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Background Whole-plant corn silage (WPCS) is an important roughage source for livestock, and have critical influences on rumen or intestinal microbiota, thus affecting the growth performance and feed efficiency. Our previous studies showed that adding WPCS to the diet of Hezuo pigs could promote the growth and fiber digestibility. While the aim of this study is to understand the effect of dietary WPCS on the colonic microflora in Hezuo pigs, which is essential for improving the roughage exploitation of pigs. Methods Thirty-two Hezuo pigs with similar body weight (7.88 ± 0.81) kg were selected and randomly divided into four groups with eight pigs in each group. Pigs in the control group were fed a basal diet, pigs in the experimental groups (Groups I, II, and III) were fed basal diet supplemented with 5%, 10%, and 15% WPCS, respectively, under 120 d experimental period. Six pigs from each group were picked for collecting colonic contents samples. 16S rRNA sequencing was performed to analyze the colonic microbiota of experimental pigs. Results The results showed that community richness indexes Chao1 and Observed_species in group III of Hezuo pig were significantly lower than that of the other three groups, community diversity indexes Shannon and Simpson were significantly higher in group I and II in comparison to the control group, and significantly lower in group III in comparison to the control group, group I and II. Adding WPCS to the diet of Hezuo pigs has no influence on the colonic dominant phylum, Clostridium sensu stricto 1 and Rikenellaceae RC9 gut group were most prevalent in the colon of Hezuo pig. When compared with the control group, the relative abundance of Streptococcus was significantly decreased in three experimental groups, while p-251-o5, Parabacteroides, Prevotellaceae UCG-003, Prevotellaceae UCG-001, and F082 exhibited significantly higher relative abundances in at least two experimental groups. Fibrobacter, Rikenellaceae RC9 gut group in group I, UCG-010 in group II, Bacteroides in group III exhibited increased relative abundance as compared with the control group. PICRUSt functional annotation indicated that the functions of cellular process and signaling were significantly increased in all WPCS-rationed groups, cancers, nervous system, immune system and environmental adaptation were all differed from groups I and II; three predominant pathways of translation, nucleotide metabolism and signal were only differed from the group II. Conclusions Feeding with 5% and 10% WPCS for Hezuo pigs could improve their colonic microflora diversity, and increase the relative abundance of fiber-digesting bacteria, which may potentially help to improve the fibre digestibility of Hezuo pigs by regulating the microbial function of cellular process and signaling, nucleotide metabolism, translation.

IntroductionUtilizing roughage resources is an effective approach to alleviate the shortage of corn-soybean feed and reducing the costs in the swine industry. Hezuo pig is one group of plateau type local Tibetan pig with strong tolerance to crude feeding. Nevertheless, current research on the roughage tolerance in Hezuo pigs and the microbiological mechanisms behind it is still minimally.This study explored the impact of various ratios of whole-plant silage (WPS) maize on the pH, cellulase activity, short-chain fatty acids (SCFAs), and intestinal microbiota in Hezuo pigs.MethodsThirty-two Hezuo pigs were randomly divided into four groups (n = 8). The control group received a basal diet, while experimental groups I, II, and III were given diets with incremental additions of 5%, 10%, and 15% air-dried WPS maize, respectively, for 120 days.ResultsThe findings revealed that compared with the control group, in Group II, the pH of cecum and colon were notably decreased (p

Clostridium perfringens (C. perfringens) type C is one of the common bacteria in piglet diarrhea, which seriously affects the swine industry’s development. The spleen plays crucial roles in the resistance and elimination of pathogenic microorganisms, and miRNAs play important roles in regulating piglet diarrhea caused by pathogens. However, the mechanism by which miRNAs in the spleen are involved in regulating C. perfringens type C causing diarrhea in piglets remains unclear. The expression profiles of the spleen miRNAs of 7-day-old piglets challenged by C. perfringens type C were studied using small RNA-sequencing in control (SC), susceptible (SS), and resistant (SR) groups. Eight-eight differentially expressed miRNAs were screened. The KEGG pathway analysis of target genes revealed that the miRNAs were involved in the MAPK, p53, and ECM–receptor interaction signaling pathways. NFATC4 was determined to be a direct target of miR-532-3p and miR-133b using a dual-luciferase reporter assay. Thus, miR-133b and miR-532-3p targeted to NFATC4 were likely involved to piglet resistance to C. perfringens type C. This paper provides the valuable resources to deeply understand the genetic basis of C. perfringens type C resistance in piglets and a solid foundation to identify novel markers of C. perfringens type C resistance.

