The Use of Enterococci as Probiotics in Poultry

INTRODUCTION

The intensive systems of broiler chicken production could be accompanied with some stressor factors (Panda et al. 2006). In the recent decades, the uncontrolled use of growth promoting antibiotics has been increasing the risk of developing of antibiotic resistant pathogens (Sorum and Sunde, 2001). In 1996, the scientific findings and public concerns, was resulted in ban of growth promoter antibiotic application in the European Union. The new situation, was triggered more intensive research to find new safe animal growth promoter alternatives, such as changing the gut microflora using live non-pathogenic microorganisms with promising effects on birds health and performance which are known as probiotics. Probiotics have shown promising effects as alternatives to growth-promoting (Awad et al. 2009). The positive effects of probiotics on immune responses (Capcarova et al. 2008; Lee et al. 2008), decreasing pathogenic flora in the intestine has been widely accepted (Crawford, 1979). The lactic acid producing bacteria are the main probiotics and in particular Lactobacillus acidophilus, L. casei, L. reuteri are the base of most lactic acid bacteria based products (Caglar et al. 2005). Poultry initiates to eat solid feed immediately after hatching, then probiotics consumption must start in the early ages when gut microflora are not still well developed (Vahjen et al. 2002). The probiotic bacteria must also have additional criteria, including resistance to gastrointestinal pH and bile salts which are prerequisites for survival, colonization and action of ingested bacteria in the intestinal tract of the host (Erkkila and Petaja, 2000; Liong and Shah, 2005). The other essential feature to screening bacteria as potential probiotic is sensitivity to antibiotics, because bacteria can contain viru lence factors and antibiotic resistance factors. The most abundant lactic acid bacteria in the intestine of chickens are Lactobacillus and Enterococcus (Mitsuoka, 2002). The present review tries to summarize the findings and reports on the Enterococcus genus a functional probiotic in poultry.

Enterococcus genus

Until 1980 s, whole the Gram-positive cocci were known as streptococci, however the microbiological progress was reported them to the new genera Enterococcus, Lactococcus and Streptococcus (Schleifer and Kilpper-Bälz, 1984; Devriese et al. 1993; Devriese and Pot, 1995). The genus Enterococcus is a member of the lactic acid bacteria family and significant bacteria in cheese production, and spoilage. The Enterococcus tolerates to high salt and pH then usually is dominant in fermented foods. More than 37 species have been recognized for the genus Enterococcus and this genus are found in the environment and many animal and human based materials (Devriese et al. 1991; Devriese et al. 2003; Franz and Holzapfel, 2006). The probiotic characteristics have been recognized for some enterococcal strains and they have effectively used in human and animals. However, there are also some photogenic enterococcal strains which are responsible for bacteraemia, endocarditis or urinary tract infections in human. The pathogen Enterococcus strains are usually antibiotic resistance and there are concerns for the secure application as probiotics (Franz et al. 2011). On the other hand, E. faecium, E. faecalis, E. hirae, E. durans, and E. cecorum are natural residents of the farm animal’s intestinal tract, which is an essential factor to probiotic survival (Devriese et al. 1991; Devriese et al. 1994; Leclercq et al. 1996). From a probiotic point of view, the facultative anaerobic bacteria, E. faecium and E. faecalis are the main enterococcal species and E. faecium is permitted by the Association of American Feed Control Officials, fed to broiler chickens as a probiotic supplement (Franz et al. 1999; Foulquié-Moreno et al. 2006; Zhao et al. 2013). The ability of chicken originated Enterococcus spp. (E. faecium EF55) to produce bacteriocins as potential antimicrobial factors could confirm their probiotic effects for poultry (Laukova et al. 2004).

Effect of enterococcal probiotics on intestinal microbial population

Vahjen et al. (2002) reported that dietary supplementation of E. faecium SF68 increased the lactic acid bacteria population in turkey small intestine. This confirms that the enterococci can tolerate turkey gastrointestinal tract condition and control pathogenic bacteria. Samli et al. (2007) found that dietary supplement of E. faecium could increase lactic acid bacteria colonization in the ileum, and increased their excretion (Samli et al. 2007). A multi species probiotic containing Enterococcus, Bifidobacterium and Pediococcus strains applied in the feed and water manipulated the cecal microbial population, such that increased the Lactobacilli, Bifidobacteria and gram-positive cocci and also reduced the Salmonella population in turkey and broilers (Mountzouris et al. 2007; Grimes et al. 2008; Capcarova et al. 2010). In the study of Samli et al. (2010), the E. faecium probiotic improved the ileal and cecal microbial population and significantly reduced the Escherichia coli population. Kralik et al. (2004) also demonstrated the effect of dietary supplementation of E. faecium on E. coli reduction in broilers. The positive effect of E. faecium on fecal microflora of broiler has also reported by Kacaniova et al. (2006). In another study, E. faecium CCM8558 effectively colonized in the intestinal tract of chickens and reduced the Campylobacter spp load (Laukova et al. 2017). Cao et al. (2013) fed E. faecium to broilers and showed lower E. coli and C. perfringens population and higher Lactobacillus and Bifidobacterium population in the cecum contents. Levkut et al. (2009) observed that the E. faecium EF55 decreased the cecal population of Salmonella in the infected broilers. Similarly, other researchers have reported that Lactobacillus acidophilus and E. faecium based probiotics, decrease the Campylobacter jejuni count in chicks (Willis and Reid, 2008; Ghareeb et al. 2012). Table 1 shows the effects of E. faecium based probiotics on intestinal microbial population.

