The 4th Experts Meeting on Harnessing the Animal Microbiome towards Sustainable Farming was held on June 10, 2021. The meeting was co-hosted by CCO Derek Butler and Radhika Bongoni, Head of Business Development at BaseClear.
With the current focus on improving sustainability in general as part of the United Nations’ sustainable development goals, farming practises are being analysed to help safeguard their production for coming generations. As one of the new frontiers of research, the microbiome is being researched for its role in improving sustainability, particularly in the area of livestock production. In the experts meeting today, current developments microbiome research for sustainability were discussed in detail.
Speakers & presentations
- KEYNOTE: Sustainability and animal health, how the research on microbiome can help?
Dr. Emmanuelle Apper – Lallemand
- Feed additive safety assessment for EFSA approval
Dr. Adalberto Costessi – BaseClear
- Early life nutritional strategies for a sustainable start of the pig microbiome
Dr. Anouschka Middelkoop – Schothorst Feed Research
- Profitability and sustainability jigsaw in animal production: Is “gut health” the missing piece?
Dr. Ajay Awati – EW Nutrition
- Pre- and probiotics supporting small intestinal function in broilers
Prof. Richard Ducatelle – Ghent University
- Protected vitamins and biofactors to support health of broiler chickens
Dr. Cristiano Bortoluzzi – Jefo
KEYNOTE: Sustainability & Animal Health, how the research on microbiome can help?
Dr Emmanuelle Apper is from Lallemand Animal Nutrition, a team dedicated to understanding the microbiome, host and holobiont. Their microbial-based products include yeast, bacteria, and their derivatives.
Microbes are the predominant lifeform on Earth, both in numbers and total biomass, and are pivotal agents for planetary health and sustainability. Different microbe-driven ecosystem services, such as the provision of food, fibre, fuel or nutrients can impact sustainability goals through deliverables such as food and water security, climate change mitigation and animal health. There are diverse options for innovation in microbiome research to contribute to sustainability.
High-throughput sequencing methods help us to understand microbes for sustainability. There has been continuous improvement in microbiome research that have unlocked the microbiome for us. The three main areas in animal research are nutrition and animal wellbeing, feed management and the animal environment. These are challenges, but also opportunities.
A key area in terms of sustainability is understanding the complex topic of the gut microbiome. Sustainability concerns such as efficient feed usage and animal health rely on a holistic view of the gut microbiome and how it impacts livestock health (see key review article from Berg et al. (2020)). Gut function and microbial composition change with different life stages. In particular, the diet can be used to modulate the gut microbiome in production animals such as pigs. Ideally, microbial-based solutions will improve the performance, well-being and health of the animals.
As an example, an experiment was conducted to determine the effect of antibiotics and probiotics using a 2×2 model in sows on the microbiota and performance of offspring. The combination of antibiotics and probiotics given as an intervention 4 weeks before farrowing resulted in the greatest weight gain and body weight of piglets over the first 35 days of life. There was also a reduction in mortality in the probiotics group. The gut microbial diversity as measured by the Shannon index after farrowing was maintained in the probiotics group, whilst in the control group it decreased according to a characteristic pattern. This showed a stabilising effect of probiotics during the critical farrowing period.
In a second article by Labussière and co-workers, the effect of a probiotic yeast on feeding behaviour, energy metabolism and faecal microbiota composition during heat stress on finishing boars was investigated. Heat stress normally causes a decrease in feed intake and a resulting reduction in growth in pigs. The addition of the probiotic to the diet resulted in an increase in body weight attained, presumably due to an increased feeding rate. Skin temperature was significantly reduced. There were some changes to the microbiota, including to Ruminococcu bromii, biomarker of energy balance during heat stress.
Silage is used as fodder for many grazing animals, however it starts to spoil after exposure to the environment, increasing waste and the risk of mycotoxin contamination by moulds. An experiment used sileage starter cultures to modulate the sileage fermentation performance, as reported by Drouin and colleagues. The aerobic stability of the animal feed 129 days after treatment with two sileage inoculants was measured. The low pH environment from the sileage starter cultures stabilised the pH during aerobic exposure and reduce mycotoxin production.
The microbiome is key to sustainability in livestock farming. A deep and holistic understanding of the structure and functions of the microbiome is crucial, and must include identifying key ecosystems, bacteria and biomarkers. Modulation of the gut, feed and environment microbiomes can support sustainable farming approaches. However, there are many challenges: a healthy microbiome is context specific. It is useful if the entire phenotype of a microbiome is characterized, from shotgun sequencing to metabolomics. Some technical challenges remain, such as bias from DNA extraction and processing, bioinformatics approaches, and the need to integrate the host response.
