Note: Descriptions are shown in the official language in which they were submitted.
~ 3~72
AVIRULENT MICROl~ES AND USES TIIEREF()R
Field of the Invention
This invention relates to avirulenk microbes,
their method of preparation, and their use in vaccines.
Backqround of the Invention
Salmonellosis of swine is one of the most
economically important of the enteric and septicemic
diseases affecting youn~ pigs, and has been described as a
significant health problem in swine. Although many
serotypes of Salmonella have been isolated from pig5, S.
choleraesuis var. kunzendorf and _. typhimurium are the
two most frequently isolated serotypes associated with
clLnical salmonellosis in swine, Wilcock, B.P., in
DISEASES OF SWINE, pp. 508-519 (Leman, A.D., et al., eds.,
1986). S. choleraesuis is host-adapted to swine and is
often the etiologic agent of fatal septicemic disease with
little involvement of the intestinal tract. This S.
choleraesuis reservoir in swine is a concern not only
bacause of its disease-causing potential for pigs, but
also because of its public health significance for humans.
The disease caused by S. choleraesuis manifests
in many clinical signs. The organism i5 inherently
invasive, and does not require the massive luminal
proliferation required by S typhimurium. Lesions and
necrosis occur in the submucosa and lamina propria of the
gut. Mortality is high, and the duration and severity of
the disease is unpredictable.
'~ . "`''~' ` '
:
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.
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Currently there is little information on vaccine
use for control of swine salmonellosis. A rough variant
of S. choleraesuis was used by Smith, H.W., J. ~yq.
63:117-135 (196S), to demonstrate protection after chal-
S lenge with virulent S. choleraesuis. However, the animalsdeveloped fever, sublethal disease, and became shedders of
the bacteria. The Smith strain is commercially available
in Europe as Suscovax~, which is manufactured and
distributed by Wellcome Laboratories. Hanna, J., et al.,
Vet. M.icrobiol. 3:303-309 tl979), reported use of Smith's
live attenuated S. choleraesuis vaccine by intramuscular
route in pregnant sows. The piglets, after birth, had
high titers of circulating maternally-derived antibodies r
and resisted intranasal challenge. Although it has been
reported that qalE mutants of S. typhimurium are avirulent
and immunogenic, in contrast, S. choleraesuis strains with
qalE mutations remain as virulent in mice as the wild-type
qal parent. S. choleraesuis auxotrophs with requirements
for aromatic amino acids due to an aroA mutation have
reduced virulence in mice, but were unable, even after
three immunizing doses, to induce protective immunity. At
present, the only S. choleraesuis vaccine licensed for use
in the United States of ~merica is a killed bacterial
bac~rin, which is not particularly efective in inducing
protective immunity.
Applicant has discovered a new method of
protecting against virulent in~ections with a Yaccine
employing transposon-induced avirulent mutants of virulent
agents in which the impairment leading to avirulence can-
not be repaired by diet or by anything supplied by ananimal host. Applicant's initial work, including a method
for creating an avirulent microbe by the introduction of
deletion mutations in the adenylate cyclase gene (cya) and
the cyclic AMP receptor protein gene (crP) of Salmonella
ty~himurlum is described in EP0 Pub. No. 315,682
(published ~7 May lg89), and PCT Pub. No. WO 88/09669
`
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(published lS December 1988). The disclosure of these
patent applications, as well as any corresponding national
patent applications, are incorporated herein by reference.
These applications teach, inter alia, recombinant DNA
methods of inactivating cya and crp genes in S.
typhimurium, introducing thereby a recombinant gene for a
heterologous pathogen in the avirulent S. typhimurium, and
thereby producing a new antigen capable of inducing an
immune response, introducing a recombinant gene for a
product capable of suppressing, modulating, or augmenting
an immune response, and vaccines and methods of
vaccination utilizing the avirulent S. typhimurium
constructs.
lS 8rief Description of the Invention
The present invention is based, in part, on new
avirulent S. choleraesuis derivatives that are not
disclosed in EPO Pub. No. 315,682. Included within the
invention is the application of these new S. choleraesuis
derivatives in, inter alia, commercial vaccines, methods
of stimulating the immune system to respond to an im-
munogenic antiqen of S. choleraesui~, and methods of
stimulating the immune system to respond to an immunogenic
antigen of a pathogen. The strains provided herein are
directly and indirectly suitable for the production of
commercial vaccines to prevent disaases caused by S.
choleraesuis, and other enteric bacteria with which anti-
bodies to S. choleraesuis cross react. These strains are
also useful as ca.rrier microorganisms for the production
of expression products encoded on recombinant genes in the
bacterial cells.
Accordingly, one embodiment of the invention is
a vaccine for the immunization of an individual comprising
an avirulent derivative of pathogenic S. choleraesuis,
said derivative being substantially incapable of producing
--4--
functional adenylate cyclase due to a mutation in a c a
gene.
Another embodiment of the invention is a method
for stimulating the immune system to respond to an im-
munogenic antigen of S. choleraesuis comprisingadministering to said individual an avirulent derivative
of pathogenic S. choleraesuis, said derivative being
substantially incapable of producing functional adenylate
cyclase due to a mutation in a cya gene.
Still another embodiment of the invention is a
method for stimulating the immune system to respond to an
immunogenic antigen of a pathogen comprising administering
to said individual an avirulent derivative of pathogenic
S. choleraesuis, said derivative being substantially in-
capable of producing functional adenylate cyclase and
cyclic AMP receptor protein (CRP) and being capable of
expressing a recombinant gene encoding the immunogenic
antigen, to produce an antigen capable of inducing an
immune response in said vertebrate against said pathogen.
Another embodiment of the invention is an
isolated avirulent strain of S. choleraesuis which is
substantially incapable of producing functional adenylate
cyclase.
Still another embodiment of the invention is a
~5 vaccine for the immunization of an individual comprising
an avirulent derivative of pathogenic S. choleraesuis,
said derivative being substantially incapable of producing
functional cyclic AMP receptor protein (CRP) due to a
mutation in a crp gene.
Yet another embodiment of the invention is a
method for stimulating the immune system to respond -to an
immunogenic antigen of S. choleraesuis comprising
administering to said individual an avirulent derivative
of pathogenic S. choleraesuis, said derivative being
. . .
substantially incapable of producing functional CRP due to
a mutation in a crp gene.
