The definition of gut health and identification of markers

Intestinal health is a term that is not yet clearly defined, despite that it has been a focus of major research efforts in the last decade, both in human and in veterinary medicine, for several reasons. First of all, in man, intestinal health problems including Crohn’s disease, ulcerative colitis, irritable bowel syndrome (IBS), small intestinal bacterial overgrowth (SIBO), are on the increase, to the point that they may evolve into the major pandemic of the 21^st century. In domestic animals, gut health is also an issue. As domestic animals have a relatively short life span, they don’t suffer from diseases like Crohn’s disease or ulcerative colitis, which are typically chronic intestinal health problems. Nevertheless, the short term effects of intestinal health problems in animals are obvious. In poultry, these problems are grouped under the headings of necrotic enteritis and dysbiosis.

Dysbiosis, which is defined as a shift in the intestinal microbiota resulting in an imbalance between commensal and pathogenic bacteria, is not generally acknowledged as a disease of poultry because it usually does not present as a clinical disease. It is still not clear what causes dysbiosis exactly, but since the ban on the use of antibiotics as growth promoters in animal feed in the EU, digestive disorders in broilers are an increasing problem. However, the problem is probably not only due to the abolishment of antibiotic growth promoters (AGP). Improving housing and feeding formulas and the genetic selection of broilers on growth rate have resulted in an earlier peak of slaughter age and a higher percentage of breast meat. To achieve this, the daily feed intake should increase and the digestive system must function better than ever. When the digestive capacity is exceeded or when the feed is not digested optimally, a disturbance of the intestinal microbiota composition arises. It is thus likely that dysbiosis groups different entities that are caused by various triggers, and cannot be considered as one disease or syndrome, and therefore the diagnosis is a difficult issue.

Macroscopic definition of gut health

Intestinal health problems in broilers are currently mainly diagnosed by the macroscopic evaluation of the condition of the intestine of the animals or by the consistency of the manure. The simplest criteria are diarrhoea and flat manure in the stables, but these criteria are obviously not specific and do not distinguish between dysbiosis/non-specific intestinal health problems and infections with pathogens that cause diarrhoea.

A macroscopic lesion scoring of the intestine, described in the paper of Teirlynck et al., 2011 is currently the only method that has been validated previously and is used by veterinarians to indicate dysbiosis in broiler chickens. In total, 10 parameters are assessed and scored 0 when absent or 1 when present during visual inspection of the intestinal wall at autopsy where after the animal will receive a total score between 0 and 10. Zero represents a normal gastrointestinal tract and 10 the most severe form of dysbiosis. The parameters are (1) ‘ballooning’ of the gut; (2) inflammation, redness, of the serosa and/or mucosal side of the gut, cranial to the Meckel diverticulum; (3) macroscopically visible and tangible fragile small intestine cranial to the Meckel diverticulum; (4) loss of turgor in longitudinal cutting of the intestine cranial to the Meckel diverticulum within the 3 seconds after incision; (5) abnormal occurrence of the intestinal content (excess mucus, orange content, gas) cranial to the Meckel diverticulum; (6,7,8,9) are identical to (2,3,4,5) but caudal to the Meckel diverticulum and (10) presence of undigested particles caudal to the ileo cecal junction. This is a time and labour intensive process since a minimum number of animals per litter should be examined, however, the macroscopic scoring system as diagnostic tool for non specific intestinal health problems or dysbiosis is the only one that distinguishes other intestinal disorders, such as coccidiosis, from dysbiosis. A simplified macroscopic definition of optimal intestinal health thus could be: the absence of parameters describing dysbiosis with a total score of as close as possible to zero after macroscopic lesion scoring.

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Morphological definition of gut health: the intestinal barrier

Barrier function is a critical aspect of intestinal health. In the traditional concept, the gastro-intestinal lumen is lined by a continuous single layer of high columnar epithelial cells. This misunderstanding was introduced by artifacts that arose from the processing of tissues for conventional microscopic examination. In reality, the intestinal lumen is lined by a mucus layer, the most superficial layer being semi-permeable mucus that is colonised by a select population of micro-organisms, while the deeper mucus layer is very dense and contains higher concentrations of antimicrobial peptides, antibodies and enzymes involved in digestion. The mucus is produced by specialised cells, the goblet cells, which are present in remarkably high numbers especially in the lower part of the intestinal tract. The dense mucus layer together with the underling epithelial cell layer form the so-called ‘intestinal barrier’. This is not a mechanical barrier, but rather a functional barrier, designed to selectively and efficiently take up the nutrients, while at the same time blocking the entry of potentially harmful substance, such as secondary metabolites of plants. The epithelial cells are multiplying in the intestinal crypts of Lieberkühn. As they ‘mature’, they move along the basement membrane up the villus. At the tip of the villus, the epithelial cells undergo apoptosis, losing their connections with neighbouring epithelial cells and with the basement membrane. Under conditions of optimal intestinal health, the intestinal villi are long and the crypts are short. The functionally most mature and active cells are at the tips of the villi. Shortening of villi and lengthening of crypts indicates faster turn-over of epithelial cells and loss of the metabolically most important epithelial cells at the villus tips. All epithelial cells in all tissues of the body are interconnected by specific bonds, named desmosomes. In the intestinal epithelium, these bonds are even more elaborate and stronger than in other epithelia, therefore they are called ‘tight junctions’.

