, respectively and resulted in the following:

[0042] FIG. 2A Campylobacter coli I (wild strain/Sm-id 5.5/Pho 1 and Pho 2);

[0043] FIG. 2B Campylobacter coli II (wild strain/Sm-id 18.1/Sm 2);

[0044] FIG. 2C Campylobacter jejuni I (wild strain/Sm-id 2.3/Pho 1 (and Pho 2));

[0045] FIG. 2D Campylobacter jejuni II (wild strain/Sm-id 2.1/Sm 2).

[0046] FIGS. 3A-3B show examples of Salmonella vaccine constructs characterized by (two), four, five or six attenuating mutations. Incubation of the plates was at 37° C. for about 20 hours and resulted in the following:

[0047] FIG. 3A Salmonella virchow:

[0048] wild strain/Sm-id I 1.4/Sm-id II 0.3/Sm 4;

[0049] wild strain/Sm-id I 1.4/Sm-id II 0.3/Sm-id III 0.x.

[0050] FIG. 3B Salmonella infantis:

[0051] wild strain/Sm-id I 4.22/Sm-id II 0.1/Rif 2;

[0052] wild strain/Sm-id I 4.22/Sm-id II 0.1/Sm-id III 1.1.

[0053] FIGS. 4A-4B show examples of Campylobacter vaccine constructs characterized by (two), four, five or six attenuating mutations. Incubation of the plates was at 39° C. for about 48 hours and resulted in the following:

[0054] FIG. 4A Campylobacter coli II wild strain/Sm-id I 18.1/Sm-id II a.1. This strain is characterized in that the second Sm-id a.1 mutation results in an Sm resistance.

[0055] FIG. 4B Campylobacter coli II wild strain/Sm-id I 17.7/Sm-id II a.1/Phol, wild strain/Sm-id I 17.7/Sm-id II a.1/Sm-id III a.1.

[0056] Thus, the present invention provides a method for the generation of a bacterial live vaccine containing stable bacteria carrying at least three (and up to six or seven) attenuating mutations, wherein said method comprises the following steps:

[0057] (a) providing a bacterial strain and growing said strain in the presence of a first antibiotic, preferably streptomycin;

[0058] (b) isolating from the strain of (a) such “mini” colonies which correspond to clones which are dependent on the first antibiotic;

[0059] (c) growing a clone of (b) in the absence of the first antibiotic and isolating attenuated revertants characterized by a colony size which is ≥50% of the colony size of the wild strain;

[0060] (d) growing a clone obtained in step (c) in a medium supplemented with a second antibiotic that may differ from the first antibiotic (e.g., an aminoglycoside such as streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, and tobramycin; rifampicin, fusidic acid, nalidixic acid, fosfomycin,) having a suitable concentration, preferably an about tenfold MIC;

[0061] (e) isolating and serially passaging colonies showing reduced size (MD A “res”); and

[0062] (f) isolating clones having the graduated reduction of the colony size as stable property.

[0063] The bacterial strain of step (a) is, preferably, obtained from “wild” virulent strains. These strains can be taken from diseased animals (e.g., chicken). The starting natural strains which are used should have a certain degree of virulence.

[0064] The choice of the antibiotic for selecting the mutants of step (a) is guided by reasons of a practical nature. For example, streptomycin is known to lead rapidly to the development of resistant and dependent strains among the micro-organisms.

[0065] Thus, in a preferred embodiment of the method of the present invention the antibiotic of step (a) is streptomycin. However, other aminoglycoside antibiotics such as neomycin, kanamycin, spectinomycin, gentamicin, amikacin, and tobramycin, and rifampicin, fusidic acid and nalidixic acid may also be suitable as the antibiotic of step (a).

[0066] It is known that resistance to the antibiotic can result from different modification mechanisms. In particular, the genetic modification may affect a chromosome of the bacterium. The chromosomal modification is a rare event which, once carried out, ensures the stability of the acquired properties.

[0067] The term “mini colonies” as used herein relates to bacterial colonies characterized by a reduction of size. Preferably, they are characterized by a size of ≤10% of the corresponding wild strain colonies.

[0068] The selection of attenuated bacterial strains as a function of growth criteria on media containing an antibiotic is an operation which has been used for various species with the object, notably, of causing the appearance of strains having reduced virulence.

