Introduction and history

In 1908 Elie Metchnikoff established the basis of the development of what we now call probiotic micro-organisms or direct fed microbials. Rusch (Reference RUSCH2002) has presented a complete overview and history of the term. The origin of the term is credited to Werner Kollath who proposed in 1953 the term ‘Probiotika’ to designate active substances that are essential for a healthy development of life. More in the line with what we now define as probiotic micro-organisms, Kolb (Reference KOLB1955) proposed probiotic therapy by administering symbiont cultures to prevent the deleterious effects of antibiotics. It was later used in an entirely different context by Lilley and Stillwell (Reference LILLEY and STILLWELL1965), and Sperti (Reference SPERTI1971) to describe substances secreted by one micro-organism which stimulated the growth of another (several species of protozoa, during their logarithmic phases of growth, produce substances that prolong the logarithmic phase in other species –more in line with the present definition of quorum sensing‑; the term probiotic was also used in contrast with antibiotic). Parker (Reference PARKER1974) made a definition closer to the present approach, and widely accepted; organisms and substances which contribute to intestinal microflora balance. Several years later, Fuller (Reference FULLER1989) redefined probiotics as ‘a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance’, excluding dead organisms and other substances from the definition.

However, the term direct‑fed microbials (DFM) was preferred in the US, and in 1989 the Food and Drug Administration (FDA) required manufacturers to use it rather than probiotic (Miles and Bootwalla, Reference MILES and BOOTWALLA1991). The FDA defined DFM as a ‘source of live (viable) naturally occurring micro-organisms’, and therefore included bacteria, fungi and yeast.

Competitive exclusion refers to the reduction of intestinal colonisation by enteric pathogens such as Salmonella and Campylobacter by the microflora already present in the gastrointestinal tract, not necessarily improving performance. According to Garlich (Reference GARLICH1999), Greenberg used the term Competitive Exclusion for the first time in 1969 to describe the control over Salmonella by using other bacteria in Calliphoridae fly larvae. Nurmi and Rantala (Reference NURMI and RANTALA1973) applied this concept in poultry and they were the first authors to apply the idea to protect chickens against Salmonella infection by inoculation with microflora from adult birds.

In the 1980's the most used probiotic micro-organisms for animal feeding belonged to three bacterial and one yeast genera: Lactobacillus spp. (several species); Streptococcus faecium, S. faecalis and S. salivarius; Bacillus cereus var. toyoi and B. subtilis; Saccharomyces boulardii, and S. cerevisiae. At least 20 different biological preparations were on the market in the European Union (EU) countries at that time, being Streptococcus faecium, Lactobacillus acidophilus and Bacillus cereus var. toyoi the most widely distributed (in at least eight countries each), with the latter the first probiotic micro-organism authorized as a feed additive in the EU (in April 1994).

The legislation in the EU on probiotic micro-organisms feed additives, including safety assessments and the Qualified Presumption of Safety (QPS) concept of micro-organisms in food and feed, were comprehensibly compiled by Anadón et al. (Reference ANADÓN, ROSA MARTINEZ-LARRAÑAGA and ARANZAZU MARTINEZ2006). The same year 2006 marked the end of the use of antimicrobials as growth promoters (AGP) in the EU, and a new opportunity for further widening the use of probiotic micro-organisms. The long term effects of this ban might be inferred from Dibner and Richards (Reference DIBNER and RICHARDS2005), who presented a review on voluntary and legislative bans of AGP, and the experience of animal producers following the 1998 ban on antimicrobials in Denmark. They concluded that replacement for AGP involves the use of multiple products in the diet and management changes to maintain animal productivity.