-
2013~72
Another embodiment of the invention is a methodfor stimulating the immune system to respond to an im-
munogenic antigen of a pathogen comprising administering
to said individual an avirulent derivative of pathogenic
S. choleraesuis, said derivative being substantially in-
capable of producing functional CRP and being capable of
expressing a recombinant gene encoding the immunogenic
antigen, to produce an antigen capable of inducing an im-
mune response in said vertebrate against said pathogen.
Yet another embodiment of the invention is an
isolated avirulent strain of S. choleraesuis which is
substantially incapable of producing functional CRP due to
a mutation in a crp gene.
Still another embodiment of the invention is a
vaccine for the immunization of an individual comprising
an avirulent derivative of pathogenic S. choleraesuis,
~aid derivative being substantiall~ incapable of producing
functional adenylate cyclase and CRP due to a mutation in
the y~ and crp genes.
Yet another embodiment of the invention is a
method for stimulating the immune system to respond to an
immunogenic antigen of S. choleraesluis comprising
administering to said individual an avirulent derivative
of pathogenic S. choleraesuis, saicl derivative being
substantially incapable of producing functional adenylate
cyclase or CRP due to a mutation in a cya gene and a crp
gene.
Another embodiment of the invention is a method
for stimulating the immune system to respond to an im-
munogenic antigen of a pathogen comprising administeringto said individual an avirulent derivative of pathogenic
S. choleraesuis, said derivative being substantially in-
capable of producing functional adenylate cyclase and CRP
and being capable of expressing a recombinant gene encod-
ing the immunogenic antigen, to produce an antigen capable
2 ~ 7 ~
--6--
of inducing an immune response in said vertebrate againstsaid pathogen.
Still another embodiment of the invention is an
isolated avirulent strain of _. choleraesuis which is
substantially incapable of producing functional adenylate
cyclase and CRP due to mutations in the cya and crp genes.
Another embodiment of the invention is a strain
selected from the group of strains ATCC 53647, ATCC 53648,
ATCC 67923, ATCC 53885, ATCC 67922, and mutants thereof,
and derivatives thereof.
Detailed Description of the Invention
Definitions:
~Vaccine,~ as used herein, means an agent used
to stimulate the immune system of a living organism so
that protection against future harm is provided. ~Im-
muni~ation" refers to the process o~ inducing a continuing
high level of antibody and/or cellular immune response in
which T-lymphocytes can either kill a pathogen and/or
activate other cells (e.g., phagocytes) to do so in an
organism, which is directed against a pathogen or antigen
to which the organism has been previously exposed.
Although the phrase ~immune system~ can encompass
responses of unicellular organisms to the presence of
foreign bodies, e.g., interferon production, in this ap-
plication the phrase is restricted to -the anatomical
features and mechanisms by which a multicellular organism
produces antibodies against an antigenic material which
in~ades the cells of the organism or the extracellular
fluid of the organism. The antibody so produced may
belong to any of the immunological classes, such as
immunoglobulins A, D, E, G or M. Of particular interest
are vaccines which stimulate production of immunoglobulin
A (~gA) since this is the principle immunoglobulin
produced by the secretory system of warm-blooded animals,
: : ~
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although vaccines of the invention are not limited to
those which stimulate IgA production. For example, vac-
cines of the nature descrihed herein arP likely to produce -
a broad range of other immune responses in addition to IgA
formation, for example, cellular and humoral immunity.
Immune response to antigens is well studies and widely
reported. A survey of immunology is given in Barrett,
James, T., Textbook of Immunoloqy: Fourth Edition, C.V.
Mosby Co., St. Louis, MO (1983), the entire of which is
herein incorporated by reference.
The term "vertebrate," as used herein r means any
member of the subphylum Vertebrata, a primary division of
the phylum Chordata that includes the fishes, amphibians,
reptiles, birds, and mammals, all of which are character-
ized by a segmented bony or cartilaginous spinal column.
All vertebrates have a functional immune system and
respond to antigens by producing antibodies. Thus, all
vertebrates are capable of responding to vaccines.
Although vaccines are most commonly given to mammals, such
as humans or dogs (rabies vaccine), vaccines for com-
mercially raised vertebrates of other classes, such as the
fishes and birds if of the nature described herein, are
within the scope of the present in~ention.
By "invertebrate" is meant any member o~ the
A~imal Kingdom, excluding the vertebrates. Such animals
constitute the Division Invertebrata and have no backbone
or spinal column. This classification includes all
animals except fishes, amphibians, reptiles, birds and
mammals. Many invertebrates are capable of eliciting a
primitive immune response to antigenic stimulation and are
susceptible to the same microorganisms which infect
vertebrates and which are disclosed herein in accordance
with this invention. Examples of such invertebrates are
shellfish and mollusks and other related animals.
Although the use of vaccines in the protection of in-
vertebrate animals have hitherto before not been well
,
~3~
documen~ed, one skilled in the art will recognize the ap-
plicability of the subject invention to said invertebrates
by use of their primitive immune systems. For example,
and in accordance with this invention, the susceptibility
of shellfish to infection by S. choleraesuis will allow
the introduction of avirulent strains of S. choleraesuis
species and thereby provide potential for the primitive
immune system to respond. Therefore, the use of an
avirulent derivative of a pathogenic microbe that is
capable of infecting an invertebrate to stimulate a
response from an immune system present in the invertebrate
against a pathogen is within the scope of this invention.
An "individual~ treated with a vaccine of the
invention, as used herein, is defined to include all
vertebrates, such as mammals, including domestic animals
and humans, and various species of birds, includin~
domestic birds, particularly those of agricultural
importance. In addition, mollusks and certain other in-
vertebrates have a primitive immune system, and are
in~luded as "individuals."
The term "avirulent", as used herein, does not
mean that a microbe of that genus c,r species cannot ever
function as a pathogen, but that the particular microbe
being used is avirulent with respect to the particular
animal being treated. The microbe may belong to a genus
or even a species that is normally pathogenic but must
belong to a strain that is avirulent. "Pathogenic~', as
used herein, means capable of causing disease or impairing
normal physiological functioning. An "avirulent strain"
is incapable of inducing the full set of symptoms of the
disease that is normally associated with its virulent
pathogenic counterpart. The term ~microbes~, as used
herein, includes bacteria, protozoa, and unicellular
fungi.