[0069] The bacterial strains according to the invention characterized by at least three (and up to six or seven) 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 only be developed satisfactorily in the presence of the antibiotic (step (b). For this reason, these strains are said to be dependent on, e.g., streptomycin (Smd mutant).

[0070] In the second place, the strains of step (c) are mutants selected from the antibiotic dependent strains and which have the particularity of being able to develop in the absence of streptomycin due to the introduction of a second attenuating mutation or marker. These strains are called Sm-id strains. Preferably, a washing step is carried out between steps (b) and (c). The preferred medium for step (c) is a Salmonella Caso (SC) medium (e.g., for Salmonella) or a Caso medium (e.g., for Campylobacter).

[0071] Step (d) allows introducing an additional MD antibiotic resistance (“res”) mutation (as a third attenuating marker). Preferably, the antibiotic is streptomycin, rifampicin or fosfomycin. The concentration of the antibiotic in step (d) can be determined by the person skilled in the art according to routine procedures. Preferably, for Salmonella the concentration of rifampicin, streptomycin (note: most Sm-id mutants are streptomycin sensitive and therefore suited for an additional MD Sm “res” marker) and fosfomycin corresponds to an MIC value of about tenfold, and for fusidic acid to an MIC value of about fourfold. Preferably, for Campylobacter, at least 200 μg fosfomycin/ml and at least 100 μg streptomycin/ml, respectively, are used.

[0072] In step (e) the Sm-id/MD antibiotic “res” strains of the previous step characterized by an additional reduced slight colony size are isolated and serially passaged in order to check stability. Preferably, at least 30 serial passages are carried out.

[0073] Finally, in step (f) the clones isolated from step (e) having the graduated reduction of the colony size as stable property are provided.

[0074] In a preferred embodiment of the method of the invention steps (a) to (c) are at least repeated once for the generation of bacteria carrying at least four attenuating mutations. This method allows to generate new attenuating mutants, e.g., according to the following schemes (shown for Sm):

[0075] (a) Smd 1.fwdarw.Sm-id I.fwdarw.Smd a.fwdarw.Sm-id II;

[0076] (b) Smd 1.fwdarw.Sm-id I.fwdarw.Smd a.fwdarw.Sm-id II.fwdarw.Smd α.fwdarw.Sm-id III;

[0077] (c) Smd 1.fwdarw.Sm-id I.fwdarw.Smd a.fwdarw.Sm-id II.fwdarw.Smd α.fwdarw.Sm-id III.fwdarw.MD antibiotic “res”.

[0078] Surprisingly, it was found that strains derived from Sm-id mutants carrying four or even six mutations do not show a higher degree of attenuation, compared to the strains having three attenuating mutations, but an even higher stability.

[0079] The choice of the antibiotic for selecting the MD antibiotic “res” mutant strains according to the present invention is guided by reasons of a practical nature and, in principle, any antibiotic capable of inducing metabolic drift (MD) mutations can be used for the purposes of the present invention, e.g., streptomycin (note: most Sm-id mutants are streptomycin sensitive and therefore suited for an additional MD Sm “res” marker), rifampicin, fosfomycin, fusidic acid or nalidixic acid.

[0080] The method of the present invention is not restricted to particular bacteria. Besides Salmonella sp. and Campylobacter sp., other bacteria such as Staphylococcus aureus, Escherichia coli, Bacillus cereus (Pseudoanthrax), Yersinia sp. such as Y. pestis, Klebsiella sp., Listeria sp., Aeromonas sp., Shigella sp., Pasteurella/Avibacterium sp., Riemerell a sp., Ornithobacterium rhinotracheale, Bordetella sp., and Pseudomonas sp. can also be used for generating bacterial live vaccine containing stable bacteria according to the methods of the present invention.

[0081] However, preferred bacteria are Salmonella and/or Campylobacter, especially Salmonella bongori, the S. enterica subspecies enterica, arizonae, diarizonae, salamae, houtenae and indica, preferably S. enterica subspecies enterica such as the following Serovars: Dublin, gallinarum (biovars gallinarum and pullorum), choleraesuis, Typhisuis, typhi, Paratyphi A,B,C, Abortusequi, Abortusovis, Abony, Enteritidis, Typhimurium, Copenhagen, infantis, virchow, hadar, agona, Newport, anatum, Heidelberg, panama, indiana, Saintpaul, Brandenburg, and Campylobacter coli, Campylobacter jejuni, and Campylobacter fetus.

