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Brazilian Journal of Poultry Science

Print version ISSN 1516-635XOn-line version ISSN 1806-9061

Rev. Bras. Cienc. Avic. vol.5 no.2 Campinas May/Aug. 2003

http://dx.doi.org/10.1590/S1516-635X2003000200001 

An alternative for antibiotic use in poultry: probiotics

 

 

Edens FW

North Caroline State University

Correspondence

 

 


ABSTRACT

Over the past 50 years, there has been increasing amounts of antibiotics used prophylactically and as growth promoters. Today, there is a consumer and governmental outcry to eliminate that practice from poultry and livestock production. Evidence has been accumulated to show that there is a link between risk of zoonotic disease and growth promoting antibiotic usage in livestock and poultry. Therefore, alternatives to the use of growth promoting antibiotics must be found to promote growth or production at or near the genetic potential of the modern day broiler, turkey, and egg producer. The use of probiotics has many potential benefits and include modified host metabolism, immuno-stimulation, anti-inflammatory reactions, exclusion and killing of pathogens in the intestinal tract, reduced bacterial contamination on processed broiler carcasses, enhanced nutrient absorption and performance, and ultimately decreased human health risk. The development of these factors generally can be ascribed to the ability of most probiotic products to balance and maintain the intestinal microflora in poultry species.

Keywords: antibiotics, antibiotic resistance, benefits, characteristics, chickens, defined culture, functions, history, human health risk, mechanism of action, probiotics, turkeys, undefined culture, zoonotic disease.


 

 

INTRODUCTION

During the past 50 years, antibiotics have been used in poultry production as therapeutic agents to treat bacterial infections that decrease performance and cause diseases. Many of the antibiotics used in the poultry industry have been used in human medicine as well. Shortly after the initiation of widespread use of antibiotics in the animal industries, they were placed under increased scrutiny because of concern over development of bacterial resistance to the usual microbiocidal effects of the antibiotics. Ever since their first usage in animals, there has been a cause for concern about the use of antibiotics in poultry and livestock production.

In June of 1999 the European Union (EU) banned the use of some growth promoting antibiotics in poultry feeds. This ban was due to very disturbing observations that potential human pathogens, frequently found on processed poultry and swine carcasses, were increasingly resistant to certain antibiotics. However, it was the determination that bacterial resistance was not due to single but to multiple antibiotics that finally resulted in the ban on the use of sub-therapeutic dosing of certain antibiotics in poultry (DANMAP 97).

In the year 2006, the EU will officially ban the usage of all antibiotics for the sole purpose of growth promotion in poultry and livestock (Halfhide, 2003). Therapeutic use of appropriate antibiotics will be allowed via prescription only through a veterinarian. The impact of this political decision will have dramatic influences on the methods used to produce broilers, turkeys and table eggs. The EU decision, to make such a drastic change in the way poultry production is practiced, was precipitated by the DANMAP 97 report (Bager, 1998). The DANMAP 97 report indicated that the use of low levels of antibiotics in food animal feed leads to the development of resistance in zoonotic organisms of animal origin. Around the world, controversy has surrounded this report, but the impact of the work has been extremely influential as it has caused unprecedented changes in the way food animal production is being conducted today.

Multiple antibiotic resistance

A major concern that resulted in the EU ban of growth promoting antibiotics in poultry was that many of the multiple antibiotic resistant strains of bacteria are capable of passing resistance factors to unrelated bacteria. Resistance develops when a bacterium survives exposure to an antibiotic that normally kills the bacterial population. Usually, a mutation occurred allowing the bacterium to survive the antibiotic exposure. That mutation could promote antibiotic resistance via (1) increased resistance to the absorption of the antibiotic through its cell wall, (2) increased metabolism of the antibiotic to a non-inhibitory form, or (3) induction of alternative metabolic products that permit circumvention of the inhibitory action of the antibiotic. Antibiotic resistance is normally passed from one bacterium to another via three different mechanisms: (a) transformation, occurring when a bacterium becomes competent and is capable of taking up DNA from surrounding fluid media, (b) transduction, occurring when there is transfer of genetic material from one bacterium to another via virus infection, and (c) conjugation, occurring when a donor bacterium joins with a recipient bacterium with DNA transfer from one bacterium to another. Whenever there is transference of DNA from one bacterium to another, the new DNA either is incorporated into the host's chromosomal DNA or it forms an element (plasmid) in the recipient's cytoplasm that is capable of replication independent of the host chromosome.

Market-based decision to replace antibiotic growth promoters

The unrelenting demand by many activist groups, calling for total cessation of the use of antibiotic growth promoters in poultry production, is not always supported by scientific justification. Nevertheless, major poultry companies (Tyson Foods, Perdue Farms, and Foster Farms) that produce one third of the chicken consumed in the United States have stated that they have voluntarily taken most if not all of the antibiotics out of the feed provided for healthy chickens. Part of the recent response by the poultry companies was due to the potential that ciprofloxacin, used to treat anthrax in humans, might contribute to antibiotic resistance in anthrax. Additionally, the poultry companies are reacting to edicts from some corporate consumers (McDonald's, Wendy's, Kentucky Fried Chicken, and Popeye's) that are refusing to buy poultry that had been treated with ciprofloxacin and now all other antibiotic growth promoters. These market-based policy developments in corporate America signaled a change of attitude in the corporate world of poultry production.

Evidence for and against antibiotic growth promoters

If one examines the scientific literature, the poultry and allied industries have long known about the potential of zoonotic diseases and the role poultry could play in their spread. Work published by Smith & Tucker (1975 a,b) clearly established a link between the prolonged shedding of Salmonella typhimurium and its development of resistance to virginiamycin, bacitracin, flavomycin, nitrovin, tylosin, sulphaquinoxaline, ampicillin, chloramphenicol, furizolidone, neomycin, oxytetracyclin, polymixin, spectinomycin, streptomycin and a mixture of trimethoprim and sulphadiazine. Smith & Tucker (1978; 1980) later studied the influence of lincomycin and avoparcin on shed rate of naladixic acid resistant (nalr) S. typhimurium and colonization of naladixic acid resistant (nalr) and naladixic acid sensitive (nals) S. typhimurium finding both increased duration of the pathogen shedding in feces and higher cecal numbers of the pathogen as compared with controls at slaughter. Since that time, an abundant amount of research with other Salmonellae has been published to support their work (Barrow et al., 1984; Barrow, 1989). Holmberg et al. (1984) also found that avoparcin-monensin treated chickens had significantly higher incidence rate of cecal and liver S. infantis than did chickens treated with avoparcin alone, but they also had a lower rate of incidence than did non-treated chickens.

However, Evangelisti et al. (1975) and Girard et al. (1976) examined oxytetracycline and neomycin each at 200 g/ton and reported that the use of growth promoting antibiotics decreases fecal shed and intestinal colonization of zoonotic organisms. Jarolmen et al. (1976) also reported that the use of a sub-therapeutic dose of chlortetracycline substantially reduced shed rate and cecal recovery of Salmonellae spp. Since the mid-1970's chlortetracycline has been banned due to its ineffectiveness in controlling bacterial infections in poultry and livestock.

