CA2131231C - Salmonella live vaccine - Google Patents

Abstract

Use of at least one attenuated immunogenic salmonella live vaccine strain comprising a marker (envelope marker) having a sensitivity towards macrolide antibiotics, for the production of a live vaccine for a specific host which, in the case of an infection caused by the specific host, facilitates an effective therapeutical treatment of the infected host by means of macrolides.

Description

Salmonella live vaccine The invention relates to a special use of a salmonella live vaccine, to new salmonella live vaccines not having been used before, to a method of producing such vaccines as well as to suitable salmonella vaccine strains.

The majority of salmonella-conditioned Gastroenteritis - 10 infectiosa of humans are caused by contaminated animal products. Especially chicken and chicken eggs, respectively, being infected with the at present pre-dominantly occurring serovar Salmonella enteritidis have increasingly been causing infections, recently.
Nevertheless, generally all food stuffs are affected which originate from animals kept in mass-rearing.
Here, normally many animals are kept in confined space, promoting the spread of infections among the animal stock.

~ 22l 31 231 The risk of a transmission of the infection from the infected animal to humans can be reduced by customary veterinary medical measures for the interruption of infection chains. Furthermore, thorough compliance with kitchen hygiene regulations during the processing of contanimated animal products can prevent a transmission to humans. However, especially the latter regulations are not always being considered during the storage and processing of food. Therefore, it is imperative to rule out the possibility of infected animals being processed right from the beginning. This can be achieved e. g.
through a vaccination of the animal stock against salmonella infections.

Suitable salmonella live vaccines have to comply with various different conditions:

1. The virulence of the vaccine strains used in the production of the vaccines has to be adjusted in a way that guarantees a non-apparent infection on the one hand, and a sufficient persistence of the ~ vaccine strains in the host ~tissue on the other hand, as a prerequisite for high immunogenicity.

2. Furthermore, the stability of the vaccine strains used with respect to their virulence and their protective properties has to be widely assured, i. e. it has to be assured that they do not mutate back into the virulent wild strain.

3. To allow for the reduction of the probability of infections it should be ascertained that the vaccine strains are not permanently being excreted alive and that they can only survice for a short period of time in the environment, respectively.

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The above-mentioned three conditions, which a live vaccine has to comply with, are to be discussed in detail in the following. As described in 1., the production of a suitable salmonella live vaccine is based on a reduction of the virulence (attenuation) of the pathongenic salmonella and simultaneous preserva-tion of their antigen structures, and thus, the immuno-genic effect in the host. One possibility is e. g. to employ deletion mutants, e. g. pur or aro auxotrophic clones, as vaccine strains. The attenuation level of these vaccine strains depends upon the lack of meta-bolites in vivo, which possibly impedes an accurate adaptation to the host to be immunized. In this respect, it is referred to the EP ~ 263 528 in which stable asp mutants of Salmonella typhimurium with different virulence reduction levels are described.
Vaccine strains with attenuation levels adapted to each of the different host species can be produced by selec-ting suitable asp mutants.
A further possibility for an attenuation consists of the employment of vaccine strains,~the virulence reduc-tion of which can be traced back to a metabolism drift mutation (called stwd mutation or marker in the following). The term "metabolism drift" comprises all essential enzymes and functionally important cell compartments, respectively, having been functionally altered by mutations, as e. g. ribosome proteins, gyrase, RNA polymerase, permease, wherein, as a result of these mutations, translation, DNA replication, DNA
transcription or permeation are more or less distinctly disturbed. Such stwd mutants, furthermore, show a resistance with respect to specific antibiotics and other substances (noxious substances). Stwd mutants can easily be obtained in laboratories as chromosomal anti-, 213123~
~ 4 -biotic resistance mutants. In this respect, from the EP 0 263 528 e. g. stwd mutants with a resistance against nalidixic acid INal), streptomycin (Sm) or rifampicin (Rif) are known. Especially if several stwd markers are incorporated into one vaccine strain (double or tripple marker vaccine strain), de facto unlimited possibilities are obtained for the production of a desired attenuation level adapted to suit every specific host species.
With respect to the prior art "attenuation by means of stwd mutations" it is referred to the following publi-cations: DD-WP 155 294; DD-~P 218 834; DD-WP 235 828;
D~-WP 253 182; DD-WP 253 184; DD-WP 281 118; DD-WP
294 ~20; EP 0 263 528.

A further (mentioned above under 2.) condition is that the attenuated vaccine strains obtained by mutation do not mutate back into the virulent wild strain. The required stability can, on the one hand, be achieved by only employing vaccine strains with which no reversions can be detected in vitro or whose3reversion ratios are < 10'. A further possibility is to employ vaccine strains comprising several mutations which indepen-dently reduce virulence. Here, the probability of aback mutation can almost be excluded.

The final condition, mentioned above under 3. in connection with the term "interruption of infection chains", especially concerns the risks with respect to a possible excretion and permanent survival of the vaccine strains outside the vaccinated host. In this respect, it is desireable to reduce the excretion and the capability of survival of the vaccine strains in the environment. To guarantee a sufficient immune 2l3l23l _ 5 response, on the other hand, the capability of temporary survival of the vaccine strains in the host tissue after e. g. oral or parenteral application should only be slightly impaired or not at all. Vaccine strain mutants complying with such requirements are known e. g. from the DD-WP 218 836, DD-WP 231 491, DD-WP 253 182, DD-WP 253 183, DD-WP 253 184, and EP
0 263 528. In the prior art it is suggested to optimize suitable vaccine strains by employing so-called anti-epidemic markers for the reduction of excretion and thecapability of survival in the environment. The term antiepidemic marker characterizes outer envelope muta-tions in a broader sense, causing a functional varia-tion of the permeability barrier in the outer membrane.
Vaccine strains can be provided with different anti-epidemic markers depending upon the intended applica-tion ~orm. The antipidemic markers known at present are divided into three groups, depending on the alterations they cause in the outer membrane of the vaccine strain.
The first group comprises the so-called hst markers.
The incorporation of an hst marke~ causes the vaccine strain to become highly sensitive towards bile, anionic detergents, macrolide antibiotics and other noxious substances. Owing to the high sensitivity towards bile, there is a reduced excretion with faeces caused by the inactivation of the vaccine strains already occurring in the intestinal lumen. If vaccine strain bacteria are excreted, they only have a shortened survival time in the environment, due to the lack of the permeability barrier in the outer membrane against tensides and macrolides and other noxious substances. Therefore, an infection can almost be excluded when using vaccine strains including hst markers. When employing the ususal doses of vaccine, vaccine strains comprising hst 2I3I23l ~ 6 .

markers can only be applied parenterally, however. If applied orally, due to the high sensitivity towards bile, the virulence is influenced to such an extent that a sufficient immune response can only be achieved by employing extremely high doses of vaccine. There-fore, the solution for an oral application would be to provide vaccine strains including an antiepidemic mar-ker from one of the other two known groups. One group comprises the so-called rbt markers (reversion to bile tolerance). The rbt marker can be obtained by mutation from the hst marker. It provides the vaccine strain with an antiepidemic potency just as the hst marker does. However, in contrast to the hst marker the vaccine strain comprising an rbt marker is tolerant towards bile, and can therefore be applied orally without a reduction of the virulence impairing the vaccination effect. The same stands for a further group, the so-called rtt marker (reversion to tenside tolerance). The rtt marker can be obtained by mutation from the rbt marker. The vaccine strain comprising the rtt marker is tolerant towards tensides and simulta-~ neously possesses a sufficient antiepidemic potency due to the remaining high sensitivity towards macrolides and other noxious substances. Also the rtt marker strain can be applied orally without any problems.