[0082] In a preferred embodiment of the method of the present invention in steps (a) and (b) Salmonella mutants are isolated from log phase cultures and as mini-colonies that start appearing after at least or more than 48 h at 37° C. incubation.

[0083] In a further preferred embodiment of the method of the present invention in steps (a) and (b) Campylobacter mutants are isolated as mini-colonies that start appearing after at least or more than 72 h at 39° C. incubation.

[0084] The present invention also provides alive bacterial strains obtainable by the method of the invention as well as a vaccine comprising alive bacterial strains of the invention and a biologically acceptable carrier. The vaccinating compositions may of course be constituted by means of freshly cultivated bacteria.

[0085] Preferably, the vaccine composition of the present invention is freeze-dried.

[0086] 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.

[0087] The vaccine of the present invention is administered in an amount suitable for immunization of an individual and may additionally contain one or more common auxiliary agents. The employed term “amount suitable for immunization of an individual” comprises any amount of bacteria with which an individual can be immunized. An “amount suitable for immunization of an individual” may be determined using methods known to one skilled in the art. The term “individual” as used herein comprises an individual of any kind. Examples of such individuals are animals (and humans).

[0088] The administration of the vaccine preferable is the oral route but also injection may be made at various sites of the individual 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.

[0089] 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/colonisation caused by Salmonella or Campylobacter.

[0090] The following strains have been deposited with the German Type Culture Collection (Deutsche Sammlung von 25 Mikrorganismen and Zellkulturen (DSMZ), Braunschweig) on Nov. 27, 2012 under the Budapest Treaty:

TABLE-US-00002 Name Accession Number Salmonella enterica ssp. enterica DSM 26682 Serovar Infantis Smid4-22/Rif2 = Campylobacter coli K2848/11 Smid18/Sm2 = DSM 26683 Campylobacter jejuni K2963/12 Smid2.1/Sm2 = DSM 26684

[0091] The below examples explain the invention in more detail.

Example 1

Materials

[0092] (A) Strains

[0093] Salmonella enterica subsp. enterica serovar virchow,

[0094] Salmonella enterica subsp. enterica serovar infantis,

[0095] Salmonella enterica subsp. enterica serovar hadar,

[0096] Salmonella paratyphi B (var. L-Tartrat+, formerly Java),

[0097] Campylobacter coli, Campylobacter jejuni (provided by Lohmann Animal Health, Cuxhaven, Germany).

[0098] (B) Media

[0099] 1000 ml Campylobacter medium (Caso-medium) contain: 35 g Caso Agar (Sifin), 3 g yeast extract, 3 g casein hydrolysate, 4 g activated carbon, 0.25 g FeSO.sub.4, 0.25 g sodium pyruvate, 5 g agar Kobe (Roth).

[0100] 1000 ml Salmonella medium (SC-medium) contain: 35 g Caso Agar (Sifin), 3 g yeast extract, 1 g glucose, 5 g agar Kobe (Roth).

[0101] (C) Antibiotics

[0102] Streptomycin (Sm) (Roth No. 0236.2), fosfomycin (Pho) (Sigma No. P5396), rifampicin (Rif) (Riemser Arzneimittel AG, Fatol Eremfat 600 mg)

[0103] (D) MIC Values of Wild Type Strains

TABLE-US-00003 Strain Streptomycin rifampicin fosfomycin Salmonella enterica 12.5 12.5 n.d. subsp. enterica serovar Virchow Salmonella enterica 12.5 12.5 n.d. subsp. enterica serovar Infantis Salmonella enterica 25 12.5 n.d. subsp. enterica serovar Hadar Salmonella paratyphi B 30 12.5 n.d. (var. L-tartrate+) Campylobacter coli WS I 1 n.d. 25 Campylobacter coli WS II 1 n.d. 25 Campylobacter jejuni WS I 2 n.d. 25 Campylobacter jejuni WS II 2 n.d. 25 n.d.: not determined

