Live salmonella vaccine having an increased stability

A live Salmonella vaccine having an increased stability is disclosed. Further, immunization methods of a host such as chickens against Salmonella diseases are carried out by the use of the vaccine. The vaccine may be administered orally or parenterally and includes one or more of the live vaccines disclosed herein. The live vaccine is produced from one or more live vaccine strains with metabolic drift attenuation via an increased generation time. The vaccine is an immunogenic, stable and single or multiple marker live vaccine strain with or without envelope mutation; and the live vaccine is a suppressor mutant of an original live vaccine strain still having the attenuation marker(s) of the original live vaccine strain but a shortened generation time and having macrolide tolerance.

The invention relates to a live vaccine having an increased stability. In 
comparison to the known live vaccines the stability is increased by at 
least the factor 10.sup.-7. The vaccine consists of at least one metabolic 
drift attenuated, immunogenic live vaccine strain with or without envelope 
mutation and an attenuation level optimally adapted to the host species. 
Possible host species are working animals and humans. One embodiment 
proposes a live vaccine for chicks and chickens for the immunization 
against Salmonella. 
The features of the vaccine strain and its production will be described. 
The safety of bacterial live vaccine strains is internationally guaranteed 
by deletions, the stability of which lying in the range of about 
10.sup.-12 (calculated from the in vivo repair of single marker Shigella 
over genetically related coli, proven by tests on volunteers (Formal et 
al., as well as DuPont et al.: Immunbiol. Standard; Karger; 
Basel/Munich/New York 1971, 15, 73-78 as well as 213-218). With the known 
double marker (spot mutation) attenuation, in the most disadvantageous 
case, a total stability (as a product of individual stabilities) of at 
least 10.sup.-14 results. According to the object of the present 
invention, this total stability is to be increased to at least 10.sup.-21, 
wherein the stability of (as a rule not usual) two (set at separate gene 
locations) attenuated deletions is achieved. 
The high requirements of the World Health Organisation and the wide-spread 
opinion about the alleged instabilities in the use of live vaccines under 
practical conditions let it appear to be necessary to enhance the already 
existing safety buffer having a stability of 10.sup.-14 by yet another 
increase of the attenuation to at least the factor 10.sup.-21, and to 
raise the stability of the antiepidemic potency provided by an envelope 
marker to at least 10.sup.-14. In the construction of such live vaccines 
having an increased stability well-established and known principles are to 
be used and elaborated. 
It is known that a special difficulty in the search for effective and 
well-tolerated vaccine strains lies in the question whether the 
marker-conditioned attenuation level ("metabolic defect") and the 
sensitivity characteristic of a host species for the possible pathogene 
are correlated such that 
still a limited but sufficient in vivo propagation of the vaccine strain is 
possible, and therefore an immunity relevant in practice results, 
in the case of a too low sensitivity and simultaneously relatively severe 
"metabolic disturbance" of the vaccine strain a more or less distinct 
overattenuation occurs, and therefore (not relevant in practice) several 
vaccinations are necessary, as e. g. in the case of S.typhi gal-E 21a, 
in the case of high sensitivity and simultaneously relatively minor 
"metabolic disturbances" of the vaccine strain the "remaining virulence" 
triggers unacceptable side effects. 
As an almost classical example of the correlation between 
marker-conditioned attenuation level, sensitivity of the host species, and 
immunogenicity the effectiveness of the vaccine Zoosalora--Salmonella 
tyhimurium His.sup.- (attenuation by co-mutation)Pur.sup.-, i. p. 
LD.sub.50 mouse 10.sup.8.2 (wild strain about 10.sup.1) cfu--in mice, 
calves and chickens: referring to one kg body weight the parenteral 
LD.sub.50 (as a degree of sensitivity) in mice is about 10.sup.3, in 
calves about 5.times.10.sup.6 and in chicks/chickens about 
5.times.10.sup.8 germs. Therefore, in the case of a single oral 
immunization Zoosaloral protects mice with &gt;95% against a lethal 
challenge, calves still with a relevance in practice (in the case of an 
LD.sub.75 challenge "only" 36% of the immunized animals die), in 
chicks/chickens, however, it fails because of overattenuation (Linde, K. 
et al.: Vaccine, 1990, 8, 278-282). 
This means that the characteristic sensitivity level of the host species to 
the respective pathogen has to be compensated by a corresponding 
attenuation level to achieve a resilient immunity with a single 
immunization. This, in itself logical (but apparently not observed and not 
formulated, respectively) rule can be met for the first time with 
relevance in practice by revealing the principle "attenuation by means of 
metabolic drift mutations" in essential enzymes and metabolic compartments 
as e. g. RNA polymerase, gyrase, ribosome proteins (isolation by means of 
chromosomal, e. g. rifampicin, streptomycin, nalidixic acid resistance). 
