Host and Microbial Biomarkers for Intestinal Health and Disease in Broilers

One of the most sustainable types of production of animal protein is chicken meat production. Chicken production needs less feed consumption per kilogram of produced meat and uses less land and water for both farming and feed production. The major reason for this is the continuous improvement of animal performance, reflected in an ever decreasing feed conversion (kg feed consumed per kg body weight) and reduced time to achieve market body weight (Zuidhof et al., 2014). Continuous improvements in performance parameters include genetic selection for high-performing chicken lines, technological developments in hatching and housing conditions, and feed optimization and management practices that support (intestinal) health. Among the latter, the use of antimicrobial growth promoters is a practice that has been banned in many countries worldwide but the use of therapeutic antimicrobials in the animal production industries is still high, though decreasing. This has created a situation in which the animal and its microbiota are experiencing a big change, as the animal breeds have been used for more than 50 years almost exclusively in a production system where antimicrobial usage was common practice. Reducing or stopping this practice has resulted in different diseases and syndromes, most of which are of intestinal origin. Indeed, about 60% of therapeutic antibiotic usage in broilers is to control intestinal diseases. The move away from antimicrobials has led to increasing concerns about gut health. Bacterial diseases, enteritis, dysbiosis, and poor digestibility are a consequence resulting in poor growth performance of birds. In fact, all these entities have common denominators in the form of microbial shifts that go hand in hand with epithelial permeability increases, inflammation and thus performance losses, and are often related to nutrient excesses in the intestine or feed-derived issues (poorly digestible nutrients, excess of energy or protein levels). The most important intestinal disease entities and syndromes in broilers, with a performance effect, are briefly described in the next paragraph.

 

The most severe example of a disease that has emerged in broiler chickens after the ban on growth-promoting antibiotics in animal feed is necrotic enteritis, which imposes a significant economic burden on the poultry industry worldwide (Skinner et al., 2010; Kaldhusdal et al., 2016). This disease is typically caused by nutritional excesses in the gut as well as predisposing epithelial defects caused by mycotoxins and coccidia, and it is occurring in animals with the highest body weight gain, so clearly related to production parameters (Moore, 2016; own unpublished data). The causative agents of necrotic enteritis are netB-toxin containing Clostridium perfringens (type G) strains (Rood et al., 2018). Necrotic enteritis can occur as an acute clinical form which is characterized by a sudden increase in mortality, and as a subclinical form which results in a lower weight at slaughter age. In both cases, macroscopic necrotic lesions are found at the mucosa of the small intestine upon necropsy, and thus the intestinal barrier is compromised and severe mucosal inflammation occurs (Prescott et al., 2016).

A disease syndrome that has clearly emerged in the EU broiler industry simultaneously with the ban of growth promoting antibiotics is the so-called ‘dysbacteriosis’. This is a poorly described condition of the gut and may or not be a synonym for conditions such as ‘wet litter’, ‘non-specific bacterial enteritis’, ‘small intestinal bacterial overgrowth’, ‘malabsorption’, and many more. The common clinical denominator is thinning and ballooning of the small intestine, increased water content of faeces and reduced digestibility of feed with undigested residues visible in the faeces (Teirlynck et al., 2011; Ducatelle et al., 2015). In many cases, this is linked to increased feed conversion, decreased body weight and thus poor performance. Moreover, wet litter leads to various additional disease conditions such as pododermatitis, breast blisters and ‘hock burn’, which are criteria used to evaluate animal welfare. It is generally believed that ‘dysbacteriosis’ is a condition in which the interaction between the gut microbiota and the host is impaired, such that the gut health is not optimal. All this is probably influenced by nutrition and it is suggested that the altered composition of the gut microbiota induces changes in the gut wall, including morphological changes (villus length decreases, crypt depth increases, epithelial cell damage, …) and inflammatory reactions (infiltration of immune cells in the wall). The combination of a suboptimal microbiota combined with effects on the gut wall would then most likely interfere with digestive processes, eventually leading to poor performance, and induce enteritis.

