Chicken gut microbiota: a brief understanding of the dynamics and interactions which govern flock performance

Introduction

The gastrointestinal tract (GIT) of a chicken harbours a diverse bacterial community in which each bacterium is adapted to its own ecological niche and synergistically lives with other bacterial species in the same community. The composition and function of these communities vary depending on the age of the birds, location in the GI tract and on the dietary components (Shang et al., 2018). The nature of the intestinal microbiota is extremely complex and is highly dependent on the physicochemical microenvironments of a specific area of the GIT. There are several factors that drive the fitness and colonisation efficiency of the microbes in the GIT of chickens of which the availability of suitable growth substrates, prevailing pH and redox potential and the antibacterial secretions of the host in a specific intestinal section are important (Apajalahti and Vienola, 2016) apart from the age of the birds which is seemingly the most important dynamic factor that determines the host-microbiota interactions and the colonisation of the latter onto the mucosal surface of the former’s small intestine. The bacteria present in the GIT find enormous scope to grow with the substrates they get from the ingested food especially when the latter remains at the upper GIT. As food passes down the substrates vanish due to digestive actions of the host animal and this makes the bacteria present in the lower GIT specialist in degradation of specific food components which remain undigested despite exposure to the digestive enzymes (Apajalahti and Vienola, 2016). Thus, the nutrients, resistant starch or proteins, the host cannot digest become the substrate for growth for certain bacterial species which might prove to be beneficial or in some instances costs a lot to the host animal.

Generally, the baby chick should come out of the egg in a ‘sterile’ condition and it is the feed that puts the baby chick in front of a cascade of microbial genera some of which the chick might find as friends and some as foes. Within a day post hatch, the bacterial densities in the ileum and caecum of the broiler chickens reach 10⁸ and 10¹⁰ per gram of digesta and the same get stable at 10¹¹ and 10⁹ per g of ileal and caecal digesta within 3 days post-hatch (Apajalahti et al., 2004). It is noteworthy that the nature and type of the bacteria that establish during the first hours of a chick’s life varies to a great extent and depend to a large extent on the diversity of the bacterial species the chick is exposed to and in absence of a parentally derived bacterial source this diversity might be enormous and unpredictable (Donaldson et al., 2017). Since the baby chick is dependent altogether on the physical contact with the environment post-hatch for their gastrointestinal microbiota to develop, extensive intra-flock and inter-flock variation with regard to intestinal microbiota population is observed and it is the initial inoculation and colonisation of the chicken GIT microbiota which can have a major influence on the growth performance and health of birds at the later age as well as flock microbiota uniformity and reproducibility (Stanley et al., 2013).

The healthy gut and some related definitions

Gut health involves a number of physiological, microbiological, and physical functions that work together to maintain intestinal homeostasis (Kogut, 2019). A healthy gut regulates not only the local (intestinal) physiological homeostasis, but also systemically other organ systems as well that supports the host ability to withstand environmental and infectious stressors. The terms microbiota and microbiome are often incorrectly used interchangeably in the literature. Microbiota refers to the microorganisms which live in an environment such as in the intestine and includes viruses, bacteria, fungi, and protozoa (Roto et al., 2015). However, in many cases, authors have been found to describe the bacterial populations residing in the GIT as the microbiota, which technically should be considered as incorrect. Similarly, microbiome is a term that describes the genome of all the microorganisms, symbiotic and pathogenic, living in and on all vertebrates. The gut microbiome is comprised of the collective genome of microbes inhabiting the gut including bacteria, archaea, viruses, and fungi (Roto et al., 2015). Hence, discussing the microbiome is far more complex than the microbiota. metagenome defines the genetic potential of the microbiota and includes the genomes of microbial populations, the genes within the microbial populations, and includes the plasmids within the different bacterial populations of the microbiota (Roto et al., 2015). In the current review, the term “microbiota” will be used frequently to describe the entire microbial community residing in the GIT. The term ”bacteria” will be used to describe the bacterial population only. Finally, the terms eubiosis and dysbiosis or dysbacteriosis should be defined. Eubiosis is defined as a state when the intestinal microbiota is theoretically “normal” and “balanced” and presumably meets all the requirements of the host animal so that the host benefits due to the presence of the microbiota in terms of their effects on health in general and in terms of metabolism, immunity, immune functions and protective barrier actions in particular. A gut microbiota in a eubiotic status is characterized by a preponderance of potentially beneficial species, belonging mainly to the two bacterial phylum Firmicutes and Bacteroides, while potentially pathogenic species, such as that belonging to the phyla Proteobacteria (Enterobacteriaceae) are present, but in a very low percentage. In case of dysbiosis “good bacteria” no longer control the “bad bacteria” which take over (Zhang et al., 2015).

