SPECIAL REPORT:
Gut health: Small window of opportunity to get it right
From 2023 Kemin Intestinal Health Symposium
By Ann Hess
The Nutrition and Animal-Microbiota EcoSystems Lab at KU Leuven defines early life gut health as “a generalized condition of gut homeostasis characterized by the establishment of a rich and stable microbiota, as well as the maturation of different intestinal cell types contributing to gut barrier integrity.”
“There should be a balance between tolerance and responsiveness,” Dr. Nadia Everaert, associate professor, KU Leuven, told attendees at the recent Kemin Intestinal Health Symposium. “It should not be sleeping, but there is some activity that we want at the immune system.”
Before examining gut-microbiota interactions in young pigs and chickens, Everaert said its important to understand the functions of the microbiota. The microbiota is involved in digestion, toxin breakdown, immune system stimulation, pathogen exclusion and endocrine activity. The microbiota also impacts an animal’s health, growth and feed conversion rate.
Studying the microbiota today also involves several sequencing techniques, through which researchers have been able to go one level deeper in identification and data utilization.
“The taxonomy is really the composition of the microbiota, but we also like to talk about alpha diversity. Alpha diversity is to look within a sample: How diverse is it inside the chicken ceca, for example? How many different species are present? So that's what we can count, and we put it into numbers,” Everaert said. “Then we're going to talk about the richness. For example, how many observed species are there in our sample? That's the value we put on for the alpha diversity.”
The values that are given to the samples show how complex the microbiota is, which leads to the question: If you take two chickens in one barn, do they have the same microbiota more or less?
“If you compare a control group with the probiotic treatment group, it's nice if we can see it results in a different microbiota, for example,” Everaert said.
The pig: A mammal
With six layers between the blood of the fetus and the blood of the mother, the epitheliochorial placenta is well protected, and that means that antigens can barely pass. It also means that immunoglobulins cannot pass.
“When pigs are born, they are really born with an immature immune system, so they don't get any immunoglobulins, and they haven’t experienced many antigens yet,” Everaert said. “So that means that really after birth, this immune system really still needs to mature as quickly as possible, because then all of a sudden, he's in contact with this environment and many pathogens.”
Colostrum intake, and the first milk, rich in immunoglobulins to give passive immunity to the piglets, is critical.
“We have big litters nowadays, and there might be competition between the piglets to get enough colostrum intake. That's another challenge,” Everaert said. “A future challenge then is that we’re weaning the piglets quite early. We're weaning them at three, four weeks of age, while normally in nature, it would not occur all of the sudden, and it would occur a lot later – two months of age, for example. We are weaning our piglets when their immune system is not mature yet.”
You then have to assume the piglet’s gut is sterile, she said. Colonization of the bacteria in the gut begins during birth, in the vaginal canal of the mother, and then through colostrum and milk, where there are some bacteria present and will also contribute to the colonization. Colonization also happens as the piglets are in contact with the excreta of the sow and then through the environment. The facultative anaerobic bacteria entering the gut will then be replaced more or less by strict aerobic bacteria. The bacteria present in the environment then becomes more dominant for colonization.
“You have some early colonizers, and then these will be followed by other bacteria. It's really a maturation in this colonization that is going on,” Everaert said. “If you would translate it into this alpha diversity that I was talking about, you see these values going up, a complexity that is increasing, more and more species are coming up. , At weaning, which is inducing stress, and you see a perturbation of the microbiota. Thereafter, the complexity of the microbiota still increases.”
Everaert said there’s a critical window for postnatal gastrointestinal development, when milk oligosaccharides can modulate the piglet’s microbiota.
“Milk is composed of so many factors. You have the macronutrients, of course, and then you have these milk oligosaccharides that are known to play an important role, but actually it's not limited only to these milk oligosaccharides,” Everaert said. “There are also growth factors inside. There are hormones in there, there are cytokines in there, and so on. It's actually this complex composition of the milk that is contributing to the maturation of the gut, microbiota and immune system.”
