Direct-fed probiotics, designed to deliver a high concentration of beneficial bacteria alive at the point of consumption, improve poultry performance.

By STEVE LERNER and PETER MURIANA*

*Dr. Steve Lerner is vice president of product development and commercialization at Nutrition Physiology Co. Dr. Peter Muriana is a professor of food microbiology at Oklahoma State University.

FOOD safety is an ongoing concern of producers, retailers and consumers. In dealing with the risk of foodborne pathogens as well as rising product demand, U.S. poultry producers must deliver healthy, robust, uniform flocks.

Research indicates that probiotics can be used to improve the health and productivity of broilers, turkeys and layers and to reduce the prevalence and concentration of potentially pathogen organisms.

A working group of experts from the World Health Organization (WHO) and U.N. Food & Agriculture Organization (FAO) defined probiotics as "live microorganisms, which, when consumed in adequate amounts, confer a health effect on the host" (2001 and 2002).

The FAO/WHO working group also identified the set of characteristics that must be in place in a bacterial strain for it to be considered a probiotic.

First, the strain must maintain its physiological characteristics and remain stable during the manufacturing and delivery processes. Second, it must reach its proposed site of action — usually the digestive tract or gut — and survive the physiological stresses and metabolic conditions following its ingestion, e.g., stomach acid/rumen fluid, intestinal pH and the presence of bile salts and digestive enzymes. Last, it must prove to be beneficial without risk to the host.

Probiotics

Lactobacilli and Bifidobacterium, both lactic acid-producing bacteria, are the genera from which the overwhelming majority of probiotic bacteria are selected for use in the food and feed industries (Gueimonde et al., 2013; Holzapel et al., 1998) and are generally known to be non-pathogenic and non-toxigenic.

Focusing specifically on lactobacilli, not a single case of Lactobacillus bacteremia has been reported in healthy animals in the literature.

The lactobacilli species Lactobacillus animalis (identified commercially as Lactobacillus acidophilus) was selected during an extensive screening of potential candidates at Oklahoma State University. The strain was chosen for its ability to thrive in the gut, to successfully colonize and achieve high numbers of viable organisms, to competitively exclude pathogenic organisms and to exert a beneficial effect on the host animal.

This species has been studied as a potential probiotic and/or as a commensal component of the gut microbiota in swine (Laycock et al., 2012), poultry (Gusils et al., 1999; Ehrmann et al., 2002; Chichlowski, 2006), dogs (Biagi et al., 2007; Murphy et al., 2009; Martin et al., 2010), monkeys (Jin et al., 2011; Gravett et al., 2012), rodents (Sarma-Rupavtarm et al., 2004; Karunasena et al., 2013) and veal calves (Ripamonti et al., 2009; Ripamonti et al., 2011; Ripamonti et al., 2011) and has been safely used in commercial beef and dairy cattle operations for 20 years (as Bovamine, Bovamine Dairy and Bovamine Defend).

How probiotics work

Effective probiotics stabilize the gastrointestinal tract and digestive function, which improves energy utilization from feedstuffs and overall health. The interaction of probiotics, gut microbiota and health was recently reviewed (Butel, 2013). In brief, there are four modes of action of probiotics:

1. Prevent or limit the colonization of pathogenic bacteria in the gut;

2. Improve the connections between cells that line the intestines, making them less "leaky";

3. Influence local (gut) and systemic immune functions, and

4. Produce enzymes that improve digestion.

Inhibiting colonization. The first mode of action is inhibiting colonization, typically exerted against pathogenic bacteria, which prevents or limits their colonization of the intestinal tract.

The bacterial inhibition manifested by probiotic organisms may be due to their production of broad-spectrum inhibitory bacteriocins, short-chain fatty acids or other metabolites, such as lactic acid, that result in a decrease in pH (which is less favorable for growth of pathogenic bacteria) or biosurfactants with direct antimicrobial activity.

This inhibition of colonization can also occur via direct physical mechanisms, such as competition for binding sites and inhibition of adhesion.

Improving the barrier function. The second mode of action concerns improvement of the barrier function of the gut mucosa. This barrier function is related to the quality of tight junctions that exist between the epithelial cells that line the intestines.