Example 2

Selection and Isolation, of Smd Mutants

[0104] (a) Practice-Orientated Isolation of Smd Mutants of Salmonella

[0105] About 10.sup.10 cfu of a 18 h/37° C. culture of Salmonella were plated on a Petri dish containing SC agar supplemented with 500 μg streptomycin/ml. Besides colonies having normal sizes and single colonies having slightly decreased sizes (virulent Sm resistant clones and MD Sm “res” clones) “mini colonies” (predominantly small colony variants=scv) with varying frequencies—depending on the strain—could be detected. After an incubation time of about ≥48 h (at 37° C.) 1 to 2 additional mini colonies (per about 30 colonies having normal sizes and colonies having slightly decreased sizes) could be detected that could not be distinguished from scv. Depending on the frequency of appearance of the scv phenotype 3% to 20% of these mini colonies could be shown to represent Smd mutants.

[0106] The calculated frequency of the Smd clones in relation to resistant mutants was ≥1%.

[0107] Note: The isolation of Smd clones is achieved by use of Sm sensitive wild type strains as the starting material. Strains preferably have a low MIC value.

[0108] (b) Practice-Orientated Isolation of Smd Mutants of Campylobacter

[0109] Bacterial material obtained from a Caso agar Petri dish culture (24 h/39° C.; about 10.sup.10 cfu) that had been inoculated in such a way that the entire surface of the disc was covered was plated on 1 or 2 Caso agar Petri dishes supplemented with 100 μg streptomycin/ml and incubated for 72 h at 39° C. Depending on the strain ≤10 colonies/plate (average value) having normal sizes and colonies having slightly decreased sizes (streptomycin resistant and MD Sm “res” clones) were detectable. In addition, colonies having a clearly reduced size (diameter is ≤25% of the normal size) with a frequency of about 20%—compared to the colonies having normal sizes and colonies having slightly reduced sizes—could be detected. About one-third of these colonies were Smd clones.

[0110] The calculated frequency of the Smd clones in relation to resistant mutants was 5%.

Example 3

Selection and Isolation of Sm-Id Mutants

[0111] (a) Isolation of Salmonella Sm-Id Mutants from Smd Clones

[0112] About 10.sup.9 cfu (per petri dish) of a washed Smd mutant were plated with SC medium and incubated for 48 h at 37° C. From the attenuated revertants obtained only such mutants were further treated that showed a colony size of about 50% compared to the wild type strain colonies (according to the objective to obtain Sm-id clones having only low attenuation).

(b) Isolation of Campylobacter Sm-Id Mutants from Smd Clones

[0113] Bacterial material obtained from a Caso agar (supplemented with 100 μg streptomycin/ml) Petri dish culture (24 h/39° C.) that had been inoculated in such a way that the entire surface of the disc was covered was subjected to one washing step, plated on Caso medium in a ratio of 1:1 (about 3×10.sup.9 cfu) to 1:4 and then incubated for 72 h at 39° C. Under these culturing conditions the majority of Smd clones showed the development of ≤10 attenuated revertants (on average). Most of these attenuated revertants were Sm sensitive. Generally, Sm-id clones showing a reduced colony size of about ≥50% compared to the wild type strain colonies were further processed.

[0114] Some strains, e.g., Campylobacter jejuni, allowed isolation only from Sm-id having a colony size of ≤50% compared to the wild type strain.

[0115] Note: Not all Campylobacter strains and the Smd mutants derived thereof allow isolation of Sm-id revertants without any problems. However, it is possible to isolate Sm-id revertants also from the problematic strains using, for example, several independent Smd mutants.

Example 4

Isolation of an Additional MD Antibiotic “Res” Mutant

[0116] The incorporation of an additional MD antibiotic “res” mutation in selected Sm-id mutants as third marker for attenuation and recognition was carried out as already described above.

Briefly,

[0117] (a) Salmonella: 10.sup.9-10 cfu of the selected Sm-id clones were incubated on SC medium supplemented with an about tenfold MIC value concentration of rifampicin or streptomycin (as regards fusidine acid the about fourfold MIC value concentration), respectively, and incubated for 48 hours at 37° C.

