The case for mannanoligosaccharides in poultry diets. An alternative to growth promotant antibiotics?

Introduction

Antibiotics have been used in the poultry industry for the past 40 years to improve growth performance of birds. They are fed during the grow-out period to prevent infection by pathogens, maintain health and improve meat quality and wholesomeness. However, antibiotics have come under increasing scrutiny by scientists, consumers and government regulators alike because of the potential development of antibiotic-resistant bacteria (including pathogenic strains) after long use of antibiotic growth promotants in livestock and poultry feed. Antibiotic resistance displayed by field Escherichia coli isolates from North Carolina commercial turkey farms has been reported, including resistance to enrofloxacin, one of the most recently approved antibiotics for poultry (Fairchild et al., 1998). Most of the antibiotics used as growth promoters have no specific claims regarding control of disease (Gustafson and Bowen, 1997). Debate over resistance seen among Gram-negative bacteria such as E. coli and salmonella has generated the strongest objection to antibiotic use (Evagelisti et al., 1975; Scioli et al., 1983; Gustafson and Bowen, 1997). It has been reported that antibiotic resistance of indigenous E. coli in poultry has remained at a relatively high level since the 1950s (Gustafson and Bowen, 1997). Furthermore, the use of these antibiotics could disrupt or destabilize normal gut microflora (Surawicz et al., 1989).


Alternatives to antibiotics: competitive exclusion

Alternatives to antibiotics such as competitive exclusion (CE) treatments have been developed to counter the growth-depressing effects that certain strains of bacteria elicit in poultry. One type of CE aims at the development of a protective barrier bacterial population in the digestive tract to prevent colonization of unfavorable (i.e., growth-depressing and/or pathogenic) microorganisms. Some cultures have included lactobacillus species. (Francis et al., 1978) or undefined normal avian gut flora (Nurmi and Rantala, 1973). Another CE approach exploits the presence of mannose-specific (Type 1) fimbriae on unfavorable Gram-negative bacteria including many strains of E. coli and salmonella. These bacteria use the fimbriae to attach to and then colonize the intestinal wall. Mannanoligosaccharides (MOS) derived from mannan on yeast cell surfaces act as high affinity ligands offering a competitive binding site for the bacteria (Ofek et al., 1977). Pathogens with the mannose-specific fimbriae adsorb to the MOS instead of attaching to intestinal epithelial cells and consequently move through the intestine without colonization. Newman (1994) reported that the presence of dietary MOS in the intestinal tract removed pathogenic bacteria that could attach to the lumen of the intestine in this manner. This might provide a more favorable environment for nutrient utilization by the bird (Savage and Zakrzewska, 1996).

In an effort to confirm the inhibitory effect of mannanoligosaccharide on pathogen colonization reported by previous research, Spring et al. (2000) screened different bacterial strains for their ability to agglutinate MOS in yeast cell preparations (Saccharomyces cerevisiae, NCYC 1026). Five of seven strains of E. coli and 7 of 10 strains of Salmonella typhimurium and Salmonella enteritidis agglutinated MOS and Sac. cerevisiae cells. However, strains of Salmonella choleraesuis, Salmonella pullorum, and campylobacter did not lead to agglutination. They also determined the effect of MOS on cecal fermentation parameters, cecal microflora and enteric pathogen and coliform colonization in chicks. After 3-day old chicks were orally challenged with 104 CFU of S. typhimurium 29E and received 4000 ppm dietary MOS, cecal S. typhimurium 29E concentrations decreased from 5.40 to 4.01 log CFU/g (P<0.05) at day 10. A similar study using S. dublin as the challenge pathogen resulted in a decrease in the number of infected birds by day 10 from 90% to 56% (P<0.05). Dietary MOS supplementation also reduced the concentration of cecal coliforms, although less significantly (P<0.10) than with the Salmonella challenges. Dietary MOS supplementation had no effect on cecal concentrations of lactobacilli, enterococci, anaerobic bacteria, lactate, volatile fatty acids or cecal pH.