Dynamics of intestinal microbial population

Donaldson et al. (2017) has made a comprehensive review on the dynamics of intestinal microbiota with age in chickens. An extensive review about the type of the bacteria present in a chicken gut is made (Rychlik 2020). From these papers it is understood that it is not only the age but also the interaction between the types of the bacterial species which govern the number and the nature of the bacterial species which will finally be established in the GIT. It will be inappropriate to assume that it is only the bacteria which are the sole inhabitants of the chicken GIT rather it is a complex microbial community consisting of bacteria, fungi, archaea, protozoa, and virus which constitute the whole microbiome (Shang et al., 2018). However, since the bacteria are the predominating creatures in this community, so it will be apt to focus on this segment to understand how performance of chickens are heavily guided by modulation in the bacterial community prevailing in the GIT.

In human beings the intestine is rapidly colonized by an array of microbes. First colonizers, facultative anaerobes, create a new environment that promotes the colonization of strict anaerobes as Bacteroides, Clostridium, and Bifidobacterium spp. (Rodríguez et al., 2015) The intestinal microbiota of neonates are less diverse and dominated by Proteobacteria and Actinobacteria. With time the Firmicutes and Bacteroidetes emerge (Eckburg et al., 2005; Qin et al., 2010; Backhed, 2011) and by first year of life, infants possess the distinct microbial profile resembling that of an adult (Palmer et al., 2007; Koenig et al., 2011; Yatsunenko et al., 2012).

Almost a similar model follows in chicken as well. Enterobacteriaceae and Lactobacillus, are the early colonisers of the GIT which are followed by the Firmicutes within 7 days of life (Carrasco et al., 2019). The facultative anaerobic properties of the former group of bacteria allow them to utilise the initial oxygen supplies in the gut during the first days of life. The subsequent depletion of this oxygen supply then creates a more favourable environment for obligate anaerobes to develop and thrive (Matamoros et al., 2013). The reduction in Enterobacteriaceae numbers over time can be influenced by the production of short chain fatty acids in the cecum of the chickens which reduce the intestinal pH and have an inhibitory effect on bacteria that are considered acid-sensitive, such as Enterobacteriaceae. This mechanism assists in the prevention of overgrowth and the potential pathogenicity that is associated with high levels of Enterobacteriaceae in the GIT (Donaldson et al., 2017). In commercial broiler chickens it generally takes approximately 3 weeks for the intestinal microbiota to reach a stable composition (Carrasco et al., 2019) although factors like genetic background of the birds, farm management practices (Oakley et al., 2014; Shang et al. 2018; Johnson et al., 2018; Jurburg et al., 2019) and seasons (Oakley et al., 2018) influence the colonization pattern to a great extent. Nevertheless, colonization of gastrointestinal tract with specific bacterial species is likely to be a stochastic process which, apart from the other factors mentioned above, are primarily driven by the contact with microorganisms coming from the rearing environment and from bacteria present in food and water.

Microbial dynamics of chicken GIT is largely dependent on the pro-inflammatory signals the GIT is exposed to in its lifetime. This will be explained in more details at a later section of this paper. In short, the cytokine profile expressed by the host intestinal cells in response to different bacterial groups play a pivotal role in controlling the microbial dynamics of the GIT. A pro-inflammatory cytokine profile may positively influence the phylum Proteobacteria which include a number of pathogens whereas e the increase in members of the phylum Firmicutes is associated with an anti-inflammatory state(Oakley and Kogut, 2016; Carrasco et al., 2019).