The Qualified Presumption of Safety (QPS) concept of micro-organisms in food and feed requires a special mention. The Scientific Committee on Animal Nutrition (SCAN) expressed its position on safety assessment and regulatory aspects of micro-organisms in feed and food applications in 2003 (SCAN, Reference SCAN2003). This system, similar in concept and purpose to the Generally Recognised As Safe (GRAS) definition used by the FDA in the USA, but adapted to the different regulatory practices in Europe, was firstly presented as a working paper for public consultation in 2003, and later debated in an European Food Safety Agency (EFSA) Scientific Colloquium on December 2004 in Brussels, Belgium. The report of this colloquium was published in 2005 (EFSA, Reference EFSA2005) and reflected that QPS might provide a mechanism to recognize and give weight to prior knowledge when assessing the safety of micro-organisms in food and feed production.

Probiotic micro-organisms for poultry

According to Jernigan et al. (Reference JERNIGAN, MILES and ARAFA1985) there are two different types of bacteria which can establish in the digestive tract. The first exists in close association with the gut epithelium and the second occurs free in the gut lumen. Therefore, the adhesion capacity of the micro-organism would not be indispensable, and probiotic micro-organisms for poultry can be designed either to establish beneficial organisms absent from the gastrointestinal tract or to provide other beneficial bacteria. Probiotic micro-organisms might be directed to act in the crop and the anterior regions of the gastrointestinal tract or mainly at the caeca. However, it is likely that either case, to some extent, they will be effective throughout the gut. Lactobacillus preparations are among the first group: they colonise the crop and small intestine, and exert their antibacterial effects against potential pathogens. Lactobacillus spp. may produce large amounts of lactate from carbohydrates and can withstand a high degree of acidity which is usually fatal to other bacteria. Lev and Briggs reported as earlier as 1956 that after feeding a lactobacillus culture to chicks a balanced lactic acid microflora was established in the GI tract within 24 hours.

The majority of microbial products for compound feeds are made up from a relatively small number of micro-organisms: Lactobacillus spp. (mainly L. acidophilus); Streptococcus faecium; Bacillus spp.; and yeasts, especially Saccharomyces species. Lactobacillus species and S. faecium are normally present in the GI tract, while Bacillus species and yeasts are only sporadically present in the gut microflora.

Direct fed probiotic micro-organism species, such as the lactic acid bacteria, are relatively fragile. They have to be technologically protected as they do not easily tolerate the heat and pressure of feed processing (without protection, lactobacilli only resist up to 52ºC, yeasts up to 63ºC, and streptococci up to 71ºC.). However, the spores of certain Bacillus species are more heat resistant, and easily survive the pelleting process during feed manufacture. Moreover, these Bacillus species seem to have growth promoting effects beyond the balancing. or stabilising effects of the lactic acid bacteria, especially in pigs (Sögaard and Suhr-Jessen, Reference SÖGAARD and SUHR-JESSEN1990).

According to SCAN (Reference SCAN2000), microbial products able to affect or stabilize the gut flora of target animals do so only when the natural flora is in some way disturbed. Adding a probiotic micro-organism becomes a preventive measure against any detrimental effect on performance originated through the intestinal flora, and it is reasonable to expect a microbial product not to affect animal performance where there is no significant disturbance to the flora.

Reviews on results obtained by the use of probiotic micro-organisms in poultry have been published recently, and readers are referred to them for an extensive overview (Simon and Jadamus, Reference SIMON and JADAMUS2002; Edens, Reference EDENS2003; Patterson and Burkholder, Reference PATTERSON and BURKHOLDER2003; Schneitz, Reference SCHNEITZ2005; Revolledo et al., Reference REVOLLEDO, FERREIRA and MEAD2006; Flint and Garner, Reference FLINT and GARNER2009). The present review focuses on the proposed modes of action.

