| Avian histomoniasis is a disease in poultry caused by the protozoan parasite Histomonas meleagridis,which mainly causes cecal mucosal lesions,intestinal wall thickening,cheesy cecal cores,and yellow-green round inflammatory necrotic foci in the liver.It severely affects the metabolism and absorption of nutrients and can lead to excessive inflammatory responses and immune dysfunction,ultimately resulting in the death of infected birds and posing a serious threat to ecological farming.Currently,the disease mainly relies on chemical treatment,as there is no effective commercial vaccine available.Although drugs have good preventive and therapeutic effects,most of them are banned due to their potential carcinogenicity.MicroRNAs(miRNAs)are a class of single-stranded,non-coding small molecule RNAs with a length of about 21～24 nt,which can bind to the 3’UTR of target mRNA.They play important regulatory roles in gene transcriptional regulation during normal development and disease occurrence and development.In recent years,with the deepening of research on miRNAs,more and more evidence has shown that the expression levels of host miRNAs change after pathogen infection.These differentially expressed miRNAs participate in a variety of biological processes,such as immune function,autophagy,apoptosis,inflammatory responses,and metabolism,by regulating the expression of target genes to inhibit or promote the development of diseases.Currently,some miRNAs have become potential targets and biomarkers for the diagnosis,prognosis,and treatment of various diseases.Based on this,in this study,using high-throughput sequencing technology,we analyzed and compared the miRNA expression profiles of host liver and cecum tissues related to early and peak stages of H.meleagridis infection in artificially infected chicks as a model of avian histomoniasis.The aim was to lay the foundation for a deeper understanding of the pathogenic mechanism of H.meleagridis from the perspective of host miRNAs.1.Analysis of pathogenicity of H.meleagridis JSYZ-F strain in white leghorn laying hens15-day-old SPF grade white leghorn laying hens with similar weight were divided into three groups of 25 each.Groups A and B were each inoculated with 2×105 of the turkey cecal coccidia JSYZ-F strain via the cloaca,while group C was inoculated with an equal amount of basic culture medium as a negative control.To evaluate the incidence and mortality rates,the chicks in group A were infected with JSYZ-F strain but not killed.On days 5,10,15,20,and 25 after infection,five birds from groups B and C were randomly killed each time for evaluation of weight gain,cecum,and liver lesions.The results showed that the chicks in group A had good mental status and no disease incidence at 5 DPI;some chicks showed symptoms such as listlessness,ruffled feathers,and standing still,with an incidence rate of 32%at 10 DPI;all chicks showed listlessness,standing still,significant decrease in appetite,and sulfur-colored feces,with an incidence rate of 100%at 15 DPI;the mental status of some chicks recovered,and the incidence rate was 48%at 20 DPI;the incidence rate was 0%at 25 DPI.During the experiment,no death occurred in group A,the mortality rate was 0.In group B,5 DPI was no cecum or liver lesion in,and the average weight gain and relative weight gain rate did not differ significantly(P>0.05);at 10 DPI,the average cecum lesion score was 1,the average liver lesion score was 0.2,and the average weight gain and relative weight gain rate differed significantly(P<0.05),with a relative weight gain rate of 84.05%;at 15 DPI,the average cecum lesion score was 2.4,the average liver lesion score was 1.4,and the weight difference was most significant,with the lowest relative weight gain rate of 62.83%.;at 20 DPI and 25 DPI,the average cecum lesion scores were 1.5 and 0.4,the average liver lesion scores were 0.4 and 0,and the relative weight gain rates were 76.11%and 88.69%,respectively.During the experiment,the chicks in group C had good mental status,no clinical symptoms and pathological changes.The results showed that the avian histomoniasis symptoms and pathological changes ared at 10 DPI,were most typical at 15 DPI,and gradually recovered thereafter.2.Analysis of miRNA expression profile in chicken cecum after infection with H.meleagridis15-day-old chicks were infected with H.meleagridis by cloacal inoculation,and cecum tissue samples were collected at 10 and 15 DPI,and from an uninfected control group at the same time points.Total RNA was extracted from the sample and subjected to miRNA sequencing.Ten miRNAs were randomly selected for validation using quantitative real-time PCR.GO and KEGG enrichment analysis were performed on the differentially expressed miRNAs,and miRNAs related to inflammatory responses were screened and analyzed.The results showed that 94 differentially expressed miRNAs(36 up-regulated and 58 down-regulated)were found at 10 DPI,and 127 differentially expressed miRNAs(61 up-regulated and 66 down-regulated)were found at 15 DPI.The expression patterns of the 10 randomly selected miRNAs were consistent with the sequencing results.Among this miRNAs,only differentially expressed at 10 DPI(C1 group,34 miRNAs),target 1393 genes,and 53 GO terms and 3 KEGG signaling pathways were enriched,which include inflammation-related terms and pathway such as apoptosis and negative regulation of cell migration,Rho protein signaling transduction,cell adhesion,and cell apoptosis;only differentially expressed at 15 DPI(C2 group,67 miRNAs),target 1742 genes,and 53 GO terms and 5 KEGG signaling pathways were enriched,which include inflammation-related terms and pathway such as angiogenesis,cytokine-mediated signaling pathways,negative