Taking this information and these publications as abasis, live vaccines can be produced which comply with all conditions required. The adaptation to the respective host can be effected, e. g. in animal test serles.

_ 7 A further problem remains to be solved. Even the compliance with all precautions does not exclude the possibility of a person dealing e. g. with the vaccination of the animals or the production of the vaccines, coming into contact with the in fact attenuated but, nevertheless, still pathogenic salmonella vaccine strains. Where healthy people are concerned, there is hardly any risk of an infection. However, if the immune system of people is weakened (e. g. by an HIV-infection), the contact with such vaccine strains can result in a salmonella infection.

Therefore, preferred objects of the invention include providing a live vaccine against salmonella infections, starting from the known prior art, being optimally attenuated for the host to be immunized, providing it with an immunity for reducing the excretion of wild strains when applied orally or parenterally, and constituting only a minor risk for people with a weakened immune system, or none at all. A further object of the invention is to provide a method for producing salmonella live vaccines optimally suited to the respective host by employing significantly less animal experiments than the conventional methods. Finally, the invention is to provide salmonella live vaccine strains for live vaccines suitable for chickens.

In one aspect, this invention relates to a salmonella live vaccine comprising an attenuated immunogenic live salmonella vaccine strain wherein the vaccine strain has an envelope marker which confers increased sensitivity towards an antibiotic selected from the group quinolons, chloramphenicols and tetracyclines wherein said antibiotic facilitates an effective therapeutical treatment of a host infected with salmonella as a result of contacting a host vaccinated with said vaccine.

In another aspect, this invention relates to a salmonella live vaccine comprising an attenuated immunogenic salmonella live vaccine strain, wherein the vaccine strain has an envelope marker which confers on the vaccine strain an increased sensitivity towards an antibiotic selected from the group quinolons, chloramphenicols and tetracyclines.
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7a Yet in another aspect, this invention relates to use of an attenuated immunogenic salmonella live vaccine strain comprising an envelope marker which confers on the vaccine strain increased sensitivity towards a therapeutically effective antibiotic, for the production of a live vaccine for a specific host wherein said antibiotic facilitates an effective therapeutical treatment of a host infected with salmonella as a result of contacting said specific host vaccinated with said vaccine.

This invention also relates to a live vaccine produced from an attenuated immunogenic salmonella live vaccine strain for immunization of poultry against salmonella infections wherein the vaccine strain is in accordance with the invention and has a generation time of about 28 to 34 minutes.

This invention further relates to a method for the production of a salmonella live vaccine optimally suited to a host serotype in its attenuation level and produced from an attenuated immunogenic salmonella live vaccine strain, comprising determining a prolonged generation time of salmonella live vaccine strains suited to the host serotype from a set of graded prolonged generation times and selecting vaccine strains having maximum possible attenuation/prolonged generation time and optimal reduction of excretion of wild strain for use as an attenuation equivalent, wherein said specifically prolonged generation time is determined in a single test.

This invention also relates to salmonella vaccine strains having generation times of 28 to 32 minutes and their use as live vaccines in accordance with the invention for immunization of poultry.

A known salmonella live vaccine strain (see e. g. EP 0 263 528) may be used for the . ~

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production of a specific live vaccine. The known salmo-nella live vaccine strains comprise an envelope marker as well as an attenuation marker (e. g. auxotrophy marker or stwd marker) providing them with an anti-epidemic potency (reduced excretion by the host andreduced survival rate in the environment, respec-tively). The envelope marker being employed there, furthermore, causes a sensitization of the vaccine strains towards macrolide antibiotics. This antibiotic sensitization has, hitherto, merely been employed for the selection of suitable envelope mutants, i. e. in the production of vaccine strains. According to the invention it has been recognized for the first time that the macrolide sensitivity of the vaccine strains comprising an envelope marker can also function as a safety mechanism when employing the vaccine strains produced in this manner. The vaccine is to be designed such that, in the case of it causing an infection of another host, an effective therapeutical treatment of the infected host can be carried out by means of macro-lides. This aim is achieved relatively easily by selec-ting only such vaccine strains (provided with an enve-lope marker) for the production of the vaccine whose propagation can be controlled by the application of justifiable doses of macrolide antibiotics.

The known salmonella live vaccine ~rains show a sensitiv-ity toward~ macrolides, due to their envelope marker.
In addition to envelope mutants having an increased sensitivity towards hydrophobic antibiotics (macro-lides, e. g. erythromycin) also other envelope mutants are described (i. a. Hancock, R. E. W.: Ann. Rev.
Microbiol. 1984, 38, 237-264), occasionally having different permeabilities with respect to a sensitivity 3 ~

towards hydropilic, hydrophobic and polycationic antibiotics.
Such envelope markers have been insignificant in the production of bacterial live vaccines, hitherto.

In a preferred embodiment, the present invention relates to salmonella live vaccines which are produced by including for the first time at least one attenuated live vaccine strain having an envelope marker providing the vaccine strain with an increased sensitivity towards a specific therapeutically effective antibiotic with the exception of macrolides. The live vaccine strains being employed in the production of the salmonella live vaccine according to the invention, therefore, preferably comprise an envelope marker generally providing them with an antiepidemic potency (interruption of infection chains) and, optionally, a sensitivity towards macrolides, but, in any case, an increased sensitivity towards another specific therapeutically effective antibiotic. The vaccine strains can be detected relatively easily by employing the selected specific therapeutically effective antibiotic, so that the production of a vaccine strain provided with a suitable envelope marker does not create a great problem.
It is understood that with respect to an optionally required possibility of treatment for an unwanted infection caused by the vaccine, preferably such antibiotics are selected which constitute good results with respect to the salmonella serovars employed.

Furthermore, it is understood that the selected live vaccine strains are derived from predominant ser~v -~

., ~, As mentioned above, there are various possibilities forthe attenuation of live vaccines. One possibility is to include an auxotrophy marker (e. g. asp-) into the vaccine strain. Preferably, the vaccine strain is provided with at least one chromo-sam~ antibiotic resistance mutation. The term chromo-somal antibiotic resistance substantially comprises the above-mentioned stwd markers. It has been proven that by employing selected stwd markers and by a specific combination of several such markers, respectively, the attenuation level of the vaccine strain can be adapted optimally. Upon the selection of such chromosomal anti-biotic resistance mutations for the attenuation of vaccine strains, it has to be secured that they do not exclude the increased sensitivity caused by the enve-lope marker towards the therapeutically effective anti-biotic. Therefore, during development of the vaccine strain it is sensible to firstly determine which thera-peutically effective antibiotic is to be employed for the possibility of treatment of the vaccine strain.
Dependent upon this, the vaccine strain and its chromo-somal antibiotic resistance mutation, respectively, are selected for the attenuation of the vaccine strain.

According to a further preferred embodiment of the invention, the envelope marker is selected such that it provides the vaccine strain (besides an antiepidemic potency and an optional macrolide sensitivity) with an increased sensitivity towards an antibiotic of the group including quinolons, chloramphenicols or tetra-cyclines. Especially preferred is an envelope marker providing the vaccine strain with an increased sensiti-vity towards the antibiotic ciprofloxacin, presently the most effective antibiotic against salmonella.
During the production of the latter vaccine strain, it is especially advantageous to provide a metabolism drift mutation, resulting in a streptomycin and/or rifampicin resistance, for the attenuation. These anti-biotic resistances do not interfere with a possible sensitivity of the vaccine strain towards the anti-biotic ciprofloxacin.