Intestinal microbiota and gut immunity

The microbes residing in the GIT develop strong interactions with the bacterial species the chicken comes across during its lifetime. The pattern of this interaction depends upon the predominance of bacteria which colonize early in the life of the chicken and how this early population changes with time. The interaction between the commensals and the potential pathogens lead to production of pathogen associated molecular pattern (PAMP) and metabolic by products which together control the immune responses of the GIT. The PAMPs are recognized by pattern recognition receptor bearing cells of the innate immune systems and many epithelial cells which is followed by release of humoral (immunoglobulins) and cell mediated immune components (heterophils). Dietary factors or supplements that triggers off the immune responses by stimulating the PAMP and cause release of cytokines in absence of any pathogen induced infection may take toll on the nutrients thus limiting growth of the bird. The complexity of the intestinal microbiome may have a dramatic influence on the gut T cell repertoire. Lactobacillus species has the capacity to induce differential cytokine expression in T cells of chicken cecal tonsils which contributes to intestinal homeostasis. After being challenged with Salmonella, broiler chickens treated with L. acidophilus, Bifidobacteriumbifidum, and Streptococcus faecalis significantly decreases the expression of interleukin (IL-1 and IL-2) and interferon-γ genes. This indicates that Lactobacillus eases out the pressure on the gut immune system and in this way spares nutrients for growth. Plausibly, this also provides the explanation for the growth promoting effects of probiotics like Lactobacillus in chickens. The complex interaction between the gut bacteria and host defence mechanism may have some paradoxical consequences as well. For example, treatments with probiotics like Lactobacillus depresses expression of β-defensin gene to keep the immune responses under control. This paves the pathway for a blunted immune response against Salmonella infection which needs to be mediated through β-defensin. These paradoxes imply that the time and type of probiotic treatments should be taken up only after a proper risk and exposure assessment of the chickens in a particular environment and the type of bacteria constituting the gut microbiota. Though this is possible under controlled experimental conditions, under commercially driven field conditions this may not be possible at all and the decisions must be driven by the wisdom and experience of the nutritionist and field veterinarians.

While it is of extreme importance that the entire GIT should remain toned up for any immune consequences any overt reaction may not be desired. Immunological tolerance to harmless commensal microbiota is essential to avoid unnecessary expression of defence functions and inflammatory reactions. Well-developed homeostasis and accurate recognition of harmful antigens and pathogens helps to reduce consumption of limited amino acid and energy resources for maintaining non-justified alertness. Microbiota regulate the rate of mucin production and its composition as well as the rate of epithelial cell proliferation. It is obvious that the active proliferation of epithelial cells and the production of mucin and antibodies help the host to defend itself against potential threats from the environment.

The cost of the intestinal microbiota

It is not always true that the host animal is only benefitted from the intestinal microbiota. Logically, the microbiota present in the GIT thrives on the nutrients consumed by the host animal and hence a nutrient cost has to be borne by the host for these microbiota. It is but obvious that an exaggerated microbial population would consume a greater amount of nutrients thus compromising the host of the essential nutrient supply. The negative impact of the microflora on bird performance is reported by Muramatsu et al. (1994). Germ-free birds offered the same diet grew substantially quicker and appeared to capture less energy from the diet than their conventional counterparts which were supposed to have a normal intestinal microbiota. The discrepancy in energy capture is a consequence of the microflora extracting a significant amount of energy from the diet thus making it unavailable to the bird. In that experiment the “energy cost” of the microflora was at least 10% of the total apparent metabolizable energy consumed by the birds (Bedford, 2000).

Intestinal microenvironment affects microbial colonization in intestine

It is the presence of available substrates that actually governs the microbial colonization pattern in the GIT (Figure 2 and Figure 3). The type of the microbiota to be colonized in different sections of GIT also depends on the substrate availability and the microenvironment. This implies, if a diet is rich in nutrients which bypass digestion in the upper GIT then substrate availability will be more in the hind gut and this should facilitate growth and colonization of bacteria which reside in the hind gut including ileum and cecum (Morgan et al., 2014). Examples of such resistant nutrients include non-starch polysaccharides, resistant starch, or resistant protein. So, plausibly, the bacterial species which use these molecules as their substrates for growth should thrive well when a greater quantity of these nutrients reach the hind gut intact. On the contrary, the proximal gastrointestinal tract (crop, proventriculus, gizzard) is characterised by low pH and hence this part is predominated by the lactic acid-producing bacteria, mainly Lactobacillus spp., Enterococcus spp. and Streptococcus spp. (Barnes et al., 1972; Salanitro et al., 1978; Lu et al., 2003; Apajalahti and Kettunen,2006; Bjerrum et al., 2006; Abbas Hilmi et al., 2007). A couple of other factors play an extremely important role in determining the type of the bacterial species in different parts of the GIT and these are the redox potential and presence of reactive oxygen species. The bacteria which reside in the proximal GIT are mainly facultative anaerobes and they can survive in presence of oxygen and are equipped with mechanisms which can quench the reactive oxygen species generated during their metabolic processes. However, the strict anaerobes, which are the typical inhabitants of the hind gut are extremely sensitive to reactive oxygen species and hence cannot survive in areas where there are scopes of development of these oxidative radicals are there. All these factors clubbed together bring about a paradigm shift in the nature of microbiota in the upper and lower GIT of chickens.