A microscopical view of villi without inflamation, healthy intact lumen. UU Gent

These tight junctions block passive diffusion of large molecules between the epithelial cells in both directions, thus avoiding loss of plasma proteins from the host into the intestinal lumen, and avoiding entry of undesirable ‘foreign’ substances from the intestinal lumen into the blood. Maintenance of tight junctions is a highly energy demanding process. Thus any energy deficit of the intestinal epithelial cells will lead to leakage of the intestinal barrier. Several harmful substances that can be present in feed, but also several pathogenic micro-organisms that can be present in the intestinal lumen, can damage the tight junctions, and, by doing this, induce leakage of plasma into the intestinal lumen, resulting in diarrhoea. In the case of broilers, this will present as wet litter. When different intestinal segments from healthy and diseased animals are examined microscopically, it is obvious that even in perfectly healthy animals, there is always some degree of inflammation in the gastro-intestinal wall. This is also acknowledged in recent years in human medicine. It is covered by the rather contradictory term of ‘physiological inflammation’. Consequently, there is no break point between ‘healthy’ and ‘diseased’ with respect to intestinal health, but the degree of inflammation in the intestinal wall can vary considerably. The severity of inflammation in the gastro-intestinal wall is defined by the interplay of three crucially important factors, i.e. the host mucosa (including the intestinal barrier and the mucosal immune system), the gastro-intestinal microbiota and the feed. A simplified morphological definition of intestinal health thus could be: long slender villi and short crypts, and an adequate number of inflammatory cells in the propria mucosae.

A clear microscopical view of inflamation of the Villi. Photo: UU Gent

Microbiological definition of gut health

The protection against micro-organisms is relying on the innate and the adaptive immunity. The innate immunity can sense the presence of micro-organisms by detecting generic recognition patterns, molecular patterns that are common to a large number of different micro-organisms, such as the lipopolysaccharide of Gram-negative bacteria. Toll-like receptors (TLR) and Nod-like receptors (NLR) are designed to carry out these tasks. Triggering of these receptors can lead to a powerful inflammatory response and an immunological reaction cascade.

In the intestinal tract, this protective task is particularly challenging, as the intestinal barrier must at the same time be highly permeable for the nutrients, while being tightly sealed for harmful substances and potentially invading micro-organisms. Fortunately, not all micro-organisms are harmful. The distinction between beneficial microbes and aggressive invading microbes is not so strict, to the point that many apparently harmless micro-organisms, can, under certain conditions, become invasive. These micro-organisms are called facultative pathogenic or opportunistic. Invasion is used by the innate immune system as the most reliable criterion to discriminate between beneficial and harmful microorganisms. For that reason, TLRs are mainly expressed at the baso-lateral sides of the intestinal epithelial cells, below the tight junctions. For all of the above mentioned reasons, the question as to which are the beneficial microbes in the intestinal tract, cannot be answered unambiguously, although multiple bacterial taxa have been shown to be health-promoting. Butyrate-producing bacteria are amongst these. Optimal intestinal health is not characterised by complete absence of (facultative) pathogenic microorganisms. One of the criteria of a healthy intestinal microbiota seems to be high diversity. Loss of diversity often goes together with poor intestinal health. A simplified microbiological definition of intestinal health thus could be: An intestinal microbiome with a high microbial and functional diversity.

Identification of markers for intestinal integrity

Dysbiosis is designated as the primary indication for antibiotic use. When it evolves into a clinical disease problem, with obvious animal suffering as a consequence, action must be taken and then treatment of the animals with antibiotics is medically safe and necessary. However, it is difficult to determine when and at what point intervention is necessary because the underlying mechanisms are unknown, and therefore there is a lack of diagnostic techniques and criteria. As long as the veterinarian does not have diagnostic tests that allow to determine objectively when antibiotic use is justified or not, the pressure will continue to preventively use massive antibiotics as a form of ‘insurance’ against outbreaks with great economic damage. When intestinal health problems appear, impaired integrity of the intestinal wall is suggested to be a parameter which could be diagnostic. Integrity disorders can result in release of cytoplasmic or cell wall molecules of epithelium into the intestinal lumen, bacterial molecules in the bloodstream, or the ability to perform certain functions is no longer optimal. Markers for these processes which are easy to measure can thus be diagnostically useful. Ghent University is currently carrying out a research project funded by the Flanders Innovation and Entrepreneurship to define dysbiosis in broilers and develop reliable biomarkers. In addition, field-like dysbiosis models have already been developed in order to test control compounds.

When dysbiosis evolves into a clinical disease problem, with obvious animal suffering as consequence, actions must be taken and then treatment of the animals with antibiotics is medically safe and necessary. Photo: Jan Zandee

Conclusion

Intestinal health is determined by several factors, including the feed composition, the intestinal barrier, and the microbiota. Since feed is one of the three factors involved, it is possible to influence intestinal health by modifying the feed formula. Several substances that are present in feed have documented effects on intestinal health, either by directly acting on the intestinal barrier and the tight junctions, or indirectly by modifying the intestinal microbiota composition.

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