In this study, ileum microbiota characteristics in L. intracellularis infected pigs under different experimental treatment regiments were defined and characterized. The ileum, as the primary site of L. intracellularis infection in pigs [37], was the selected anatomical location in this study. Our approach consisted of collecting ileum content from 9-week-old pigs, 16 days after experimental infection with L. intracellularis and 44 days post commercial L. intracelluaris vaccination (VAC-LAW) or the start of three different probiotics (T01-LAW, T02-LAW, T03-LAW) which were added to the regular base diet given to pigs in all groups. This differs from other L. intracellularis studies which have focused on fecal [11] or caecum and colon microbiota [16], or analyzed the distal ileum content using terminal restriction fragment length polymorphism [15] and provides a broad snapshot of L. intracellularis infection dynamics at the site of colonization. We specifically examined the bacterial community as earlier L. intracellularis experiments in gnotobiotic pigs suggested a certain required microbial background for L. intracellularis infection to establish [37]. The factor “treatment” was used to analyze the changes in ileum microbiota using 16S rRNA gene sequencing. We then linked observed changes to clinical phenotypes of L. intracellularis infection including fecal shedding and histopathology in the ileum.
Overall, we observed a strong association between treatment and changes in ileum microbiota structure based on alpha diversity indices. These changes were significant and not limited to richness (i.e. the number of unique OTUs) but also to evenness and relative abundance. For example, richness was highest in T03-LAW and lowest in VAC-LAW (Fig. 1c). However, VAC-LAW had a higher average Simpson index compared to T01-LAW even though the unique OTU count in VAC-LAW was significantly lower. It remains puzzling why, in this study, the vaccinated group had the lowest OTU count but a more even distribution of bacteria and how this impacted the overall outcome. A more even and diverse ileum microbiota may be beneficial for pigs infected with L. intracellularis as has been observed in relation with other pathogens [38, 39]. Microbiota changes were associated with specific clinical phenotypes as shown in the downstream analysis; specifically, increased diversity was associated with reduced histopathology lesions and fecal shedding. It is likely the presence of specific organisms in T03-LAW pigs which results in the desired effect on clinical presentation. It has been previously shown that area under the curve measurements and shedding duration were significantly increased in VAC-LAW pigs compared to T02-LAW and T03-LAW pigs, 40% of the VAC-LAW pigs had severe ileum lesions compared to 10% of T02-LAW and 0% of T03-LAW animals [24].
Similarly, we exploited the subtle methodological differences in analyzing beta diversity to define a range of variation likely attributable to treatment. Specifically, in this study, treatment explained between 26%-36% of the population variation. To put this in context, the variation attributable to treatment is comparable to what is caused by the physiological impact of increased pH due to bile in the small intestines [40]. In this regard, the largest change in beta diversity was seen for T03-LAW while changes for T01-LAW and T02-LAW were similar, hence the co-clustering.
Our approach assumed that clustering by richness and abundance corresponding to treatment is indicative of a strong taxonomic effect for the experiment. The extent to which treatment impacted the ileal microbial structure differed for each group. These differences potentially define the exploitable usefulness of these treatments, specifically in augmenting clinical outcomes to control L. intracelluaris in pig herds. For example, significant differences were observed between POS-CONTROL and NEG-CONTROL on the alpha diversity indices as well as discernible clusters on the beta diversity indices. However, intriguingly, the microbial structural changes between POS-CONTROL and VAC-LAW were comparable. T03-LAW almost exclusively grouped in cluster 1 (Fig. 3) which accounts for a considerable amount of the overall observed richness. This dramatic increase in richness was associated with a neutralizing effect on hedding and histopathology, specifically, low shedding and low histopathology scores. T03-LAW therefore appears to break the direct relationship (positive correlation) observed in all other L. intracellularis infected pigs between shedding and histopathology. It may be beneficial to increase the microbiota richness for the robustness of a niche in warding off a pathogen invasion. In this particular context further research is required to identify the microbial combination of probiotics responsible for this effect.
