Since piglets are born immunologically naive, one critical component of immune system development is the colonization of the gastrointestinal tract. With commensal microbes, a correctly developed immune system is essential for protection from pathogens, as well as to ensure that the piglet reaches its full growth potential, says Tausha Prisnee.

"The gastrointestinal tract is home to the largest reservoir of immune tissue anywhere in the body and this is because essentially the GI tract is in contact with the outside environment and must prevent pathogens from making it to other places in the body," Prisnee says. "So, these microbes will come in contact with immune cells, and it will then stimulate these immune cells during the first days and weeks of life. These immune cells will then be educated as to whether something is commensal or pathogenic and some of these educated immune cells will then stay in the gut to promote gut homeostasis and some will travel to other parts of the body. For example, microbes educated in the gut may travel to the lungs where they will be able to recognize microbes that they've either seen before or ones that are very similar and protect against some types of respiratory pathogens."

To date, most research in the microbiome and immune system development has focused on bacteria, despite the fact that fungi themselves are immunomodulatory. A graduate research assistant with the University of Alberta, Prisnee says work in humans has actually shown that early life contact with fungi can shape long-term host health trajectories. Within the pig, the most common fungi is yeast, a cellular structure that is more similar to humans’ than it is to that of bacteria.

While supplemental yeast, such as Saccharomyces cerevisiae or Saccharomyces boulardii, has long been fed to livestock and has been shown to improve growth performance, prevent diarrhea, and to improve immune response to pathogens, the mechanism through which this happens is not quite clear, says Prisnee.

The most common yeast in the gastrointestinal tract of the pig is Kazachstania slooffiae, a microbe that can only be found in pigs and has no known environmental reservoir. It is found across many different geographic locations and in all types of production systems and is known to interact with bacteria.

Previous work in the University of Alberta’s laboratory has shown that piglets who are colonized with large amounts of K. slooffiae typically come from a sow who also has large amounts of K. slooffiae. This time the research team wanted to see what impact K. slooffiae plays on the early life immune system development of pigs. The objectives of their project were to:

In order to study immune system development early in life, the team created a "bubble pig" or a gnotobiotic piglet model.  Piglets (n=12) were derived via c-section and reared in sterile isolators for eight days where they were inoculated with a consortium of bacteria commonly found in the piglet gastrointestinal tract, and with or without K. slooffiae. Contents from the jejunum, ileum, cecum and colon were collected and plated for yeast and total anaerobes. The impact of yeast on the bacterial community was assessed by 16S rRNA gene sequencing. Blood and mesenteric lymph nodes were collected for cytokine analysis.

"The first thing we wanted to do was to make sure that our K. slooffiae did indeed colonize … it was the lowest in the jejunum and was about the same in the ileum, cecum and colon … and we also plated to make sure that there was no contamination in the defined bacterial community group and there was not," Prisnee says. "Next, we wanted to look at the number of total anaerobes in both the ilium and the cecum … the K slooffiae group had almost a log higher of total anaerobes, and we believe that this is because Kazachstania slooffiae is reducing the oxygen in the gut and allowing more anaerobes to grow. This also means that the piglets have a more mature microbiome which may be beneficial to the pig for pathogen protection."

Next, the team examined how K. slooffiae may alter bacterial community composition. Prisnee says they were surprised that they did not see a change in Bacteroides, which is the main anaerobic genera, even though they did see an increase in total anaerobes.

"It's important to point out here that in 16S sequencing we can only look at relative abundance and we can't quantify the total amount that is present, so in the future we will be doing quantification of both total bacteria and bacteroides using qPCR," Prisnee says. An increase in E. coli as well as a decrease in lactobacillus was also observed.

The team also examined the effect K. slooffiae has on immune phenotype. Total T-cells, as well as helper T-cells, were decreased in the pigs inoculated with K. slooffiae. However, Prisnee points out that even though they were lower, they were still within a normal range.

"This suggests that they're not immunocompromised or anything, that they're just having less of an immune response to the defined bacterial community," Prisnee says.

The researchers also look at activated cells and again found, activated T-cells, as well as activated T helper cells, were lower.

In the K. slooffiae piglets, the team also studied their immune function, specifically the level of cytokines and plasma. They found an increase in the IL-2 cytokine that promotes the differentiation of T-cells into regulatory T-cells. Prisnee says, "this will inhibit some of an immune response in order to maintain homeostasis as well as prevent autoimmune disease."

They also found that TNF alpha, which is a pro-inflammatory cytokine, increased in the defined bacterial community group.

"This means that K. slooffiae may be preventing some of this immune response to the defined bacterial community," Prisnee says.

Finally, the researchers measured how the immune cells would respond to a challenge, when stimulated with lipopolysaccharide. They observed an increase in IL-18, a pro-inflammatory cytokine, as well as an uptick in IL-10, an anti-inflammatory cytokine.  "You often see both a pro and anti-inflammatory cytokine at the same time and this is just the immune system trying to regulate itself," Prisnee says.

The research team concluded that K. slooffiae is able to promote an inflammatory response towards potential pathogens, however there's still a great deal of work to do in studying the benefits of this yeast.

"Next, we will be looking at intestinal development where we will use gene expression to look at tight junction proteins as well as we will do histology. We will perform metabolomics … we will quantify total bacteria as well as bacteroides via qPCR," Prisnee says.

Prisnee was a finalist in the R.O. Ball Young Scientist Grad Student Competition at the 2023 Banff Pork Seminar.