The effects of hen age, Escherichia coli, and dietary Bio-Mos and bambermycin on poult performance from 1 to 21 days were studied previously in our laboratory (Fairchild et al., 1999). Day-of-hatch BUTA (BIG-6) male poults were gavaged (1 ml) with 108 CFU/ml E. coli composed of four serotypes or sterile carrier broth. A mixture of the same E. coli cultures was added to the drinking water (106 CFU E.coli/ml drinking water) on a weekly basis to ensure a continuous bacterial challenge. Within each E. coli split plot treatment group, poults from hens of different ages (33 and 58 wk of age) were fed diets containing Bio-Mos (2 lb/ton feed) and bambermycin (2 g active ingredient/ton feed), alone and in combination, in a randomized complete block design. At week 1 and week 3, one bird per pen (n=128) was randomly chosen for bacterial sampling of liver and intestinal tissue for coliforms, aerobic bacteria, and lactobacilli. Individual bodyweight and feed consumption by pen were recorded weekly and poult mortality was recorded daily. E. coli isolates from tissue samples were serotyped. Feed conversion and weight gains were calculated. Under E. coli challenge, dietary Bio-Mos and bambermycin improved (P#0.05) poult bodyweight gains. When poults were not challenged with E. coli, dietary Bio-Mos improved (P#0.05) poult growth during week 2 while dietary bambermycin improved (P#0.05) poult growth through week 3. Cumulative three week bodyweight gains for unchallenged poults were improved (P#0.05) by both Bio-Mos and bambermycin. Two of the four E. coli serotypes administered were recovered in cultures of tissue samples. Several serotypes were recovered that were not administered. This work demonstrated that dietary Bio-Mos can improve the overall performance of poults, especially when they are faced with an E. coli challenge, as well as traditionally used antibiotics.


Effect of Bio-Mos, bambermycin and virginiamycin on growth performance of turkeys

MATERIALS AND METHODS

Recently, research at North Carolina State University was conducted to evaluate the effects of Bio-Mos and two popular in-feed antibiotics (bambermycin and virginiamycin) on the growth and performance of market turkeys. Poults were randomly assigned to 48 pens on day of hatch and raised from 1 to 140 days of age. There were eight replicate pens per dietary treatment with 20 birds per replicate placed at the start of the trial, and randomly culled to 12 birds per pen at 12 weeks of age. Dietary treatments were randomly assigned to each of 48 pens within blocks of 12 pens each. This experimental design was used to account for any variation in bird performance due to pen location in the turkey house, which might be affected by the environment such as light or temperature differences. Each treatment was replicated twice in each block of pens. The six dietary treatments were as follows:

1. Control: typical US corn/soybean meal diet
2. Bio-Mos (2 lbs/ton to 6 weeks of age then 1 lb/ ton)
3. Bambermycin (2 g/ton active ingredient)
4. Virginiamycin (20 g/ton active ingredient)
5. Bio-Mos + bambermycin (as in Treatments 2 and 3)
6. Bio-Mos + virginiamycin (as in Treatments 2 and 4)

The experimental diets are presented in Table 1. Typical US corn-soy based diets were employed to minimize possible variability due to ingredient digestibility. All diets met or exceeded NRC (1994) nutrient recommendations for turkeys. No coccidiostats were added to the feed in order to avoid any confounding effects with the dietary treatments. Dietary feed additives were added at the expense of washed builder’s sand as inert filler to avoid any differences in dilution effects among the diets. All feed was pelletprocessed and fed in crumble form up to 6 weeks of age and subsequently as 5/16” pellets.


Table 1. Composition of treatment diets fed from 1 to 140 days of age.




Facilities used in this experiment consisted of an industry-standard curtainsided house containing 48 pens each with nine square meters of floor space. Pen floors were covered with clean, soft pine shavings top-dressed with used litter from a previous flock to supply some microbial challenge to the birds. Caked litter was removed as necessary throughout the trial. Ventilation was provided by natural air movement through appropriately adjusted curtain sides and air mixing fans located on the ceiling throughout the house. Birds were offered feed ad libitum using one 22 kg capacity tube feeder per pen and water by one Plasson drinker per pen. Lighting was provided 23 hrs out of every day for the first week and subsequently by natural length daylight. Standard gas brooders provided heat for each pen. House temperature was kept at approximately 29.5°C during the first two weeks and then gradually stepped down by 2.75°C per week until brooders were no longer used. Brooder temperatures were kept at approximately 35°C at poult level and stepped down to 2.75°C each week in conjunction with house temperature. Ambient temperature outside the turkey house ranged from 37.8°C in the late summer to -4°C in the late fall. High and low ambient temperature within the house was recorded at two places daily throughout the duration of the trial.

Feed consumption (by pen) and individual bird body weights were recorded at 0, 3, 6, 9, 12, 15, 18, and 20 weeks of age. Mortality and culled birds were recorded as they occurred and their weights were used to adjust feed conversion. Body weight, weight gain, feed consumption and adjusted feed conversions were determined for each of these periods. An attending veterinarian (Rollins Diagnostic Laboratory, NCDA, Raleigh, NC) determined cause of mortality.