Background Breast carcinoma amplified sequence 2 (BCAS2) participates in pre-mRNA splicing and DNA damage response, which is implicated in spermatogenesis and meiosis initiation in mouse. Nevertheless, the physiological roles of BCAS2 in the testes of large mammals especially boars remain largely unknown. Methods In this study, testes were collected from Hezuo pig at three development stages including 30 days old (30 d), 120 days old (120 d), and 240 days old (240 d). BCAS2 CDS region was firstly cloned using RT-PCR method, and its molecular characteristics were identified using relevant bioinformatics software. Additionally, the expression patterns and cellular localization of BCAS2 were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry and immunofluorescence. Results The cloning and sequence analysis indicated that the Hezuo pig BCAS2 CDS fragment encompassed 678 bp open reading frame (ORF) capable of encoding 225 amino acid residues, and possessed high identities with some other mammals. The results of qRT-PCR and Western blot displayed that BCAS2 levels both mRNA and protein were age-dependent increased (p < 0.01). Additionally, immunohistochemistry and immunofluorescence results revealed that BCAS2 protein was mainly observed in nucleus of gonocytes at 30 d testes as well as nucleus of spermatogonia and Sertoli cells at 120 and 240 d testes. Accordingly, we conclude that BCAS2 is critical for testicular development and spermatogenesis of Hezuo pig, perhaps by regulating proliferation or differentiation of gonocytes, pre-mRNA splicing of spermatogonia and functional maintenance of Sertoli cells, but specific mechanism still requires be further investigated.

LncRNAs play important roles in resisting bacterial infection via host immune and inflammation responses. Clostridium perfringens (C. perfringens) type C is one of the main bacteria causing piglet diarrhea diseases, leading to major economic losses in the pig industry worldwide. In our previous studies, piglets resistant (SR) and susceptible (SS) to C. perfringens type C were identified based on differences in host immune capacity and total diarrhea scores. In this paper, the RNA-Seq data of the spleen were comprehensively reanalyzed to investigate antagonistic lncRNAs. Thus, 14 lncRNAs and 89 mRNAs were differentially expressed (DE) between the SR and SS groups compared to the control (SC) group. GO term enrichment, KEGG pathway enrichment and lncRNA-mRNA interactions were analyzed to identify four key lncRNA targeted genes via MAPK and NF-κB pathways to regulate cytokine genes (such as TNF-α and IL-6) against C. perfringens type C infection. The RT-qPCR results for six selected DE lncRNAs and mRNAs are consistent with the RNA-Seq data. This study analyzed the expression profiling of lncRNAs in the spleen of antagonistic and sensitive piglets and found four key lncRNAs against C. perfringens type C infection. The identification of antagonistic lncRNAs can facilitate investigations into the molecular mechanisms underlying resistance to diarrhea in piglets.

In this study, we investigated the effects of the dietary inclusion of different proportions of whole-plant corn silage on growth performance, serum biochemical indexes, and intestinal microorganisms in Hezuo pigs. Thirty-two two-month-old Hezuo pigs (body weight: 7.88 ± 0.81 kg) were randomly divided into four groups of eight pigs (half male, half female) each. The control (CON) group received a basal diet, while the three experimental groups were fed the basal diet, part of which had been replaced with 5%, 10%, and 15% whole-plant corn silage, respectively. The experiment lasted for 127 days, including 7 days of pre-testing and 120 days of formal testing. At the end of the experiment, blood and fecal samples were collected. Compared with the CON group, the feed-to-gain ratio was significantly lower in the 10% test group (p < 0.05), whereas the total protein, albumin, triglyceride, and glucose contents were significantly higher (p < 0.05). No significant differences in total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, creatinine, urea, aspartate aminotransferase, and alanine aminotransferase were observed among the groups (p > 0.05). The addition of whole-plant corn silage to the diet significantly increased alpha diversity in the pig gut based on 16S rRNA gene sequencing. The principal coordinate analysis results showed significant clustering of the different groups (p < 0.05). At the phylum level, the addition of whole-plant corn silage to the diet significantly decreased (p < 0.05) the relative abundance of Firmicutes and significantly increased (p < 0.05) that of Bacteroidetes. At the genus level, the relative abundance of Streptococcus significantly decreased (p < 0.05) with increasing silage supplementation levels, whereas species diversity significantly increased (p < 0.05). In conclusion, 10% is the recommended inclusion ratio for whole-plant corn silage in the diets of pigs.

