Combined Transcriptomic and Proteomic of Corynebacterium pseudotuberculosis Infection in the Spleen of Dairy Goats.

Corynebacterium pseudotuberculosis (C. pseudotuberculosis) is a zoonotic chronic infectious disease. It mainly occurs in dairy goats reared in herds, and once it invades the dairy goats, it is difficult to completely remove it, causing great harm to the development of the sheep industry. This study mainly was based on TMT-based quantitative proteomics and RNA-seq methods to measure the spleen samples of infected dairy goats at different time periods. Nine four-month-old dairy goats were divided into three groups, with three goats in each group. The dairy goats in the first group (NC group) were inoculated with 1.0 mL of sterilized normal saline subcutaneously, and the second (72 h group) and third groups (144 h group) were inoculated with 1.0 mL of 1 × 107 cfu/mL bacterial solution subcutaneously in the neck. Significant changes in the protein and mRNA expression were observed in different infection and control groups. In the 72 h group, 85 genes with differential genes and proteins were up-regulated and 91 genes were down-regulated in this study. In the 144 h group, 38 genes with differential genes and proteins were up-regulated and 51 genes were down-regulated. It was found that 21 differentially expressed genes and proteins were co-up-regulated in the two groups. There were 20 differentially expressed genes and proteins which were co-down-regulated in both groups. The 72 h group were mainly enriched in protein processing in the endoplasmic reticulum, lysosome, amino sugar and nucleotide sugar metabolism and the estrogen signaling pathway. In the 144 h group, they were protein processing in the endoplasmic reticulum pathway which was enriched by mRNA-proteins pairs co-upregulated by the five pairs. The combined transcriptomic and proteomic analyses were performed to provide insights into the effects of C. pseudotuberculosis through several regulatory features and pathways. We found that in the early stage of infection (72 h), the co-upregulated gene-protein pairs were enriched in multiple pathways, which jointly defended against a bacterial invasion. However, in the later stages of infection (144 h), when the disease stabilizes, a few co-upregulated gene-protein pairs played a role in protein processing in the endoplasmic reticulum pathway. In addition, the mRNA and protein expressions of dairy goats infected with the bacteria at different periods of time indicated the adaptability of dairy goats to the bacteria. At the same time, it guides us to carry out a corresponding treatment and feeding management for dairy goats according to different periods of time.

Molecular Characterization of Anaplasma spp. among Dairy, Cashmere, and Meat Goats in Shaanxi Province, Northwestern China.

Anaplasma spp. are important tick-borne pathogens endangering the health of humans and various animals. Although several studies have reported Anaplasma infection in livestock in China, little is known about the impact of production categories on the occurrence of Anaplasma species. In the present study, PCR tools targeting the 16S rRNA and msp4 genes were applied to investigate the prevalence of Anaplasma spp. in 509 blood samples of dairy (n = 249), cashmere (n = 139), and meat (n = 121) goats from Shaanxi province. The prevalence of Anaplasma spp. was 58.5% (298/509) in goats, and significant differences (p < 0.001) were identified in the prevalence among production categories, with the highest in meat goats (84.3%, 102/121), followed by cashmere goats (58.3%, 81/139) and dairy goats (46.2%, 115/249). Significant differences (p < 0.001) in prevalence were also found among sampling sites and age groups. Meanwhile, the prevalence was 36.9% (188/509) for A. phagocytophilum, 36.1% (184/509) for A. bovis, and 11.0% (56/509) for A. ovis, and significant differences (p < 0.001) in prevalence of A. phagocytophilum, A. bovis and A. ovis were recognized among production categories and sampling sites. A. phagocytophilum, A. bovis and A. ovis were dominant species in meat, dairy, and cashmere goats, respectively, and A. ovis was absent in meat goats. Co-infections were found in 124 (24.4%) investigated samples. Goats aged < 2, 3−6, and 7−12 months, and goats from Qingjian and Zhenba were risk factors associated with the occurrence of Anaplasma. Phylogenetic analysis indicated separate clades for the distribution of A. phagocytophilum from different ruminant, reflecting potential host adaption within this species. This study reported the colonization occurrence of Anaplasma spp. among production categories in goats in Shaanxi province and enriched our knowledge on the transmission of Anaplasma spp. in goats in China. Considering the existence of zoonotic A. phagocytophilum in goats in this study and previous reports, interventions based on One Health are needed to be developed to control the transmission of Anaplasma spp. between humans and animals.

