**7. Vaccination for control of** *Salmonella* **in poultry**

Killed whole-cell bacterins and live attenuated vaccines are the most common types of vaccines currently used in the poultry industry. Vaccination programs depend on the recognition of specific antigens, called epitopes, by the immune system of the host to prevent or reduce the spread of pathogenic viruses and bacteria. Because there are a large

Higgins and co-workers (2005) successfully treated turkey carcasses at a processing facility with bacteriophages specific to the *Salmonella* to which they were infected. This process was effective when either an autogenous bacteriophage treatment targeted to the specific *Salmonella* strain infecting the turkeys was used, or a cocktail of nine wide host-range *Salmonella*-targeting bacteriophage were used. Similarly, a bacteriophage treatment for cattle carcass contamination has been effective at reducing the *E. coli* 0157:H7 load at processing has been developed and commercially licensed in the United States. These successes avoid development of bacteriophage resistance by applying treatment at a single point during production, in an environment where proliferation of the target organism is extremely limited. In this way, since the target organism is never intentionally exposed twice to the same treatment, resistance is unlikely to ever increase beyond the naturally-occurring

One of the most well documented successes of published treatment of enteric Enterobacteriaceae infections with bacteriophages was the study of Smith and Huggins (1983) as described above. It is notable that in this successful study, the bacteriophage cocktail used was a combination of two bacteriophages, but the second was isolated using the target organism which was resistant to the first bacteriophage. This approach of selecting for bacteriophage isolates using target bacteria that are resistant to sequential bacteriophage treatments was not used in the work of Higgins et al (2007), or in several other published studies. Higgins and co-workers (2007) used a collection bacteriophages, independently isolated from different sources and with several different plaque morphologies, suggesting that a number of different bacteriophages were employed – and

It is possible that one of the most notable exceptions to the many failures to treat enteric Enterobacteraceae infections during recent years, that of Smith and Huggins (1983), provides a singular clue as to the potential for enhancing the likelihood of enteric Enterobacteriaceae efficacy. It is possible that selection of multiple bacteriophages for the same target cell phenotype results in selection of bacteriophages that are effective through identical mechanisms of adhesion, penetration, replication, and release. When new bacteriophages are isolated for efficacy against sequentially resistant isolates of the target bacteria, and these are combined for administration as a cocktail, the ability of the target cell to shift phenotype may be severely limited, resulting in a much larger proportion of target

Clearly, widespread bacteriophage treatments with Enterobacteriaceae have not been adopted for any animal species during the last 60 years and successful research in this area has been modest and sporadic. Nevertheless, the occasional reports by reputable scientists in solid journals must indicate that there is potential for improved therapeutic efficacy of bacteriophages for this purpose. With the diminution of new antimicrobial pharmaceuticals and the widespread resistance among many pathogenic enteric Enterobacteriaceaes, a

Killed whole-cell bacterins and live attenuated vaccines are the most common types of vaccines currently used in the poultry industry. Vaccination programs depend on the recognition of specific antigens, called epitopes, by the immune system of the host to prevent or reduce the spread of pathogenic viruses and bacteria. Because there are a large

cell reduction, thereby increasing the probability of elimination or cure.

resistance to the bacteriophage (or cocktail) used.

failed to persistently reduce enteric colonization.

breakthrough in this area is sorely needed.

**7. Vaccination for control of** *Salmonella* **in poultry** 

number of *Salmonella* serovars, each with individual epitopes that do not elicit crossprotection against other serovars, there has been little traditional emphasis on development of generic *Salmonella* vaccines. Primarily, killed vaccines, which generally must be administered parenterally (through injection), have been applied to protect against systemic infections, and although they have been shown to reduce colonization and shedding, the protection provided by these vaccines has limited ability to stop intestinal colonization. They predominantly stimulate both humoral (circulating IgM and IgG) and cell-mediated responses, but are quite ineffective at generating mucosal immunity as secretory IgA antibody stimulation is very low through this type of vaccination. This is important because, whereas both systemic (humoral and cell-mediated) and mucosal immunity can reduce the chances of disease and mortality, only the mucosal portion of this adaptive immune response is capable of protecting animals from infection. The key to inducing both an adaptive systemic and mucosal response has traditionally been through the use of the mucosa as a "portal of entry" for live but weakened (attenuated) vaccines. However, the use of such vaccines for protection against *Salmonella* infection have been tremendously limited due to the very large number of different antigens presented by the more than 200 serotypes that can infect domestic animals and man, with more than 38 of these commonly infecting poultry within the United States, as discussed below (Hargis et al., 2010).