The data were subjected to the General Linear Models procedure for ANOVA (SAS, 1992). The pen served as the experimental unit. Variables having a significant F-test were compared using the LS Means function of SAS (SAS, 1992) and considered to be significant at P#0.05.


RESULTS

Dietary supplementation with Bio-Mos, bambermycin, and virginiamycin all resulted in the improvement of body weight (Table 2) and feed utilization (Tables 3 and 4) when fed to male turkeys. Overall, there were no further improvements in body weights when either of the antibiotics was fed in conjunction with the Bio-Mos. Bio-Mos and virginiamycin fed individually resulted in increased body weights at week 20 when compared to the control diet while birds fed the combination treatment were intermediate in body weight.

Bio-Mos, bambermycin and virginiamycin all improved feed:gain (Table 3) from 0-3 weeks of age. Virginiamycin also improved feed:gain from 3-6 weeks of age. There was a significant improvement in feed:gain from 3-6 weeks of age when Bio-Mos and bambermycin were fed in combination indicating a possible synergistic action between the two compounds. Bio-Mos and virginiamycin also improved feed:gain from 15-18 weeks of age. Cumulative feed:gain (Table 4) was significantly improved for the virginiamycin treatment from 0-6 and 0-9 weeks of age. The combination of Bio-Mos and bambermycin also resulted in improved cumulative feed:gain during these same time periods. Cumulative feed:gain from 0-18 weeks of age was slightly improved for birds fed Bio-Mos or bambermycin compared to control fed birds although these differences were not significant. However, birds fed the combination treatment of Bio-Mos and bambermycin had significantly improved feed:gain compared to the feed:gain of birds fed the control diet. Cumulative feed:gain from 0-20 weeks of age was not affected by any treatment. This may have been a result of increased variability due to uncontrollable feed wastage as the large birds became more crowded within the pens at this late age. In addition, during the 18 to 20 week period the outside temperature was much colder than average and may have resulted in increased consumption of feed for thermal regulation purposes, which may not have been available for body weight gain. There were no significant differences due to treatment observed in cumulative feed consumption, mortality, or cull rate from 0 to 20 weeks of age (Table 5).


Table 2. Effects of Bio-Mos, bambermycin and virginiamycin on the body weights of maleturkeys.




Table 3. Effects of Bio-Mos, bambermycin and virginiamycin on the period feed: gain ofmale turkeys.




Table 4. Effects of Bio-Mos, bambermycin and virginiamycin on the cumulative feed: gainof male turkeys.




Table 5. Effects of Bio-Mos, bambermycin and virginiamycin on the cumulative performance (weeks 0-20) of male turkeys.




Comparative modes of action: antibiotics and mannan oligosaccharides

The practice of feeding subtherapeutic levels of antibiotics to livestock and poultry has been in use for over fifty years. Their prevalence has become widespread because of the benefits afforded to the producer, the animal and the environment, as well as the consumer (Gadd, 1997). Antibiotics are fed to poultry because they improve the utilization of feed and increase body weight gain. Gastrointestinal disorders caused by the unhealthy microflora can influence feed intake, feed conversion, weight gain and overall animal health (Gedek, 1999). Feed-grade antibiotics may serve to improve performance by several different mechanisms, all of which center on the influence of the gut microflora on the host animal. Research has shown that it is possible to achieve superior performance when rearing birds germ-free versus under normal conditions (Visek, 1978).

The body is specifically designed to withstand the colonization and translocation of microorganisms that can cause harm or dysfunction. Skin, mucous membranes and the inflammatory response to a wound are all designed to defend the animal against microbial attack. The gastrointestinal tract is a front-line defense against the constant invasion of foreign microbes. Antigenic characteristics are deflected by the gut-associated lymphatic tissue (GALT) when challenged by one of any number of microbial agents, which in turn leads to mounting a specific immune response. Two important organs associated with the GALT are the cecal tonsils and bursa. The epithelial tuft cells of the bursa are phagocytic and can present antigens to lymphocytes within the basal cortex, which ultimately allows immunological protection against the organism (Tizzard, 2000). The cellular and humoral complements are stimulated into a defensive mode in order to fend off the attack on the host’s internal system. Along with this reaction comes an acute systemic response to infection or disease challenge (Klasing, 1988; Klasing, 1998; Cook, 2000). The acute systemic immune response can be a great consumer of metabolic energy causing alterations such as increased gluconeogenesis, increased lipolysis and an increase in the turnover rate of body proteins and amino acids (Klasing, 1988). Immunologically-challenged birds have been shown to produce several classes of cytokines that can increase metabolic rate, decrease appetite and possibly re-direct nutrients needed for immune response instead of skeletal muscle growth (Ferket and Qureshi, 1999). These actions of mounting an acute immune response may be a greater energetic drain than those of the specific immune responses, such as the manufacturing of B-cells, T-cells, macrophages and neutrophils.