Mode of action of probiotic micro-organisms

One of the main interests in animal production is the relationship between nutrition and gut health, especially in the small intestine. Digestion, absorption and intestinal barrier (the first line of defence against pathogens) in monogastric animals should be optimised to spend the minimal amount of nutrients for immune or anti-inflammatory responses while achieving the maximal production performance. The quality of the barrier function of the intestinal epithelium (the mucus layer, the glycocalyx and the enterocytes) warrants an optimal first line of defence. This quality is determined by host's genetics and the intestinal environment with its microflora. A normal gastrointestinal tract requires balance within its bacterial populations. This balance is challenged when animals are subjected to stressful conditions such as hot weather and humidity, feed changes or imbalances, mycotoxin contamination, transportation, and moulting. Pathogenic bacteria become harmful either through mucosal invasion or toxin production or both. Feeding probiotic micro-organisms continuously to animals has been found to maintain a beneficial intestinal microflora. For instance, several studies have demonstrated the in vivo efficiency of B. toyoi in modulating the intestinal microflora, sustaining beneficial bacteria such as lactobacilli and decreasing the presence of potential pathogens like E. coli and S. enteritidis (Jadamus et al., Reference JADAMUS, VAHJEN and SIMON2000, Reference JADAMUS, VAHJEN, SCHÄFER and SIMON2002; Simon et al., Reference SIMON, JADAMUS and VAHJEN2002; Taras et al., Reference TARAS, VAHJEN, MACHA and SIMON2005; Vilà et al., Reference VILÀ, FONTGIBELL, BADIOLA, ESTEVE-GARCIA, JIMENEZ, CASTILLO and BRUFAU2009). These findings were confirmed in vitro and the mechanisms of action elucidated (Calvo et al., 2007): B. toyoi exhibited a wide range of enzymatic activities that possess a destructive activity upon gram-negative bacteria (esterases, hydrolases, phosphatases, etc.) and possibly interfere with protein synthesis (leucine arylamidase, valine arylamidase).

The gastrointestinal tract in broilers is sterile at hatching, and immediately bacteria from the environment or the diet colonise it. After this first colonisation, new bacterial species have more difficulties to establish themselves. A wide range of dietary factors affect the composition of the microflora. This leads to new micro-ecological conditions that allow a better colonisation of some species due to improved adhesion or growth rate. Ingested bacterial species could colonise the gastrointestinal tract, and this is the case when probiotic micro-organisms are administered to the animals. Using probiotic micro-organisms shortens the period needed to stabilize the microflora. This microflora regulation may serve three purposes:

• • Improve feed conversion and weight gain

• • Improve the intestinal health and immune competence of the animals

• • Suppress food-borne pathogens such as Salmonella and Campylobacter species, which is interesting for the production of safe meat and meat products.

Under natural conditions, the microflora colonising the gastrointestinal tract few days after birth consists of 400 to 500 different bacterial strains for a total count of 10¹⁴ bacteria. The microflora consists of transient bacteria which temporarily reside in the tract, and indigenous bacteria that colonise the intestinal tract permanently. Colonisation by a bacterial species is defined as ‘a bacterial population in the gastrointestinal tract which is stable in size and occurrence over time, without the need for periodic reintroduction of bacteria by repeated oral doses or other means’ (Snel et al., Reference SNEL, HARMSEN, WIELEN and WILLIAMS2002). Therefore, colonising bacteria multiply in a particular niche, at a rate equal or superior to their rate of washout or elimination. Certain species of the microflora can influence the expression of glycoconjugates of epithelial cells that may serve as receptors for adhesion of other bacteria, positively or negatively influencing in this way colonisation by other species.

Snel et al. (Reference SNEL, HARMSEN, WIELEN and WILLIAMS2002) gave a detailed list of the mechanisms by which the microflora can contribute to intestinal health of animals and man: growth promotion; improvement of the mucosal architecture; degradation of unfermentable substrates into digestible components; improvement of intestinal and general health; breakdown of cytotoxic substances; production of vitamins; suppression of pathogens; competition for nutrients; competition for adhesion sites at the mucosal epithelium; stimulation of intestinal motility; stimulation of the immune system; production of volatile fatty acids; production of antimicrobial substances.