As broilers often have gut barrier integrity issues (increased permeability), toxins, feed antigens, but also bacterial products and bacteria can cross this barrier and spread systemically. This also aids locomotory diseases that are a consequence of both the high body weight gain and the pressure this puts on the skeleton of the animal, but also of bacteria that attach to bones at different sites in the body. Indeed, lameness in broiler chickens is a significant animal welfare problem, which is increasingly occurring (up to 1% of all animals). Bacterial chondronecrosis with osteomyelitis (BCO) is a disease characterized by bacterial infection in rapidly growing bones under repeated mechanical stress and typically occurs in tibiae, femora and the thoracic vertebrae (Wideman, 2016). The terminology is often confusing and names such as ‘kinky back’, spondylitis, spondylolisthesis, femoral head necrosis and others are given to describe similar or the same syndromes. It is assumed that bacteria cross the intestinal barrier, enter the bloodstream and hematogenously spread to osteochondritic clefts or to microfractures at the growth plates. When colonizing the growth plates, the bacteria are rather inaccessible to antibiotics and the host immune system, enabling them to induce necrosis. Bacteria that are found in BCO lesions are commensal intestinal bacteria that have translocated through the intestinal epithelium and have spread systemically. Bacterial genera and species that are isolated from BCO cases are, amongst others, opportunistic bacteria including staphylococci, Escherichia coli, and enterococci. These kind of disease entities are thus again originating from high performance and at least partly have an intestinal origin. It has been shown that probiotics can affect BCO, again pointing to the intestine as origin of the bacteria that cause the disease (Wideman et al., 2015).

 

Intestinal health is a term that is not yet clearly defined, despite being a focus of major research efforts in the last decade, both in human and in veterinary medicine. It can be described at different levels. In the past, one has used different indirect systems to measure gut health, such as the water content of faecal material. At macroscopic level, optimal gut health can refer to a condition in which there are no observable changes in gut wall appearance as compared to a normal condition. While this is very clear in conditions in which gross lesions are seen, such as in necrotic enteritis and coccidiosis cases, this is less clear and even invisible in conditions that might cause microscopic alterations that affect performance. A method to score gut wall appearance has been validated previously (Teirlynck et al., 2011) and is used by veterinarians for broiler chickens. In this system, in total, 10 parameters are assessed and scored 0 when absent or 1 when present during visual inspection of the intestinal wall at autopsy after which 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) is presence of undigested particles caudal to the ileo-cecal junction. A low gut wall appearance score thus indicates good gut health. This system however is rather subjective, as it depends on the person who performs the scoring and it is influenced by specific factors such as diet type (e.g. meal vs pellet with regard to the presence of undigested feed particles). It has, however, been described that the score is associated with histological parameters under certain conditions, including villus length and infiltration of immune cells in the gut wall (Teirlynck et al., 2011). These parameters are much more objective, quantitative and clearly associated with gut health, as they relate to the epithelial surface (villus length) and thus digestibility, and with inflammation. As such, they are associated with intestinal insults that damage the epithelial lining and thus affect performance. Although histological parameters have value in evaluating gut health, these are mainly of importance under experimental conditions (e.g. when testing interventions) and are more difficult to use in field conditions, given their invasive and time-consuming nature. Optimal gut health could thus also be defined as a condition in which no microscopically visible alterations are seen. Other invasive biomarkers are mainly ones that can be found in blood. Acute phase protein (APP) production in the liver can be the result of intestinal bacteria that cause inflammation in the gut, and thus cytokine production by epithelial and immune cells that is sensed by liver cells that produce APP. It can also be the consequence of translocation of bacteria and their products through the gut wall reaching the liver, so that hepatocytes secrete the APP. APP can be measured in serum, but the production of APPs can be triggered anywhere in the body of the animal and not specifically the gut (Langhorst et al., 2008; Eckersall and Bell, 2010). Other biomarkers that could potentially be found in the serum are of microbial origin. Increases in intestinal permeability in poor gut health conditions would lead to translocation of bacteria, LPS and even metabolites such as D-lactate, that can be found in serum. These markers however do not seem to be very reliable because of multiple reasons, including intrinsic differences in the intestinal concentrations on itself that may cause variability in the serum levels (Ducatelle et al., 2018 for more detailed references).