Effect of enterococcal probiotics on poultry performance

Samli et al. (2007) reported that dietary supplementation of E. faecium NCIMB 10415 improved broiler chickens weight gain and feed efficiency. The same findings have been reported by Mountzouris et al. (2007) and Awad et al. (2009). In another study using a multi species containing Lactobacillus, Pediococcus, Bifidobacterium and Enterococcus strains in feed and water, Mountzouris et al. (2007) found a growth rate comparable to feeding 2.5 mg/kg avilamycin antibiotic. Surprisingly, probiotic included in drinking water was more effective than dietary route. Demeterová et al. (2009) studied the supplementation of E. faecium DSM 7134 and natural humic substances in broiler chickens. The improved feed conversion ratio in birds fed both the supplements together was attributed to the increased phagocytes (Demeterová et al. 2009). Capcarova et al. (2010) used E. faecium probiotic in the diet of broiler chickens and found a decrease in feed intake without any change in feed conversion ratio. Luo et al. (2013) reported a normal growth rate in broiler chickens fed E. faecium supplement, and Zheng et al. (2015) found that dietary E. faecium inclusion did not influence the weight gain and feed intake of broilers, however, the feed conversion ratio was improved.

Table 1 The effects of Enterococcus probiotic on poultry intestinal microbial population

Cao et al. (2013) found that E. faecium probiotic improved the growth rate of chickens experimentally infected with pathogenic E. coli K88. In the study of Majidi-Mosleh et al. (2017), in ovo injection of E. faecium had no effect on hatchability our growth performance in broiler chickens. Zheng et al. (2016) suggested that dietary E. faecium feeding may change the partitioning of nutrients, consequently, improve nutrient utilization. Table 2 shows the effects of E. faecium based probiotics on performance traits of broilers.

Effect of enterococcal probiotics on poultry meat quality

Zheng et al. (2015) used 2D-DIGE-based proteomics to study the proteome changes in the meat of broilers fed E. faecium probiotic. The E. faecium supplement increased pH, water holding capacity and meat colour of pectoral muscle, however reduced abdominal fat content. They suggested that meat quality alterations following E. faecium feeding were due to changes in expression of 22 proteins in the pectoral muscle, such that dietary E. faecium probiotic improved meat quality of broilers. This was due to the changes in expression of proteins responsible for energy and carbohydrate metabolism, cytoskeleton, and also molecular chaperones. These proteins are the main controllers of pH and water holding capacity of meat. The pectoral muscle of broiler chickens fed E. faecium supplement had also reduced the cooking loss and drip loss (Zheng et al. 2015).

Effect of enterococcal probiotics on intestinal morphology in poultry

The gastrointestinal tract is responsible for the uptake of nutrients, remove pathogens and immune response. Luo et al. (2013) reported that dietary supplementation of E. faecium increased gut microvilli and influenced immune organ development and mucosal structure chickens. They also showed that dietary supplement of E. faecium had a pronounced effect on genes expression related to the intestinal tissue development and epithelium maturation, and genesresponsible for digestion and absorption of nutrients. The mucin is a very glycosylated protein that is synthesized by the goblet cell in epithelial tissues and acts as a protective barrier (Marin et al. 2008). In the study of Luo et al. (2013), E. faecium supplementation led to down-regulation of mucin-2 which is a member of mucin protein family, which can combine to pathogens as part of the immune response (Johansson et al. 2011). Samli et al. (2007) also reported that dietary supplementation of E. faecium increased the villus height in jejunum and ileum of broilers, which could enhance the digestive and absorptive capacity of the intestinal tract because of a higher absorptive surface area, up-regulation of brush border enzymes and enhancing nutrient transport mechanisms (Amat et al. 1996). In the study of Cao et al. (2013), dietary inclusion of E. faecium increased villi height and decreased crypt depth in the jejunum and the same effect was observed using an antibiotic. Chichlowski et al. (2007) found that a multi-strain probiotic containing Lactobacilli, Thermophilum, Bifidobacterium, and E. faecium increased villus height and decreased the crypt depth in jejunum, compared with the control group or birds fed salinomycin. Therefore, it seems that dietary E. faecium probiotic could play a positive role in the small intestinal morphology of broilers.