Links between antibiotic growth promoters and human disease

Even though there are numerous publications showing evidence for reduction in the number of zoonotic organisms in chickens fed antibiotics, and publications also showing increased shedding and colonization rates of zoonotic organisms. The study of antibiotic resistance per se has been limited to relatively small laboratory studies. However, there are only limited data on continuous surveillance of antimicrobial resistance in farm animals. In Europe, the DANMAP 97 report (Bager, 1998) was the first and the most influential showing for the first time that there was a linkage between antibiotic growth promoter use in food animals and antibiotic resistance in pathogenic, zoonotic, and indicator bacteria in food and humans. The DANMAP 97 report was developed over an extended time and provided a comprehensive and systematic evaluation of antimicrobial resistance among farm animals and allowed scientists and medical workers to study changes in microbial resistance patterns. In France, a similar approach with cattle has been published (Martel et al.,1995). In the United Kingdom, a comprehensive review on the impact of antibiotic resistance in all stages of the food chain was published (MAFF, 1998). These reports very clearly and definitively conclude that the links between sub-therapeutic usage of antibiotic growth promoters and antimicrobial resistance among zoonotic bacteria really do exist.

A report by the US Food and Drug Administration (2000) resulted in an inconclusive summation of available data for the United States. The report suggested that (1) there was no evidence for an increased pathogen load in processed animals if they had been fed antibiotics with the exception of penicillin in swine, (2) avoparcin effects on Salmonella infection was age dependent, (3) a sufficient body of literature exists but is applicable only for Salmonella spp. infection limited to swine and poultry, and (4) a concern does exist that sub-therapeutic use of antibiotics in poultry and livestock might contribute to the prevalence of antimicrobial resistance.

An earlier report issued by the US National Research Council (1999) also considered the risks of the use of antibiotics in animal production and concluded that existing data were fragmentary and that a total ban of their use was not mandated. Coffman (2000) further reported on those conclusions and recommendations emanating from the US National Research Council work and summarized that there was no fulminating hazards from drug residues in processed meat and that sufficient efforts to limit microbial resistance existed in the USA. However, a second conclusion was even more important because the US National Research Council found that antibiotic use in food and animals is related to antibiotic resistance and development of a set of diseases that exhibit resistance in humans. This represented the first US report that found a direct link between the use of antibiotics in food animals, anti-microbial resistance, and human disease. Nevertheless, the report was tempered by a statement that the incidence of resistance was very low.

Recent evidence from scientists around the world show that the link between the use of antibiotic growth promoters in food animals and antimicrobial resistance is increasing (van den Bogaard, 1998; van den Bogaard et al. 1997; van den Bogaard & Stobberingh, 2000; Caprioli et al., 2000). Kolár et al. (2002) reported antibiotic resistance among 128 E. coli strains and found resistance to 21 of 23 antibiotics used in the poultry industry. The percentage resistance ranged between a low of 6% to a high of 97%. Additionally, Kolár et al. (2002) found 88 strains of Staphylococcus spp. and 228 strains of Enterococcus to be resistant to 13 antibiotics used in the poultry industry (Table 1). Edens et al. (1997b) studied poult enteritis and mortality syndrome (PEMS) in turkey poults and observed multiple antibiotic resistance profiles for two E. coli isolates thought to be involved in the PEMS etiology. Those two atypical E. coli isolates also were noted to alter their biochemical properties after exposure to sarafloxacin and enrofloxacin. Additionally, Edens looked at several E. coli isolates and some Salmonella commonly found in PEMS infected poults and found multiple antibiotic resistance profiles for those isolates against 10 antibiotics that had been used in the turkey industry (Table 2). The observations noted by those scientists cited here suggest that antibiotic resistance is not an uncommon event, but it is a problem of greater proportions than that already acknowledged by governmental agencies and the poultry industry.

 

 

 

 

There is growing evidence that the use of sub-therapeutic antibiotic growth promoters is associated with increased anti-microbial resistance and increased risks for human health. Sub-therapeutic usage of antibiotic growth promoters is a problem because many of the multiple antibiotic resistant strains of bacteria are capable of passing resistance factors to unrelated bacteria. Resistance develops when a bacterium survives exposure to an antibiotic that normally kills the bacterial population. Usually, a mutation occurs allowing the bacterium to survive the antibiotic exposure. Additionally, we know that antibiotic resistance develops when there is transformation, transduction or conjugation within a population of bacteria that allows transference of DNA leading to plasmid formation.

Maintenance of antibiotic resistance is an energetically expensive process for a bacterium. Removal of antibiotics that promote resistance and seeding the bird with antibiotic sensitive probiotic organisms gradually leads to development of a bacterial population that is sensitive to antibiotics. The bacteria that are used as probiotic organisms have an ecological advantage in the gastrointestinal tract because they can multiply more efficiently than the antibiotic resistant forms that must expend extra energy for maintenance of the resistance factors rather than for reproduction. Use of probiotic bacteria that have a competitive advantage constitutes the basis of the competitive exclusion (CE) concept. However, it is important to understand that bacteria with plasmid(s) bearing antibiotic resistance factors do not simply go away. Those bacteria can survive for long periods of time before they lose resistance-bearing plasmid(s) and continue to pose a potential problem to poultry production as well as to human health. As long as there are populations of bacteria in the gastrointestinal tract that express a competitive advantage, the potential pathogens can be kept in check.

This principle has been demonstrated in Sweden and in Denmark where there has been long-term poultry production without the use of sub-therapeutic antibiotic growth promoters (van den Bogaard & Stobberingh, 2000). In 1986, Sweden banned the usage of antibiotic growth promoters in animal feeds. The prevalence of resistance against antibiotic growth promoters or related compounds in fecal samples of Swedish pigs in 1997 was significantly lower than in Dutch pigs (van den Bogaard & Stobberingh, 2000), and even as early as 1994 E. coli resistance to ampicillin, streptomycin, apramycin, tetracycline, trimetoprim-sulpha, and enrofloxacin was lower in food animals in Sweden than in France and Belgium ( MAFF, 1997). In Denmark (Bager et al., 1999), Germany (Klare et al., 1999), and Italy (Pantosti et al., 1999) a decrease in the prevalence of vancomycin resistance in poultry was reported after discontinuation of its use. Thus, it is possible that antibiotic resistance profiles might be eliminated after discontinuation of antibiotic usage in sub-therapeutic levels in poultry and other livestock.

In 2001, the Animal Health Institute reported that roughly 10 million kilograms of antibiotics were used in the USA, and in that usage statistic about 13 to 17% (1,3 to 1,7 million kilograms; some groups estimate that more than 9,0 million kilograms entered animal feeds) was considered to have been used for animal growth promotion. In 1999, about 15% of all antibiotics used in the EU (about 30% in all of Europe) went into animal feeds, and this amounted to an estimated 3,52 million kilograms of antibiotics entering into the human system via pork and chicken alone. The EU has already taken action to deal with that public issue by the year 2006 when use of antibiotic growth promoters will be banned. The USA has begun to ban the use of antibiotics that are no longer effective in poultry production and has issued a warning that a risk does indeed exist for human consumers. In Brazil, with its large export markets in the EU and other parts of the world, there is a demand for poultry products that are produced in an antibiotic growth promoter-free environment.

The proceedings of a recent symposium on the application, future prospects and alternatives to the use of antimicrobials in poultry has been published. Brief articles dealing with historical development of antimicrobials for poultry (Jones & Ricke, 2003), antibiotic residues in poultry tissues and eggs (Donoghue, 2003), monitoring and identification of antibiotic resistance mechanisms (Roe & Pillai, 2003), use of probiotics and prebiotics in poultry (Patterson & Burkholder, 2003), organic acids and short chain fatty acid use for pathogen control (Ricke, 2003), and alternatives to antibiotics (Joerger, 2003) were presented. The conclusions drawn from that symposium were that (1) there is no clear and unbiased estimate of antibiotic use in the poultry and livestock industries in the United States [range 9.28 to 13.91 million kg in 1999], (2) there are few, if any, excessive levels of antibiotics in the poultry produced in the United States, (3) antibiotic resistance in bacteria can be acquired via transformation, plasmid exchange, and transduction, but a new concept is that resistance factors are transferred via integrons, (4) the concept of probiotic and prebiotic usage in poultry as an alternative to antibiotics is interesting but requires additional research, (5) organic acids, if properly utilized, are effective in suppressing bacterial growth but less than adequate use presents a risk that targeted bacteria can develop resistance, and (6) bacteriocins, antimicrobial peptides and bacteriophages may have a role in poultry production, but much work is required before they are used commercially.