Derivatives of avirulent S. choleraesuis
microbes are also contemplated to be within the scope o~
, :,
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this invention. By ~derivativell is meant sexually or
asexually derived progeny and mutants of -the avirulent
strains including single or multiple base su~stitutions,
deletions, insertions or inversions which retain the in-
ability to produce functional adenylate cyclase and cAMPreceptor protein with or without naturally occurring
virulence plasmids. For example, strains such as Chi4062
and Chi4064 carry the qyrA mutation conferring nalidixic
ac.id resistance which has been used herein as a convenient
marker to follow strains following oral inoculation.
E~owever, drug resistance is not a desirable attribute for
strains to be used as vaccines. Thus, the qyrA+ mutation
can be easily removed by transducing the qyrA+ (conferring
sensitivity to nalidixic acid) gene into strains by
selecting for inheritance of a closely linked TnlO and
then removing TnlO by selection for fusaric acid resist-
ance.
The term "recombinant gene", as used in this
application, refers to genetic material that has been
transferred from one organism into a second in such a man-
ner that reproduction of the second organism gives rise to
descendants containing the same genetic material.
Techniques for transferring genetic material from a first
organism to a second organism that normally does not
exchange genetic material ~ith the first organism have
become widely available as the result of rapidly expanding
recombinant DNA technology.
The term ~gene~ is used herein in its broadest
sense to represent any biological unit of heredity. It is
not necessary that the recombinant gene be a complete gene
as present in the parent organism, which was capable of
producing or regulating the production of a macromolecule,
for example, a functioning polypeptide. It is only neces-
sary that the gene be capable of serving as the template
in the production of an antigenic product. The product
will not necessarily be found in that exact form in the
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~ 3~7~
--10--
parent organism. For example, a functional gene coding
for a polypeptide antigen comprising 100 amino acid
residues may be transferred in part into a carrier microbe
so that a peptide comprising only 75, or even 10, amino
acid residues is produced by the cellular mechanism of the
host cell. However, if this gene product is an antigen
that will cause formation of antibodies against a similar
antigen present in the parent organism, the gene is
considered to be within the scope of the term "gene~ as
defined herein. Alternatively, if the amino acid sequence
of a particular antigen or fragment thereof is known, it
is possibla to chemically synthesize the DNA fragment or
analog thereof by means of automated gene synthesizers or
the like and introduce said DNA sequence into the ap-
propriate expression vector. At the other end of thespectrum is a long section of DNA coding for several gene
products, one or all of which can be antigenic. Thus, a
gene as defined and claimed here is any unit of heredity
capable of producing an antigen. I~he gene ma~ be of
ch~omosomal, plasmid, or viral ori~in.
The term "gene expressio~", as used here means
that the information inherent in the structure of the gene
is transformed into a physical product in the form of an
RNA molecule, polypeptide or other biological molecule by
the biochemical mechanisms of the cell in which the gene
is located. The biological molecule so produced is called
the gene product. The term "gene product" as used herein
refers ~o any biological product or products produced as a
result of the biochemical reactions that occur under the
control of a gene. The gene product may be, for example,
an RNA molecule, a peptide, or a product produced under
the control of an enzyme or other molecule that is the
initial product of the gene, i.e., a metabolic product.
For example, a gene may first control the synthesis of an
RNA molecule which is translated by the action of
ribosomes into an enzyme which controls the formation of
: . , ~.,
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2~3~'7~
--11--
glycans in ~he environment external to the original cell
in which the gene wa~ found. The RNA molecule, the
enzyme, and the glycans are all gene products as the term
is used here. Any of these as well as many other types of
gene products, such as glycoproteins and polysaccharides,
will act as antigens if introduced into the immune system
of an animal. Protein gene products, including
glycoproteins and lipoproteins, are preferred gene
products for use as antigens in vaccines.
The term "allergensl', as used herein, means
substances that cause allergic reaction, in this case in
the animal which will be vaccinated against them. Many
different materials may be allergens, such as animal
dander and pollen, and the allergic reaction of individual
animals will ~ary for any particular allergen. It i3 pOS-
sible to induce tolerance to an allergen in an animal that
normally shows an allergic response. The methods of
inducing tolerance are well-known and generally comprise
administering the allergen to the animal in increasing
dosages. Further discussion of tolerance induction is
given in the Barrett textbook previously cited.
The Invention:
This invention is predicated on the discovery
that certain mutations can render a microbe avirulent
without substantially affecting its immunogenicity. More
specifically, this invention is made possible by microbial
vaccines in which the microbe carries the deletion (delta)
mutations delta-cya and delta-crp. These deletions
eliminate the ability to synthesize adenylate cyclase (ATP
pyrophosphate lyase (cyclizin~) EC 4.6.1.1) and the cyclic
AMP receptor protein (CRP), respectively, as described in
~PO Pub. No. 315,682.
EPO Pub. No. 315,862 described how the elimina-
tion of cyclic-3',5'-adenosine monophosphate (cA~IP),
adenylate cyclase and the cyclic AMP receptor protein
' ~
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2~33~
through delta-cya and delta-crp mutants rendered S.
typhimurium avirulent. The procedure for obtaining those
mutants was followed in the current invention, and applied
to a new strain, S. choleraesuis, to obtain avirulent
mutants thereof which can be used as the immunogenic
component of a vaccine to induce S. choleraesuis immunity.
In another embodiment of the invention, the
avirulent S. choleraesuis derivative can be used as a car-
rier bacteria to deliver selected antigens to the gut-
associated lymphoid tissue (GALT), for example -to the
Peyer's patches of the ileum. Methods for expression of a
recombinant gene from a pathogenic organism in S.
typhimurium to induce antibody production against the
pathogen in the host were reported in EPO Pub. No.
315,682. That publication also explains how the carrier
microbe may present the recombinant pathogen-derived
antigen to the host.
