Intestinal integrity and inflammation: how does this affect the microbiota composition and what is the relevance for production?

I. INTRODUCTION

Animals used for food production have been genetically selected for feed intake and muscle development and are therefore divergent from their ancestors. In addition, these animals are reared in conditions that favor fast spread of pathogens. The high uptake of feed and the fast growth make these animals prone to intestinal disorders. This has been neglected in the past because of the use of low doses of specific antimicrobial compounds, called antimicrobial growth promoters (AGPs). These were used worldwide to maintain the profitability of the broiler industry. These antibiotic substances, added at sub-therapeutic level as feed additives, increased animal performance.

A ban on the use of AGPs, mainly driven by consumer concerns about increases in antimicrobial resistance, was instigated in the EU in 2006, followed by global concerns that led to decreased use or ban, depending on the region. The mode of action of the AGPs is still under debate, but a variety of mechanisms have been proposed, including a reduction in total bacterial counts in the gut (and consequently less competition for nutrients), a reduction of specific pathogens (e.g. Clostridium perfringens), a decreased abundance of specific harmful bacterial properties (e.g., bile salt hydrolase activity and thus poor fat digestion), and reduced inflammatory reactions because of the decreased pathogen load, amongst others (Butaye et al., 2003; Knarreborg et al., 2004).

Also, direct immune-modulatory effects by AGPs have been suggested. Whatever the mechanism of action is, it is evident that host-microbiota interactions are involved. The gutmicrobiota interactions are very complex since the gut is an organ that contains multiple cell types that fulfill many functions and hosts a diverse microbiota that carries out many functions as well, including breakdown of dietary molecules and consequently production of absorbable end products, and maturation and development of the (mucosal) immune system. The term ‘gut ecosystem’ is used to describe that the gut and the gut microbiota form one organ, with specific functions that are derived from both the gut microbiota’s genetic potential (the microbiome), and the functions of the host gut wall. Novel technologies (-omics technologies) have been used recently to get a better understanding of host-microbiota interactions. More specifically, various studies using 16S rDNA sequencing led to the identification of microbial taxa that are associated with beneficial or harmful host responses, and metabolomics has been used to identify microbial metabolites that trigger these effects. The production of microbial metabolites can be steered using nutritional factors, creating an excellent opportunity to make animals more resilient against non-infectious and infectious challenges, using dietary additives.

II. THE HOST SIDE: EPITHELIAL CELLS AS MAJOR SIGNAL SENSORS

The luminal side of the intestinal wall is lined with absorptive epithelial cells, whose major task is water and nutrient uptake, and secretion of enzymes. They form a semi-permeable barrier between the outside world (the gut lumen) and the internal host tissues. The semipermeable barrier is not only formed by the cell membranes of the epithelial cells, but also by tight junctions that connect neighboring epithelial cells (Piche, 2014). These connections are regulated at different levels (e.g., by cytokines). The permeability of the intestinal epithelial cell layer can be affected by epithelial cell death but also by luminal signals that increase the epithelial layer permeability by affecting the tight junctions or inducing cell death, and thus causing loss of integrity of an important barrier between the ‘inside’ and the ‘outside’ of the gut (Hooper, 2015). When epithelial cells are killed or when the tight junctions between epithelial cells are damaged, some opportunistic pathogens can benefit by gaining access to the basolateral side of the epithelial cells and induce inflammation.

Nutrient leakage and inflammation cost energy for the animal, and cause villus shortening or blunting, thus decreasing performance. Loss of intestinal epithelial integrity can cause losses of host proteins (‘leaky gut’) into the lumen and can allow luminal molecules (including toxins) and micro-organisms to reach the gut submucosa under the epithelial layer. If these components have pro-inflammatory properties, this can yield massive infiltration of immune cells, which is energy-demanding for the host. Inflammation is mediated by binding of pathogen associated molecular patterns (e.g., lipopolysaccharide, peptidoglycan lipoproteins, flagellin) to receptors (e.g., Toll like receptors) that transmit signals in a cascade ultimately leading to inflammatory cell infiltration in the mucosa (Brown et al., 2011). Although this is a protective response, this inflammatory cascade should be brought back to normal conditions when the trigger is eliminated. Also, intracellular receptors (NOD-like receptors) can sense bacterial compounds and can even induce tolerance (e.g., peptidoglycanderived muramyldipeptides).