Since in this principle the logarithm of the LD.sub.50 (measured in the 
sensitive test animal) correlates linearly with the (prolonged) generation 
time, also the (prolonged) generation time can serve as an attenuation 
equivalent for pathogens where a suitable (sensitive) test animal is 
missing. The principle "attenuation by means of metabolic drift mutations" 
is described comprehensively in DD 155294, DD 218834, DD 235828, DD 
253182, DD 253184, DD 281118, DD 294420, EP 0263528, as well as in the 
publications: 
Linde, K.: Zbl. Bakt. Hyg. I. Abt. 1981, 249, 350-361 
Linde, K.: Dev. Biol. Standard 1983, 53, 15-28 
Linde, K.: Arch. exper. Vet. med. 1982, 36, 647-656 
Linde, K. et al.: Arch. exper. Vet. med. 1983, 37, 353-360 
Linde, K. et al.: Vaccine 1990, 8, 25-29 
Linde, K. et al.: Vaccine 1990, 8, 278-282 
Linde, K. et al.: Vaccine 1991, 9, 101-1105 
Linde, K. et al.: Vaccine 1992, 10, 337-340 
Linde, K. et al.: Vaccine 1993, 11, 197-200 
Marakusha, B. I. et al.: Z. Microbiol. Epidemiol. Immunol. 1987/4, 3-8. 
These vaccine strains in which one of the attenuating metabolic drift 
markers is usually a chromosomal nalidixic acid resistance (gyrase) 
mutation can be optimized by means of hst (high sensitivity to tensides 
and macrolide antibiotics), rbt (reversion to bile tolerance) or rtt 
(reversion to tenside tolerance) markers. These markers reduce the 
excretion and the capability of survival of the vaccine strains in the 
environment without a significant influence on the parenteral virulence 
behaviour. 
The antiepidemic markers are described comprehensively in DD 218836, DD 
231491, DD 253182, DD 253183, DD 252184, and EP 0263528, as well as in the 
publications: 
Linde, K.: Arch. exper. Vet. med. 1982, 36, 657-662 
Linde, K.: Dev. Biol. Standard 1983, 53, 15-28 
Linde, K. et al.: Vaccine 1990, 8, 278-282. 
Finally, an envelope marker was suggested which combines the features of 
the antiepidemic markers with those of a safety-/and therapy marker. The 
prototype of such an envelope marker is the so-called Ssq 
(supersensitivity to (fluor-) quinolones)-marker which, in the Sm/Rif 
metabolic drift combination, next to the antiepidemic potency, a. o. 
provides a supersensitivity to the antibiotic ciprofloxacin, presently the 
most effective against salmonella. 
The common feature of vaccine strains produced by using two separately 
attenuated metabolic drift markers as well as a combination of metabolic 
drift and envelope and antiepidemic markers, respectively, is that their 
reversion frequency with respect to the attenuated markers is usually 
restricted to a factor of .ltoreq.10.sup.-14, and with respect to the 
envelope marker-conditioned antiepidemic potency of selected clones to a 
factor of .ltoreq.10.sup.-8, which is generally sufficient under practical 
conditions. 
Therefore, it is the object of the invention to increase the stability of 
the vaccine serving for the production of the vaccine strain(s) such that, 
with respect to the attenuation, a stability of at least 10.sup.-21 and, 
with respect to the antiepidemic potency, of at least 10.sup.-14 is 
achieved. Here, known advantageous principles for the adaptation of the 
attenuation depending on the sensitivity of the host species and the 
influence on the excretion and capability of survival of the vaccine 
strains in the environment are to be kept and improved further. 
This object is attained by providing a live vaccine, a special live vaccine 
for chickens and chicks, a method for the production of such live 
vaccines, and live vaccine strains as well as their use.

In one embodiment, a live vaccine produced from at least one metabolic 
drift attenuated, immunogenic, stable single or multiple marker vaccine 
strain with or without envelope mutation and an optimal attenuation level 
adapted to the sensitivity of the host species is used. According to the 
invention, as vaccine strain its revertant, as a suppressor mutant still 
having the attenuation or/and the envelope marker, is used. 
In a preferred embodiment, as vaccine strains its revertant having a 
shortenend generation time is used, wherein this revertant, as a 
suppressor mutant, has one or more attenuating metabolic drift markers. 
The revertant has a similar as or an only slightly lower attenuation level 
than the original vaccine strain. 