Depending on the desired effective spectrum, the salmo-nella live vaccine according to the invention can be produced from one or several vaccine strains of diffe-rent serovars of the O-groups B (e. g. Salmonella typhimurium), D (e. g. Salmonella enteritidis), C
(e. g. Salmonella infantis) and E (e. g. Salmonella anatum) as mono, bi, tri or tetra-vaccine.

A further proble~, already mentioned above, is consti-tuted in that, due to different sensitivities, a spe-cifically adapted live vaccine has to be provided forevery host. In this respect, it is referred to the already traded Salmonella typhimurium vaccine "Zoo-saloral Dessau" which sufficiently immunizes calves after a single oral application, however, does not protect chickens against an i. p. toxic infection until three times oral vaccination (Linde, R. et al.: Vaccine 1990, 8, 278-282). With respect to the sensitivity towards Salmonella typhimurium, dependent upon the host species, a hierarchy mice > calves > chickens can be constituted. From this it follows that e. g. Salmonella typhimurium vaccine strains for chicks and chickens have to have a lesser attenuation level (in comparison with mice) for the compensation of the lesser sensiti-vity. It can be assumed that the same correspondingly stands for other salmonella serovars.

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It has been found that the generation times of salmonella live vaccines generally correlate with their attenuation levels. This correlation between generation time and attenuation level especially occurs if the vaccine strains have been provided with a chromosomal resistance mutation for the attenuation.

The relation between generation time and attenuation level permits a relatively reliable selection of vaccine strains being suitable for a specific host without prior animal experiments. Salmonella live vaccine strain of the present invention for the immunization of chicks and chickens, have a generation time of about 28 to 34 minutes. Vaccine strains with such generation times have a lower attenuation level (in comparison with the vaccine strains of calves and mice), compensating the small sensitivity of chicks/chickens towards e. g. Salmonella typhimurium and other salmonella serovars.

As mentioned above, an effective salmonella vaccine is especially interesting with respect to the host species chicks/chickens. The Salmonella typhimurium stwd mutants, having been deposited/published hitherto, have no direct relation to this host species. Preferred embodiments of the present invention are directed to vaccine strains which can be optimally attenuated for chicks/chickens, or have already been optimally attenuated. In this respect, the vaccine strain S.tm Nal 2/Rif 9/Rtt is emphasized which is optimally attenuated for chicks/chickens. The same stands for the vaccine strain S.tm Nal 2/Rif 9 which has not been deposited, yet, with respect to this application, but still is to be commented on. The vaccine strains cited have a generation time of about :' :

32 minutes. From this fact the "attenuation equivalent generation time" for the selection of optimally attenuated other strains of the same or other serovars has been derived. Nevertheless, with respect to the selection of suitable vaccine strains for chicks/
chickens generation times vary from 28 to 34 minutes.
This diversity acknowledges the fact that chicks/
chickens have different sensitivities towards different strains and strain-dependent differences in virulence of each serovar, respectively. By means of a few test series, from the preselected vaccine strains having generation times between 28 and 34 minutes those can be chosen which are optimally attenuated for chicks/
chickens. Advantageously, this at least allows for the animal experiments required in the preselection to be dropped.

Furthermore, with respect to the possible host chicks/
chicken it is especially interesting if, as proposed according to the invention, a salmonella live vaccine is used comprising a vaccine strain with an increased sensitivity towards a specific t~erapeutically effec-tive antibiotic. Chickens have a relatively short life span in comparison with other host species, as e. g.
humans, calves or piglets. In general, they are slaugh-tered after a comparatively short rearing period and sold as frozen or fresh meat. Since the vaccination has not taken place very far in the past, it can be assumed that there are still live salmonella vaccine strain bacteria within the chickens at the time of slaughter.
These can then find their way into the environment. For the normal population the small amounts of germs in question are insignificant. The risk of an infection can almost be excluded in this respect. Nevertheless, in individual cases risk patients with weakened immune , CA 02131231 1998-09-29 systems (e.g. people wlth the HIV-vlrus) can be lnfected and react wlth clinical symptoms. Especially with respect to these people, it is requlred that an lnfectlon caused by the vacclne strain may be controlled rapidly and without any problems. With respect to this, the lncorporatlon of a sensitivlty towards therapeutically effective antibiotics as a safety and therapy marker is a sensible alternative, excludlng all theoretlcal reservatlons.

The prototype of such a safety and therapy marker is the so-called ssq marker. Vaccine strains having an ssq marker possess a hypersensltlvity towards qulnolons, especlally towards clprofloxacin, at present, being the most effectlve antibiotic agalnst salmonella. Also the ssq marker, llke the above-mentloned hst, rbt and rtt-mutations, is an envelope mutant and, therefore, also has a more or less distinctive antiepidemic potency - dependent upon lts bile and anionic detergent tolerance and sensltlvlty, respectlvely.

The lnvention not only deals with the production of especially safe llve vaccines. A further ob~ect is to provlde a method for the productlon of a llve vaccine optimally attenuated for a specific host employlng hardly any anlmal experiments.

The principle of the method of the present invention ls based on the fact that an attenuatlon of vacclne strains (especially wlth respect to the lncorporatlon of stwd markers) leads to a prolongatlon of the generatlon tlmes ln comparlson wlth the wlld strain. It has already been mentioned above that the prolonged generation tlmes may allow for a concluslon ~_ 15 ~ 7 ~

with respect to the attenuation level of the vaccine strains. In conformity with the method according to the invention, it is therefore sufficient to determine the specific prolonged generation time of a vaccine strain suitable for a host in a single test (e. g. an animal experiment). The prolonged generation time determined such can then serve as an approximate figure for the following selection of further vaccine strains (of the same serotype), or it can be transferred to other sero-types of the same genus as an attenuation equivalent.

For the production of a vaccine optimally suited to chicks/chickens, e. g. a vaccine strain is selected, the generation time thereof beinq prolonged to about 28 to 34 minutes in comparison with the wild strain (22 minutes). The vaccine strain employed may e. g. be attained from a wild strain having been provided with a streptomycin (Sm) or nalidixic acid (Nal) marker.
Through this, a prolongation of the generation time from 22 minutes to 2S to 29 minutes is achieved. Subse-quently, a rifampicin (Rif) marker is incorporated as a further marker prolonging the generation time.

Preferred methods of the invention are directed to the different possibilities with respect to the selection of the serovars, the stwd markers used for the attenuation, and the sequence of the marker incorporation.

According to such methods a vaccine can be produced - in a first step, by isolating stwd mutants of a phenotype having a specific antibiotic resistance ~ . .;

21~1231 ~ ~ - 16 -and showing a generation time being prolonged by about 3 to 6 minutes in comparison with salmonella wild strains, for the purpose of producing strains with small or moderate attenuation, - in a second step, by again isolating stwd mutants of another phenotype with an antibiotic resistance from these lesser or moderately attenuated single marker strains, again having a generation time being prolonged by an additional 3 to 6 minutes, whereby a set of vaccine strain candidates is being achieved having prolonged generation times graded from about 28 to 34 minutes (wild strain about 22 minutes) - which with respect to S.tm in the mouse model ~since the logarithm of an LD~o correlates linearly with the (prolonged) generation time) corresponds to graded LD~o values of about 10~ to 10' (wild strain about 101) cfu - then once orally immunizing s 36 hours old chicks with 109 cfu of the individual vaccine strains of this set, after two weeks orally infecting the immunized animals with 106 cfu of the wild strai~, and finally favou-ring the S.tm Nal 2/Rif 9 strain with a (prolonged) generation time of about 32 minutes ~and an i. p.
LD~o mouse of about 106 cfu3 as the favourite prototype vaccine strain with respect to the relation of "maximum possible prolongation of gene-ration time/attenuation level and yet optimal reduction of the excretion of wild strain", for the determination of the "attenuation equivalent (prolonged) generation time" for other strains of the same serovar and serovars with only moderate or lacking virulence towards mice, respectively.