Nutrient digestibility determines gut health

Oviedo-Rondón (2019) is of the opinion that it is the presence of the undigested nutrients in the GIT is the main cause of dysbacteriosis which leads to proliferation and colonization of pathogens like Clostridium perfringens, E. coli, or Salmonella spp. the same hypothesis has been propounded by other workers as well (Bedford, 1995; Apajalahti and Bedford, 1999; Brown et al., 2012; Chan et al., 2013). Proliferation of these potential pathogens results in disruption of gut microbiota-host equilibrium (Round and Mazmanian, 2009; Weiss and Hennet, 2017) causing the metabolic, pathogenic, or sterile inflammation (Kogut et al., 2018). The excess of nutrients in the hindgut may be due to either high nutrient levels in the diet or suboptimal digestion (Brown et al., 2012) and in absence of interventions which reduce the supply of such nutrients, bacterial proliferation and dysbacteriosis is the most likely consequence. This also explains the why antibiotic growth promoters (AGPs) remained the most successful digestibility enhancers in poultry industry with the most consistent effects involving the least cost. The AGPs, which have a broader spectrum of activities, basically reduce the overall load of bacteria in a rather nonspecific manner thus creating enteric conditions with fewer bacteria which could negatively impact the small intestinal milieu. The consequence is further extended to effects which include less competition for nutrients with the host, a blunted pro-inflammatory response and timid cytokine stimulation in the local tissues. The latter two effects are further responsible for sparing a substantial amount of nutrients which, would otherwise be wasted, but now can be used for growth of the birds. However, selection of the AGPs is of extreme importance in gaining the optimum response from the AGPs. The evolution of species in the GIT of a chicken depends on the type of the bacteria the baby chick will be exposed to at the beginning of their life. This is dependent upon the dominant species at the outset in the environment of the cage, pen or shed and, in particular, the hatchery (Bedford, 2000). The order in which the chick gets exposed to the bacterial species is further important to determine the bacterial colonization pattern at the beginning of the life. Moreover, the bacterial population will not be a “static” one rather it will be highly “dynamic” because the environment in the intestine becomes increasingly hostile to ’new’ invaders for reasons of space, the presence of toxins and the availability of nutrients(Bedford, 2000). This explains why the bacterial colonization pattern may altogether be different in adult birds compared to that in a baby chick. Selection of the type of the AGPs depending on the type of bacterial colonization becomes crucial in order to achieve optimum growth. For example, if the chick is found to be colonized predominantly by bacterial genera which are responsive to a particular class of AGPs, like the bacitracin, then it is likely that this particular class of AGP will outperform the other classes like colistin. However, the class of bacterial species inhabiting the intestine and their interactions with a particular class of AGP is extremely complex and with hitherto incomplete knowledge in this field it is always difficult to pin point towards a specific class of AGPs for optimum growth promotion and hence commercial practices involve supplementation of diets with more than one AGP class in order to achieve a more comprehensive effects, albeit with some overlapping.

Exogenous enzymes and intestinal microbiota

If it is accepted that undigested nutrients are the main cause for establishment and proliferation of potential pathogens in the GIT, then any attempt that can reduce the supply of such nutrients to the resident bacteria by shifting the site of digestion to the anterior gut should ideally “starve” them of available nutrients thus controlling their growth (Bedford, 2000; Cowieson and Kluenter, 2019). While Bedford and Cowieson (2012) opined that enzymes like phytase improve the integrity of intestinal mucin, increase gastric residency of feed, reduced inflammatory responses and immune functions, Cowieson and Roos (2016) extended the effects to exogenous protease with the hypothesis that which the authors claim to be associated with the reduction in undigested dietary and endogenous protein flow to the caudal gut, reduced inflammatory effects associated with proteinaceous antinutrients and improved gut tensile strength and tight junction integrity. Thus, exogenous enzymes may improve the stability of the gut by reducing substrate for putrefactive organisms, increasing substrate for beneficial fermentative organisms, and enhancing the ability of the intestine to defend itself against unwanted bacterial ingress (Cowieson and Kluenter, 2019). Like the selection of AGPs to achieve an optimum response from the treated species, deployment of enzymes to gain the maximum advantage is of enormous importance. For this the primary objective should be to judge the ingredients being used in the dietary regimens and identifying the extent to which potentially undigested nutrients are present there. The dynamic nature of small intestinal milieu is the other factor that needs to be taken care of while selecting the enzyme for intervening the digestive processes. The consequences of the presence of undigested materials in the GIT should be known to the nutritionist in order to select the enzyme (and other intervening candidates) which should be apt to counteract with the likely consequences. A good number of works and hypothesis have been propounded by several authors in the last century and there is an excellent compilation of these references by (Bedford, 2000). When the quantity of undigested materials in GIT increases, the birds try to compensate for the unabsorbed nutrients by increasing the size of the GIT (Brenes et al., 1993), a process which, at least theoretically should increase the digestive capacity of the gut. In doing so the villi enterocytes of the intestine grow and move up the villus more rapidly (Silva and Smithard, 1996). Such enterocytes are more immature and, as a result, less able to absorb nutrients efficiently as a result of having a limited range and concentration of digestive and absorptive enzymes. With a change in surface glycoprotein structure of the newly developed enterocytes, the GIT environment changes altogether, and this leads to proliferation of hitherto dormant bacterial species some of which may be potential pathogens. If AGPs are there in the feeding regimen then the change in bacterial species despite the adaptive modifications in the physical structure of the GIT gets controlled. Though a similar effect is unlikely with exogenous enzyme supplementation, it is possible to partially achieve a similar goal if selection of the enzyme is appropriate and the enzyme targets the specific dietary fractions which can escape digestibility by the endogenous enzymes.

