Cecal microbial populations and litter E. coli content: effects of Bio-Mos®, purified lignin or virginiamycin
Published: November 9, 2007
By: CIRO A. RUIZ-FERIA and BUSHANSINGH BAURHOO (Courtesy of Alltech Inc.)
At the same time, food safety remains a major public health concern worldwide. The principal pathogenic bacteria causing food-borne illnesses are Campylobacter, Salmonella and E. coli (Mead et al., 1999). The critical point of bacterial contamination of poultry products occurs at the slaughterhouse when pathogens from the intestinal contents make contact with chicken carcasses (Heyndrickx et al., 2002). Different strains of antibiotic-resistant E. coli have been isolated from poultry and poultry meat products in several countries (Zhao et al., 2001; Mayrhofer et al., 2004). Furthermore, E. coli has been identified as the main cause of cellulitis, which has become the major cause of carcass condemnations at poultry processing plants in Canada (Kumor et al., 1998).
The threat of antibiotic resistant bacteria and global demands for safe poultry products have prompted the need for effective biological modulators of enteric microflora in the poultry industry, and in this context, there is increased interest in the use of prebiotics.
These dietary additives have the potential to limit or eliminate intestinal pathogenic bacteria. The objectives of this study were to determine the effects of dietary addition of a mannan oligosaccharide (Bio-Mos®) or purified lignin (Alcell® lignin) to replace virginiamycin. Microbial populations in the ceca and litter, and cecal E. coli after an in vivo challenge with known pathogenic strains of E. coli, were monitored.
MATERIALS AND METHODS
BIRD MANAGEMENT AND EXPERIMENTAL DESIGN
Eight hundred one-day-old male broilers (Cobb 500) were grown over a 42-day experimental period. The chicks were raised on fresh pine wood shavings and brooded following standard temperature regimes, under a 20:4 light:dark cycle. Procedures for bird management and care were approved by the Animal Care Committee of McGill University.
Birds were randomly assigned to five treatments (four pen replicates; 40 birds per pen). The five experimental diets included: 1) antibiotic free, negative control diet (CTL-); 2) positive control diet (CTL+, 11 mg/kg virginiamycin); 3) CTL- diet plus Bio-Mos® (0.2% of the starter diet and 0.1% of the grower diet); 4) CTL- diet plus Alcell lignin (Alcell® Technologies Inc., QC, Canada) at 1.25% of the diet (LL); 5) CTL- diet plus Alcell® lignin at 2.5% of the diet (HL). The diets were formulated to meet or exceed NRC (1994) requirements. The ingredient composition and nutrient content of the diets are shown in Table 1.
MICROBIAL POPULATIONS OF CECAL DIGESTA AND LITTER
At 28 and 42 days of age, cecal contents (1 bird per pen replicate) were aseptically collected into sterile plastic bags and stored at -20 ºC for later microbiological analysis. Samples of the cecal contents were serially diluted in 0.85% sterile saline solution and used to assay lactobacilli, bifidobacteria, and E. coli. Lactobacilli were anaerobically assayed using lactobacilli MRS Agar (Fischer Scientific, ON, Canada). Enumeration of bifidobacteria was performed using Wilkins-Chalgren agar (Oxoid, ON, Canada) supplemented with glacial acetic acid (1 ml/L) and mupirocin (100 mg/L, Rada et al., 1999). E. coli was assayed using Rapid E. coli 2 agar (Bio-Rad laboratories, ON, Canada) modified using E. coli supplement (Bio-Rad) to be selective for E. coli.
Litter samples from each pen were taken at five different locations, in the middle and at equidistant points at each end both longitudinally and vertically. The five sub-samples were thoroughly mixed by hand and sealed into sterile Whirlpak microbiological bags and sealed (Rybolt et al., 2005). All samples were kept at -20 oC for later microbiological analysis. A 10 g sample was serially diluted in sterile saline solution and E. coli was enumerated as previously described.