In a further preferred embodiment, as vaccine strain its macrolide tolerant 
revertant is used, wherein this revertant, as a suppressor mutant, still 
has the envelope marker. The revertant has a similar as or an only 
slightly higher antiepidemic potency than the original vaccine strain. 
In a third preferred embodiment, as vaccine strain its double revertant is 
used, wherein this revertant having a shortened generation time and a 
macrolide tolerance, as a suppressor mutant, has one or more attenuating 
metabolic drift markers and also still has the envelope marker. 
Surprisingly, it has been found that, starting from metabolic drift 
attenuated vaccine strains with or without additional envelope marker, the 
revertants having a shortened generation time or/and a macrolide tolerance 
possess an unchanged attenuation and antiepidemic potency, which proves 
that the metabolic drift attenuation or/and envelope marker of the 
original vaccine strains still exist in the revertant. Furthermore, it has 
been shown that during the transition from the original vaccine strains to 
their revertants/suppressor mutants the attenuation in the sense of an 
adaptation to the sensitivity of the host species can still be varied to a 
small extent. The same stands for the antiepidemic potency. 
As an example for the live vaccine the following Salmonella vaccine strains 
for a live vaccine for the oral immunization of chicks as well as the oral 
or parenteral immunization/boostering of chickens against Salmonella 
infections are proposed, which have been derived from original vaccine 
strains having a generation time of about 31 to 32 minutes and/or an MIC 
of erythromycin of .ltoreq.5 .mu.g/ml. The revertants having a shortened 
generation time: 
S.tm Ssq/Sm 60/Rif 42/-GVR II 30 generation time about 26.0 minutes 
S.ent Ssq/Sm 24/Rif 12/-GVR II 30 generation time about 27.0 minutes 
S.inf Ssq/Sm 153/Rif 7/-GVR II 30 generation time about 25.0 minutes 
S.ana Ssq/Sm 81/Rif 21/-GVR II 30 generation time about 26.5 minutes 
show a generation time of .ltoreq.27 minutes. In the case of revertants 
having a macrolide tolerance: 
S. tm Ssq-MTR 16/Sm 60/Rif 42 
S. ent Ssq-MTR 25/Sm 24/Rif 12 
S. inf Ssq-MTR 12/Sm 153/Rif 7 
S. ana Ssq-MTR 23/Sm 81/Rif 21 
the MIC of erythromycin was increased from .ltoreq.5 .mu.g/ml to .gtoreq.20 
.mu.g/ml. 
The listed revertants/suppressor mutants have been derived from the 
following Salmonella vaccine strains, deposited at the DSM--Deutsche 
Sammlung von Mikroorganismen und Zellkulturen GmbH, D-38124 Braunschweig, 
Germany: 
______________________________________ 
S. tm Ssq/Sm 60/Rif 42 
DSM 8433 
S. ent Ssq/Sm 24/Rif 12 
DSM 8435 
S. inf Ssq/Sm 153/Rif 7 
DSM 8434 
S. ana Ssq/Sm 81/Rif 21 
DSM 8441. 
______________________________________ 
The revertants/suppressor mutants according to the invention represent a 
new generation of increasingly stable (total stability=product of the 
markers of the original vaccine strains and the suppressor mutants) 
vaccine strains. They use the surprisingly found rule that 
reversions/suppressor mutations to the pseudo wild type with unchanged or 
only slightly lower attenuation level and/or unchanged or only slightly 
varying antiepidemic potency occur significantly more often than back 
mutations. It was tested in how far this can also be used for testing the 
actual stability of the metabolic drift attenuation and envelope markers, 
and, at the same time, for gaining a new generation of extremely stable 
vaccine strains which comply ideally with the stability-/and safety 
requirements postulated by the WHO (Report of the WHO Scientific Group: 
Oral Enteric Bacterial Vaccines, World Health Organisation, Technical 
Report Series No. 500, 1972), and which, in view of these now triple 
marker attenuated vaccine strains, reduces the discussion about the 
stability of double marker vaccine strains impeding the vaccine 
introduction to absurdity. The safety of bacterial live vaccine strains is 
internationally guaranteed by deletions (see above), the stability of 
which lying in the range of about 10.sup.-12 (calculated from the in vivo 
repair of single marker Shigella over genetically related coli), proven by 
tests on volunteers (Formal et al., as well as DuPont et al.: Immunbiol. 
