Diarrhoea associated with enteric diseases caused by infectious bacterial and viral pathogens is a common problem in grow-finishing pigs, even on farms with excellent herd management practices (Karuppannan and Opriessnig, 2018). These infectious pathogens affect the health and production efficacy of pigs, leading to negative economic impacts on swine production. Among bacterial pathogens, the most important and prevalent pathogen worldwide is Lawsonia intracellularis (L. intracellularis) occurring mainly in post-weaned and growing pigs (Lawson and Gebhart, 2000). A study from Sweden showed that up to 67% of faecal samples collected from pigs with diarrhoea were positive for L. intracellularis (Jacobson et al., 2003). Similarly, up to 31% of enteric infections in diarrhoea in pig herds in Brazilian pig farms were also caused by L. intracellularis (Viott et al., 2013).L. intracellularis is an anaerobic obligate intracellular bacterium infecting actively dividing cells in the crypt of the small intestine of pigs and is responsible for the porcine proliferative enteropathy (PPE), also referred to as ileitis. The disease occurs either as an acute or chronic infection. The acute form, also known as proliferative haemorrhagic enteropathy (PHE), is characterized by bloody diarrhoea due to severe damage to the small intestine. For reasons not yet clear, the acute form of PPE is common in healthy herds, resulting in the sudden death of healthy adult pigs (Gilts and Boars). On the other hand, the chronic form of PPE, also known as Porcine Intestinal Adenomatosis (PIA), is characterized by weight loss and chronic diarrhoea. The diarrhoea is greyish in colour due to death and sloughing off the proliferating intestinal cells. During postmortem, the typical findings are thickened mucosa in the distal ileum and loss of goblet cells (Lawson and Gebhart, 2000; Kroll et al., 2005).

The economic impact of ileitis could be important, particularly in grow-finish pigs, resulting in a reduction in daily weight gain of approximately 3% to 19%, depending on the severity of the disease, and a decrease in refeed efficiency up to 7%. In acute cases, the disease may also kill 1,3% to 6,7% of the pigs within infected herds, leading to fewer and lighter finishing pigs. In addition to production loss, the cost incurred for treatment and prevention is considerable for farmers (Fourchon and Chouet, 2000; Holtkamp, 2019). In general, the monetary value of the diseases is estimated to be around $2.00 to $3.00 per pig in mild and subclinical cases, and as high as $15.00 per pig in severe cases (McOrist et al., 1997). Due to the ban of antibiotics in many countries and the limitations that currently available vaccines have to fully control ileitis, the swine industry is looking for alternative products to control infection or increase the potency of vaccines.

Recently, it was shown that the innate immune system could build innate immune memory responses after exposure to pathogens and, therefore, developed stronger and enhanced immune responses during secondary exposure. Innate immune memory, also called trained immunity, is non-specific and can mount protection against a cognate antigen or an unrelated pathogen (Netea et al., 2011; Netea et al., 2016). B-glucans (BG), an abundant branched polysaccharide located in the yeast cell wall, has been described as a strong inducer of trained immunity. In vitro studies using porcine monocytes exposed to BG showed stronger immune responses after exposure to microbial pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS) (Byrne et al., 2020). Furthermore, in vivo mouse studies using BG have demonstrated that macrophages develop better protection against Staphylococcus aureus (Quintin et al., 2012). Intestinal homeostasis is crucial for good performance of animals and is achieved by fine regulation of the intestinal barrier to reduce the risk of infectious or inflammatory conditions. Intestinal macrophages, which can be infected by L. intracellularis (Pereira et al., 2020), play an important role in host defence against invading pathogens (Bain and Schridde, 2018). Therefore, taking advantage of the ability of BG to induce trained immunity in intestinal macrophages could be an interesting strategy to reduce the damage caused by pathogens.