Time for probiotic usage in poultry production

The ban of growth promoting antibiotic usage in the EU will ultimately affect every poultry exporting country because poultry products found to have antibiotic residues of EU-banned products or to harbor bacteria such as S. typhimurium DT104, Staphylococcus aureus, Acinetobacter, Listeria moncytogenes, Enterococcus faecalis, pathogenic Escherichia coli, or Camplyobacter jejuni, that have multiple antibiotic resistance profiles, likely will be refused. Provision of a healthy food supply is the primary goal of the global poultry industry, and in order for the global industry to remain viable in the 21st century, it must be able to make changes to meet the demands of the consumers of its products. When consumers of a product develop an idea or perception about a product, no matter that there is no scientific data to support or refute the perception, that perception becomes real for that consumer. If large numbers of consumers accept those perceptions, then pressures are placed upon the producer to make the product conform to the standards set by the consumer. Thus, alternatives to sub-therapeutic antibiotic growth promoters must be developed. Therefore, this review will address the concept of probiotics for use in the poultry industry as an alternative to antibiotic growth promoters.

What are probiotics?

Havenaar & Huis in't Veld (1992) modified the definition for probiotics offered by Fuller (1992), and that definition is as follows: "a mono-or defined mixed-culture of live microorganisms which, applied to animal or man, beneficially affect the host by improving the properties of the indigenous gastrointestinal microbiota, but restricted to products that (a) contain live microorganisms (e.g., as freeze-dried cells or in fresh or fermented product), (b) improve the health and well-being of animals or man (including growth promotion of animals), and (c) can have their effect on all host mucosal surfaces, including the mouth and gastrointestinal tract (e.g., applied in food, pill, or capsule form), the upper respiratory tract (e.g., applied as an aerosol), or in the urogenital tract (local application)". The definition is very broad and provides a basis for the use of numerous bacteria and yeast for the enhancement of health and well being in host animals. However, there might be some misunderstanding of the definition because there are other terms that describe similar concepts and these include direct-fed microbials, competitive exclusion agents, and synbiosis.

Direct-fed microbials (DFM), originally described as probiotics, have been defined by the US FDA as a source of live, naturally occurring microorganisms. Under this definition, a DFM does not have to be defined with identification of each organism in the mixture. This definition then applies to use of undefined cultures from the cecal contents of healthy chickens for the expressed purpose of facilitating early colonization of the chicken's intestinal tract with bacteria that will inhibit the growth and colonization of harmful bacteria. This definition incorporated the Nurmi concept of competitive exclusion (CE). The term "competitive exclusion" was applied first in poultry by Lloyd et al. (1974) but in actuality Nurmi & Rantala (1973) and Rantala & Nurmi (1973) were the first to use the concept in poultry production.

In traditional terms, CE in poultry has implied the use of naturally occurring intestinal microorganisms in chicks and poults that were ready to be placed in brooder house. Nurmi & Rantala (1973) and Rantala & Nurmi (1973) first applied the concept when they attempted to control a severe outbreak of S. infantis in Finnish broiler flocks. In their studies, it was determined that very low challenge doses of Salmonella (1 to 10 cells into the crop) were sufficient to initiate salmonellosis in chickens. Additionally, they determined that it was during the 1st week post-hatch that the chick was most susceptible to Salmonella infections. Use of a Lactobacillus strain did not produce protection, and this forced them to evaluate an unmanipulated population of intestinal bacteria from adult chickens that were resistant to the S. infantis. On oral administration of this undefined mixed culture, adult-type resistance to Salmonella was achieved. This procedure later became known as the Nurmi or CE concept.

Synbiosis is a term that encompasses two different concepts, specifically, provision of a prebiotic and a probiotic in the same product. First, a prebiotic is an indigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon (Gibson & Roberfroid, 1995). The definition of prebiotic overlaps with that of a dietary fiber. Thus, a synbiotic must contain, as an example, fructooligosaccharides (FOS) that are naturally occurring indigestible short chain fructose polymers found in artichokes, chicory root, garlic, banana, onion, barley, wheat, rye, tomato, asparagus root, brown sugar and honey constituting the fiber used for Bifidobacteria fermentation resulting in lactic and acetic acid production that will kill acid sensitive bacteria and promote the growth of acid loving bacteria such Lactobacillus (Gibson & Roberfroid, 1995). A synbiotic relationship between a prebiotic substance and a probiotic organism suggests synergism, and in this case, provision of FOS would promote indigenous Bifidobacteria and indirectly promote Lactobacillus spp. resulting in direct benefit for the host (Schrezenmeir & de Vrese, 2001). Thus, provision of FOS will selectively promote healthful Bifidobacteria and Lactobacillus through acid production in the intestine, and these events will tip the balance of the gut microecology in favor of beneficial bacteria away from E. coli, Salmonella, Clostridium, Campylobacter, Citrobacter, and other potential pathogens. Maiorka et al. (2001) have shown that the use of a synbiotic composed of Saccharomyces cerevisiae cell walls and the spore forming Bacillus subtilis was an alternative to the use of antibiotics in broiler feed. They reported improvement in feed conversion and weight gain compared with antibiotic and control treatments at 45 days of age. Additionally, Fukata et al. (1999) looked at the use of probiotics and FOS singly and in combination in chicks and its effect on Salmonella enteritidis. At 1 day and 7 days post inoculation in both FOS and FOS plus probiotic groups, there were significantly less S. enteritidis than in the control group. In 7- and 21-day-old chicks, few changes were noted in the number of total bacteria, Bifidobacterium, Bacteroides, Lactobacillus, and E. coli in the cecal contents of treated groups compared with the control group. Low-dose feeding of FOS in the diet of chicks with a low dose of probiotic reduced susceptibility to Salmonella colonization.

History of probiotics

The idea that intestinal bacteria played a role in maintenance of health was originated by Metchnikoff (1907) when he studied "lactic acid bacteria" in fermented milk products and their use to increase longevity and maintenance of youthful vigor in humans. His landmark publication sparked research efforts around the world, and by the 1930s, evidence was accumulating to show that normal intestinal microflora inhibited the growth of intestinal pathogens.

However, the work by Greenberg (1969) in a report dealing with total exclusion of S. typhimurium from maggots of blow flies, although not related to either human or food animals, demonstrated that one species of bacterium more vigorously competed for receptor sites in the intestinal tract than did another species. He observed that S. typhimurium would only survive if there was a reduction or elimination of the normal intestinal microflora. Since that time, several terms have been developed to describe the concept of competitive exclusion through the use of defined probiotics or undefined mixtures from adult chickens (van der Waaij et al., 1971).