Study on the SHP2-Mediated Mechanism of Promoting Spermatogenesis Induced by Active Compounds of Eucommiae Folium in Mice.

Spermatogenesis directly determines the reproductive capacity of male animals. With the development of society, the increasing pressure on people's lives and changes in the living environment, male fertility is declining. The leaf of Eucommia ulmoides Oliv. (Eucommiae Folium, EF) was recorded in the 2020 Chinese Pharmacopoeia and was used in traditional Chinese medicine as a tonic. In recent years, EF has been reported to improve spermatogenesis, but the mechanisms of EF remain was poorly characterized. In this study, the effect of EF ethanol extract (EFEE) on spermatogenesis was tested in mice. Chemical components related to spermatogenesis in EF were predicted by network pharmacology. The biological activity of the predicted chemical components was measured by the proliferation of C18-4 spermatogonial stem cells (SSCs) and the testosterone secretion of TM3 leydig cells. The biological activity of chlorogenic acid (CGA), the active compound in EF, was tested in vivo. The cell cycle was analysed by flow cytometry. Testosterone secretion was detected by ELISA. RNA interference (RNAi) was used to detect the effect of key genes on cell biological activity. Western blotting, qRT-PCR and immunofluorescence staining were used to analyse the molecular mechanism of related biological activities. The results showed that EFEE and CGA could improve spermatogenesis in mice. Furthermore, the main mechanism was that CGA promoted SSC proliferation, self-renewal and Leydig cell testosterone secretion by promoting the expression of SHP2 and activating the downstream signaling pathways involved in these biological processes. This study provided strong evidence for elucidating the mechanism by which EF promotes the spermatogenesis in mice and a new theoretical basis for dealing with the decrease in male reproductive capacity.

Genetic Features of Plasmid- and Chromosome-Mediated mcr-1 in Escherichia coli Isolates From Animal Organs With Lesions.

The genomic context of the mcr-1 gene in Escherichia coli from animal feces has been widely reported. However, less is known about the mcr-1-carrying plasmid characteristics and other functional regions of Escherichia coli isolates from animal organs with lesions. The present study investigated the antimicrobial resistance, population structure, and genetic features of mcr-1-positive Escherichia coli strains isolated from animal organs with lesions. The antimicrobial susceptibility testing indicated that 24 mcr-1-positive Escherichia coli isolates were resistant to at least three or all antimicrobial categories. MLST analysis suggested that the dominant clone complexes (CC) were mainly CC156, CC448, and CC10. In addition, ST10596, a newly discovered sequence type in swine, failed to be classified. Meanwhile, the mcr-1 gene located on the different plasmids was successfully transferred to the recipients, and whole-genome sequencing indicated the mcr-1 gene was embedded in mcr-1-pap2 cassette but not flanked by ISApl1. The mcr-1 gene is located on the chromosome and embedded in Tn6330. Furthermore, NDM-5 was found on the IncX3-type plasmid of J-8. The virB6 and traI gene of type IV secretion system (T4SS) were truncated by IS2 and IS100 and located on the IncX4- and the IncHI2/HI2A/N-type plasmids, respectively. The multidrug-resistant (MDR) region of IncHI2/HI2A/N-type plasmids contained two class 1 integrons (In0, In640) and four composite transposons (Tn4352, Tn6010, cn_4692_IS26, cn_6354_IS26). Overall, 24 mcr-1-positive Escherichia coli isolates in our study showed MDR, or even extensively drug resistant (XDR), and exhibited population diversity. The T4SS gene truncation by the insertion sequence may affect the efficiency of plasmid conjugative transfer. Furthermore, the class 1 integrons and composite transposons in the MDR region of IncHI2/HI2A/n-type plasmid contributed to the multireplicon plasmid formation, the acquisition, and transfer of antimicrobial resistance genes (ARGs).

