The Importance of Gut Microbiota in Chickens with Particular Emphasis on The Field Situation

I. INTRODUCTION

The recent major drop in the cost of DNA sequencing, combined with advances in the annotation of 16S rRNA gene microbial databases and analysis tools, have all contributed to the “golden age of microbial ecology” (Oakley et al., 2014). Poultry researchers have leveraged and applied these advances to define approaches that may be taken to manipulate microbiota to improve a number of poultry production steps. One of the major issues in poultry microbiota research is the extent of flock to flock variability which makes both health and nutritional treatments difficult to reproduce among the flocks (Stanley et al., 2013b). A key contributor to this variation is removal of maternal microbiota transfer via use of modern hatching practices. Removal of antibiotic growth promoters left the industry looking for alternative approaches to control pathogen growth, and new methods for improving intestinal microbiota membership. It is very difficult to change a mature intestinal microbial community; therefore, the first days of bird’s life are likely to be the key intervention point to permanently reducing pathogen load and encouraging beneficial bacteria (Stanley et al., 2014). In addition, maintaining low levels of stress and operating production systems close to optimal are prerequisites for better bird health. As in other symbiotic relationships, host and microbiota have a two way relationship, if the host suffers the microbiota will not be left unchallenged.

II. FIRST DAYS OF LIFE

Until recently we assumed that chicks are fully sterile in ovo. Recently, that opinion has been challenged and a low level colonisation before birth is being suggested for both chickens (Kizerwetter-Swida and Binek, 2008) and humans and other mammals (Perez-Munoz et al.,, 2017) alike. Moreover, if the in ovo colonisers of the bird embryo include opportunistic pathogens, the embryo is likely to die before hatch (Jahantigh, 2010), similar to the spontaneous abortion due to foetal infection in humans (Perez-Munoz et al., 2017). There is an abundance of studies that have used in ovo probiotic injection to improve immunity and vigour of hatchlings without any negative effects on hatching rate and with a number of health benefits reported (Pender et al., 2017; de Oliveira et al., 2014).

Despite possible low-level colonisation in ovo, major steps towards formation of gut microbiota starts immediately post hatch and is determined by the bacteria present in the chick’s immediate environment during the first days of life. Unlike in humans where the microbiota is considered stable and formed by the toddler age, the timeline for microbiota establishment in broiler chickens is significantly reduced and well established during the first week post-hatch (Apajalahti et al., 2016), remains reasonably constant until 30 days of age (Lu et al., 2003); however, slowly increasing in richness and still capable of acquiring new members (Donaldson et al., 2017). Mature microbiota attains a level of stability and the ability to resist change, unlike the immature developing microbiota where significant perturbations, such as antibiotic administration or pathogen exposure, may lead to lifelong health consequences (Zhou et al., 2018). For example, very low levels of Salmonella are needed to permanently colonise the intestinal tract at a young age while old birds show significant resistance to Salmonella colonisation. However, transfer of microbiota from older to young chicks increases their resistance to colonisation (Oakley et al., 2014).

The knowledge of rapid colonisation and maturation of microbiota in chicken strongly suggests that it is of utmost priority to reduce pathogen load and increase beneficial bacteria load during the first week of chick’s life. Both of these aspects are crucial for intestinal and overall health; just reducing pathogens via maintenance of a very clean environment (Stanley et al., 2013b) is more damaging to the microbiota than early overloading of the bird with beneficial probiotic strains without any pathogen control, which, depending on strains used, can even be beneficial (Baldwin et al., 2018). The inherent variability of poultry microbiota is much more pronounced than in mammalian species due to nearly complete removal of maternal influence. Hatching birds in very clean hatcheries without the presence of poultry adapted microbiota leads to random colonisation and high flock to flock variation (Stanley et al., 2013b). There are currently no published data on the dynamics of natural microbiota seeding from mother hen to the chicks.

Exposure of birds to the microbiota of previous well performing flocks reduces the effects of random colonisation and microbiota variability among the birds but does not fully transfer donor microbiota and performance, mostly depending on the ability of specific core microbiota species to colonise and persist (Donaldson et al., 2017). In the United States, bacterial diversity is encouraged by litter re-use from the previous flock, providing adequate and rich inoculum to the new flock. The litter carryover would be detrimental if the previous flock was of mildly compromised heath due to latent low level pathogen infection which could then be amplified in the subsequent flock due to early exposure. Regardless, high levels of hygiene in the first days and depletion of maternal microbiota adapted to the chicken host over thousands of years appears to be an unforeseen and undesirable outcome of modern day hatchery practices.

III. SPATIAL AND TEMPORAL FLUCTUATIONS

Introducing significant changes to microbiota does not really need a very influential variable. Even the most insignificant treatments will cause temporary change in gut microbiota. It is well recognized in human research that gut microbiota of each individual changes slightly on a daily bases. In chickens, faecal microbiota is so variable (Stanley et al., 2015; Stanley et al., 2013b) that even replicates of a single sample can show variation and faecal matter is often visibly non-homogenous. Faecal microbiota from the same bird will vary slightly due to periodic emptying of different gut sections; however, core faecal microbiota remains fairly constant (Stanley et al., 2015). The caeca is the gastrointestinal site with the most diverse microbiota in birds and is more stable in composition than the faecal microbiota; it has therefore been a preferred sampling site when birds are sacrificed at the end of a trial.