Lecture 2: Feed additive safety assessment for EFSA approval
Dr Adalberto Costessi, Product Manager Genomics and Regulatory Affairs at BaseClear, gave a presentation on the regulatory environment for microbe-based feed additives, which are important if introducing a sustainability-based feed additive to the market.
EU regulations guide the safety of the food chain “from farm to fork”. The current framework are the regulations from 2002. Microorganisms can be used in the food chain as feed additives, plant protection products, food enzymes, flavourings, food additives and as novel foods and for health claims. Any chemicals that can enter the food chain are regulated. Prior authorisation is required to put products on the market, and the requirements vary slightly by country. The authorisation is based on a science-based safety assessment, and the requirements aim to determine whether the product is safe and effective. The two key regulations are 1831/2003 on additives for use in animal nutrition, and detailed rules for these regulations in 429/2008, and are supported by scientific and administrative guidance documents.
The authorisation process is complex, requiring the applicant to send samples, a technical dossier and the final application to different organisations to receive the final approval. The technical dossier follows a standard structure and contains information on the identity, safety and efficacy of the product. Safety is assessed in target animals, the consumer, the environment, and the person who uses it on the farm. Open consultations are a good way to find out specifics in a developing field.
When registering a microorganism as feed additive, whether they are bacteria, yeasts, fungi (or perhaps phages or algae in the near future), it’s important to know the history of the strain and how it was put together or where it was found. Types of products include probiotics or silage agents, which may not be genetically modified, or derived from microorganisms.
A new requirement was introduced in March 2021 containing recommendations on how to perform and report whole genome sequencing analysis. This requirement has improved some guidance by establishing clear thresholds for antibiotic markers and virulence gene searches in bacteria, and for bacterial taxonomic identification. However, it now requires the submission of raw sequencing data and alignment files, which could cause problems in terms of protection of proprietary data.
There are several challenges for applicants remaining. The interpretation of antibiotic resistance can be problematic when comparing phenotypic minimum inhibitory concentrations (MICs) with antibiotic marker gene searches. Good reference genomes are required for taxonomic identification based on whole genome sequencing. Some of the requirements continue to be unclear, such as those for the presence or absences of known metabolic pathways in toxigenicity.
A recent development was an application for the use of a bacteriophage in poultry. Although it was deemed to be safe, efficacy data were still lacking.
In another example for animal feed, the absence of antibiotic markers was established through solid interpretation of MICs and literature searching. For example, a survey of Bacillus species showed that an antibiotic resistance gene was naturally-acquired, and therefore a wild type feature of the strain (see Agersø 2018).
Genomics plays a crucial role in safety assessment, although it is a rapidly developing field. All people working in the area have challenges in the requirements, methods, interpretation of results and new organisms. The transparency regulations are likely to have a large effect on microbial feed additives for the coming years.
Lecture 3: Early life nutritional strategies for a sustainable start of the pig microbiome
Dr. Anouschka Middelkoop from Schothorst Feed Research gave a presentation about how the early life microbiome in pigs can have life-long consequences. For the pig microbiome, there is large variation between individuals. Tremendous variation occurs after weaning, with an increase in gut microbial diversity as pigs age. The change at weaning is due to change in diet from milk to the normal porcine diet, which cause a major shift in the microbiome. Dysbiosis can occur at weaning, with negative effects on pigs’ overall health. Early life stress, such as early weaning, gut inflammation or infection during the first 3 months of life determines whether the pig will have normal gut development or an elevated risk of disease.
A study intending to apply the Western-style diet to pigs investigated early life nutritional programming, as reported in Clouard (2016). Western diets promote obesity in humans. This study showed that an 8-week postnatal exposure to a high fat, high sugar diet could improve feed efficiency later in life, proving the effect of early life feed efficiency programming.
Through early life programming, the animal microbiome features prominently in sustainable pig farming. Gastrointestinal problems are the main cause of sickness and mortality in pig production. Large litter sizes that are found in commercial pig breeds increase the risk of intra-uterine growth restriction (IUGR) in piglets, which has an adverse effect on weight gain, susceptibility to disease and death. IUGR piglets have a different gut microbiome compared to normal piglets.