Standard; Karger; Basel/Munich/New York 1971, 15, 73-78 as well as 
213-218). With the known double marker (spot mutation) attenuation, in the 
most disadvantageous case, a total stability (as a product of individual 
stabilities) of at least 10.sup.-14 results. An additional suppressor 
mutation therefore increases the total stability to at least 10.sup.-21, 
and thus the stability of (as a rule not usual) two (set at separate gene 
locations) attenuating deletions is achieved. Upon coupling two 
attenuating metabolic drift markers with an envelope mutation and 
subsequent production of the suppressor mutation of both principles a 
total stability of .ltoreq.10.sup.-35 would even result, which is not 
necessary as such--since according to the WHO's definition a mutation 
interrupting the infection chains of a vaccine strain is to be seen as an 
attenuation marker (WHO Technical Report Series No. 500, 1972). 
Depending on the markers, two kinds of revertants are differentiated 
between and will be described in the following. 
REVERTANTS HAVING A SHORTENED GENERATION TIME (GVR) 
The stability of the metabolic drift (antibiotic resistance mutation) 
attenuation together with a corresponding (increased in Salmonella vaccine 
strains for chicks/chickens from 22 to 31-32 minutes) prolongation of the 
generation time can only be proven indirectly by the received resistance 
and the defined prolonged generation time. 
Slowly growing metabolic drift attenuated vaccine strains, however, offer a 
selective advantage for clones where further mutations (because there is 
no genetic stability as such) lead to shorter generation times by means of 
"corrected" metabolic sequences. This means, that an enrichment of GVR 
strains can be reckoned with under the corresponding selection pressures 
in a vaccine strain population growing more slowly. 
The conclusion based on the known prior art that "prolongation of 
generation time leads to attenuation" and therefore "shortening of 
generation time (inversely proportional) leads to increased virulence" is 
now proven wrong in the knowledge about the importance of 
reversions/suppressor mutants (which occur significantly more often than 
back mutations) (Linde, K.: Arch. exper. Vet. med. 1978, 32, 943). 
In the following example 1 by means of the well-established model 
S.typhimurium/mouse direct proof will be offered for the fact that the 
GVR's attenuation (in relation to the original vaccine strain) is 
unchanged or only slightly lower and, therefore, they still have their 
original attenuation markers. These revertants/suppressor mutants having a 
shortened generation time therefore represent a new type of extremely 
stable vaccine strains, the reversion frequency of which being at maximum 
.ltoreq.10.sup.-21 (total stability=product of the individual stabilities 
(when assuming an unlikely high scale of .ltoreq.10.sup.-7 per single 
marker): Sm.times.Rif.times.suppressor mutation). 
MACROLIDE TOLERANT REVERTANTS (MTR) 
The reversion frequency of the envelope mutation taken from the number of 
20 .mu.g erythromycin tolerant clones in general lies between 10.sup.-6 
and 10.sup.-8 and only in the case of a few selected strains is 
.ltoreq.10.sup.-8. 
It has been found that the macrolide tolerant clones--despite a regained 
wild strain tolerance to erythromycin as well as generally the loss of the 
supersensitivity to ciprofloxacin, doxycycline, chloramphenicol--in their 
antiepidemic function (reduced capability of survival in the environment 
and shortened excretion in chicks) approximately correspond to the 
original strain or only vary slightly around its values and therefore 
still have the original envelope mutation. 
Details can be taken from the following example 2. 
These macrolide tolerant revertants/suppressor mutants therefore represent 
a new type of extremely stable vaccine strains, the reversion frequency of 
which being at maximum 10.sup.-14 in the antiepidemic potency (total 
stability=product of the original envelope marker and the suppressor 
mutation). 
Therefore, the revertants having shortened generation times and/or a 
macrolide tolerance which have been derived from metabolic drift 
attenuated single or multiple marker original vaccine strains with or 
without additional antiepidemic envelope marker lead to vaccine strains 
which, to a great extent, comply with the strict requirements for the use 
of live vaccines under practical conditions. 
The object of producing a live vaccine having an increased stability is 
attained by using at least one metabolic drift attenuated, immunogenic, 
stable single or multiple marker live vaccine strain with or without 
envelope mutation and an attenuation level optimally adapted to the host 
species. According to the invention, starting from a metabolic drift 
attenuated original vaccine strain without or with additional envelope 
mutation and an attenuation adapted to the host species and, at the same 
time, a defined prolonged generation time, the revertant having a 
shortened generation time and a macrolide tolerance is isolated. This 
suppressor mutant, still having the attenuation or/and envelope marker of 
the original vaccine strain, is used as increasingly stable vaccine 
strain, from which the live vaccine is produced by means of methods known 
as such. 
In further embodiments of the method according to the invention, the 
conditioning features in comparison to the original vaccine strain can 
possibly still be influenced by means of the suppressor mutant. 