Effect of enterococcal probiotics on immune responses in poultry

There have are several reports in the literature of the positive effects of enterococcal probiotics on poultry immune response. Zheng et al. (2016), suggested that the improved production efficiency in the broiler chickens fed with E. faecium supplement could be attributed to lower nutrient costs for immune response and more available nutrient for growth of birds.

Table 2 The effects of Enterococcus probiotic on performance of broilers

In the study of Luo et al. (2013), the relative weights of intestine, spleen and Bursa Fabricius were heavier in chickens fed E. faecium probiotic, they concluded that dietary supplementation of E. faecium could improve immune organ development. They also found that the E. faecium probiotic decreased the inflammation and oxygen stress conditions in the intestinal mucosa of broilers. This means less energy costs and probably explains the improved feed conversion ratio. In the study of Majidi-Mosleh et al. (2017), the antibody titre against Newcastle disease virus, antibody titre against sheep red blood cells and cell-mediated immune response was not influenced in E. faecium fed broilers. Cao et al. (2013), studied the pattern of immune system related gene expression in response to dietary E. faecium supplementation. They observed an up-regulation of Interleukin 4 (IL-4) which has a key role in the immune responses, in the jejunal mucosa of chicks fed E. faecium probiotic. In birds treated with E. faecium, the expression of tumor necrosis factor alpha (TNF-α), a key cytokine responsible for systemic inflammation and is one of the cell signaling proteins involved in the acute phase reaction, was also increased in the jejunal mucosa. Birds fed E. faecium probiotic had higher levels of secretory immunoglobulin A (S-IgA) in jejunal mucosa, which is an important factor in protecting organs such as oral cavity, intestine, and lungs from invading pathogens. As a matter of fact, the majority of invading pathogens makes first contacts with the host at mucosal level, particularly in the agasreo-intestinal tract, and S-IgA is known as the first protective ban (Muir et al. 1998). Phagocytic action is an important constituent of the cellular innate immunity system and has a vital role in host protection against pathogens. Laukova et al. (2017) reported that Phagocytic activity was considerably increased in chickens fed E. faecium probiotic and attributed it to the ability of E. faecium CCM8558 strain to promote the toll-like receptors (TLRs). The TLRs are pattern detection receptors that act as pathogens invading sensors and are vital for the start the innate inflammatory and adaptive immune reactions (Shang et al. 2008). There are also reports on the effects of E. faecium probiotic on up-regulation of MIF, IFN-β, MD-2, and CD14 immune system related proteins in chickens (Karaffova et al. 2017). Table 3 shows the effects of E. faecium based probiotics on immune system of broilers.

Effect of enterococcal probiotics on blood parameters in poultry

There are reports on the effects of probiotics in altering the chicken blood lipid fractions as an index of body metabolism (Panda et al. 2006). Capcarova et al. (2010) showed that the E. faecium M 74 probiotic reduced levels of total cholesterol and also total lipids in blood plasma. The blood cholesterol concentration is an important factor to prevent atherosclerosis, and it’s known that atherosclerosis could be controlled by adjusting the blood cholesterol level (Kapila et al. 2009). De Smet et al. (1994) found that probiotics increase the production of unconjugated bile acids, therefore involve in the regulation of blood cholesterol, which is the precursor substance of bile acids. Capcarova et al. (2008) reported that blood bilirubin concentration increased in broilers fed E. faecium M 74 and addition of a probiotc containing L. fermentum and E. faecium caused in an increase in serum calcium and iron concentration and a lower blood triglyceride level. The higher serum calcium concentration could be a positive effect for the animals to reach a more strength bone and growth rate. Capcarova et al. (2010) studied the effect of E. faecium M74 strain on blood parameters of laying hens, and found a reduced levels of calcium, lipids, cholesterol, haematocrit values and leucocyte counts in plasma, although the triglyceride level was not altered and the erythrocyte counts were greater than before.

Table 3 The effects of Enterococcus probiotic on immune system responses of broilers

Probiotic application was not changed the egg production parameters. The effect of E. faecium CCM8558 strain on blood lipid fractions could be attributed to the fact that the probiotic is a lactic acid producing bacteria, release bile degrading enzymes, deconjugates bile, and reduces pH. These alterations could decrease blood triglycerides or cholesterol levels (Bovdisova and Capcarova, 2015). In the experiment of Capcarova et al. (2008), supplementation of E. faecium M 74 strain in drinking water had no effect on serum hepatic enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT) and glutamate dehydrogenase (GLDH). However, the antioxidant potential of E. faecium M 74 strain added was proved, such that multivalent anti-oxidativity (TAS) was increased, which is an index to inhibit the exogenous and endogenous oxidative stress.

CONCLUSION

To date, the Enterococcus faecium are known as safe probiotic microorganisms which enhance the immune system and performance of broiler chickens. However, various factors may affect the potential probiotic effects and more investigations may be needed to conclusively reveal the involved mechanisms.