Microbial management practices aim to stimulate beneficial bacteria and/or suppress detrimental bacteria. This is done by suppression of certain species by including antibiotics (no longer allowed in the EU), and alternatively short chain fatty acids. Another option is to promote beneficial species in the microflora by feeding the animal suitable substrates such as oligosaccharides or other prebiotic fibres; or directly by adding beneficial bacteria to the diet of the animals.

Fuller (Reference FULLER1977) established that Lactobacillus spp. in the crop were important in maintaining the microbial balance and also exerted its influence on the small intestine, the inhibitory effect against E. coli not being solely due to pH alone, as organic acids (such as lactic acid and acetic acid) affecting membrane structure and oxidative metabolism; other antibacterial factors have been found to be produced in vitro by lactobacilli such as hydrogen peroxide, antibiotic and bacteriocin‑like substances. Bacillus species also produce a large number of antimicrobials, including bacteriocin and bacteriocin‑like substances. Once swallowed, food is temporarily stored in the crop where a predominantly lactic acid fermentation takes place. The pH is fairly low, and a simple microflora is present compared with that of the caeca: predominant micro-organisms are lactobacilli, that produce lactic and acetic acids decreasing the pH of crop contents to 4‑5 in a healthy chicken. The pH of the proventriculus and gizzard is much lower (pH 1‑2) and microbial survival depends on acid tolerance.

The relatively high flow rate of the fluid content of duodenum implies little multiplication of the micro-organisms. The caeca contain a thick viscous fluid, and allow the highest viable bacterial counts (counts of 10¹¹ g^‑1 of contents) and most complex microflora. Most of the micro-organisms present are obligate anaerobes: gram-positive, anaerobic cocci, comprise up to one third of the total; other major components include gram-negative, non-sporing rods such as the Bacteriodaceae (one fifth of the total); Clostridium spp. and bifidobacteria only represent one tenth of the total; while lower numbers of facultative anaerobes including E. coli, Salmonella, and Klebsiella spp. are frequently present. Diet has more influence on the flora of the first part of the gastrointestinal tract, while little changes occur in the caeca. In the absence of stress or major selective pressures, the adult intestinal flora is relatively stable and difficult to change simply by oral administration of micro-organisms. It would be easier to establish a beneficial organism soon after hatching before other organisms are able to colonise (Barrow, Reference BARROW1992).

Therefore, the intestinal micro-ecology is very complex as the gastrointestinal tract is not uniform and consists of small ecological niches within which most bacteria grow in colonies enclosed in the glycocalyx, forming biofilms on the surfaces of both tissue and digesta. Bacteria are attached to their nutritive substrates by chemotaxis or form colonies in locations with high concentrations of nutrients. Tissues exposed to extreme concentrations of acids may be colonised by single species of bacteria (lactobacilli) or yeast, as special adhesion mechanisms or acid resistance are required. However, most non secretory epithelia are colonised by a rich mixture of bacteria, the majority being associated with the viscous layer of the mucosa. They must be able to enzymatically digest the mucinous glycoproteins and to use the degradation products such as carbon, energy and nitrogen in situ. Also, they have to overcome peristalsis in the small intestine, as well as the turnover of epithelial cells, colonising new surfaces. The digestion of the mucus layer represents a metabolic expense for the host, as it has to replace it by continuously secreting more mucus.

As explained above, animals reared under natural conditions have, in their gastrointestinal tract a few days after birth, a population of micro-organisms that protects them against disease. However, commercial production tends to limit the contact with the mother and provides unnatural environmental conditions. Modern animal husbandry, intensive or semi-intensive, brings numerous stresses. Chicks have no access to the mother and when they hatch they might be challenged by potential pathogens in the hatchery. Then they go into the brooding stage where challenge from microflora may vary from almost nil, due to extremely hygienic conditions, or an excessive one if the environment is dirty.