Apart from absorptive epithelial cells, also other epithelial cell types are present in the lining of the gut wall. These include mucin-producing goblet cells and antimicrobial peptide producing Paneth cells (in the crypts, not present in all animal species), important in innate defenses (Muniz et al., 2012). Entero-endocrine cells can secrete peptide hormones at the basal side of the cells that can reach the bloodstream. These peptide hormones have a variety of functions, including effects on epithelial cell proliferation, inflammation, and consequently intestinal integrity, even at distant segments of the intestine. One of the key hormones is glucagon-like peptide 2 (GLP-2), a hormone that is important in maintaining epithelial integrity (Baldassano and Amato, 2014). Below the epithelial lining, many other cell types are present that form the lamina propria of the intestinal mucosa. These are immune cells, fibroblasts, nerve cells and muscle cells, amongst others. Intestinal integrity, inflammation, and gut function are all influenced by pathogens and their products (coccidia, toxins, bacterial pathogens, viruses) and by luminal signals, of which many are produced by the microbiota (Havenaar, 2011). The above-mentioned cells sense microbial signals and transmit these signals to other cell types and to other parts in the body of the animal. The microbiota composition and the metabolites produced by the bacteria are thus crucial for health and productivity.

III. THE MICROBIAL SIDE: THE MICROBIOTA AS SIGNAL PRODUCERS

The microbiota composition in the gut varies with age and with the gastrointestinal segment (Stanley et al., 2014; Song et al., 2017; Sun et al., 2019; Yang et al., 2019). In general, the diversity of the microbiota increases with age. In industrial animal production, often the birth or hatching environment is as sterile as possible. This situation can be considered as unfavorable because the establishment of a protective microbiota is delayed, and the young animals are more prone to colonization by pathogens.

In general, low numbers of bacteria are found in the proximal parts of the gut while the numbers increase towards the distal ileum, caecum, and colon. The diversity generally increases significantly towards the distal gut, and while in the small intestine a limited variability is found, with lactobacilli often dominant, the distal intestinal tract harbors a huge number of different bacterial groups. The distal intestinal tract of healthy subjects is mostly dominated by bacteria from the phyla Bacteroidetes and Firmicutes (together comprising more than 80% of the microbiota), the former containing many polysaccharide degrading bacterial species, while the latter contains a variety of bacterial families, including Ruminococcaceae and Lachnospiraceae families, that are considered important health-promoting populations, due to butyrate production. Also, members of the phylum Proteobacteria are usually present, although in lower numbers. These include Enterobacteriaceae, such as Escherichia coli, thus Gram-negative bacteria that contain opportunistic pathogens and often are associated with harmful inflammatory effects. The bacterial community has the genetic potential to carry out an enormous number of physiological functions. The number of microbial genes in the gut, the microbiome, exceeds the number of animal genes, and together they form a ‘hologenome’ (Rosenberg and Zilber-Rosenberg, 2011). The variety of bacterial functions includes degradation of complex substrates (polysaccharides, proteins, fat), fermentation of substrates to yield acidic compounds, immunomodulation, communication with other bacteria, and many more. The metabolites produced by the bacterial community are of vital importance for maintaining gut health and controlling pathogen colonization.

Polysaccharide breakdown is performed by the microbiota in a cascade in which different bacterial members take care of specific catalytic steps in degrading the substrates (Flint et al., 2012). Complex substrates (such as polysaccharides, including arabinoxylans, pectins, and cellulose) are converted to oligosaccharides by specific bacterial populations (e.g., lactobacilli, some Bacteroides species, and others), and these oligosaccharides (e.g., arabinoxylanoligosaccharides (AXOS)) are further used by other bacterial groups to produce short-chain fatty acids (SCFAs, i.e., acetic, propionic, and butyric acid), lactate and gases. The most important butyric acid producing bacteria belong to the Ruminococcaceae (Clostridial cluster IV) and Lachnospiraceae (Clostridial cluster XIVa) families (Pryde et al., 2002). These families contain strictly anaerobic bacteria that are highly abundant in the distal gut. Some of the Lachnospiraceae consume lactic acid to produce butyric acid (Duncan et al., 2004). Butyric acid is a major energy source for enterocytes and has a variety of beneficial properties, including pathogen control, anti-inflammatory effects, increased mucin, and antimicrobial peptide production, strengthening of the epithelial barrier, etc. (Guilloteau et al., 2010).

Fermentation to butyrate in the distal gut can affect small intestinal function by stimulating GLP-2 secretion by entero-endocrine cells in the blood stream (Tappenden et al., 2003). This GLP-2 can have effects on various cell types in the small intestine, leading to antiinflammatory effects, effects on the integrity of the epithelial barrier and increased cell proliferation (Rowland and Brubaker, 2011). Typically, inflammation is associated with a loss of anaerobes, including butyric acid producing bacteria, and an increase in oxygen-tolerant opportunistic pathogenic Enterobacteriaceae, such as E. coli. This exacerbates the inflammation, as the anti-inflammatory signal butyrate is decreased.