Recombinant DNA techniqu~s, whereby genetic
material from one organism is tran~;ferred to and becomes a
permanent part of the genetic material of a second organ-
ism, are now sufficiently well known and widespread so as
to be considered routine. Usually, a small piece of DNA
from the parent organism is obtained either from a plasmid
or a pa.rent chromosome. A plasmid (also called an
extrachromosomal element~ is a hereditary unit that is
physically separate from the chromosome of the cell. The
DN~ may be of any size and is often obtained by the action
of a restriction endonuclease enzyme which acts to split
DNA molecules at specific basepair sites. Following liga-
tion to plasmid, phage or cosmid vectors, ~he new re-
combinant molecules may be transferred into a host cell by
various means such as transformation (uptake of naked DNA
from the external environment, which can be artificially
induced by the presence of various chemical agents, such
as calcium ions) or transduction (recombinant DNA packaged
and introduced within a phage such as transducing phage or
.:
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cosmid vectors). Recombinant DNA in the carrier cell may
continue to exist as a separate piece of DNA or it may
insert into the host cell chromosome and be reproduced
with the chromosome during cell division. This invention
sometimes utilizes transposons as the transferred
material. Transposons are highly movable pieces of DNA
that insert in DNA and may also be excised. The-excision
may carry off surrounding genetic material, causing
deletion mutations. Presence or absence of transposons is
then monitored by antibiotic-residue genetic markers.
Selection for organisms that have received
transferred genetic material is performed by a ~shotgun~
approach when pathogen-derived antigens are desired. The
"shotgun~ approach is described in EPO Pub. No. 315,682,
and in detail in Maniatis, T., et al., Molecular Clonin~,
Cold Spring Harbor Laboratories (1982), which are herein
incorporated by re~erence. The techniques of gene
transfer are not considered to be part of this invention,
and any method capable of producin~ recombinant organisms
comprising genes from an organism t:hat are expressed in
avirulent microbes will suffice. ~:n cases where the spe-
cies normally exchange genetic information, classical
methods of gene transfer such as conjugation, transforma-
tion or transduction may be employed.
In another embodiment, when the immunogenic
component of the vaccine is an allergen of the host such a
vaccine may be used in an exposure regimen designed to
specifically desensitize an allergic host.
Yet another embodiment of this invention
provides a vaccine for the immunization of a vertebrate or
invertebrate animal comprising a live avirulent derivative
of S. choleraesuis incapable of producing functional
adenylate cyclase and cAMP receptor protein, and capable
of e~pressing a recombinant gene derived from an organism
that is a pathogen of or that produces an allergen of said
animal.
~ 3~
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In an embodiment which contemplates ~11 of the
above, a subject of the invention is avirulent strains of
S. choleraesuis, which carry mutations in the cya and/or
crp genes.
Administration and Use:
In order for a vaccine to be effective in
producing antibodies, antigenic material must be released
in such a way that the antibody-producin~ mechanism of the
vaccinated animal can come into play. Therefore, the S.
choleraesuis carrier of the gene product must be properly
introduced into the animal~ In order to stimulate a
preferred response of the GALT or bronchus-associated
(BALT) cells as discussed previouslyl introduction of the
microbe or gene product directly into the gut or bronchus
is preferred, such as by oral administration, gastric
intubation or in the form of aerosols, althou~h other
methods of administering the vacci.ne, such as intravenous,
intramuscular, subcutaneous injection or intramammary or
intrapenial or vaginal administration, are possible.
La~tly, the host organism itself can serve as a source of
genetic material when immunoregulatory ~enes or genes for
other pharmacologically actlve substances are being
expressed by the vectors.
Administration of a live vaccine of the type
disclosed above to an animal may be by any known or
standard technique. These include oral ingestion, gastric
intubation, or bxoncho-nasal spraying. ~11 of these
methods allow the live vaccine to easily reach the GALT or
BALT cells and induce antibody formation and are the
preferred me~hods of administration. Other methods of
administration, such as intravenous injection, that allow
the carrier microbe to reach the animal s blood stream may
be acceptable. Intravenous, intramuscular or intramammary
3S injection are also acceptable with other embodiments of
the invention, as is described later.
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~3.~72
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The dosages required will vary with the
antigenicity of the gene product and need only be an
amount sufficient to induce an immune response typical of
existing vaccines. Routine experimentation will easily
establish the required amount. Multiple dosages used as
needed to provide the desired level of protection.
The pharmaceutical carrier in which the vaccine
is suspended or dissolved may be any solvent or solid or
encapsulated in a material that is nontoxic to the
inoculated animal and compatible with the carrier organism
or antigenic gene product. Suitable pharmaceutical carri-
ers include liquid carriers, such as normal saline and
other nontoxic salts at or near physiological concentra-
tions, and solid carriers, such as talc or sucrose and
which can also be incorporated into feed for farm animals.
Ad~uvants may be added to enhance the antigenicity if
desired. When used ~or administering via the bronchial
tubes, the vaccine is preferably presented in the form o~
an aerosol.
Immunization with a pathogen derived gene
product can also be used in conjunction with prior im-
munization with the avirulent derivative of a pathogenic
microorganism acting as a carrier to express the gene
product specified by a recombinant gene from a pathogen.
Such parenteral immunization can serve as a booster to
enhance expression of the secretory immune response once
the secretory immune system to that pathogen-derived gene
product has been primed by immunization with the carrier
microbe expressing the pathogen derived gene product to
stimulate the lymphoid cells of the ~ALT or BALT. The
enhanced response is known as a secondary, booster, or
anamnestic response and results in prolonged immune
protection of the host. Booster immunizations may be
repeated numerous times with beneficial results.
The above disclosure generally describes the
present invention. ~ more complete understanding can be
: .
2~:~3~72
-16-
obtained by reference to the following specific example
which is provided herein for purposes of illustration only
and is not intended to be limiting unless otherwise
specified.
Example
This example illustrates the construction of
delta-~y~, dèlta-crP, and delta-cya delta-crP derivatives
of S. choleraesuis, the virulence properties of the
mutants after peroral (p.o.) inoculation, and the
immunogenicity of these derivatives.
Bacterial strains.
The S. choleraesuis strains used in this Example
are listed in Table 1. Bacterial strains were grown at
37C in L broth and on L agar (Lennox, Viroloqy 1:190-206
~3~i7~
T~le 1 l~on~ tralns.
~rcln No. ~ n~ gano~yp~ ~ou~
~, ~ atr~l~s
X3000 ~ld typ- L~2-2 Obt~l~ed ~ro- C. Su~nbou~h
~ZInd~r sn~ L-d~rberg, 1952
X30~2 ~lld typ- sL13~4 H~13eth und Stoc~r, 1981
~ull~ and Cu~tl~, 1997
~P~ a ~ sTR10 ~rD11223 }'O~t~ et ~1., 19116~
Cur~l~J ~nd ~elly, 1987
PP1037 ~D7732:~nlo trD322~ ~o~t~o ~t ~ J
Cu~t~ n~ ~lly, l9
X3339 ~lld ty~ S~1344 ~o w e-pa~ 4 ~ X3o~2
~ ` CU1l~ AA6 Curtlss, 1
X338S ~ 496 h-dLb h9d5A29 re~trlct~on~, ~odltlo~lon~
d-rlv-d ~o~ AS60 ~ T.