The result is that the gut microflora is deficient in some of the normal components that could provide resistance to disease. Even the microflora of more adult birds can be affected by diet, coccidiostats or antimicrobial products, and stress. Stress affects the animals from the very beginning: travel with temperature stress or dehydration; overcrowding that leads to excessive bacterial challenge; vaccination; deficient supplies of feed or water; chilling or poor ventilation in the house; bad litter; sudden environmental fluctuations and airborne challenges that bring clinical and sub-clinical infections, etc. The use of probiotic micro-organisms tries to repair these deficiencies restoring the full protective capacity of the microflora.

Probiotic micro-organisms may be administered to the animal in several forms, either directly or through feed or water; continuous or multiple dosing is essential to obtain the full effect. In order to be effective, probiotic micro-organisms have to be stable for long periods under normal storage and feed production conditions, and must be able to survive in the intestine of the target species to produce its beneficial effect.

In relation to the nutritional, metabolic and immunological point of view, according to Vanbelle et al. (Reference VANBELLE, TELLER and FOCANT1990), an ideal probiotic micro-organism must fulfil the following requirements:

• • Be resistant against digestive enzymes, lysozyme, the low pH in the stomach for a few hours, also to bile salts

• • Produce a sufficient decrease in the pH of the gut to avoid the development of pathogens and reduce the production of toxins

• • Produce antibiotics and be resistant to in feed antimicrobials (coccidiostats)

• • Attach to the brush border cells or colonisation of mucous and glycocalix, although this characteristic is not strictly necessary

• • Be present in a viable state resistant to product/feed processing and storage; and confer immune stimulation to the host.

From a practical standpoint, Delbecque (Reference DELBECQUE1991) pointed out that the adhesion capacity of the micro-organism is not indispensable, as adhesive strains disappear one week after finishing their administration; also, bioregulation by the probiotic micro-organism requires an adaptation period of at least two weeks.

Secretion of bacteriocins

Lactobacilli and B. cereus have been reported to produce various types of antibiotics (Fox, Reference FOX1988; Oscáriz et al., Reference OSCÁRIZ, LASA and PISABARRO1999; Risøen et al., Reference RISØEN, RØNNING, HEGNA and KOLSTØ2004). Lactobacillus acidophilus produces acidophilin, lactocidin, and acidolin, and L. plantarum produces lactolin. Nisin and diplococcin are among the anti-metabolites produced by streptococci. Bacillus cereus produces bacteriocin-like substances that inhibit closely related Bacillus spp. and species such as Staphylococcus aureus and Micrococcus luteus; and presents high activity in the pH range of 2.0‑9.0 (Risøen et al., Reference RISØEN, RØNNING, HEGNA and KOLSTØ2004). Additionally, some of the lactobacilli produce sufficient hydrogen peroxide to inhibit various micro-organisms. Acidophilin, acidolin, lactobacillin, and lactocidin have demonstrated an in vitro inhibitory activity against Bacillus, Klebsiella, Pseudomonas, Proteus, Salmonella, Shigella, Staphylococcus, and Vibrio species and enteropathogenic E. coli.

In any case, probiotic micro-organisms are not an alternative to antibiotic treatment for acute diseases and should not be considered a curative medicine against any specific disorder. Probiotic micro-organisms should only be used to aid feed conversion and prophylactically against enteritis. In the truest sense they are not growth promotants, but rather growth permitants, allowing the host to best express its genetic potential.

Immunomodulation

The normal microflora of an animal has a significant impact on the body's immune system. The numbers of intraepithelial lymphocytes, plasma cells, and Peyer's patches are lower in germ-free animals than in conventional animals.