Competitive exclusion against Salmonella can be induced by exposure of day-old chicks to fecal and cecal bacteria from healthy adults. Indeed, in some European countries, fecal and cecal contents have been used to induce competitive exclusion in growing poultry (Wierup et al., 1988). Several attempts were made to isolate pure cultures of protective microflora (Impey et al., 1982, 1984) and mixed cultures (Impey et al., 1982, 1984; Stavric, 1987; Stavric et al., 1985; Gleeson et al., 1989) to induce competitive exclusion of Salmonella, but Mead & Impey (1986) reported that there were many problems associated with this method because at that time there was a lack of adequate selective isolation media that would permit the detailed analysis of the cecal microflora, a lack of clear means to determine in vitro whether a particular strain was protective, and the need to identify all component cecal microorganisms. However, during the past decade a considerable amount of work has been done and significant advances in our knowledge of probiotic organisms has been made. Nevertheless, Mead (2000) pointed out that less than 25% of the intestinal microflora have been characterized in healthy chickens and that valid in vitro selection criteria for probiotic usage remain to be demonstrated.

In recent years, defined cultures have become increasingly important for use as probiotics. A competitive exclusion culture containing a mixture of 29 different bacterial isolates obtained from the cecae of healthy broiler chickens was developed utilizing continuous-flow culture techniques (Nisbet, 2002). This culture (CF3) was shown to be efficacious in controlling gut colonization by enteropathogens in both experimentally infected broilers and under commercial field conditions. In day-old broiler chicks provided CF3 and challenged with 10,000 CFU S. typhimurium, greater than a 99% reduction in Salmonella cecal colonization levels was observed compared to control chicks. Similarly, CF3 was shown to protect experimentally infected broiler chicks from cecal colonization by S. enteritidis phage types 4 and 13 (Nisbet, 2002), S. gallinarum (Nisbet et al., 1998), Listeria monocytogenes (Hume et al., 1998), and E. coli O157:H7 (Nisbet, 2002). A commercial product was developed from CF3 and is sold under the tradename PREEMPT. In a Food and Drug Administration approved, double blinded, pivotal field trial, chicks treated with PREEMPT had significantly fewer Salmonellae than untreated chicks at the end-of-grow-out. This product is the first of its kind available to the U.S. poultry industry. However, the first truly successful commercial probiotic CE product (Broilact®) was developed in Finland (Schneitz et al., 1990). Broilact® is a selected mixture of 22 strictly anaerobic rods and cocci and 10 different facultatively anaerobic rods and cocci derived from the ceca of an adult healthy hen. Broilact® has been used to protect chicks from intestinal colonization and invasion of the heart, liver and spleen by S. enteritidis and S. typhimurium (Schneitz et al., 1990; Bolder et al., 1992; Cameron & Carter, 1992; Nuotio et al., 1992; Methner et al., 1997; Schneitz & Hakkinen, 1998). It decreased Campylobacter jejuni numbers in the cecal contents (Bolder et al., 1995; Hakkinen & Schneitz, 2000), and it provided good protections against E. coli and E. coli O157:H7 (Hakkinen & Schneitz, 1997).

Before development of these products for the poultry industry, there were numerous probiotic products with either single or multiple organism composition. Edens et al. (1997a) reviewed the in ovo and ex ovo use of Lactobacillus reuteri, which was the basis for a single organism product that has the unique distinction of being the only probiotic that can be applied directly to the chicken or turkey embryo. Lactobacillus reuteri is now used in several probiotic preparations made in the USA and in Japan. Casas et al. (1998), Edens et al. (1997a), and Dobrogosz et al. (1989) have suggested that L. reuteri, through its ability in vivo to produce reuterin, confers an ecological advantage that allows L. reuteri to play a modulating role in the growth of all enteric microflora (Axelsson et al., 1989; Chung et al., 1989; Talarico & Dobrogosz, 1989; Talarico et al., 1988). It is known that: (1) L. reuteri normally resides in the GI tract of healthy chickens with the highest numbers found in the crop and ceca (Casas et al., 1993). Inclusion of lactose (whey) as a prebiotic in the diet increases the number of L. reuteri found in the ceca (Edens et al., 1991; Parkhurst et al., 1991), and dietary lactose supplements have been reported to reduce the numbers of Salmonella found in the GI tract of chickens and turkeys (Corrier et al., 1990a,b; Edens et al., 1991; Parkhurst et al., 1991; Hinton et al., 1990). (2) L. reuteri cells are able to convert the natural substrate glycerol into reuterin, which is secreted by L. reuteri and has potent antimicrobial activity. (3) Reuterin in concentrations as low as 10 to 30 µg/mL can kill Salmonella, Escherichia, and Campylobacter (and other bacteria) within 30 to 90 minutes. (4) The number of chickens and turkeys that test positive for Salmonella increases significantly when they are harvested and delivered for processing, but L. reuteri fed birds had fewer numbers of pathogens.

Among all of the intestinal microflora in the avian intestinal tract, the Lactobacillus genus predominates similar to the condition found in the mammalian gastrointestinal tract (Fuller, 1977, 1978; Soerjadi et al., 1981; Sarra et al., 1985; Axelsson & Lindgren 1987). In the chicken, there are three predominant species of Lactobacillus (L. reuteri, L. salivarius, and L. animalis), but only L. reuteri has the potential to produce reuterin, a clearly defined antimicrobial substance that is an intermediary metabolite of glycerol (Axelsson et al., 1989; Chung et al., 1989; Talarico & Dobrogosz, 1989). This observation is significant as one considers the importance of Lactobacillus in the process of competitive exclusion of organisms such as the Salmonella, Campylobacter, Listeria, Enterococci, and E. coli from the intestine of the domestic fowl.

Even though there is ample evidence for beneficial bacteria competing and excluding potential pathogens in the intestinal tract of chickens and other animals, there is evidence that competition among pathogens also occurs. Casas et al. (1994) reported that chickens given L. reuteri in ovo or as a pre-hatch spray onto the hatching eggs had improved livability when challenged in-hatcher with S. typhimurium and given Staphylococcus HI post hatch. When S. typhimurium and Staphylococcus HI were administered concurrently, mortality was reduced by approximately 50% compared with S. typhimurium alone (Figure 1). The Staphylococcus HI challenge resulted in very low levels of mortality, but the persistence of that organism in the ceca could possibly influence respiratory and wound healing functions later in the life of the turkey.

 

 

Recently, Rabsch et al. (2000) reported that S. gallinarum competitively excluded S. enteritidis. In fact, those investigators suggest that S. gallinarum was the primary agent that competitively excluded S. enteritidis early in the 20th century, but when S. gallinarum was eradicated from commercial poultry, S. enteritidis replaced S. gallinarum in its niche. Thus, competition for receptor sites in the intestinal tract is a complicated process and depends upon the ability of a species or strain to produce substances that will kill other similar or dissimilar bacteria or produce adhesins that allow that bacterium to bind to the intestinal mucosa more tightly than another bacterium. In essence, competitive exclusion occurs daily in the lives of poultry and other animal species. Therefore, it is important that the mode of action of a probiotic be determined, and it is important to use that knowledge to select organisms that will actively compete against known pathogens.

Mechanism of action of probiotics

Much of our perception about the function of probiotic organisms in poultry is based upon work done in mammals, specifically humans (Kopp-Hoolihan, 2001), but the same principles might not always be the same in the avian species. Nevertheless, a delicate balance among microbes in the gastrointestinal tract of chickens provides the necessary protection that prevents invasion of a multitude of potential bacterial and protozoan pathogens that can disrupt the normal body functions of poultry. Animals and humans alike have developed an elaborate defense strategy whereby a symbiotic relationship has evolved in which commensial microorganisms actually protect and provide to the host certain benefits. Among these beneficial effects is modification of the host immune system (Table 3). In order for this mutual relationship to flourish, a complex physiological and host defense mechanism must be established. Once established, the microbiota of the gastrointestinal tract prevent colonization by other bacteria. The mechanisms used by one species of bacteria to exclude or reduce the growth of another species are varied, but Rolfe (1991) determined that there are at least four major mechanisms involved in the development of a microenvironment that favors beneficial microorganisms. Beneficial microorganisms possess certain favorable characteristics that allow for the expression of several mechanisms that prevent pathogens from colonizing the intestinal tract (Table 4). These mechanisms are listed as follows: (1) creation of a microecology that is hostile to other bacterial species, (2) elimination of available receptor sites, (3) production and secretion of antimicrobial metabolites, and (4) competition for essential nutrients.