P~ CUl~ n6 Cu~a~, l9~a
X3~7~ ~lt!l -uvrPl-1005 h~ W hs~s~29 GUll~ ~nd Cur~la~ 2
X3~8S ~y~::TnlO lE~96 h~dL~ h9~2~ ~22~ 1At(mOO2) ~ X33~S
X34@6T q~::TnlO ~ 49a ha~L~ h~dsA29 X3~5 ly pnl~ w~h
X~52~ a~ -uvr~l-100~ crprr~ n~O
hiaS~2~ P22~r Int~7~10~ X3~
~p~04 cY~ 0 P2211T int~PtlO02) ~ X3339
X360~ cr~773~tTnlO P22~ lnt(PP10~7~ ~ X3339
CYo-~2 ~u--rlc ~cl~r, ~-tr-cycllneJ
d-rl v~tlv- of X3~0
X~2~ u~ c el~r, ~ctrccYCllne~
der~v-tiv- of ~3~05
X3656 le h~L6 hcdLT hudCl h~dSS N~k~ganc et al... l9B8
~l~o--
X3~70 (p6DllO) t~496 h~dL6 hc~5~29 X33BS transfo~ d ~lth pSDI10
X3711 ~cy~ c~d-6~stTnltl P22~T lrt~x3t~3) ~ )~61~
~ 3~;72
--18--
~ atruln~ or~ml-d~.
9e~n No. R~lævl~ne IE~no~y~ ~ourc~
S. t~phlmllrlu~ et~ain~
..
,t3738 ~id 62:~Tn1~ P22~ ~nlt(l~2104) ~ X3000
S~nder90~ a33d ROth, 1983
X3741 2hb~Ta~ 22~ nttDUU02) ~ XgOW
S~UtdQr907~ ~ 0th~ 1t`83
~a157 ~Y--12 ~ ,~_3:'rr310 P221~T 1nttX371l) ~ X338S
~ ls9~L6 ~
X~273 ~ler~-ll Zhb::r~10 P2Urr ~nt~?~ )Q623
X3819 ~Tn10 ~ 115496 P22~ 1nt~)t3773)
S. ChO1~a~DU1~ Str~in~
X3246 54~1~84 9V1nO 1~01ate V~ Un1Ve~91tY
d tYPa 0~ OU~1, CO1UObilt
X3492 ~ tTnlO Pl clr ~l~t~X3~T) ~ X324
X~7~ ttnlQ PlU~x~24) ~ X3245
X37~2 ~c~Q-19 Pw-rl~ a~ld~, t~ cycllna~
d~lntl~ 0~ X375~
X3733 ~ 24 ~Utl~t~C Acl~rt t-8~acyc~ cJ
d~riY~tlYe of X34g~
X37~3 (pSOllO) ~crp-19 PlL4~,~6~0) ~ ~37~2
X37~9 ~S~IiO) k~ ~c~ P~4~X37~7) ~ X373
8~-6~: ~Tn
~pSDllO) ~C~p-lg ~c~ Pu~arlc ~cl~', te~rseyel~ne~
a6~vat~l~e o~ X~7~9
X3781 ~p 19 ~c~ X~ c.urlid o~ p9Dllo
X~820 ~ Zhb::TnlO PlL4t~ 9~ ~ X32~
X3~58 ~e~,a-12 ~ld-~2:~hl0 nL4(~p757) ~ X32~6
: . .
;
,
.~ .
~3~
--19--
tltb~ t~ on~nu~
No. R~l~v~nt ~ typc~ So~r~:~
X3859 ~y~.12 Fu3~rle ~cldr, t~tr~cyclln~
derl~ tiv~ o~ X3858
X3~16~ ~c~ Pu~lc acldr, ts~a~yelln~J
d~lva~ of X3820
X3gO3 1~ld tYlpe X~2-6 eurQd of 50 kb
Yl~'UlgR~e p~541tl11
. ., . ~ . , ,
' , ' ` ~ ` :
:
~`' ' `` :,
~3~
-20-
(1965); Luria and Burrous, J. Bacteriol. 74:461-476
(1957)), Penassay agar (Difco antibiotic media #3 + 1.5
BBL agar, Becton Dickinson Microbiology Systems,
Cockeysville, MD) and MacConkey Base agar (Difco
Laboratories) with 1~ final concentration of an appropri-
ate carbohydrate. Media were supplemented with MgSO4 (10
mM), CaC12 (5 mM), tetracycline (12.5 micro~grams/ml) and
ampicillin (100 micrograms/ml) when required. Synthetic
media were minimal liquid and minimal agar supplemented
with nutrients at optimal levels as previously described
(Curtiss, J. Bacteriol. 89:28-40 (1965)). Buffered saline
with gelatin (BSG) was used as a diluent (Curtiss, 1965,
supra).
The pertinent references in Table 1 are the fol-
lowing:
Zinder and Lederberg, J. Bacteriol. 64:679 (1952).
Hoiseth and Stocker, Nature 291:238 (1981).
Gulig and Curtiss, Infect. Immun. 55:2891 (1987).
Postma et al., J. Bacteriol. 168:1107 (1986).
Curtiss and Kelly, Infect. Imnlun. 55:3035 (1987)
Gulig and Curtiss, Infect. Immun. 56:3262 (1988).
Genetic manipulations.
Transductions were performed with bacteriophages PlL4
or P22 HT int with standard methods and media, as
described in Curtiss, M~NUAL OF METHODS FOR GENERAL
BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, p. 243
(Gerhardt et al., eds., 1981), Schmeiger, Mol. Gen. Genet.
119:75 (1972), and Davis et al., ~ MANUAL FOR GENETIC
ENGINEERING: ADVANCED BACTERIAL GENETICS (Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1980). The
methods and media described by Maloy and Nunn, J.
Bacteriol. 145:1110 (1981) were used for fusaric acid
selection for deletion mutations in strains with Tnl0
insertions. Transformations were performed by the method
of Dagert and Ehrlich, Gene 6:23 (1979). The plasmid,
~13~
-21-
pSDllO, which carries the crp and Ampr genes from E. coli,
which was described in Schroeder and Dobrogosz, J.