One approach to solving the problem of serotype variation among the common paratyphoid strains of *Salmonella*, which are often not a disease-causing problem for poultry but rather create a source of foodborne illness for consumers, is the identification of "universal epitopes" that are shared among all *Salmonella* isolates. This concept has been established for a number of pathogens and is based on the identification of a minor surface structure (antigen or epitope) which does not cause robust immune reaction during infection, but which can be targeted for protection if the antigen is presented in a way that tricks the animal into responding robustly. Some of these are relatively minor antigens which are highly conserved among related organisms – usually because they involve biological function. Since small peptide sequences that are biologically functional cannot vary in sequence, organisms that carry a mutation for such sequences are often either lethal or sufficiently detrimental to cause these to not be successful over time (Neirynck et al., 1999).

A well-described example of this phenomenon is a small 23 amino acid peptide on the surface of Type A Influenza viruses named M2e. This peptide is part of an ion transport channel which is necessary for viral activation. Mutations in this sequence undoubtedly occur frequently, but since the 1918 Spanish Influenza outbreak, all Type A Influenza isolates share a highly conserved core sequence for this peptide (Layton et al., 2009). Although natural influenza infection does not result in a robust immune response to this peptide sequence, tricking the animal into producing a robust response has resulted in protective immunity in several animal species (Neirynck et al., 1999; Mozdzanowska et al., 2003; Fiers et al., 2004; Zou et al., 2004). In recent years, the rapid increase in molecular biological techniques has led to the development of more sophisticated vaccines, of which live recombinant bacterial vectored vaccines are one of the most promising (Ashby et al., 2005; Zhang et al., 2006; Duc et al., 2007; Kajikawa et al., 2007; Uyen et al., 2007; Yang et al., 2007; Huang et al., 2008; Liu et al., 2008; Ceragioli et al., 2009; Deguchi et al., 2009).

This type of vaccine uses a genetically modified bacterium to express a heterologous antigen. Oral live attenuated *Salmonella* vaccine vectors expressing recombinant foreign antigens have previously been shown to stimulate systemic, mucosal, humoral, and cellmediated immune responses against *Salmonella* (Mollenkopf et al., 2001; Koton and

Alternative Strategies for *Salmonella* Control in Poultry 271

mutants have been proposed (Zhang-Barber et al., 1999; Sydenham et al., 2000). Day-ofhatch chicks vaccinated with this type of attenuated *Salmonella* vaccine have been shown to have serological protection to homologous and heterologous *Salmonella* serotypes, possibly through a mechanism similar to competitive exclusion (Hassan and Curtiss, 1994; Hassan and Curtiss, 1997; Dueger et al., 2003; Holt et al., 2003; Bohez et al., 2008). Furthermore, maternal antibodies can be demonstrated in eggs and chicks from breeders vaccinated with this vaccine. These antibodies were reported to reduce *Salmonella*e colonization and to provide protection to laying hens up to 11 months post-inoculation (Hassan and Curtiss, 1997). However, susceptibility to antimicrobial agents commonly used in poultry production can reduce or eliminate the efficacy of live vaccines, and these vaccines are

Autogenous vaccines provide for yet another mechanism for vaccinating poultry. In many (but not all) countries, there are regulatory provisions under certain circumstances for production of specific killed vaccines using the specific isolate plaguing a given poultry flock or complex. These "autogenous" *Salmonella* isolates are typically grown, killed and mixed with an adjuvant (a chemical that potentiates the immune response) for parenteral administration. Some veterinarians associated with valuable breeder flocks believe that these vaccines are highly preferred for vaccination against endemic and common serotypes

Taken together, there are tremendous future opportunities for manipulating the acquired immune response, particularly the mucosal secretory IgA response, for reduceing *Salmonella*  infections in poultry. However, current vaccine availability is limited and progress is greatly needed on two fronts: 1) improving mucosal immune responses for *Salmonella* vaccines; and 2) targeting shared protective epitopes for broad-spectrum serotype coverage for the paratyphoid *Salmonella*e that currently plague poultry producers world-wide. Currentlyavailable commercial vaccines are enjoying significant popularity due to the intense regulatory pressures facing meat and egg producing poultry, although applications are

generally limited to breeder or layer flocks except under intense regulatory pressure.

The interest in digestive physiology and the role of microorganisms has generated data whereby human and animal well-being can be enhanced and the risk of disease reduced. New molecular techniques that allow an accurate assessment of the flora composition, resulting in improved strategies for elucidating mechanisms. Given the recent international legislation and domestic consumer pressures to withdraw growth-promoting antibiotics and limit antibiotics available for treatment of bacterial infections, probiotics can offer alternative options. New advances in the application of probiotics, are directed to produce significant changes in gut physiology and provide even higher levels of health as well as increase

Metchnikoff founded the research field of probiotics, aimed at modulating the intestinal microflora (Dobrogosz, Peacock, & Hassan, 2010; Schmalstieg & Goldman, 2010; Weissmann, 2010). However, other parts of the body containing endogenous microflora or problems relating to the immune system may also be candidates for probiotic therapy. Research has shown that probiotics have potential for human health issues such as: vaginal candidiasis (Ehrstrom et al., 2010; Ya, Reifer, & Miller, 2010); dental caries (Chen & Wang, 2010; Stamatova & Meurman, 2009); allergies (Gourbeyre, Denery, & Bodinier, 2011; Schiavi, Barletta, Butteroni,

subject to the serotype limitations as discussed above.

for which no commercial vaccine exists.

performance parameters in poultry.