IV. INTERFERING WITH BACTERIAL SIGNAL PRODUCTION AND HOST SENSING, BY NUTRITIONAL INTERVENTIONS, RESULTING IN ANTI-INFLAMMATORY RESPONSES

A variety of feed additives are used nowadays as either antimicrobial growth promotor alternatives or gut health stabilizers. Some might inhibit certain bacterial groups, but most are supposed to steer the microbiota composition to a more favorable one, and have important host effects, either direct or indirect, the latter through the microbiota. As discussed above, feed formulas or feed additives should improve intestinal epithelial integrity, stimulate tolerance responses towards non-harmful bacteria, avoid excess inflammation, stimulate host antibacterial responses (mucin and antimicrobial peptide production), and bring the host to a steady state of mutualism with its microbiota. This means that these feed additives or formulae should favor beneficial microbes and inhibit the microbes that produce harmful metabolites or reduce pathogen colonization. This will result in reductions of inflammatory responses, and increased animal performance. Below a short overview is given on dietary additives that affect gut health and inflammation.

a) Feed composition and enzymes

Gut inflammation and villus shortening can be induced by feeding a diet containing high amounts of non-starch polysaccharides (NSP) without NSP-degrading enzymes (Teirlynck et al., 2009). AGPs are able to reverse the inflammatory changes and villus shortening induced by the high NSP containing diet, in association with a shift in the microbiota (Teirlynck et al., 2009). It appears that the use of AGPs in the past has masked the dysbiosis-inducing effects of many feed formulas used in monogastrics. Also, the feed structure, protein source and the choice of ingredients can affect gut health. Enzymes such as xylanases convert large polysaccharides to shorter oligosaccharides and thus perform one of the initial steps in the breakdown of these substrates, as is done in the gut by bacterial species in cross-feeding pathways. This also reduces viscosity and bacterial overgrowth in the small intestine. More information on the effects of feed constituents and gut health can be read in a review paper by Choct (2009).

b) Probiotics

Probiotics are defined as live micro-organisms that, when consumed in adequate amounts, confer a health effect on the host. The most widely used bacterial probiotics are bacilli, as they are stable in formulation (spores) and produce antibacterial compounds, apart from beneficial metabolites. As an example, recently it was shown that specific Bacillus species produce high concentrations of niacin in vivo. Niacin is sensed by the receptor Gpr109a, that is also activated by butyrate, and activates anti-inflammatory responses (Singh et al., 2014). Apart from Bacillus species, other single strain probiotics are marketed, including lactobacilli. Multi-strain products are on the market as well.

Also, competitive exclusion products, containing a freeze-dried mixture of gut content, are marketed. In the scientific literature, reports on the effect of probiotics on intestinal inflammation and animal performance have been published, and reports on protection against pathogen colonization and disease are available. The question remains how many studies are not published because of inconsistent, no, or negative effects observed. Data from our laboratory show that the efficacy of probiotics is highly dependent on the model used and not all studies show clear, reproducible beneficial results. Instead of empirically developing and marketing probiotics only because of their genus name, we should rethink the system and develop probiotics based on their mode of action. For example, based on the above-described data, attempts could be made to evaluate strains that stimulate butyrate production by strains of Clostridial cluster IV and XIVa, or use these butyrate-producing strains as probiotics. These are, however, strict anaerobes and do not form spores consistently, making them difficult to formulate and use, while this is not a problem for Bacillus species, which are usually incorporated in feed as heat resistant spores (Shivaramaiah et al., 2011).

c) Prebiotics

Prebiotics are defined as natural or processed functional foods which contain biologically active compounds that have documented benefits on health by altering the interactions between beneficial and pathogenic bacteria (Gibson and Roberfroid, 1995). Most prebiotics are oligosaccharides such as, fructooligosaccharides, galactooligosaccharides, AXOS, and xylan oligosaccharides (XOS). Mannanoligosaccharides are often not considered as prebiotics because they may not be fermented but have direct immunomodulatory effects. Prebiotics are complex molecules because of the chain length, the nature of the sugar bounds, and the nature of the side chains on the saccharides. All this can affect function. The scientific literature reports various studies in which prebiotics have beneficial effects on broiler performance, inflammation, and pathogen control.