Bacteriol. 167:616 (1986), was generously provided by C.
Schroeder. Curing of the virulence plasmid was
facilitated by use of pYA2028 which has the Inc/Par region
of the virulence plasmid cloned into the high copy number
plasmid pUC18, as described below.
Animal infectivity studies.
Female BALB/c mice ~Harlan Sprague-Hawley,
Indianapolis, IN) were used for all in~ectivity and im-
munization studies. Seven-week-old mice were held for one
week in a quarantined room prior to being used in
experiments. Animals were housed in Nalgene filter-
covered cages with raised wire floors. Food and water
were given ad lihitum. The animal room was maintained at
22C to 23C with 12 h of illumination daily.
Virulence of S. choleraesuis strains was determined
after p.o. inoculation. Bacteria Por inoculation in mice
were grown overnight as static cultures at 37 C in L
broth. All cultures were diluted 1:20 into prewarmed L
broth and aerated at 37C for approximately 5 h to an
optical density at 600 nm of a~ouk 0.8 to 1Ø The cells
were concentrated 50-fold by centrifugation at 8,000 x g
for 10 min at room temperature, followed by suspension in
BSG. Dilutions were plated on MacConkey agar with 1%
maltose to verif~ the Cya or Crp phenotype and to enumer-
ate cells.
Prior to p.o. inoculations, mice were deprived of
food and water for 4 h before infection. They were then
given 30 microliters of 10% (wt/vol) sodium bicarbonate 5-
10 min before being fed a 20 microliter aliquot of S.
choleraesuis cells suspended in BSG. Food and water were
returned 30 min after the inoculation. Data on morbidity
and mortality of mice were collected daily.
: :
.
:. .. '~
2~ 3~2
- -22-
Evaluation of protective immunLty.
Groups of five mice/cage were perorally immunized
with various doses of avirulent mutants and then chal-
lenged 30 days later with various doses of the wild-type,
virulent parent~ Chi3246. Morbidity and mortality condi-
tions were observed for at least 60 days.
Construction of S. choleraesuis strains with cya and
-
crp mutat ons.
The highly virulent strain Chi3246 was used as the
parent in the construction of all the ~accine strains used
in these studies (the peroral LDSo value for Chi3246 is
presented in Table 2, infra.)
The strains which were constructed which were derived
from Chi3246 are shown in Table 1. Also shown in Table 1
are the relevant genotypes of the strains, and a descrip-
tion of the method by which the strain was derived,
utilizing the methods for transposon insertion via
transduction, transposon deletion, as well as the selec-
tion methods described in Example 1 of EPO Pub. No.315,682, with the modifications described below.
Introduction of cya::TnlO, c~::TnlO, delta-cya-12,
and delta-crp-ll Erom the S. typhimurium strains PP1002,
PP1037, Chi3615, and Chi3623, respectively (see Table 1),
into S. choleraesuis strain Chi3246 was facilita~ed by
PlL4 transduction (via intermediate S. typhimurium hosts
Chi3385 and Chi3477) and transformation with pSD110. S.
choleraesuis strain Chi3246 which is P22HTint resistant
is, however, PlL4 immune. Thus, PlL4 or P1 clr clm
bacteriophage can adsorb to and inject DNA into the cells
of all S. choleraesuis strains used in this example, but
are unable to replicate their DNA in these bacterial
cells.
Since delta-cya-12 zid-62::TnlO, delta~
zhb::TnlO, crp773::TnlO and ~y~::TnlO mutations are in S.
typhimurium strains Chi 3711, Chi3773, PP1037 and PP1002,
.
.-, ;
20~3~
-23-
Table 2 ~MorealitY of ~ALB/c mice 30 days after peroral inoculation
~ith wil~-type Salmonella choleraesuis and its derivative~a
Strain Inoculating Survival
number Genotype dose (CFU) live/total
X3246 Uild-type 1.9 x 105 0/5
8.0 x 104 4J5
X3492 cya::TnlO 7.8 x 108 5/5
7.8 x 106 5/5
7.8 x 104 S/5
1.5 x 1~9 5~5
1.5 x 108 5/5
1.5 x 107 5/S
X3751 cre::TnlO 1.3 x 109 3/S
1.3 x 107 5/S
1.3 x 105 5/5
1.3 x 109 ~/S
1.3 x 108 4/5
1.3 x 107 5/5
X3752 ~crp-19 1 3 x 107 5/5
1.3 x 105 5/5
X3~53 osy~ 1.0 x 109 5/5
1.0 x 107 5J5
1.0 ~ 105 5/5
9.6 ~ 108 5/5
9.6 x 107 5/5
9.6 ~ 106 5/5
X3820 ocr ~ ~hb::TnlO 5.6 x 108 15~15
aPollo~ing a 4-hour ~ood and water fast, eight^~eek-old fe0ale mice
were inoculated with the indicated strains and doses. Daily ~orbidity
and mortality data ~cre observed for 30 days post-immunization.
.~i
~ 3~ ~?
-24-
respectively (see Table 1), it was necessary to move the
mutations from the smooth-LPS S. typhimurium background
into an intermediate Salmonella host in which both
P22HTint and PlL4 could be propagated. The bacteriophages
P22HTint and PlL4 are specific for strains with smooth and
rough LPS coats, respectively. The hosts, Chi3385 and
~hi3477, are restriction-deficient, modification-
proficient ~ S. typhimurium strains. Growth of Chi3385
and Chi3477 in media with low concentrations of galactose
permits synthesis of UDP-galactose, resulting in normal
levels of LPS side chains; these conditions are essential
for attachment and infection by P22HTint. Growth of
Chi3385 and Chi3477 in media containing glucose and lack-
ing galactose permits synthesis of a rough or incomplete
LPS, and enables the adsorption and replication of PlL4 or
Pl clr clm in these rough strains. By these means, the S.
choleraesuis strains Chi3492, Chi3751, Chi3755, Chi3759,
Chi3820, and Chi3858 were constructed. In the latter two
~trains, the TnlO linked to the delta-cYa or delta-crp
mutation was eliminated by selection for fusaric acid
resistance to yield Chi3860 and Chi3659, respectively.