Background S100 calcium-binding protein A9 (S100A9) is a commonly known pro-inflammatory factor involved in various inflammatory responses. Clostridium perfringens (C. perfringens ) type C is known to cause diarrhea in piglets. However, the role of S100A9 in C. perfringens type C-induced infectious diarrhea is unclear. Methods Here, the S100A9 gene was overexpressed and knocked down in the IPEC-J2 cells, which were treated with C. perfringens beta2 (CPB2) toxin. The role of S100A9 in CPB2 toxin-induced injury in IPEC-J2 cells was assessed by measuring the levels of inflammatory cytokines, reactive oxygen species (ROS), lactate dehydrogenase (LDH), cell proliferation, and tight junction-related proteins. Results The results showed elevated expression of S100A9 in diarrhea-affected piglet tissues, and the elevation of S100A9 expression after CPB2 toxin treatment of IPEC-J2 was time-dependent. In CPB2 toxin-induced IPEC-J2 cells, overexpression of S100A9 had the following effects: the relative expression of inflammatory factors IL-6, IL8, TNF-α, and IL-1β was increased; the ROS levels and LDH viability were significantly increased; cell viability and proliferation were inhibited; the G0/G1 phase cell ratio was significantly increased. Furthermore, overexpression of S100A9 reduced the expression of tight junction proteins in CPB2-induced IPEC-J2 cells. The knockdown of S100A9 had an inverse effect. In conclusion, our results confirmed that S100A9 exacerbated inflammatory injury in CPB2 toxin-induced IPEC-J2 cells, inhibited cell viability and cell proliferation, and disrupted the tight junctions between cells. Thus, decreased S100A9 expression alleviates CPB2 toxin-induced inflammatory injury in IPEC-J2 cells.

BackgroundLong non-coding RNAs (lncRNAs), as key regulators, are closely associated with the development of a variety of disease. However, the mechanisms by which lncRNAs regulate Clostridium perfringens type C induced piglet diarrhea are unclear.MethodsIn the present study, we explored the expression and characterization of lncRNAs in a C. perfringens beta2 (CPB2) toxin-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) using RNA-sequencing (RNA-seq).ResultsA total of 6,558 lncRNAs were identified, of which 49 lncRNAs were significantly differentially expressed between the control and CPB2 groups. Functional enrichment analysis showed that the target genes of differentially expressed lncRNA EN-90756 were mainly associated with defense response to virus, and negative regulation of apoptotic process. LncRNA EN-90756 was significantly up-regulated in IPEC-J2 cells at different time points after CPB2 treatment. Functionally, knockdown of lncRNA EN-90756 might regulate the proliferation and apoptosis of IPEC-J2 cells by affecting the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. LncRNA EN-90756 may be involved in CPB2 toxin-induced piglet diarrhea by regulating the expression of its target gene MX1 (encoding MX dynamin like GTPase 1).ConclusionLong non-coding RNA EN-90756 affected the antiviral ability of IPEC-J2 cells by regulating the expression of MX1. Meanwhile, lncRNA EN-90756 might regulate cell proliferation and apoptosis by affecting JAK-STAT signaling pathway activation. These findings provide novel perspectives and directions for further exploration of the regulatory mechanisms of lncRNAs on CPB2 toxin-induced diarrhea in piglets.