- in a third step, by incorporating an "ssq safety and therapy" marker into the double marker vaccine strains attenuated by means of stwd mutation, for the optimization of the vaccine strains/increase of the acceptance, the safety and therapy marker approximately quadrupling the sensitivity towards ciprofloxacin (chloramphenicol, doxycycline, etc.) and simultaneously insignificantly reducing the excretion and capability of survival in the envi-ronment.

The sequence described is arbitrary, and noxious sub-stance resistance phenotype~ (DD-WP 235 828) may also be used.
Further~ore, the invention preferably relates to salmonella vaccine strains, having generation times of 28 to 32 minutes, as well as to variants on these vaccine strains having higher or lower attenuation levels and generation times between 28 and 34 minutes. Finally, the invention also relates to the use of such vaccine strains for the oral immunization of chicks as well as the oral and parenteral immunization of chickens against salmonella infections.
The vaccine strains (with or without ssq markers) referred to in the claims and the following examples (as well as having been deposited) are examples for all the vaccine strain candidates with graded attenuation levels which may be produced. They are suitable for the production of immunogenic live vaccines, especially for chicks/chickens according to propagation methods known as such. With respect to the specifically stated va~cine strains having ssq markers (and deposit members 8433, 8435, 9362, 8434, 8441, 8432), generation times of -respect to generation time is not to be understood a~ a restriction. Taking strain-dependent differences in virulence (invasive capacity/coloni2ation activity) into account, generation times of 2 8 to 34 minutes are also possible as attenuation equivalents.

In the following table the salmonella vaccine strains favoured for the different serovars are listed.

salmonella vaccine ~trains laboratory deposit serovar clone number number*~
typhimuriu~ Ssq/S~ 60/Rif 42 4242 DSM 8433 enteritidi S q/Sm 24/Rif 12 4266 DSM 8435 Ssq/Sm 24/Rif lZk 4298 DSM 9362 Ssq/Sm 24/Rif 12g 4297 D5M 9361 S~Sm 24/Rif 3 4296 D5M 9360 infantis Ssq/S~ 153/Rif 7 4289 DSIS 8434 anatun Ssq/S~ 81/Rif 21 4279 DSM 8441 typhimuriu~ Nal 2/Rif 9/Rtt 4223 DSM 8432 *) the microorgani~ ere depositet at:
DSM - Deutsche Sa~ lung von ~ ro-organi~e~ unt Zell~ulturen G~b~
Mascheroder ~eg 1 B
D-38124 Braun~ch~eig The deposits were made on August 4, 1993 for DSM 8432, 8433, 8434, 8435 and 8441, and on August 11, 1994 for DSM 9360, 9361 and 9362 In the fo~lowing the invention is to be described in detail by several examples of embodiments.

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ExamPles of embodiments Material and method Strains used - wild strains . S.typhimurium (S.tm) 415 (Metschnikov-Institute, Moscow~
i.p. LD~o mouse ~ 10' cfu, generation time ~ 22 min.
. S.enteritidis (S.ent) 318 (Prof. Selbitz, Univer-sity of Leipzig) i . p . LDw mouse ~ 10~ cfu, generation time ~ 22 min.
. S.infantis (S.inf) (Dr. Beer, Veterinarunter-suchungsamt Chemnitz) generation time ~ 22 min.
. S.anatum (S.ana) (Dr. Beer, Veterinarunter-suchungamt Chemnitz) generation time ~ 22 min.

- wild strains having a neutral nalidixic acid and streptomycin resistance for the detection of the reduced excretion upon immunized chicks/chickens . S.tm Nal/Sm, generation time ~ 22,5 minutes . S.ent Nal/Sm generation time ~ 22,5 minutes . S.inf Nal/Sm generation time ~ 22,5 minutes . S.ana Nal/Sm generation time ~ 22,5 minutes - wild strains as examples for other double or tripple marker mutants with lower (or higher) attenuation/
correspondingly less or more prolonged generation time . S.tm Ssq/Sm 60/Rif 42, generation time ~ 31 min.
laboratory no. 4242; deposit no. DSM 8433 . S.ent Ssq/Sm 24/Rif 12, generation time ~ 32 min.
laboratory no. 4266; deposit no. DSM 8435 . ~.~n~ ~q/~M 2~ 2~ n~ n ~im~_ ~2 m~n.
laboratory no. 4298; deposit no. DSM 9362 213123~L
_~ l9a . S.ent Ssq/Sm 24/Rif 12g, generation time ~28 min.
laboratory no. 4297; deposit no. DSM 9361 . S.ent Ssq/Sm 24/Rif 3, generation time ~30 min.
laboratory no. 4296; deposit no. DSM 9360 . S.inf Ssq/Sm 153/Rif 7, generation time x31 min.
laboratory no. 4289; deposit no. DSM 8434 2I~123 ~
~ 20 . S.ana Ssq/Sm 81/Rif 21, generation time ~ 32 min.
laboratory no. 4279; deposit no. DSM 8441 . S.tm Nal 2/Rif 9/Rtt, generation time ~ 32 min.
laboratory no. 4223; deposit no. DSM 8432 as a prototype strain (with optimal attenuation for chicks/chickens) for the determination of the - "attenuation equivalent (prolonged) generation time".

Nutrient media - Nutrient agar (SIFIN, Berlin-Wei~ensee) - Tryptose phosphate broth (Difco, U.S.A.) Antibiotics - nalidixic acid (CHINOIN, Budapest) wild strains MHK 6.2 ~g~ml - streptomycin (Jenapharm) wild strains MHR 6.2 ~g/ml - rifampicin (UBM, Bucarest) wild strains MHR 12.5 ~ug/ml - ciprofloxacin (Bayer) wild strains MHR 0.05 ~ug/ml - chloramphenicol (Berlin-Chemie) wild strains MHR 2.0 ,ug/ml~5 - doxycycline (Jenapharm) 3 wild strains MHR 4.0 ,ug/ml - erythromycin (Abbott) wild strains MHR 60.0 llg/ml Test animals and conditions of keePinq Chicks of hens laying brown eggs from different breeding stations were kept in cages by 5-10 animals and fed with turkey feed as well as water ad libitum.

Oral immunization Groups of 10 chicks each had a single dose of 109 cfu, partly 108 cfu, of the respective vaccine strain applied orally into their gullets, either 36 hours after hatching or (for the purpose of comparison and 2I3123 ~

determination of the optimal immunization age) on the fourth day of their lives. The immunization under practical conditions is also possible via the drinking water (after water deprivation for 4 hours an amount of drinking water of 2 ml/chick is taken in within 3 hours).

Oral infection Two to four weeks after the oral immuni~ation the chicks orally received 106 cfu (or 107 cfu) of the respective neutrally marked homologous wild strain by means of a pipette.

Detection of the excretion of the - vaccine strain S.tm Ss~/Sm 60/Rif 42, S.ent Ssq/Sm 24/Rif 12, S.inf Ssq/Sm 153/Rif 7 and S.ana Ssq/Sm 81/Rif 21:
nutrient media containing 100 ~g rifampicin and 200 ,ug streptomycin/ml;
- vaccine strain S.tm Nal 2/Rif 9 with or without rtt marker:
nutrient media containing 1~0 ~g rifampicin and 12.5 ~g nalidixic acid/ml;
- neutral Nal/Sm marked wild strains:
~ 25 nutrient media containing 100 ,ug nalidixic acid and 200 ~g streptomycin.