In the case of strain dependent preferred separation of a revertant having 
a shortened generation time with a slightly lower attenuation level, an 
original vaccine strain with a higher (possibly up to the limit of 
overattenuation) attenuation is used for the isolation of an optimally 
attenuated supressor mutant. 
For the production of a vaccine having an increased stability in the 
antipidemic potency, a revertant having a similar as or a slightly higher 
antiepidemic potency than the original vaccine strain is used. 
By using this method in an arbitrary order, from a metabolic drift 
attenuated original vaccine strain having a macrolide sensitivity 
(envelope) marker, a revertant having a shortened generation time as well 
as a macrolide tolerance is obtained. 
As an example for the method described above, one of the following methods 
for the production of a vaccine optimally adapted to chicks or chickens is 
proposed according to the invention. 
For the production of the vaccine an increasingly stable vaccine strain is 
selected, the original vaccine strain of which having a generation time 
prolonged from about 22 minutes to about 31 to 32 minutes in comparison to 
the wild strain which, in the case of the revertant having a shortened 
generation time, is shortened to up to .ltoreq.27 minutes. 
A further method is characterized by the selection of a vaccine strain 
having an increased stability in the antiepidemic potency, the original 
vaccine strain of which having a macrolide sensitivity which, in the case 
of the macrolide tolerant revertant, results in an increase of the MIC of 
erythromycin from .ltoreq.5 .mu.g/ml to .gtoreq.20 .mu.g/ml. 
Finally, both methods can also be combined in an arbitrary order by 
selecting a revertant having a shortened generation time as well as a 
macrolide tolerance from a metabolic drift attenuated original vaccine 
strain with macrolide sensitivity (envelope) marker for the production of 
a live vaccine having an increased stability. As vaccine strain one or 
more of the vaccine strains are used. 
In the following examples the live vaccine and the method for the 
production of the live vaccine are to be described in detail. 
MATERIAL AND METHOD 
Strains Used 
wild strains 
Salmonella (S.) typhimurium, S.enteritidis, S.infantis, S.anatum 
sets of metabolic drift double (triple) marker (total stability as a 
product of the individual stabilities) vaccine strains with an additional 
envelope marker and a graded attenuation/graded prolonged generation time 
(GT) (as attenuation equivalent) as well as further deposited vaccine 
strains as e. g. 
S.typhimurium Ssq/Sm 60/Rif 42 GT:.apprxeq.31 min. deposit no. DSM 8433 
S.enteritidis Ssq/Sm 24/Rif 12 GT:.apprxeq.32 min. deposit no. DSM 8435 
S.infantis Ssq/Sm 153/Rif 7 GT:.apprxeq.31 min. deposit no. DSM 8434 
S.anatum Ssq/Sm 81/Rif 21 GT:.apprxeq.32 min. deposit no. DSM 8441 
S.typhimurium Nal 2/Rif 9/Rtt GT:.apprxeq.32 min. 
(location of deposit: DSM--Deutsche Sammlung von Mikroorganismen und 
Zellkulturen GmbH Braunschweig) 
Used test and working animals for testing the respective mutants of the 
sets by means of methods known as such 
chicken chicks (laying brown eggs, vaccinated against Marek) 
ICR and Prob 01 mice. 
EXAMPLE 1 
Revertants Having a Shortened Generation Time (GVR), derived from metabolic 
drift attenuated Salmonella original vaccine strains (with envelope 
marker) for chicks/chickens, as vaccine strains having an increased 
stability in the attenuation, 
The original vaccine strains (with envelope marker) are vaccinated in 50 ml 
of nutrient bouillon (or in any other liquid medium), and are then 
passaged continuously with the dilution factor.ltoreq.1:1000. 
Marker Controls: 
After each time four passages the received growth on nutrient agar with 
(12.5 .mu.g nalidixic acid/ml or) 200 .mu.g streptomycin/ml as well as 200 
.mu.g rifampicin/ml (as well as the missing growth with 20 .mu.g 
erythromycin) is tested by means of a vaccine swab. 
Within the 30 passages controlled the markers were stable. 
Determination of Generation Time (GT) in the MS-2 Test System (Abbott): 
In liquid cultures (nutrient, LB and minimal medium bouillon) the GTs do 
not change significantly up to the fifth passage. From the sixth passage 
on, faster growing GVR clones (graded shortening of GT) accumulate in the 
original vaccine strain populations (GT about 31 to 32; wild strain about 
22 minutes). This process takes place in separate steps with different 
speed. The GTs between the 10th and the 15th passage differ between 29 and 
27 minutes, up to the 30th passage GT of .ltoreq.25 minutes can occur. 
With further passages clones with even shorter GTs are likely. 