 

 

 

 

Creating a gut microecology favorable to beneficial microorganisms

The balance among the gut microflora and the host in both mammals and birds can be challenged on a daily basis because there are potential invasive microorganisms living in our common environments. Those potential invasive microorganisms can be commensial (they live in the intestinal tract but cause no problems when there is a normal balance among microbiological species) or nosocomial (opportunistic invaders living outside the body). The water we drink, the food we eat and the air we breathe have these potential invaders present and ready to challenge the symbiotic relationship between the host and the gut microbiota. Because of this constant state of siege, elaborate defense mechanisms have evolved to cope with the potential invaders (Table 4). All food, once ingested must be subjected to gastric pH in the range of 2.0 to 4.0, which can cause a 10 to 100 fold killing of bacteria in the food being digested in the upper part of the gastrointestinal tract. The microecology of the intestinal tract is the determining factor in the viability of specific microorganisms. The production of volatile fatty acids at a pH below 6.0 is known to decrease the populations of Salmonella and Enterobacteriacea (Maynell, 1963). Disruption of the normal intestinal microbial populations with antibiotics will abolish this protective mechanism because the concentrations of volatile fatty acids produced by the intestinal bacteria will decrease and gut pH will increase toward a more alkaline range. In newly hatched chicks in commercial hatcheries, the volatile fatty acid concentration and pH are not sufficient to chemically suppress pathogens (Barnes et al., 1979, 1980a,b), and therefore, supplementation of probiotic microorganisms will be very beneficial.

A good balance of beneficial microorganisms provided through supplemental probiotic bacteria prevents adaptation of ingested and transient pathogenic microbes. It is critical to apply probiotic products as early as possible to achieve the best results in poultry (Casas et al., 1993, 1998; Edens et al., 1997a). Furthermore, some products must be provided constantly for the best results, and some products can be provided as a bolus at the time of placement for excellent but possibly transitory effects in the exclusion of certain pathogens.

As soon as a chicken hatches into an environment that is heavily contaminated by bacteria, viruses, and protozoans, it must begin to develop protective gut microflora. The gastrointestinal tract of the chicken and turkey is practically void of beneficial bacteria at the time of hatching, and in some cases, a period of five to seven days after hatching is required to establish a healthy population of lactic acid bacteria in the gut. Because the lactic acid bacteria can survive and grow in both aerobic as well as anaerobic environments, they become the dominant bacteria throughout the gastrointestinal tract from the crop through the large intestine. Due to the abundance of substrates, the lactic acid bacteria thrive in the gut and produce lactic acid and hydrogen peroxide in addition to antibacterial substances such as bacteriocins, reuterin, nisin, or lactococcins (Table 5) all of which are known to have inhibitory effects on enterobacteriacea genera such as E. coli and Salmonella spp., and other bacteria such as Staphylococci spp., Clostridium spp., Listeria spp. both in vitro and in vivo.

 

 

Before the development of lactic acid bacterial populations in the gut, the newly hatched chicken begins to pick-up coliforms and streptococci from its environment. These bacteria can be beneficial or they can be pathogenic. Because there is a delay in the development of a population of beneficial bacteria, the potential for colonization by pathogenic strains can be elevated, but usually, if the dam has done her job properly, maternal antibodies can help to prevent pathogen colonization. Nevertheless, under normal conditions, a three to five week period is required for development of a stable population of gut associated bacteria, and it is in the ceca where the greatest numbers reside (Sarra et al., 1992).

In the ceca, an anaerobic environment develops and favors the growth of organisms such as Bifidobacterium spp. and Bacteriodes spp. In this strictly anaerobic environment those named bacteria along with other lactic acid bacteria create a microecology that can be characterized by an acid pH resulting from the production of volatile fatty acids (acetic, butyric, propionic, and lactic acids) and antimicrobial substances (Table 5) that effectively exclude or kill many different pathogens.

Elimination of available receptor sites

The adhesion of microorganisms to the gut epithelium is mediated through polysaccharide-containing components attached to the cell wall (Soerjadi et al., 1982). An acidic polysaccharide cell wall component mediates adherence of common bacteria to each other and to the intestinal epithelium preventing other bacteria from attaching to the epithelium, effectively blocking all receptor sites (Fuller, 1975). However, a multitude of other mechanisms also exist. Recently, it has been shown that it is possible for healthful organisms to express complex carbohydrates similar to the cell surface adhesins found on potential pathogens. Neeser et al. (2000) demonstrated that Lactobacillus johnsonii La1 had two major carbohydrate-binding specificities. These were the O-linked oligomannosides and the gangliotriosylceramide and gangliotetraosylceramide (asialo-GM1). Similar carbohydrate-binding specificities are known to be expressed on several enteropathogens. Thus, L. johnsonii can inhibit the binding of the pathogens to the mucosal epithelial mannan receptors. Gusils et al. (2000) have shown that chicken L. animalis and L. fermentum utilize a lectin-like structure that has glucose/mannose as specific sugars of binding. Addition of mannose or sialic acid to culture media reduced adhesion of chicken L. fermentum to host specific epithelial cells. Chicken L. fermentum decreased adhesion to host-specific epithelial cells of S. pullorum by 77%, and L. animalis reduced adhesion by S. pullorum, S. enteritidis, and S. gallinarum by 90%, 88%, and 78%, respectively.

However, a report by Lee et al. (2000) suggested that even though probiotic bacteria such as L. rhamnosus GG and L. casei Shirota have similar carbohydrate-binding specificities compared with E. coli, they do not prevent binding of the pathogen to intestinal cells even if adequate probiotic cell numbers are present. If adequate numbers of probiotic bacteria are present, the probiotic bacteria appeared to inhibit E. coli adhesion to intestinal cells. The competition among probiotic and pathogenic bacteria is complex and very competitive. In the intestinal lumen, the Lactobacilli can be displaced by pathogens if the numbers of Lactobacilli decline. The mucus layer on the intestinal cells plays a significant role in the adhesion of probiotic and the pathogenic bacteria. Some probiotic bacteria have very high affinities for mucus binding sites and others have low affinity. Furthermore, pathogenic bacteria have variable affinities for binding sites on the mucus layer. If a probiotic bacterium has multiple binding sites in mucus and on the intestinal cell surface, its ability to exclude pathogens might be improved. Thus, it is important to provide the highest number of probiotic bacteria as possible and as soon as possible to achieve the best results in the control of pathogenic bacteria.

Competition for available binding sites on the intestinal mucosa is also influenced by the pH of the luminal contents. Fuller (1977, 1978) has demonstrated that an acid pH favors the survival of acid loving bacteria such as the Lactobacilli. Therefore, larger numbers of the Lactobacilli will bind to the intestinal mucosal epithelial cells and exclude pathogens such as Salmonella and E. coli. Furthermore, the composition of the medium in which the probiotic is growing will influence the adhesion of the organism to the mucosal epithelium and affect its resistance to acid (Fuller, 1975).