Precocious puberty is closely related to testicular development and spermatogenesis, and there is increasing evidence that miRNAs are involved in regulation of testicular development and spermatogenesis. However, little is known about the regulation of microRNAs (miRNAs) during precocious maturation in Hezuo (HZ) boars. In this study, serum Testosterone (T), Estradiol (E2), Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) levels were detected in HZ and Landrace (LC) boars in the postnatal period at 30, 90, 120, 180, and 240 days, and the testes of HZ and LC boars at 30 and 120 days were used for histological observation. In addition, we performed small RNA-Seq to identify miRNA at sexual immaturity (30-days-old) and maturity (120-days-old) of HZ boar testis (using LC boar as control) to reveal the key miRNA in regulation of precocious puberty. Hormone assay results showed that high levels of T, E2, FSH, and LH may be related to precocious sexual maturity of HZ boars, and that FSH may play an important function before sexual maturity. Histological observation showed that HZ boars developed earlier than LC boars and had reached sexual maturity at 120 days. Small RNA-Seq yielded a total of 359 exist miRNAs, 767 known miRNAs and 322 novel miRNAs in 12 samples; 549, 468, 133, and 247 differentially expressed (DE) miRNAs were identified between Ha vs. Hb, La vs. Lb, Ha vs. La, and Hb vs. Lb (log2 fold change >1 and p < 0.05). Enrichment analysis showed that target genes of these DE miRNAs were enriched in many gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways (such as PI3K-Akt, Hippo and Rap1 signaling pathways) were related to testicular development and spermatogenesis. Further screening, some miRNAs (such as ssc-miR-29b, ssc-miR-199b, ssc-miR-383, ssc-miR-149, ssc-miR-615, and ssc-miR-370) were possibly associated with precocious puberty. These results provide new light on miRNA regulatory mechanisms involved in precocious puberty.

Objective microRNAs (miRNAs) can play a role in a variety of physiological and pathological processes, and their role is achieved by regulating the expression of target genes. Our previous high-throughput sequencing found that ssc-miR-185 plays an important regulatory role in piglet diarrhea, but its specific target genes and functions in intestinal porcine epithelial cell (IPEC-J2) are still unclear. We intended to verify the target relationship between porcine miR-185 and cell division cycle 42 (CDC42) gene in IPEC-J2 and to explore the effect of miR-185 on the proliferation of IPEC-J2 cells. Methods The TargetScan, miRDB, and miRanda software were used to predict the target genes of porcine miR-185, and CDC42 was selected as a candidate target gene. The CDC42-3′ UTR-wild type (WT) and CDC42-3′UTR-mutant type (MUT) segments were successfully cloned into pmirGLO luciferase vector, and the luciferase activity was detected after co-transfection with miR-185 mimics and pmirGLO-CDC42-3′UTR. The expression level of CDC42 was analyzed using quantitative polymerase chain reaction and Western blot. The proliferation of IPEC-J2 was detected using cell counting kit-8 (CCK-8), methylthiazolyldiphenyl-tetrazolium bromide (MTT), and 5-ethynyl-2′-deoxyuridine (EdU) assays. Results Double enzyme digestion and sequencing confirmed that CDC42-3′UTR-WT and CDC42-3′UTR-MUT were successfully cloned into pmirGLO luciferase reporter vector, and the luciferase activity was significantly reduced after co-transfection with miR-185 mimics and CDC42-3′UTR-WT. Further we found that the mRNA and protein expression level of CDC42 were down-regulated after transfection with miR-185 mimics, while the opposite trend was observed after transfection with miR-185 inhibitor (p

Clostridium perfringens beta2 (CPB2) toxin, one of the virulence factors of Clostridium perfringens (C. perfringens), can cause necrotizing enterocolitis in piglets. Accumulating pieces of evidence indicate that microRNAs (miRNAs) refer to the regulation of inflammatory processes. Previously, we have discovered that miR-30d was differentially expressed between the ileum of normal piglets and C. perfringens type C-infected diarrheal piglets. Here, we found that miR-30d expression was lowered in CPB2 toxin-treated intestinal porcine epithelial cells (IPEC-J2) at different time points. Subsequently, we determined that miR-30d inhibitor attenuated CPB2 toxin revulsive inflammatory damage in IPEC-J2 cells and promoted cell proliferation and cell cycle progression, whereas miR-30d mimic had opposite results. In addition, we confirmed that Proteasome activator subunit 3 (PSME3) was a downstream target gene of miR-30d via a dual luciferase reporter assay, qPCR, and western blot. We also found that overexpression of PSME3 suppressed CPB2 toxin-induced inflammatory damage and promoted cell proliferation and cycle progression. Our results demonstrate that miR-30d aggravates CPB2 toxin revulsive IPEC-J2 cells inflammatory injury via targeting PSME3, thereby providing a novel perspective for the prevention and treatment of piglet diarrhea at the molecular level.