Additionally, crosstalk between the microbiota, epithelial cells, and immune system is key for the development and regulation of intestinal immune processes. (Rajput et al., 2022).- Therefore, L. intracellularis infection may result in microbial dysbiosis and decrease the immune response of the host to pathogens, thereby exposing the host to other enteric infections, such as Salmonella (Leite et al., 2018). We hypothesized that by stimulating the innate immune response (trained immunity) and through its ability to modify the gut microbiota of pigs, yeast-extracted BG could mitigate the negative impact of L. intracellularis on pig health and performance. In addition to demonstrating the efficacy of yeast BG in decreasing the negative impact of L. intracellularis on performance parameters and its positive effect in reducing faecal shedding of the pathogen in fattening pigs protective immunity induced by BG, we aimed to outline the specific changes in faecal microbiota composition related to yeast-derived BG supplementation during L. intracellularis infection.

In this study, we reported that yeast-extracted BG ameliorates the negative impact of L. intracellularis infection on the health and performance of grow-finishing pigs. Previous studies in different animal species have shown that BG were able to enhance growth and immunity in Nile Tilapia (Selim and Reda, 2014) and pigs (Vetvicka and Oliveira, 2014), as well as to decrease lesions in Eimeria-challenged broiler (Cox et al., 2010). In agreement with all these previous findings, our current study has also shown significant improvement of zootechnical performances of pigs under L. intracellularis pressure when supplemented with BG. Overall, this study showed that the administration of yeast BG resulted in an increase of BW and ADG, compared to the animals from the control group.

The performances improvement can be explained by the decrease of L.intracellularis shedding, itself explained by the known effect of BG on inducing the innate immune memory. Indeed, we observed a lower proportion of positive animals for L.intracellularis among those having received BG, at D81 as well as at D97, compared to the control group. In addition to this, our results have also shown a significant decrease in bacterial L.intracellularis shedding in the faeces of BG-supplemented pigs as compared to control. Those results are in line with a recent finding that reported a limited translocation of Salmonella Typhimurium to lymph nodes and decrease bacterial shedding in Salmonella challenged nursery piglets fed with BG diet (Bearson et al., 2023). As L. intracellularis infects macrophages, one of the most represented innate immune cells of the intestinal tract (Pereira et al., 2020), BG would reinforce this macrophage line of defence helping the animals to eliminate more efficiently the pathogen. These could be explained by the impact of BG in stimulating innate immune cells (through immune training) or their role in modifying the gut microbiota of piglets towards beneficial bacteria. To support this last hypothesis, we are discussing here below the positive impact of yeast BG in modifying the gut microbiota of pigs in the context of L. intracellularis infection.

Gut microbiota α-diversity decreases with increasing L. intracellularis burden. This observation is supported by the one made by Muwonge and collaborators who observed a difference in the α-diversity of the luminal ileum microbiota (Muwonge et al., 2021) according to the presence of L. intracellularis, and an increased α-diversity associated with reduced histopathology lesions and faecal shedding. In our study, the reduction in α-diversity was mainly observed for animals with the highest L. intracellularis burden (i.e. low copy number per mg of faeces). In addition, at D97, according to the infectious status of the animals, a slight modulation of the faecal microbiota has been observed. Sarcina and Catenibacterium were found in higher proportions in animals carrying L. intracellularis. Sarcina species are fastidious Gram-positive anaerobic bacteria that occur in cubic packs of eight or more cells. In 2006 and 2007, they were associated with cases of acute abomasal bloat in young lambs and calves (Edwards et al., 2008), whereas Catenibacterium, a genus of Gram-positive and obligate anaerobe bacteria, are mainly involved in the biosynthesis and metabolism of fatty acids (Choudhury et al., 2021; Loo et al., 2022).

Although, L.intracellularis has little impact on the faecal microbiota, some taxa can modulate the outcome of the infection. The biomarker defined by the ratio Fibrobacter/Lachnospiraceae AC2044 group at a time when L. intracellularis infection is not yet detectable by qPCR, could be a predictor of future infection and/or a marker of susceptibility to the infection. Indeed, before the infection occurs, this ratio was higher than in the ‘future’ positive animals’ group. Interestingly, a previous study by Leite (Leite et al., 2018) noted that a challenge with L. intracellularis decreased the relative proportion of Fibrobacter. On the contrary, Lachnospiraceae AC2044 group could have a preventive character in agreement with the positive correlation found between Lachnospiraceae AC2044 group and the contents of SCFAs, fat, and crude ash, as well as the negative correlation with amino acids in different gut locations of pigs (Song et al., 2022). Our results seem to be in line with these study as when the infection is declared, this ratio decreased in the positive animals throughout the trial until reaching the same level as that of the negative animals ( Supplementary Data …).

