Channel: ANIMAL SCIENCE·VETERINARY SCIENCE https://www.chinaagrisci.com EN-US https://www.chinaagrisci.com/EN/0578-1752/current.shtml https://www.chinaagrisci.com 0578-1752 https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2023.12.013 【Objective】Heat stress has seriously impaired the production and health of dairy cows, causing the subsequent limitation in sustainable development of dairy industry. DNA methylation is an important epigenetic regulatory mechanism involved in an animal’s heat stress response, but the potential functions and molecular mechanisms of which are not clear. The current study was conducted to detect the DNA methylation related to heat stress in dairy cows and to identify target genes related to DNA methylation, so as to provide a better insight into the epigenetics mechanism of heat stress in dairy cows.【Method】In the study, 24 Chinese Holstein lactation cows (same lactation stage and same parity) in Sanyuan dairy farm were used for the blood samples collection in heat stress period (July in the summer of 2017) and non-heat stress period (April in spring 2017), respectively, followed by DNA extraction. To explore the DNA methylation differences in dairy cows from different heat stress period, 15 of 24 animals were randomly assigned to 3 groups (N=5 animals/group), 5 DNA samples in one group were mixed together to get a single pooled DNA sample, thus 6 pooled DNA samples including 3 from spring and 3 from summer were used for the DNA methylation detection by the whole-genome bisulfite sequencing (WGBS), then the differential methylation region (DMR; 1000 bp windows, 500 bp overlap, P<0.05) and key gene were identified. PROMO and Methprimer software were used to predict transcription factor binding sites and CpG islands, respectively. Then, the bovine mammary gland epithelial cells (Mac-T) were treated at 39 ℃ for 24 h, 48 h, and 72 h, and the cell viability were detected by MTT method. Finally, using the bisulfite sequencing PCR (BSP), the methylation levels of target gene promoter in 24 dairy cows in spring and summer and Mac-T cells treated in 39 ℃ were examined, respectively. 【Result】Based on the DNA methylation analysis of WGBS data, 49 861 differential methylation regions (DMRs) associated with heat stress were identified. One of DMRs was attributed to the promoter area of GNAS complex locus (GNAS), whose methylation level significantly increased in heat-stressed animals (P<0.001). Also, there was a 352 bp CpG island in the promoter of GNAS containing potential binding sites for Sp1, C/EBP and other important transcription factors. Further the methylation status of the GNAS gene promoter region in heat stressed dairy cows were verified by BSP, and the average methylation level in all cytosine of 31 CpG sites was higher in heat stress cows than that in control groups (P<0.05), which corresponding to the above WGBS results. Moreover, the 21 (-113 bp, Chr13:57532733) and 27 (-63 bp, Chr13:57532683) CpG sites showed significant differences between the spring and summer groups (P<0.05). In Mac-T cells, after 48 h and 72 h heat treatment, the cell viability decreased significantly (P<0.01), but the overall CG methylation level of 31 CpG sites in the GNAS gene promoter region increased significantly (P<0.05), and also the similar significant methylation changes appeared in the site 21 and 27 CpG in cell. 【Conclusion】 Heat stress increased the methylation levels of the promoter region of the GNAS in dairy cows as well as in cells, which indicated that GNAS was a potential target gene regulated DNA methylation in heat stress response of dairy cows.

]]> https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2023.12.014 【Objective】 To improve the efficiency of the reprogramming process of Bactrian camel fibroblasts and to reduce the risk of tumorigenesis caused by the introduction of proto-oncogenes. In this experiment, valproic acid (VPA) was added to the fibroblast reprogramming process to explore the effect of small molecules on the reprogramming of Bactrian camel fibroblasts. 【Method】 Given this, March-aged Bactrian camel fetal fibroblasts were used as test materials, combined with classic induction combination OSKM (Oct4, Sox2, Klf4, and c-Myc) and EGFP five retrovirus reprogramming of Bactrian camel fibroblasts (OSKM group), and the cells by adding VPA treatment for 7 days after the second viral infection (OSKM+VPA group) were collected. Endogenous and exogenous genes were examined by using PCR to confirm the modification effect of retrovirus on Bactrian camel fibroblasts. Eight genes were randomly selected from those more significantly affected by VPA. According to RNA-seq data, whether their trends before and after VPA addition were consistent with the trends of RNA-seq data was checked to verify the accuracy of RNA-seq data. The transcriptome sample genes were classified by GO analysis and significant enrichment pathways for target genes were clarified by using KEGG pathway enrichment analysis and hypergeometric validation analysis. Total RNA was extracted from the collected cells, and then, combined with RNA-seq and Real time-quantitative interpretation (RT-qPCR) techniques to detect the effect of VPA on the reprogramming of Bactrian camel fibroblasts. 【Result】 It was detected by using PCR that the expression of endogenous and exogenous genes in different groups. The results showed that nSox2, Sox2, Oct4, Klf4, and c-Myc genes were expressed in both OSKM and OSKM+VPA groups, and the expression in OSKM+VPA group was higher than that in the OSKM group, while they were not expressed in BCEFs group. Eight genes were randomly selected for testing, and the results showed that: three genes of TP53, CCNB1, and CCD20 were down-regulated in expression after the addition of VPA, which were related to the cell cycle signaling pathway. S100A4, CKS2, VIM, and MMP9 genes signaling were down-regulated in expression, which was related to the phenotypic characteristics of cancer; VEGFC gene expression was up-regulated in the PI3k-Akt signaling pathway. This expression trend was consistent with the trend of the histological data. The results showed that the expression of proliferation genes Mki67 and PCNA were down-regulated, while the expression of apoptosis gene CASP7 was up-regulated after the addition of VPA. KEGG and hypergeometric validation analyses of the transcriptome data were performed, and 959 differentially expressed genes were screened according to the analysis results, which were enriched in 276 signaling pathways, including eight signaling pathways with Q values less than 0.05: steroid biosynthesis, cell cycle, PPAR signaling pathway, progesterone-mediated oocyte maturation, fatty acid metabolism, ECM-receptor interactions, cell adhesion molecules, and cholesterol metabolism. The 26 differentially expressed genes related to cell cycle, fatty acid metabolism, cell adhesion molecule, and cholesterol metabolism were screened, and four of which were randomly selected for testing, showing that VPA upregulated the expression of L1CAM, CNTN1 and NFASC genes in the Bactrian camel fibroblast adhesion molecule signalling pathway and enhanced intercellular interactions. It was also upregulated that the expression of CD36 gene in the fatty acid signaling pathway. 【Conclusion】 The results showed that the VPA blocked cell before the split phase to reduce risk differentiation during the process of reprogramming. Meanwhile, VPA affected several signaling pathways in the reprogramming process of Bactrian camel fibroblasts, and regulated the expression trend of related genes in the signaling pathways, which effectively improved the reprogramming efficiency of the cells and played an important role in the reprogramming of Bactrian camel fibroblasts.