E. COLI CHALLENGE
At day 21, randomly selected birds (one from each pen replicate) were transferred to individual cages in environmentally controlled rooms. Two E. coli serotypes (O2 and O88) were used based on pathogenicity to poultry (Menao et al., 2002), and agglutination to Bio-Mos® (Mirelman et al., 1980). Prior to the challenge, litter samples were screened for E. coli to confirm that birds were free from the administered O2 and O88 serotypes. At day 29, birds were orally challenged with a mixed culture of E. coli (O2 and O88 serotypes) at a concentration of 1 x 107 CFU/mL of sterile PBS (pH = 7.2).
At nine days post-inoculation, the birds were euthanized and the ceca aseptically removed and collected for enumeration of total E. coli. Samples of the fresh cecal contents were serially diluted for identification and quantification of E. coli. Samples of E. coli isolates were sub-cultured on Sheep Blood Agar and O serotyped (PCR) to verify that the serotypes recovered in the ceca matched those administered. Isolates were further genotyped (PFGE) to verify that they were identical to the serotypes used in the challenge.
STATISTICAL ANALYSIS
Data were analyzed as a one-way ANOVA, using the General Linear Models (GLM) procedure of SAS (SAS Institute, 2003). Treatment means were separated using the Bonferroni’s Multiple Comparison test. Statistical significance was declared at a probability of P<0.05. All microbiological concentrations were subject to log10 transformation prior to analysis.
RESULTS AND DISCUSSION
The cecal population of lactobacilli at day 42 of the study was highest in birds fed Bio-Mos® (Figure 1). At this age, adding virginiamycin to the diet caused a major reduction in lactobacilli population. Fernandez et al. (2002) and Denev et al. (2005) reported increases in the cecal populations of lactobacilli and bifidobacteria in broilers fed mannan oligosaccharides compared to AGP-free diets. Sims et al. (2004) observed an increased cecal population of bifidobacteria in turkeys fed Bio-Mos® compared to an AGP-free diet, but there were no differences in cecal load of lactobacilli. Spring et al. (2000) also reported no effect of Bio-Mos® on the cecal population of lactobacilli in broilers. Factors contributing to variability in the effects of Bio-Mos® on populations of beneficial bacteria in the gut may include differences in experimental conditions, diet formulation, seasonal effects, and health status of the flock.
Published data on the effects of lignin on the populations of lactobacilli and bifidobacteria in the chicken gut are not available. Lignin had no effect on cecal populations of lactobacilli and bifidobacteria when compared to the AGP-free diet; however, when compared to the diet containing virginiamycin, lignin had a positive effect on the population of lactobacilli.
However, the higher level of lignin inhibited the growth of bifidobacteria (Figure 2).
Lactobacilli and bifidobacteria promote gut health by competing against potential pathogens for nutrients and binding sites (Rolfe, 2000), and by producing bacteriocins that act as antimicrobial compounds to control intestinal pathogens (Gibson and Wang, 1994; Kawai et al., 2004). The use of Bio-Mos® and low levels of lignin in the diet create a favorable environment that limits the proliferation of pathogenic bacteria and therefore may be an effective strategy to maintain the integrity and health of the gut in chickens.
Chicken litter is a potential reservoir and transmission vehicle for pathogens and potential pathogens and represents a major source of E. coli (Garrido et al., 2004; Schrader et al., 2004). Our results revealed that the litter from birds given Bio-Mos® had reduced population of E. coli when compared to birds fed the virginiamycin or CTL- diet at both day 28 and 42 (Figure 3). According to Newman (1994), Gramnegative bacteria that possess the mannose-specific Type-1 fimbriae, such as E. coli, adsorb to Bio-Mos® in the chicken gut and less is excreted in the feces. It may also be possible that E. coli remains bound to Bio-Mos®, thereby limiting E. coli proliferation in the litter. This may explain the reduced population of E. coli in the litter of Bio- Mos®-fed birds. The effect of Bio-Mos® in reducing E. coli load in the litter is consistent with the results of Stanley et al. (2000). Compared to the CTL- diet, adding virginiamycin had no effect on the litter E. coli load. Gram-negative pathogenic bacteria, such as E. coli, are resistant to most of the AGPs used in poultry production (Page, 2003), explaining our finding.