Background Clostridium perfringens (C. perfringens) type C is the principal pathogenic clostridia of swine, frequently causing hemorrhagic diarrhea, even necrotic enteritis in piglets, leading to severe economic loss for swine industr ies worldwide. However, there are no specific and effective prevention measures. Therefore, clarifying the molecular mechanisms of hosts against pathogenesis infection is very important to reduce the incidence of C. perfringens type C infected piglet diarrhea disease. Methods We performed an TMT labeling-based quantitative spleen proteomic analysis of the control group (SC), tolerance group (SR) and susceptible group (SS) to identify the differentially expressed proteins (DEPs), and screened potential molecular markers of piglet spleen tissues in response to C. perfringens type C infection. Results In this study, a total of 115, 176 and 83 DEPs were identified in SR vs SC, SS vs SC, and SR vs SC, respectively, which may play the important regulatory roles in the process of piglet spleens in response toC. perfringens type C-infected diarrhea diseases. GO enrichment analysis revealed that the DEPs were mostly significantly enriched in acute inflammatory response, defense response, antimicrobial response, transporter activity, cellular metabolic process and so on, and KEGG pathway enrichment analysis showed that the significantly enriched immune related pathways of the PPAR signaling pathway, IL-17 signaling pathway, antigen processing and presentation, which hints at the immune defense process of piglet spleen against C. perfringens infection. This study helps to elucidate the protein expressional pattern of piglet spleen against C. perfringens type C-infected diarrhea disease, which can contribute to the prevention and control for pig diarrhea disease and the further development of diarrhea resistant pig breeding.

Clostridium perfringens type C (Cp) is one of the principal microorganisms responsible for bacterial diarrhea in neonatal and pre-weaning piglets. To better understand the molecular effects of Cp infection, we performed a genome-wide comparison of the changes in DNA methylation and gene expression in Cp infected resistant and susceptible piglets. We characterized the pattern of changes in methylation and found 6485, 5968, and 6472 differentially methylated regions (DMRs) of piglets infected with Cp in IR vs. IC, IS vs. IC, and IS vs. IR groups, respectively. These methylation changes for genes mainly involved in immune and inflammatory responses, cell adhesion, and activation of transcription factors. Gene ontology and KEGG pathway analyses showed that the differentially methylated genes (DMGs) were associated with negative regulation of transcription, apoptotic processes, protein binding, and kinase activity. In addition, they were enriched in immunity-related pathways, such as MAPK signaling pathway, Toll-like receptor signaling pathway, and NF-kappa B signaling pathway. Integrative analysis identified 168, 198, and 7 mRNAs showing inverse correlations between methylation and expression with Cp infection. Altered DNA methylation and expression of various genes suggested their roles and potential functional interactions upon Cp infection, 14 immune-associated mRNAs with differential methylation and transcriptional repression were identified in IS vs. IR, commonly revealing that the differentially expressed genes (DEGs) LBP, TBX21, and LCN2 were likely involved in the piglets against Cp infection. The present results provide further insight into the DNA methylation epigenetic alterations of C. perfringens type C infected piglet ileum tissues, and may advance the identification of biomarkers and drug targets for predicting susceptibility to and controlling C. perfringens type C-induced piglet diarrhea.

Piglet diarrhea caused by Clostridium perfringens (C. perfringens) type C (CpC) seriously endangers the development of the pig production industry. C. perfringens beta2 (CPB2) toxin is a virulent toxin produced by CpC. Long non-coding RNAs (lncRNAs) are key regulators in the immune inflammatory response to bacterial infection. Nevertheless, the functional mechanism of lncRNAs in bacterial piglet diarrhea is unclear. Herein, a novel lncRNA lnc001776 expression was confirmed to be substantially elevated in the ileum tissue of CpC-infected diarrhea piglets and in CPB2 toxin-treated porcine small intestinal epithelial cells (IPEC-J2). lnc001776 knockdown restrained CPB2 toxin-induced apoptosis, inflammatory injury, barrier dysfunction and activation of JNK/NF-kB pathway in IPEC-J2 cells. Additionally, ssc-let-7i-5p was identified as sponge for lnc001776. Overexpression of ssc-let-7i-5p repressed CPB2-induced injury in IPEC-J2 cells. Interleukin 6 (IL-6), a target gene of ssc-let-7i-5p, was enhanced in CPB2 toxin-treated IPEC-J2 cells. Rescue experiments demonstrated that a ssc-let-7i-5p mimic reversed the effect of lnc001776 overexpression on CPB2 toxin-induced IPEC-J2 cell injury and JNK/NF-kB pathway, whereas IL-6 overexpression partially restored the impact of lnc001776. Overall, lnc001776 overexpression exacerbated CPB2 toxin-induced IPEC-J2 cell damage by sponging ssc-let-7i-5p to regulate IL-6 to activate JNK/NF-kB pathway, indicating that lnc001776 could be a key target for piglet resistance to CpC-induced diarrhea.