Starting from birth, the piglet obtains its microbiome from the vaginal tract, colostrum and milk, environment, sow faeces and saliva from nose-to-nose contact. Could rectifying microbiome changes improve piglet health for IUGR piglets?
Streptococcus suis is a common inhabitant of pig tonsils, where it causes septicaemia, meningitis and other infections that both reduce animal welfare and profitability. It is also responsible for disease in humans. There are few nutritional strategies against this pathogen, for which colonisation of the tonsils begins rapidly after birth. Early life microbiota modulation shows potential in reducing colonisation of the tonsils with S. suis. The recent publication of the entire genome sequence of S. suis by Gaiser et al. can help discern its virulence factors.
One hint that the microbiome is important for improving sustainability is through experiments whereby the faeces of lactating sows is removed from the pen so that it does not colonise the piglet microbiome. Faeces removal hampers later life performance in terms of weight gain, with significant differences seen after 73 days, as shown by Aviles-Rosa and co-workers. Beneficial microbes are being consumed resulting in gut health and better performance.
Gut microbial colonisation in new born piglets affects later life diarrhoea susceptibility, which is a cause of low weight gain and poor feed conversion. The Avant Project (see https://avant-project.eu/) is looking into developing alternatives to antimicrobials in piglets, particularly during the post-weaning period. Gut microbiome modulators are a key part of the strategy. Dietary supplements around the time of weaning can affect diarrhoea incidence. For example, altering the fibre content of weaned piglet food can reduce diarrhoea. Colostrum or faecal microbiota transplantation reduces intestinal diseases. Supplemental milk or “creep feed” (pre-weaning solid food) can also reduce the relative abundance of pathogens such as Escherichia coli or accelerate post-weaning changes in the gut microbiota.
There are many potential early life nutritional strategies that can reduce disease and death, improve immune function, and improve pig performance. These contribute to sustainability by reducing inputs required for meat production.
Lecture 4: Profitability and Sustainability Jigsaw in Animal Production: Is “Gut Health” the Missing Piece?
Dr Ajay Awati from EW Nutrition gave a presentation on how animal gut health can improve the profitability and sustainability of livestock production. A major worldwide development goal is achieving food security for the entire population. Improvements in livestock production efficiencies has resulted in greater meat production with the use of fewer resources. The annual compound growth rate has continued to improve, even in recent decades, and has outpaced human population growth. We produce more meat from a similar number of animals.
The last decades have seen a shift in focus. Over the last half century, we have had an “era of increased production,” with improved animal performance paramount. Future directions will see factors such as animal welfare, health, meat quality, public health and the environment. Thus, it will be an “era of responsible production”.
One way that microbes contribute to sustainability is by increasing nutrient availability of animal feed. For example, volatile fatty acid production from microbial fermentation contributes up to 28% of the total energy requirement of the pig, and 70% of the energy needed by the gut epithelium. It also contributes to the production of nutrients such as vitamin K and several B vitamins. The gut microbiome improves water re-absorption in the colon and is associated with improved mineral absorption and bone mineralisation.
When animal gut health is compromised, overall animal health is affected. For example, chickens with microbial overgrowth in the small intestine have decreased nutrient digestibility. More than 70% of total antibody production is from IgA secreted across the gastrointestinal tract. In chickens, one fifth to one third of whole body energy expenditure is used by the gut metabolism, mainly due to cell turnover. Gut diseases such as necrotic enteritis cost the poultry industry US$5-6 billion per year.
Some animal welfare issues can result in higher costs for producers. For example, foot pad lesions form in poultry in contact with wet litter, and are considered an animal welfare problem. Gastrointestinal microbes cause painful lesions on the foot pads. A flock foot pad score, indicating the extent to which lesions affect the foot, draws a financial penalty in Scandinavia at the time of slaughter.
Gut microbes can also affect meat quality. For example, skatole produced by microbial degradation of L-tryptophan in the large intestine causes an off flavour in pork.
Protein fermentation by the microbes in the large intestine produces ammonia and other odours. Particularly on pig farms, the health and wellbeing of people working on the farm and people who live nearby can be affected.
Increasing rates of antimicrobials on farms is contributing to global antibiotic resistance. Despite the ban on the use of antibiotic growth promotors in the EU and other countries, antibiotic use is increasing worldwide. Reducing dependency on antibiotics requires improvements in animal management practices, housing and worker training. Antimicrobials are used to treat problems that overlap with the microbiome. Managing livestock gut health is also an option. The strategic use of feed additives can be most effective here. Solutions that are tailored to particular situations are most effective and profitable.