Per chick group and day of examination, five fresh stool samples each were suspended in 2 ml physiological sodium chloride solution and, additionally, dilutions from 10-1 to 10-4 were produced.

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- quantitative determination: 0.1 ml of the original suspension and the dilutions were applied onto nutrient agar with each 1% lactose and saccharose, 0.015% bromthymol blue (determination of the number of coli/enterobacteria germs) by means of a spatula. Parallel to this, both were applied onto the same medium containing the respective anti-biotic. As a quantitative measure for the excretion and the reduction of salmonella colonization occur-ring in immunized chicks in comparison with the controls, the number of salmonella colonies vs. the number of enterobacteria colonies was determined in thousandths.
- qualitative determination: an antibiotic bouillon was added to the remaining original suspension.
After incubation for 24 hours at 37~C, the salmo-nella were transferred onto nutrient agar contain-ing the respective antibiotic additives.

The confirmation of the grown salmonella was effected serologically, biochemically, and through the determi-nation of the markers.

As a quantitative measure for the excretion and the reduction of salmonella colonization occurring in immu-nized chicks in comparison with the controls, the number of salmonella colonies vs. the number of entero-bacteria colonies was determined in thousandths.

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Example 1 S.tm: Isolation of spontaneous chromosomal antibiotic resistance clones as (Nal-twd single and) Nal/Ri-stwd double marker strains having graded prolonged genera-tion times between about 29 to 34 (wild strain about 22) minutes for the isolation of a prototype vaccine strain for the determination of the Nattenuation equivalent (prolonged) generation time" for strains of the same serovar and serovars having only moderate (~.ent~ or lacking (S.inf and S.ana) virulence towards mice, respectively.

109 (and 101~) cfu of the wild strain are transferred onto nutrient agar containing 100 ug (or in two steps of 50 ~g and afterwards 400 llg) nalidixic acid/ml by means of a spatula and incubated for approximately two days at 37~C. The resistance clones are transferred onto nutrient agar, controlled with respect to obtained resistance, and the less or more reduced extinction is determined (Spekol with tube samples, Zeiss-Jena, wave length 650 nm, starting germ count 107 cfu, incubation in shaking water bath for 3 hours ~t 37~C). The genera-tion times of clones appearing to be suitable are measured by means of the Abbott MS-2 test system. As described above, on nutrient agar with 400 ~g rifam-picin/ml, resistance clones are attained from clone Nal 2 having a generation time of about 28 minutes. The generation times of these resistance clones are deter-mined, and the Nal 2/Rif clones having graded prolonged generation times between about 29 and 34 minutes are used for the determination of the approximate figure (prolonged) "generation time as an attenuation equiva-lent" ~see example 3).

~ _ 24 -Example 2 Detection of obtained colonization activity of the neutrally Nal/Sm marked S.t~, S.ent, S.in~ and S.ana wild strains in 17 to 25 days old chicks.

17 to 25 days old chicks orally received 106 (or 10') cfu of neutrally marked wild strains by means of a pipette. During a period of 10 to 15 days the quantita-tive salmonella colonization density was determined in comparison with the number of enterobacteria germs.

In the selected infection model (dose 106 to 10' cfu), after oral infection, either the excretion of the neutrally marked wild strains with high germ counts already occurs after 24 hours, or the salmonella germ counts gradually only reach their highest figures in the stool between the third and the sixth day (maximum figures at a 50 thousandth of the total enterobacteria flora). After eight to ten days, the number of salmo-nella colonies drops to a 1.0 to 0.1 thousandth of the enterobacteria flora, and remains in this range for a longer period of time.

This colonization dynamism demonstrates that the ~ 25 neutrally Nal/Sm marked wild strains are suitable for the determination of a reduced wild strain colonization in immunized chicks.

~ _ 25 =

Example 3 Determination of the S.tm Nal 2/Rif prototype vaccine strain having a (prolonged) generation time/attenuation level optimally suited to chicks/chickens by means of the relation of maximum possible prolonged generation time/attenuation level and yet optimal reduction of excretion of wild strain, for the purpose of the deter-mination of the "attenuation equivalent (prolonged) generation time " for strains of the same serovar and of serovars having only moderate or lacking virulence towards mice, respectively.

Chicks at the age of s 36 hours were immunized orally with 109 cfu of the different strains from the set of S.tm double marker strains having graded generation times between 29 and 34 minutes and, after two weeks, were infected orally with 106 cfu of the wild strain.
Parallel to this, control chicks of the same age were infected orally.
In comparison with the controls, the immunized chicks which received the vaccine strains having graded gene-ration times between 29 and 34 minutes, show a signifi-cantly reduced excretion of the wild strain (determined ~25 from the enterobacteria colonization density in thousandth), especially in the first five to ten days after the oral challenge. The variation (reducing with time) lies within the range of one to two tenth powers.
From the sixth to the tenth day after the oral challenge the salmonella colonization densities in immunized chicks come into closer alignment with those of the controls (in the 0.1 thousandth range of entero-bacteria germ counts). In individual cases, however, immunized chicks may show a reduced excretion in the range of one log step, even after the tenth day.

~I31231 .

With respect to vaccine strains having generation times of 2 33 minutes the excretion of wild strain is reduced significantly less, wherein the difference to the controls generally does not exceed a tenth power. On the basis of the relation: "maximum possible prolonged generation time/attenuation level and yet optimal reduction of excretion of wild strain", the S.tm Nal 2/Rif 9 having a generation time of about 32 minutes (and an i. p. LDw mouse of a~out 106 cfu) was deter-mined as a prototype vaccine strain, and its (pro-longed) generation time of about 32 minutes was used as the approximate figure "attenuation equivalent".

Example 4 Recognition/detection of the Salmonella typhimurium clone S.tm Nal 2/Rif 9 (i. p. LD~o mouse about 106 (wild strain about 101) cfu; generation time prolonged from 22 to 32 minutes as an attenuation equivalent), optimally attenuated for chicks/chickens, as the favou-rable vaccine_ strain over the ~quivalent protective effect against a toxic infection conveyed by means of an i. p. and oral immunization;
~ 25 (with respect to a corresponding transfer of these regularities to further "enteritidis" salmonella):

a. Preliminary test of the extremely differentiated sensitivity of chicks/chickens and mice towards Salmonella typhimurium by determining the i. p.
LD~o rate of the S.tm wild strain and the S.tm Nal 2/Rif 9 vaccine strain for chicks and mice.

21312~ ~

For mice and two-day chicks the different i. p.
LD~o rates of the S.tm wild strain and the S.tm Nal 2/Rif 9 vaccine strain as well as, additionally, the i. p. LDoo rate of the wild strain for 17 days old chicks, are depicted in table 1.

Table 1 S.tm wild strain and S.tm Nal 2/Rif 9 vaccine strain: comparative i.p. LDw rates of mice and chicks 2-day 17-day ICR
chicken chicken mice S.tm (cfu) (cfu) (cfu) wild strain lo6 1o Nal 2/Rif 9 107 n.t. lo6 The LDoo rates for chicks and mice according to table 1 show the lesser sensitivity of chicks towards S.tm, which has to be compensated by a lesser attenuation level of the vaccine strains.

b. Recognition of the Salmonella' typhimurium vaccine strain optimally suited to chicks/chickens (with or without an rtt marker as an envelope mutation opti-mizing the vaccine strain) over the equivalent protective effect against a toxic infection conveyed by means of an i. p. or oral i'munization.

The equivalent protective effects against an LD,~
toxic infection, effected two weeks later, which can be attained through a single - i. p. immunization with the vaccine strain S.tm Nal 2/Rif 9 and the strains S.tm Pur- (i. p.
LD~o mouse 107-~ cfu) and Zoosaloral (i. p. LD~
108-2 cfu) being overattenuated for chicks, on the second day after hatching: see table 2;
- oral immunization with S.tm Nal 2/Rif 9; S.tm Nal 2/Rif 9/Rtt; as well as the calf vaccine Zoosaloral being overattenuated for chicks, within ~ 36 hours or on the fourth day after hatching: see table 3;
served as the criterion "optimal attenuation".

Table 2 Table 3 Attainable immunity Attainable immunity against a against a toxic infection toxic infection in the case of in the case of a single a single oral immunization with i.p. immunization with 109 cfu of the vaccine stralns 106 cfu of mutants having and Zoosaloral, being over-different attenuation attenuated for chicks; challenge levels on the second dày with i.p. 3X108 cfu of the S.tm of life; challenge with wild strain on the 16th day of 3X108 cfu of the wild life (mean of 4 experiments) strain on the 16th day of life (mean of 3 exp.) S.tm oral immunization on vaccine J 2.day 4.day i.p immunization: 106 cfu strain/ with i.p. challenge test ~.tmi.p. challenge strain (cfu) mortality (%) - vaccine strain/mortality Nal 2/ 109 ~i 35 test Rif 9 108 27 n.t.
strain (%) Nal 2/ 109 24 40 Rif 9/
Nal 2/Rif 9 25 Rtt lo8 26 n.t.
Pur-~l 25 Zoosaloral30 Zoosal- 109 42 60 oral 108 47 65 control 75 control 75 2I31 23~

As shown in table 2, the single i. p. immunization with all vaccine strains reduces the mortality of an unphysiological toxic infection from about 75%
to ~ 30%.
s As shown in table 3, this reduction in mortality -as opposed to Zoosaloral and metabolism drift mutants having generation times of 2 33 minutes and an i. p. LD~o > 106-~ cfu - can also be obtained by a single oral immunization with the vaccine strain S.tm 2/Rif 9 ~with or without rtt marker), i. e., therefore, this vaccine strain is optimally attenuated for chicks.

Furthermore, table 3 shows the - overattenuation of the calf vaccine Zoosaloral - less protectively effective immunization against a toxic infection on the fourth day of life.
Presumably, this is for reasons of the higher colonization resistance on the fourth day of life, which should influence the translocation and penetration rates of t~e vaccine strains.

2l3l23~

-Example 5 S.tm, S.ent, S.inf and S.ana: Isolation of spontaneous antibiotic resistance clones as (single and) double marker vaccine strains being optimally attenuated for chicks/chickens by means of the "attenuation equivalent (prolonged) generation time" tsee also example 1).

109 to 101~ cfu of the wild strain are transferred onto nutrient agar containing 400 ~g streptomycin/ml by means of a spatula and incubated for approximately two days at 37~C. The resistance clones are transferred onto nutrient agar, controlled with respect to obtained resistance, in the preliminary test the less or more reduced extinction in comparison with the wild strain is determined (Spekol with tube samples, Zeiss-Jena, wave length 650 nm, starting germ count 10' cfu, incu-bation in shaking water bath for 3 hours at 37~C) (see example 1), and the generation times of clones appear-ing to be suitable are measured by means of the Abbott MS-2 test system. As described above, in the second step, from clones having generation times being pro-~ longed by about 3 to 6 minutes ~ifampicin resistance clones (nutrient agar containing 400 ~ug rifampicin/ml) are obtained, the generation times thereof are deter-mined, and Sm/Rif clones having generation times of about 28 to 32 minutes are favoured as double marker vaccine strains. (The use of the Sm-stwd attenuation instead of the Nal-stwd attenuation is prefera~le, since the ssq marker (see example 6) as an envelope mutation - e. g. in Sm/Rif vaccine strains (upon abstaining from an Nal-stwd attenuation as a gyrase mutation) - also provides a hypersensitivity against the at present most effective antibiotic cipro-floxacin.) 213123~

Example 6 Additional incorporation of an ssq ("safety and therapy") marker, optimizing the vaccine strain and increasing its acceptance, into the wild and vaccine strains, respectively.

A fresh culture is suspended in P~S and treated with 100 ,ug/ml N-methyl-N-nitro-N-nitrosoguanidine (MNG, ZIMET, Jena) up to a survival rate of 10%. Then, it is incubated for 2 hours at 37~C in nutrient bouillon containing 0.4 ,ug chloramphenicol, and treated in the conventional manner with 1000 IU penicillin/ml. Clones lacking growth are obtained by means of the stamping technique on nutrient agar containing 0.4 ,ug chlor-15amphenicol (0.01 ,ug ciprofloxacin, 1.0 ~ug doxy-cycline)/ml. As ssq tsupersensitivity to quinolons) strains such clones are determined and used as safety and therapy markers optimizing the vaccine strain and increasing its acceptance, which - do not grow on nutrient agar containing 0.4 ~ug chloramphenicol, 0.01 ,ug ciprofloxacin or 1.0 ~ug ~ doxycycline (as well as, mo~tly, 30 ~ug erythro-mycin~/ml, - show a desirable reversion frequency of < 10-7.
The reversion fre~uency of the envelope mutation is better determined on nutrient agar containing 20 or 30 ~g erythromycin/ml, since, upon the high germ counts, the rest growth shifts towards a two steps higher concentration of ciprofloxacin, chloramphenicol and doxycycline.

_ ~ - 32 ..

Suitable as vaccine strains are clones having an ssq marker, which, through mutagen treatment, undergo no or only a minor generation time prolongation/attenuation caused by co-mutation.

Example 7 Recognition of the S.typhimurium, S.enteritidis, S.infantis and S.anatum vaccine strain being optimally attenuated for chicks/chickens by means of the detec-tion of the obtained or residual invasive capacity of the metabolism drift (stwd) single marker mutants and double marker vaccine strains for chicks.
Chicks at the age of s 36 hours were infected orally with 109 cfu of the respective wild strains, stwd single marker mutants and stwd double marker mutants, respectively. After five to eight days the chicks were killed, the liver extracted asepticly, was homogenized, transferred onto nutrient agar, and the remaining material was mixed with nutrient~bouillon. The grown colonies and cultures, respectively, were controlled with respect to O-group identity and marker.
With respect to S.typhimurium and S.enteritidis, after five days the germ counts/gram liver are in the range of about 103 cfu, after eight days they are in the range of about 102 cfu. In contrast to that, with respect to S.infantis and S.anatum, the germ counts are in the border area of quantitative determination at about 101 to S 102 cfu, and after eight days the detec-tion often can only be obtained by means of propa-gation. These lower germ counts/gram liver, found with respect to S.infantis and S.anatum, apparently corre-, 21~12~
~ 33 .

late with U. Methner's observation (doctoral thesis, University of Leipzig, Veterinary Medical Faculty, 1991) that S.infantis is less invasive for chicks.

The generally obtained invasive capacity of the favou-red vaccine strains (and single marker mutants) which -determined from the numbers of colonies incubated -does not quite reach the figures of the homologous wild strains, apparently is essential for the vaccination success (Barrow, P. A. et al., Res. Microbiol., 1990, 141, pp. 851-853).

Example 8 Determination of the frequency in percentages and the duration of excretion of the S.tm Nal 2/Rif 9 vaccine strain (with or without rtt marker) after oral immuni-zation of < 36 hours old chicks, and of the S.tm Nal 2/Rif 9 after oral immunization of five days old chicks.