]]> https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2023.12.015 【Objective】The aim for this study was to identify the predominant antigen of Salmonella and to develop a sensitive, specific and universal iELISA assay method for the rapid and accurate detection of Salmonella antibodies in equidae.【Method】For the purpose of screening out dominant antigens for Salmonella Abortusequi, the immunoprecipitation (pull down) tests were performed using Salmonella Abortusequi positive/negative sera with whole bacterium antigens of Salmonella Abortusequi. Then, amino acid sequence alignment of the dominant antigen were compared with Salmonella Typhimurium, Salmonella Dublin, and Salmonella Enteritidis to verify itsconservative. Three pairs of specific primers were designed and synthesized according to the nucleotide sequence of the full ompA gene published in GenBank. Three ompA genes with different lengths were amplified by PCR, and then cloned into pET28a vector and transformed Escherichia coli Rosetta (DE3) competent cell. The expressed products were analyzed by SDS-PAGE electrophoresis and Western-blot test after induced by IPTG. The reactivity of the purified protein was verified using S. Abortusequi, Salmonella Typhimurium, Salmonella Dublin (S. Dublin), and S. Enteritidis serums, and one negative serum by Western blot. An indirect ELISA method for the diagnosis of equine abortion salmonellosis was developed by optimizing the amount of coating antigen, serum and secondary antibody concentrations using the purified ompA3 protein as the coating antigen, and evaluating the specificity and sensitivity of the iELISA, and finally applying the iELISA to detection of clinical samples.【Result】In this study, the ompA dominant antigen of S.Abortusequi was screened. S.Abortusequi ompA was conservative and showed 99.4%-100% identical with Salmonella Typhimurium, Salmonella Dublin and S.Enteritidis strains at the amino acid level. Three target genes were successfully obtained by PCR amplification. Three recombinant plasmids, including pET28a-ompA1, pET28a-ompA2, and pET28a-ompA3 were successfully constructed. The expressed products were analyzed by SDS-PAGE electrophoresis and Western-blot test after induced by addition of IPTG to a final concentration of 0.6 mmol·L^-1 for 5 h at 24 ℃. The recombinant ompA1 and ompA2 were obtained in an inclusion and soluble recombinant ompA3 proteins. The soluble recombinant ompA3 were identified by Western blot, which had a specific reaction with four Salmonella positive serums, therefore, the ompA3 was considered as a potential target candidate for serological detection of Salmonella. An iELISA method was developed in a maximum P/N ratio using the coating antigen at a concentration of 1 μg·mL^-1, a serum dilution of 1﹕200 and secondary antibody was 1﹕10000. The cutoff value was 0.143, and an OD₄₅₀ value over 0.143 was considered as positive. The specificity test showed that the coated antigen did not cross-react with the positive serum of common equine infectious diseases. The iELISA provided better sensitivity by detecting antibodies in intravenously infected horses, as the iELISA could continue to monitor antibody positivity up to 116 days, 47 days longer than microagglutination test (69 days). The established iELISA method was used to detect antibodies in 180 serum samples from 8 different farms. The average positive rate of iELISA antibody was 63.3%, which was 53.9% higher than that of micro agglutination test.【Conclusion】The soluble ompA3 protein was successfully expressed, and a universal indirect ELISA antibody method was established for the diagnosis of equine abortus salmonellosis. The method enables detection of antibodies to Salmonella Abortusequi in clinical samples.The method has good specificity and sensitivity and could be a promising candidate tools for use in the monitoring of the equine abortus salmonellosis epidemic.

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