This invention relates to a biologically pure culture of a smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Saimone! la strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, a process of preparing a smooth aromatic-dependent (ar_o~) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, vertebrate vaccines, comprising a live smooth aromatic-dependent (aro-) non-reverting, avirulent Salmonella strain of GΓOUD C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, methods of inducing immunity against Salmonellosis in a vertebrate comprising administering o the vertebrate an effective amount of a vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonel la strain of Group C2;0-6,8, C3;0-8 or C3;0-8,2_0, as herein defined, an antibody raised against a smooth aromatic-dependent (aro-) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, a biologically pure culture of a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0-j_,13,22, as herein defined, a process of preparing a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonel la strain of Group G2,0-1,13,23 or Gl ;0-l, 13,22, as herein defined, vertebrate vaccines comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0-l, 13,22, as herein defined, methods of inducing immunity against Salmonellosis in a vertebrate, comprising administering to the vertebrate an effective amount of a vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonel la strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, an antibody raised against a smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella strain of

Group G2,0-1,13,23 or Gϊ;0-χ,13,22, a method for inducing immunity against

Salmonellosis in a foetus carried by a pregnant vertebrate, a diagnostic kit for the detection of species of Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, a diagnostic kit for the detection of species of Salmonella strain of Group G2,0-1,13,23 or Gl;0-1,13,22, as herein defined, a neonatal vertebrate vaccine and a method for inducing immunity against Salmonellosis in a neonatal vertebrate.

BACKGROUND ART

Salmonellosis of ruminant animals is recognised as a significant disease in most countries because of the serious economic losses it causes (Wray and Sojka, 1977), particularly in animals subjected to stressful conditions such as those encountered during shipping over long distances, e.g. in the export of live sheep to the Middle East (Jelinek et a_l_. , 1982) and intensively-managed calf-rearing enterprises. Further, it constitutes a serious public health hazard not only for the consumer of meat and meat products but also, for the worker associated with the handling of these products such as the abattoir worker (Smeltzer et §_ _. , 1980; Samuel et a_. , 1980).

Although the range of Salmonella serotypes isolated from outbreaks of clinical Salmonellosis can be multiple and variable in percentage of isolations for different serotypes, most of the episodes can be attributed for the most part to a few major serotypes. In Australia, the most common isolates in order of significance from cases of Salmonellosis in cattle are Salmonel la dublin≥ Sal onella typhimurium and Salmonella bovismorbifleans, whereas in sheep they are Salmonel la typhimurium. Salmonel la bovismorbifleans and Salmonell havana (Richards et ___]_. , 1989).

It has been shown that sheep immunised with Salmonella typhimurium aro ^" (bovine origin) by either the oral or iintramuscular route were

protected against oral challenge with virulent organisms (Mukkur e_t aJL ,

1987). Similar results were previously reported in cattle (Robertsson et a_ , 1983). Non-reverting aro ^" mutants are non-virulent since they are unable to synthesize essential aromatic compounds which are not obtainable from vertebrate tissues.

A useful experimental model for Salmonellosis in sheep and cattle is provided by mouse Salmonellosis. Experiments carried out in this laboratory revealed that mice immunized with Salmonell typhimurium aro ^" of bovine origin and challenged with some virulent Salmonell typhi urium strains of ovine origin were not protected as efficiently as those challenged with virulent organisms of bovine origin (Mukkur e_t aj.. , 1987).

It was also discovered that while mice immunized with Salmonel la typhimurium aro ^" (bovine or ovine origin), were only partially protected against experimental challenge infection with virulent Salmonel la bovismorbificans (Begg, Walker, Love and Mukkur, unpublished), sheep immunized as above were completely unprotected (Mukkur and Walker, unpublished). On the other hand, mice immunized with Salmonella typhimurium aro ^" and challenged with Salmonella havana were not protected at all (Begg, Walker, Love and Mukkur, unpublished). Therefore it was considered necessary to generate aromatic-dependent mutants of the above organisms by genetic manipulation and evaluate their vaccine potential in mice and sheep. The 0-specific phage, P22, has been used to transduce genes from one Salmonel la strain to another within or between Groups A, B and D. However phage P22 will only adsorb to smooth strains of these 0-serotypes. The generalized transducing phage PI has been used for transduction of genes from Salmonella typhimurium aro ^" (Group 3) made galE to Salmonel la choleraesui s (Group CD made qalE (Nnalue and Stocker, 1987). Phage PI does not act on smooth Salmonella of 0 group B but is

adsorbed by mutants making the R (galactose-deficient) lipopolysaccharide (LPS), including those with mutations at qalE which specifies the enzyme UDP-galactose epimerase.

OBJECTS OF THE INVENTION

It is an object of the invention to provide a biologically pure culture of a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group C20-6,8, C3;0-8 or C3;0-8,20, as herein defined.

Another object is to provide a biologically pure culture of a smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;(M 3,22, as herein defined.

Further objects are to provide a process of preparing a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, vertebrate vaccines, comprising a live smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonell strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, methods of inducing immunity against Salmonellosis in a vertebrate comprising administering to the vertebrate an effective amount of a vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, an antibody raised against a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, a process of preparing a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, vertebrate vaccines comprising a live smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonell strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, methods of inducing immunity against Salmonellosis in a vertebrate, comprising

administering to the vertebrate an effective amount of a vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, an antibody raised against a smooth aromatic-dependent

(aro ^" ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, a method for inducing immunity against Salmonellosis in a foetus carried by a pregnant vertebrate, a diagnostic kit for the detection of species of Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, a diagnostic kit for the detection of species of Salmonel la strain of Group G2,0-1,13,23 or Gl ;( ,13,22, as herein defined, a neonatal vertebrate vaccine and a method for inducing immunity against Salmonellosis in a neonatal vertebrate.

DISCLOSURE OF THE INVENTION

The following definitions apply throughout the specification and claims:

A deletion is defined as a mutation resulting from loss of a DNA segment.

An inversion is defined as a rearrangement of a DNA segment in relation to neighbouring sequences.

The term Enterobacterial strain I has been used to denote members of the family Enterobacteriaceae. Examples of these organisms that can be used as donors of cjal genes include Salmonella typhimurium [(Hfr K20) serA 13 rfa 3058], S. typhimurium [(Hfr K25) serA 15 rfx], S. abony [(Hfr 3) str-5013, S. choleraesuis (F'-8-gal), Escherichia coli K-12 (JC 12 Hfr) and Escherichia coli K-12 (R5 Hfr ⁾ etc.

Salmonella strains of Group C2;0-6,8 are those serotypes possessing somatic (0) antigens 6 and 8 and Group C3;0-8 or 0-8,20 are those serotypes possessing somatic (0) antigens 8 and 20 or 8 alone (see Table A).

Salmonella strains Group G2 are those serotypes possessing somatic

(0) antigens 1,13 and 23 or 13 and 23 and Group Gl ;0-l 13,22 are those serotypes possessing somatic (0) antigens 1,13, and 22 or 13 and 22 (see

Table B).

Avirulent Salmonella species are defined as those organisms which upon inoculation into vertebrates do not cause clinical Salmonellosis.

Non-reverting Salmonella organisms are those that do not regain their virulence (disease producing capability) upon culture in vitro or in vivo.

Non-reverting aro ^" hosts are those bacteria which are non-reverting because of their dependence on aromatic compounds.

The expressions "smooth" and "rough" relate to the surface antigens of the specific microorganisms herein described and are defined as such: mutants unable to synthesize 0-antigenic polysaccharides or to transfer them to the core lipopolysaccharides (LPS) are considered rough as opposed to smooth organisms where the above defect is not existent. Other mutations can lead to defective biosynthesis of the rough or R core itself and R mutants have been classified into chemotypes R to R on the basis of the carbohydrate composition of their core LPS.

With specific reference to the non-switenable, aro ^" Salmonella bovismorbificans (CS404/SL5785) , precursor of the final mutant CS405/SL5794, the lipopolysaccharide (LPS) was of the R chemotype which is deficient in having no galactose. This was demonstrated by LPS extraction from the final mutant derivative. This is also explained by the sensitivity of the above strain to rough specific phages C21 , 6SR and Br60.

The present inventors surprisingly found that a smooth Salmonella field isolate (Salmonella havana) could be transduced with phage PI without it having to be made galE and additionally that completely rough (non-switchable) Salmonella species (Salmonella bovi morbificans) could be

made into a smooth (switchable) strain by the introduction of deoxyribonucleic acid (DNA) carrying genes coding for the g _ operoπ.

According to a first embodiment of this invention there is provided a biologically pure culture of a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonel la strain of Group C2;0-6,8, C3;0-8 or

C3;0-8,20, as herein defined, said strain being characterised as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable].

Advantageously, there is provided a biologically pure culture of a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Sal onel 1 bovismorbificans strain of Group C2;0-6,8, characterised as follows:

[aroA deletion or deletion inversion mutation; gal ^"1" ; switchable].

More advantageously, there is provided a biologically pure culture of a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonel la bovismorbificans strain of Group C2;0-6,8, said strain having the characteristics of the Salmonella bovismorbificans strain deposited under accession number N90/000304 at the Australian Government Analytical

Laboratories said strain being characterised as follows:

[Zbj-903::TnlO(Tc ^s )3 [aroA554 qalE::Tn10 (Tc ^s , non-rev); F'8-gal; switchable].

Typically, the biologically pure culture of a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain is grown on Brain Heart Infusion medium containing 0.5 percent D-galactose at a temperature in the range 20-45°C and within the pH range of 5.0 to 9.0 or on a growth medium containing para-aminobenzoic acid, 2,3-dihydroxybenzoic acid and D-galactose at a temperature in the range 20-45"C and within the pH range of 5.0 to 9.0.

According to a second embodiment of this invention there is provided a process of preparing a. smooth aromatic-dependent (aro~) non-reverting,

avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being characteri ed as follows:

[aroA deletion or deletion inversion mutation; gal ^"1' ; switchable], said process comprising, transducing a Salmonel la strain of Group C2;0-6,3, C3;0-8 or C3;0-8,20, as herein defined, with phage PI, propagated on an aro ^" host to form a rough (aro ^" ,gal ^" ) Salmonella strain; and conjugating the rough (aro ^" ,gal ^" ) Salmonel la strain with a Enterobacterial strain I, as herein defined, carrying gal*, to form the smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain.

Typically in the process of the second embodiment the smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain is a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella bovismorbificans strain characterised as follows:

[aroA deletion or deletion inversion mutation; gal ^" * ^" ; switchable] or a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella bovismorbificans strain of Group C2;0-6,8, said strain having the characteristics of the Salmonella bovismorbificans strain deposited under accession number N90/000304 at the Australian Government Analytical Laboratories said strain being characterised as follows:

[Zb,i-903::Tn10(Tc ^s )3 [aroA554 qalE::TnlO (Tc ^s , non-rev); F'8-gal; switchable].

According to a third embodiment of this invention there are provided the following vertebrate vaccines:

(i) A vertebrate vaccine, comprising a live smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being

characterized as follows:

[aroA deletion or deletion inversion mutation; gal ^'1' ; switchable] together with a live aro ^" Salmonella typhimurium strain, and a live aro ^" Salmonella dublin strain, together with a pharmaceutically/veterlnarily acceptable carrier, diluent, excipient and/or adjuvant.

(ii) A vertebrate vaccine, comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

CaroA deletion or deletion inversion mutation; gal ^"1" ; switchable] together with a live aro ^" Salmonel la dubl in strain, together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(iii) A vertebrate vaccine, comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation; gal ^'1' ; switchable] together with a live aro ^" Salmonella typhimurium with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(iv) A vertebrate vaccine, comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonel la strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation; gal ^"1" ; switchable] together with a live aro ^" Salmonella havana strain and a (aro ^" )

Salmonella dublin strain together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(v) A vertebrate vaccine, comprising a live smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation; gal ^'1' ; switchable] together with a pharmaceutically/ veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

Typically, in the vaccines of the invention the smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella strain is a aro ^" Salmonella bovismorbificans strain characterised as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable] or a smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella bovismorbificans strain of Group C2;0-6,8, said strain having the characteristics of the Salmonella bovismorbificans strain deposited under accession number N90/000304 at the Australian Government Analytical Laboratories said strain being characterised as follows:

[Zb,j-903::TnlO(Tc ^s )] [aroA554 ga1E::TnlO (Tc ^S , non-rev); F'8-gal; switchable].

Advantageously the vertebrate is a monkey (eg chimpanzee), ape, bovine, human, ovine, equine, caprine, Leporine, domestic fowl, feline or canine vertebrate.

Typically, the vertebrate is a bovine.

According to a fourth embodiment of this invention there is provided the following methods of inducing immunity against Salmonellosis in a vertebrate:

⁽ i ⁾ A method of inducing immunity against Salmonellosis in a

vertebrate, comprising administering to the vertebrate an effective amount of a vertebrate vaccine comprising a live smooth aromatic-dependent

(aro ^" ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8,

C3;0r8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable] together with a live aro ^" Salmonella typhimurium strain, and a live aro ^" Salmonella dub! in strain, together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant

(ii). A method of inducing immunity against Salmonellosis in a vertebrate, comprising administering to the vertebrate an effective amount of a vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable] together with a live aro ^" Salmonella dublin strain, together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(iii) A method of inducing immunity against Salmonellosis in a vertebrate, comprising administering to a vertebrate an effective amount of a vertebrate vaccine comprising a live smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable] together with a live aro ^" Salmonella typhimurium strain together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or

adjuvant.

(iv). A method of inducing immunity against Salmonellosis in a vertebrate, comprising administering to a vertebrate an effective amount of a vertebrate vaccine, comprising a live smooth aromatic-dependent (aro~) non-reverting, avirulent Sal onella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable] together with a live aro ^" Salmonella havana strain and an aro ^" Salmonel la dub!in strain together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(v). A method of inducing immunity against Salmonellosis in a vertebrate, comprising administering to a vertebrate an effective amount of a vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^" ) non-reverring, avirulent Sal onella strain of Group C2;0-6,8,- C3;0-8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable] together with a pharmaceutically/ veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

Typically, in the methods of the fourth embodiment the smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella strain is a (aro~) Salmonella bovismorbificans strain characterised as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable] or a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella bovismorbificans strain of Group C2;0-6,8, said strain having the characteristics of the Salmonella bovi morbificans strain deposited under accession number N90/000304 at the Australian Government Analytical Laboratories said strain being characterised as follows:

[Zbj-903::Tnl0(Tc ^s )] [aroA554 ga1E::Tn10 (Tc ^s , non-rev); F'8-gal;

switchable].

Advantageously the vertebrate is a monkey (eg chimpanzee), ape, bovine, human, ovine, equine, caprine, Leporine, domestic fo l, feline or canine vertebrate.

Typically, the vertebrate is a bovine.

Generally the effective amount is in the range of 1x10 to 1x10 colony forming units and the total accumulative amount is in the range of

1x10 to 1x10 colony forming units.

According to a fifth embodiment of this invention there is provided an antibody raised against a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonel la strain of Group C2;0-6,8, C3;0-8 _. or

C3;0-8,20, as herein defined, said strain being characterised as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable].

Typically, the smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, is a aro ^" Salmonella bovismorbificans strain characterised as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable] or a strain having the characteristics of the Salmonella bovismorbificans strain deposited under accession number N90/000304 at the Australian

Government Analytical Laboratories said strain being characterised as follows:

[Zbj-903::Tnl0(Tc ^s )] [aroA554 galE::Tn10 (Tc ^S , non-rev); F'8-gal; switchable].

According to a sixth embodiment of this invention there is provided a biologically pure culture of a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or

Gl ;0-l,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation].

Advantageously there is provided a biologically pure culture of a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella havana strain of Group G2,0-1,13,23, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation].

More advantageously there is provided biologically pure culture of a smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella havana strain of Group G2,0-1,13,23, said strain having the characteristics of the Salmonella havana strain deposited under accession number N90/000303 at the Australian Government Analytical Laboratories said strain being characterized as follows:

[Zb.i-903::TnlO(Tc ^s )][aroA554::TnlO(Tc ^s ,non-rev)3.

Typically the smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonel la strain of Group G2,0-1,13,23 is grown on-Brain Heart Infusion medium containing 0.5 percent D-galactose at a temperature in the range 20-45 ^β C and within the pH range of 5.0 to 9.0 or a growth medium containing para-aminobenzoic acid, 2,3 di ydroxybenzoic acid and D-galactose at a temperature in the range 20-45°C and within the pH range 5.0 to 9.0.

According to a seventh embodiment of this invention there is provided a process of preparing a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, said strain being characterized as follows: [aroA deletion or deletion inversion mutation], said process comprising: transducing a Salmonella strain of Group G2,0-1,13,23 or Gl;0-l,13,22, as herein defined, with phage PI, propagated on a non-reverting aro ^" host, as herein defined, to form the smooth

aromatic-dependent (aro ^" ) Salmone la strain.

Typically the smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group G2, 0-1, 13,23 is a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella havana said strain being characterized as follows:

[Zb.i-903::TnlQ(Tc ^s )][aroA554::TnlQ(Tc ^S , non-rev)] or a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella havana strain of Group G2 0-1,13,23, said strain having the characteristics of the

Salmonel la havana strain deposited under accession number N90/000303 at the

Australian Government Analytical Laboratories, said strain being characterized as follows:

[Zb.i-903::Tn10(Tc ^s )][aroA554::Tn10(Tc ^s ,non-rev)].

According to an eighth embodiment of this invention there is provided the following vertebrate vaccines:

(i) A vertebrate vaccine comprising a live smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonel la strain of

Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation] together with any live aro ^" Salmonella typhimurium strain and a live aro ^" Salmonella bovismorbificans strain, together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(ii) A vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonel la strain of

Group G2,0-1,13,23 or Gl ;0-χ,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation]

together with a live aro ^" Salmonell typhimurium strain together with a pharmaceutically/ veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

^• (iii) A vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of

Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation] together with a live aro ^" Salmonella bovismorbificans strain together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(iv) A vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation] together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

Typically the smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 is a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella havana said strain being characterized as follows:

[Zb,i-903::TnlQ(Tc ^S )][aroA554::TnlO(Tc ^s ,non-rev)] or a smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella havana strain of Group G20-1,13,23, said strain having the characteristics of the Salmonella havana strain deposited under accession number N90/000303 at the Australian Government Analytical Laboratories, said strain being characterized as follows:

[Zb.i-903: : Tn10(Tc ^s )][aroA554: :Tπl0(Tc ^s , non-rev) ] .

Advantageously the vertebrate is a monkey (eg chimpanzee), ape, bovine, human, ovine, equine, caprine, Leporine, domestic fo l, feline or canine vertebrate.

Typically, the vertebrate is a ovine.

According to a ninth embodiment of this invention there is provided the following methods of inducing immunity against Salmonellosis in a Vertebrate:

(i) A method of inducing immunity against Salmonellosis in a vertebrate, comprising administering to the vertebrate an effective amount of a vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation] together with any live aro ^" Salmonella typhimurium strain and a live aro ^" Salmonella bovismorbificans strain, together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(ii) A method of inducing immunity against Salmonellosis in a vertebrate, comprising administering to the vertebrate vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0~1,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation] together with a live aro ^" Salmonella typhimurium strain together with a pharmaceutically/ veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(iii) A method of inducing immunity against Salmonellosis in a vertebrate, compri ing administering to the vertebrate a vertebrate vaccine comprising a live smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;(M,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation] together with a live aro ^" Salmonella bovismorbificans strain together with a pharmaceuti ally/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

(iv) A method of inducing immunity against Salmonellosis in a vertebrate, comprising administering to the vertebrate a vertebrate vaccine comprising a live smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation] together with a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

Typically, the smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group G2,0-l,13,23 is a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella havana said strain being characterized as follows:

[Zb.i-903::Tnl0(Tc ⁾ ][aroA554::Tnl0(Tc ^s ,non-rev)] or a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella havana strain of Group G20-1,13,23, said strain having the characteristics of the Salmonel la havana strain deposited under accession number N90/000303 at the Australian Government Analytical Laboratories, said strain being characterized as follows:

[Zbj-903::Tnl0(Tc ^S )][aroA554::Tn10(Tc ^S ,non-rev)].

Advantageously the vertebrate is a monkey (eg chimpanzee), ape, bovine, human, ovine, equine, caprine. Leporine, domestic fowl, feline or canine vertebrate.

Typically, the vertebrate is a ovine.

Generally the effective amount is in the range of 1x10 to 1x10 colony forming units and the total accumulative amount is in the range of

1x10 to 1x10 colony forming units.

According to a tenth embodiment of this invention there is provided an antibody raised against a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonel la strain of Group G2,0-1, 13,23 or

Gl ;0-l,13,22, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation].

Typically, the smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain is a smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella havana strain of Group G2,0-1,13,23, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation] or a smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella havana strain of Group G20-1,13,23, said strain having the characteristics of the

Salmonella havana strain deposited under accession number N90/000303 at the

Australian Government Analytical Laboratories, said strain being characterized as follows:

[Zb _. i-903::TnlO(Tc ^s )][aroA554::TnlQ(Tc ^s ,non-rev)].

According to an eleventh embodiment of the invention there is provided a method for inducing immunity against Salmonellosis in a foetus carried by a pregnant vertebrate, comprising administering to said vertebrate an immunological ly effective amount of at least one of the vaccines of the third or eighth embodiments.

According to a twelfth embodiment of this invention there is provided a diagnostic kit for the detection of species of Salmonella strain of Group

C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, comprising at least one of the antibodies as defined in the fifth embodiment together with a diagnostically acceptable carrier and/or diluent.

According to a thirteenth embodiment of this invention there is provided a diagnostic kit for the detection of species of Salmonella strain of Group G2,0-1,13,23 or Gl ;0-l,13,22, as herein defined, comprising at least one of the antibodies as defined in the tenth embodiment together with a diagnostically acceptable carrier and/or diluent.

According to a fourteenth embodiment there is provided a neonatal vertebrate vaccine comprising at least one of the antibodies of fifth or tenth embodiments together with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

According to a fifteenth embodiment of this invention there is provided a method for inducing immunity against Salmonellosis in a neonatal vertebrate, comprising administering to said vertebrate an immunological ly effective amount of the vaccine as defined in the thirteenth embodiment.

According to a further embodiment of this invention there is provided a vertebrate vaccine, comprising a live smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable] together with a live smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl;0-l,13,22, as herein defined, said strain being characterized as follows:

[aroA deletion or deletion inversion mutation], together with a pharmaceutically/veterinarily acceptable carrier,

diluent, excipient and/or adjuvant.

Typically the smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20 is a aro Salmonella bovismorbificans strain characterised as follows:

[aroA deletion or deletion inversion mutation; gal*; switchable].

Typically the smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, :C3;0-8 or C3;0-8,20 is a smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonella bovismorbificans strain of Group C2;0-6,8, said strain having the characteristics of the Salmonella bovismorbificans strain deposited' unαer accession number N90/000304 at the Australian Government Analytical

Laboratories said strain being characterised as follows:

[Zbj-903::TnlO(Tc ^s )] [aroA554 qalE::Tn10 (Tc ^s , non-rev); F'8-gal; switchable].

Advantageously the smooth aromatic-dependent (aro ^~ ) non-reverting, avirulent Salmonel la strain of Group G2,0-1,13,23 is a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella havana said strain being characterized as follows:

[Zb.i-903: :Tn10(Tc ^S )][aroA554: :Tn10(Tc ^s .non-rev)] .

More advantageously the smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonel la strain of Group G2,0-1,13,23 is a smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella havana strain of Group G2 0-1,13,23, said strain having the characteristics of the Salmonella havana strain deposited under accession number N90/000303 at the Australian Government Analytical Laboratories, said strain being characterized as follows:

[Zb.i-903: :Tn1Q(Tc ^S )][aroA554::TnlO ⁽ Tc ^s ,non-rev)].

The vaccines of the invention are typically formulated for

administration by either the oral or parenteral route, preferably the intramuscular or subcutaneous route of administration.

In a process of preparing a vertebrate vaccine of the invention a typical protocol includes: washing the icrobial growth free of nutrient medium, harvesting and resuspension of the microorganisms in a pharmaceutically/ veterinarily acceptable carrier, diluent, excipient and/or adjuvant. Oral vaccination can then be performed by delivering the microorganisms in a biodegradable polymer capsule, composed of gelatin or the like, or an appropriate slow release device(s) as are well known in the art. The vaccine can also be suitably diluted to a manageable volume with an appropriate pharmaceutically/veterinarily acceptable carrier, diluent and/or adjuvant and delivered orally using devices such as those used for the delivery of anthelminthics.

In delivery systems utilizing the parenteral route it is preferred that the live microorganisms are suitably washed, harvested and resuspended in a pharmaceutically/veterinarily acceptable carrier, diluent and/or adjuvant suitable for injection, utilizing methods of administration as are well known in the art.

In the administration of the live vaccine formulations herein disclosed there are preferred non-toxic pharmaceutical and veterinary carriers, diluents, excipients and/or adjuvants which are also microbiologically acceptable. For administration of the vaccine the organisms to be used are admixed with these non-toxic carriers, diluents, excipients and/or adjuvants and may be in the form of capsules, aqueous or oily suspensions, emulsions, syrups, elixirs or injectable solutions.

Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxy ethylcellulose, ethylcellulose, sodium alginate, gum acacia, gum

tragacanth, dextrose, sucrose, sorbitol, annitol, gelatin and lecithin.

In addition these oral formulations may contain suitable flavouring and colouring agents. When used in capsule form the capsules may be coated with compounds such as glyceryl onostearate or glyceryl distearate which delay disintegration.

For administration as an injectable solution or suspension non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline.ethanol and 1,2 propylene glycol .

When the vaccine composition is administered the dosage rate is generally in the range of 10 to 10 colony forming units (cfu) of each microorganism found in the vaccine composition. Specifically for each animal undergoing either oral or parenteral vaccination the preferred dosages are 10 cfu's per mouse, 3.3x10 cfu per sheep and TO cfu's per calf.

A typical vaccination regime is to deliver the vaccine in multiple doses generally one, two or three equal doses, where the total number of microorganisms delivered correlates with the total dosage.

In general to induce the production of antibodies to the bacterial compositions of smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strain of Group C2;0-6,8, C3;0-8 or C3;0-8,20, as herein defined, or a smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella strain of Group G2,0-1,13,23 or Gl ;0~1,13,22, as herein defined, they can be oleogenous or aqueous suspensions formulated in accordance with known methods in the art using suitable dispersing, suspension and/or wetting agents. Examples of suitable dispersing, suspension and wetting agents include Freund's complete/ incomplete adjuvant, Montenide Marcol adjuvant and phosphate buffered saline.

The antibodies raised as described herein may be used to provide a boosted immune response to neoπates unable to receive such antibodies through the normal route of feeding colostrum, as typically supplied by the πeonates mother, by feeding a milk supplement containing these antibodies to the neonate. Additionally a pregnant vertebrate host may receive a vaccine formulation to induce antibody production specifically to induce immunity in the unborn foetus.

Advantages of the bacterial vaccine herein disclosed include its safety threshold for both microorganisms described herein. Large numbers of colony forming units up to 10 12 cfu's in sheep and cattle, for both microorganisms, can be administered without any deleterious effects.

Furthermore the longevity of both mutant organisms in various sites within the body is shorter than the virulent parent strains.

A further advantage of the invention is the non-reversion of both aro ^" mutants. Specifically these aro ^" Salmonell bovismorbificans strains and aro ^" Salmonella havana strains have been shown not to revert to aro* forms at a detectable frequency (that is < 10 per bacterium per generation).

A final advantage of the invention is that both mutant microorganisms are serological ly indistinguishable from their respective virulent parent strain, thus enabling an identical cellular and humoral immune response to be elicited thus providing a more efficient vaccine, due to the indistinguishable identity of wild-type and mutant organisms.

BEST MODE AND OTHER MODES FOR CARRYING OUT THE INVENTION

The first step is to generate a non-reverting, deletion or deletion-inversion aro ^" (preferably aro ^~ A) mutant of Salmonella species (preferably Salmonella typhimurium). Growth of these mutants, is dependent upon the availability in the nutritional medium of aromatic compounds at

least two of which viz., para-aminobenzoic acid (PABA) and

2,3-dihydroxybeπzoic acid (DHB) are not available in vertebrate tissues which requirement makes these strains avirulent. A transducing phage lysate is then prepared by propagation of an appropriate phage, preferably

PI, in the above aro ^" mutant. This phage lysate is then used for transduction of either the smooth, target recipient Salmonella species as in the case of Salmonella havana or those with one of the incomplete - LPS types including those with mutations affecting either part or whole of the gal operon to produce either the q lE mutant which lacks the enzyme

UDP-galactose- epi erase, or mutants in which the (jalK and/or qalT functions are also affected to yield aro ^" gal ^" rough mutant organisms as in the case of Salmonella bovismorbificans. The aro ^" gal ^" rough

Salmonel la bovismorbificans is then made smooth by transfer of DNA coding for £a_l genes, preferably by conjugation with a Salmonella species harbouring the gal operon, preferably as F'-qal .

The derived organisms, aro ^" Salmonel l bovismorbificans and aro ^"

Salmonel la havana can be homogeneously mixed with other aro ^" derivatives of Salmonella typhimurium and Salmonella dublin or with each other or together with the other aro ^" derivatives or individually with any pharmaceutically/veterinarily acceptable carriers, diluents, excipients or adjuvants to form appropriate suspensions for use as vaccine formulations.

These vaccine formulations can then be administered to a selected . vertebrate host by either the oral or parenteral route, dependent upon the specific formulation mixture and recipient host. When administered parenteral ly the route of administration may be either intramuscular or subcutaneous.

The vaccine formulations are specifically to prevent the onset of

Salmonellosis by inducing an effective cellular and humoral immune response

in a vertebrate host. The vaccines are of particular benefit to sheep and cattle destined to be subjected to stress, such as during feedlot holding, live export or intensive farming.

Further to their use as vaccines the two aro ^" strains of Salmonella bovismorbificans and Salmonella havana may be used to raise specific antibodies against themselves which can then be used as a colostrum substitute in neonates deprived of their natural source of colostrum i.e. the mothers milk. The antibodies can also be used in a diagnostic kit for the detection of these organisms in susceptible vertebrate hosts.

Specific examples of the invention follow:

(A) Salmonella bovismorbificans Bacterial strains and phage lysates

Virulent Salmonella bovi morbificans (CS400/SL5747) used in this investigation was isolated from a dead sheep. Other bacterial strains and phage lysates used in the generation of Salmonella bovismorbificans aro ^" are shown in Table 1.

Example 1 Generation of aromatic-dependent (aro~) Salmonella bovismorbificans

The wild-type strain of Salmonella bovismorbificans, CS400/SL5747, was prototrophic, smooth (as indicated by resistance to rough-specific phages and by reaction with Salmonella 0-6 and 0-8 sera) and sensitive to Felix.0 phage; it was resistant to tetracycline. Salmonella of groups other than A,B and D do not adsorb phage P22 which therefore cannot be used to transduce genes to or from such strain . Another general tranducing phage, PI, is, in general, ineffective on smooth Salmonella but active on strains with mutation galE, because when grown on medium not supplemented with galactose they make "rough" LPS of type Re (galactose-deficient) , appropriate for adsorption of phage PI (Ornellas and Stocker, 1974).

Galactose-negative (non-fermenting) mutants of the wild-type Salmonella bovismorbificans strain were therefore isolated by selection for ability to grow on defined medium containing 2-deoxygalactose, which inhibits the growth of galactose-positive strains. Two of 20 such mutants tested were resistant to Felix 0 phage (indicating LPS core defect) and sensitive to phage C21, which like phage PI acts efficiently on strains making type Re

LPS. These two mutants were sensitive also to rough-specific phages 6SR and Br60. The presence of galactose (plus glucose, to prevent galactose toxicity) in the medium used for testing phage sensitivities restored the phage sensitivity pattern of the wild-type parent strain, indicating loss of g_a!E function but retention of qalK and qalT functions, so that exogenous galactose could be used for synthesis of UDP-galactose, the precursor of the galactose units of LPS. One of the two aJ_E mutants,

CS401/SL5750, was plated on the autoclaved-chloramphenicol/fusaric-acid medium of Bδchner et al , (1980), which impedes the growth of tetracycl ine-resistant Salmonella and Escherichia col i . Nine of ten resistant mutants from this selection were found to be tetracycline- sensitive. One of them, CS401/SL5752, was treated with phage PI grown on a

Salmonella typhimurium strain, SL2328, which is galE and has a transposon-generated non-reverting mutation at aroA and an adjacent silent

TnlO insertion, Zb.i-903: :TnlO (Nnalue and Stocker, 1987). Selection was made for tetracycl ine-resistance. One of 34 tetracycl ine-resistant colonies tested was found to be aromatic-dependent, by co-transduction of the aroA of the donor with its TnlO insertion. This transductant,

CS403/SL5780, had the same phage sensitivity pattern as its parent, which showed that it was still cjalE, but its phage sensitivities were not altered when tested on galactose-supple ented medium. This probably indicates that it had lost galK and/or qalT function by a second mutation affecting the

cj ! operon. As tetracycline-resi tance is not desirable in a live-vaccine strain a tetracycline-sensitive mutant, CS404/SL5758, was next isolated from CS403/SL5780 by the same method as before. Finally, to restore wild-type LPS character (smooth) strain CS404/SL5780 was given an F' factor, F'8, which includes the gal* operon of Escherichia coli, by a conjugation cross with an Salmonella typhimurium strain carrying this plasmid. A galactose-positive transconjugant, now smooth by the criterion of phage sensitivity pattern and 0 antigen character and still aromatic-dependent, was labelled CS405/SL5794. This strain was employed as a live vaccine in the trials. No reversion to aromatic-independence has been detected in strain CS405/SL5794, as was to be expected since its transposon-generated aroA mutation, originally used to construct an

Salmonella typhimurium live-vaccine strain, SL1479, has given no revertants in extensive trials of that strain (Smith et al . , 1984).

Example 2

In vivo safety of aromatic-dependent aro ^" Salmonella bovismorbificans

(CS405) in Mice and Sheep

The LD50 of the parent virulent Salmonella bovismorbificans

(CS400/SL5747) for mice was calculated to be lxlO ⁷ cfu (Litchfield and

Wilcoxon, 1949). As can be seen from Table 2, a significant percentage of mice survived when challenged with a 200 LD50 dose-equivalent of that g calculated for the wild-type Salmonella bovismorbificans viz. 2x10 cfu with the qalE (CS402/SL5752) non-switchable ^aT, aro ^" (CS404/SL5785) or gal* aro ^" (CS405/SL5794) smooth derivatives of Salmonella bovismorbificans.

Oral challenge of sheep with virulent Salmonella bovismorbificans

(CS400/S15747) with 5xl0 ^1] to lxlO ¹² cfu induced severe clinical

Salmonellosis whereas the same dose of the mutant (CS405/SL5794) was

uneventful. Intramuscular administration of CS405/SL5794 into the gastroc muscle of sheep resulted in a transient rise in temperature, returning to within the normal range within 2 to 3 days.

The j_n vivo safety of Salmonella bovismorbificans aro ^"

(CS405/SL5794) was further substantiated by examining its j_n vivo multiplication kinetics with that of its virulent parent, upon g administration by the oral (5x10 cfu per mouse) or intraperitoneal routes (1x10 cfu per mouse). It is clear from the date presented in Table 3 that Salmonella bovismorbificans aro ^" (CS405/SL5794) was present in the liver, spleen, mesenteric lymph nodes (MLN) and intestine (only in small numbers) regardless of the route of administration. By day 27 post-administration by the oral route, the aromatic-dependent mutant organisms were detectable in (small numbers) in the intestine but not in liver, spleen or MLN. However, when the organisms were administered by the intraperitoneal route, the mutant aro ^" organisms were detectable in 1 out of 3 mice from the spleen and MLN (not liver or intestine), but only by enrichment cultures. When mice were inoculated with the virulent parent organisms (CS400/SL5747) by the oral route, the organisms were detectable by direct plating in all organs and the total cfu showed a significant increase on day 7. On day 14, although the total cfu showed a decline, 1 out of 3 mice had died. The remaining inoculated mice died on day 16 and no more animals were available for enumeration of organ counts. On the other hand, when inoculation of mice with the virulent parent was by the intraperitoneal route, the organisms were detected by direct plating on day 1 from the liver, spleen, MLN and intestine. The total number of cfu increased in these organs on day 4 and on day 7 post-inoculation all but 2 mice were dead (Table 4).

The safety of this vaccine strain (CS405/SL5794) was further

demonstrated by inoculation of these organisms in mice immuπocompromised by injection of either silica or cyclophαsphami e, when no deaths were recorded.

Following inoculation of sheep with 10 cfu of aromatic-dependent Salmonella bovi morbificans (CS405/SL5794) in the gastroc muscle, the mutant organism could be recovered from a combination of sites including muscle, spleen, lymph nodes (popliteal, medial iliac and meseπteric) and faeces. By day 21 post-inoculation, mutant organisms were recorded only following enrichment in a selective medium (cysteine-maπnitol-selenite broth) but were not detectable at day 28. Upon administration by the oral route (10 cfu per sheep), the mutant organism was also detected from a combination of sites including rumen contents, intestinal contents (jejunal, ileal, caeeal), faeces and lymph nodes (anterior mediastinal and esenteric) up to day 21 post-inoculation. However, again it -was not detectable at day 28 post-inoculation. Further, this vaccine strain has been passaged through sheep by the oral route 3 times and no revertants have been observed.

Example 3 Immunization and Challenge Experiments in Mice Protection Studies

Immunization of mice (6-8 week-old) with Salmonella bovismorbificans aro ^" (CS405/SL5794) was carried out either by the oral (one or two doses of 10 cfu per mouse administered one week apart) or intraperitoneal

5 5 6 route (one does of 10 cfu per mouse or two doses of 1C and 10 cfu administered one week apart). Additional groups of mice were also immunized intraperitoneally with formalin-killed Salmonella bovismorbificans (3 does given one week apart). All mice including unimmunized control mice were orally challenged at 4 weeks

post-immunisation with 20, 100 or 200 LD50 dose equivalents of virulent

Salmonella bovismorbificans (CS4QQ/SL5747) by administering 0.5 ml of a culture, grown in brain heart infusion broth (BHI) for 18 hours (pH adjusted to 8.5), by gavage. These mice were observed for 15 days post-challenge and mortality recorded. The number of mice used per group in these experiments ranged from 8 to 18.

It can be seen from data presented in Table 5, that immunization of mice with 3 doses of formalin-killed virulent Salmonella bovismorbificans imparted no protection against oral challenge with virulent organisms whereas those immunized with the live Salmonella bovismorbificans aro ^"

(CS405/SL5794) by the oral or intraperitoneal routes were significantly protected (Table 5). Protection was apparently better in mice immunized with 2 rather than one dose only and this was particularly evident at challenge doses of 100 and 200 LD50 (Table 5). On the other hand, the percentage survival of unimmunized control mice in the above experiments ranged from 0 to 13 percent. It is also of interest to mention that while mice surviving immunization with the qalE mutant of Salmonel la bovismorbificans (CS402/SL5752) were significantly protected against challenge-infection with the virulent parent, those immunized with the non-switchable gal ^" , mutant aro ^" strain (CS404/SL5785) were not (Table

5).

Immunological Procedures or Responses

Antibody titres were determined using an enzyme-linked immunosorbent assay (ELISA) as described elsewhere (Engvall and Perlmann, 1971). Antigen

(100 μg protein) used for coating the ELISA plates (M129B, Dynatech) was prepared by ultrasonicating virulent Salmonella bovismorbificans followed by centrifugation at 27,000 g to remove the cell debris. The ELISA titres were expressed as end-point antibody titres, the cut-off point being

determined by an average of the optical densities at 405 nm for four antigen-free wells, using specific aπti-Salmonella bovismorbificans hyperimmune serum at a dilution identical to the starting dilution of individual test samples.

Delayed-type hyperseπsitivity (DTH) was measured by injecting ultrasonicated antigen preparation described above (10 μg protein per 5 μl saline) into the foot pads of 4 mice and measuring their thickness with Vernier calipers at 48 hours post-inoculation (Mukkur et aj_. , 1988).

Student's t test was used to determine the significance of differences between mean values for ELISA titres and DTH measurements. Significance of protection in the mouse immunization and challenge experiments was assessed by Fisher's exact test.

Mice immunized with either 2 doses of live mutant (CS405/SL5794) or 3 doses of formalin-killed virulent organisms by the intraperitoneal route developed high antibody ELISA titres whereas those immunized with one dose of live mutant organisms by the intraperitoneal route had low antibody titres (Table 6).

On the other hand, in mice immunized orally with one or two doses of the mutant aro ^" strain no antibody titres were detectable. Further, while mice immunized with the live, Salmonella bovismorbificans aro ^" (CS405/SL5794) by the intraperitoneal route (one or two doses) developed a significant delayed-type hypersensitivity response, those immunized by the oral route or with formalin-killed virulent parent organisms did not (Table 6).

The fact that mice immunized with the formalin-killed vaccine by the intraperitoneal route, were' not protected is indicative of the importance of DTH in protection against Salmonellosis. However, mice immunized by the oral route which did not develop any DTH were also protected against oral

challenge with virulent organisms. Although the nature of these local protective factors is currently under investigation, similar observations were made previously in sheep immunized with Salmonella typhimurium aro ^" by the oral route (Mukkur et __Λ_. , 1987).

Example 4

SHEEP

Protection studies

While a vaccine dose of greater than 10 colony forming units (cfu) g can result in a protective immune response, the preferred dose is 3.3x10 cfu per sheep. Sheep (8 per group or interval) were immunised with one dose of aro ^" Salmonell bovismorbificans (CS405/SL5794) by either the

12 intramuscular or oral route and challenged with 10 cfu of the virulent parent organisms (CS400/SL5747) by the oral route. Immunised sheep were significantly protected against virulent challenge at 7, 14 or 21 days post-immunisation whereas those immunised by the oral route were not (Table

7). However, if sheep were immunised with 3 doses of aro ^" Salmonel la bovismorbificans (as a component of the composite vaccine constituted of aro ^" strains of Salmonella bovismorbificans, Salmonella typhimurium and

Salmonella havana) and challenged at 7 days after administration of the first dose, partial protection was observed (Table 8). But sheep immunized as above were still susceptible to oral challenge with virulent Salmonella typhimurium. These results were in contrast to those observed in sheep immunized with aro ^" Salmonel la typhi urium (CS332) and challenged orally with the virulent parent organisms. Sheep were significantly protected at day 21 post-immunisation but not at days 7 or 14 (Table 7). Sheep in the unimmunized groups died of septicaemia and/or acute enteritis within 7 - 10 days of challenge.

Immunological Procedures or Responses

Antibody levels against the somatic (0-group) and flagellar (H) antigens were measured using an agglutination test as described previously

(Mukkur et a_L , 1987). Delayed type skin reactions were measured with

Vernier calipers before (0 hour) and after (48 hours) intradermal injection of purified lipopolysaccharide (100 μg) or flagellin (100 μg) prepared from the mutant strains.

As observed previously (Mukkur et a_k , 1987), serum antibody titres against 0 and H antigens were significantly higher in sheep immunized by the intramuscular route than those immunized orally. Similarly, only those sheep immunized by the parenteral (intramuscular) route developed significant indurated skin swellings characteristic of delayed-type hypersensitivity (DTH) at days 7, 14 or 21 post-immunization with one dose of the aro ^" Salmonella bovismorbificans, thus affirming the suggestion of a positive correlation between DTH and protection.

(B) Salmonella havana Bacterial Strains and Phage Lysates

The smooth strain of Salmonella havana used in this investigation represented a faecal isolate from an acutely scouring sheep held in a feedlot in Perth, Western Australia prior to being loaded aboard ships destined for the Middle East. Other bacterial strains used in this investigation are listed in Table 9.

Example la Generation of aromatic-dependent (aro~) Salmonella havana

As shown in Table 9 the wild-type Salmonella havana isolate of Australian origin (CS4/SL5749) was sensitive to ampicillin, kenamycin, streptomycin, naladixic acid and tetracyeline and susceptible to the action of Felix-0 (F0) phage.

Phage P22, which has been used to convert virulent strains of

Salmonella typhimurium. Salmonella dub!in or Salmonella typhi into non-virulent, candidate live-vaccine strains by transducing in a mutant aro ^" alleie (Hoiseth and Stoeker, 1981; Smith et al- , 1984a,b), adsorbs only to smooth strains or 0 groups B, D or A and so could not be used to transduce a mutant aro al 1ele to Salmonella havana of group G2 (0-1, 13,

23). Phage PI does not act on smooth Salmonella of 0 group B but is adsorbed by mutants making type Re (galactose-deficient) lipopolysaccharide

(LPS), including those with mutations at ___d*_]_E , which specifies the enzyme

UDP-galactose epimerase; this phage has been used to transduce genes from galE strains of Salmonella typhimurium (0 group B) to ___§_]_ strains of

Salmonella choleraesuis (group Cl , 0-6, 7) and the reverse (Nnalue and

Stoeker, 1987a). We therefore attempted to isolate a alE mutant from the wild-type (smooth) Salmonella havana strain, for use as a recipient in

Pl-mediated transduction but were unsuccessful. Surprisingly however,

Salmonel 1a havana CS4/SL5479) could adsorb phage PI. This was shown by the appearance of colonies (Ca. 50 per 0.01ml drop of undiluted lysate) after overnight incubation at 30°C of plates of blood agar base with chloramphenicol (12μg/ml) flood-inoculated with Salmonella havana wild-type strain and spotted with phage PI Cm Ct-s (which when it lysogenizes converts its host to chloramphenicol-resistance and is temperature-sensitive for induction). The yield of chloramphenicol-resistant colonies from Salmonel la havana was only about

1/200 of that from a galE strain of Salmonel la typhimurium.

In view of this result, we next tried to transfer a previously characterized non-reverting mutation at aroA to our wild-type Salmonel la havana strain by cotransduction with an adjacent silent insertion of tranposon Tn 10, designated Zbj-903::Tn 10 (Hoiseth and Stoeker, 1981;

Nnalue and Stoeker, 1987b), with selection for tetracycline-resistance.

The strain used as donor, SL2838, is a Salmonella typhimurium line with a galE mutation which has been given by transduction, mutation Zbj-903::Tn 10 and the linked TnlO-generated non-reverting aroA mutation of a Salmonella typhimurium live-vaccine strain, SL1479 (Smith et al . , 1984; Nnalue and

Stoeker, 1987b). Drops of a PI Cm Ct-s lysate of strain SL2838, obtained by thermal induction of SL2838 converted to chloramphenicol-resistance by lysogenization with this phage, were applied to plates selective for tetracyeline resistance which had been flood-inoculated with the Salmonella havana recipient. After 3 days incubation at 37°C, nine tetracycline-resistant colonies were observed. Eight of these gave prototrophic growth, like the parent strain; they are presumed to have resulted from incorporation of donor Zb.i-903: :Tn 10 without the adjacent aroA mutation or from transportation of Tn 10 from a transduced chromosomal fragment into some unrelated site in the recipient. One tetracycline-resistant clone designated CS233/SL5770 was aromatic-dependent but unaltered in serological character and phage sensitivity pattern. This aromatic-dependent transductant was now plated on the medium devised by

Bochner and his colleagues (Bochner et al., 1980) which inhibits growth of tetracycline-resistant strains. All of 14 clones tested were tetracycline-sensitive but still aromatic-dependent and unaltered in other characters tested; one such isolate, numbered CS234/SL5775, constitutes our candidate Salmonel la havana live-vaccine strain. No reversion of this strain to aromatic-independence was detected; this was to be expected, since it has the aroA mutation of the Salmonella typhimurium live vaccine strain, SL1479, which gave no revertants in extensive trials (Smith et §_]_. ,

1984a).

The aro ^" Salmonella havana, as expected, was non-reverting and had

9 7 an LD50 in mice of 1x10 cfu as compared with 1x10 cfu for its

wild-type parent. Studies on the in vivo persistence of aro ^" Salmonel la havana revealed that it could not be detected in either liver, spleen or mesenteric lymph nodes by day 15 post-inoculation by the intraperitoneal route. This antigenic exposure was apparently sufficient to elicit a protective immune response. The fact that mice immunized intraperitoneally with either the live or formalin-killed aro ^" Salmonella havana had high antibody titres but only those immunized with the live mutant organisms by the intraperitoneal route developed significant delayed-type hypersensitivity and were protected against challenge with the wild-type organisms, clearly suggesting that development of delayed-type hypersensitivity to Salmonella antigens following vaccination was an indicator of resistance to Salmonellosis.

Example 2a

Determination of Fifty percent lethal dose (LD50)

The LD50 of wild-type and aromatic-dependent aro ^" Salmonella havana was determined by inoculating Balb/c mice with varying doses of colony

4 11 forming units (10 to 10 cfu) by the intraperitoneal or oral routes

(by gavage). While the LD50 dose for the former route was calculated according to Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949), that for the oral route could not be established as the organisms were found to be nonpathogenic when administered by this route.

The LD50 of the parent wild-type strain of Salmonella havana when administered by the intraperitoneal route was 1x10 cfu whereas that of g the aro derivative was 1x10 cfu per mouse. However, the wild-type organisms were apparently nonpathogenic when up to 10 cfu per mouse were administered by the oral route.

Example 3a

Immunization-chal leπqe Experiments

(A) MICE

Two groups of 12-week-old Balb/c mice (27 per group) were immunized by either the intraperitoneal (group I) or oral routes (group II) with lxlO ⁷ or 10 ¹⁰ cfu of aro ^" Salmonella havana (CS234/SL5775) respectively. A third group (III) of 27 mice were immunized with two doses of formalin-killed aro ^" Salmonella havana (CS234/SL5775) by the

8 8 intraperitoneal route, administered one week apart (10 cfu and 5x10 cfu respectively). A fourth group (IV) of 27 mice served as unimmunized controls. Four weeks post-immunization, 10 mice from each of the groups were challenged with 2 LD50 or 10 LD50 doses of wild-type Salmonella havana by the intraperitoneal route and mortality recorded for 15 days post-challenge. At the same time, 3 mice from each group were blood sampled individually for collection of sera and determination of antibody titres, whereas the remaining 4 mice were used for assessing the development of delayed-type hypersensitivity. Delayed type hypersensitivity (DTH) was measured by injecting sonicated Salmonella havana preparation (lOμg protein per 5μl saline) into the foot-pads of

4 mice and measuring their thickness with Vernier calipers at 48 h post-inoculation.

Mice immunized with live aro ^" Salmonella havana by the intraperitoneal route were protected against an intraperitoneal challenge with 2xl0 ⁷ (2 LD50) or lxlO ⁸ (10 LD50) cfu of the wild-type organisms.

On the other hand, mice immunized orally with one dose (10 cfu of the live mutant organism or two doses of formalin-killed Salmonella havana,

8 8 administered one week apart (10 and 5x10 cfu respectively) were not protected (Table 3). Mice immunized with live or killed aro ^" Salmonella havana by the intraperitoneal routes developed high ELISA antibody titres whereas those immunized with live aro ^" organisms by the oral route

developed low antibody levels. However, only those mice immunized with the live organisms by the intraperitoneal route showed significant development of delayed-type hypersensitivity (Table 11).

In vivo multiplication of aromatic-dependent (aro~) Salmonella havana.

Two groups of 12 Balb/c mice were inoculated with 5x10 cfu of the wild-type parent strain or of its aro ^" derivative by the intraperitoneal route; another group of 12 mice were inoculated with 10 cfu by the oral route. On days 3, 6, 9 and 15 post-inoculation, 3 mice from each group were euthanised and live bacteria in the liver, spleen and mesenteric lymph nodes were enumerated by crushing the individual organs using a Stomacher (400), then spread-plating serial 10-fold dilutions.

On day 3 post-inoculation with wild-type Salmonella havana (CS4/SL5749) in mice by the intraperitoneal route, the number of live bacteria recovered from liver, spleen and mesenteric lymph nodes was significantly greater than that observed for its aro ^" derivative (CS234/SL5775) (Table 10). Further, there was a significant increase in the live wild-type or aro ^" bacteria recovered from the liver and spleen on day 6 post-inoculation. By day 9, all mice inoculated with wild-type Salmonella havana died. In contrast, there was a significant decline in the numbers of live aro ^" Salmonella havana recovered from the liver, spleen and mesenteric lymph nodes on day 9 post-inoculation. However, by day 15, the aro ^" organisms could not be recovered even by enrichment cultures (Table 10). Wild-type organisms could not be detected in the liver and spleen of mice inoculated by the oral route, although they were detectable in the intestine and mesenteric lymph nodes by enrichment cultures, on day 3 post-inoculation. By day 6, no organisms were detectable in either the intestine or mesenteric lymph nodes (data not

shown). It should be pointed out that several individual colonies of

Salmonella havana isolated from the liver, spleen or mesenteric lymph nodes of mice inoculated with aro ^" Salmonella havana at various intervals were tested for dependence on aromatic compounds and were all found to be aromatic-dependent. Similar results have also been recorded for several individual colonies of Salmonella havana isolated from the iliac and popliteal lymph nodes of sheep following inoculation of aro ^" Salmonell havana in the gastroc muscle (data not shown). The safety of this aro ^"

Salmonella havana vaccine strain (CS234/SL5775) was further demonstrated by inoculation of these organisms in mice immunocompromised by injection of either silica or cyclophosphamide, when no deaths were recorded.

Immunological parameters

Salmonella havana-specific antibody titres in sera from individual mice were measured using an enzyme-linked immunosorbent assay (ELISA) as described previously (Engvall and Perlmann, 1972). An ultrasonicate preparation of the parent Salmonella havana (lOOμg protein per ml) was used as antigen to coat the microtiter ELISA plate (M129B Dynatech).

Example 4a SHEEP Experimental reproduction of clinical Salmonellosis in sheep

Four groups of 2 sheep each, housed in an isolation facility, were infected with 10 ⁹ , 10 ¹⁰ or 10 ¹² cfu of the parent wild-type Salmonella havana (CS4/SL5749) by the oral route, using gelatin capsules as described previously (Mukkur et al. , 1987). Various clinical parameters including rectal temperature and degree of scouring were monitored daily for 2 weeks post-infection. All sheep were weighed before infection and at 5 and 14 days post-infection.

Sheep infected with the wild-type Salmonella havana by the oral route

did not develop any signs of clinical Salmonellosis. There was neither an elevation of rectal temperature nor any signs of the onset of scours. At the termination of the experiment, while the organisms were recovered from the intestinal contents and mesenteric lymph nodes by direct plating or enrichment cultures, they were absent from the liver and spleen. However, since Salmonella havana has been isolated consistently both ante- and post-mortem from cases of clinical Salmonellosis in feedlots and during transit aboard ships (personal communication from Dr. R. B. Richards,

Department of Agriculture, Western Australia), it was considered relevant to include aro ^" Salmonel la havana in any vaccine formulation for use in sheep as an appropriate dose would be expected to promote an effective protective. immunity in vivo. This consideration would still be valid if the pathogenicity of Salmonella havana for sheep was subsequently demonstrated to be limited biologically or entirely conditional on stresses imposed by transport of sheep through port accessible feedlots and aboard ships, and/or intercurrent disease or nutritional factors e.g., parasitism, vitamin B12 deficiency etc.

In-vivo multiplication of aromatic-dependent (aro ^" ) Salmonella havana

Upon immunisation of sheep with aro ^" Salmonella havana

(CS234/SL5775) by the intramuscular route (gastroc muscle) with 10 cfu per sheep, the mutant organisms were recovered from the inoculation site in the muscle, and the popliteal and medial iliac lymph nodes, up to day 14 post-immunisation. On day 21, the mutant could be recovered again from the popliteal lymph node but only upon enrichment in a selective medium. At no time was aro ^" Salmonella havana recovered from the liver, spleen, mesenteric lymph nodes or faeces. When sheep were vaccinated by the oral route (10 cfu per sheep), the mutant organisms could be recovered from a combination of sites including contents from rumen, jejunum, ileu and

**** _*

- 42 - caecum, and ileal and caeca! lymph nodes up to day 21. However, by day 28 post-immunisation, aro ^" Salmonella havana was not detectable at all. The mutant organisms were never recovered from the heart blood, bile, liver, spleen or anterior mediastinal lymph nodes at any of the specified intervals. Further, this vaccine strain has been passaged through sheep by the oral route 3 times so far and no revertants have been observed.

Immunisation-challenge experiment

Two groups of vaccinated and non-immune sheep (3 sheep per interval) were challenged orally with a field isolate of Salmonel la havana and bacterial counts enumerated/detected in the mesenteric lymph nodes and faeces. No challenge organisms were detectable in vaccinated sheep by day 21 post-challenge, whereas in the non-immune, they could be still detected in significant numbers in the mesenteric lymph nodes at day 28 post-challenge. However, the challenge strain was not detected in the faeces of both treatment groups by day 21 post-challenge. These studies confirmed the potential benefit to be derived by inclusion of aro ^" Salmonella havana in the composite salmonella vaccine. Immunological procedures or responses

As observed previously (Mukkur et al-, 1987), sheep vaccinated by the intramuscular route developed a significant delayed-type hypersensitivity measured 48 hours after an interdermal injection (0.05ml) of an ultrasonicate (100 μg protein per ml; 35 μg carbohydrate per ml) prepared from the challenge strain of Salmonella havana. No delayed-type skin response was observed in sheep immunized by the oral route.

Example 5 COMPOSITE VACCINES

The most common isolates in order of significance from cases of Salmonellosis in sheep are Salmonella typhimurium. Salmonella

bovismorbificans and Salmonella havana whereas in cattle they are

Salmonella dubl . n≥ Salmonel la typhimurium and Salmonella bovismorbificans.

Nonreverting, aromatic-dependent aro ^" mutants of Salmonella typhi urium and Salmonella dublin were generated and their protective potential determined in mice and sheep or cattle. Generation of (aro ^~ ) Salmonella typhimurium and Salmonella dublin

While the procedure used for the generation of aro ^" mutant strains of Salmonella typhimurium and Salmonella dublin was essentially that described by Hoiseth and Stoeker (1981), appropriate bacterial strains and phage P22 were obtained through the courtesy of Dr. John Roth, University of Utah, Salt Lake City, U.S.A.

In vivo safety of (aro ^~ ) Salmonella typhimurium (CS332) and (aro~) Salmonella dublin (CS101)

The in vivo safety of the above aro ^" strains (CS332 and CSIOI) was first determined in mice by oral or intraperitoneal inoculation. The vaccine strains were not detectable in liver, spleen, mesenteric lymph nodes or intestinal contents at week 5 post-inoculation by the intraperitoneal route although they could still be detected from some of the enrichment cultures at week 6 post-inoculation by the oral route. However, in sheep the aro ^" Salmonella typhimurium vaccine strain (CS332) was not detectable in any of the organs/tissues examined on day 28 post-inoculation by the intramuscular route liver, spleen, muscle, popliteal, medial iliac and mesenteric lymph nodes, and faeces) or oral (rumen and intestinal contents including faeces, and mesenteric lymph nodes) route. In calves, all the aro ^" strains tested viz., Salmonella typhimurium (CS332), Salmonella bovismorbificans (CS405/SL5794) or Salmonella dublin (CS101) were not detectable in the faeces at 2 weeks post-administration by the oral or intramuscular route. Further no

revertants were observed and the recovered vaccine strains were still aromatic-depeπdent.

Example 6

Utilization of Composite Vaccines in Sheep

While the vaccine dose of the composite vaccine for sheep (containing aro ^" derivatives of Salmonella typhimurium, Salmonella bovismorbificans and Salmonella havana) of 3x10 cfu can promote effective immunity, the g preferred dose is 3.3x10 cfu of each mutant organism per sheep in the vaccine.

Immunisation-challenge experiments

Sheep immunized with the composite vaccine by a parenteral route, preferably intramuscular, and challenged orally with either virulent Salmonella typhimurium or Salmonella bovismorbificans at 7 and 14 days post-immunisation, significant protection was observed (Table 8). The response of these sheep to oral inoculation with the field isolate of Salmonella havana was similar to the that described earlier in Example 4a. Oral immunisation of sheep with three doses of the composite vaccine by the oral route, while imparting partial protection against challenge with virulent Salmonella bovismorbificans at 7 days post-immunisation, was ineffective at protecting sheep against challenge with virulent Salmonell typhimurium (Table 8). Immunological procedures or responses

The antibody responses of immune sheep to the component mutant organisms in the composite vaccine (measured by agglutination tests) and delayed-type skin responses to the ultrasonicates of the component mutant organisms were similar to those described earlier in this document for sheep immunized with individual mutant organisms.

Example 7

Utilization of Composite Vaccines in Cattle

Previous studies revealed that calves immunized with aromatic- dependent aro ^" Salmonella typhimurium by the oral (Robertssoπ et al., 1983; Smith et al., 1984) routes were significantly protected against oral challenge with virulent organisms. Similar results were later reported for calves immunised with aro ^" Salmonella dubl in by the intramuscular route and challenged with virulent homologous organisms by the oral route. However, neither the protective potential of aro ^" Salmonella dublin administered by the oral route nor the composite salmonella vaccine (containing aro ^" strains of Salmonella typhi urium or Salmonel la dubl in) administered by the oral or intramuscular routes, has been reported to date. Further, no information on the protective potential of another significant Salmonella species viz., Salmonella bovismorbificans, in cattle is available. However, since immunisation of sheep, another ruminant animal, with aro ^" Salmonella bovismorbificans (CS405/SL5794) has been shown to impart significant protection against oral challenge with the virulent parent organisms, it is expected that cattle immunized with the above aro ^" strain will also be protected against homologous virulent challenge. Since it has already been demonstrated that the inclusion of aro ^" Salmonella bovismorbificans (CS405/SL5794) in the composite vaccine for sheep did not compromise the immune response and protection induced by the other components in the vaccine, it was considered necessary only to evaluate the protective potential of the composite vaccine containing aro ^" strains of Salmonella typhimurium (CS332) and Salmonella dubl in (CS101) in calves. Protective potential of (aro~) Salmonella dublin vaccine

The protective potential of aro ^" Salmonel la dubl in (CS101) in calves (3-4 week old) administered by the intramuscular versus oral route

was evaluated. One group of seven calves each were immunised with 10 cfu aro ^" Salmonella dublin by the intramuscular route (administered 7 days apart in two equal doses) while a second group of seven calves were immunized by the oral route, receiving the same dose but equally divided into 3 doses administered 4 days apart.

A third group of six unimmunized calves were used as controls.

12 Calves in the above three groups were then challenged orally with 10 cfu of the virulent parent organisms (CS50), two weeks after administration of the first immunising dose and observed clinically for three weeks post-challenge. Five of the seven calves in the vaccinated groups survived the challenge infection (71 percent protection) whereas all six unimmunized calves died of acute enteritis and septicaemia within 10 days of the challenge infection. Serum anti-0 and -H titres measured by agglutination tests were significantly higher in calves immunized by the intramuscular route than in those immunized orally. It should be pointed out, however, that while an immunizing dose of 10 cfu of aro ^" o

Salmonella dublin was used in this experiment, a dose as low as 10 cfu per calf can promote an effective immune response. Protective potential of the composite salmonella vaccine

A total of forty five (2-4 week old) calves divided into seven groups (5 to 8 calves per group) were used in this experiment. Five of the seven groups were immunized intramuscularly or orally with a total of 10 cfu of each organism. The composite vaccine containing equal numpers of aro ^" strains of Salmonella typhimurium (CS332) and S. dublin (CS101), administered in two or three equally-divided doses. Two groups (I,II) of calves were immunized intramuscularly receiving two vaccinations one week apart while another two groups (III,IV) were immunized orally using the same regime. A fifth group (V ⁾ of calves was immunized with three doses

administered orally 4 days apart. The remaining two groups of calves

(VI, II) were used as unimmunized controls. While calves in groups I, III,

12 V and VI were challenged orally with 10 cfu of virulent Salmonella typ.himuriu of bovine origin (CS12), those in groups II, IV and VII were

12 challenged with 10 cfu virulent Salmonella dublin (CS50). These calves were then observed for the development of clinical Salmonellosis for a period of 3 weeks post-challenge. As shown in Table 12, calves immunized with the composite salmonella vaccine were significantly protected against challenge with virulent Salmonella typhimurium or Salmonella dubl in as compared with the unimmunized controls.

Example 8 CROSS-PROTECTION STUDIES

In order to determine the potential range of cross-protection imparted by the salmonella vaccine, use was made of the experimental mouse Salmonellosis as the model system and aro ^" Salmonel la typhimurium as a model organism. Balb/c mice (4-6 week old) were immunized with 10 cfu of aro ^" Salmonella typhimurium (CS332) and challenged with virulent partially and completely heterologous salmonella organisms by either oral or intraperitoneal route as dictated by pathogenicity determined previously. It was discovered that if the 0-group antigen 4 was also present on the challenge strain, significant cross-protection was observed. If the 0-group antigens on the challenge strains were completely different from those on the immunizing aro ^" strain(s), no cross- protection was observed.

Sharing of one or more flagellar antigens, on the other hand, was of no consequence. These studies demonstrated that immunisation of mice (and other species) was capable of imparting protection against other Salmonella species as long as critical 0-group antigens were shared.

Surprisingly, mice immunised parenterally or orally with aro ^"

Salmonella typhimurium (CS332) were partially protected against oral challenge with virulent Salmonella bovismorbificans (CS2). However, sheep immunized with the aro ^" Salmonella typhimurium vaccine strain (CS332) by the intramuscular or oral route and challenged orally with the virulent parent were not protected at all, thus justifying the need for inclusion of the aro ^" Salmonella bovismorbificans strain (CS405/SL5794) in the vaccine formulations proposed in this application.

Example 9

Response of pregnant animals to vaccination with aromatic-dependent salmonella species

The aro ^" salmonella vaccine strains viz., CS234/SL5775,

CS405/SL5794 and CS332 upon inoculation by the oral or parenteral routes in sheep and cattle (10 cfu per animal) 4 weeks pre-partum were-found to be quite safe and did not result in any side effects, including abortion.

Using sheep as a model animal and aro ^" Salmonella typhimurium (CS332) as a model organism, it was discovered that antibody levels in the colostrum of sheep immunized with one dose of the live mutant organisms (10 cfu per sheep) were significantly higher than those immunized with the same dose of nonliving organisms. Because of the recent suggestion that feeding of salmonella-specific immune colostrum to calves caused a reduction in the incidence of neonatal Salmonellosis (Jones et ah, 1988), it is self evident that for production of immune colostrum with a high specific activity, immunisation of pregnant dam(s) with live aro ^" organisms is to be preferred.

INDUSTRIAL APPLICABILITY

This invention provides smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strains of Group C2;0-6,8, C3;0-8 or C3;0-8,20

especially Salmonella bovismorbificans. The invention also provides smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella strains of Group G2,0-1,13,23 or Gl;0-i,13,22 especially Salmonella havana. Composite vaccines containing one or both of these strains are of utility in causing a reduction in the mortality of sheep and cattle aboard boats during transit over long distances to other countries, in intensively-managed sheep and cattle properties by the prevention of

Salmonellosis. These vaccines can also be used for the prevention of

Salmonellosis in other species including humans, equines, porcines, canines, avians etc., and other vertebrate species such as fish. These vaccines can also be used for the prevention of Salmonellosis in a foetus by immunisation of the pregnant dams/mothers or in neonates by feeding colostrum or milk rich in specific antibodies raised against the strains of the invention.

DEPOSIT OF MICROORGANISMS

A smooth aromatic-dependent (aro~) non-reverting, avirulent Salmonella bovismorbificans strain (CS405/SL5794) of Group C2;0-6,8 was deposited at the Australian Government Analytical Laboratories on 3rd January 1990 and accorded Accession NumberN90/000304.

A smooth aromatic-dependent (aro ^" ) non-reverting, avirulent Salmonella havana strain (CS234/SL5775) of Group G20-1,13,23 was deposited at the Australian Government Analytical Laboratories on 3rd January 1990 and accorded Accession Number N90/000303.

REFERENCES

Bochner Br, Hui-Chuan Huang, Schieven GL, Ames BN (1980). Positive selection for loss of tetracyeline resistance.

J Bacteriol 143: 926-933

Engvall E, Perlmann P (1972). Enzyme-linked immunosorbent assay, ELISA. Ill Quantitation of specific antibodies* by enzyme-labeled anti-immunoglobul in in antigen-coated tube.

J Immunol 109: 129-135

Hoiseth SK, Stoeker BAD (1981). Aromatic-dependent Salmonella typhi urium are non-virulent and effective as live vaccines. Nature (Lon) 291: 238-239

Jelinek PD, Franklin DA, Iveson JB (1982). The recovery of salmonella from sheep that died during transporation by ship. Aust Vet J 58: 170-171

Jones PW, Collins P, Aitken MM 1988. Passive protection of calves against experimental infection with Salmonella typhimurium. Vet Rec: 536-541

Litchfield JT, Wilcoxon F (1949). A simplified method of evaluating dose-effect experiments.

J Pharmacol Exp Ther 96: 99-113

Mukkur TKS, McDowell GH, Stoeker BAD, Lascelles AK (1987). Protection against experimental Salmonellosis in mice and sheep by

immunisation with aromatic-dependent Salmonella typhimurium.

J Med Microbiol 24: 11-19

Nnalue NA, Stoeker BAD (1987a). Tests of virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis.

Infect Immun 35: 955-962

Nnalue NA, Stoeker BAD (1987b). The effects of 0 antigen character and enterobacterial common antigen content on the in vivo persistence of aromatic-dependent Salmonella sp. live-vaccine strains.

Microb Pathog 3: 31-44

Ornellas E P, Stoeker B A D 1974 Relation of lipopolysaccharide character to PI Sensitivity in Salmonella typhimurium Virology 60: 491-502

Richards R B, Norris R T, Dunlop R H, McQuade N C 1989. Causes of death in sheep exported live by sea.

Australian Veterinary Journal. 66_: 33-38

Robertsson JA, Lindberg AA, Hoiseth S, Stoeker BAD (1983). Salmonella typhimurium infection in calves: protection and survival of virulent challenge bacteria after immunisation with live or inactivated vaccines.

Infect Immun 41: 742-750.

Samuel J L, Eccles J A, Frances J 1981. Salmonella in the intestinal tract and associated lymph nodes of sheep and cattle. J Hyg 87: 225-232

S eltzer T, Thomas R, Collins G 1980. Sal onellae on posts, hand rails and hand in a beef abattoir.

Australian Vet J 56: 184-186

Smith BP, Reina-Guerra M, Hoiseth SK, Stoeker BAD, Habasha F. Johnson E, Merritt F (1984a). Aromatic-dependent Salmonella typhimurium as modified live vaccines for calves.

Am J Vet Res 45: 59-66

Smith BP, Reina-Guerra M, Stoeker BAD, Hoiseth SK, Johnson E (1984b). Aromatic-dependent Salmonel la dubl in as a parenteral modified live vaccine for calves.

Am J Vet Res 45: 2231-2235

Wray C, Sojka WJ (1977). Reviews of the progress of dairy science: bovine Salmonellosis.

J Dairy Res 44: 383-425

TABLE A SALMONELLA SPECIES OF GROUPS C2 AND C3

Somatic (0) Flaqellar (H) Antigens

Serovar antigens Phase 1 Phase 2

..contd.

Table A contd. laqellar (H) Antigens Serovar hase 1 Phase 2

S. molade

S. II e,π,x:z ₄₂

S. tamale [e,n,z,,-]

S. angers z.

Any of the organisms of the above table may be genetically altered by processes other than those described herein. Examples of said other processes may include: radiation mutagenesis (e.g. X-rays, thermal neutrons, ultraviolet, etc.) chemical mutagenesis (e.g. acridine half mustards).

TABLE B

.contd.

Table B contd.

Somatic (0) Flaqellar (H) Antigens

Serovar antigens Phase 1 Phase 2

[e,n,χ]

1,5 ^Z 42

1,5 e,n,x ^z 42

15

15

"42 1,6 e.n.z 15

1,7 hw

^Z 6

1,5

1,6 hw

...contd.

Table B contd.

Somatic (0) Fla ellar (H) Anti ens

Any of the organisms of the above table may be genetically altered by processes other than those described herein. Examples of said other processes may include: radiation mutagenesis (e.g. X-rays, thermal neutrons, ultraviolet, etc.) chemical mutagenesis (e.g. acridine half mustards).

TABLE 1. Bacterial Strains

Strain Number Description Origin or Reference

(a) Salmonella bovismorbificans CS400/SL5747 Wild-type, tetracycline- From fatal enteritis resistant in sheep CS401/SL5750 As CS400/SL5747 but galE From SL5747 by selection for resistance to 2- deoxygalactose

CS402/SL5752 As CS401/SL5750 but tetra¬ From SL5750 by selection cycl ine-sensitive on Bochner & medium (Bochner et ah, 1980 3 Bact 143: 926-933

CS403/SL5780 As CS402/SL5752 but CRR426 From SL5752 by trans¬ [aroA554::Tn10(Tc- duction from SL2838, sens, non-rev)3Zb.i- with selection for 903: :TnlO gal ^"2 tetracycl ine-resi stance

CS404/SL5758 As CS401/SL5752 but CRR[aroA3 From SL5780 by selection C CRRRR((Zb.i-903::TnlO ⁽ Tc ^s> >] on Bochner & medium qal ^" (Bochner et ah, 1980 3 Bact 143: 926-933

CS405/SL5794 As CS404/SL5758 but gal ⁺ From SL5758 by con- because carrying F'-8 jugational cross with qal SL3550, with selection for Gal*

(b ⁾ Salmonella typhimurium SL1479 ³ UCD108-11 CRR426[aroA554 S. typhimurium UCD aroA

::TnlO(Tc ^s , non-rev)] live-vaccine strain Smith et ah, 1984 Am 3 Vet Res 45J.: 59-66

SL2838 7471 g lE705 CRR426[aroA554 Pi-sensitive stable aroA : :Tn10(Tc ^s , non-rev)] zbj::TnlO strain used as Zb.i-903::TnlO tranductional donor (Nnalue and Stoeker 1987 Microb Pathog 3: 31-44)

SL3550 LT2 trβ metA qa1E541 T-t Kuo, Ph.d. thesis, /F'8 gal Stanford, 1969

h CRR, TnlO-gener ted complex rearrangement mutation causing phenotypic characters indicated in parentheses. T ^s -seπs, sensitive to tetracyeline. non-rev, unable to revert to aro ^" ' ^' . Zb -903: :Tπ10, silent insertion of TnlO at minute 20 of chromosome (cotransducible with aroA). Gene symbols abbreviated after first mention.

Additional chracters of these strains, not here relevant, omitted. 2. The failure of strain SL5780 to recover wild-type phage sensitivity pattern when grown with galactose (and glucose) is presumed to result from a coincidental mutation in the ________ operon, affecting function of gene galT or galK or both, indicated gal~

TABLE 2. Rel tive in-vivo safety of Salmonella bovismorbificans derivatives administered to mice by the oral route

Derivative Challenge dose (cfu) Total number of mice surviving/total number challenged

galE (CS402/SL5752) 2.0x10 τ 8/12 (75*) gal ^" , aro ^" non- switchable (CS404/SL5785) 2.0x10 ^' 12/12 (100) ga1 ⁺ , aro ^" (CS405/SL5794) 2.0x10 ^" 12/12 (100 ⁾ wild-type, parent 2.0x10 ^" 0/12 (0)

τ 2.0x10 organisms represent 200 LD50 dose-equivalent of that calculated for the wild-type parent organism.

* Numbers in parenthesis represent the percentage of mice surviving.

TABLE 3. In vivo multiplication of Salmonella bovismorbificans gal ^"1" , aro ^" strain (CS405/SL5794) irr mice inoculated by oral or intraperitoneal routes

*E denotes isolation of salmonella organisms using an enrichment procedure whereas the proportion shown within parenthesis represents number of mice in which the isolation of salmonella organisms from organs by enrichment culture divided by the total number of mice used on various days post-inoculation.

TABLE 4. In vivo multi lication of the arent virulent Salmonella

τ average of 2 mice only since no more mice were alive after this interval .

*E denotes isolation of salmonella organisms using an enrichment procedure whereas the proportion shown within parenthesis represents number of mice in which the isolation of salmonella organisms from organs by enrichment culture divided by the total number of mice used on various days post-inoculation.

TABLE 5. Protection in mice iiraπunized with Salmonella bovi morbificans qalh aro ^" strain (CS405/SL5794) against oral challenge with virulent organisms (CS400/SL5747) compared with other derivatives.

No. of Number of mice surviving/ vaccine total number of

Vaccine Route of doses mice challenged preparation immunization admin- LD50 istered 20 100 200

None

(Unimmunized) N.A. N.A. 2/15 (13) 0/16 (0) 0/16 (0) Formalin-killed virulent parent Intraperit¬ 3 2/8 (25) 0/8 (0 ⁾ 0/8 (0) (CS400/SL5747) oneal Live derivative, aro ^" Intraperit¬ 1 τ!5/18 (83) *8/18 (44) *7/18(39) (CS405/SL5794) oneal

Intraperit¬ 2 τ8/8 (100) §12/10(75) 12/17(71) oneal Oral 1 *6/8 (75) 3/10 (30) 2/10(20) Oral 2 *7/8 (87) §6/8 (75) 3/8 (38)

Live derivative, gal~,aro~ nonswitenable Intraperit¬ 2 3/8 (38) 0/8 (0) 0/8 (0) (CS404/SL5785) oneal Oral 2 1/8 (13) 0/8 (0) 0/8 (0)

Live derivative, galE Intraperit¬ 2 τ8/8 ⁽ 100) *4/8 (50) 4/8 (50)

(CS402/SL5752) oneal Oral 2 τ8/8 ⁽ 100 ⁾ 3/8 (38) 3/8 (38)

τ Numbers in parenthesis denote percentage survival in various groups

N.A. Not applicable.

Significance of protection in immunized vs. unimmunized mice; p < 0.001 (§), p < 0.01 (τ), p < 0.05 (*)

TABLE 6. Immunological response of mice immunized with either live

Salmonella bovismorbificans gal ^"1- , aro ^" strain (CS405/SL5794) or formalin-killed virulent parent organisms four weeks post-immunization

Number of ELISA titre Delayed-type

Vaccine Route of vaccine (units per ml) hyperseπsitivityτ strain immunization doses ± SEM (mm) ± SEM*

1126 ^α ± 236 57 ± 0.12

20,094 ^u ±9860 0.86 ^{^} ± 0.08

Not detectable N.D.

Not detectable 0.17'± 0.02

11,767 ^C ±3958 0.20 ⁹ ± 0.07 Not detectable 0.13± 0.05

τ Delayed-type hypersensitivity represents mean increase in the foot-pad thickness (mm) following antigen-injection in one hind foot minus that obtained following injection of saline in the second hind foot, measured at

48 hours post-injection.

* SEM denotes standard error around the mean.

N.D. Not done

N.A. Not applicable

Values significantly different from each other; b and c vs. a (p < 0.05), d vs. f and g (p < 0.05), e vs. f and g (p

< 0.01).

TABLE 7. Ontogeny of protection in sheep immunized with aro ^" Salmonella species by the intramuscul r or oral routes

Vaccine Challenge Route Total No. of sheep Control strain strain of surviving oral Groups and group immun- challenge with ization virulent organisms administered at days post-immunisation 7 14 21

I aro ^" Virulent

S. typhimurium S. typhimurium (CS332)

II aro ^" Virulent S. bovis- S. bovis- morbificans morbificans (CS405/SL5794)

a: One of the surviving sheep had severe diarrhoea at the time of post-mortem.

TABLE 8. Ontogeny of protection in sheep immunized with the composite ⁴ aro ^" Salmonella vaccine by intramuscular or oral routes

Vaccination Route and Challenge Total No.of sheep Unimmunized group No. of doses organism surviving oral controls used for challenge at day immunization post-immunization

7 14

9 a: Composite vaccine contained 3.3x10 cfu of each of aro strains of

S. typhimurium (CS332), S. bovismorbificans (CS405/SL5794) and S. havana

(CS234/SL5775). b: Immunizing doses administered on days 0,2 and 4.

TABLE 9. Bacterial Strains

Strain Number Description & Origin or reference

SL1479 S. typhimurium UCD live- Smith et ah, 1984 vaccine strain; CRR426 Am 3 Vet Res [aroA554::Tn 10 (Tc ^s , 45: 59-66 non-rev)]

SL2838 S. typhimurium M7471 Nnalue and Stoeker CRR426 [aroA554::Tn 10 1987 Microb Pathog (Tc ^s , non-rev.)] 3: 31-44 Zb.i-903: :Tn 10 galE

CS4/SL5749 S. havana wi 1d-type From scouring sheep CS233/SL5770 S. havana Zb.i-903: :Tn 10 CRR426 [aroA554::Tn 10 From CS4/SL5749 by (Tc ^s , non-rev)] transduction

CS234/SL5775 S. havana CRR. [Zb.i-903: :Tn From CS233/SL5770 by 10 (Tc ^S )]CRR426 [aroA554:: selection for tetra¬ Tn 10 (Tc ^s , non-rev)] cyeline sensitivity

a Additional mutant characters of strain SL1479 and SL2838, not here relevant, omitted. CRR, complex rearrangement mutation, here Tn 10-generated deletion or deletion-inversion causing loss of tetracycl ine-resistance. Tc ^s , tetracycl ine-sensitive. non-rev., unable to revert by "clean excision" of transposon.

TABLE 10. In-vivo survival of virulent or aromatic-de endent (aro ^~ )

τ CFU: colony forming units ττ MLN: mesenteric lymph nodes τττ ND: not detectable

TABLE 11. Antibody titres, delayed-type hypersensitivity and protection in mice immunized with live or killed aromatic de endent Salmonella havana

τ CFU: colony forming units ττ DTH: delayed-type hypersensitivity τττ ND: not detectable

TABLE 12. Protective efficacy of the composite aromatic-dependent salmonella vaccine in calves

a: Figures in parenthesis denote percentage of protection.