Remark: On solid media the GTs remain unchanged until the 20th passage 
tested (lower number of divisions (overvaccination with loop)=dilution 
down to population level or other selective pressure?). 
Unchanged or Only Slightly Lower Attenuation Level of S.typhimurium GVR 
Strains in Comparison to the Original Vaccine Strain: 
original strain and GVR strains: five mice each (Prob 01: laboratory of the 
University of Leipzig) receive i. p. 10.sup.5, 10.sup.6 and 10.sup.7 cfu 
wild strain: five mice each receive i. p. 10.sup.3 and 10.sup.5 cfu. 
The animals which died within a period of two weeks are determined. 
______________________________________ 
pas- GT No. dead/ 
S. typhimurium GVR strains 
sages (min) 15 mice 
______________________________________ 
Nal 2/Rif 9-GVR: Mm A 11/Rtt 
11 28,0 3 Wh 3 
.sup. GVR: Mm C 11/Rtt 
11 28,5 1 
.sup. GVR: Mm E 11/Rtt 
11 27,0 7 
.sup. GVR: Nb A 15/Rtt 
15 29,0 4 
.sup. GVR: Nb B 15/Rtt 
15 28,5 7 Wh 4 
.sup. GVR: Nb B 25/Rtt 
25 27,0 2 
.sup. GVR: Nb C 15/Rtt 
15 28,5 0 Wh 0 
.sup. GVR: Nb D 15/Rtt 
15 28,5 2 
.sup. GVR: Nb D 25/Rtt 
25 28,0 1 
.sup. GVR: Nb E 15/Rtt 
15 28,5 2 
.sup. GVR: LB A 15/Rtt 
15 28,5 3 
.sup. GVR: LB B 15/Rtt 
15 28,5 5 
.sup. GVR: LB C 15/Rtt 
15 28,0 7 Wh 4 
.sup. GVR: LB C 25/Rtt 
25 26,0 7 
.sup. GVR: LB D 15/Rtt 
15 28,5 0 Wh 0 
.sup. GVR: LB D 25/Rtt 
25 26,5 0 
.sup. GVR: LB E 15/Rtt 
15 27,0 7 Wh 4 
*Ssq/Sm 60/Rif 42-GVR III 12 
12 29,0 7 
GVR III 16 16 28,0 8 
GVR I 30 30 28,5 8 
GVR II 30 30 26,0 9 
Nal 2/Rif 9/Rtt 
three parallel tests 
32,0 4 Wh 6, 7 
prototype four tests 1992-1990 5, 8, 5, 6 
wild strain control 
i.p. 10.sup.3 cfu = .gtoreq. 14; 
i.p. 10.sup.5 cfu = 15 
______________________________________ 
Mm: liquid minimum medium 
Nb: nutrient bouillon 
LB: LBBouillon 
Wh: animal test repeated with same strain 
*S. tm Ssq/Sm 60/Rif 42 original strain; GT: 31 minutes 
Apart from a few apparently slightly higher (or slightly lower) attenuated 
clones, the GVR strains tested show a variation in their attenuation 
behavior which corresponds to the variation of the original strain values 
determined in seven separate approaches 1993-1990, and which are within 
the variation under test conditions. Even if a few GVR strains are 
attenuated slightly lower, this is irrelevant for the practice: the 
differences in the LD.sub.50 mouse in comparison to the wild strain (i. p. 
LD.sub.50 mouse about 10.sup.1 cfu) of about five logarithmic steps are 
retained. 
In the case of strain-dependent separation of GVRs with perhaps 
significantly lower attenuation level (and possibly not leading to 
acceptable side effects) preferrably vaccine strains with a higher (up to 
the border of overattenuation) attenuation are used for the isolation of 
optimally attenuated suppressor mutants. 
In other words: The GVRs are (like Sm-id and T-tol strains, Linde. K.: 
Arch. exper. Vet. med. 1978, 32, 943-949) no back mutants but apparently 
intra and intergenetical suppressor mutants still having their attenuation 
markers and an attenuation level which varies around the original value of 
the vaccine strain. 
For the detection of the unchanged invasive capacity in comparison to the 
original vaccine strains .ltoreq.36 hours old chicks orally received 
10.sup.9 cfu of the GVR. After five and eight days the germ count/gram 
liver was determined quantitatively (see example 2) as well as 
qualitatively by means of the accumulation (nutrient media with 100 .mu.g 
streptomycin and rifampicin/ml in the case of the GVRs of the Ssq/Sm/Rif 
strains, or 100.mu.g rifampicin and 12.5 .mu.g nalidixic acid/ml in the 
case of the GVRs of the Nal/Rif/Rtt strain): the germ counts of the GVRs 
in the chick's liver more or less correspond to the values of the original 
vaccine strains. With respect to S. infantis and S. anatum lower germ 
counts are found in the wild strain, the original strain as well as in the 
selected GVRs in comparison to S. enteritidis and S. typhimurium, which 
corresponds to U. Methner's observation (doctoral thesis, University of 
Leipzig, Veterinary Medical Faculty, 1991) that S. infantis is less 
invasive for chickens. 