The contents of the digestive tract are always moving. The transit of the intestinal contents is influenced by the microbes, both free and attached, that exist in the intestinal lumen, and the motility or peristalsis of the intestinal tract affects the ability of pathogens and probiotic bacteria to attach to the epithelial cells in the lumen (Savage, 1977). Many of the beneficial microbiota can stimulate lower gut motility via production of short chain fatty acids and decreasing pH (Ohashi et al., 2002). The involvement of mucus in the ability of microbe to attach to the underlying epithelial cells is influenced by the carbohydrate and protein content of the mucin (Mikelsaar et al., 1987). It is apparent that Lactobacilli require the mucin for their attachment, and if the mucin content decreases, the beneficial Lactobacilli numbers also decrease (Mikelsaar et al., 1987). However, some pathogens have evolved to take advantage of this response in the gut and even increase the rate of mucin degradation (Mikelsaar et al., 1987). Additionally, the beneficial Lactobacilli also metabolize both protein and sugar content of the mucin and use it for energy and growth.

There has been a significant amount of speculation regarding modulation of mucosal immunity in animals given probiotic microorganisms. The influence of probiotic microorganisms has been reviewed extensively (Marteau & Rambaud, 1993; McCracken & Gaskins, 1999; Perdigón et al., 1995). Because the gastrointestinal tract contains the majority of all of the immuno-competent cells in humans and other animals, local stimulation of gut associated lymphoid tissues can provoke a generalized systemic response (McCracken & Gaskins, 1999). Sanders (1999) has summarized numerous immuno-modulator events in human and animal models given probiotics. Probiotic bacteria are capable of enhancing both specific and nonspecific immune responses by activating macrophages, increasing cytokine production by intraepithelial lymphocytes (IEL), and increasing levels of immunoglobulins especially IgA (see Table 3). The immunoglobulin IgA is the most active in the gut and inhibits bacterial colonization via agglutination and direct binding to attachment sites. Cross et al. (2002) have shown enhanced production of Th1 and Th2 cytokines in ovalbumin primed mice fed L. rhamnosus HNOO1 bacteria. In rats, L. casei has been shown to induce mucosal IgA levels thereby improving the surface epithelial immunological barrier (Malin et al., 1996). However, it has been shown that all probiotic organisms do not act to induce the same immunological functions in the gastrointestinal tract and that proper strain selection or probiotic product with the desirable probiotic strains will affect the outcome of treatment (Maassen et al., 1998).

The poultry literature concerning these processes is very meager. Casas et al. (1998) reported that turkey poults given L. reuteri had enhanced humoral antibody levels against S. typhimurium, and this appeared to be highly correlated with increased numbers of ileum IEL CD4+ (helper) T-cells that function to expand the humoral immune response. On the other hand, the number of ileum IEL CD8+ (cytotoxic) T-cells were not different in L. reuteri-fed poults. The ileum CD4+/CD8+ ratio in L. reuteri-fed poults increased from 2 to 3.5, but in the duodenum, where few to no L. reuteri reside, the CD4+/CD8+ ratio was not affected. Dalloul et al. (2003) report that a Lactobacillus-based probiotic treatment given to chickens challenged with Eimeria acervulina sporulated oocysts resulted in larger numbers of IEL CD3+, CD4+, CD8+, and a b TCR than those on a control diet. Probiotic-fed chickens also shed fewer oocysts than controls.

Laying hens given probiotics have given variable results. Balevi et al. (2001) reported that probiotic treatment had no significant influence on peripheral immune response. Panda et al. (2003) reported that 64 weeks old Leghorn hens, given probiotic therapy, had increased cutaneous basophilic hypersensitivity responses against phytohemagglutinin and had higher antibody titers against sheep red blood cells. Casas et al. (1998) actually observed a decreased cutaneous basophilic hypersensitivity to phytohemagglutinin antigen, but attributed the smaller response to intensive recruitment of T-cells to the ileum in L. reuteri-fed broilers.

Production and secretion of antimicrobial metabolites

Many of the probiotic organisms that produce antimicrobial substances often times will have an advantage over organisms that grow and compete vigorously for intestinal sites for colonization. Antimicrobial substances produced and secreted by natural inhabitants of the intestinal tract can either kill or inhibit growth of pathogens (Rolfe, 1991). Generally, most bacteria produce agents that either kill or inhibit related species or even different strains of the same species of bacteria (Iglewski & Gerhardt, 1978). Some of the inhibitory products produced by probiotic bacteria can be seen in Table 5. These products include the short chain volatile fatty (lactic, propionic, butyric, and acetic acids), hydrogen peroxide, and diacetyl and each has a different mode of action.

Additionally, there are metabolic products frequently classified as bacteriocins (Table 5) to distinguish them from antibiotics. Bacteriocins are produced by a large variety of organisms and the bacteriocins are frequently mediated through plasmids (Mishra & Lambert, 1996). Bacteriocins are proteinaceous compounds of bacterial origin that are lethal to bacteria other than the producing strain. It is assumed that some of the bacteria in the intestinal tract produce bacteriocins as a means to achieve a competitive advantage, and bacteriocin-producing bacteria might be a desirable part of competitive exclusion preparations (Joerger, 2003). In this capacity, the acid-loving Lactobacilli have shown that as a group, they produce significant amounts of bacterial growth inhibitory substances such as nisin and reuterin. Nisin is generally recognized as safe. Its mode of action is as a targeted membrane-permeabilizing peptide that induces pore formation in bacteria (Breukink et al., 2003). Reuterin, a product of glycerol metabolism that is secreted by L. reuteri, has broad-spectrum killing abilities in the intestinal tract of chickens (Dobrogosz et al., 1989; Talarico et al., 1988, 1990; Talarico & Dobrogosz, 1989, 1990). Bacillus subtilis now used as an oral probiotic organism has a wide range of antimicrobial activities associated with a serine protease called subtilisin. It has been demonstrated that Bacillis subtilis facilitates the growth of another probiotic organism, L. reuteri, through production of catalase and subtilisin (Hosoi et al., 2001). Colicin is produced by E. coli to enhance their competitiveness in the gut of animals. Colicins are plasmid-encoded polypeptide toxins produced by and active against E. coli and closely related bacteria. The channel-forming colicins are transmembrane proteins that depolarize the cytoplasmic membrane, leading to dissipation of cellular energy (Parker et al., 1992; Braun et al., 1994).

Competition for essential nutrientes

Competition for available nutrients as a means to control intestinal bacterial populations is probably not the most effective means for CE. Rolfe (1991) indicated that there were many environmental factors that come into play that either enhance availability of nutrient from the diet of the host or through manipulation of dietary ingredients that enhances the growth of certain microbial populations which may result in exclusion of other bacterial species. A normal balance of bacteria in the gastrointestinal tract is capable of utilizing all of the potential carbon sources in the environment (Freter et al., 1983). It has been shown that by manipulating the lactose concentration in the diets of chicks and poults, one can selectively provide an advantage for the enhancement of L. reuteri (Casas et al., 1993, 1998). Behling & Wong (1994) gave day old chickens an E. coli (O75:H10) with 2.5% dietary lactose and found that there was significant protection against S. enteritidis. Using this method of deduction, provision of certain types of feed ingredients may also enhance the presence of certain other types of gut microflora. Oyofo et al. (1989a) studied in vitro the effect of mannose on the colonization of S. typhimurium in chickens. They incubated intestinal sections, isolated from one-day-old chickens, with either radiolabeled-S. typhimurium strains ST-10 and ST-11 (mannose-sensitive), or strains Thax-1 and Thax-12 (non-yeast-agglutinating strains), or with only phosphate buffered saline in the presence of D-mannose, arabinose, methyl-a-D-mannoside, or galactose. The incubation of intestinal sections with bacteria and mannose resulted in a significant reduction of S. typhimurium adherence. This same group of investigators also confirmed this result in vivo (Oyofo et al., 1989b). When they gave mannose orally to chickens and subsequently challenged the chickens with S. typhimurium, they reported that mannose inhibited S. typhimurium colonization to the intestine. In a different study, Oyofo et al. (1989c) tested other carbohydrates such as dextrose, sucrose, and maltose with little if any inhibition of colonization.