Dunham et al. (Reference DUNHAM, WILLIAMS, EDENS, CASAS and DOBROGOSZ1993) reported that birds treated with L. reuteri had longer ileal villi and deeper crypts than control birds, which is a response associated with enhanced T-cell function, and increased production of anti‑Salmonella IgM antibodies. Nahashon et al. (Reference NAHASHON, NAKAUE, SNYDER and MIROSH1994) found that Lactobacillus supplementation of layers diets increased cellularity of Peyer's patches in the ileum indicating a stimulation of the mucosal immune system that responded to antigenic stimuli by secreting immunoglobulin (IgA).

Khajarern and Ratanasethakul (Reference KHAJARERN and RATANASETHAKUL1998) stated that when used continuously, probiotic micro-organisms also served to reinforce the non specific immune system of animals, decreasing the need of anti-infectious treatments. In trials with broiler breeders, they showed that a supplementation of B. toyoi in feed under practical farming conditions improved not only some zootechnical variables, but also the humoral immune response. They detected higher titres and average mean values with the Newcastle Disease Haemagglutination Inhibition test (ND HI) and with Infectious Bursal Disease Virus (IBD) for the probiotic-fed birds during the four-month study. B. toyoi have been also demonstrated to improve humoral response in mice and piglets (Coppola et al., Reference COPPOLA, CONCEIÇÃO and GIL-TURNES2005; Scharek et al., Reference SCHAREK, GUTH, FILTER and SCHMIDT2007b) and improve systemic and intestinal immunity in piglets (Scharek et al., Reference SCHAREK, ALTHERR, TÖLKE and SCHMIDT2007a; Schierack et al., Reference SCHIERACK, WALK, REITER, WEYRAUCH and WIELER2007).

Zulkifli et al. (Reference ZULKIFLI, ABDULLAH, AZRIN and HO2000) assessed the effect of antimicrobial growth promoters and probiotic micro-organisms in two strains of broiler chickens (Shaver and Hubbard) also on antibody production against Newcastle disease vaccine. They supplemented feed by either 50 mg/kg oxytetracycline or 10⁶ CFU/g of a lactobacillus culture and submitted birds to heat stress (36±1ºC for 3 hours daily from day 21 to 42). Before heat exposure, antibody production was not influenced by chickens’ strain or feeding treatment. In contrast, after heat exposure, a significant interaction was observed: Hubbard chicks fed probiotic micro-organism exhibited a greater antibody response than those given the control diet; while feeding treatments had no effect on antibody response of Shaver chickens.

Kabir et al. (Reference KABIR, RAHMAN, RAHMAN, RAHMAN and AHMED2004) found a significantly (P<0.01) higher antibody production against SRBC, and higher spleen and bursa weights, in experimental birds provided with a multi‑strain probiotic through drinking water (a product containing nine strains of several bacterial, fungal and yeast species: L. plantarum, L. bulgaricus, L. acidophilus, L. rhamnosus, Bifidobacterium bifidum, Streptococcus thermophilus, Enterococcus faecium, Aspergillus oryzae and Candida pintolopessi) as compared to control birds (6.0 log₂vs. 2.8 log₂ respectively).

Khaksefidi and Ghoorchi (Reference KHAKSEFIDI and GHOORCHI2006) evaluated the influence of dietary supplementation of probiotic Bacillus subtilis on performance and immunocompetence in broiler chicks. At 7, 14, 21 and 28 days of age, twenty birds per dietary group were injected intravenously (brachial vein) with 0.1 ml of 0.5% sheep red blood cell (SRBC). The probiotic micro-organism had positive effect on production and persistency of antibody in response to SRBC antigen. Also, antibody production against Newcastle disease virus in probiotic micro-organism supplemented group was significantly higher at 10 days post immunization compared to control. The results suggested that the use of probiotic containing Bacillus subtilis had positive effect on performance and immune system of broiler.