Therefore, the revertants having a shortened generation time, as e. g. S. 
tm Ssq/Sm 60/Rif 42/-GVR II 30, S. ent Ssq/Sm 24/Rif 12/-GVR II 30, S. inf 
Ssq/Sm 153/Rif 7/-GVR II 30, S. ana Ssq/Sm 81/Rif 21/-GVR II 30, are ideal 
as salmonella vaccine strains having an extreme stability in the 
attenuation (total stability of the attenuation markers reducing virulence 
at least .ltoreq.10.sup.-21 : Sm.times.Rif.times.suppressor mutation) (see 
also table at the end). 
EXAMPLE 2 
Macrolide Tolerant Revertants (MTR), Derived from the Ssq (Envelope) Marker 
of the Sm/Rif Metabolic Drift Attenuated Vaccine Strains for 
Chicks/chickens, as Salmonella Vaccine Strains Having an Increased 
Stability in the Antiepidemic Potency. 
10.sup.8 to 10.sup.9 cfu of the Ssq/Sm/Rif original strains are transferred 
onto nutrient agar containing 20 .mu.g erythromycin/ml (or in the two step 
procedure at first onto 5 to 10 .mu.g erythromycin/ml and from this 
culture onto 20 .mu.g erythromycin/ml) by means of a spatula, and the 
grown colonies/clones are tested with respect to the macrolide tolerance 
they obtained after about ten passages on nutrient agar. The 
tolerance/sensitivity of these macrolide tolerant revertants to 
Na-desoxycholate, Na-dodecylsulphate as well as a. o. ciprofloxacin 
(presently the most effective antibiotic against Salmonella), 
chloramphenicol and doxycycline generally corresponds to the values of the 
wild strain, individual revertants of S. enteritidis, however, still have 
the up to four times higher (super)-sensitivity of the original strain to 
ciprofloxacin. 
The death kinetics (10.sup.6 cfu/ml starting germ count) in water at 37 
.degree. C. (as an indirect measure for the environmental resistance) does 
not show the values of the wild strain, since there is a variation (in 
comparison to the wild strain/vaccine strain without envelope marker) in 
the life span of the vaccine strain, which is shortened to about two 
thirds. 
A selection of these revertant--with respect to a similar as or even 
shorter survival span in water in comparison to the original vaccine 
strain and, with respect to S. enteritidis, the remaining supersensitivity 
to ciprofloxacin--was tested in the chick model in comparison to the 
vaccine strain with respect to invasion and excretion behavior. For this, 
.ltoreq.36 hours old chicks orally received 10.sup.9 cfu of the respective 
original vaccine strains and of the macrolide tolerant revertants derived 
from those: 
Detection of invasive capacity: After five and eight days the germ 
count/gram liver was determined quantitatively (liver homogenate in 5 ml 
PBS, 3.times.0.1 ml spread by means of a spatula; border of detection 
.ltoreq.20 germs/gram) as well as qualitatively by means of the 
accumulation in nutrient bouillon containing an antibiotic additive (see 
example 1). 
Testing the excretion behavior: Over a period of twelve days the salmonella 
germ counts are determined in the faeces as a thousandth of the coli 
population, and these values are compared to those of an additional 
comparative vaccine strain S. tm Nal 2/Rif 9 without envelope marker (with 
the same i. p. LD.sub.50 mouse and similar prolongation of generation time 
as e. g. the S. tm Ssq/Sm 60/Rif 42). 
Also in this model, the macrolide tolerant revertants show a similar 
behavior to that of the original vaccine strain (with envelope marker); 
Invasive capacity: The germ counts of the revertants in the chicken liver 
correspond to the values of the original vaccine strain. With respect to 
S. infantis and S. anatum lower germ counts are found in the wild strain, 
the original vaccine strain as well as in the selected revertants in 
comparison to S. enteritidis and S. typhimurium, which, however, 
corresponds to U. Methner's observation (doctoral thesis, University of 
Leipzig, veterinary Medical Faculty, 1991) that S. infantis is less 
invasive for chickens. 
Excretion behaviour: The quantitative excretion behavior of the macrolide 
tolerant revertants, determined from the thousandth part of 
enterobacteria, approximately corresponds to that of the original vaccine 
strains and drops below the 0.1 thousandth border within five to six days. 