As with probiotics, it is difficult to estimate the bias that is present using data derived from scientific papers, because only beneficial effects are mostly reported, and no or negative effects are seldom published. It is anyhow the case that the prebiotics need to be converted by the microbiota to metabolites. Because prebiotics are saccharides, the end products will be SCFAs, lactate, and gases and thus the beneficial effect can theoretically be evaluated or predicted by measuring the ratio of beneficial versus harmful bacterial groups or metabolites. As such, prebiotics that increase colonization of butyrate-producing Clostridial cluster IV and XIVa bacteria are considered to be beneficial. Other parameters could include reductions in Enterobacteriaceae. Also, in the case of prebiotics we thus need to proceed in the future towards a science-driven development in which the mechanism of action plays a central role, instead of empirically developing prebiotics. For example, our group has shown that XOS administration to a broiler diet increased the number of lactobacilli and Clostridial cluster XIVa strains in the distal gut, hereby stimulating cross-feeding of lactate to produce butyrate (De Maesschalck et al., 2015).

d) Phytochemicals (essential oils, oleoresins, etc.)

Phytochemicals are also well-known feed additives in the broiler production industry. Biologically active constituents of plants include terpenoids (mono-and sesquiterpenes, steroids, etc.), phenolics (tannins), glycosides, and alkaloids (present as alcohols, aldehydes, ketones, esters, ethers, lactones, etc.). Many of these, but not all, have antibacterial activity (Penalver et al., 2005; Barbosa et al., 2009). Effects on immune function have also been described. According to Adams (1999) the antimicrobial activity is rather weak for ginger and pepper, medium for cumin (p-cymene), coriander (lialol), oregano (carvacrol), rosemary (cineol), sage (cineol) and thyme (thymol) and strong for clove (eugenol), mustard (allylisothiocyanate), cinnamon (cinnamaldehyde) and garlic (allicin). Also, here the dosage, purity, extraction method from the plant (in case of mixtures, thus phytobiotics) or synthetic production method will determine the success of the products.

It is clear that the antibacterial essential oils will affect the gut microbiota composition, and there is a need to clarify which ones promote beneficial bacterial species, using in vivo studies. Resin acids have recently been studied and seem to alter matrix metalloproteinase activity in the gut mucosa, that could be highly relevant in restoration of intestinal damage (Aguirre et al., 2019). Indeed, matrix metalloproteinase upregulation has been shown in gut inflammation models and are likely involved in extracellular matrix breakdown.

f) Short chain fatty (and other) acids

Drinking water and feed additives containing SCFAs, medium chain fatty acids and even aromatic acids (e.g. benzoic acid) are widely used in the animal production industry. While drinking water acidification is mainly for sanitation purposes, feed additives are used mainly for optimizing animal performance and for pathogen control (Van Immerseel et al., 2006). It is difficult to compare the relative efficacy of commercial products because they differ in the nature of the acids used (often combinations are used), the concentration, and even more importantly, the delivery method (pure, on a carrier, encapsulated, etc.). The latter determines the site of release in the gut and can affect the outcome. Butyric acid has a strong antiinflammatory effect in the gut. While SCFAs are more considered as signaling molecules for the microbiota and the host, the medium-chain and aromatic acids are more antibacterial.

V. FINAL CONSIDERATIONS

A huge number of experimental and field trials have been carried out in broilers, using a variety of feed additives. The most commonly measured outcome parameter is performance, either or not under challenge conditions. Some studies have been undertaken to determine the effect on pathogen colonization and inflammatory responses. The approach so far has been mostly empirical and the products are thus mainly developed without a clear understanding of the reasons for the expected beneficial effects. Many feed additives that are meant to replace AGPs have variable activities. The only way to develop a product with an enhanced activity as compared to the already existing products will be based on a thorough understanding of the intestinal ecosystem, and the way the gut wall responds to the microbiota and their metabolites. Identifying the microbiota components that are crucial for gut health is ongoing and is essential for proper development of additives that affect gut health. This needs to be done by identifying both the beneficial ones and the harmful ones. In fact, current knowledge indicates that butyrate-producing bacteria need to be boosted or maintained while Enterobacteriaceae and specific pathogens such as C. perfringens need to be suppressed, and the inflammation is reduced. These are easy to measure criteria and are well known to correlate with a good morphological structure of the gut. In fact, studies that have recently been carried out and future studies using –omics technologies will be of value to identify potential performance-related beneficial gut microbiota components and metabolites (Dehau et al., 2022a,b).

Presented at the 35th Annual Australian Poultry Science Symposium 2024. For information on the latest and future editions, click here.