BG used in this study also influenced the relative proportion of several taxa. The genus Prevotellaceae UCG-004 and Megasphaera, which are positively modulated in animals receiving BG, are known as polysaccharides degraders and SCFAs producers (Heinritz et al., 2016). In contrast, animals in the control group exhibited a much higher relative abundance in Sarcina, Lachnospiraceae UCG-007, Subdoligranulum, three taxa belonging to the Clostridia class, and to a lesser degree Streptococcus and Intestinibacter.

Here, the data led to the identification of a potential biomarker allowing to discriminate the microbiota pattern of BG-fed animals from the one associated with the control group. Although this potential gut microbiota pattern displayed the sum of Sarcina, Lachnospiraceae UCG-007, Subdoligranulum and Streptococcus as the denominator, as expected, these are Terrisporobacter and Clostridia UCG-014 that appeared in the numerator. The positive correlation between their relative abundance and the BW highlighted at the end of the trial is in accordance with previous studies. Interestingly, these two genera are considered to be plant fibre degrading bacteria (Li et al., 2021). In addition, other studies showed that relative abundance of Terrisporobacter is also positively correlated to feed efficiency in young pigs as well as amino acids metabolism, including tryptophan, a metabolite of which (indoleacetic acid) that can positively modulate intestinal homeostasis and inflammatory response (McCormack et al., 2019; Yan et al., 2021; Shen et al., 2022).

Even though it could be tricky to distinguish between the consequence from the cause in correlation studies between phenotype and microbiota, we can nevertheless make a hypothesis considering the nature of the additive. β-1,3/1,6-glucans are essential components of a prebiotic-rich diet that promotes growth and metabolism of the gastrointestinal microbiota (Metzler-Zebeli et al., 2011). Indeed, in the hypothesis of a prebiotic-type treatment favouring an increase in the relative abundance of certain taxa at the expense of others, BG used in this study would favour the taxa Terrisporobacter and Clostridia UCG-014, which are themselves associated with a higher weight gain, decreasing consequently Sarcina, Lachnospiraceae UCG-007, Streptococcus and Subdoligranulum relative abundance. In our study, Sarcina and Lachnospiraceae UCG-007 were relatively independent of body weight, although Sarcina has been associated with poorer performance in terms of weight gain in previous studies as well as Lachnospiraceae (Levesque et al., 2014; Bergamaschi et al., 2020; Quan et al., 2020; Holanda and Kim, 2022). But, interestingly, these two taxa were strongly associated with the level of L. intracellularis contamination. Two other genera, Streptococcus and Subdoligranulum, do not appear to have a clear association with the BW or burden of L. intracellularis.

In conclusion, some specific yeast branched BG were able to improve health, by reducing bacteria shedding and by increasing body weight in pigs. Regarding biomarker and differential analysis, this study showed that taxa, such as Clostridia UCG-014 and Terrisporobacter, are preferentially related to animals fed with BG and with an improved BW whereas the taxa Subdoligranulum, Streptococcus, Sarcina and Lachnospiraceae UCG-007 are more present in animals in the control group and are partly related to the L. intracellularis load ( Figure 11 ). This dual analytical approach led to the establishment of a ratio with these taxa, that should be validated in future studies, allowing to distinguish the faecal microbiota of an animals fed or not with BG. Those findings open up also the possibility of having microbial biomarkers to evaluate high performing and L. intracellularis-resistant animals.

The original contributions presented in the study are included in the article/ Supplementary Files , further inquiries can be directed to the corresponding author/s.

The animal study was approved by spanish Royall Decree 1135/2002. The study was conducted in accordance with the local legislation and institutional requirements.

GE, CA performed the microbiota analysis and analysed the data. LR coordinated the microbiota analysis. GE, CA, SL-R, LR, TK, JS wrote the manuscript. JS designed the study, organised and conducted the study, analysed the performance data. GK found the CRO and dealed with the administrative work. All authors contributed and approved the submitted version.