Spleen proteome profiling of dairy goats infected with C. pseudotuberculosis by TMT-based quantitative proteomics approach.

Corynebacterium pseudotuberculosis (C.pseudotuberculosis) is a zoonotic pathogen that can cause cheese lymphadenitis in goats. In order to obtain detailed information about the pathogenesis and host immune response of goats infected with C.pseudotuberculosis, we used tandem mass tag (TMT)-labeling proteomic analysis to detect differentially expressed proteins (DEPs) in dairy goats infected with C.pseudotuberculosis, and confirmed the altered proteins with western blot. A total of 6611 trusted proteins were identified, and 126 proteins were differentially abundant. Gene ontology (GO) analysis showed that all DEPs were annotated as biological processes, cell composition, and molecular functions. Biological processes mainly involve acute inflammation and immune response; cell components mainly involve extracellular areas and high-density lipoprotein particles; molecular functions are mainly antigen binding, ferric iron binding, and iron ion binding. KEGG analysis showed that a total of 102 pathways were significantly enriched, mainly lysosomes, phagosomes, and mineral absorption pathways. Our findings provided the relevant knowledge of spleen protein levels in goats infected with C.pseudotuberculosis and revealed the complex molecular pathways and immune response mechanisms in the process of C.pseudotuberculosis infection. SIGNIFICANCE: C.pseudotuberculosis is the most fatal infectious disease in dairy goats, causing huge economic losses. However, the molecular pathways and immune response mechanisms of C.pseudotuberculosis infection in goats remain unclear. Therefore, we conducted a comparative quantitative proteomics study on dairy goats infected with C.pseudotuberculosis. The results provide a basis for better understanding the complexity of C.pseudotuberculosis infection, reveal the complex molecular pathways and immune response mechanisms in C.pseudotuberculosis infection, and provide some clues for identifying potential therapeutic targets. This is the first report to show that C.pseudotuberculosis infection in dairy goats can disrupt the immune response mechanism and lead to massive immune cell death. The study provided new findings on the interaction between C.pseudotuberculosis and the host.

Bioactivity-guided isolation of immunomodulatory compounds from the fruits of Ligustrum lucidum.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruits of Ligustrum lucidum (FLL) W.T. Aiton (Oleaceae) is included in the 2020 "Chinese Pharmacopoeia" and is widely used in traditional Chinese medicine as a tonic. In recent years, FLL has been reported to improve immune function, but the bioactive compounds and mechanisms of FLL remain poorly characterized. AIM OF THE STUDY: To identify FFL compounds with strong immune activity and explore their molecular mechanisms. MATERIALS AND METHODS: The phagocytic activity of RAW264.7 macrophages and proliferation activity of spleen lymphocytes were used to guide the isolation of bioactive compounds from FLL extracts. Lymphocyte subpopulations, Ca2+ concentrations, and surface molecule expression were analyzed using flow cytometry. Cytokine secretion was examined using ELISA. FITC-OVA uptake was observed using fluorescence microscopy. NF-κB activation was analyzed using western blotting. RESULTS: The extraction and isolation produced ten compounds, namely oleuropeinic acid, nuezhenide, isonuezhenide, salidroside, isoligustrosidic acid, ligulucidumosides A, 8(E)-nuezhenide, hydroxytyrosol, oleuropein, and p-hydroxyphenethyl 7-ß-D-glucosideelenolic acid ester were isolated and identified from FLL-Bu-30%. Immunoactivity experiments showed that hydroxytyrosol had the strongest macrophage phagocytotic and lymphocyte proliferation-promoting activities. Further studies showed that hydroxytyrosol could significantly enhance lymphocyte subsets CD3+, CD4+/CD8+, and CD3+CD4-CD8-, promote IL-4, IFN-γ, and TNF-α secretion, and increase intracellular Ca2+ concentrations. In addition, the results from RAW264.7 macrophages showed that hydroxytyrosol increased FITC-OVA uptake, induced TNF-α and IL-1ß production, upregulated MHC-II, CD80, and CD86 expression, promoted cytoplasmic IκB-α degradation, and increased nuclear NF-κB p65 levels. CONCLUSION: Our study provides substantial evidence regarding the mechanism of the immunomodulatory effects of compounds from FLL.

