**5.** *Bacillus* **spore***-***based probiotic for** *Salmonella* **control and performance enhancement in poultry**

In spite of the success showed by the development of the LAB probiotic for use in commercial poultry as described above, there is still an urgent need for commercial probiotics that are shelf-stable, cost-effective and feed-stable (tolerance to heat pelletization process) to increase compliance and widespread utilization. Among the large number of probiotic products in use today some are bacterial spore formers, mostly of the genus

<sup>1</sup>Adapted from Tellez et al., 2006

Alternative Strategies for *Salmonella* Control in Poultry 265

a potential mechanistic action of these new *Bacillus* candidates at least partially involve rapid activation of innate host immune mechanisms (system or responses) in chickens and turkeys (unpublished data). This data provides an exciting possibility for identification of

**6. Prospects of bacteriophage therapy to control gastrointestinal disease** 

During the last approximately 60 years, there have been sporadic published reports of efficacy in treating Enterobacteriaceae infections systemically and within the gastrointestinal tract. While a number of reports have rather consistently indicated that systemic or tissueassociated infections were treatable by parenteral administration of appropriate bacteriophage cocktails, reports of successful treatment of enteric Enterobacteriaceae are much more sporadic, and are interspersed with a number of reports of failed attempts for enteric treatment. The following sections will discuss selected successes and failures and describe the possible differences in these studies and the potential for development of more

The bacteriocidal effects of bacteriophages have long been studied for their usefulness in treating gastrointestinal infections. Early studies originating from the former Soviet Union, Eastern Europe, and Eastern Asia suggested bacteriophages could prevent and treat *Vibrio cholera* infections (Dubos et al, 1943; Dutta, 1963; Sayamov, 1963; and Marčuk et al, 1971). In the 1980s Slopek and co-workers (1983a-b, 1984, 1985a-c, 1987) published numerous papers showing the promising results of treating septic patients with bacteriophages. While the validity of these studies has been questioned, in part due to relaxed scientific rigor in these regions during the time when these studies were completed (Merril et al, 2003; Alisky et al, 1998) and are not often cited by bacteriophage researchers in recent years, they have served as an inspiration for continued research into the possibility that bacteriophages can cure

Smith and Huggins (1982) compared the efficacy of phages with that of antibiotics in treating both generalized and cerebral infections in mice. They isolated anti-K1 bacteriophages that were able to lyse K1-positive *E. coli*. These bacteriophages were able to cure infection caused by K1-positive, even when used at a low titer. The bacteriophages were more effective than several antibiotics for curing mice. Smith and Huggins (1983) also successfully used bacteriophage therapy to treat calves, pigs, and lambs that had been infected with *E. coli*. Perhaps key to their success, they selected a bacteriophage that would lyse *E. coli* and also selected a second bacteriophage that would lyse the target *E. coli* that had become resistant to the first bacteriophage. In 1987, Smith and Huggins used bacteriophages to treat calves with *E. coli*-caused diarrhea. They selected their bacteriophages by administering *E. coli* to a calf followed by a bacteriophage cocktail. Bacteriophages able to survive the gastrointestinal tract were collected in the feces 24 hours post-administration. These bacteriophages were used to treat subsequent calves. Calves given bacteriophages within 24 hours of the onset of diarrhea recovered within 20 hours. Also, sick calves placed on litter that had been sprayed with bacteriophages recovered from diarrhea. Smith and Huggins noted that during the period of disease, bacteriophages continued to persist in the feces, but after recovery, bacteriophage

vastly superior and more potent probiotics in the near future.

gastrointestinal diseases in humans and animals.

numbers dropped dramatically.

**6.1 Overview** 

effective strategies.

**6.2 Successes** 

*Bacillus*. Used primarily in their spore form, some (though not all) have been shown to prevent selected gastrointestinal disorders and the diversity of species used and their applications are astonishing. While not all *Bacillus* spores are highly heat tolerant, some specific isolates are the toughest life form known on earth (Vreeland, Rosenzweig, & Powers, 2000) and can be used under extreme heat conditions. Several studies have shown that either live vegetative cells or endospores of some isolates can prevent colon carcinogenesis (Parket al., 2007) or discharge antimicrobial substances against Grampositive bacteria, such as *Staphylococcus aureus*, *Enterococcus faecium*, and *Clostridium difficile*  (O'Mahony et al., 2001). These results provided evidence of colonization and antimicrobial activity of probiotic bacteria, thus, products containing *Bacillus* spores are used commercially as probiotics, and they offer potential advantages over the more common LAB products since they can be used as direct feed microbials (Anadón et al., 2006; Barbosa et al., 2005; Duc le et al., 2004; Hong et al., 2005; Hong et al., 2008a; Hong et al., 2008b; McNulty et al., 2007; Osipova et al., 2003; P. Williams, 2007a; Wolken et al., 2003). There is scientific evidence suggesting that some but not all isolates of ingested *B. subtilis* spores can, in fact, germinate in the small intestine (Casula & Cutting, 2002; Casula & Cutting, 2002; Duc le & Cutting, 2003; Hoa et al., 2001). Together, these studies not only show that spores are not transient passengers in the gut, but they have an intimate interaction with the host cells or microflora that can enhance their potential probiotic effect. Several commercial sporeforming *Bacillus* cultures have been shown to reduce food borne pathogens (Aureli et al., 2010). However, cost issues associated with achieving necessary concentrations of spores in feed have greatly limited commercial acceptance in the animal industry (Hong et al., 2005).

While the majority of clear-cut research with regard to beneficial probiotic cultures has focused on LAB, as discussed above, a major question in several laboratories is whether or not selected spore-former bacteria (genus *Bacillus* or related) can be as effective as the best known LAB cultures. Recently, one *Bacillus subtilis* spore isolate was as effective as a wellestablished LAB-based probiotic for *Salmonella* reduction in poultry (Wolfenden R.E. et al., 2010; Shivaramaiah et al., 2011), and was equal to bacitracin for prevention of experimental necrotic enteritis, and was able to markedly reduce necrotic enteritis issues in large scale feed trials (unpublished from the author's laboratory).

Other isolates or combinations of isolates with increased potency and efficacy may be identified with continued research. Some of these environmental *Bacillus* isolates have been evaluated *in vitro* for antimicrobial activity against selected bacterial pathogens, heat stability, and the ability to grow to high numbers. Unpublished experimental evaluations have confirmed improved body weight gain as well as *Salmonella sp.* or *Clostridium perfringens* reduction in commercial turkey and broiler operations when compared with medicated (nitarsone) or control nonmedicated diets respectively. Indeed, preliminary data suggests that these isolates could be an effective alternative to antibiotic growth promoters for commercial poultry.

Importantly, improved efficiency of amplification and sporulation is absolutely essential to gain widespread industry acceptance of a feed-based probiotic for ante mortem foodborne pathogen intervention, as well as cost effectiveness. Recently, both vegetative growth and sporulation rates have been optimized, which may lead to new efficiencies for commercial amplification and manufacture of a cost-effective product at very high spore counts (Wolfenden R.E. et al., 2010). In order to select even more effective isolates, current research is focused on the mechanistic action of new *Bacillus* candidates. Preliminary studies indicate a potential mechanistic action of these new *Bacillus* candidates at least partially involve rapid activation of innate host immune mechanisms (system or responses) in chickens and turkeys (unpublished data). This data provides an exciting possibility for identification of vastly superior and more potent probiotics in the near future.
