**3. Mechanism of action of probiotics against** *Salmonella*

Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Amongst the many benefits associated with the consumption of probiotics, modulation of the immune system has received considerable attention (Borchers, Keen, & Gershwin, 2002; Borchers, Selmi, Meyers, Keen, & Gershwin, 2009).

Previously, it was thought that administration of bacteria such as probiotics to neonates directly reduced infection by pathogens due to competition amongst the bacteria for attachment sites and nutrients and, that beneficial bacteria would out-compete pathogens within the GIT. This competition, coined as "competitive exclusion" was first described in 1973 by Nurmi and Rantala (Nurmi& Rantala, 1973). Their data indicated that early administration of beneficial bacteria to chicks prevented infection by pathogens. Since Nurmi and Rantala proposed competitive exclusion could be used as a method to prevent *Salmonella* infection, numerous researchers have reported the ability of live bacterial cultures to also reduce colonization of opportunistic microorganisms in the gastrointestinal tract (Callaway et al., 2008; Wagner et al., 2003; Hollister et al., 1999; Corrier et al., 1998; Hume et al., 1998; Nisbet et al., 1998) and probiotic organisms (J. P. Higgins et al., 2010; S. E. Higgins et al., 2008; Vicente et al., 2008; J. P. Higgins et al., 2007; Bielke et al., 2003; Patterson & Burkholder, 2003). Yet, understanding of how probiotics mediate these health benefits, specifically reduction of *Salmonella* infection, is very limited.

Balanced gastrointestinal microflora and immune-stimulation are major functional effects attributed to the consumption of probiotics (Amit-Romach, Uni, & Reifen, 2010; Boirivant & Strober, 2007; Boirivant, Amendola, & Butera, 2008; Flint, O'Toole, & Walker, 2010; Flore, Francois, & Felicite, 2010; Ibrahim et al., 2010; Klein, Sanders, Duong, & Young, 2010; Nayak, 2010). Many probiotic effects are mediated through immune regulation, particularly through balance control of pro-inflammatory and anti-inflammatory cytokines (Di Giacinto, Marinaro, Sanchez, Strober, & Boirivant, 2005; Foligne et al., 2010; Hacini-Rachinel et al., 2009; Jobin, 2010; Li, Xia, & Li, 2009). However, several animal and human studies have provided unequivocal evidence that specific strains of probiotics are able to stimulate multiple aspects of innate immunity (Amit-Romach et al., 2010; Boirivant & Strober, 2007; Boirivant et al., 2008; Farnell et al., 2006; Romanin et al., 2010; Weiss et al., 2010) as well as to increase humoral immunity (Fang, Elina, Heikki, & Seppo, 2000; Galdeano, de Leblanc Ade, Carmuega, Weill, & Perdigon, 2009; Leblanc, Fliss, & Matar, 2004; Nermes, Kantele, Atosuo, Salminen, & Isolauri, 2011).

Using a *Salmonella* challenge model, an effective LAB probiotic, administered 2 hours after *Salmonella* challenge, had no effect during the first 12 hours on increasing cecal colonization by this pathogen, although marked and rapid decreases were observed between 12 and 24 hours post-challenge (J. P. Higgins et al., 2007; J. P. Higgins et al., 2010). Later, using the same model and microarray analysis of gut mRNA expression, gene expression differences in birds treated with a *Lactobacillus-*based probiotic were compared to saline treated birds. At 12h postprobiotic treatment, 170 genes were significantly different (P<0.05), but by 24h post treatment, the number of differentially regulated genes were 201. Pathway analysis revealed that at both time points, genes associated with the NFκB complex were significantly regulated, as well as genes involved in apoptosis. Probiotic-induced differential regulation of the genes *GAS2* and *CYR61* may result in increased apoptosis in the ceca of chicks. Because *Salmonella* is an intracellular pathogen, it was suggested that increased apoptosis may be a mechanism by which B11 reduces *Salmonella* infection (S. E. Higgins et al*.,* 2011).

Alternative Strategies for *Salmonella* Control in Poultry 263

**<sup>24</sup>***Weissella confusa Lactobacillus casei Lactobacillus casei Clostridium* 

**27** *Weissella confusa Lactobacillus casei Lactobacillus casei Weissella confusa* 

**Midi system ID Microbial ID Inc.** 

*Lactobacillus* 

*Lactobacillus delbreuckiibulgaricus* 

*cellobiosus Lactobacillus casei Weissella confusa* 

*Pediococcus* 

*cellobiosus* 

*Lactobacillus* 

*Lactobacillus sanfranciscensis* 

*Lactobacillus gasseri* 

**Biolog ID Dept. of Poultry Sc. U. of Arkansas** 

*clostridiiforme* 

*Lactobacillus hamsteri* 

*Weissella paramesenteroides* 

> *Lactobacillus salivaruis*

> *Lactobacillus salivarius*

*gasseri Unable to identify* 

*ruminis Unable to identify* 

*fermentum Unable to identify* 

*cellobiosus Unable to identify* 

**Midi system ID Micro Test Lab Inc.** 

> *Enterococcus cecorum*

> *Lactobacillus delbreuckiibulgaricus*

> *Lactobacillus*

*acidilactici* 

*Lactobacillus fermentum* 

*Lactobacillus helveticus* 

*Lactobacillus helveticus* 

*parvulus Unable to identify Lactobacillus* 

Table 1. Comparisons between MicroSeq , MIDI, and Biolog identifications of FloraMaxTM

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

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>40</sup>***Weissella confusa Lactobacillus casei Lactobacillus* 

**LAB ID** 

**16S RNA Sequencing (FIRST 500 bp) Microbial ID Inc.** 

*parvulus* 

*parvulus* 

*salivaruis* 

*paramesenteroides* 

*salivaruis* 

*salivarius* 

**37B** *Weissella confusa Pediococcus* 

**<sup>18</sup>***Pediococcus* 

**<sup>29</sup>***Pediococcus* 

**<sup>36</sup>***Lactobacillus* 

**<sup>44</sup>***Weissella* 

**<sup>46</sup>***Lactobacillus* 

**<sup>48</sup>***Lactobacillus* 

**<sup>52</sup>***Pediococcus* 

**enhancement in poultry** 

1Adapted from Tellez et al., 2006

lactic acid bacteria1