The frequency in percentages in the tested stool samples and the maximum detectable duration of excre-tion of the vaccine strain with or without rtt marker, dependent upon the chicks' age upon oral immunization is shown in Fig. 1.

Figure 1 Frequency (percentage of positive samples after propa-gation) and duration ~last positive result, border of detection about 10 germs/gram stool) of excretion of the vaccine strains S.tm Nal 2/Rif 9 without (- ) and with rtt marker (- - -) after a single oral immu-nization of < 36hours old chicks with 109 cfu as well _ ~ 34 as with S.tm Nal 2/Rif 9 (individual figures not shown, (- )) on the fourth day of life.
(Mean of four to five experiments) per~entage of pos. stool samples fOO~

50 ~\ , \

. , . ,,I,,,~I,............................ .
5 xt 15 20 25 30 days after immunization The following facts are shown in F~ig. 1:
- upon immunization within S 36 hours after hatching, the S.tm Nal 2/Rif 9 is excreted over the 32 days - 15 tested. After incorporating the rtt marker, the vaccine strain can rarely be detected three weeks after the immunization.
- upon immunization on the fourth day, the S.tm Nal 2/Rif 9 (without rtt marker) can only be detected occasionally after the 18th day, apparently caused by the already existing colonization resistancy caused by anaerobic bacteria.
- the frequency in percentages of positive accumu-lation cultures is analogous to this. With respect to the oral immunization within 5 36 hours, the S.tm Nal 2/Rif 9 can still be detected in about 90%

21~1231 -of the samples, with respect to the vaccine strain having been optimized with the rtt marker, only about 40% of the stool samples are still positive.

The colonization dynamism of the favo~Led vaccine strains S.tm Ssq/Sm 60/Rif 42, S.ent Ssq/Sm 24/Rif 12 g, S.in~ Ssq/Sm 153/Rif 7 and S.ana Ssq/Sm 81/Rif 21 was determined in that, after oral application of 109 cfu within 5 36 hours after hatching, the portion of the respective salmonella vaccine strain in the entero-bacteria flora of the stool samples over a duration of two weeks was tested, wherein the favoured vaccine strains (see above) showed an excretion behaviour comparable to the vaccine strain S.tm Nal 2/Rif 9/Rtt.
_ Example 9 Reduction of the quantitative excretion of homologous neutrally Nal/Sm marked wild strains in immunized chicks in comparison with controls.
-The ~ 36 hours old chicks which received a monovalent oral immunization by the vaccine strains S.tm Nal 2/Rif 9/Rtt, S.tm Ssq/Sm 60/Rif 42, S.ent Ssq/Sm 24/Rif 12, S.ent Ssq/Sm 24/Rif 12k, S.ent Ssq/Sm 24/Rif 12g, S.ent Ssq/Sm 24/Rif 3, S.inf Ssq/Sm 153/Rif 7 and S.ana Ssq/Sm 81/Rif 21, respectively were infected with 106 (partly 107) cfu of the respective homologous wild strain on the 17th day of life, parallel to controls of the same age.

In comparison with the controls, the immunized chicks -in compliance with the results obtained with respect to the S.tm prototype vaccine strain Nal 2/Rif 9 - show a 35a significantly reduced excretion of the wild strain, especially in the first five t.o ten days after the oral 213123~

challenge (determined from the enterobacteria coloniza-tion density in thousandth). The variation (reducing with time) lies in the range of up to two tenth powers.
From the sixth to the tenth day after the oral challenge, the salmonella colonization densities in immunized chicks come into closer alignment with those of the controls (in the 0.1 thousandth range of entero-bacteria germ counts). In individual cases, however, immunized chicks may show a reduced excretion in the range of one log step, even after the tenth day.

A complete elimination of the salmonella bacteria within the individual infected animal can hardly be expected, since, apart from S.gallinarum pullorum, these pathogenes apparently behave like a "normal flora" within chicks. The highly reduced excretion with respect to immunized chicks/chickens should, in the medium term, lead to a salmonella free chic~en stock, in combination with hygiene measures.