Since bacteria use lectins on their cell surface to bind to mannan on the intestinal epithelial cells to initiate attachment and colonization, it has been suggested that mannanoligosaccharide (MOS), a yeast cell wall derivative, might inhibit the colonization of bacteria to the intestine by binding to bacterial mannan-binding lectin. Spring et al. (2000) report that MOS (BioMos, Alltech, Inc., Nicholasville, KY USA) acts to bind and remove pathogens from the broiler chicken intestinal tract and stimulate the immune system. Swanson et al. (2002) investigated whether supplemental BioMos influenced microbial populations in dogs. Dogs treated with BioMos were shown to have a higher number of Lactobacilli that produce lactic acid as their major end product during fermentation of carbohydrates. Not only does BioMos inhibit the attachment of some enteropathogenic bacteria to the intestinal epithelium, but it also alters the numbers of the broiler chicken intestinal microflora (Spring et al., 2000). Fernandez et al. (2002) investigated the effect of BioMos on the number of microflora in chickens and showed that there was increased numbers of Eubacterium spp. and Enterococcus spp. while the number of Bacteroides spp. were found to be decreased. The increased number of these bacteria probably indirectly inhibited the colonization of pathogenic bacteria by preventing their attachment to the gastrointestinal epithelial cells. In a study in young turkeys fed BioMos, Bradley et al. (1995) observed improved body weight and altered ileum morphology. In the ileum, the crypt depth was less and the number of goblet cells per mm of villus were increased significantly. Edens et al. (1997a) reported an increase in goblet cell numbers and mucus secretion in the intestine of chickens challenged with S. typhimurium, but this condition was corrected by the application of a probiotic.

A recent study in mice has shown that Saccharomyces cerevisiae var. boulardii in mice stimulated secretory IgA production (Rodrigues et al., 2001). Saccharomyces cerevisiae NCYC 1026 is the basis for BioMos. BioMos also has been reported to exert an immuno-stimulatory characteristic. The levels of IgG in serum and IgA in bile and cecum were elevated in turkeys and rats, respectively, fed with BioMos compared to control (Kudoh et al., 1999). In addition, pigs fed BioMos had an increased number of blood lymphocytes (Spring & Privulescu, 1998). The elevated levels of IgA may be associated with increased rate of bacterial clearance via antibody-mediated phagocytosis.

Use of prebiotics such as fructooligosaccharide (FOS) can serve as a fiber source for certain microbial populations and enhance production of organic acids in the gut. Furthermore, use of mannanoligosaccharide (MOS) can bind to receptors on many bacterial pathogens themselves preventing their attachment to epithelial binding sites and modify intestinal commensial microorganisms.

Stress factors affecting probiotic performance

Use of probiotics for poultry production is not without certain risks and limitations. There are many stress factors in the environment of newly hatched poultry species that could reduce the effectiveness of the maternal antibody defense mechanism and normal colonization of the gut by beneficial microorganisms effectively allowing the colonization of pathogens during the early post-hatch stage. This seems to be somewhat ironic because there is evidence that probiotics can limit the consequences of exposure to stressors of many types. Some of the stress factors and causes of the stress are listed in Table 6.

 

 

The factors listed in Table 6 show that there are high probabilities that newly hatched chickens and turkeys will face a situation in commercial as well as in experimental settings that will alter the development of natural gut-associated beneficial microorganisms. The primary factor affecting this development can be the feed source and quality. Under-formulated diets result in nutritional stress and decrease the growth of beneficial organisms. Molds and mycotoxins further add to the problem of nutritional stress and can cause the loss of essential nutrients for the gut microbes. However, nutrient degradation may be the most important factor to affect the gut microbes. This can be caused by numerous factors such as oxidized dietary fat and lipid peroxidation, vitamins, amino acids and proteins also influence the populations of beneficial organisms in the gut, but in this era of concern about microbial contamination of feed, higher and higher pelleting temperatures in feed manufacturing causes the destruction of not only pathogenic but beneficial organisms as well. The only probiotic organism that can tolerate relatively high temperatures associated with the pelleting of chicken and turkey feed are the spore-forming Bacilli. All other probiotic organisms will die as a result of pelleting. Therefore, most probiotics must be applied via drinking water or as a top dressing to pelleted feed.

Exposure of chickens and turkeys to extreme conditions in the environment can induce nonspecific stress responses leading to depressed immuno-responsiveness that will influence gut microbial populations. Unfortunately, the depression in the production of immunoglobulins, specifically IgA, tends to influence pathogen growth more than beneficial microbes. Many managerial stressors such as beak and claw trimming and other hatchery processes such as vaccinations and handling for sexing and high population densities after placement contribute to immuno-suppression in poultry. However, we always come back to antibiotic use/abuse in the poultry industry. Over use of antibiotics can have very negative effects in the young bird. In some commercial operations, it is common practice to add high levels of antibiotics to the first feed given to chickens and turkeys. Usually, in the USA, this medicated feed can be available for as long as 10 days after placement. This medicated feed is replaced then with feed that does not contain antibiotics. Within a few days after the new feed has been provided, the chickens and turkey poults may begin to refuse feed and to develop signs of an enteritis that is now frequently called "off-feed enteritis". When the intestinal tracts are analyzed for bacterial populations, there are usually low numbers of beneficial bacteria such as Lactobacilli and extraordinary numbers of potentially pathogenic E. coli, Salmonella, Clostridium, and others. Naturally, the producers revert to an antibiotic treatment, and sometimes they also think about the possibility of a probiotic. Unfortunately, there is a limited number of products that can be used along with certain antibiotics. Among the commonly used antibiotics, Bacitracin has been shown to have the least influence on Lactobacilli (Casas et al., 1998). Therefore, we as producers of commercial poultry have created a situation that appears to be feeding upon itself and continuing to grow. The end result of prolonged use of antibiotics is antibiotic resistant bacteria and inhibition of growth of beneficial bacteria in the intestinal tract of poultry and other livestock.

Nevertheless, we can break this chain of events by (1) reducing antibiotic use on a prophylactic basis, and (2) we can develop a managerial plan that incorporates the use of probiotics into flock management programs.

Performance of poultry given probiotics

Body weight gain, feed conversion and reduced mortality are characteristics of performance that ultimately dictate whether managerial and company practices will be altered for acceptance of a new way of managing poultry. Mead (2000) described field experiences with competitive exclusion usage for control of Salmonella in poultry and clearly states that it is possible to control pathogen infection without sub-therapeutic antibiotic application, which was incompatible with probiotics. In field trials with market turkeys, we have demonstrated that Lactobacillus reuteri improved weight gain at 120 days of age by 4.8% (Casas et al., 1998). In ovo Lactobacillus reuteri-treated broiler chickens given a S. typhymurium challenge, body weights were improved by 206 g at 40 days of age and mortality was reduced by 32% (Edens et al., 1997a). Lan et al. (2003) reported that broiler chickens given Lactobacillus agilis JCM 1048 and Lactobacillus salavarius subsp. salicinius JCM 1230 significantly increased weight gain by 10.7%. Use of Bacillus subtilis (Calsporin; Calpis Corporation, Tokyo, Japan) did not improve body weight (Calsporin 2416 g vs. control 2407 g) at 42 days of age but feed conversion was improved (Calsporin 1.741 vs. control 1.773) (Edens, unpublished), but Fritts et al. (2000) have shown that Calsporin will improve broiler body weight gain and fed conversion. There is only one report on a probiotic product based upon the presence of Bacillus subtilis in Calsporin, that demonstrates the effectiveness of Bacillus subtilis in significantly reducing carcass contamination from enteric bacteria that have the potential to become human pathogens (Fritts et al., 2000). However, there are earlier reports indicating that Bacillus subtilis can effectively reduce the numbers of potential pathogens in feces from broiler chickens (Maruta et al., 1996a) and from swine (Maruta et al., 1996b).