Higgins et al. (Reference HIGGINS, ERF, HIGGINS, HENDERSON, WOLFENDEN, GAONA-RAMIREZ and HARGIS2007) hypothesised that the innate immune system of chickens, specifically macrophages, played a role in reduction of Salmonella enteritidis colonisation with probiotic treatment (lactobacillus-based probiotic culture FM-B11). Chicks were challenged or not with S. enteritidis at day of hatch and treated or not with the probiotic culture 1 hour later in a factorial design. Probiotic micro-organism treatment on the day of hatch reduced (P<0.05) caecal S. enteritidis recovery as compared with the control treatment, but the modest differences detected in two out of four experiments, and the fact that those differences were not repeatedly detectable, suggested them that the macrophage-related changes were not solely responsible for the reductions of S. enteritidis following probiotic micro-organism treatment.

Interference with quorum sensing signalling agents

Bacteria communicate to each other using chemical signalling molecules (called auto-inducers). This phenomenon is known as quorum sensing, which allows bacteria to measure the population density, nutrient concentration and other ecological characteristics, and to co-ordinate the gene expression of the entire community in response to changes in cell number or niche conditions (Schauder and Bassler, Reference SCHAUDER and BASSLER2001).

Highly specific as well as universal quorum sensing languages exist which enable bacteria to communicate within and between species. In addition, prokaryotic and eukaryotic mechanisms that interfere with bacterial quorum sensing have evolved. Specifically, the secretion of enzymes that degrade the auto-inducers, or the production of auto-inducer antagonists, are recourses used by competitor bacteria and susceptible hosts to render quorum sensing bacteria mute and deaf, respectively. Analogous synthetic strategies are being explored for the development of novel antimicrobial therapies (Schauder and Bassler, Reference SCHAUDER and BASSLER2001) and might also be used by probiotic micro-organisms.

Most gram negative bacteria use N‑acylhomoserine lactone (AHL) signals to monitor their own population density. A luxR-like protein is responsible for recognition of the AHL auto-inducer; this protein of the luxR type have a domain for binding AHL and a second domain for binding DNA, and subsequently activates the transcription of downstream target genes. The proteins of the luxI type catalyze the final step in AHL synthesis, each luxI homolog makes a specific AHL, which differ primarily in the acyl chain length and the nature of the substituents at the C‑3 position. In addition to the luxI family of AHL synthases, other synthases types have been described (Michael et al., Reference MICHAEL, SMITH, SWIFT, HEFFRON and AHMER2001).

In general, each bacterial cell in a population produces AHL, and as the population density increases, the concentration of AHL also increases. Above a threshold concentration, the LuxR homolog binds AHL and activates transcription of target genes (one of the target genes is often the luxI homolog, which results in a positive feedback) (Michael et al., Reference MICHAEL, SMITH, SWIFT, HEFFRON and AHMER2001).

Many bacterial behaviours have been shown to be regulated by AHLs, including plasmid conjugal transfer, protein secretion, synthesis of exoenzymes, cytotoxins, antibiotics, and capsular exopolysaccharide, biofilm formation, and motility (Michael et al., Reference MICHAEL, SMITH, SWIFT, HEFFRON and AHMER2001).

Escherichia coli and Salmonella enterica serovar typhimurium encode a single luxR homolog named sdiA. Virulence functions in γ‑proteobacterial pathogens are controlled by a transcription factor encoded by uvrY orthologs. However, Escherichia spp., Salmonella spp., and Klebsiella spp., are the only genera that present this gene downstream of sdiA. It is also surprising that although these three genera possess a copy of sdiA, they are not known to synthesize the AHLs that are typically detected by luxR homologs. In fact, there are no AHL synthase genes (luxI or luxLM homologs) in any of the available genome sequences for these organisms. Therefore, Escherichia, Salmonella, and Klebsiella spp. appear to be unusual with regard to quorum sensing in that they encode a putative AHL receptor, SdiA, but not an AHL synthase (Michael et al., Reference MICHAEL, SMITH, SWIFT, HEFFRON and AHMER2001).