Shifts in the ileal core genera were associated with changes in the threshold for shedding level, i.e. the clusters with the most depleted core (cluster 5) and the most enriched cores (clusters 1 and 3). This suggests that depleting the ileal core genera results in a higher threshold for shedding. It is difficult to compare the core genera across studies. Besides age, commonly studies are done in pigs around weaning [41, 42], housing and diet may also impact core genera. Moreover, it has been recently determined that when defining the core microbiome in pigs, differences in study protocols have a significant impact and standardization of experimental techniques appear to be important [43]. Nevertheless, using a meta-analysis of 20 data sets, several shared genera such as Prevotella, Clostridium, Alloprevotella, and Ruminococcus were identified [43]. A more recent study, based on analysis of freshly collected feces, identified a pig core microbiome of 69 bacterial features present in all growth stages. Besides confirming the earlier core genera findings, at the family level the top three families were Prevotellaceae, Ruminococcaceae, and Lactobacillaceae [44]. The analysis in this study does not suggest a major effect of the core microbiota on clinical presentation other than threshold differences in shedding detection (cluster 3 enriched and cluster 5 depleted in Fig. 4b).
Having established that in this study a direct relationship exists between pathology and shedding, identifying microbial parameters that disrupt it provides a foundation for limiting the impact of this infection. The precise mechanism of this microbial interaction is unknown. The data obtained here will be useful to design future experiments in an attempt to further reduce bacterial shedding and pathology. This perhaps could advance probiotic supplementation to limit the impact of infectious diseases in food production. Limiting pathology arguably reduces not only the adverse effects on an animal’s productivity but also shedding and thus limits transmission to other pigs. In this regard, T03-LAW (cluster 1) appears to be the only treatment group in which the shedding dynamics were reduced. At the family level, a log increase in abundance of Clostridiaceae, Streptoccaceae or Erysipelatricheae was associated with 3.9, 3.1 and 3.4 log reduction in copies of L. intracellularis shedding and pathology rank, respectively. In particular, reduction in shedding and pathology in the VAC-LAW group appeared to be influenced by the increase in abundance of Clostridiaceae. A similar but weaker relationship was noted for T01-LAW and T02-LAW. In a previous study, analysis of the microbiota of L. intracellularis vaccinated pigs showed that vaccination led to changes in the abundance of Clostridium species, including Clostridium butyricum [11] which is in agreement with the results of this study. However, an increase in abundance of the three aforementioned families was not associated with shedding or pathology (reduction or increase) for T03-LAW.
An increase of one unit in the Shannon diversity index resulted in a 2.8 log reduction in shedding in this study. In other words, by increasing diversity one could reduce shedding of L. intracellularis. In addition to T03-LAW, a reduction in shedding associated with increased diversity was also evident for T01-LAW and T02-LAW. An increase in diversity was however associated with increased shedding among VAC-LAW pigs, this still requires an explanation. Perhaps this could reflect the effect of a longer term dysbiosis as the VAC-LAW group was first exposed to attenuated L. intracellularis before being exposed to pathogenic L. intracellularis challenge. In addition, the VAC-LAW group did manifest associated changes in the ileal microbiota after L. intracellularis challenge (Additional file 3) which warrants further studies. Crucially, the comparison between clinical phenotypes and ileal microbiota diversity showed that both control groups (NEG-CONTROL, POS-CONTROL) were not affected by this relationship. The direction and strength of the relationships observed with a treatment, i.e. the associated microbial community, therefore reflects its true impact.
A potential application of the study findings includes microbiota augmentation to modify pathology, shedding and, by extension, L. intracellularis transmission. This may have profound implications for the dependence on antibiotic use to control infectious diseases in livestock production systems. However, extensive research will be required to optimize probiotics (and likely also prebiotics) outside the realm of controlled experimentation. Ultimately such scalable innovation means livestock will be raised without adverse environmental effect while also minimizing the contribution to the evolution of antibiotic resistance. In this regard, our findings indicate the potential usefulness of microbial families such as Clostridiaceae, Streptoccaceae, Erysipelatricheae, Rummincococeae, Coriobacteriaceae and Peptostreptococaceae in augmenting clinical outcomes during L. intracellularis infections.