**8. Conclusions** 

Hohmann, 2004; Ashby et al., 2005). *Salmonella* vectors have the potential advantage of being extremely inexpensive to manufacture and, because they do not have to be injected and can be administered by spray or drinking water, they are much more acceptable for widespread administration to commercial poultry.

Currently, some laboratories are exploiting this concept by identifying candidate antigens/epitopes that are evolutionarily conserved between the many different serotypes of *Salmonella* and which do not elicit a robust response when animals are infected with wild type *Salmonella* (or vaccinated with conventional vaccines), but which may protect against infection when delivered in an appropriate way using a recombinant vaccine platform (Wolfenden, RE et al., 2010; Kremer et al., 2011). Recently, bacterial carriers of antigens (vectors), including *Salmonella* Enteritidis and *Bacillus subtilis*, have been manipulated to express protein antigens to protect against bacterial, viral, and protozoal pathogens (Layton et al., 2009; O'Meara et al., 2010; Kremer et al., 2011; Layton et al., 2011). These vaccines have an advantage over many other types of vaccines in that they are able to be delivered directly to a mucosal surface via nasal, ocular, or oral administration. Because most pathogens invade the host through a mucosal surface, an enhanced mucosal immune response is the only portion of acquired immunity that can markedly reduce the probability of an animal or flock to become infected, as discussed above. While prevention of morbidity and mortality alone are useful traits of conventional vaccines for most poultry disease-causing agents, in the case of the common *Salmonella* serotypes which cause foodborne illness, these isolates generally cause little or no disease in the animals. Thus, recombinant vaccines that are able to provide wide-range protection against common *Salmonella* serotypes of poultry, by mucosal presentation, may be a critical component for controlling this problem in the next few years.

Along with presentation of conserved antigens through mucosally-administered recombinant vaccines, there is a need to trick the immune system of the animal to respond robustly to these recombinant bacteria that are not capable of infecting or causing disease. Co-expression of molecules that may enhance the immune response or may be recognized by receptors located on the mucosal surface of the gastrointestinal tract is a promising area of work. Several such molecules may enhance the response to these recombinant vaccines (Layton et al., 2009; O'Meara et al., 2010; Wolfenden et al., 2010).

Presently, there are no broad-spectrum recombinant vaccines approved for use in agricultural animals to protect against the wide range of serotypes which plague poultry producers worldwide. Specific serotype vaccines, such as *S.* Enteritidis or *S.* Gallinarum, have gained considerable acceptance in countries with endemic problems with these more devastating serovars, particularly in breeders and table egg production chickens (see Shivaprasad, 1997, for a review). These vaccines generally do not provide robust protection against infection with even the identical serotype, and even less protection against heterologous serotypes (Hargis et al., 2010). However, there is a general consensus that some protection is provided and for valuable birds, these vaccines may offer a much-needed modicum of protection, though often through reduced persistence and shedding of the organism, thus limiting spread. For example, studies have shown that oil emulsion *Salmonella* Enteritidis bacterins administered to breeders caused a three log10 cfu/g cecal content reduction in recovery from progeny chicks (Inoue et al., 2008), and a two log10 cfu/g cecal content reduction in breeders after molting (Nakamura et al., 2004). Thus, these vaccines have value at the present time, especially for breeders and at-risk laying hens.

Live-type vaccines with gene deletions assuring avirulence while allowing immunogenicity have been reported (Curtiss and Kelly, 1987; Dueger et al., 2003), and other specific deletion

Hohmann, 2004; Ashby et al., 2005). *Salmonella* vectors have the potential advantage of being extremely inexpensive to manufacture and, because they do not have to be injected and can be administered by spray or drinking water, they are much more acceptable for widespread