There is a range of host factors that influence development and stability of intestinal microbiota. The majority of studies about poultry microbiota have been done in broilers, generally in birds less than 42 days of age, so the available information represents the microbiota of younger chickens. Considerable difference is reported between broilers and layers. However, to our knowledge, there has only been one published study comparing broilers and layers hatched together and reared in the same facility, using randomised design, on the same feed and environment (Han et al., 2016), showing that there are significant genetic breed influences between layers and broilers, on intestinal microbiota composition (Ranjitkar et al., 2016; Videnska et al., 2014). Bird sex is also a strong variable when considering microbiota studies, extremely so in sexually mature birds (Wilkinson et al. 2016).

Environmental factors (reviewed by Kers et al., 2018) such as housing, including individual rearing conditions and ambient environmental conditions as well as the type of production system, can influence microbiota composition. Access to free range opens a path for ingestion of soil and insect associated microbiota. Other factors shown to influence microbiota profiles in poultry include litter quality, type and management, lighting schedule, access to feed, climate and quality of temperature and humidity control.

IV. MANIPULATION THROUGH NUTRITION

Feed is the source of nutrients and energy for both host and microbiota growth and homeostasis, and consequently a very strong microbiota modifier. Macronutrients have a very significant influence on microbiota composition: proteins induce expansion of bacteria taxa that are very different to the ones that prefer to grow on carbohydrates. Poultry nutrition is already well-designed for the optimum productivity in both layers and broilers and manipulation of poultry health through manipulation of poultry microbiota can be achieved via feed additives. The most notable example is addition of Antibiotic Growth Promoters (AGPs), a practice that was used in the poultry industry for over 6 decades (Dibner and Richards, 2005). The primary function of AGP addition was demonstrated productivity improvement and control of enteric pathogens like Clostridium perfringens. Although AGPs were used in low, sub-therapeutic doses and were different from antibiotics used in human medicine, recent concerns over possible linkage between AGP use in the livestock industries and the rising level of antibiotic resistance in human pathogens has resulted in a ban on the use of AGPs in many countries and the Australian poultry industry is voluntarily removing AGPs from a majority of farms. The need for pathogen control, especially fear of necrotic enteritis outbreaks, is encouraging the industry to look for alternatives to AGPs with proven ability to control major poultry pathogens. Pathogen control leads to better bird health and often results in better performance. Herbs and spices with known antimicrobial properties (Cetin et al., 2016; Scocco et al., 2017) as well as other natural products such as biochar, bentonite and zeolites (Prasai et al., 2016) are also being tested as pathogen controlling feed additives and giving promising results. However, the major microbiota modifying additives in poultry feed are still probiotics and prebiotics.

VI. PROBIOTICS AND PREBIOTICS

Probiotics are widely used in agriculture for their proven health benefits; however, individual farmers report very variable success using commercial products. This is expected knowing that intestinal microbiota can vary at a phylum level even in flocks of the same genetics, reared under same conditions using the same feed batch (Stanley et al., 2013b). Flocks with microbiota differences at such high magnitude would respond differently to probiotic administration in feed. Commercial probiotics targeting the livestock market include Lactobacillus, Enterococcus, Bifidobacterium and Streptococcus species (Fuller, 1989); however Bacillus is emerging as a more reliable alternative because of its favourable resistance to feed preparation procedures, and independent of viability issues due to its sporulation nature.

One of the major benefits of probiotic action is their ability to assist the host microbiota during pathogen invasion. In order to launch successful infection, pathogens must either colonise or, if latently present, activate and expand. The host, including the symbiotic microbiota, will also launch a range of defences (Patterson & Burkholder, 2000). Probiotics may also assist via the mechanism of competitive exclusion and the production of antibacterial products such as very efficient antibacterials– bacteriocins as well as toxic metabolites such as hydrogen peroxide. The host will launch immune responses to prevent pathogen translocation from the gut and often amplify rapid periodic emptying of the small intestine which leads to pathogens being washed out (Patterson and Burkholder, 2003).

There is an abundance of research showing beneficial effects of probiotics in in vitro assays against pathogens and in controlled bird trials. However, running a chicken trial in a research facility environment often uses administration of freshly grown probiotics or a batch validated by the industry for high probiotic isolate viability. Birds are generally grown under optimal, ethics committee approved conditions, which exclude extreme stress. Sometimes those birds are challenged with a pathogen to inspect the efficiency of the probiotic strain/product. In reality, on the farm, birds are often not under such ideal conditions, exposed to more stress, and harbouring very different microbiota in their gut. These differences, especially in layers, in our own experience from sampling a number of major layer farms, are of a very high magnitude. Probiotics thus immediately encounter very different intestinal environments in terms of the inhabitants and their metabolite driven chemistry. In addition to that, the viability of commercial probiotic products and the ability to withstand acid and bile varies greatly due to the intense processing, such as freeze drying (Dodoo et al., 2017) which can alter not just viability but also their probiotic properties (Archacka et al., 2019).