Profitability and sustainability are linked. Managing the gut microbiome contributes to a health gut and lower rates of disease, which is a key measure in sustainable farming without antibiotic growth promotors. “Act inside the gut, but think outside of it!” is an important concept.
Lecture 5: Pre- and probiotics supporting small intestinal function in broilers
Professor Richard Ducatelle from Ghent University gave a presentation on an area of animal microbiome research that does not attract much attention: overgrowth in the small intestine of broilers. The small intestine has a low density of microorganisms, particularly in the duodenum, and the microbes there are poorly culturable. The microbiome composition of the small intestine resembles that of dental plaque or saliva. Small intestinal bacterial overgrowth is an undesirable condition that is linked to the microbiome. For example, Leite and co-workers show that the duodenal microbiome is altered during bacterial overgrowth in humans. On the other hand, bacteria that are present in the small intestine are often unculturable and metabolically inactive, and cannot take up nutrients so they don’t come into competition with the host.
The host has many defence mechanisms against microbes. For example, lipopolysaccharides are a cell wall component of gram negative bacteria. The enzyme alkaline phosphatase (produced by the intestinal cells) is capable of breaking down lipopolysaccharides, thus destroying the cell wall of gram negative bacteria.
An example of a probiotic targeting the poultry small intestine is Bacillus subtilis 29784. The probiotic is supplied as spores, and it becomes active in the anaerobic environment of the small intestine. The abundance of functional modules related to the ileum microbiota was reduced after this probiotic was given, including those that are harmful for the host. A major product was hypoxanthine (see article by Choi and colleagues), which allows the Bacillus to compete with other microbes on the epithelium, thus preventing small intestinal bacterial overgrowth.
Xylooligosaccharides (XOS) are a component of human breast milk, and can affect the microbiota of both the small and large intestine (see Liu et al.). It was added to the diet of broilers to see if could have an effect on performance, as described in De Maesschalck (2015). There was a significant improvement in the feed conversion ratio with an increase in body weight at day 26 compared to a control diet without XOS. Microbiota analysis indicated increases in Lactobacillus crispatus and Anaerostipes butyraticus in the XOS group. Cross-feeding experiments with A. butyraticus and L. crispatus showed that butyrate production increased, which could explain the improvement in performance via the gut.
In summary, bacteria can compete with the host for nutrients in the small intestine, however there is a solid defence system against bacterial colonisation, particularly via the intestinal epithelial cells. Avoiding small intestinal bacterial overgrowth can be achieved by reducing overall density of microbes in the small intestine and supporting Lactobacilli in the ilium.
Lecture 6: Protected Vitamins and Biofactors to support health of broiler chickens
Dr Cristiano Bortoluzzi, Research Scientist from Jefo Nutrition gave a presentation on how protected vitamins and other biofactors can assist in the health of broilers. The main challenges for chicken flocks include temperature stress, intestinal disorders including dysbiosis, vaccination stress, transportation and errors by management. Research into the effect of early life conditions on final weight show that each 10g above average for 7-day old chicks results in an increase of 40 to 60 g at 35 days. Vitamins and other biofactors within a matrix improves nutrient absorption and reduces inflammation, and allows the nutrients to be delivered to the site where they are needed the most (see Bortoluzzi et al.).
This concept was tested in 308 broiler chickens. They were given base diet, or base diet supplemented with a complex of biofactors and antioxidants. They were double vaccinated against infectious bronchitis and subjected to cold shock on day 3. After 21 days, feed conversion ratio and body weight improved. General anti-inflammatory and antioxidant response in chickens undergoing early life stress were supported.
The kinome array is a tool that measures phenotypic changes of the tissue by the action of kinases. Arsenault and Kogut provide a useful review of the technology. In the broiler chicken study, the protected complex of biofactors and antioxidants induced gene expression and antioxidant functions as measured by kinome analysis.
This supplementation concept can be used to target particular sections of the digestive tract of chickens. By modulating pro- and anti-inflammatory molecules, the immune system of chickens can be supported. Nutrients that help balance the microbiome and produce certain short chain fatty acids could be added. The function of the intestinal epithelium can also be assisted via targeted nutrients.