There was a tendency for lignin to reduce E. coli load in the litter. Although not statistically different from the control diets, the effects were comparable to that of Bio- Mos®. Research conducted in vitro with the lignin product used in this study demonstrated that it has inhibitory effects on growth of E. coli, Staphylococcus aureus, and Pseudomonas (Nelson et al., 1994). Although the exact mechanism of lignin action remains unclear, it has been suggested that the phenolic compounds in lignin cause cell membrane damage and bacterial lysis (Jung and Fahey, 1983).
E. coli is the principal pathogenic organism implicated in cellulitis, the major cause of carcass condemnation at the processing plants in Canada (Kumor et al., 1998). Cellulitis is characterized by subcutaneous inflammatory reaction resulting from an infection by E. coli associated with litter (Schrader et al., 2004). The findings from this study indicate that Bio-Mos®, and to a lesser extent lignin, can be used to reduce E. coli proliferation in poultry litter, and potentially to control the incidence of cellulitis.
Results of the E. coli challenge study clearly show that Bio-Mos® was effective in suppressing cecal growth of E. coli compared with the virginiamycin diet (Figure 4). Fernandez et al. (2002) reported similar findings when broilers given the mannan oligosaccharide were orally challenged with Salmonella enteritidis (PT4). The synergistic effect of Bio-Mos® in increasing the intestinal populations of lactobacilli and bifidobacteria and by acting as a decoy agent against E. coli, might explain the reduced cecal E. coli population in the challenge study. Therefore, diets containing Bio-Mos® offered a significant advantage over virginiamycin in improving the microbial ecology of the gut, reducing E. coli proliferation in the ceca and reducing the amount shed into the environment.
The challenge study indicates that both the low and high levels of lignin addition reduced the cecal population of total E. coli compared to the CTL- diet. However, the effects were more pronounced with the high lignin diet, suggesting that the inhibition of E. coli by lignin is dose dependent. Other phenolic compounds, such as carvacrol, thymol and cinnamaldehyde, have been shown to exert antimicrobial effects against lactobacilli, bifidobacteria and E. coli (Lee et al., 2004; Bozin et al., 2006).
Intestinal E. coli contaminates poultry carcasses during processing at the slaughterhouse (Heyndrickx et al., 2002), and this represents an important cause of food-borne illnesses in humans (Mead et al., 1999). Therefore, the addition of Bio-Mos® or low levels of lignin to poultry diets could be a useful dietary strategy to improve the safety of poultry products. However, in addition to a marked reduction in the cecal population of total E. coli, the HL diet inhibited the cecal growth of lactobacilli and bifidobacteria. Such an outcome is not desirable and would militate against the use of lignin in poultry diets.
In conclusion, the dietary addition of Bio-Mos® increased the cecal populations of lactobacilli and bifidobacteria, and resulted in a major reduction in E. coli load in the litter as compared with the addition of virginiamycin. A lower load of E. coli in the litter may have implications for the control of cellulitis in chickens. In E. coli challenged birds, the Bio-Mos® and 1.25% lignin treatments reduced the cecal populations of total E. coli. High levels of lignin (2.5 % of DM) caused a reduction in cecal E. coli, but the same levels of lignin inhibited the growth of the beneficial bacteria, lactobacilli and bifidobacteria. Thus, the addition of Bio-Mos® to poultry diets is an advantageous alternative to the use of virginiamycin, because Bio-Mos® promotes a better gut ecology, reduces the proliferation of E. coli in the ceca, and reduces the shedding and prevalence of E. coli in the litter, while eliminating the threat of developing antibiotic resistant strains of E. coli.
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Authors: CIRO A. RUIZ-FERIA and BUSHANSINGH BAURHOO
McGill University, Ste. Anne de Bellevue, Quebec, Canada
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