In other words: The similar or partly even faster death rate in water with 
respect to the tested macrolide tolerant revertants in comparison to the 
original vaccine strains as well as the de facto identical excretion 
behaviour (and the unchanged invasive capacity) in chicks indirectly 
proves that these strains are no back mutants in the envelope marker and, 
therefore, the Ssq marker in the original strains has a much higher 
stability than the reversion frequency of .ltoreq.10.sup.-8 given in the 
phylogenetic pass. 
Therefore, the macrolide tolerant revertants, as e. g. S. tm Ssq-MTR 16/Sm 
60/Rif 42, S. ent Ssq-MTR 25/Sm 24/Rif 12, S. inf Ssq-MTR 12/Sm 153/Rif 7 
and S. ana Ssq-MTR 23/Sm 81/Rif 21, are ideal as vaccine strains having an 
increased stability in the antiepidemic potency (total stability=product 
of the individual stabilities of the Ssq marker and the suppressor 
mutations) (see following table). 
SUPPLEMENT: TABLE WITH RESPECT TO EXAMPLES 1 AND 2 
GVR derived from metabolic drift attenuated double marker (original) 
vaccine strains with envelope marker as well as MTR*: De facto unchanged 
invasive capacity (germ count/gram liver five and eight days after oral 
application of 10.sup.9 cfu to .ltoreq.36 hours old chicks); remaining 
antiepidemic potency of the MTR* (death kinetics of 10.sup.6 cfu/ml in 
water at 37.degree. C.) 
__________________________________________________________________________ 
.0. germ count/gram liver 
cfu water 
Salmonella 
strains GT (min) 
fifth day 
eight day 
ninth day 
__________________________________________________________________________ 
typhimurium 
wild strain 
60/Rif 42 
22,0 6,0 .times. 10.sup.3 
nt 4 .times. 10.sup.5 
Ssq/Sm 31,0 1,5 .times. 10.sup.3 
2,8 .times. 10.sup.2 
4 .times. 10.sup.3 
GVR II/30 
26,0 2,5 .times. 10.sup.3 
6,3 .times. 10.sup.2 
GVR III/16 
28,0 1,5 .times. 10.sup.3 
2,8 .times. 10.sup.2 
MTR 16 1,0 .times. 10.sup.3 
1,4 .times. 10.sup.2 
1 .times. 10.sup.4 
MTR 40 1,5 .times. 10.sup.3 
5,6 .times. 10.sup.2 
1 .times. 10.sup.4 
enteritidis 
wild strain 
24/Rif 12 
22,0 5,0 .times. 10.sup.3 
nt 3 .times. 10.sup.5 
Ssq/Sm 32,0 4,0 .times. 10.sup.3 
nt 2 .times. 10.sup.4 
GVR II/30 
28,0 4,5 .times. 10.sup.3 
5,3 .times. 10.sup.2 
GVR IV/16 
27,0 2,0 .times. 10.sup.3 
nt 
MTR 2 2,5 .times. 10.sup.3 
1,9 .times. 10.sup.2 
5 .times. 10.sup.3 
MTR 25 2,5 .times. 10.sup.3 
5,6 .times. 10.sup.2 
7 .times. 10.sup.3 
anatum 
wild strain 
81/Rif 21 
22,0 1,5 .times. 10.sup.2 
nt 4 .times. 10.sup.4 
Ssq/Sm 32,0 3,5 .times. 10.sup.1 
accumulation 
1 .times. 10.sup.4 
GVR II/30 
26,5 1,0 .times. 10.sup.2 
mostly 
GVR III/16 
27,5 2,0 .times. 10.sup.2 
positive 
MTR 5 3,5 .times. 10.sup.1 
1 .times. 10.sup.4 
MTR 23 7,0 .times. 10.sup.1 
1 .times. 10.sup.4 
infantis 
wild strain 
153/Rif 7 
22,0 2,0 .times. 10.sup.2 
nt 5 .times. 10.sup.5 
Ssq/Sm 31,0 1,0 .times. 10.sup.2 
accumulation 
6 .times. 10.sup.4 
GVR II/30 
25,0 7,0 .times. 10.sup.1 
mostly 
GVR III/24 
28,5 1,0 .times. 10.sup.2 
positive 
MTR 12 2,0 .times. 10.sup.2 
7 .times. 10.sup.4 
MTR 28 2,0 .times. 10.sup.2 
6 .times. 10.sup.4 
__________________________________________________________________________ 
*MTR: Doctoral theses of Adler, T. and Jaensch, C. (1993/94)