Diarrhea is a leading cause of increased mortality in neonatal and young piglets. Aberration of the gut microbiota is one important factor in the etiology of piglet diarrhea. However, information regarding the structure and function of the gut microbiome in diarrheic neonatal piglets is limited. To investigate the composition and functional potential of the fecal microbiota in neonatal piglets, we performed 16S rRNA gene sequencing on 20 fecal samples from diarrheic piglets and healthy controls, and metagenomics sequencing on a subset of six samples. We found striking compositional and functional differences in fecal microbiota between diarrheic and healthy piglets. Neonatal piglet diarrhea was associated with increases in the relative abundance of Prevotella, Sutterella, and Campylobacter, as well as Fusobacteriaceae. The increased relative abundance of Prevotella was correlated with the reduction in Escherichia coli and the majority of beneficial bacteria that belonging to the Firmicutes phylum (e.g., Enterococcus, Streptococcus, Lactobacillus, Clostridium, and Blautia) in diarrheic piglets. The differentially functional gene abundances in diarrheic piglets were an increase in bacterial ribosome, and contributed primarily by the genera Prevotella, this indicates a growth advantage of the Prevotella in diarrheic conditions. Additional functional gene sets were associated with the reduction of polyamine transport, monosaccharide and sugar-specific PTS transport, amino acid transport, and two-component regulatory system. These profiles likely impact the ability to transport and uptake nutrients, as well as the ability to fight microbial infections in the piglet gut ecosystem. This work identifies a potential role for Prevotella in the community-wide microbial aberration and dysfunction that underpins the pathogenesis of piglet diarrhea. Identification of these microbial and functional signatures may provide biomarkers of neonatal piglet diarrhea.

Diarrhea is one of the most common enteric diseases in young piglets. Diverse factors such as an unstable gut microenvironment, immature intestinal immune system, early supplementary feeding, and weaning often induce dysfunction of gut microbiota, thus leading to a continuing high incidence of diarrhea in piglets. However, few studies have characterized the gut microbiota of diarrheic piglets following changes in diet and during the development of intestinal physiology. In this study, we used 16S rRNA gene sequencing to analyze the dynamic establishment of fecal microbiota in six healthy piglets in response to age‐related changes in the diet: sow‐reared, early supplementary creep‐feeding (sow‐reared + starter diet), and weaning (solid nursery diet). We compared the gut microbiota of these six healthy piglets with those of diarrheic piglets during each of the three dietary stages (n = 10 sow‐reared, n = 10 early supplementary creep‐feeding, and n = 5 weaning). We found that weaning (solid nursery feeding) was the primary factor leading to dynamic colonization by microbiota in healthy piglets, and diarrhea primarily affected the microbial communities of piglets before weaning. Healthy piglets showed a continuous decrease in Lactobacillus and Escherichia, as well as a gradual increase in Prevotella with the transition to solid food. An altered relationship between Prevotella and Escherichia may be the main cause of diarrhea in preweaned piglets, whereas reduced numbers of Bacteroides, Ruminococcus, Bulleidia, and Treponema that are responsible for the digestion and utilization of solid feeds may be related to the onset of postweaning piglet diarrhea. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) functional analysis indicated that a reduction in genes involved in carbohydrate metabolism induced by intestinal dysbacteriosis in diarrheic piglets was one of the major causes of diarrhea at the three dietary stages. These findings provide insights into developing an intervention strategy for better management of diarrhea in piglets.