Agersø Y, Stuer-Lauridsen B, Bjerre K, et al. Antimicrobial Susceptibility Testing and Tentative Epidemiological Cutoff Values for Five Bacillus Species Relevant for Use as Animal Feed Additives or for Plant Protection. Appl Environ Microbiol. 2018;84(19):e01108-18. Published 2018 Sep 17. https://dx.doi.org/10.1128%2FAEM.01108-18
Arsenault RJ, Kogut MH. Immunometabolism and the Kinome Peptide Array: A New Perspective and Tool for the Study of Gut Health. Front Vet Sci. 2015;2:44. Published 2015 Oct 13. https://dx.doi.org/10.3389%2Ffvets.2015.00044
Aviles-Rosa EO, Rakhshandeh A, McGlone JJ. Preliminary Study: Depriving Piglets of Maternal Feces for the First Seven Days Post-Partum Changes Piglet Physiology and Performance before and after Weaning. Animals (Basel). 2019 May 23;9(5):268. https://doi.org/10.3390/ani9050268
Berg G, Rybakova D, Fischer D, Cernava T, Vergès M-CC, Charles T, et al. Microbiome definition re-visited: old concepts and new challenges. Microbiome. 2020;8:103. https://doi.org/10.1186/s40168-020-00875-0
Bortoluzzi C, Lahaye L, Perry F, Arsenault RJ, Santin E, Korver DR, Kogut MH. A protected complex of biofactors and antioxidants improved growth performance and modulated the immunometabolic phenotype of broiler chickens undergoing early life stress. Poult Sci. 2021 Apr 20;100(7):101176. https://doi.org/10.1016/j.psj.2021.101176
Choi P, Rhayat L, Pinloche E, Devillard E, De Paepe E, Vanhaecke L, Haesebrouck F, Ducatelle R, Van Immerseel F, Goossens E. Bacillus Subtilis 29784 as a Feed Additive for Broilers Shifts the Intestinal Microbial Composition and Supports the Production of Hypoxanthine and Nicotinic Acid. Animals (Basel). 2021 May 8;11(5):1335. https://doi.org/10.3390/ani11051335
Clouard C, Gerrits WJ, Kemp B, Val-Laillet D, Bolhuis JE. Perinatal Exposure to a Diet High in Saturated Fat, Refined Sugar and Cholesterol Affects Behaviour, Growth, and Feed Intake in Weaned Piglets. PLoS One. 2016 May 18;11(5):e0154698. https://doi.org/10.1371/journal.pone.0154698
De Maesschalck C, Eeckhaut V, Maertens L, De Lange L, Marchal L, Nezer C, De Baere S, Croubels S, Daube G, Dewulf J, Haesebrouck F, Ducatelle R, Taminau B, Van Immerseel F. Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota. Appl Environ Microbiol. 2015 Sep 1;81(17):5880-8. https://doi.org/10.1128/aem.01616-15
Drouin P, Tremblay J, Renaud J, Apper E. Microbiota succession during aerobic stability of maize silage inoculated with Lentilactobacillus buchneri NCIMB 40788 and Lentilactobacillus hilgardii CNCM-I-4785. Microbiologyopen. 2021 Jan;10(1):e1153. https://doi.org/10.1002/mbo3.1153
Gaiser RA, Zomer AL, Wells JM, van Baarlen P. Draft Genome Sequence of Streptococcus suis S10, a Virulent Strain Used in Experimental Pig Infections. Microbiol Resour Announc. 2019 Jun 6;8(23):e00227-19. https://doi.org/10.1128/mra.00227-19
Labussière E, Achard CS, Dubois S, Combes S, Castex M, Renaudeau D. Saccharomyces cerevisiae boulardii CNCM I-1079 supplementation in finishing male pigs helps to cope with heat stress through feeding behavior and gut microbiota modulation. Br J Nutr. 2021 May 27:1-35. https://doi.org/10.1017/s0007114521001756 Epub ahead of print. PMID: 34039449.
Leite G, Morales W, Weitsman S, Celly S, Parodi G, Mathur R, Barlow GM, Sedighi R, Millan MJV, Rezaie A, Pimentel M. The duodenal microbiome is altered in small intestinal bacterial overgrowth. PLoS One. 2020 Jul 9;15(7):e0234906. https://doi.org/10.1371/journal.pone.0234906
Liu Z, Subbaraj A, Fraser K, Jia H, Chen W, Day L, Roy NC, Young W. Human milk and infant formula differentially alters the microbiota composition and functional gene relative abundance in the small and large intestines in weanling rats. Eur J Nutr. 2020 Aug;59(5):2131-2143. https://doi.org/10.1007/s00394-019-02062-w