regulation of NFkappaB transcription factor activity,and T cell-mediated immunity,as well as phagosomes,lysosomes,and Salmonella infection;differentially expressed at both 10 DPI and 15 DPI(C3 group,60 miRNAs),target 1030 genes,and 52 GO annotations and 5 KEGG signaling pathways were enriched,which include inflammation-related terms and pathway such as positive regulation of interleukin 2,cell response to interleukin 6,and B cell activation.Functional analysis of the inflammatory miRNAs showed that miRNAs that inhibited inflammation were up-regulated in the C1 group,while those that promoted inflammation were down-regulated.In contrast,miRNAs that promoted inflammation were up-regulated in the C2 group,while those that inhibited inflammation were down-regulated,and the expression patterns of inflammation-related miRNAs were consistent with the inflammatory process in the cecum of turkeys infected with H.meleagridis.These findings lay a foundation for a deeper understanding of the pathogenesis of avian histomoniasis in chicks from the perspective of host miRNAs.3.Analysis of miRNA expression profile in chicken liver after infection with H.meleagridis15-day-old chicks were infected with H.meleagridis by cloacal inoculation,and liver tissue samples were collected at 10 and 15 DPI,and from an uninfected control chicks at the same time points.Total RNA was extracted from the sample and subjected to miRNA sequencing.Eight miRNAs were randomly selected for validation using quantitative real-time PCR.GO and KEGG enrichment analysis were performed on the differentially expressed miRNAs,and miRNAs related to inflammatory responses were screened and analyzed.The results showed that 120 differentially expressed miRNAs(66 upregulated and 54 downregulated)were found at 10 DPI,while 118 differentially expressed miRNAs(66 upregulated and 52 downregulated)were found at 15 DPI.The expression patterns of the randomly selected eight miRNAs were consistent with the sequencing results.Among this miRNAs,only differentially expressed at 10 DPI(L1 class,45 miRNAs),target 1646 genes,and 89 GO terms and 3 KEGG signaling pathways were enriched,which include inflammation-related terms and pathway such as negative regulation of the Wnt signaling pathway,endothelial cell proliferation;only differentially expressed at 15 DPI(L2 class,43 miRNAs),target 2257 genes,and 87 GO terms and 4 KEGG signaling pathways were enriched,which include inflammation-related terms and pathway such as response of cells to tumor necrosis factor and gamma interferon,activation of MAPK activity,positive regulation of T-cellmediated immunity,differentiation of cytotoxic T cells,and positive regulation of STAT protein tyrosine phosphorylation;differentially expressed at both 10 and 15 DPI(L3 class,75 miRNAs),target 1623 genes,and 41 GO terms and 5 KEGG signaling pathways were enriched,which include inflammation-related terms and pathway such as Wnt signaling pathway,integrin-mediated signaling pathway,cell chemotaxis,and T-cell-mediated immunity.The functional analysis of inflammatory miRNAs showed that the expression pattern of L1 class differentially expressed miRNAs favored the promotion of balanced inflammatory responses,while the miRNAs in L2 class,which had the ability to inhibit immune inflammatory responses,were downregulated,possibly related to a more severe inflammatory phenotype at 15 DPI.The expression pattern of inflammatory-related miRNAs was consistent with the liver inflammatory process after artificial infection.These results provide a basis for in-depth analysis of the pathogenic mechanism of H.meleagridis from the perspective of host miRNA.4.Screening of key host miRNAs and validation of their target genes during H.meleagridis infection in chicksTo investigate the key host miRNAs during H.meleagridis infection in chicks,this study performed integrated analysis of miRNA expression profiles in the cecum and liver tissues at 10 DPI and 15 DPI.This study identified miRNAs that were differentially expressed in both cecum and liver tissues after infection and predicted target genes using miRanda,RNAhybrid,MIRDB,and TargetScan.This chapter also used TargetScan to predict the interaction patterns between gga-miR-155 and RNF123.Subsequently,we used qPCR to measure the expression levels of gga-miR-155 and its candidate target genes in the cecum and liver tissues.Our results showed that 25 miRNAs were differentially expressed both in the cecum and liver tissues at different time points after infection.RNF123 was predicted to be a target gene of gga-miR-155,and the seed sequence of gga-miR-155 was found to be completely complementary to the 5’ UTR of RNF123.The qPCR results showed that the expression patterns of ggamiR-155 and RNF123 were opposite in the cecum and liver tissues at 10 and 15 DPI.These findings suggest a potential targeting relationship between gga-miR-155 and RNF123,and indicate that they play a crucial role in the host inflammatory response induced by H.meleagridis infection in chicks.Our study provides a basis for further investigation of the mechanisms underlying the host inflammatory response to H.meleagridis infection in chicks.In summary,our study not only elucidated the progression of H.meleagridis JSYZ-F strain infection in chicks,but also analyzed the miRNA expression profiles in the cecum and liver tissues of infected chicks using high-throughput sequencing technology.This study preliminarily revealed the host inflammatory response mechanism caused by differentially expressed miRNAs at different time points,laying a foundation for further investigation of the interaction mechanism between H.meleagridis and chicks. |
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