In order to determine whether di~ferent deletion
mutations generated by the excision of TnlO from cya or
crp exhibit different levels of virulence and/or
immunogenicity, two different deletion mutants of cya and
crp were constructed in S. choleraesuis Chi3246. Two
deletions were isolated in, and transduced from S.
typhimurium as described above, and are represented by S.
choleraesuis strains Chi3859 (delta-cYa-12) and Chi3860
.,
(delta-crp-ll). The other two deletion mutations were
isolated by excision of TnlO inserted into the S.
choleraesuis cya and crp genes in Chi3492 and Chi3751,
respectively, usin~ selection for fusaric acid resistance
to yield Chi3752 (delta-crp-l9) and Chi3753 (delta-cya-
24), respectively.
. .
.
- ~
,
. . :
" ' ~
~ :
20~3-.~72
.
-25-
Construction of a S. choleraesuis strain with
deletion mutations in both cya and crp genes was
facilitated by using pSD110, which encodes the E. coli
crp gene. pSD110 was thus transduced into Chi3752
(Table 1), and this was followed by transducing in the
delta-~y~-12 mutation, which is closely linked to zid-
62::Tnl0 by selection for tetracycline resistance and
screening for inability to ferment maltose, which is
indicative of the introduction of the cya mutation. Fol-
lowing selection of Chi3759 for fusaric acid resistance toeliminate Tnl0, pSD110 was cured by growth at 43C to
yield Chi3781 (See Table 1). The serotype of all
constructs was confirmed by agglutination with Salmonella
group C1 O-antigen antisera.
Phenotypic anAlysis of cya and crp mutants.
The ~y~ mutants (Chi3492, Chi3753, Chi3~59), the crp
mutants (Chi3751, Chi3752, Chi3820) and the cya crp mutant
(Chi3781) were subjected to phenotypic analysis. These
strains failed to ferment maltose, mannitol, sorbitol, and
melibiose, and slowly fermented galactose. The phenotypes
were as expected based on known requirement for cAMP, and
for CRP for catabolic activities. The requirements for
cAMP, and for CRP for regulation of gene expression are
described in the following references. Perlman and
Pastan, Biochem. Biophys. Re_. Comm. 37:151 (1969~; Pastan
and Perlman, Science 169:339 (1970); Schwartz and
Beckwith, THE lac OPERON (1970, Zipser and Beckwith,
eds.); Pastan and Adhya, Bacteriol. Rev. 40:527 (1976);
30 and Scholte and Postma, J. Bacteriol. 141:757 (1980).
Construction of ~irulence plasmid-cured derivatives
of S. choleraesuis and of its delta-cya and/or delta-crp
strains.
We have discovered that the Inc/Par region af the
virulence plasmid of S. typhimurium, which encodes in-
:
` ~...: ..
~ ~ 3 3 r.~- P~ 2
-26-
compatibility and partition functions, hybridizes to and
exhibits incompatibility with the virulence plasmid of S.
choleraesuis. Based upon this finding, pYA2028, which
contains the Inc/Par region of the virulence plasmid of S.
typhim rium, high copy number pUC vector was used to
facilitate the curing of the virulence plasmid from S.
choleraesuis. Introduction of pYA2028 into _.
choleraesuis was accomplished using essentially eLther the
-
transformation methods described in Dagert and Ehrlich
1979, supra, or the electroporation methods of Feidler and
Worth, Anal. Biochem. 170:38 (1988). Transformants were
selected for, and maintained by, the inclusion of
ampicillin in the medium. After several generations of
growth, the virulence plasmid is lost due to incompat-
ibility exclusion. Growth of the cured S. choleraesuisstrain for subsequent cycles in media without ampicillin
leads to a rapid loss of pYA~028. The resul-ting cells
lack both the virulence plasmid and pYA2028. Chi3903
represents a virulence plasmid-cur~3d derivative of the
wild-type S. choleraesuis strain Chi32~6. Loss of the
virulence plasmid reduces the abil:Lty of Salmonella to
reach and/or colonize deep tissues such as the liver and
spleen, but is without effect in terms of colonization of
the intestinal tract and the GALT. Elimination of the
virulence plasmid thus reduces virulence without diminish-
ing immunogenicity. Removal of the virulence plasmid from
the double cya crp mutants would follow the same
procedure.
Virulence of c~a and crp mutant strains in mice.
In order to study the virulence of the S.
choleraesuis strains listed in Table 1, groups of mice
were orally inoculated with lO0-fold varying doses of each
strain. The results are shown in Table 2. The p.o. LD50
of the parental wild-type strain (Chi3246), which was
about l x 105 CFV, was determined by the method of Reed
" . ~
~3~
and Muench (1938). This LDSo is much lower than those of
the respective ~y~ and crp strains.
The data in Table 2 show that the mutant strains are
avirulent at at least 6,000 times the LDSo of the parental
wild-type strain, Chi3246. Mice orally inected with 10
CFU of Chi3751 (crp::TnlO) and Chi3752 (delta-crp) became
scruffy, lethargic, inappetent, and some died. However,
all mice infected with the same dose of Chi3492
(cya::TnlO) and Chi3753 (delta-cya-24) became slightly
ill, recovered, and survived the dose. This dose
represents 12,500 times the LDSo of the wild-type parent
strain. It is therefore evident that the ~y~::TnlO and
delta-~y~ mutants are more avirulent than the crp::TnlO
and delta-~ mutants. This result is somewhat surprising
since the opposite has been found with S. typhimurium
delta-c a and delta-crp infections in mice.
The results also showed that there were no obvious
differences in the health of the animals inoculated with
delta-crp-l9 compared to dalta-~-ll strains, and delta-
cya-12 as compared to delta-cya-24 strains.
Virulence of the strains with both delta-cYa and
delta-crp may also be established using the above
described procedure.
In~unoqenicit~ of cya and crp mutant strains.