Transcriptome analysis of Corynebacterium pseudotuberculosis-infected spleen of dairy goats.

Caseous lymphadenitis is a chronic disease of goats caused by Corynebacterium pseudotuberculosis (C.pseudotuberculosis) which causes great harm to the dairy goats industry. In order to obtain detailed information about the pathogenesis and host immune response in C.pseudotuberculosis-infected goats, in this study, the gene expression difference of spleen tissue after infection with C.pseudotuberculosis was analyzed by high-throughput sequencing. Transcripts obtained over 412 700 462 clean reads after reassembly were 21 343 genes detected, of which 14 720 were known genes and 7623 new genes were predicted. There were 448 up-regulated and 519 down-regulated differentially expressed genes (DEGs). Gene Ontology (GO) analysis indicated that all of the DEGs were annotated into biological process, cellular component and molecular function. Most of these unigenes are annotated in cellular processes, the cell and binding. KEGG analysis of the DEGs showed that a total of 8733 DEGs unigenes were annotated into 459 pathways classified into 6 main categories. Most of these annotated unigenes were related to immune system response to the infectious diseases pathways. In addition, 14 DEGs were verified by quantitative real-time PCR. As the first, in vivo, RNAseq analysis of dairy goats and C.pseudotuberculosis infection, this study provides knowledge about the transcriptomics of spleen in C.pseudotuberculosis-infected goats, from which a complex molecular pathways and immune response mechanism are involved in C.pseudotuberculosis infection.

Development of a multiplex TaqMan qPCR assay for simultaneous detection and differentiation of four DNA and RNA viruses from clinical samples of sheep and goats.

Mixed infections with different pathogens are common in sheep and goats under intensive production conditions. Quick and accurate detection and differentiation of different pathogens is necessary for epidemiological surveillance, disease management and import and export controls. Multiplex TaqMan qPCR protocols were developed and subsequently evaluated as effective tools in simultaneously detecting single and mixed infections in sheep and goats. Four pairs of primers and four probes labeled with Rox/BHQ2, Cy5/BHQ2, Hex/BHQ1 and Fam/BHQ1 for peste des petits ruminants virus (PPRV), foot and mouth disease virus (FMDV), goat pox virus (GTPV) and orf virus (ORFV), respectively, were used in the multiplex TaqMan qPCR assay. The assay was shown to be sensitive with detection limits of 9.17 × 101, 1.69 × 102, 9.41 × 101 and 7.46 × 101 copies/µL for PPRV, FMDV, GTPV and ORFV from a mixture of four viruses in a reaction, respectively. The assay was highly specific in its ability to detect one or more viruses in various combinations in the specimens. 38 clinical samples collected from sheep and goats were detected among 43 samples tested by multiplex TaqMan qPCR, showing highly effective identification. Overall, the multiplex TaqMan qPCR panel provides a fast, specific, and sensitive diagnostic tool for the accurate detection of multiple viral pathogens in sheep and goats.

Full-length genome sequence analysis of enzootic nasal tumor virus isolated from goats in China.