Viability as well as acid/bile tolerance is overcome by the use of sporulating Bacillus probiotics and the research on benefits of such probiotic formulations intensified in the last few years. Despite the obvious Bacillus production advantage, there is no need to give up on the development of beneficial lactic acid bacteria and bifidobacteria species. Preservation methods need to be further optimised towards the targeted place of delivery (Dodoo et al., 2017), for example, although they may be spread through multiple gut sections, Lactobacillus often targets the small intestine while Bifidobacterium use less digestible polysaccharides and the more anaerobic environment in the large intestine. Probiotics are suggested to be more effective when combined with prebiotics – so called synbiotics (Mookiah et al., 2014). Prebiotics are dietary carbohydrates, particularly oligosaccharides and polysaccharides, resistant to intestinal enzymatic digestion but fermentable by intestinal microbiota. They selectively modulate the microbial populations or activity of intestinal bacteria, promoting health and wellbeing of the host (Gibson et al., 2004; Gibson and Roberfroid, 1995). Prebiotics are a nutritional source for beneficial intestinal microbiota that produce short chain fatty acids (SCFAs). Appropriate delivery combined with specific probiotic optimised prebiotic mixes will result not only in better gut health and pathogen reduction but will also benefit the productivity of birds (Angelakis, 2017). Healthy intestinal microbiota is characterised by a high beneficial/pathogenic species ratio. Probiotics administration can help maintain that ratio, especially if the probiotics are delivered from the first days of life, which can allow them to colonise first and, most importantly, colonise intestinal mucosa and remain permanently protective (Baldwin et al., 2018).

VII. MICROBIOTA AND HOST METABOLISM

The demonstrated correlation between intestinal microbiota and growth and productivity indicates that the microbiota can positively influence the metabolism of the host (Stanley et al., 2012; Stanley et al., 2013a; Stanley et al., 2016). Short Chain Fatty Acids (SCFAs) (Byrne et al., 2015) and bile salts (Krogdahl, 1985; Maldonado-Valderrama et al., 2011) are two of the major metabolites mediating the impact of microbiota on the host metabolism. Intestinal microbiota produces SCFAs by fermentation of digestive-enzyme-resistant non-starch polysaccharides. Although SCFAs affect body weight gain and adiposity in mice (Ridaura et al., 2013; Liou et al., 2013), possibly through appetite regulation and energy homeostasis, their effects on feed intake and energy metabolism in chicken are yet to be fully revealed. However, the beneficial effects of SCFAs as antimicrobials and growth promoters have been studied to some extent.

Bile salts (bile acids conjugated to glycine or taurine) also affect energy homeostasis (Watanabe et al., 2006; Watanabe et al., 2012). Host and intestinal microbiota, through their bile salt hydrolase (BSH) activity, together determine the size of the bile salts pool in the intestine. Although the role of bile salts in lipid, cholesterol and glucose metabolism in mammals has been well studied (Thomas et al., 2008; Joyce and Gahan, 2016), these metabolites have received less attention in poultry research. One of the suggested modes of action of AGPs to improve growth rate in poultry is lowering of BSH activity in the intestine (Guban et al., 2006; Feighner and Dashkevicz, 1987). As we are gradually phasing out AGPs from poultry diets, non-antibiotic BSH inhibitors could be another area of research to find alternatives for AGPs.

VIII. STRESSFUL LIFE OF CHICKENS

Intestinal microbiota influences the gut-brain axis and can be altered when the host is exposed to periods of stress. Both broilers and layers can be exposed to periods of metabolic stress by factors such as oscillations in environmental conditions, sudden loud noises, transportation, beak trimming, intestinal pathogens, feed change, to name a few. Broilers are, however, harvested at a young age, while in layers environmental challenges can persist over longer periods of time. Living in a large flock, small disturbances in social hierarchies, inability to use a preferred nesting box, and other social behaviour induced anxiety can be a problem with layers. Based on the abundance of human research on the influence of anxiety on gut microbiota, as well as certain microbiota causing and amplifying anxiety (Neufeld et al., 2011), it is reasonable to suggest that this connection is valid in chickens. Stresses can also lead to intestinal permeability issues and this has recently becoming a topic of interest in poultry research with a number of gut permeability models being investigated by poultry researchers (Gilani et al., 2017; Kuttappan et al., 2015).

VIII. CONCLUSIONS

The last decade of intensive research into poultry microbiota has resulted in a large scientific literature and knowledge repository now being available to poultry researchers and the industry. Despite the inherent complexity of microbiota-host interactions, we are now well into a stage of transition towards applying this knowledge in solving some major issues in poultry production, such as pathogen control, bird immunity and AGP removal. Removing or controlling flock to flock and bird to bird microbiota variation via early post-hatch microbiota remodelling, appears to be one of the ways towards growing healthier more productive birds.

Abstract presented at the 30th Annual Australian Poultry Science Symposium 2019. For information on the latest edition and future events, check out https://www.apss2021.com.au/.