Laying hens have needs that differ from broilers. Among the problems the laying hen encounters is S. enteritidis that contaminates eggs. As indicated already, it is possible to use probiotic bacteria to reduce or eliminate the S. enteritidis problem. However, there are other benefits to the egg producer. Pedroso et al. (1999) have reported that the use of probiotics (Bacillus subtilis) improved feed conversion and eggshell thickness. Improvement of these two factors alone will result in significantly improved profit margins for the egg producer.

Commercial poultry production must receive some benefit in order for it to make the move from prophylactic/growth promoting antibiotic use to the application of probiotic bacteria in its management programs. It has been shown in this article that probiotics can reduce the number of potentially pathogenic bacteria in the intestinal tract of production broilers, layers and turkeys. In healthy flocks little benefit can be seen in the use of probiotics, but in the real world of the broiler house and in the cage layer house, there are numerous potential challenges from bacteria and even protozoan parasites that will compromise the performance of poultry. Thus, use of probiotics has the potential for reduction of risk of infection from pathogens and also totally eliminates the possibility for induction of antibiotic resistance among pathogenic organisms. Furthermore, the potential for carcass contamination from gut-associated pathogens appears to be reduced and therefore public health concerns are decreased. If these were the only benefits that could be derived from the use of probiotics, it would be worth the cost of change from prophylactic use of antibiotics. However, there are other benefits derived from the use of probiotics that affect the bird. Specifically, immuno-stimulation as indicated by improved production of immunoglobulins IgA, IgM, and IgG, improved phagocytosis, improved cytokine production, promotion of natural killer T-cells, CD3+, CD4+ and CD8+ T-cells, and ultimately faster shed of intestinal pathogens. Additionally, there is evidence in the literature to demonstrate improved growth and feed efficiency in chickens and in turkeys. The improved performance of chickens and turkeys fed probiotics can be correlated with microstructures in the intestine where villus height is increased, goblet cell numbers increase, and crypt depth is decreased. Thus, probiotics improve the morphology of the intestinal tract leading to improved absorption of nutrients. The cost of probiotics is competitive with the use of antibiotic growth promoters making them just as attractive as the growth promoters.

Use of probiotics as a routine practice in meat and egg producing poultry species has had a slow start, but it appears that increasing pressures from consumers will force the industry to adapt or fail. Adaptation simply means that antibiotics are no longer acceptable by the consumer of poultry products. If there its a small increase in the cost of production due to some alternative to antibiotic growth promoters, the consumer appears to be prepared to pay the additional cost. Market driven decisions by corporate consumers of poultry products, in response to pressures from is customers, will also drive the poultry industry to adapt to a new standard of probiotics.

Thus, the company that adapts will survive in the near future, but the company that does not adapt will fail and ultimately will cease to exist. Therefore, the benefit, that the commercial poultry industry will derived from the change from antibiotic growth promoters to probiotics, will be survival. Survival is then measured in terms of maintenance of market shares and continued domestic and international sales. However, the industry will also derive the benefit of improved welfare status of their flocks. It is not uncommon to find that improved welfare also is associated with improved performance and improved profit margins. In time, it appears that antibiotic resistance factors might be lost from potentially zoonotic bacteria, and this would result in less difficulty in maintenance of flock health and public health of the consumers of poultry products. Therefore, the commercial poultry industry has numerous benefits to be gained from the use of probiotics and much to lose if it does not adapt to the new era where consumers tell producers how to produce the product that will be purchased.

Future for probiotic application

Public concerns about food animal production and food safety will drive decision-making processes in the future. As illustrated in this review, the public will demand specific standards in the food production system, and if companies desire to remain in business, those companies will respond positively to consumer demands. Thus, the future for probiotics appears to be very strong for the poultry industry. Already, there have been many demonstrations of beneficial effects of probiotic use in decreasing many different bacterial pathogens in live poultry and on carcasses of processed poultry. Products that cause the reduction of human pathogens on poultry meat will be demanded to a greater extent in the future. This is especially true with regard to the export of products to the European Union. However, that is only the beginning. It is anticipated that other major import markets will also demand antibiotic growth promoter-free products. In response to those demands, poultry production centers such as those in the United States, Brazil, Thailand, and in China will respond with production of more products with reduced microbiological risks for humans.

Conclusions

Increasing knowledge of bacterial resistance to antibiotics used by both humans and livestock has contributed to development of perceptions in consumers that an alternative to antibiotics must be identified. An understanding of the importance of the intestinal microflora in the maintenance of health and the prevention of disease in poultry has led those consumers to demand that in lieu of antibiotics, poultry producers should utilize probiotics or prebiotics that stimulate the growth of probiotic bacteria in the intestinal tract of meat animals and layers. Because perceptions are real in the mind of the consumer, it is important that the poultry industry respond to the demands of their consumers and provide them with the types of products they desire. Probiotics provide the dietary means to balance the intestinal bacteria in poultry and promote a responsive and improved immune system that can detect and eliminate certain potential pathogens from the intestinal tract. Probiotics also stabilize the intestinal mucosa making it more difficult for pathogens to colonize and cause damage in the intestinal tract, and they also promote a condition in which less contamination occurs on processed meat and meat products thereby decreasing the risk of compromised human health status. With the ultimate end of prophylactic and antibiotic growth promoter usage in the European Union and less antibiotic usage in other poultry producing centers in the world, the future for application of probiotics appears to be growing.

Even though a great amount of research is still required to understand the mode(s) of action of many defined and undefined probiotic cultures and commercial products, evidence is mounting up in strong support of the benefits that can be derived from the use of probiotics. Probiotic products are not to be considered as antibiotics. Instant responses to the use of probiotics can never be seen, but the use of probiotics seems to improve the longer the product is utilized in broiler growing and egg laying facilities. Additionally, every probiotic product is different and efficacy against specific organisms is not always the same. Thus, the producer must be able to very specifically identify the production problem for which specific probiotics must be applied. A single product may not solve the problem, but products used in combination may be more effective.

The poultry industry is entering into uncharted areas now that antibiotics are being banned. Antibiotic replacement with probiotics will create some challenges for the commercial producer, but the rapid pace of research today often times can address those problems in short order. The ultimate outcome of this consumer driven change to probiotics may actually become advantageous to the producer in times to come.

However, an integrated, rational approach to the development and application of probiotics in the poultry industry remains an issue of high priority. It is understood that all probiotics do not act the same in the intestinal tract of poultry, and due to this diversity of action among the many commercial products and those still in development, there is a need to characterize each probiotic according to its influence on the physiological status of the intestinal tract. Even though significant work has already been reported in this area, our understanding of the symbiotic relationship between the host and the intestinal microorganisms still is very limited. Thus, systematic investigations are required to better understand the host's response to the probiotic and the response of the probiotic to the micro-ecology of the intestinal tract of the host.

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Correspondence to
Frank W. Edens
North Caroline State University
Department of Poultry Science
Raleigh, NC 27695-7635 USA
Telephone: +919 515 2649
Fax: +919 515 2625
E-mail: fwedens@mindspring.com

Arrived: july 2003
Approved: september 2003

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