Example 10 Increased sensitivity towards detergents The vaccine strains S.tm Ssq/Sm 60/Rif 42, S.ent Ssq/
Sm 24/Rif 12, S.ent Ssq/Sm 24/Rif 12k, S.ent Ssq/Sm 24/
Rif 12g, S.ent Ssq/Sm 24/Rif 3, S.inf Ssq/Sm 153/Rif 7, S.ana Ssq/Sm 81/Rif 21, S.tm Nal 2/Rif 9/Rtt show in comparison to wild strains an increased sensitivity towards anionic detergents, especially towards sodium dodecyl sulphate (SDS). So the vaccine strains ~ not show growth on appropriate nutrient media (approximately without proteins) at a concentration of 0,5 mg SDS/ml the wild strains grow at 5 mg SDS/ml.

~~~ ~ 36a Vaccine strains suspended in physiological solution of sodium chloride become lysed at room temperature within 30 minutes to about 90 % whereas wild strains show only a lysis rate of about 10 %.

It can be concluded from these observation,that the vaccine strains have an increased sensitivity resp.
reduced capability of survival in the environment especially if measures for hygienic cleaning are taken.

Example 11 Separation of vaccine strains from wild strains.
The separation of wild strains with respect to - S.tm Ssq/Sm 60/Rif 42, S.ent Ssq/Sm 24/Rif 12, S.ent Ssq/Sm 24/Rif 12k, S.ent Ssq/Sm 24/Rif 12g, S.ent Ssq/Sm 24/Rif 3, S.inf Ssq/Sm 153/Rif 7 and S.ana Ssq/Sm 81/Rif 21 is obtained by their resistance against 100 ~g rifampicin and 200 ~g streptomycin/ml nutrlent agar~as well as the missing growth on nutrient agar containing 0.4 ~g chloramphenicol (or 0.01 ~g ciprofloxacin, 1.0 ~g doxycycline and 30 ~g erythromycin, respectively)/ml.

~_ _ 37 - S.tm Nal 2/Rif 9/Rtt is obtained by its resistance against 100 ~g rifampicin and 12.5 ~g nalidixic acid*/ml nutrient agar as well as the missing growth on nutrient agar containing 30 ~ug erythro-mycin/ml.

-* (The reduced resistance of the rtt strain against nalidixic acid in comparison with the initial Nal 2/Rif 9 strain is a consequence of 10the rtt mutation (incorporated later) leading to a varied permeability of the outer membrane.) Example 12 Mass culture, preparation and use of vaccine strains.

For the production of live vaccines (double marker vaccine strains without ssq marker or) triple marker vaccine strains with ssq marker are incubated in a suitable full medium as a liquid culture until the end ~ of the logarithmic phase. The bact~rial suspensions are mixed with a conventional stabilizer and lyophilized.

The vaccine strains obtained in this manner are usually applied to ~ 36 hours old chicks in a single dose of 108 to 109 cfu. Chickens usually receive a single immu-nization/boostering of 109 cfu orally, or about 108 cfu parenterally, prior to the laying period.

Claims (28)

1. A salmonella live vaccine comprising an attenuated immunogenic live salmonella vaccine strain wherein the vaccine strain has an envelope marker which confers increased sensitivity towards an antibiotic selected from the group quinolons, chloramphenicols and tetracyclines wherein said antibiotic facilitates an effective therapeutical treatment of a host infected with salmonella as a result of contacting a host vaccinated with said vaccine.

2. A salmonella live vaccine comprising an attenuated immunogenic salmonella live vaccine strain, wherein the vaccine strain has an envelope marker which confers on the vaccine strain an increased sensitivity towards an antibiotic selected from the group quinolons, chloramphenicols and tetracyclines.

3. The live vaccine according to any one of claims 1 and 2, wherein the vaccine strain comprises a chromosomal antibiotic resistance mutation for attenuation.

4. The live vaccine according to any one of claims 1 to 3 wherein the envelope marker confers an increased sensitivity towards the antibiotic ciprofloxacin.

5. The live vaccine according to any one of claims 1 to 4 wherein the vaccine strain additionally includes a metabolism drift mutation for attenuation.

6. The live vaccine according to claim 5 wherein said metabolism drift mutation confers on the vaccine strain streptomycin resistance and/or rifampicin resistance.

7. The live vaccine according to any one of claims 1 to 6, comprising one vaccine strain or two, three or four vaccine strains as mono, bi, tri or tetra-vaccine selected from vaccine strains of a serovar of the O-groups B, D, C and E.

8. The live vaccine according to claim 7 wherein the vaccine strain of a serovar of the O groups B, D, C and E is Salmonella typhimurium, Salmonella enteritides, Salmonella infantis, and Salmonella anatum.

9. The live mono vaccine according to claim 7 wherein the vaccine strain is selected from the group of vaccine strains consisting of S.tm Ssq/Sm 60/Rif 42 (No. 4242; deposit no. DSM
8433), S.tm Nal 2/Rif 9/Rtt (No. 4223; deposit no. DSM 8432), S.ent Ssq/Sm 24/Rif 12 (No. 4266; deposit no. DSM 8435), S.ent Ssq/Sm 24/Rif 12k (No. 4298; deposit no. DSM 9362), S.ent Ssq/Sm 24/Rif 12g (No. 4297; deposit no. DSM 9361), and S.ent Ssq/Sm 24/Rif 3 (No. 4296; deposit no. DSM 9360).

10. The live bi-vaccine according to claim 7 wherein the vaccine strains are selected from the group of vaccine strains consisting of S.tm Ssq/Sm 60/Rif 42 (No. 4242; deposit no. DSM
8433), S.tm Nal 2/Rif 9/Rtt (No. 4223; deposit no. DSM 8432);
and the group of the vaccine strains consisting of S.ent Ssq/Sm 24/Rif 12, S.ent Ssq/Sm 24/Rif 12k, S.ent Ssq/Sm 24/Rif 12g and S.ent Ssq/Sm 24/Rif 3.

11. The live tri-vaccine according to claim 7 wherein the vaccine strains are selected from the group of vaccine strains consisting of S.tm Ssq/Sm 60/Rif 42 (No. 4242; deposit no. DSM
8433), S.tm Nal 2/Rif 9/Rtt (No. 4223; deposit no. DSM 8432);
the group of the vaccine strains consisting of S.ent Ssq/Sm 24/Rif 12, S.ent Ssq/Sm 24/Rif 12k, S.ent Ssq/Sm 24/Rif 12g and S.ent Ssq/Sm 24/Rif 3; and the group of the vaccine strains consisting of S.inf Ssq/Sm 153/Rif (No. 4289; deposit no. DSM
8434) and S.ana Ssq/Sm 81/Rif 21 (No. 4279; deposit no. DSM
8441).

12. The live tetra-vaccine according to claim 7 wherein the vaccine strains are selected from the group of vaccine strains consisting of S.tm Ssq/Sm 60/Rif 42 (No. 4242; deposit no. DSM
8433), S.tm Nal 2/Rif 9/Rtt (No. 4223; deposit no. DSM 8432);
the group of the vaccine strains consisting of S.ent Ssq/Sm 24/Rif 12, S.ent Ssq/Sm 24/Rif 12k, S.ent Ssq/Sm 24/Rif 12g and S.ent Ssq/Sm 24/Rif 3; S.inf Ssq/Sm 153/Rif (No. 4289; deposit no. DSM 8434); and S.ana Ssq/Sm 81/Rif 21 (No. 4279; deposit no. DSM 8441).

13. Use of an attenuated immunogenic salmonella live vaccine strain comprising an envelope marker which confers on the vaccine strain increased sensitivity towards a therapeutically effective antibiotic, for the production of a live vaccine for a specific host wherein said antibiotic facilitates an effective therapeutical treatment of a host infected with salmonella as a result of contacting said specific host vaccinated with said vaccine.

14. A live vaccine produced from an attenuated immunogenic salmonella live vaccine strain for immunization of poultry against salmonella infections wherein the vaccine strain is according to any one of claims 1 or 14 and has a generation time of about 28 to 34 minutes.

15. A method for the production of a salmonella live vaccine optimally suited to a host serotype in its attenuation level and produced from an attenuated immunogenic salmonella live vaccine strain, comprising the steps of determining a prolonged generation time of salmonella live vaccine strains suited to the host serotype from a set of graded prolonged generation times and selecting vaccine strains having maximum possible attenuation/prolonged generation time and optimal reduction of excretion of wild strain for use as an attenuation equivalent, wherein said specifically prolonged generation time is determined in a single test.

16. The method according to claim 15 wherein the prolonged generation time is transferred to other serotypes of the same genus as an equivalent for attenuation.

17. The method according to claim 15 for the production of a vaccine optimally suited to poultry, wherein the generation time of the vaccine strain used is prolonged from about 22 minutes to about 34 minutes, in comparison with the wild strain.

18. The method according to claim 17, wherein the vaccine strain used is obtained from a wild strain firstly by providing the wild strain with a streptomycin (Sm) or nalidixic acid marker to cause a prolongation of the generation time from 22 minutes to 29 minutes and then by incorporating a rifampicin (Rif) marker to further prolong the generation time, and finally by isolating clones suitable as vaccine strains having a generation time of 28 to 34 minutes.

19. The method according to claim 18 wherein the wild strain is selected from the group Salmonella typhimurium (S.tm), Salmonella enteritidis (S.ent), Salmonella infantis (S.inf.) and Salmonella anatum (S.ana).

20. The method according to claim 19 wherein, - S.tm vaccine strain having a generation time of about 31 minutes (and an i. p. LD50 mouse of about 10 5 (wild strain about 10 1) cfu), - S.ent vaccine strain having a generation time of about 28 minutes (and an i. p. LD50 mouse of about 10 7 (wild strain about 10 5) cfu), - S.inf. vaccine strain having a generation time of about 31 minutes, - S.ana vaccine strain having a generation time of about 32 minutes, is employed as a vaccine optimally attenuated for poultry.

21. The method according to any one of claims 18 to 20 wherein the vaccine strain comprises an envelope marker which approximately quadruples the sensitivity of the vaccine strain towards ciprofloxacin and simultaneously insignificantly reduces the excretion and capability of survival in the environment.

22. The method according to claim 21 wherein ciprofloxacin is selected from chloramphenicol and doxycycline.

23. The method according to claim 21 or 22 wherein the marker increasing the sensitivity towards ciprofloxacin is either incorporated into the wild strain or following the incorporation of the Sm/Rif double marker by means of mutagenesis, followed by conventional penicillin screening.

24. The method according to claim 23 wherein the sequence of the marker incorporation is arbitrary.

25. The method according to any one of the claims 15 to 20, wherein vaccine strains without envelope markers are used.

26. Salmonella vaccine strains having generation times of 28 to 32 minutes.

27. The salmonella vaccine strains according to claim 26 having deposit nos. DSM 8432, 8433, 8434, 8435, 9360, 9361, 9362 and/or 8441 and their more or less attenuated variants having generation times of 28 to 34 minutes.

28. Use of the vaccine strains according to claim 26 or 27 as the live vaccines according to claim 1 or 2 for immunization of poultry against salmonella infections.