Feeding antibiotics suppresses microbial growth in the gut, thereby reducing the immunological stress described above. Furthermore, antibiotics likely reduce the microbial by-products and toxins that impart negative effects on the energy needs of the animal. Zimber and Visek (1972) reported that microbial products such as ammonia (NH3) and lactic acid can increase enterocyte cell division and alter mucosal barriers. The animal must then maintain more intestinal tissue with a higher turnover rate when microbes are present, thus robbing it of metabolic energy and possibly inhibiting the maximum absorption of vital nutrients (Visek, 1978). Indeed, the feeding of antibiotics has been shown to reduce the relative weight and length of the intestines (Visek, 1978; Postma et al., 1999). This reduction in gut mass due to dietary supplementation of antibiotics is similar to that observed in germ-free birds. Stutz et al. (1983) reported reduced amounts of lamina propria, lymphoid tissue, reticuloendothelial cells, intestinal weight and moisture in germ-free birds.

Antibiotics produce their effects on the microflora by acting on the biochemical machinery of the microbial cell. Bambermycin specifically inhibits the synthesis of the bacterial cell wall by blocking the incorporation of the muramyl-pentapeptide into the cell wall peptidoglycan structure (Huber and Nesemann, 1968). In contrast to bambermycin, virginiamycin functions by inhibiting bacterial protein synthesis by acting on or binding to the 50s ribosomal subunit which blocks normal peptide bond formation (Parfait et al., 1978; Cocito, 1973; Cocito, 1978). These antibiotics have a broad spectrum of activity and act mainly on Gram-positive microbes such as lactobacillus, bifidobacterium, and streptococcus. Antibiotics have been shown to primarily reduce lactic acid-producing bacteria which predominate in the upper gastrointestinal tract of the fowl (Cummings, 1995). Lactic acid is largely responsible for many of the detrimental effects associated with the gut microflora (Cummings, 1995). Both bambermycin and virginiamycin have been shown in numerous reports to increase weight gain and improve feed efficiency when fed to poultry (Waldroup et al., 1970; Caston and Leeson, 1992; Waibel et al., 1991; Buresh et al., 1986). Much of the previously mentioned research agrees with the findings in our study. Overall, the addition of both bambermycin and virginiamycin improved body weights and feed:gain although virginiamycin was more influential on feed conversion than was bambermycin. It is noteworthy that both antibiotics and Bio-Mos improved feed:gain of poults from 0-3 weeks of age; a period when the gut
microflora is not fully developed and stabilized. Therefore, these feed additives may convey benefit by stabilizing the gut microflora and limiting the colonization of pathogens.

Controversy over the feeding of antibiotics as growth promotants in Europe has increased because of fears concerning the development of antibiotic resistant microbes stemming from the use of these substances in food animals. Trade with the European Union along with the uncertain future of feeding antibiotics in the US poultry industry makes it prudent to seek alternatives to antibiotics as growth promotants.

In contrast to the mode of action of antibiotics, which limit or suppress growth of common Gram-positive microflora, mannanoligosaccharide (Bio-Mos) and other oligosaccharides can serve to prevent attachment of Gramnegative pathogens, thereby preventing attachment onto enterocytes and subsequent enteric refection. Bio-Mos is an oligosaccharide with a terminal mannose moiety. These mannose units mimic the receptors found on enterocytes on which the Gram-negative pathogens possessing Type-1 fimbriae are able to bind. In most cases, the ability of the pathogenic microbe to attach to the host cell surface is crucial for it to colonize, produce toxins and cause enteric disease. For example, unless Vibrio cholerae can successfully attach itself to the enterocyte of the intestinal wall, it is unable to initiate disease conditions despite the presence of large numbers of bacteria (Freter, 1969). Previous research has shown that bacterial adhesion can be mediated by fimbriae on the bacterial cell surface and that mannose and similar sugars can inhibit the agglutination of bacterial cells in culture by occupying cell surface binding sites necessary for bacterial adhesion (Sharon and Lis, 1993; Duguid et al., 1966). Bacteria that bind to the mannose units on the oligosaccharide are ultimately excreted as these oligosaccharides cannot be digested by the host. Before excretion, however, the host may be still able to mount an immunlogical defense against the bacteria bound to the mannanoligosachoride because the barrier would still possess its antigenic properties as detected by the GALT.

The unique mode of action of Bio-Mos may make its application in commercial field conditions more effective than can be observed in university trials. While broad-spectrum antibiotics limit the growth of both beneficial and pathogenic bacteria in the gut, Bio-Mos may be more beneficial by being more specific. Therefore, the use and activity of Bio-Mos differ depending on the specific circumstances of the gut ecosystem. Indeed, Olsen (1995) concluded that turkeys raised under commercial farm conditions showed improvements in livability, performance, efficiency and condemnations when fed a diet supplemented with Bio-Mos. Turkeys fed Bio-Mos during a specific challenge from Salmonella typhimurium had decreased incidence of fecal contamination, while broilers fed Bio-Mos had reduced fecal counts of Salmonella dublin and E. coli (Spring et al., 1996). Schoeni and Wong (1994) also showed a reduction in Campylobacter jejuni colonization when birds were fed Bio-Mos. Fairchild et al. (1999) observed improved performance of poults challenged with field isolates of E. coli and fed Bio-Mos. These responses may not be limited only to growth. Choi et al. (1994) reported a reduction in colony-forming units of Salmonella typhimurium in broiler chicks challenged while being fed a diet supplemented with fructo-oligosaccharides (FOS) (Fairchild et al., 1999).

In contrast to antibiotics, Bio-Mos has been shown to enhance immune response. Cotter (1997) observed improved secondary and tertiary humoral immune responses during the PHA test in birds fed Bio-Mos while Savage et al. (1996) observed an enhancement of immunoglobulin production of both circulatory (plasma IgG) and secretory (IgA) levels in the turkey. The mechanism by which Bio-Mos enhances immunity is not fully understood, although it may be associated with enhanced stimulation of the GALT. The degree of immunological response differs in various studies of oligosaccharide supplementation of poultry diets. Waldroup et al. (1993) demonstrated inconsistent performance results when FOS was fed to broilers, while Kumprecht and Zobac (1997) reported improved performance of broilers fed mannanoligosaccarides (Bio-Mos). Signs of bacterial infection and immune challenge may also be a function of numbers and not just the presence of pathogens. There may exist a certain concentration threshold necessary for blatant infection to occur (Garlich, 1999). Raibaud (1992) reported that clostridium species may be well-tolerated at log 107 within the gastrointestinal tract but become potentially toxic at levels of about log 108.

In addition to inhibiting bacterial colonization and immune stimulation, Bio-Mos may also improve the structural integrity of the gastrointestinal tract. Choi et al. (1994) reported a significant increase in the length of the ileal microvilli in birds challenged with Salmonella typhimurium while being fed FOS. Increases in the jejuna villi length (Iji and Tivey, 1999) as well as the length of the small intestine (Trevino et al., 1990) have also been reported in birds fed FOS. Reasons for this response are unknown, although certain biochemical alterations due to the oligosaccharide molecule may be responsible for the long-term regulation of the gastrointestinal tract structure (Iji and Tivey, 1999).

The finding of a possible synergism between Bio-Mos and the antibiotic bambermycin is also an important aspect of this trial. Feeding the Bio-Mos in conjunction with bambermycin improved cumulative feed conversion from 0-12 and 0-18 weeks of age (Table 4). One positive role of the gastrointestinal microflora is that of competitive exclusion. The establishment of the normal microflora serves to occupy binding sites need by pathogenic bacteria to colonize the gut lining. Antimicrobial compounds produced by these microbes may also serve as a pathogen control mechanism. The feeding of antibiotics may increase the host susceptibility to pathogenic colonization because of their growth limiting effects on the normal microflora. George et al. (1982) concluded that bambermycins had no effect on the microbial resistance of Salmonella typhimurium of experimentally infected broiler chicks. Feeding Bio-Mos may limit the attachment capability of pathogens during this time when more attachment sites are available on the enterocyte, thus providing a secondary mode of protection during Gram-negative pathogenic challenge.


Summary

The bird’s gastrointestinal ecosystem is an important aspect of poultry performance and flock health. Antibiotics improve performance by modifying the normal gut microflora, although their future use is questionable. We must increase our understanding of those mechanisms governing gut health and develop novel and alternative gut health management techniques. Oligosaccharides such as Bio-Mos have been demonstrated to improve poultry performance in numerous trials, including the one reported herein. This improvement in performance may be due in part to the reduction of the pathogenic load encountered by the bird under farm conditions. Future research must continue to evaluate the effectiveness of such alternatives and help us learn more about the effects they have on health, metabolism, and nutritional status of the bird.


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