BackgroundThe n6-methyladenosine (m6A) modification is present widely in mRNAs and long non-coding RNAs (lncRNAs), and is related to the occurrence and development of certain diseases. However, the role of m6A methylation in Clostridium perfringens type C infectious diarrhea remains unclear.MethodsHere, we treated intestinal porcine jejunum epithelial cells (IPEC-J2 cells) with Clostridium perfringens beta2 (CPB2) toxin to construct an in vitro model of Clostridium perfringens type C (C. perfringens type C) infectious diarrhea, and then used methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify the methylation profiles of mRNAs and lncRNAs in IPEC-J2 cells.ResultsWe identified 6,413 peaks, representing 5,825 m6A-modified mRNAs and 433 modified lncRNAs, of which 4,356 m6A modified mRNAs and 221 m6A modified lncRNAs were significantly differential expressed between the control group and CPB2 group. The motif GGACU was enriched significantly in both the control group and the CPB2 group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis showed that the differentially methylated modified mRNAs were mainly enriched in Hippo signaling pathway and Wnt signaling pathway. In addition, the target genes of the differentially m6A modified lncRNAs were related to defense response to virus and immune response. For example, ENSSSCG00000042575, ENSSSCG00000048701 and ENSSSCG00000048785 might regulate the defense response to virus, immune and inflammatory response to resist the harmful effects of viruses on cells.ConclusionIn summary, this study established the m6A transcription profile of mRNAs and lncRNAs in IPEC-J2 cells treated by CPB2 toxin. Further analysis showed that m6A-modified RNAs were related to defense against viruses and immune response after CPB2 toxin treatment of the cells. Threem6A-modified lncRNAs, ENSSSCG00000042575, ENSSSCG00000048785 and ENSSSCG00000048701, were most likely to play a key role in CPB2 toxin-treated IPEC-J2 cells. The results provide a theoretical basis for further research on the role of m6A modification in piglet diarrhea.

Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as “hyper-down” genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

Unlike in many mammals, poultry testes are found in the abdominal cavity where they develop and perform spermatogenesis at high body temperature. Scarce reports among current publications detail the growth of testes and ST morphometry among juvenile chicks. Therefore, this study aims to investigate changes in components occurring in Gallus domesticus testes, by assessing the GSI and morphologically and histologically evaluating the testes and ST morphometry from 1-wk- to 4-mo-old. Right and left testes were collected from 70 healthy chickens divided into seven age-related groups (n = 10) and then immersed into the alcoholic acetate formalin (AAF) fixative solution. Hematoxylin- and eosin-stained tissues were used for microscopic observations. The findings revealed that both testes exhibited smooth features from 1-wk-old to 1-mo-old, and thereafter showed a consistent increase in vascularization until 4-mo-old. Histologically, both testes exhibited unclear ST, with ST apoptotic resorption observed in the 1-wk-old chicks. Until 1-mo-old, ST formed and few spermatogonia differentiated into primary spermatocytes, with all spermatogenic cells observed at 3-mo-old, i.e., sexual maturity. These findings suggest that both testes develop in analogy, and their sizes including increases in length and diameter are related to the spermatogenic activity in the ST. Subsequently, ST resorption by apoptosis is assumed to participate in the physiological mechanism regulating germ cells (GC). Finally, the GSI tended to increase with growth.