Production of autoinducers is not limited to pathogenic bacteria. Many commensal and potentially probiotic bacteria such as lactobacillus, bifidobacterium, or B. cereus strains, i.e., possess a luxS homologue and can produce autoinducers (Auger et al., Reference AUGER, E., AYMERICH and GOHAR2006; Lebeer et al., Reference LEBEER, DE KEERSMAECKER, VERHOEVEN, FADDA, MARCHAL and VANDERLEYDEN2007). The toxicity of E. coli O157:H7 is developed once the bacteria have attached to host intestinal epithelial cells, and genes involved in attachment are directly activated by quorum sensing. The (human) probiotic Lactobacillus acidophilus La‑5 secretes a molecule that acts inhibiting the quorum sensing signals or directly interacts with bacterial transcriptionals regulators, controlling the transcription of E. coli O157 genes involved in colonisation and avoiding bacteria toxicity (Medellin-Peña et al., Reference MEDELLIN-PEÑA, WANG, JOHNSON, ANAND and GRIFFITHS2007). Degradation of AHL by B. cereus has also been described by Medina‑Martinez et al. (2007). Cerdà‑Cuéllar et al. (2009) also demonstrated the ability of B. toyoi to degrade AHL, partly explaining the action mechanisms of this probiotic micro-organism.

From the previous papers it can be concluded that quorum sensing regulates the virulence expression in some micro-organisms and probiotics may interfere with this signalling system avoiding the onset of virulence.

Implications

The demonstration of the importance for animal's health of a normal microflora has been vital for the development of probiotic micro-organisms in poultry production. In practice it is difficult to keep flocks clean of pathogens, even if chicks come free of infection from the hatchery. In the case of Salmonella, it may be that some birds are undetected carriers and start shedding when stressed, or that Salmonella remained in the house or came through the feed, trucks, visitors, air, wildlife or personnel. Problems may arise if a challenge with these micro-organisms happens before a normal microflora has been established. Additionally, if birds require treatment for any disease, the chemotherapeutic or antibiotic used may cause a disruption in the intestinal microflora and Salmonella or Campylobacter may be allowed to infect or emerge.

Feeding probiotic micro-organisms on a continuous basis from the very beginning helps to develop and stabilise (after hatching or disruption) a competent microflora that should successfully avoid the proliferation of pathogens, especially when these stresses might arise.

The establishment of a correct microflora in the gastrointestinal tract of the chickens is crucial to avoid colonisation by potential pathogens. Interference with quorum sensing signals may play an important role in that. The principal locations of risk are the crop - the first site for colonisation following ingestion, and the caeca - the main colonisation site for most pathogens including Salmonella and Campylobacter. Although Salmonella is firstly thought as the food poisoning cause in the public mind, Campylobacter species actually cause more outbreaks in man than Salmonella.

Meat and meat products may pose a risk if contaminated with pathogenic micro-organisms such as Salmonella and Campylobacter. To improve food safety, the industry is requested to decrease the level of contamination to zero or at least to acceptable levels. Several intervention strategies are been applied starting at the breeding and farm level through the final product. Part of these intervention strategies are the use of probiotic micro-organisms and competitive exclusion microflora, used for prophylactic and curative purposes.

Gut microfloral enzymes are beneficial to the nutrition of the host because they increase the digestion of nutrients, especially in the lower intestine, and suppress ammonia production and urease activity, which in turn can improve animal health and enhance growth because ammonia produced by ureolysis in the intestinal mucosa may significantly damage the surface of the cells.

Other effects of probiotic micro-organisms include enterotoxin neutralisation or synthesis inhibition by interfering with quorum sensing signals and stimulation of the immune system. Enterotoxins produced by pathogenic bacteria may be neutralized by substances produced by a probiotic micro-organism or signalling molecules for transcription might be degraded before reaching its target. Lactobacilli could be important in the development of immune competence in animals, especially when protection must be acquired against antigens that will probably cause gut inflammatory reactions.