BACKGROUND: Enzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in China. METHODS: To evaluate the genetic diversity of ENTV-2 from Shaanxi province of China, the complete genome sequence of four isolates from Shaanxi province was determined by RT-PCR. These sequences were analyzed to evaluate their genetic relatedness with other small ruminant betaretroviruses. Phylogenetic analyses based on the gag gene and env gene were performed. RESULTS: The ENTV-2-Shaanxi1 genome shared 97.0% sequence identity with ENTV-2-SC (accession number HM104174.1), and 89.6% sequence identity with the ENTV-2 sequences (accession number AY197548.1). ENTV-2 is closely related to the ENTV-1 and jaagsiekte retrovirus (JSRV). The main sequence differences between these viruses reside in LTR, two small regions of Gag, Orf-x, and the transmembrane (TM) region of Env. A stretch of 6 consecutive proline residues exists in VR1 of the ENTV-2-Shaanxi1 ~ 4 isolates. All the ENTV-2-Shaanxi isolates have the YXXM motif in the cytoplasmic tail of the Env. Phylogenetic analysis by nucleotide sequences showed that ENTV-2-Shaanxi1 ~ 4 isolates were closest related to two ENTV-2 isolates published in NCBI, especially with ENTV-2-SC strain. CONCLUSIONS: This finding indicates that ENA most likely was introduced to Shaanxi province by the movement of contaminated goats from other areas in China. This study adds to understand the circulation, variation and distribution of ENTV-2, and may prove beneficial in future control or eradication programmes.

Development of multiplex PCR for simultaneous detection and differentiation of six DNA and RNA viruses from clinical samples of sheep and goats.

Multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and subsequently evaluated for its effectiveness in detecting simultaneously single and mixed infections in sheep and goats. Specific primers for three DNA viruses and three RNA viruses, including foot and mouth disease virus (FMDV), Bluetongue virus (BTV), peste des petits ruminants virus (PPRV), sheeppox virus (SPPV), goatpox virus (GTPV) and orf virus (ORFV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive because it could detect at least 100pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty seven clinical samples collected from sheep and goats were detected among forty three samples tested by both uniplex and multiplex PCR, showing highly identification. As results of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in sheep and goats with a reaction.

Characterization of the Transcriptional Complexity of the Receptive and Pre-receptive Endometria of Dairy Goats.

Endometrium receptivity is essential for successful embryo implantation in mammals. However, the lack of genetic information remains an obstacle to understanding the mechanisms underlying the development of a receptive endometrium from the pre-receptive phase in dairy goats. In this study, more than 4 billion high-quality reads were generated and de novo assembled into 102,441 unigenes; these unigenes were annotated using published databases. A total of 3,255 unigenes that were differentially expressed (DEGs) between the PE and RE were discovered in this study (P-values < 0.05). In addition, 76,729-77,102 putative SNPs and 12,837 SSRs were discovered in this study. Bioinformatics analysis of the DEGs revealed a number of biological processes and pathways that are potentially involved in the establishment of the RE, notably including the GO terms proteolysis, apoptosis, and cell adhesion and the KEGG pathways Cell cycle and extracellular matrix (ECM)-receptor interaction. We speculated that ADCY8, VCAN, SPOCK1, THBS1, and THBS2 may play important roles in the development of endometrial receptivity. The de novo assembly provided a good starting point and will serve as a valuable resource for further investigations into endometrium receptivity in dairy goats and future studies on the genomes of goats and other related mammals.

Identification and profiling of microRNAs in goat endometrium during embryo implantation.

BACKGROUND: MicroRNAs (miRNAs) are short, highly conserved small noncoding RNAs that had fundamental roles in post-transcriptional gene expression, and they are crucial for proper control of biological processes and known to participate in embryo implantation. However, miRNA expression profiles in the pre-receptive and receptive phases of the goat endometrium during embryo implantation are unknown. RESULTS: A total of 1,069 and 847 miRNAs were expressed in receptive (R) and pre-receptive (P) goat endometrium, and 632 miRNAs were co-expressed in both phases. We identified 545 (50.98%) known miRNAs in the R library and 522 (61.63%) in the P library. There were 110 up-expressed miRNAs and 33 down-expressed miRNAs in receptive endometrium compared with the pre-receptive endometrium meeting the criteria of P-values< 0.05. Moreover, GO and KEGG analysis of the target genes of the differentially expressed miRNAs revealed some candidate miRNAs, genes and pathways that may involve in the formation of the receptive endometrium. Based on stem-loop RT-qPCR, 15 miRNAs were detected and the results suggested that the majority of the miRNA expression data measured by Solexa deep sequencing could represent actual miRNA expression levels. CONCLUSIONS: Our data revealed the first miRNA profile related to the biology of the goat receptive endometrium during embryo implantation, and the results suggested that a subset of miRNAs might play important roles in the formation of endometrial receptivity. Thus, elucidating the physiological roles of endometrial miRNAs will help us better understand the genetic control of embryo implantation in goats.