[0118] (b) Campylobacter: The material of a Petri dish culture (Caso medium) that was inoculated with the Sm-id mutant in such a way that it covered the whole surface and incubated for 24 h at 39° C. was plated at a ratio of 1:4 to 1:8 on Caso medium supplemented with 200 μg fosomycin/ml or 100 μg streptomycin/ml and incubated for ≥72 h at 39° C.

[0119] The colonies showing (more or less) reduced sizes were isolated and subjected to serial passages. About 20% of these clones maintained the clone specifically graded reduction of colony size as a stabile feature.

Example 5

Generation of Vaccine Strains Having 4 or 6 Attenuated Mutations

[0120] The generation of vaccine strains having 4 or 6 attenuated mutations was achieved by sequentially incorporating a second and, optionally, a third Sm-id suppressor mutation into a basic Sm-id I clone: Sm-id I/Sm-id II/Sm-id III.

[0121] (a) Salmonella: About 10.sup.10 cfu of the basic Sm-id I mutant (or the Sm-id II starting strain) were plated on SC medium supplemented with 500 μg streptomycin/ml and incubated for 48 h at 37° C. About 5% of Sm-resistant colonies are Smd mutants (now growing primarily as colonies having “normal sizes”). By use of these Smd clones derived from Sm-id I strains and Sm-id II strains, respectively, Sm-id mutants were again isolated according to the approach described in Example 3a. Clones having the desired reduction of colony size were treated further.

[0122] (b) Campylobacter: The material obtained from a Caso medium Petri dish culture that was inoculated with an Sm-id mutant in such a way that the entire surface was covered and incubated for 24 h at 39° C. was plated at a ratio of 1:4 on Caso medium supplemented with 100 μg streptomycin/ml and incubated for 72 h at 39° C. Besides the about 15 Sm resistant colonies having a “normal size” 2 to 3 small colonies could be detected. 50% of these colonies are Smd clones. These Smd clones (derived from Sm-id I strains and Sm-id II strains, respectively) were used as starting clones—according to Example 3(b)—for again isolating Sm-id mutants. Clones showing the desired reduction of colony size were treated further.

Example 6

[0123] Isolation of an MD Antibiotic “Res” Mutant from Selected Sm-Id II Mutants

[0124] (a) Salmonella: The incorporation of an advantageous MD antibiotic “res” mutation into selected Sm-id If Sm-id II mutants as an additional 5.sup.th attenuation- and recognition marker was carried out analogously according to the approach described in Example 4(a).

[0125] (b) Campylobacter: The incorporation of an advantageous MD antibiotic “res” mutation into selected Sm-id I/Sm-id II mutants as an additional 5.sup.th attenuation- and recognition marker was carried oat analogously according to the approach described in Example 4(b).

[0126] Note: The approach described above can also be used for the additional incorporation of an MD antibiotic “res” mutation into selected Sm-id III mutants (having six attenuated mutations) as 7.sup.th marker for attenuation and recognition. However, this might result in over-attenuation, which might interfere with relevancy to practice.

Example 7

Colony Sizes Converted to Bar Graphs for Prospectively Oriented Evaluation of the Probable Degree of Attenuation

[0127] Suspensions of the corresponding wild type strains and the MD mutants derived from these strains are diluted logarithmically and then plated on culture medium in such a way that per Petri dish 10 to 50 well definable single colonies can be obtained. At least 5 Petri dishes per grade of dilution are prepared in order to compensate for differences in growth due to the medium. Single colonies grown under standardized conditions (e.g., identical times of incubation, identical layer thicknesses of the medium) are photographed. Digital photographs are processed with the CellProfiler program (Broad Institute): The diameters of the individual colonies were determined and saved. After averaging of the values the data are plotted as bar graphs in relation to the sizes of the wild type strain colonies (given as 100%).

Example 8

[0128] Preparation of Vaccines from Suitable Vaccine Strains and Use for Vaccination of Chicks/Chicken and Further Hosts to be Protected, Respectively

[0129] For the preparation of live vaccines vaccine strains harbouring three (four, five and six, respectively) attenuating mutations were grown in common liquid media up to logarithmic phase. Vaccine suspensions and vaccine sediments, respectively, were supplemented with a suitable stabilisator and subsequently lyophilized. The vaccines obtained were administrated (according to the kind of indication one, two or three doses) by oral or parenteral administration.