Long non-coding RNAs (lncRNAs) have been shown to play important roles in regulating host immune and inflammatory responses to bacterial infection. Infection with Clostridium perfringens ( C. perfringens ), a food-borne zoonotic pathogen, can lead to a series of inflammatory diseases in human and piglet, greatly challenging the healthy development of global pig industry. However, the roles of lncRNAs involved in piglet immune response against C. perfringens type C infection remain unknown. In this study, the regulatory functions of ileum lncRNAs and mRNAs were investigated in piglet immune response to C. perfringens type C infection among resistance (IR), susceptibility (IS) and sham-inoculation (control, IC) groups. A total of 480 lncRNAs and 3,669 mRNAs were significantly differentially expressed, the differentially expressed lncRNAs and mRNAs in the IR and IS groups were enriched in various pathways of ABC transporters, olfactory transduction, PPAR signaling pathway, chemokine signaling pathway and Toll-like receptor signaling pathway, involving in regulating piglet immune responses and resistance during infection. There were 212 lncRNAs and 505 target mRNAs found to have important association with C. perfringens infectious diseases, furthermore, 25 dysregulated lncRNAs corresponding to 13 immune-related target mRNAs were identified to play potential roles in defense against bacterial infection. In conclusion, the results improve our understanding on the characteristics of lncRNAs and mRNAs on regulating host immune response against C. perfringens type C infection, which will provide a reference for future research into exploring C. perfringens -related diseases in human.

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Clostridium perfringens type C is a pathogen that causes necrotizing enteritis ( NE ), which is an intestinal tract disease in piglets. The pathogenesis of C. perfringens type C‐induced NE is still unclear, leading to a lack of effective therapies. Earlier studies have reported that circular RNA s (circ RNA s) are involved in the pathogenic processes of various diseases. However, it is not known if circ RNA s in spleen play a role in C. perfringens type C infection in NE . To address this question, we infected 7‐day‐old piglets with C. perfringens type C to induce NE . Hematoxylin and eosin staining of small intestine revealed inflammation, atrophy and shedding of intestinal villi, and intestinal mucosal necrosis. We observed increased expression of cytokine genes (such as IL ‐1β and IL ‐6 ) and inflammation in the spleen. In addition, we used RNA ‐seq and bioinformatics analysis to examine changes in circ RNA expression. A total of 103 circ RNA s were found to be differentially expressed in NE , and Gene Ontology analysis revealed that the genes producing differentially expressed circ RNA s were enriched in regulation of the cellular metabolic process protein binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the genes producing differentially expressed circ RNA s were involved in the tumor necrosis factor signaling pathway, T cell receptor signaling pathway and nuclear factor‐κB signaling pathway. Finally, we found eight circ RNA s (including circ_0002220 and circ_0000821) that are related to NE . Therefore, our study provides new insights into the mechanisms underlying C. perfringens type C infection in piglets.

BackgroundClostridium perfringens (C. perfringens) type C is the most common bacteria causing piglet diarrheal disease and it greatly affects the economy of the global pig industry. The spleen is an important immune organ in mammals; it plays an irreplaceable role in resisting and eradicating pathogenic microorganisms. Based on different immune capacity in piglets, individuals display the resistance and susceptibility to diarrhea caused by C. perfringens type C. Recently, long non-coding RNA (lncRNA) and mRNA have been found to be involved in host immune and inflammatory responses to pathogenic infections. However, little is known about spleen transcriptome information in piglet diarrhea caused by C. perfringens type C.MethodsHence, we infected 7-day-old piglets with C. perfringens type C to lead to diarrhea. Then, we investigated lncRNA and mRNA expression profiles in spleens of piglets, including control (SC), susceptible (SS), and resistant (SR) groups.ResultsAs a result, 2,056 novel lncRNAs and 2,417 differentially expressed genes were found. These lncRNAs shared the same characteristics of fewer exons and shorter length. Bioinformatics analysis identified that two lncRNAs (ALDBSSCT0000006918 and ALDBSSCT0000007366) may be involved in five immune/inflammation-related pathways (such as Toll-like receptor signaling pathway, MAPK signaling pathway, and Jak-STAT signaling pathway), which were associated with resistance and susceptibility to C. perfringens type C infection. This study contributes to the understanding of potential mechanisms involved in the immune response of piglets infected with C. perfringens type C.

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