Natrium fluoride influences methylation modifications and induces apoptosis in mouse early embryos.

This study epigenetically examined the effect of fluoride on early embryos of Kunming mice administered with 0, 20 (low), 60 (medium), and 120 mg/L (high) sodium fluoride (NaF). The results showed that NaF repressed oocyte maturation, fertilization and blastocyst formation in all NaF-treated groups. Meanwhile, TUNEL assay showed that embryo apoptosis was induced dramatically in blastocyst stage at either low or medium doses, and in 8-cell stage at high dose, compared to the control, suggesting a dose-dependent effect. Furthermore, the immunostaining displayed global increases of DNA methylation, H3K9m2 and H3K4m2 with increasing dose, which were consistent with gene expression results, exhibiting general increases of DNMT1, DNMT3a, G9a, LSD1, and MLL1 and a reduction of JHDM2a in transcription and protein levels. More closely, the differential methylation domain in parentally imprinted gene H19 showed low methylation, while materanlly imprinted gene IGF2 showed high methylaiton in NaF-treated groups compared to the control group, which corresponded with high expression of H19 and low expression of IGF2 confirmed by qPCR. Collectively, we demonstrated that fluoride epigenetically impaired mouse oocyte maturation and embryonic development, supplying a better knowledge of fluoride in toxicology and a deeper evaluation of its potential influence in physiological and clinical implications.

The effect of deposition Se on the mRNA expression levels of GPxs in goats from a Se-enriched county of China.

Previous studies revealed that Se was an important regulatory factor for glutathione peroxidase (GPx) genes. However, the relationship between Se concentrations and mRNA expression levels of GPxs were unclear in goats, especially the goats living in natural Se-enriched area. Thus, the aim of this study was to determine the Se concentrations and the mRNA expression levels of GPx-1, GPx-2, GPx-3, and GPx-4 in goats from Ziyang County (ZY-H and ZY-L goats) and Baoji City (BJ-P goats), which were Se-rich region and Se-poor region in China, respectively. Atomic fluorescence spectrometry was used as an essential method to determine the Se concentrations in heart, liver, spleen, lung, kidney, longissimus, biceps femoris, and serum, and the gene expressions were quantified in mRNA samples extracted from the above tissues by real-time quantitative reverse transcription-polymerase chain reaction. We found that the Se concentrations in ZY-H and ZY-L goats were higher than that in BJ-P goats significantly (P < 0.05), and the pertinence relations of Se levels between serum and heart, liver, spleen, and kidney were significant (P < 0.05). The mRNA levels of GPx-1 in ZY-H and ZY-L goats were higher than that in BJ-P goats very significantly (P < 0.01) except for longissimus (P < 0.05). Our results indicated a significant trend for GPx-2 in the direction of increasing mRNA levels with increasing Se concentrations in goats but had no statistical significance (P > 0.05) in our experimental conditions. As to GPx-3, its mRNA expression in spleen, lung, and kidney (P < 0.05) were upregulated and were consensual to high Se contents in ZY-H goats, but no significant effects were observed in heart, liver, longissimus, and biceps femoris among our three groups (P > 0.05). The mRNA levels of GPx-4 in heart, liver, lung, and kidney of ZY-H and ZY-L goats were higher than that of BJ-P goats (P < 0.05), and the difference was very significant in lung especially (P < 0.01), but no change in spleen, longissimus, and biceps femoris (P > 0.05). In summary, these data suggested that the goats living in Ziyang County were rich in Se, and the deposition Se played important roles in the mRNA expression of GPx-1, GPx-3, and GPx-4 in certain tissues of goats differentially.