The ability of the different S. choleraesuis mutants
to induce immunity to subsequent oral challenge with the
wild-type virulent parent, Chi3246, was examined. Mice
were inoculated p.o. with the S. choleraesuis delta-cya
and delta-crp strains shown in Table 3, at the indicated
doses. Thirty days after immunization with the attenuated
strains, mice were challenged p.o. with Chi3246. Morbid-
ity and mortality were observed daily for 30 days post-
challenge. The results in Table 3 reveal the apparent
differences in the degrees of immunogenicity of the dif-
ferent strains. Mice immunized with 107, 108, or 109 CFU
-28-
Table 3 Effectivenes-~ of i~unl~atlon with attenuated Salmonella
cho eraesuis vaccine strains in BALa/c mice to chall~nge v~h-vlld-type
vlrulent S. choleraesuisa
__
Strain I~munizing ~ilt-type Survival
number Genotypedose (cfu)Challenge (cfu) live/~otal
_ _ . . _ _ . _ _ _ _ _ _ r ~
X3492 cya::TnlO 7.8 x 108 1.1 x 109 2/5
7.8 x 106 1.1 x 109 0/5
7.8 x 104 1.1 x 1~9 0/~
1.5 x 109 2.4 x 109 2/5
1.5 x lQ~ 2.4 x lO9 1/5
1.5 x 107 2.4 x 1~9 0/5
X3751 crp::TnlO 1.3 x 107 1.1 x 109 3/3
1.~ x 10 l.1 x 109 2/5
1.3 x 105 1.1 x 109 0~5
1.3 x 109 2.4 x 1~9 4/4 :
1.3 x 108 2.4 x 109 3~4
~ 1.3 x 107 2.4 x 109 2~5
X3752 acrp~ 3 x 109 1.1 x 109 3/3
1.3 x 10~ 1.1 x 109 4/5
1.3 x 105 1.1 x 109 0/5
X3753 ~cya-241.0 x 109 1.1 x 109 2/5 1.0 x 107 1.1 x 109 0~5
1.0 x 105 l.l x 109 O~S
9.6 x 108 2.4 x 109 2~5
~.6 x lO~ ~.4 x 109 2/5
9.fi x 106 2.~i x lOg 2/5
X3820 ~crp-11 zhb::10 5.6 x 108 1.0 x 109
aPollo~ing a 4-hour food and v~ter fast, eight-veek-old fe~ale ~ic~ vere
inoculated ~ith th~ indicate~ strain~ and doses. Thlrty day~ a~ter
im~unizatlon vith th~ a~enuated strains, mice vere challen~ed vlth a
p~roral dose of ~ild-type virulent X3246- Daily morbidiey and mortality
data vere ob~er~ed ~or an additlonal 30 day~ post-ch~ nge.
' ' ' ,.., ' -,~' -
.
--`` 20 ~ 3~2
~29-
of the somewhat more virulent strains with mutations in
_~e exhibited better health and a higher rate of survival
after oral challenge with Chi3246 than did mice immunized
with the less virulent cya mutant strains. Thus, animals
immunized with 109 CFU of the cya mutants
had a rate of sur ival below 50~ whereas 100% of the mice
immunized with 10 cells of the crp strain survived chal-
lenge; in fact, those immunized with 107 or 108 cells of
the crp strain survived challenge with 10 cells of the
wild-type strain.
The immunogenicity of cya crp mutants of S.
choleraesuis is determined by immunizing the mice with a
high, but sublethal dose (~ x 108 CFU) of the attenuated
strains. Thirty days later, survivors are challenged with
Chi3246 at lOl, 102, and 103 times the LD50 value of
Chi3246. Morbidity and mortality conditions are observed
as described above.
Construction of asd cya crp mutants of S.
choleraesuis
Salmonella vaccine strains can serve as carriers to
deliver a forei~n antigen to the GALT of an animal host by
introduction of a gene encoding the antigen into the vac-
cine strains. NaXayama et al., B:io!Tech 6:693 (1988)
described a unique system where an Asd expression-cloning
vector was constructed for the purpose of high-level
stable expression of foreign antigen genes in delta-c~a
delta-crp delta-asd S. typhimurium. The avirulent proper-
ties of the delta-~y~ delta-crp mutations have been
consistently proven with doses administered to mice at
approximately 1000 times the LD50 of the wild-type parent
in all Salmonella species previously tested. These
avirulent strains have also stimulated a protective immune
response in the immunized animals as demonstrated by chal-
lenge with approximately lO00 times the wild-type parent.
Repeated animal experiments have confirmed that the S.
.::
2~13~'i'2
-30-
choleraesuis delta-cYa and delta-crp strains are avirulsnt
and immunogenic in mice. It can be deduced from earlier
results obtained fxom studies on avirulence and
immunogenicity that a S. choleraesuis construct with
delta ~y~ and delta-~ mutations would be avirulent and
immunogenic. The addition of the delta-asd mutation to
the S. choleraesuis delta-cya delta-crp Chi3781 is
facilitated by bacteriophage P22 HT int transduction to a
restriction-deficient, modification-proficient intermedi-
ate S. typhimurium with subsequent propagation ofbacteriophage plL4 on the intermediate S. typhimurium host
and final transduction of delta-asd into the _.
choleraesuis vaccine strain.
S. typhimurium Chi3656 is grown in L broth containing
5 mM CaC12, and infected with PlL4 to propagate a high
titer lysate. The PlL4(Chi3656~ lysate is then used to
transduce S. choleraesuis delta-cya delta-crp Chi3781;
transductants are screened for by tetracycline resistance.
A portion of the tetracycline-resistant transductants are
screened for the Asd phenotype. As a final step, selec-
tion on fusaric acid media is performed to identify a
tetracycline-sensitive derivative of the S. choleraesuis
delta-cYa delta-~ delta-asd strain. Additional
characterization of the final construct is completed by
verifying the markers and presence of a complete LPS coat,
and by the nonacquisition of additional auxotrophic
phenotypss.
Deposits of Strains
The following listed materials are on deposit under
the terms of the Budapest Treaty, with the American Type
Culture Collection, 12301 Parklawn Drive, Rock~ille,
Maryland. The accession number indicated was assigned
after successful viability testing, and the requisite fees
were paid.
203.3~72
-31-
Strain De~osit Date ATCC No.
.. . __
Chi4062 July 15, 1987 53647
Chi4064 July 15, 1987 53648
Chi3781 delta-crp-l9March 29, 1989 67923
delta-cya-12
Chi3903 (wild-type, 50kbMarch 29, 1989 53885
plasmid-cured)
Chi3246 (wild-type)March 29, 1989 67922
Commercial Utility
The strains provided herein are directly and
indirectly suitable for the production of commercial vac-
cines to prevent diseases caused by _. choleraesuis, and
other enteric bacteria with which antibodies to S.
choleraesuis cross react. These strains are also useful
as carrier m.icroorganisms for the production of expression
products encoded on recombinant genes in the bacterial
cells.
.:
