WO2005001069A1

WO2005001069A1 - Genetic marker for live bacterial vaccines

Info

Publication number: WO2005001069A1
Application number: PCT/US2003/035696
Authority: WO
Inventors: Kenneth L. Roland; Sandra M. Kelly-Aehle
Original assignee: Avant Immunotherapeutics, Inc.
Priority date: 2002-11-08
Filing date: 2003-11-07
Publication date: 2005-01-06
Also published as: AU2003304255A1; AR042015A1

Abstract

Genetically altered attenuated bacteria deficient in the ability to produce hydrogen sulfide (H2S) are described for use as live vaccines or live vectors that may be administered to a host organism to provide immunity to a disease or to provide another beneficial gene product or function. Genetically altered bacteria of the invention may be readily distinguished from other bacteria by an H2S negative phenotype when incubated on or otherwise contacted with a medium used to detect H2S production in bacteria.

Description

GENETIC MARKER FOR LIVE BACTERIAL VACCINES

This application claims priority to U.S. provisional application no. 60/424952, filed 8 November 2002.

Field of the invention This invention is generally in the field of bacterial genetics, i particular, the invention relates to a genetic marker for the detection or identification of bacteria, especially bacteria used as live vaccines to elicit in a host organism an immune response to a pathogen or in bacteria used as live vectors to provide a host organism with a polypeptide for a purpose other than immunization against a disease.

Background of the Invention Salmonella bacteria are one of the major causes of food-born illness in the United States (Mead et al., Emerg. Infect. Dis., 5: 607-625 (1999)). Poultry products are considered to be a major source of salmonellae in the food supply (Portillo, F.G., in Microbial Food Borne Diseases: Mechanisms of Pathogenesis and Toxin Synthesis, Cary, Linz, Bhatnagar, eds. (Technomic Publishing Co., Inc., Lancaster, PA, 2000), p. 7). One approach to controlling salmonella contamination in poultry has been through the use of live salmonella bacteria that have been attenuated in virulence through mutation so that such mutants are no longer pathogenic in a host vertebrate organism. Such attenuated bacteria have thus been employed as live, attenuated bacterial vaccines. Attenuated salmonellae can also prevent colonization by other wild type, potentially pathogenic, salmonella bacteria (see, e.g., Zhang-Barber et al., Vaccine, 17: 2538-2545 (1999)). Specific attenuating mutations such as Acya Acrp (see, e.g., Hassan et al., Res. Microbiol, 141: 839-850 (1990); erratum in Res. Microbiol, 142: 109 (1991)), aroA (Cooper et al., Infect. Immun., 62: 4747-4754 (1994), and nuoG (Zhang-Barber et al, Vaccine, 16: 899- 903 (1998)) have been used successfully in bacteria employed as live vaccines for poultry, as well as relatively uncharacterized mutations, such as those derived from heterophil-passaged bacteria (Kramer, Avian Dis., 42: 6-13 (1998)) and temperature- sensitive bacteria (Cerquetti et al., Vet. Microbiol, 76: 185-192 (2000)). Attenuated salmonella bacteria have also been used successfully as vaccines for food safety in other species, including pigs (Kennedy et al., Infect. Immun., 67: 4628- 4636 (1999)) and cattle (Jones et al., Vaccine, 9: 29-34 (1991)), and for animal health in pigs and dogs (McNey et al., Vaccine, 20: 1618-1623 (2002)). In addition, live S. typhi vaccines have been used to protect humans from typhoid fever (Hohmann et al, J. Infect. Dis., 173: 1408-1414 (1996)) and S. typhimurium has been used as a vector for antigens from other human pathogens (see, e.g., Angelakopoulos et al., Infect. Immun., 68: 2135- 2141 (2000)). A growing number of attenuated bacteria are being developed not only as live vaccines against the analogous pathogenic form of the bacteria, but also as live vaccines that express and present heterologous immunogenic molecules (e.g., polypeptides, carbohydrates) to elicit an immune response in a vertebrate host organism against other pathogens and thereby prevent or treat other diseases (see, e.g., Roland et al., Avian Dis., 43: 429-441 (1999); Hohmann et al., J. Infect. Dis.,173: 1408-1414 (1996)). With increasing use of live, attenuated bacteria as vaccines and therapeutic vectors, it is becoming increasingly important to be able to distinguish a particular live attenuated vaccine bacterium from other non-vaccine bacteria (e.g., wild type bacteria) for clinical, regulatory, pharmaceutical, and commercial purposes. Clinically, for example, it is desirable to have the ability to rapidly distinguish a potential pathogen from a vaccine in a patient being treated or at risk for a bacterial infection. Environmental and public health studies of live bacterial vaccines would also be facilitated by the ability to rapidly identify a live, attenuated vaccine bacterium, e.g., to follow dispersion of a live bacterial vaccine in a population at risk for the disease targeted by the vaccine. In addition, it is desirable that a live, attenuated vaccine bacterium can be identified as to its institutional or commercial origin to ensure that patients are provided with a live vaccine of the highest quality as mandated by regulatory agencies and also to avoid unauthorized use and distribution of such live bacterial vaccines. Accordingly, for reasons such as described above, there is a need for means and methods to rapidly distinguish live, attenuated bacteria used as live vaccines from other bacteria, e.g., wild type bacteria.

Summary of the Invention The invention provides a solution for the problem of distinguishing a live, attenuated bacterium useful as a live vaccine or live vector from other non-attenuated, non-vaccine or non- vector bacteria by using a particular genetic marker, which renders the attenuated bacterium deficient in the ability to produce hydrogen sulfide (H₂S). The invention provides a genetically altered bacterium deficient in the ability to produce hydrogen sulfide (H₂S) comprising: (a) a first mutation which attenuates virulence of said bacterium toward a host organism, wherein said first mutation does not affect the ability of said bacterium to produce H S, and (b) a second mutation, wherein said second mutation renders the bacterium deficient in the ability to produce H₂S. As noted above, a genetically altered bacterium of the invention comprises one or more mutations that specifically attenuate virulence so that the bacterium is no longer capable of causing disease in at least one host organism (e.g., a mammalian or avian species) of the bacterium. A "first mutation" that attenuates virulence of a bacterium of the invention also does not render the bacterium deficient in the ability to produce H₂S from thiosulfate. An attenuated bacterium useful in the invention may be obtained from a virulent population of bacteria by selection or by specifically mutating one or more virulence genes using any of a variety of mutagenic methods available in the art. Accordingly, a first mutation affecting attenuation in a genetically altered bacterium of the invention maybe uncharacterized, partially characterized, or well defined, e.g., as to the exact alteration in nucleotide sequence of a particular genetic locus. In another embodiment, a mutation present in a bacterium of the invention may be a mutation in any of a variety of genetic loci (including coding sequences, genes, operons, regulons, and regulatory sites) known to affect virulence including, without limitation, phoP,phoQ, aroA, aroC, aroD, cdt,poxA, rpoS, htrA, nuoG,pmi, galE,pabA,pts, damA, purB, gua, cadA, rfc, rfb, rfa, ompR, cya, crp, and combinations thereof. As described herein, a mutation in such genetic loci, except the cya and/or crp, are particularly preferred for use as a "first mutation" in a bacterium of the invention. A genetically altered bacterium of the invention comprises an additional, different "second mutation" that renders the bacterium deficient in the ability to produce H₂S from thiosulfate, thereby conferring an "H₂S negative" phenotype. Relevant to the invention is the production of H₂S in bacteria that typically occurs under anaerobic conditions such as exist in the center of a bacterial colony and in other anaerobic environments. The H₂S negative phenotype of a bacterium of the invention may be readily distinguished from other bacteria that produce H₂S (i.e., an "H₂S positive" phenotype) by using any of a variety of assays, e.g., various solid media, that detect H S production in bacteria or by detecting the presence in bacteria of genetic information encoding a functional enzyme essential for H₂S production. Although mutation of the cya and/or crp genetic loci will attenuate virulence in salmonellae, it also typically renders such bacteria deficient in the ability to produce H₂S, thereby disqualifying its use as a "first mutation" of a bacterium of the invention. However, mutations of the cya and/or crp genetic loci that result in attenuation and deficiency in H₂S production may be used as a "second mutation" of a bacterium of the invention. In a preferred embodiment, a second mutation of a genetically altered bacterium of the invention is present in a bacterial phs operon such that the bacterium does not express a functional thiosulfate reductase. Bacterial thiosulfate reductase is an oligomeric enzyme that catalyzes reduction of thiosulfate to hydrogen sulfide. In H₂S-producing salmonellae, for example, a number of genes associated with H₂S production are organized in an operon, which is designated "phs" for "production of hydrogen sulfide". Preferably, the wild type version of a bacterial phs operon useful for making a bacterium of the invention comprises the six phs genes: phsA, phsB, phsC, phsD, phsE, stndphs F, wherein the phs A gene encodes the catalytic subunit of thiosulfate reductase. A particularly preferred bacterium of the invention comprises a mutation at one or more of phsA, phsB, oxphsC. Other non-Salmonella, H S-producing bacteria may use an enzyme that is a functional equivalent of thiosulfate reductase to generate H₂S from thiosulfate. Hence, at the enzymological level, a bacterium useful in the invention is attenuated in virulence and deficient in H₂S production owing to a deficiency in the expression of functional thiosulfate reductase activity compared to H₂S positive bacteria. Any type of mutation in the nucleotide sequence of one or more genetic loci of a bacterium's genome may be used in the invention to provide a genetically altered bacterium that is attenuated in virulence and displays an H₂S negative phenotype. Examples of the type of mutations that maybe used in the invention include, but are not limited to, a deletion mutation, an insertion mutation, a point mutation, a frame-shift mutation, and combinations thereof. A deletion mutation is a particularly preferred type of mutation in the invention because deletion mutations are among the least likely to spontaneously revert. Any of a variety of assays for H₂S production or the presence of genetic information conferring the ability to produce H₂S in bacteria may be used to readily distinguish an H₂S negative bacterium of the invention from H₂S positive bacteria. Such assays include any of a variety of standard media commonly used to detect H S production by bacteria. When contacted with (e.g., incubated on or grown in) such media, a genetically altered bacterium of the invention fails to produce H₂S and forms a white colony (indicating H₂S negative phenotype) in contrast to H₂S-producing bacteria, which form dark, black, or dark-centered colonies (indicating H₂S positive phenotype). Preferred media, which may be used to distinguish or identify an H₂S negative bacterium of the invention from H₂S positive bacteria, include but are not limited to, Triple Sugar Iron medium (TSI), Lysine Iron Agar (LIA), XLT4 agar medium, XLD, bismuth sulfate agar, Salmonella-Shigella agar, and modified forms thereof that are supplemented with one or more additional ingredients that do not alter the property of the medium to detect H S production in bacteria. Another convenient method for detecting H₂S production by thiosulfate reductase activity in bacteria is to incubate bacteria in or on a standard bacteriological detection strip, such as an AP120E bacteriological detection strip (Bio Merieux Vitek, Hazelwood, MO). Still other methods of detecting H₂S production from thiosulfate in a bacterium include but are not limited to, assays for production of H₂S gas and standard enzymatic assays for thiosulfate reductase activity. In addition, any of a variety of assays may be employed to detect a mutation(s) in the genomic nucleotide sequence that confers an H₂S negative phenotype in a bacterium of the invention. Such assays include, but are not limited to, polymerase chain reaction (PCR) and various hybridization protocols using nucleic acid primers or probes to identify specific mutated sequences of DNA or RNA associated with H₂S production in a bacterium. Particularly preferred bacteria useful in the invention are attenuated serovars of Salmonella enterica, i.e., salmonellae, selected from the group consisting of Salmonella enterica serovar Typhimurium (S. typhimurium), Salmonella enterica serovar Hadar (S. hadar), Salmonella enterica serovar Typhi (S. typhi), Salmonella enterica serovar Paratyphi B (S. paratyphi B), Salmonella enterica serovar Paratyphi C (S. paratyphi C), Salmonella enterica serovar Enteriditis (S. enteriditis), Salmonella enterica serovar Kentucky (S. kentucky), Salmonella enterica serovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S. pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonella enterica serovar Muenchen (S. muenche ), Salmonella enterica serovar Anatum (S. anatum), Salmonella enterica serovar Dublin (S. dubliή), Salmonella enterica serovar Derby (S. derby), and Salmonella enterica serovar Choleraesuis var. kunzendorf In further embodiments of the invention, non-salmonellae H₂S-producing bacteria may be genetically altered to produce an attenuated, H₂S negative bacterium of the invention. Particularly preferred non-salmonellae include but are not limited to,

Wollinella succinogenes, Proteus vulgaris, Proteus mirabilis, Edwardsiella tarda, most known isolates of Citrobacter freundii, species of Pasteur ella, and certain species of Haemophilus. Owing to the attenuated virulence and easily identifiable H₂S negative phenotype, genetically altered bacteria of the invention are particularly useful as live vaccines against diseases in a host organism, such as in a mammalian or avian species. Accordingly, in a preferred embodiment, a bacterium of the invention may be used as a live vaccine, which is administered to a host organism to elicit an immune response to treat a disease or to provide protection to the host organism from a disease caused by the corresponding virulent form of the bacterium. In another embodiment, a bacterium of the invention may be further modified using recombinant DNA methodologies to express a heterologous antigen from a selected heterologous pathogenic organism. Such a recombinant bacterium of the invention may be employed as a live vaccine to treat or protect a host organism against a disease caused by the heterologous pathogenic organism. Particularly preferred bacterial strains of the invention useful as live vaccines in poultry include S. hadar strain MGN4902 (ATCC accession No. PTA-4777) described herein. Other preferred bacterial strains of the invention useful as live vaccines include but are not limited to S. typhimurium UK-1 strain MGN5501, S. kentucky strain MGN5367, S. infantis strain MGN5504, and S. infantis strain MGN5507. h yet another embodiment, a genetically altered bacterium of the invention may be genetically engineered for use as a non-vaccine, live bacterial vector that may be administered to a host organism to provide a gene product or function that is beneficial to the host but unrelated to eliciting an immune response, to treat or protect against a particular disease or otherwise benefit the host organism. As in the case of a live vaccine, the distinguishing characteristics of a bacterium of the invention permit the bacterium to be readily distinguished from other bacteria in samples from the host organism or from the environment.

Brief Description of the Drawings Figure 1 is a diagram of the nucleotide sequence (SEQ ID NOJ) of _ρhsA gene cloned from S. typhimurium, including the encoded amino acid sequence (SEQ ID NO:2) for thiosulfate reductase. Underlined portions of nucleotide sequence indicate polymerase chain reaction (PCR) primers. Figure 2 is a diagram of the cloning of the phsA gene from S. hadar and construction of a deletion in the cloned copy of ϋxephsA gene. Figures 3 A-3D are diagrams depicting the construction of a S. hadar bacterial vaccine strain carrying a chromosomal deletion mutation (AphsA) of the phs A gene by homologous recombination and resolution of integrant structures. Figure 3 A is a diagram of the >tr-dependent suicide vector plasmid pMEG-865, which encodes resistance to chloramphenicol (cam) as a selectable marker, a sacBR operon (sacBR) as a counter- selectable marker, and ΑphsA deletion mutation (AphsA), and which has been introduced into S. hadar strain MGN4636 by conjugation. Figure 3B diagrams the crossover event between the AphsA mutation in plasmid pMEG-865 and the wild type chromosomal phs A gene of S. hadar strain MGN4636. Figure 3C diagrams the plasmid-chromosomal integrant structure at phs . Figure 3D diagrams resolution of the integrant structure upon LB-sucrose counter-selection for a chromosomal AphsA marker. The resolved plasmid is subsequently lost by dilution from the population of bacteria carrying the chromosomal AphsA. Figure 4 shows a gel of nucleic acid products of a polymerase chain reaction (PCR) to detect wild type phsA or AphsA mutations in attenuated mutant S. infantis strains MGN5433 (lane 2), MGN5504 (lane 3), MGN5434 (lane 4), MGN5507 (lane 5), and MGN5508 (lane 6). Samples containing PCR products were run on a 0.7% agarose gel and stained with ethidium bromide. DNA from the bacteria with a wild type copy of phsA yielded a PCR fragment of 1,787 base pairs (bp) (lanes 2, 4, and 6 in Figure 4), while DNA from bacteria with the AphsA deletion mutation yielded a PCR fragment of 486 bp (lanes 3 and 5 in Figure 4). Lane 1 (size markers) contains a set of standard nucleic acid size markers, and the actual size of several of the markers is shown in base pairs (bp) to the left of lane 1.

Detailed Description of the Invention In order that the invention may be more fully understood, the following terms are defined. As used herein, "attenuated", "attenuation", and similar terms refer to elimination or reduction of the natural virulence of a bacterium in a particular host organism. "Virulence" is the degree or ability of a pathogenic microorganism (including infective particles, see below) to produce disease in a host organism. A bacterium may be virulent for one species of host organism and not virulent for another species of host organism. Hence, as used herein, an "attenuated" bacterium or strain of bacteria is attenuated in virulence toward at least one species of host organism that is susceptible to infection and disease by a virulent form of the bacterium or strain. Attenuation of virulence of a bacterium is not restricted to the elimination or inhibition of any particular mechanism and may be obtained by mutation of one or more genes in the genome (which may , include chromosomal and non-chromosomal genetic material) of a bacterium. Thus, an "attenuating mutation" may comprise a single site mutation or multiple mutations that may together yield a phenotype of attenuated virulence toward a particular host organism. Furthermore, according to the invention, an attenuating mutation that serves as the "first mutation" of two essential mutations of a bacterium of this invention cannot also render the bacterium deficient in the ability to produce hydrogen sulfide (H₂S) from thiosulfate. As used herein, the term "genetic locus" is a broad term and comprises any designated site in a genome or nucleotide sequence of an organism, including but not limited to a nucleotide coding sequence, gene, operon, regulon, regulatory site, intercistronic region, and portions thereof. A genetic locus may be identified by any of a variety of in vivo and/or in vitro methods available in the art, including but not limited to, conjugation studies, crossover frequencies, transformation analysis, transfection analysis, restriction enzyme mapping protocols, nucleic acid hybridization analyses, polymerase chain reaction (PCR) protocols, nuclease protection assays, and direct nucleic acid sequence analysis. As used herein, the term "infection" has the meaning generally used and understood by persons skilled in the art and includes the invasion and multiplication of a microorganism or particle in or on a host organism (or simply "host") with or without a manifestation of a disease (see, "virulence" above). Infective microorganisms comprise infective prokaryotic and eukaryotic microorganisms (e.g., bacteria, protozoan parasites) as well as infective particles (e.g., viruses, prions, and the like). An infection may occur at one or more sites in or on a host organism and an infection may be unintentional (e.g., unintended ingestion, inhalation, contamination of wounds in a host, etc.) or intentional (e.g., administration of a live vaccine or live vector bacterium to a host). In a vertebrate host organism, such as a mammalian or avian host, a site of infection includes, but is not limited to, a circulatory system, a respiratory system, skin, nails, bone, epithelial tissue, endothelial tissue, musculature, surfaces of body orifices, alimentary canal, an endocrine system, a neural system, an organ, and intercellular spaces. Some degree and form of replication or multiplication of an infective microorganism is required for the microorganism to persist at a site of infection. However, replication may vary widely among infecting microorganisms. Accordingly, replication of an infecting microorganism comprises, but is not limited to, continuous multiplication of microorganisms (progeny); transient or temporary production of microorganisms; single- copy per host cell replication; and even less than single-copy per host cell replication.

Single-copy per host cell replication of a microorganism permits the microorganism or its genome to persist in most cells of a particular tissue or population and to emerge at a later time, as in the case of a latent virus (e.g., herpes simplex, human immunodeficiency virus, etc.). Less than single-copy per host cell replication of an infecting microorganism provides a relatively small reservoir of the infecting microorganism to persist in only a sub-population of susceptible cells or in an intercellular space of a host organism. Whereas "infection" of a host organism by a pathogenic microorganism is undesirable owing to the potential for causing disease in the host, an "infection" of a host organism with a live vaccine or live vector comprising a genetically altered bacterium as described herein is desirable on account of the ability of the bacterium to elicit a protective immune response or to provide a beneficial gene product or function to the host. As used herein, the terms "disease" and "disorder" have the meaning generally known and understood in the art and comprise any abnormal condition in the function or well being of a host organism, including mammalian and avian species. A diagnosis of a particular disease or disorder by a healthcare professional may be made by direct examination and/or consideration of results of one or more diagnostic tests. A "live vaccine", "live bacterial vaccine", and similar terms refer to a composition comprising a bacterium that expresses or otherwise presents at least one antigen of a pathogenic microorganism such that when administered to a host organism the bacterium will elicit an immune response in the host organism against the pathogenic microorganism. Preferably the immune response elicited by administration of the vaccine renders the host protected against disease caused by the pathogenic microorganism. The pathogenic microorganism may be a virulent form of the bacterium of the live vaccine or a different species of pathogenic microorganism. Preferably, when administered to a host organism, a live vaccine comprising a bacterium of the invention provides at least partial protection against a disease caused by a pathogenic microorganism, including but not limited to, amelioration of one or more disease symptoms, fewer disease symptoms, shorter duration of illness, diminution of tissue damage, regeneration of healthy tissue, clearance of pathogenic microorganisms from the host organism, and increased sense of well being by the host organism. Although highly desired, it is understood by persons skilled in this art that no vaccine is expected to induce complete protection from a disease in every host organism that is administered a vaccine. Furthermore, it is understood that a live vaccine comprising a bacterium of the invention may be, at the discretion of a healthcare professional, administered to an individual host organism that is at risk of infection, is suspected of being infected, or is known to already have been infected with a particular pathogenic microorganism. In the following sections, the term "recombinant" is used to describe non-naturally altered or manipulated nucleic acids, host cells transfected with exogenous nucleic acids, or polypeptides expressed non-naturally, through manipulation of isolated nucleic acids, especially DNA, and transformation of host cells. Recombinant is a term that specifically encompasses nucleic acid molecules that have been constructed in vitro using genetic engineering techniques, and use of the term "recombinant" as an adjective to describe a molecule, construct, vector, cell, polypeptide, or polynucleotide specifically excludes naturally occurring such molecules, constructs, vectors, cells, polypeptides or polynucleotides. As used herein, the term "salmonella" (plural, "salmonellae") refers to a bacterium that is a serovar of Salmonella enterica. Salmonellae particularly useful in the invention include, but are not limited to, the group of serovars consisting of Salmonella enterica serovar Typhimurium (S. typhimurium), Salmonella enterica serovar Hadar (S. hadar), Salmonella enterica serovar Typhi (S. typhi), Salmonella enterica serovar Paratyphi B (S. paratyphi B), Salmonella enterica serovar Paratyphi C (S. paratyphi C), Salmonella enterica serovar Enteriditis (S. enteriditis), Salmonella enterica serovar Kentucky (S. kentucky), Salmonella enterica serovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonella enterica serovar Muenchen (S. muencheή), Salmonella enterica serovar Anatum (S. anatum), Salmonella enterica serovar Dublin (S. dublin), Salmonella enterica serovar Derby (S. derby), and Salmonella enterica serovar Choleraesuis var. kunzendorf The genomes of salmonellae are so closely related in both gene organization and nucleotide sequence that the same nucleotide sequences from one serovar may be employed in all serovars to carry out various genetic manipulations and in vitro procedures, such as mutagenesis, nucleic acid hybridizations, and PCR in order to produce, analyze, and/or detect attenuated, H₂S-deficient salmonellae of the invention. Thus, relevant mutated sequences in different genetically altered serovars of the invention may be produced or detected using nucleic acid molecules having nucleotide sequences from the genome of a strain of, e.g., S. typhimurium or of S. hadar, which have been genetically altered according to the invention. As used herein, the terms "strain" and "isolate" are synonymous and refer to a particular isolated bacterium and its genetically identical progeny. Actual examples of particular strains of bacteria developed or isolated by human effort are indicated herein by specific numerical and/or lettered designations. The definitions of other terms used herein are those understood and used by persons skilled in the art and/or will be evident to persons skilled in the art from usage in the text. In recent years, a variety of bacteria, and particularly salmonella bacteria, have been developed that are attenuated for pathogenic virulence in a host organism. Such attenuated bacteria have found use as live vaccines against bacterial infections, e.g., against salmonella-mediated infection to treat or prevent disease in a vertebrate subject, such as an avian or mammalian subject (see, e.g., U.S. Patent Nos. 5,389,368; 5,468,485; 5,387,744; 5,424,065; Zhang-Barber et al., Vaccine, 17; 2538-2545 (1999); all incorporated herein by reference). Furthermore, as described herein, an attenuated bacterium of the invention ma be genetically engineered for use as a live bacterial vector to provide a host organism with a polypeptide that is beneficial to the host for a reason other than immunity to a particular disease. As the use of attenuated bacteria, especially attenuated salmonellae, for live vaccines or live vectors increases there is an attendant need to identify such attenuated bacteria from other bacteria (e.g., wild type bacteria) in various biological and environmental samples. Means and methods to identify attenuated live vaccine or vector bacteria permit the identification and tracking of such bacteria in intended host organisms that were administered the bacteria. Furthermore, such means and methods also permit the identification and tracking of such attenuated bacteria that have been dispersed into the environment, including but not limited to, on various environmental surfaces, soil, air, water, and into other species of organisms that were not the originally intended host (e.g., by ingestion of the original host). To address the need to identify bacteria of live vaccines and vectors, the invention provides a genetically altered bacterium deficient in the ability to produce hydrogen sulfide (H₂S) comprising: (a) a first mutation which attenuates virulence of said bacterium toward a host organism, wherein said first mutation does not affect the ability of said bacterium to produce H₂S, and (b) a second mutation, wherein said second mutation renders the bacterium deficient in the ability to produce H₂S.

Attenuation of virulence A genetically altered bacterium useful in the invention is attenuated in virulence so that the bacterium is no longer capable of infecting and causing disease in at least one host organism, which host organism is susceptible to infection and disease by the corresponding virulent (un- attenuated) form of the bacterium. Preferably, the host organism is a vertebrate organism, such as a mammalian or an avian species. Numerous examples of attenuated bacteria and mutations at genetic loci causing attenuation of virulence in bacteria are known in the art. An attenuated bacterium maybe selected from a virulent population of bacteria. Standard tests are available in the art for determining whether a strain of bacteria is attenuated in virulence. Preferably, by using any of a variety of site-specific, mutagenic protocols, a mutation(s) conferring attenuation on a bacterium of the invention is defined down to the particular alteration in nucleotide sequence of a genetic locus. However, the exact identity or location of an attenuating mutation in a bacterium need not be so well characterized for use in the invention provided the mutant bacterium clearly demonstrates an attenuated phenotype that permits its use in an intended host organism. Furthermore, to serve as the "first mutation" of two essential mutations in a bacterium of the invention, an attenuating mutation must not render the bacterium deficient in the ability to produce hydrogen sulfide (H₂S) from thiosulfate (see below). Particularly preferred for the invention is an attenuated bacterium that carries a known attenuating mutation in one or more genetic loci in the bacterium's genome including, without limitation, the following genetic loci which have been well- characterized in the genome of salmonellae: phoP, phoQ, cya, crp, aroA, aroC, aroD, cdt, poxA, rpoS, Mr A, nuoG,pmi, galE,pabA,pts, damA,purB, gua, cadA, rfc, rfb, rfa, ompR., and combinations thereof. It is noted that due to the global control of cyclic AMP (cAMP) regulation on many bacterial genes, operons, and regulons, strains of salmonellae attenuated by mutation of the cya and/or crp genes (e.g, AcyalAcya mutant strains) are also deficient in expression of a sufficient level of thiosulfate reductase from the cAMP -regulated phs operon to produce H₂S, and therefore exhibit an H₂S negative phenotype. According to the invention, a mutation(s) that produces attenuation and an H₂S negative phenotype does not qualify as a "first mutation", but maybe employed as the "second mutation" (see below). In such cases, a genetically altered bacterium of the invention must also comprise a "first mutation", which attenuates virulence independently without negatively affecting H S production (e.g., AphoP). Deficiency in hydrogen sulfide production addition to attenuated virulence, a bacterium of the invention also is genetically marked to exhibit a phenotype that is particularly useful for distinguishing or identifying the bacterium from other non- vaccine bacteria. In particular, a bacterium of the invention is genetically altered with a "second mutation" that renders the bacterium deficient in the ability to produce hydrogen sulfide (H S) by enzymatically reducing thiosulfate under anaerobic conditions as exist in the center of a bacterial colony and in other anaerobic environments. Bacteria that are able to reduce thiosulfate to form H₂S exhibit an "H₂S positive" phenotype and those that are deficient in this ability have an "H₂S negative" phenotype, as readily detected or determined using any of a variety of assays for H S production (see below). In addition, if the nucleotide sequence or genetic arrangement of a mutated genetic locus is known to cause a deficiency in the ability to produce H₂S in a bacterium of the invention, such a mutation(s) may be detected using a standard nucleic acid-based technique, such as a polymerase chain reaction (PCR) protocol or nucleic acid hybridization protocol, which employs a nucleic acid primer or probe to detect the specific mutation(s) in the nucleic acid of a bacterium. Many H₂S positive bacteria express a functional bacterial thiosulfate reductase, which catalyzes the reduction of thiosulfate to H S under anaerobic conditions, such as exists in the center of bacterial colonies or in various other anaerobic environments. Other H₂S positive bacteria may express an enzyme which is not classified as a prototypical bacterial thiosulfate reductase, but which is able to reduce thiosulfate to H₂S and thereby confer the H₂S positive phenotype. Hence, it is understood that at the enzymatic level a genetically altered bacterium of the invention is deficient in H₂S production and exhibits an H₂S negative phenotype owing to a deficiency in the expression of sufficient levels of functional thiosulfate reductase activity compared to H₂S positive bacteria from which it can be readily distinguished as described herein. Preferably, a genetic marker that confers an H₂S negative phenotype on a bacterium of the invention is a mutation in the nucleotide sequence of a bacterial phs operon. In the genome of salmonellae (which are prototypical H₂S positive bacteria) the phs operon comprises six genes (phsA, phsB, phsC, phsD, phsE, phs F), which encode subunits for a functional thiosulfate reductase. For example, the wild type phs A gene specifically encodes the catalytic subunit of thiosulfate reductase. Mutating the phsA gene to prevent expression of a functional thiosulfate reductase prevents the resulting mutant bacterial cell from reducing thiosulfate to H₂S under anaerobic conditions. Bacteria that cannot reduce thiosulfate to H S under anaerobic conditions may be conveniently identified as white bacterial colonies (H₂S negative phenotype) when contacted with or incubated on any solid (e.g., agar, agarose) medium designed to detect H S production (thiosulfate reductase activity), contrast, bacteria that are able to produce a functional thiosulfate reductase produce dark, black, or dark-centered colonies (H S positive phenotype) on the same medium. A bacterium isolated from a white colony may then, as necessary, be subjected to further virulence, biochemical, molecular, and/or genetic analyses to confirm the identity of the particular H₂S negative bacterium. Any medium or test used to detect (i.e., screen for) H₂S production or functional thiosulfate reductase activity in bacteria may be used to distinguish a mutant H₂S negative bacterium of the invention from H₂S positive bacteria. A medium used to test for H₂S production by bacteria is normally employed as a solid diagnostic medium, as in agar or agarose plates, or in a battery of diagnostic media (e.g., API20E bacteriological detection strips, Bio Merieux Nitek). Examples of media useful for screening bacteria for H₂S phenotype include, but are not limited to, Triple Sugar Iron medium (TSI; see, e.g., Difco Manual, 11th edition, (Sparks, Maryland), pp. 521-523), Lysine Iron Agar (LIA; see, e.g., Edwards and Fife, Appl. Microbiol, 9: 478), and XLT4 medium (Miller and Tate, The Maryland Poultryman (April 2-7, 1990)), all available commercially (e.g., from Difco Laboratories, Detroit, Michigan) or readily prepared using published recipes for such bacteriological growth media. Such media may also be used in various modified forms by addition of one or more additional ingredients or alteration of a component to optimize the medium in a particular protocol. For example, Modified Lysine Iron Agar (MLIA, Remel, Lenexa, Kansas) is LIA modified according to Rappold et al., (Appl. Environ. Microbiol, 38: 162-163 (1979) and Bailey et al., Avian Dis., 32: 324-329 (1988)).

Double Modified Lysine Iron Agar (DMLIA) is MLIA medium supplemented with the antibiotic novobiocin. Accordingly, any modified form of a medium described herein may also be used to identify a bacterium of the invention as long as the modified form of the medium retains the property of the original medium to detect H₂S production in bacterial colonies. While the aforementioned examples of media are commonly employed to screen for H₂S production (or lack thereof) in bacterial colonies, it is understood that any other medium may be employed that provides a distinguishing phenotype based on H₂S production (or lack thereof) or the presence (or absence) of thiosulfate reductase activity in bacteria. Samples to be screened for an attenuated, H₂S deficient bacterium according to the invention may be obtained from any source that is suspected or presumed to contain the bacterium, including but not limited to, blood, urine, lymph, cerebrospinal fluid, tissue biopsies, skin samples, swabs from body orifices, feces, mucosal samples, swipes of environmental surfaces, food, air, water, soil, and bacterial cultures. Typically, a sample presumed to contain a particular attenuated bacterium of the invention is applied and dispersed using standard bacteriological technique to the surface of sterile, solid agar or agarose medium (e.g., sterile agar plate) that permits identification of H₂S phenotype in bacterial colonies. The medium with the applied sample is then incubated under the appropriate conditions that normally would permit wild type bacteria to express thiosulfate reductase activity and exhibit an H₂S positive phenotype. In some cases, it may be useful to include a bacterium having known positive and/or negative H S phenotype as a control to compare with the bacteria from a sample in a test for the presence of a bacterium of the invention. Any mutation in the nucleotide sequence of the phs operon (or equivalent genetic locus conferring the ability to produce H₂S) that prevents production of a functional thiosulfate reductase (or equivalent enzyme) maybe used to produce a bacterium of the invention, provided the mutation does not diminish any other trait (e.g., immunogemcity, attenuation, expression of a desired heterologous molecule) that could disqualify the resulting mutant bacterium from use in a particular composition or method of the invention. The kind of mutations that may be used in the invention includes, but is not limited to, a deletion mutation, an insertion mutation, a point mutation, a frame-shift mutation, and combinations thereof. A deletion mutation is particularly preferred for disrupting expression of a functional thiosulfate reductase (or equivalent enzyme) in a mutant bacterium according to the invention because a deletion mutation is particularly refractory to reversion (i.e., has a particularly low reversion frequency).

Mutagenesis for H₂S negative phenotype and attenuation ■ Any of a variety of methods of mutating bacterial genes known in the art may be used to produce a mutation that provides an H₂S negative phenotype. embodiments involving attenuated salmonella strains, the mutation preferably is in the nucleotide sequence of a. phs operon and disrupts expression of a functional thiosulfate reductase. In bacteria that rely on an enzyme that is a functional equivalent of thiosulfate reductase for H₂S production, the preferred mutation disrupts expression of that enzyme. As noted above, mutant H₂S negative bacteria may be readily distinguished from H₂S positive bacteria that carry a non-mutated (wild type) or otherwise functional phs operon (or equivalent genetic locus) by the color of bacterial colonies incubated on (or otherwise contacted with) a solid medium formulated to detect H₂S production. A method of mutagenesis used to produce a bacterium of the invention may be an in vitro method or a combination of in vitro and in vivo methods. Methods for producing specific mutations in a nucleotide sequence of a nucleic acid molecule may include protocols in which a chemically synthesized nucleic acid molecule comprising an altered (mutant) nucleotide sequence is inserted into a plasmid or other nucleic acid cloning molecule or into a chromosome of a bacterial cell. Particularly useful are methods of producing specific mutations employing polymerase chain reaction (PCR). Currently available site-specific methods for mutating a nucleotide sequence of a nucleic acid molecule substantially decrease the time and steps that would otherwise be necessary to produce or select a particular mutant bacterium by non-specific mutagenic methods. Nevertheless, a general or non-specific method of mutagenesis maybe employed to generate a mutant bacterium that exhibits the desired attenuation and H S negative phenotype according to the invention. A variety of non-specific mutagens are available in the art for generating mutations in bacteria and include without limitation, ultraviolet radiation, chemical mutagenesis (such as ethyl methane sulfonate (EMS) treatment), mutagenic phage (such as Mu phage), or mutagenic elements such as insertion sequences or transposons. Although uncharacterized mutations resulting in attenuation and/or an H S negative phenotype obtained using such mutagens may very well be due to a mutation in a known genetic locus (e.g.,phoP for attenuation, phs operon for H₂S negative phenotype), knowledge of the exact genetic alteration for the first and/or second mutations of a bacterium of the invention would provide an unambiguous genotype for identification purposes as well as a better understanding of any possible genetic, physiological, or environmental implications of using such a mutated bacterium in the compositions and methods of the invention. As noted above, particularly preferred for salmonellae of the invention are second mutations that reside in the salmonella phs operon encoding thiosulfate reductase, e.g., in the nucleotide sequence of phs A. Accordingly, it is more preferable, but not required, to employ methods of mutagenesis that generate defined mutations in the nucleotide sequence of a desired genetic locus to produce a bacterium of the invention. In an example of a preferred general protocol, a cloned or synthesized nucleic acid molecule containing aphsA coding sequence is deleted in vitro to remove all or a portion of the nucleotide structural coding sequence for the catalytic subunit of thiosulfate reductase using polymerase chain reaction (PCR) or a limited nuclease digestion. The resulting deleted (mutated) nucleic acid molecule may then be placed in an appropriate nucleic acid vector molecule, which enables the mutated sequence to replace the functional, wild type phs A sequence in the chromosome of a bacterium by a crossover recombination event. Methods for replacing a specific nucleotide sequence present at a genetic locus on a chromosome of a bacterial cell with a modified or mutated nucleotide sequence are well known in the art and include the Cre-lox system of homologous recombination (see, e.g., Sauer, Methods In Enzymol, 225: 890-900 (1993); plasmid pMEG-865 in Example 1, below). Bacteria carrying the deleted phsA gene sequence may then be identified in white colonies (H S negative phenotype) on plates of lysine iron agar or any other medium formulated for the detection of H₂S production in bacterial colonies.

Bacterial species useful in the invention Bacteria that may be mutated to produce a genetically altered bacterium of the invention include salmonellae, i.e., any serovar of Salmonella enterica. A particularly preferred salmonella useful in the invention includes, but is not limited to, a serovar selected from the group consisting of Salmonella enterica such as Salmonella enterica serovar Typhimurium (S. typhimurium), Salmonella enterica serovar Hadar (S. hadar), Salmonella enterica serovar Typhi (S. typhi), Salmonella enterica serovar Paratyphi B (S. paratyphi B), Salmonella enterica serovar Paratyphi C (S. paratyphi C), Salmonella enterica serovar Enteriditis (S. enteriditis), Salmonella enterica serovar Kentucky (S. kentucky), Salmonella enterica serovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S. pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonella enterica serovar Muenchen (S. muenchen), Salmonella enterica serovar

Anatum (S. anatum), Salmonella enterica serovar Dublin (S. dublin), Salmonella enterica serovar Derby (S. derby), and Salmonella enterica serovar Choleraesuis var. kunzendorf Other non-salmonella H₂S producing bacteria may also be used to produce an attenuated, detectable bacterium of the invention. Particularly preferred among non- salmonella bacteria useful in the invention include but are not limited to Wollinella bacteria (e.g., W. succinogenes), Proteus bacteria (e.g. P. vulgaris, P. mirabilis), Edwardsiella bacteria (e.g., E. tarda), Citrobacter bacteria (e.g., C. freundii), Pasteurella bacteria, and Haemophilus bacteria. As noted above, any of a wide variety of standard, non-specific or specific mutagens and methods may be employed to generate mutations to produce a genetically altered bacterium of the invention. In addition, there are also available a variety of reagents and molecular genetic techniques particularly useful for manipulating nucleic acids and genetic loci of the preferred non-salmonella bacteria described herein. Examples of such reagents and techniques include, but are not limited to, nucleic acid electroporation and suicide plasmids for Proteus bacteria (see, e.g., Zhao et al, Infect. Immun., 66: 330-335 (1998); Dufour et al., Mol. Microbiol, 29: 741-751 (1998)), suicide plasmids for Citrobacter bacteria (see, e.g., Schauer et al., Infect. Immun., 61: 4654-4661 (1993)), transposon mutagenesis for Edwardsiella bacteria (see, e.g., Mathew et al., Microbiol, 147: 449-457 (2001)), and deletion mutagensis and suicide plasmids for Pasteurella bacteria (see, e.g., Homchampa et al., Mol. Microbiol, 6: 3585-3593 (1992); Homchampa et al, Vaccine, 15: 203-208 (1997); Hunt et al., Infect. Immun., 69: 3004-3012 (2001)).

Use of attenuated, H₂S negative bacteria of the invention Genetically altered bacteria described herein are particularly useful for providing the live bacterial species in live bacterial vaccines and live bacterial vectors. Such genetically altered bacteria are readily distinguishable from other bacteria in biological and environmental samples. As a live vaccine, a bacterium of the invention maybe administered to a host organism to elicit in the host an immune response to the bacterium to provide immunity to a disease caused by a virulent form the live vaccine bacterium. Alternatively, a bacterium of the invention may be further genetically engineered by standard recombinant DNA techniques to expresses an antigen of a heterologous pathogenic species. In this way, a recombinant bacterium of the invention may be used as a live vaccine against any number of diseases for which an antigen has been cloned and can be genetically engineered for expression in the bacterium. Suitable such antigens may include, for instance, anthrax antigens (especially protective antigen, lethal factor or edema factor, or immunogenic portions thereof, B. anthracis capsular proteins, etc.), plague antigens (e.g., Y. pestis antigens, such as FI or V proteins), Shigella antigens (particularly O-antigen), E.coli antigens (particularly ETEC), hepatitis B and C antigens (e.g., hepatitis B surface antigen), Campylobacter antigens, viral antigens of, e.g., HIV or rotavirus, and many other immunogenic bacterial, parasitic, and viral antigens that have been fully characterized in the art. Such genetic engineering typically comprises a nucleic acid expression vector comprising a nucleotide sequence encoding an antigenic polypeptide operably linked to a promoter that will function in a bacterium of the invention. A variety of nucleic acid expression vectors are available for expressing a heterologous polypeptide in a bacterium, including but not limited to plasmid expression vectors and bacteriophage expression vectors. Also, a variety of promoters are known and available in the art that may be operably linked to nucleotide coding sequence to direct expression of a heterologous polypeptide in a salmonella and/or non-salmonella species described herein. Preferably, a regulated promoter is used in combination with a recombinant expression vector, to permit control over when and to what extent the recombinant polypeptide is expressed in a bacterium of the invention. Such recombinant vectors may be inserted into a bacterium of the invention using any of a variety of methods including but not limited to transformation, electroporation, conjugation, and transfection. As with most vaccines against disease, it is understood by those skilled in the art that a live vaccine of the invention may not provide complete immunity to a disease, nor provide immunity to all individuals of a species of host organisms. Nevertheless, even partial immunity to a disease may be desirable in a host organism, e.g., as evidenced by an amelioration of one or more disease symptoms, shortening the duration of illness, regeneration of tissue, increasing a feeling of well-being in the immunized subject, and the like. Furthermore, vaccines may be desirable if some immunity is provided to even a portion of a population of susceptible host organisms, h addition, it is understood by those skilled in the art that a live vaccine of the invention may be administered to a host organism that already has a particular disease or is at risk of developing the disease as deemed appropriate by a healthcare professional or appropriate regulatory agency. In addition to use in live vaccines as described above, a bacterium of the invention may also be used as a live bacterial vector that expresses a polypeptide that is beneficial to a host organism for a reason other than eliciting immunity to a disease. Such beneficial polypeptides may include but are not limited to enzymes, growth factors, peptide hormones, peptide nutrients, and structural polypeptides (e.g., collagen). As noted above with respect to heterologous antigenic polypeptides, a variety of standard recombinant DNA methods and nucleic acid expression vectors (e.g., plasmid or bacteriophage expression vectors) are available that maybe used to express a polypeptide of interest in a bacterium of the invention. A live bacterial vaccine or live bacterial vector of the invention may comprise a genetically altered bacterium described herein alone or in combination with one or more other component substances, including but not limited to, a physiologically compatible buffer, a therapeutic agent, a nutritional supplement, and an immunostimulatory agent. A physiologically compatible buffer is any aqueous buffering solution that is compatible with the physiology of the intended host organism that will be administered a live vaccine or vector of the invention. Therapeutic agents useful in the live vaccine and vectors of the invention may include but are not limited to other vaccines, antibiotics, anti-cancer drugs, blood-enhancing agents, growth factors, and combinations thereof. Nutritional supplements useful in the invention may include but are not limited to vitamins, minerals, anti-oxidants (e.g., lazaroids), and combinations thereof. Various immunostimulatory agents are known in the art and may be particulalrly useful in live vaccines of the invention. Immunostimulatory agents useful in the invention may include but are not limited to broad range or universal T cell epitopes, interleukins, adjuvants, and combinations thereof. A live bacterial vaccine or live bacterial vector comprising a bacterium described herein may be administered to a host organism by any of a variety of routes, including but not limited to: intravenously, intramuscularly, sub-cutaneously, intravaginally, sub- lingually or buccally, by inhalation, and orally. Oral administration is preferred, particularly for attenuated salmonellae of the invention. Procedures of optimizing compositions and protocols for administering a live vaccine or live vector to a particular host organism are within the skill of the art. Additional aspects of the invention will be further understood and illustrated in the following examples. Specific parameters included in the following examples are intended to illustrate the practice of the invention and various features thereof, and are not presented to in any way limit the scope of the invention. Examples

Example 1. Construction of Salmonella hadar phsA deletion strain MGN4902 Luria Bertani (LB) broth was prepared by standard methods (see, Ausubel et al., Protocols in molecular biology, vol. 1 (Greene Publishing Associates & Wiley

Interscience, New York, 1991)). The recipe used to prepare LB + sucrose medium is basically the recipe for LB medium, which is supplemented with sucrose (5%), but which contains no NaCl. A 1932 base pair (bp) DNA fragment containing a portion of the Salmonella hadar phsA gene was cloned by the polymerase chain reaction (PCR) (Mullis et al., Methods Enzymol, 155: 335-350 (1987)) using primers based on the S. typhimurium DNA sequence (Heinzinger et al, J. Bacteriol, 177: 2813-2820 (1995)). The nucleotide sequence (SEQ ID NOJ) of the S. typhimurium DNA fragment is shown in Figure 1, which also provides the encoded amino acid sequence for thiosulfate reductase (SEQ ID NO:2). The DNA sequences of the primers used in the cloning (underlined nucleotide sequences in Figure 1) were: 5'-GTATGGCGATGGAGATCAC-3' (SEQ LD NO:3) and 5'-CATCGCAGCTGACCACCAG-3' (SEQ LD NO:4). The template DNA was wild type S. hadar strain MGN2453. The PCR fragment was cloned into plasmid pCR-BluntJJ-TOPO, a commercially obtainable cloning vector ( ivitrogen Corporation, Carlsbad, CA) to yield plasmid pMEG-857 (see Figure 2). Plasmid pMEG-857 was digested with restriction enzyme Sspl and re-ligated to yield plasmid pMEG-858. The resulting deletion removed 1,301 bp from thephsA gene, including the start codon. Plasmid pMEG-375 (Figure 2) is a jp/r-dependent suicide plasmid, which encodes resistance to chloramphenicol as a selectable marker and the sacBR operon as a counter-selectable marker (Kaniga et al., Gene, 1090: 137-141 (1991); Reyrat et al., Infect. Immun., 66: 4011-4017 (1998)). P/r-dependent plasmids and the use ofp/r-dependent plasmids for allelic exchange have been described (Miller et al., J. Bacteriol, 170: 2575-2583 (1988)). A 700-bp BamHI-Xbal fragment carrying AphsAl was subcloned from pMEG-858 into plasmid pMEG-375 to yield plasmid pMEG-865. Plasmid pMEG-865 was electroporated into Escherichia coli strain MGN617 (Roland et al., Avian Dis., 43: 429-441 (1999)). E. coli strain MGN617 is a conjugation donor strain, which expresses the pir gene. For routine laboratory maintenance and plasmid production, plasmid pMEG-865 was typically maintained in and isolated from E. coli strain MGN4891, which is not a derivative of strain MGN617, but rather a derivative of MGN026, which in turn is a pir-expressing derivative of the standard laboratory E. coli strain DH5. The construction of a chromosomal deletion of phs A is outlined in Figure 3. The

AphsA mutation was then moved into S. hadar MGN4636, a vaccine strain that is attenuated with a ΔphoP mutation. Strain MGN617 (pMEG-865) was mated with MGN4636. Transconjugants were selected by plating onto LB agar plates containing chloramphenicol (Cm). A chloramphenicol-resistant (Cm¹) isolate was picked and grown in liquid LB media containing Cm and plated onto LB + sucrose plates. The LB + sucrose plates were incubated at room temperature for 36 hours (hr). A sucrose-resistant, Cm-sensitive isolate was chosen and designated MGN4902. Strain MGN4902 was confirmed phenotypically to be AphsA by producing white colonies on lysine iron agar (Difco Laboratories, Detroit, Michigan) and H₂S negative on an API20E diagnostic strip (Bio Merieux Vitek, Hazelwood, MO). The genotype was confirmed by PCR with the same primers used to clone the gene.

Example 2. Evaluation and comparison of AphoP24 and AphoP24 AphsA 1 attenuated strains of Salmonella hadar for use as live avian vaccines in chickens

Fertile eggs from specific-pathogen-free (SPF) chickens obtained from Sunrise Fanns (Catskill, NY), were incubated and hatched in a Humidaire™ incubator/hatcher located in the Department of Biology at Washington University (St. Louis, MO). All chickens used in this study were white leghorns. Chickens were provided food and water ad libitum after day-of-hatch vaccinations. Groups of vaccinated and/or challenged birds were housed in Horsfall isolators. Bacteria were grown in Luria Bertani (LB) broth (Ausubel et al., Protocols in molecular biology, vol. 1 (Greene Publishing Associates & Wiley h terscience, New York, 1991)). Group C specific antisera were obtained from Difco Laboratories (Detroit, Michigan). Strains with mutations inphoP were identified by an agar overlay method (Kier et al., J. Bacteriol, 138: 155-161 (1979)). The phenotype of attenuated bacteria carrying a AphsA was seen as white colonies of the bacteria when grown on plates of Lysine Iron Agar medium (LIA, Difco) or modified forms of LIA medium. Modified Lysine Iron Agar (MLIA, Remel, Lenexa, Kansas) is LIA modified according to Rappold et al., (Appl. Environ. Microbiol, 38: 162-163 (1979) and Bailey et al., Avian Dis., 32: 324-329 (1988)). Double Modified Lysine Iron Agar (DMLIA) is MLIA medium supplemented with 15 mg/1 novobiocin. A standard peptone glycerol medium was prepared by the method of Curtiss and Kelly (Infect. Immun., 55:3035-3043 (1987)) and contains 1% peptone and 5% glycerol. All salmonella bacterial strains used to inoculate birds were grown as follows: Overnight static broth cultures of each strain were inoculated directly from frozen peptone glycerol stocks and grown at 37° C in LB broth. Two ml of the static overnight was used to inoculate 48 ml of pre-warmed LB broth in a 250-ml flask and grown at a shake rate of 200 rpm at 37° C to an optical density of 1 J measured at 600 nm. Cells were concentrated by centrifugation, resuspended in one-half volume of peptone glycerol, and stored at -70° C in 1 ml aliquots. The titer of each bacterial strain was determined by thawing and pooling two or three of the tubes, preparing five independent serial dilution sets, and plating on LB agar. Tubes were thawed and diluted to the desired concentration in buffered saline with gelatin (BSG) (Curtiss UI, J. Bacteriol. , 89 :28-40 (1965) as needed. Exact titers were measured again when vials were thawed and used to inoculate chickens.

Vaccine strain construction S. hadar strain SA970135, a PT10 chicken heart isolate (phage type 10) was provided by Dr. Cornelius Poppe (O.LE. Reference Laboratory for Salmonellosis,

Guelph, Ontario). Strain SA970135 was passaged three times in chicks to yield strain MGN2453. Strain MGN2453 was passaged once on fusaric acid plates (Bochner et al, J. Bacteriol, 243: 926-933 (1980)) to select for the tetracycline sensitive isolate, strain MGN2674. Strain MGN2674 was used for all subsequent genetic manipulations. An attenuated strain of S. hadar containing a deletion mutation in the phoP gene

(AphoP24) was constructed basically as described previously (see, international application PCT/USOl/31606 (WO 02/30457)). The starting material for the construction of a AphoP mutation in S. hadar was plasmid pEG5381, containing the S. typhimurium phoP gene, which was provided by Eduardo Groisman (see, Groisman et al., Proc. Natl. Acad. Sci. USA, 86:7077-7-81 (1989)). To construct the deletion, inverse PCR was performed using pEG5381 as the template with primers 5'-GATCTAAGAAAAAGAGGGTGAGGCAG-3' (SEQ ID NO:5) and 5'-AATTCATGAATAAATTTGCTCGCCATTTT-3' (SEQ ID NO:6). The resulting deletion removes 775 nucleotides of DNA, including the phoP coding region and 100 nucleotides upstream of the start codon. These primers were designed with EcoRI and Bgiπ sites to allow directional insertion of the trpA terminator (Behlau et al., J.BacterioL, 175:4475-4484 (1993)) at the point of deletion. This was accomplished by annealing the following two DNA oligonucleotides:

5'-GATCGCGGCCGCCCGCCTAATGAGCGGGCTTTTTTTGCC-3' (SEQ ID NO:7), and

5'- AATTGGCAAAAAAAGCCCGCCTCATTAGGCGGGGCGGCCGC-3' (SEQ ID

NO: 8), thereby creating the trpA terminator sequence modified to include aNotl site. The inverse PCR product of pEG5381 was digested with EcoRI and BglU, and then ligated to the annealed oligonucleotides, which had single-stranded DNA overhangs compatible with the single-stranded DNA overhangs generated by EcoRI and BglU digestion. The resulting plasmid carried a complete deletion of the phoP gene with the trpA terminator inserted just upstream of the phoQ gene. This construct, designated AphoP24, was subcloned into a ?z>-dependent suicide vector, which encoded resistance to chloramphenicol as a selectable marker and the sacBR operon as a counterselectable marker (Reyrat et al, Infect. Immun., 66:4011-4017 (1998)) yielding plasmid pMEG-368. Plasmid pMEG-368 was electroporated into E. coli strain MGN617 (Roland et al., Avian Dis., 43:429-441 (1999)) to construct strain MGN1358. Strain MGN1358 was conjugated with S. hadar strain MGN2674 as described (Roland et al., id.), except that the antibiotic selection was ampicillin (100 mg/1) instead of tetracycline and counter- selection was done using LB + sucrose (5 %, no NaCl) according to Reyrat et al. (Infect. Immun., 66:4011-4017 (1998)), instead of fusaric acid. The resulting AphoP24 S. hadar strain was designated MGN4636. The AphoP24 mutation was confmned phenotypically by an agar overlay test (Kier et al, J. Bacteriol, 138: 155-161 (1979)) and the chromosomal deletion was confirmed by PCR, using the original cloning primers. Assessment of vaccines in chickens In separate experiments, white leghorn SPF chicks were vaccinated with S. hadar strains MGN4636 (AphoP ) or MGN4902 (AphoP, AphsA ) at a dose of approximately 1 x 10⁸ CFU (colony forming units). Control chicks were mock-vaccinated with buffer only. Five birds from each vaccinated group were necropsied at day 6 (MGN4636) or day 9 (MGN4902) to evaluate colonization. The results from various tissues are shown in Table 1, below, which shows the recovery of vaccine bacterial strains 6 days (MGN4636) or 9 days (MGN4902) post- vaccination from tissues of birds vaccinated with S. hadar strains MGN4636 or MGN4902 (colony forming units/gram of chick; "CFU/g",). Table 1.

- , no vaccine bacteria isolated after enrichment in tetrathionate-hajna broth;

+, vaccine bacteria isolated after enrichment (Roland et al., 1999, Avian Dis., ¥3:429-441) The results for strain MGN4902 are similar to the results seen for strain MGN4636, with the exception of bird 4, in which the spleen and liver were colonized with greater than 1 x 10⁵ colony forming units (CFU) of vaccine. Colonization of these tissues at such a high level was unexpected and probably results from the genetic variability between individual birds. The remaining birds were boosted at 14 days of age with 1.0 x 10 CFU of the same strain used to vaccinate on day 1. All birds were challenged at 28 days of age. Birds vaccinated with MGN4636 were challenged with 7.5 x 10³ CFU S. hadar, and birds vaccinated with MGN4902 were challenged with 8.0 x 10⁴ CFU of wild-type S. hadar. All birds were necropsied 6 days post-challenge to evaluate colonization of the challenge strain. The results (Table 2, below) show that both vaccinated groups showed a significant reduction in cecal colonization by the challenge strain, indicating that the addition of the AphsA mutation did not reduce efficacy. Table 2. Results from birds vaccinated with MGN4636 and MGN4902, and challenged with S. hadar Group Number of birds Cecal counts S.D. (x loglO, CFU/g)^a

Non-vaccinates 23 5.92^B 6.59

Vaccinates 19 2.38^c 2.53 (MGN4636)

Non-vaccinates 20 6.08° 1.62

Vaccinates 20 2.08^e 0.35 (MGN4902) a, for statistical calculations, samples in which no salmonella was detected were assigned a value of 100 CFU. 100 CFU was the limit of detection. b, different from c, P= 0.0012; d, different from e, P= 0.0001. S.D., standard deviation

The above results show that vaccination of chickens with either attenuated salmonella strain MGN4636 (AphoP) or strain MGN4902 (AphoP, AphsA) provided significant protection against challenge with virulent salmonella bacteria. Thus, the genetic mutation used as a marker (AphsA) did not destroy the effectiveness of the attenuated strain.

Example 3. Utility of AphsA as a vaccine marker Nineteen SPF leghorns were inoculated with S. hadar strain MGN4902 (AphoP AphsA), followed by a wild type S. infantis (PhsA⁺). At necropsy, cecal samples were plated onto brilliant green medium (Difco, Detroit, Michigan), which does not permit differentiation between PhsA^~ and PhsA⁺ salmonella bacteria. Six to fourteen isolates were picked from each bird and streaked onto DMLIA medium. On DMLIA medium, PhsA⁺ salmonella bacteria form black colonies (i.e., H S positive phenotype), while PhsA- bacteria form white colonies (i.e., H₂S negative phenotype). The results from this screen are shown in Table 3. Table 3. Identification of AphsA vaccine salmonella bacteria from the ceca of chickens inoculated with both vaccine and wild-t e salmonella bacteria.

Strain MGN4902 (S. hadar) can also be differentiated from the S. infantis by a slide agglutination test (Lam, J. S., and L. M. Mutharia, "Antigen- Antibody Reactions", pp. 118-119, in Methods for General and Molecular Bacteriology, P. Gerhardt, ed. (American Society for Microbiology, Washington, D.C., 1994)). When this test was performed, all of the white colonies were confirmed to be S. hadar and all of the black colonies were confirmed to be S. infantis.

Taken together, the results from Examples 2 and 3 show that mutation of the phsA gene to prevent expression of a functional thiosulfate reductase is a useful genetic marker, which can provide a convenient and readily detectable phenotypic marker for attenuated strains of salmonellae used as live vaccines, and that the AphsA genetic marker does not destroy the effectiveness of the attenuated strain as a vaccine. Example 4. Detection of the AphsA mutation by polymerase chain reaction (PCR) To demonstrate that the AphsA mutation is detectable by the polymerase chain reaction (PCR), the following S. infantis strains were grown: MGN5433 AphoP MGN5504 AphoP AphsA MGN5434 AphoP MGN5507 AphoP AphsA MGN5508 AphoP. Bacterial cultures were grown overnight in LB broth at 37° C. The next day, 100 μl aliquots from each culture were used to prepare DNA, which was subjected to PCR using the following primers: 5'- GTATGGCGATGGAGATCAC-3' (SEQ LD NO:3), and 5'- TTACCAGGTCCGAACGACAGG-3' (SEQ ID NO:9). Conditions for the PCR were as follows: 5 minutes at 95° C, followed by 30 cycles of 30 seconds at 95° C, 30 seconds at 57.9° C and 2 minutes at 72° C. The thirty cycles were followed by an additional 4 minutes at 72° C. Samples were then run on a 0.7% agarose gel as shown in Figure 4. DNA from the bacteria with a wild type copy of phsA yielded a PCR fragment of 1,787 bp (lanes 2 and 5 in Figure 4), while DNA from bacteria with the AphsA deletion yielded a PCR fragment of 486 bp (lanes 3 and 5 in Figure 4). These results show that the AphsA deletion can easily be detected by PCR.

Example 5. Production of additional Aphs salmonellae Other attenuated salmonella bacteria useful in vaccines were marked with a deletion mutation in the phs A gene (AphsA) to provide an easily detected H S negative phenotype as indicated by formation of white bacterial colonies on a medium, such as DMLIA, used to detect H S production in bacteria. All constructions were carried out in the same manner, using the S. hadar phs A sequence. In addition to S. hadar, examples of attenuated salmonella bacteria marked with AphsAl mutation were obtained from S. kentucky and S. typhimurium following the general scheme for producing AphsA mutations as diagrammed in Figure 3. Example 6. Use of the AphsAl marker to identify salmonella PhoP vaccines from samples collected from poultry environment

Two groups of white leghorn chickens were reared on built-up litter in closed chambers and administered one of two live salmonella vaccine strains, each strain carrying a AphsAl marker. The standard bedding for rearing broilers was prepared by applying approximately 2-3 inches of wood shavings on top of used litter derived from non-treated 46-week-old hens. Chickens were placed in the rooms to simulate a colony house where flock density is approximately 0.8 ft² per bird. Table 4, below, describes the characteristics of each strain of live salmonella vaccine used in this study. Each vial of salmonella vaccine was diluted to achieve 10⁷ CFU/0J ml and administered by coarse spray at less than one day of age. The same dosage of each of strain of salmonella vaccine was administered in the drinking water at 14 days of age in approximately 10 ml.

Table 4. Vaccine strain descriptions

Vaccine organisms were detected in the litter 3 days post spray vaccination. The litter used in this study was derived from the manure pits of 46-week old hens and was heavily colonized with bacteria. XLT4 medium was used to differentiate H₂S negative phenotype (H₂S^~) of vaccine isolates from the H₂S positive phenotype (H₂S⁺) of native flora. H₂S negative phenotype isolates were confirmed to be vaccine bacteria by slide agglutination with antisera to O antigen Group B and C₃. Example 7. Use of the AphsAl marker in attenuated S. typhimurium PhoP^" live vaccines for isolation and differentiation from H₂S producing (H₂S+) salmonellae The original protocol of this study called for two sets of eggs to be set in an incubator/hatcher and to receive an injection of one of two vaccines as described in Table 5, below, on the day 18 of incubation. On the day 21 of incubation, birds were to be placed in separate isolators and subsequently vaccinated with the same vaccine at 14 days of age. Table 5. Vaccine strain descriptions

H₂S , H₂S negative phenotype; H₂S , H ₂S positive phenotype

The birds were euthanized and ceca removed for bacterial culture analysis. Samples were plated onto XLT4 medium. After overnight incubation at 37° C, both white and black colonies arose from both sets of birds. It was later determined that cross- infection had occurred in the hatcher and that the birds in both treatment groups were co- colonized with both MGN-5501 and MGN-738 attenuated salmonella strains. Nevertheless, the H₂S negative strain MGN-5501 was easily differentiated from the H S positive strain MGN-738 using this plating technique. Slide agglutination with anti-sera to O antigen group B was used to confirm presence of the H₂S negative and H₂S positive vaccine strains in cecal samples. The results indicated that of the sixteen birds originally vaccinated with the H₂S positive strain MGN-738 in Group 2, eleven had been co- colonized with the H₂S negative strain MGN-5501. Likewise, the results indicated that birds originally vaccinated with H₂S negative strain MGN-5501 in Group 1 were found to be co-colonized with the H₂S positive strain MGN-738. As further support of the invention, the following strains were deposited under the Budapest Treaty with the American Type Culture Collection (Manassas, Virginia, U.S.A.) on October 25, 2002: Salmonella hadar strain MGN4902 (ATCC accession No. PTA-4777), as described above, is an attenuated, hydrogen sulfide negative salmonella that carries the AphoP24 AphsAl mutations, and Escherichia coli strain MGN4891 (ATCC accession No. PTA-4776), as described above, is a pir-expressing strain, which carries plasmid pMEG-865, which in turn carries the AphsAl mutation.

All patents, applications, and publications cited in the above text are incorporated herein by reference.

Other variations and embodiments of the invention described herein will now be apparent to those skilled in the art and may be produced and practiced without exceeding the full scope of the invention as defined by the claims that follow.

Claims

CLAIMS:

1. A genetically altered bacterium deficient in the ability to produce hydrogen sulfide (H₂S) comprising: (a) a first mutation which attenuates virulence of said bacterium toward a host organism, wherein said first mutation does not affect the ability of said bacterium to produce H₂S, and (b) a second mutation, wherein said second mutation renders the bacterium deficient in the ability to produce H₂S.

2. The genetically altered bacterium according to Claim 1, wherein said first and second mutations are, independently, mutations selected from the group consisting of a deletion mutation, a point mutation, an insertion mutation, a frame-shift mutation, and combinations thereof.

3. The genetically altered bacterium according to Claim 1, wherein said bacterium is a salmonella.

4. The genetically altered bacterium according to Claim 3, wherein said salmonella is a serovar selected from the group consisting of Salmonella enterica serovar Typhimurium (S. typhimurium), Salmonella enterica serovar Hadar (S. hadar), Salmonella enterica serovar Typhi (S. typhi), Salmonella enterica serovar Paratyphi B (S. paratyphi B), Salmonella enterica serovar Paratyphi C (S paratyphi C), Salmonella enterica serovar Enteriditis (S. enteriditis), Salmonella enterica serovar Kentucky (S. kentucky), Salmonella enterica serovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S. pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonella enterica serovar Muenchen (S. muenchen), Salmonella enterica serovar Anatum (S. anatum), Salmonella enterica serovar Dublin (S. dublin), Salmonella enterica serovar Derby (S. derby), and Salmonella enterica serovar Choleraesuis var. kunzendorf

5. The genetically altered bacterium according to Claim 3, wherein said first mutation is a mutation in a salmonella genetic locus selected from the group consisting of phoP,phoQ, aroA, cdt,poxA, rpoS, htrA, nuoG,pmi, galE,pabA,pts, damA,purB, gua, cadA, rfc, rfb, rfa, ompR, and combinations thereof.

6. The genetically altered bacterium according to Claim 3, wherein said second mutation is a mutation in a salmonella genetic locus selected from the group consisting of phsA, phsB, phsC, phsD, phsE, phsF, and combinations thereof.

7. The genetically altered bacterium according to Claim 6, wherein said second mutation is a mutation in phs A.

8. The genetically altered bacterium according to Claim 7, wherein said mutation in phs A is a deletion mutation (AphsA).

9. The genetically altered bacterium according Claim 3, wherein said second mutation is a mutation in a salmonella genetic locus selected from the group consisting of cya, crp, and combinations thereof.

10. The genetically altered bacterium according to Claim 9, wherein said second mutation is a deletion mutation.

11. The genetically altered bacterium according to Claim 3, wherein said salmonella is S. hadar, said first mutation is a AphoP, and said second mutation is a AphsA.

12. The genetically altered bacterium according to Claim 11, wherein said genetically altered bacterium is S. hαdαr strain MGN4902 (ATCC accession No. PTA-4777).

13. The genetically altered bacterium according to Claim 3, wherein said salmonella is S. typhimurium UK-1, said first mutation is a AphoPQ, and said second mutation is a AphsA.

14. The genetically altered bacterium according to Claim 3, wherein said salmonella is S. kentucky, said first mutation is a AphoP, and said second mutation is a AphsA.

15. The genetically altered bacterium according to Claim 3, wherein said salmonella is S. infantis, said first mutation is a AphoP, and said second mutation is a AphsA.

16. The genetically altered bacterium according to Claim 1, wherein said bacterium is a species of bacteria selected from the group consisting of species of Wollinella, Proteus, Edwardsiella, Citrobacter, Pasteurella, and Haemophilus.

17. The genetically altered bacterium according to Claim 16, wherein said bacterium is a species of Wollinella.

18. The genetically altered bacterium according to Claim 17, wherein said species of Wollinella is W. succinogenes

19. The genetically altered bacterium according to Claim 16, wherein said bacterium is a species of Edwardsiella bacteria.

20. The genetically altered bacterium according to Claim 19, wherein said species of Edwardsiella is E. tarda.

21. The genetically altered bacterium according to Claim 16, wherein said bacterium is a species of Proteus.

22. The genetically altered bacterium according to Claim 22, wherein said species of Proteus is selected from the group consisting of P. vulgaris and P. mirabilis.

23. The genetically altered bacterium according to Claim 16, wherein said bacterium is a species of Citrobacter.

24. The genetically altered bacterium according to Claim 23, wherein said species of Citrobacter is C. freundii.

25. The genetically altered bacterium according to Claim 1, further comprising a recombinant nucleic acid sequence coding for a heterologous polypeptide, wherein said coding sequence is operably linked to a promoter that permits expression of said heterologous polypeptide in said bacterium.

I

26. The genetically altered bacterium according to Claim 25, wherein said heterologous polypeptide is an antigen of a pathogenic organism.

27. The genetically altered bacterium according to Claim 25, wherein said heterologous polypeptide is a polypeptide other than an antigen of a pathogenic organism.

28. The plasmid pMEG-865.

29. A bacterium harboring plasmid pMEG-865.

30. The bacterium according to Claim 29, wherein said bacterium is a salmonella or Escherichia coli.

31. The bacterium according to Claim 30, wherein said bacterium is Escherichia coli strain MGN4891 (ATCC accession No. PTA-4776).

32. A genetically altered bacterium comprising: (a) first mutation which attenuates virulence of said bacterium toward a host organism, wherein said first mutation does not affect the ability of said bacterium to produce H₂S, and (b) a second mutation, wherein said second mutation renders the bacterium deficient in the ability to produce H₂S; and (c) a recombinant expression vector comprising a nucleotide sequence coding for a heterologous polypeptide operably linked to a promoter that permits expression of said heterologous polypeptide in said genetically altered bacterium.

33. A therapeutic composition comprising a bacterium according to Claim 32, and an additional component selected from the group consisting of a physiologically compatible buffer, a therapeutic agent, a nutritional supplement, an immunostimulatory agent, and combinations thereof.

34. The composition according to Claim 33, wherein said bacterium is a salmonella.

35. The composition according to Claim 34, wherein said salmonella is a serovar selected from the group consisting of Salmonella enterica serovar Typhimurium (S. typhimurium), Salmonella enterica serovar Hadar (S. hadar), Salmonella enterica serovar Typhi (S. typhi), Salmonella enterica serovar Paratyphi B (S. paratyphi B), Salmonella enterica serovar Paratyphi C (S. paratyphi C), Salmonella enterica serovar Enteriditis (S enteriditis), Salmonella enterica serovar Kentucky (S. kentucky), Salmonella enterica serovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S. pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonella enterica serovar Muenchen (S. muenchen), Salmonella enterica serovar Anatum (S. anatum), Salmonella enterica serovar Dublin (S. dublin), Salmonella enterica serovar Derby (S. derby), and Salmonella enterica serovar Choleraesuis var. kunzendorf.

36. The composition according to Claim 34, wherein said first mutation is a mutation in a salmonella genetic locus selected from the group consisting of phoP,phoQ, aroA, cdt,poxA, rpoS, htrA, nuoG,pmi, galE,pabA,pts, damA,purB, gua, cadA, rfc, rfb, rfa, ompR, and combinations thereof.

37. The composition according to Claim 36, wherein said second mutation is a mutation in a salmonella genetic locus selected from the group consisting of phsA,phsB, ph C, phsD, phsE, phsF, and combinations thereof.

38. The composition according to Claim 37, wherein said second mutation is a mutation in phs A.

39. The composition according to Claim 38, wherein said mutation inphsA is a deletion mutation (AphsA).

40. The composition according to Claim 34, wherein said second mutation is a mutation in a salmonella genetic locus selected from the group consisting of cya, crp, and combinations thereof.

41. The composition according to Claim 40,' wherein said second mutation is a deletion mutation.

42. The composition according to Claim 34, wherein said salmonella is S. hadar, said first mutation is a AphoP, and said second mutation is a AphsA.

43. The composition according to Claim 34, wherein said genetically altered bacterium of said composition has the characteristic features of S. hadar strain MGN4902 (ATCC Accession No. PTA-4777).

44. The composition according to Claim 34, wherein said salmonella is S. typhimurium UK-1, said first mutation is a AphoPQ, and said second mutation is a AphsA.

45. The composition according to Claim 34, wherein said salmonella bacterium is S. kentucky, said first mutation is a AphoP, and said second mutation is a AphsA.

46. The composition according to Claim 34, wherein said salmonella bacterium is S. infantis, said first mutation is a AphoP, and said second mutation is a AphsA.

47. The composition according to Claim 33, wherein said genetically altered bacterium is a species of bacteria selected from the group consisting of species of Wollinella, Proteus, Edwardsiella, Citrobacter, Pasteurella, and Haemophilus.

48. A method for making a genetically altered salmonella deficient in the ability to produce hydrogen sulfide (H₂S) and attenuated for virulence comprising: (a) introducing into a salmonella a first mutation that attenuates virulence of said salmonella toward a host organism, wherein said first mutation does not affect the ability of said salmonella to produce H₂S, (b) introducing plasmid pMEG-865 into said salmonella to insert a AphsAl mutation into the chromosome of said salmonella to render said salmonella deficient in the ability to produce H₂S, and (c) identifying said salmonella from step (b) as carrying said AphsAl mutation.

49. The method according to Claim 48, wherein step (c) is carried out by screening for an H₂S-deficient phenotype of said salmonella from step (b) by contacting said salmonella from step (b) with a medium used to detect H₂S production.

50. The method according to Claim 48, wherein step (c) is carried out by identifying the nucleotide sequence for the AphsAl mutation in the chromosome of said salmonella

51. The method according to Claim 48, wherein step (c) is carried out by detecting a deficiency in thiosulfate reductase activity in said salmonella from step (b).

52. A method for eliciting an immune response to a pathogenic organism in a host organism comprising: administering to a host organism a genetically altered bacterium deficient in the ability to produce hydrogen sulfide (H₂S) comprising: (a) a first mutation which attenuates virulence of said bacterium toward a host organism, wherein said first mutation does not affect the ability of said bacterium to produce H₂S, and (b) a second mutation, wherein said second mutation renders the bacterium deficient in the ability to produce H₂S, wherein said genetically altered bacterium expresses an antigen of a pathogenic organism such that administration of said genetically altered bacterium expressing said antigen elicits an immune response in said host organism that is directed toward said expressed antigen and toward said pathogenic organism.

53. The method according to Claim 52, wherein said host organism is a mammalian species or an avian species.

54. The method according to Claim 52, wherein said pathogenic organism is a virulent form of said genetically altered bacterium.

55. The method according to Claim 52, wherein said pathogenic organism is a species that is different from said genetically altered bacterium.

56. The method according to Claim 52, wherein said bacterium is a salmonella.

57. The method according to Claim 56, wherein said salmonella is a serovar selected from the group consisting of Salmonella enterica serovar Typhimurium (S. typhimurium), Salmonella enterica serovar Hadar (S. hadar), Salmonella enterica serovar Typhi (S. typhi), Salmonella enterica serovar Paratyphi B (S. paratyphi B), Salmonella enterica serovar Paratyphi C (S. paratyphi C), Salmonella enterica serovar Enteriditis (S. enteriditis), Salmonella enterica serovar Kentucky (S. kentucky), Salmonella enterica serovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S. pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonella enterica serovar Muenchen (S. muenchen), Salmonella enterica serovar Anatum (S. anatum), Salmonella enterica serovar Dublin (S. dubli ), Salmonella enterica serovar Derby (S. derby), and Salmonella enterica serovar Choleraesuis var. kunzendorf.

58. The method according to Claim 56, wherein said first mutation is a mutation in a salmonella genetic locus selected from the group consisting of phoP, phoQ, aroA, cdt, poxA, rpoS, htrA, nuoG,pmi, galE,pabA,pts, damA,purB, gua, cadA, rfc, rfb, rfa, ompR, and combinations thereof.

59. The method according to Claim 58, wherein said second mutation is in a mutation in a salmonella genetic locus selected from the group consisting of phs A, phsB ', phsC, phsD, phsE, phsF, and combinations thereof.

60. The method according to Claim 56, wherein said salmonella is S. hadar, said first mutation is a AphoP, and said second mutation is a AphsA.

61. The method according to Claim 56, wherein said genetically altered bacterium of said method has the characteristic features of S. hadar strain MGN4902 (ATCC accession

No. PTA-4777).

62. The method according to Claim 56, wherein said salmonella is S. typhimurium UK-1, said first mutation is a AphoPQ, and said second mutation is a AphsA.

63. The method according to Claim 56, wherein said salmonella is S. kentucky, said first mutation is a AphoP, and said second mutation is a AphsA.

64. The method according to Claim 56, wherein said salmonella is S. infantis, said first mutation is a AphoP, and said second mutation is a AphsA.

65. The method according to Claim 52, wherein said genetically altered bacterium is a species of bacteria selected from the group consisting of species of Wollinella, Proteus, Edwardsiella, Citrobacter, Pasteurella, and Haemophilus.

66. A method for providing a polypeptide to a host organism comprising: administering to a host organism a genetically altered bacterium deficient in the ability to produce hydrogen sulfide (H₂S) comprising: (a) a first mutation which attenuates virulence of said bacterium toward a host organism, wherein said first mutation does not affect the ability of said bacterium to produce H₂S, and (b) a second mutation, wherein said second mutation renders the bacterium deficient in the ability to produce H₂S, wherein said genetically altered bacterium expresses a polypeptide.

67. The method according to Claim 66, wherein said host organism is a mammalian species or an avian species.

68. The method according to Claim 66, wherein said bacterium is a salmonella.

69. The method according to Claim 68, wherein said salmonella is a serovar selected from the group consisting of Salmonella enterica serovar Typhimurium (S. typhimurium), Salmonella enterica serovar Hadar (S. hadar), Salmonella enterica serovar Typhi (S. typhi), Salmonella enterica serovar Paratyphi B (S. paratyphi B), Salmonella enterica serovar Paratyphi C (S. paratyphi C), Salmonella enterica serovar Enteriditis (S. enteriditis), Salmonella enterica serovar Kentucky (S. kentucky), Salmonella enterica serovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S. pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonella enterica serovar Muenchen (S. muenchen), Salmonella enterica serovar Anatum (S. anatum), Salmonella enterica serovar Dublin (S. dubliή), Salmonella enterica serovar Derby (S. derby), and Salmonella enterica serovar Choleraesuis var. kunzendorf.

70. The method according to Claim 68, wherein said first mutation is a mutation in a salmonella genetic locus selected from the group consisting of phoP, phoQ, aroA, cdt, poxA, rpoS, htrA, nuoG,pmi, galE,pάbA,pts, damA,purB, gua, cadA, rfc, rβ, rfa, ompR, and combinations thereof.

71. The method according to Claim 70, wherein said second mutation is in a mutation in a salmonella genetic locus selected from the group consisting of phsA, phsB, phsC, phsD, phsE, phsF, and combinations thereof.

72. The method according to Claim 66, wherein said genetically altered bacterium is a species of bacteria selected from the group consisting of species of Wollinella, Proteus, Edwardsiella, Citrobacter, Pasteurella, and Haemophilus.