She pointed to studies where piglets given bovine colostrum were compared to piglets given milk replacer and piglets only on sow’s milk. The bovine colostrum-fed piglets had less mucosa-associated E. coli and haemolytic bacteria and fewer cases of diarrhea. The immune system of those piglets matured better compared to the ones that were reared on artificial milk.
However, Everaert noted that there are other factors in play in impacting the gut microbiota. Gut colonization and filtering requires a persistence of present microbes through nutritional input. Feed intake and retention time will determine outflow. Persistence also depends on interactions with other microorganisms (such as metabolic cross-feeding), and microbes must survive host immune responses.
Microbiota is also present in different sections of the digestive tract. For example, in the lumen some bacteria present are attached to the epithelium, some bacteria are attached to the mucus, and then some bacteria are attached to feed particles. While all of these result in a different microbiota composition, they are highly interconnected and influence each other’s community composition, Everaert said.
“If you consider as well what's happening in the upper digestive tract to understand what's happening lower in the digestive tract, that's all important for these cross-feeding,” Everaert said.
The microbiota also likes to use undigested nutrients, or fibers, as an energy source.
“They produce some metabolites which can be used by other bacteria. They might produce lactate, for example, and the lactate can be used by other bacteria, and they can use propionate, or lactate can be used by other bacteria to produce acetate, and that can be converted then by other bacteria into butyrate,” Everaert said. “So, it's this intercross connections of different microbiota in different sections of the digestive tract. For example, more lactate producing bacteria, and then downstream, they are using this lactate that was produced, and they're converting it into butyrate.”
The microbiota composition changes with age, but also through different diets. Some bacteria will use mucus as nutrients, and they have enzymes that can digest the mucus which they will use as an energy source. On the other hand, the thick mucus is composed of glycocalyx, so to synthesize this mucus, there are other bacteria affecting the enzyme activity of the host to produce the mucus.
Everaert said that is just one of the existing interactions. Secretory immunoglobulin A can neutralize toxins, for example. There is a positive correlation between the abundance of Prevotella bacteria and the amount of secretory immunoglobulin, and Prevotella is also known to have a positive correlation with growth performance.
There is thus a symbiose of the microbiota with the host, and other bacteria like lactobacillus can adhere to the mucus and use the mucus to protect against certain pathogens. While symbiose is important, Everaert said it needs to be balanced.
Considering the metabolites produced by the bacteria, host-microbiota interactions can be divided into three categories. Bile salts are present in the gut helping in emulsification of the lipids and are important for efficient lipid digestion and absorption. But then there are also bacteria present, and they can modify these salts, producing certain metabolites that are signaling molecules for the host.
“For example, there are certain signaling molecules that can affect the gut integrity, so they are stimulating this tight junction proteins, or they can affect the villous heights, increasing the absorptive capacity of the epithelium,” Everaert said. “There are other signaling molecules that are affecting the release of GLP 1 and GLP 2, which is an endocrine factor that has an effect on glucose metabolism.”
Second is short-chain fatty acids, or the end products of the energy metabolism of the bacteria. Acetates, propionates and butyrate are very important, and they can be absorbed by the host.
“About 15% of the energy actually comes from these short chain fatty acids, so that's an important amount, and then propionate can also be used for glucose metabolism by the liver,” Everaert said. “But besides that they also play a role on the immune system where you can see a big effect of butyrate that is going to the dendritic cells that has receptors that can cause a modification at the gene expression level.”
Epigenetic modification can then impact the maturation of the dendritic cells.
“Regulatory T-cells are important to have this balanced immune system,” Everaert said. “The immune system should not be in the inflammatory status, but it should be there to be awake and readywhen a pathogen attacks.”
The third category is amino acids. In the past, it’s been said if you don't have good protein digestion, then amino acids will end up in the large intestine, and fermentation will occur, which is negative for gut health. While that is all true, Everaert said we can turn amin acid fermentation into a positive thing.