Highly specialized Paneth cells line the crypts between intestinal villi. They produce peptides, specifically defensins and lysozyme that have antimicrobial properties. Last, mucus cells, as the name suggests, produce a protective layer of mucus, which prevents any direct contact with bacteria found in the intestinal lumen.

Probiotic bacteria can interact with these cell types at the level of cell-to-cell signaling pathways, leading to an increase in the mucus layer, to increased production of defensins or to a change in the protein configurations of tight junctions, thereby improving these barrier functions.

Modulating the immune system. The third mode of action is modulation of the immune system. More than 70% of immune cells are located in the small intestines; these are commonly referred to as the gut-associated lymphoid tissue.

Activating an immune response is highly complex and requires a receptor-mediated interaction of the cells that confer innate immunity (epithelial cells, dendritic cells and macrophages) with structural components that are present and repeated on the surface of microorganisms.

These structural components, called microbial-associated molecular patterns, interact with the epithelial cells of the intestine and stimulate the immune system at the level of the lamina propria — the thin layer of connective tissue that lies beneath the cellular lining of the intestines (Round and Mazmanian, 2009).

The outcome of this interaction is the activation of specialized classes of white blood cells (regulatory T cells and T-helper cells) that leads to the production of pro-inflammatory or anti-inflammatory signaling molecules called cytokines.

Probiotic bacteria have well-documented effects that differ depending on the profile of the cytokines secreted in response to their presence (Menard et al., 2008). The effects may be local and limited to stimulation of an immune response in the gut, or they may be systemic.

Direct digestive aids. Probiotic strains also can have direct beneficial effects by providing enzymes that improve digestive function. This effect has not been documented for the majority of lactobacillus strains studied.

Chicks and poults

Under typical commercial conditions, chicks and poults can be exposed to pathogenic bacteria on their first day of hatch, before they develop a mature microbiota in their gastrointestinal tract.

If pathogenic organisms successfully colonize the guts of these naive birds, then poor performance will likely follow. Producers with these flocks would be expected to see relatively high early mortality, runting and stunting, high feed conversions, poor rates of gain and persistent morbidity associated with other opportunistic infections (e.g., clostridium leading to necrotic enteritis, coccidiosis, etc.).

Time and time again, published research points to the relative effectiveness of individual strains and combinations of strains and to the daily delivery of an effective concentration of viable bacteria on feed as requisite conditions to achieve success with probiotics.

Antibiotic resistance

Currently, the biggest concern about the use of probiotic organisms in feeds and foods isn't the inherent resistance of those organisms to antibiotics but, rather, the transfer of antibiotic resistance via mobile genetic elements from the probiotic organisms to potentially pathogenic organisms.

Gueimonde et al. (2013) and Devirgiliis et al. (2013) reviewed this subject and concluded that these mobile elements constitute a reservoir of resistance for potential food or gut pathogens and, thereby, constitute a serious safety issue.

The erythromycin ribosome methylation type B genotype (erm(B)) was found in six of 14 strains of L. animalis isolated from the tonsillar and nasal flora of piglets (Martel et al., 2003). The methylase produced by this gene alters the 50S subunit of ribosomes, rendering them less capable of binding erythromycin and other macrolide antibiotics (Devirgiliis et al., 2013).

From a recent review of the subject, no other antibiotic resistance elements have been reported to be found in L. animalis in other species (Thumu and Halami, 2012).

Using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information, the erm(B) nucleotide sequence can be found and aligned with a high percentage of consensus sequences to 100 entries in the GenBank database. Use of accession number JX535233.1 — for Listeria monocytogenes strain LM78 rRNA methylase (ermB) gene, complete cds — in a BLAST search will retrieve the list of sequences with significant alignments.

An attempt to align the complete genomic sequence of L. animalis found with the erm(B) sequences revealed no significant alignment. Therefore, it can be concluded that this strain does not contain the erm(B) gene, making it like the majority of the L. animalis strains isolated from piglets (Martel et al., 2003) and, thus, making it safe, with no threat of antibiotic resistance.

Conclusion

Gaining an understanding of the science of probiotics should enable an informed producer to weigh the opinions and to choose the best product for the operation. That choice should be based on the demonstrated efficacy of the probiotic bacteria contained in the product, as well as its safety and potential for long-term use without risk.

Direct-fed probiotics, which are designed to deliver a high concentration of beneficial bacteria alive at the point of consumption, have been demonstrated to improve poultry performance.

References

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