Non-invasive markers are preferred in the field and ideally they should be based on faecal material as this is easy to collect. In addition, mixed faecal samples can be taken so that the gut health status of the whole flock can be evaluated. These markers can be microbial or host-derived. Microbial markers originate from the observation that gut health problems often are associated with shifts in the microbial composition. This has been described in detail for human inflammatory bowel disease, in which Enterobacteriaceae have been associated with inflammation and butyrate producing bacteria from the Ruminococcaceae family, such as the genus Faecalibacterium, have been shown to be depleted in the faeces of diseased individuals (Machiels et al., 2018; Rivera-Chavez et al., 2017). While changes in microbial composition are clear in the case of severe intestinal inflammation, differences can be much more subtle in intestinal disorders with a much less clear phenotype, such as irritable bowel syndrome in humans. The same is true for chickens, in which rather well-described microbial composition shifts have been described in the gut of animals with necrotic enteritis, but, despite numerous studies, it is not easy to identify OTUs that are correlated with intestinal health and animal performance (Stanley et al., 2016). Our group has conducted a number of studies using intestinal inflammation models to describe 16S rDNA sequences that have a correlation with intestinal health (i.e. villus length, immune cell infiltration in the gut wall, and performance) and some general patterns of beneficial and harmful microbial groups can be extracted from these data. Examples are correlations between the reduced abundance of Faecalibacterium prauznitzii and Butyricicoccus pullicaecorum and conditions that increase the villus length and decrease the CD3⁺ T-cell infiltration in the small intestinal wall of broilers under experimental challenge conditions (unpublished), but many more relevant OTU changes occur. In addition to the use of microbial composition and taxa, one could also use functional genes or metabolites as markers. The most well-known example of a beneficial microbial metabolite is butyrate, and functional genes such as the butyryl-CoA:acetate CoA-transferase can be used to quantify the abundance of butyrate producing bacteria in faecal samples (Onrust et al., 2015; De Maesschalck et al., 2015). A lot of other metabolites are involved in gut health and this is a domain in which much progress can be made. Measurements of epithelial permeability can be done using oral administration of compounds that pass through the epithelial layer when damaged and thus can be measured in serum (e.g. FITC-dextran, lactulose/rhamnose) (Ducatelle et al., 2018 for references). This is not applicable to field conditions. Host biomarkers for gut health should ideally be associated with gut function, such as digestibility, cellular damage and inflammation, amongst others. In humans, calprotectin, a neutrophil granule protein, is used to quantify gut inflammation and is very useful to assess the severity of intestinal inflammation (Ayling and Kok, 2018). For poultry, our group recently identified a similar protein biomarker in colonic content of animals from an inflammation model. Other markers were identified and were related to inflammation, serum leakage, epithelial cell and tight junction damage. Currently, these are being brought to a practical field assay using ELISA or dipstick assays. An example is ovotransferrin, a protein that is produced in the liver and thus only reaches the faeces when serum leaks through the epithelial cells. The concentration of ovotransferrin increases with the severity of necrotic enteritis and coccidiosis infections, and has been associated with gut damage in not yet published dysbiosis models (Goossens et al., 2018).

 

Easy-to-measure biomarkers in faecal samples are of value for the poultry industry for various reasons. First, they can be used to measure gut health in field conditions and justify interventions to promote gut health. These can be the administration of antimicrobials when animals suffer from a diagnosed bacterial infection but can also be the supplementation of a gut-health promoting feed additive when the animals are not having symptoms. The latter is often the case, and prediction of poor performance will likely be the most important driver for using gut health biomarker tools. Ideally, diagnostic tests for gut health result in the choice of specific feed additives or changes in feed management, depending on the parameter that is affected. Another important application of gut health biomarkers is efficacy testing of newly developed feed additives by the industry. While faecal biomarker proteins (host proteins) and microbial markers (taxa and metabolic pathway genes) have been identified in experimental models (necrotic enteritis, gut inflammation, and more), field applications are likely not to be straightforward for various reasons. Apart from the technical aspects (dipstick or ELISA development), field conditions are very different in different regions worldwide and it might be complex as a lot of preventive antimicrobials are still used. It will be a challenge to convince poultry producers to use a diagnostic tool to justify use of antimicrobials, as the latter are often a cheap and easy certainty for production performance. Educating the poultry production industry on the risks of antimicrobial usage, antimicrobial resistance, and the need to use a strategy of measuring gut health and using non-antibiotic prevention methods is already occurring but needs further effort.

 

ACKNOWLEDGEMENTS: We thank all scientists, technicians and personnel from the Department of Pathology, Bacteriology and Avian Diseases, all funding agencies and industrial partners, and all research groups that have contributed to the data used to produce the paper.