“The bacteria, they need amino acids also to grow, and they turn it into some metabolites that actually are positive for gut health,” Everaert said. “So, it's all about the balance and about quantity.”
The chick: A precocial birdSince nutrients are deposited in the egg before incubation, a maternal core microbiota might not be completely transferred to hatching chicks. Some researchers have measured microbiota present in the meconium, when the egg was laid, and found some bacteria present there. Everaert notes bacteria could come through the pores of the eggshell and then colonize the gut of the embryo.
“It's really during these first five days that the microbiome load is increasing tremendously. So, from five days on, you have a maximum concentration that is already reached, but it's the richness and the complexity that is still changing a lot,” Everaert said.
For chickens, there are three defined stages of colonization, and the first week after hatch is critical to the broiler microbiome.
“There are more and different bacteria that are taking over this complexity. So, you have this difference in concentration,” Everaert said. “Again, you have to think about the retention time – how long the nutrients are staying in that place of the gut. For example, the crop is there, and it's a storage system; there are quite some bacteria actually there – a lot of lactobacilli that are present there.”The small intestine also holds lactobacilli, but the highest density and the biggest complexity is present in the ceca. There are also differences in age and thus maturation in the different sites of the guts. For example, the highest concentration of short-term fatty acids will be present in the ceca.
Everaert said there are internal and external factors that impact the chicken’s gut microbiota. Researchers have found differences between gender and age.
“Nowadays we're hatching chickens on farm,” she said, “they hatch not in the hatchery, but we're transporting eggs on embryonic day 18, and then we let them hatch on farm. We're looking into the difference in the microbiota there. Again, it's something that is transient. There are some differences in the beginning, but then they disappear later on.”
As for passive immunity, chickens get it during embryonic development, so their immunoglobulins are present in the yolk and in the albumen (egg white).
“The concentration of immunoglobulins is actually high in the first days post hatch. Then it will decrease,” Everaert said. “They don't have a long half-life. These immunoglobulins break down quickly, but, while present, they do provide a passive immunity.”
Maternal immunoglobulin A is also present in the albumen. Chickens will swallow the amniotic fluids, and the immunoglobulin A is then transferred, so some is present in the digestive tract already at the end of the embryonic development. The B-cells will produce their own immunoglobulins, and that process starts in the neonatal period. While they are not active yet in these first days, Everaert said they are maturing.
“We see already some activity at four days of age, but that's still maturing,” Everaert said; it needs seven to 10 days of age before there’s an active B-cell population.
On day one, the energy needed for the functioning and the survival of the gut epithelium is actually coming from the host.
“There are some enzymes that are present there that can use glucose, but then on day five, you already have a microbiota in the gut, so they're producing short-term fatty acids and butyrate, for example, will serve already as an energy source in the large intestine,” Everaert said.
Also active on day five, genes that are expressed, that prevent pathogens, can attach to the epithelium. From day 15 on, immunoglobulins are produced, and immunoglobulin A is secreted to neutralize the toxins. However, Everaert said gut health is truly a balance between tolerance and activation.
“We see already during embryonic development that there are some antimicrobial peptides that are expressed, toll-like receptors that are also important to recognize the possible pathogens,” Everaert said. “Mucus production is then more stimulated once a microbiota is present at the mucus – a protective mucus layer. Then post-hatch you see maturation through the increase of gene expression of some cytokines.”
There are different strategies to use, dietary or management-wise, for good gut development and colonization. For example, some researchers have tried spraying lactic acid bacteria to modify early life programming and alter the microbiome. From this, they have seen reduced pathogen presence. They’ve also been trying to spray mature microbiomes to chicks early in life by using adult fecal content.
Finally, when it comes to gut health and gut homeostasis, Everaert said we need to get it right early in life.
“We're looking into focusing on good colonization, gut colonization and maturation,” Everaert said. “There are different factors that you can try to use, dietary factors and management factors, to have a good and positive start and good early life programming for these young animals.”
Watch the full presentation from Dr. Everaert and access all 2023 Kemin Intestinal Health Symposium content at kemin.com/symposium.