General comments

Intestinal health is necessary to maintain efficient and sustainable physiology of the GIT which has digestive, absorptive, metabolic, immunological and endocrinological functions (Oviedo-Rondón, 2019). Any disruptions of intestinal health can affect the overall performance of a flock and it is the main reason that research on chicken GIT attracts researchers to a great extent. Due to increasing demands for economic efficiency, animal welfare, food safety, reduction in environmental impacts, and a ban on or avoidance of AGP use a better understanding of the chicken gut microbiota with regard to the its dynamics and the interaction between the microbial population and the host animal has become extremely important. It is not possible to cover the entire subject within a short span of a review article since the subject is a vast one and involves micro as well as macro environment surrounding an individual chicken as well as the entire flock. Quality of feed, water, management, and external stressors play enormous role in controlling the microbial homeostasis of chicken GIT. Health conditions of the parents the microbiological environment of the hatcher are the other factors having influence on the gut microbiota.

General comments

Conclusions

Intestinal health impacts poultry productivity, animal welfare, food safety, and the environmental impact of poultry production. The intestinal health is largely dependent on the complex interactions between the members of the microbiota community. This microbiota is a complex and dynamic system that influences the local immunity of the small intestine thus influencing the bird’s immune responses to the pathogens invading through the GIT. In this way the gut microbiota serves as a functional organism to control bird’s performance. Given the increasing evidence demonstrating the importance of the microbiota in maintaining intestinal homeostasis, which is the pivot for maintenance of flock performance, and tools available to modulate it with impact in chicken productivity, it is likely that monitoring the composition of gut microbiota in the farms could be routinely included in the future.

Chicken gut microbiota: a brief understanding of the dynamics and interactions which govern flock performance - Image 1

The intestinal microbiome is a signalling hub that integrates environmental inputs, such as diet, with genetic and immune signals to affect the host’s metabolism, immunity, and response to infection. The haematopoietic and non-haematopoietic cells of the innate immune system are located strategically at the host–microbiota interface. These cells have the ability to sense microorganisms or their metabolic products and to translate the signals into host physiological responses and the regulation of microbial ecology.

Figure 2: Protein flow in small intestine

Chicken gut microbiota: a brief understanding of the dynamics and interactions which govern flock performance - Image 2

In an ideal case the protein uptake by the host prior to the ileum remains at its optimal level thus leaving little undigested protein for the bacteria to feed on. However, in such “ideal” cases also, a substantial amount of proteins in the form of mucins, sloughed off epithelial cells, unspent enzymes, antibodies etc. get the chance to pass to the cecum. These proteinaceous materials become the substrates for the growth of the bacteria which survive mainly by utilizing protein as their main substrate for growth. These include potential pathogens like the clostridia and the proteobacteria like E. coli (Apajalahti J and Vienola K, 2016).

Figure 3: Protein fermentation in ileal bypass protein

Chicken gut microbiota: a brief understanding of the dynamics and interactions which govern flock performance - Image 3

Once the protein escaping digestion in the upper GIT reaches the large intestine it becomes the substrate for growth of number of bacteria. These bacteria produce compounds like short chain fatty acids, branched chain fatty acids, amines, ammonia, indoles, and cresols and all these substances collectively elicit sufficient proinflammatory responses. A part of the above-mentioned metabolites passes through the excreta but some go back to the small intestine and elicit substantial inflammatory changes and even dysbiosis.

Figure 4: Photographs showing poor intestinal health caused by dysbacteriosis

Chicken gut microbiota: a brief understanding of the dynamics and interactions which govern flock performance - Image 4

Intestinal lumen filled up with undigested feed particles, blood tinge in excreta, flaccid intestine, orange tinges in digesta are some of the common indications of dysbiosis.