Currently, some laboratories are exploiting this concept by identifying candidate antigens/epitopes that are evolutionarily conserved between the many different serotypes of *Salmonella* and which do not elicit a robust response when animals are infected with wild type *Salmonella* (or vaccinated with conventional vaccines), but which may protect against infection when delivered in an appropriate way using a recombinant vaccine platform (Wolfenden, RE et al., 2010; Kremer et al., 2011). Recently, bacterial carriers of antigens (vectors), including *Salmonella* Enteritidis and *Bacillus subtilis*, have been manipulated to express protein antigens to protect against bacterial, viral, and protozoal pathogens (Layton et al., 2009; O'Meara et al., 2010; Kremer et al., 2011; Layton et al., 2011). These vaccines have an advantage over many other types of vaccines in that they are able to be delivered directly to a mucosal surface via nasal, ocular, or oral administration. Because most pathogens invade the host through a mucosal surface, an enhanced mucosal immune response is the only portion of acquired immunity that can markedly reduce the probability of an animal or flock to become infected, as discussed above. While prevention of morbidity and mortality alone are useful traits of conventional vaccines for most poultry disease-causing agents, in the case of the common *Salmonella* serotypes which cause foodborne illness, these isolates generally cause little or no disease in the animals. Thus, recombinant vaccines that are able to provide wide-range protection against common *Salmonella* serotypes of poultry, by mucosal presentation, may be a

Along with presentation of conserved antigens through mucosally-administered recombinant vaccines, there is a need to trick the immune system of the animal to respond robustly to these recombinant bacteria that are not capable of infecting or causing disease. Co-expression of molecules that may enhance the immune response or may be recognized by receptors located on the mucosal surface of the gastrointestinal tract is a promising area of work. Several such molecules may enhance the response to these recombinant vaccines

Presently, there are no broad-spectrum recombinant vaccines approved for use in agricultural animals to protect against the wide range of serotypes which plague poultry producers worldwide. Specific serotype vaccines, such as *S.* Enteritidis or *S.* Gallinarum, have gained considerable acceptance in countries with endemic problems with these more devastating serovars, particularly in breeders and table egg production chickens (see Shivaprasad, 1997, for a review). These vaccines generally do not provide robust protection against infection with even the identical serotype, and even less protection against heterologous serotypes (Hargis et al., 2010). However, there is a general consensus that some protection is provided and for valuable birds, these vaccines may offer a much-needed modicum of protection, though often through reduced persistence and shedding of the organism, thus limiting spread. For example, studies have shown that oil emulsion *Salmonella* Enteritidis bacterins administered to breeders caused a three log10 cfu/g cecal content reduction in recovery from progeny chicks (Inoue et al., 2008), and a two log10 cfu/g cecal content reduction in breeders after molting (Nakamura et al., 2004). Thus, these vaccines have value at the present time, especially for breeders and at-risk laying hens. Live-type vaccines with gene deletions assuring avirulence while allowing immunogenicity have been reported (Curtiss and Kelly, 1987; Dueger et al., 2003), and other specific deletion

critical component for controlling this problem in the next few years.

(Layton et al., 2009; O'Meara et al., 2010; Wolfenden et al., 2010).

administration to commercial poultry.

mutants have been proposed (Zhang-Barber et al., 1999; Sydenham et al., 2000). Day-ofhatch chicks vaccinated with this type of attenuated *Salmonella* vaccine have been shown to have serological protection to homologous and heterologous *Salmonella* serotypes, possibly through a mechanism similar to competitive exclusion (Hassan and Curtiss, 1994; Hassan and Curtiss, 1997; Dueger et al., 2003; Holt et al., 2003; Bohez et al., 2008). Furthermore, maternal antibodies can be demonstrated in eggs and chicks from breeders vaccinated with this vaccine. These antibodies were reported to reduce *Salmonella*e colonization and to provide protection to laying hens up to 11 months post-inoculation (Hassan and Curtiss, 1997). However, susceptibility to antimicrobial agents commonly used in poultry production can reduce or eliminate the efficacy of live vaccines, and these vaccines are subject to the serotype limitations as discussed above.

Autogenous vaccines provide for yet another mechanism for vaccinating poultry. In many (but not all) countries, there are regulatory provisions under certain circumstances for production of specific killed vaccines using the specific isolate plaguing a given poultry flock or complex. These "autogenous" *Salmonella* isolates are typically grown, killed and mixed with an adjuvant (a chemical that potentiates the immune response) for parenteral administration. Some veterinarians associated with valuable breeder flocks believe that these vaccines are highly preferred for vaccination against endemic and common serotypes for which no commercial vaccine exists.

Taken together, there are tremendous future opportunities for manipulating the acquired immune response, particularly the mucosal secretory IgA response, for reduceing *Salmonella*  infections in poultry. However, current vaccine availability is limited and progress is greatly needed on two fronts: 1) improving mucosal immune responses for *Salmonella* vaccines; and 2) targeting shared protective epitopes for broad-spectrum serotype coverage for the paratyphoid *Salmonella*e that currently plague poultry producers world-wide. Currentlyavailable commercial vaccines are enjoying significant popularity due to the intense regulatory pressures facing meat and egg producing poultry, although applications are generally limited to breeder or layer flocks except under intense regulatory pressure.