N6-methyladenosine (m6A) modification can accommodate mRNA processing, stability, and translation in mammals, and fat mass and obesity associated protein (FTO) is a vital demethylase in the m6A modification pathway. Clostridium perfringens type C (C. perfringens type C) causes diarrhea in piglets and has a serious impact on the pig industry. However, our understanding of the effect of m6A in the process of C. perfringens type C infectious piglet diarrhea (CPTCIPD) is limited. Here, an in vitro model of CPTCIPD was constructed by treating the intestinal porcine epithelial cell line-J2 (IPEC-J2) with Clostridium perfringens beta2 (CPB2) toxin, and the role of FTO was analyzed using quantitative real-time polymerase chain reaction, Western blotting, and flow cytometry. The results revealed that the overall RNA m6A contents at the tissue and cell levels were significantly up-regulated after C. perfringens infection (p < 0.05). FTO expression was significantly reduced in CPB2-treated IPEC-J2 cells. Functionally, FTO knockdown in the treated cells inhibited their proliferation and promoted apoptosis and the inflammation phenotype, whereas FTO overexpression had the opposite effects. Inhibiting FTO prolonged the half-life and up-regulated the expression of Caspase 3, leading to apoptosis. Therefore, this work explored the regulation of FTO in IPEC-J2 cells after CPB2 treatment and enhanced our understanding of the effect of the m6A modification in CPTCIPD.

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 RNAs (circRNAs) are involved in the pathogenic processes of various diseases. However, it is not known if circRNAs 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 circRNA expression. A total of 103 circRNAs were found to be differentially expressed in NE, and Gene Ontology analysis revealed that the genes producing differentially expressed circRNAs 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 circRNAs were involved in the tumor necrosis factor signaling pathway, T cell receptor signaling pathway and nuclear factor‐κB signaling pathway. Finally, we found eight circRNAs (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.

Chinese HZ boars are typical plateau miniature boars characterized by precocious puberty, which is closely related to testicular development and spermatogenesis. Accumulating evidence indicates that lncRNA is involved in the testicular development and regulation of spermatogenesis. However, little is known about the lncRNA precocious regulation in testicular development and spermatogenesis on early sexual maturity of HZ boars. Thus, we investigated the expression and characterization of lncRNA and mRNA in 30-day-old and 120-day-old HZ boar testes using transcriptome to explore precocious puberty. Landrace (LC) boar was treated as the control. Histological analyses indicated that HZ boar underwent puberty development at an earlier stage than LC boar and had achieved sexual maturity at 120 days old. RNA-Seq yielded a total of 187 lncRNAs and 984 mRNAs; these molecules were identified as possible candidates for precocious puberty. GO terms and KEGG pathways enrichment analyses revealed that the differentially expressed lncRNA and their targeted genes were involved in metabolic pathways regulating testis development and spermatogenesis, such as the PI3K-Akt, TGF-beta and Wnt pathways. Further screening, some lncRNA (such as LOC102166140, LOC110259451, and MSTRG.15011.2), and mRNA (such as PDCL2, HSD17B4, SHCBP1L, CYP21A2, and SPATA3) were found to be possibly associated with precocious puberty, which would add to our understanding of the molecular regulatory mechanisms of precocious puberty. This study provided valuable information for further study of the role of lncRNA and mRNA in the process of precocious puberty.

Abstract 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.

Background Clostridium 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. Methods Hence, 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. Results As 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.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in translational regulation of the messenger RNA molecules. Sequence variations in the genes encoding miRNAs could influence their biogenesis and function. MiR-15b plays an important role in cellular proliferation, apoptosis and the cell cycle. Here, we report the identification of a C58T mutation in porcine pre-miR-15b. Through in vitro and in vivo experiments, we determined that this mutation blocks the transition from pri-miRNA to pre-miRNA, alters the strand selection between miR-15b-5p and miR-15b-3p, and obstructs biogenesis of the downstream miR-16-1. These results serve to highlight the importance of miRNA mutations and their impacts on miRNA biogenesis.

C. perfringens type C can induce enteritis accompanied by diarrhea and annually causes significant economic losses to the global pig industry. The pathogenic mechanisms of C. perfringens type C in pigs are still largely unknown. To investigate this, we challenged seven-day-old piglets with C. perfringens type C to cause diarrhea. We performed hematoxylin & eosin (H&E) staining of the small intestine (including duodenum, jejunum, and ileum) and assessed gene expression in the ileal tissue. H&E staining of the duodenum, jejunum, and ileum demonstrated inflammation and edema of the lamina propria and submucosa. A total of 2181 differentially expressed genes (DEGs) were obtained in ileal tissues. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of DEGs indicated that the main pathways were enriched in the T cell receptor signaling pathway, NF-kappa B signaling pathway, and (tumor necrosis factor) TNF signaling pathway. These results provide insights into the pathogenicity of C. perfringens type C and improve our understanding of host−bacteria interactions.