Development and verification of a 10K liquid chip for Hainan black goat based on genotyping by pinpoint sequencing of liquid captured targets.

BACKGROUND: China has thousands years of goat breeding and abundant goat genetic resources. Additionally, the Hainan black goat is one of the high-quality local goat breeds in China. In order to conserve the germplasm resources of the Hainan black goat, facilitate its genetic improvement and further protect the genetic diversity of goats, it is urgent to develop a single nucleotide polymorphism (SNP) chip for Hainan black goat. RESULTS: In this study, we aimed to design a 10K liquid chip for Hainan black goat based on genotyping by pinpoint sequencing of liquid captured targets (cGPS). A total of 45,588 candidate SNP sites were obtained, 10,677 of which representative SNP sites were selected to design probes, which finally covered 9,993 intervals and formed a 10K cGPS liquid chip for Hainan black goat. To verify the 10K cGPS liquid chip, some southern Chinese goat breeds and a sheep breed with similar phenotype to the Hainan black goat were selected. A total of 104 samples were used to verify the clustering ability of the 10K cGPS liquid chip for Hainan black goat. The results showed that the detection rate of sites was 97.34% -99.93%. 84.5% of SNP sites were polymorphic. The heterozygosity rate was 3.08%-36.80%. The depth of more than 99.4% sites was above 10X. The repetition rate was 99.66%-99.82%. The average consistency between cGPS liquid chip results and resequencing results was 85.58%. In addition, the phylogenetic tree clustering analysis verified that the SNP sites on the chip had better clustering ability. CONCLUSION: These results indicate that we have successfully realized the development and verification of the 10K cGPS liquid chip for Hainan black goat, which provides a useful tool for the genome analysis of Hainan black goat. Moreover, the 10K cGPS liquid chip is conducive to the research and protection of Hainan black goat germplasm resources and lays a solid foundation for its subsequent breeding work.

Immune Cells in the Spleen of Mice Mediate the Inflammatory Response Induced by Mannheimia haemolytica A2 Serotype.

(1) Background: Mannheimia haemolytica (M. haemolytica) is an opportunistic pathogen and is mainly associated with respiratory diseases in cattle, sheep, and goats. (2) Methods: In this study, a mouse infection model was established using a M. haemolytica strain isolated from goats. Histopathological observations were conducted on various organs of the mice, and bacterial load determination and RNA-seq analysis were specifically performed on the spleens of the mice. (3) Results: The findings of this study suggest that chemokines, potentially present in the spleen of mice following a M. haemolytica challenge, may induce the migration of leukocytes to the spleen and suppress the release of pro-inflammatory factors through a negative feedback regulation mechanism. Additionally, an interesting observation was made regarding the potential of hematopoietic stem/progenitor cells congregating in the spleen to differentiate into immune cells, which could potentially collaborate with leukocytes in their efforts to counteract M. haemolytica invasion. (4) Conclusions: This study revealed the immune regulation mechanism induced by M. haemolytica in the mouse spleen, providing valuable insights into host-pathogen interactions and offering a theoretical basis for the prevention, control, and treatment of mannheimiosis.

Integrated Analysis of Transcriptome and Metabolome Profiles in the Longissimus Dorsi Muscle of Buffalo and Cattle.

Buffalo meat is gaining popularity for its nutritional properties, such as its low fat and cholesterol content. However, it is often unsatisfactory to consumers due to its dark color and low tenderness. There is currently limited research on the regulatory mechanisms of buffalo meat quality. Xinglong buffalo are raised in the tropical Hainan region and are undergoing genetic improvement from draught to meat production. For the first time, we evaluated the meat quality traits of Xinglong buffalo using the longissimus dorsi muscle and compared them to Hainan cattle. Furthermore, we utilized a multi-omics approach combining transcriptomics and metabolomics to explore the underlying molecular mechanism regulating meat quality traits. We found that the Xinglong buffalo had significantly higher meat color redness but lower amino acid content and higher shear force compared to Hainan cattle. Differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were identified, with them being significantly enriched in nicotinic acid and nicotinamide metabolic and glycine, serine, and threonine metabolic pathways. The correlation analysis revealed that those genes and metabolites (such as: GAMT, GCSH, PNP, L-aspartic acid, NADP+, and glutathione) are significantly associated with meat color, tenderness, and amino acid content, indicating their potential as candidate genes and biological indicators associated with meat quality. This study contributes to the breed genetic improvement and enhancement of buffalo meat quality.

Overexpression of Pasteurella multocida OmpA induces transcriptional changes and its possible implications for the macrophage polarization.

Pasteurella multocida (P. multocida) is a highly infectious, zoonotic pathogen. Outer membrane protein A (OmpA) is an important virulence component of the outer membrane of P. multocida. OmpA mediates bacterial biofilm formation, eukaryotic cell infection, and immunomodulation. It is unclear how OmpA affects the host immune response. We estimated the role of OmpA in the pathogenesis of P. multocida by investigating the effect of OmpA on the immune cell transcriptome. Changes in the transcriptome of rat alveolar macrophages (NR8383) upon overexpression of P. multocida OmpA were demonstrated. A model cell line for stable transcription of OmpA was constructed by infecting NR8383 cells with OmpA-expressing lentivirus. RNA was extracted from cells and sequenced on an Illumina HiSeq platform. Key gene analysis of genes in the RNA-seq dataset were performed using various bioinformatics methods, such as gene ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, Gene Set Enrichment Analysis, and Protein-Protein Interaction Analysis. Our findings revealed 1340 differentially expressed genes. Immune-related pathways that were significantly altered in rat alveolar macrophages under the effect of OmpA included focal adhesion, extracellular matrix and vascular endothelial growth factor signaling pathways, antigen processing and presentation, nucleotide oligomerization domain-like receptor and Toll-like receptor signaling pathways, and cytokine-cytokine receptor interaction. The key genes screened were Vegfa, Igf2r, Fabp5, P2rx1, C5ar1, Nedd4l, Gas6, Cxcl1, Pf4, Pdgfb, Thbs1, Col7a1, Vwf, Ccl9, and Arg1. Data of associated pathways and altered gene expression indicated that OmpA might cause the conversion of rat alveolar macrophages to M2-like. The related pathways and key genes can serve as a reference for OmpA of P. multitocida and host interaction mechanism studies.

Association between INHA gene polymorphisms and litter size in Hainan black goats.

Background: The inhibin alpha (INHA) gene is one of the important genes affecting the reproductive traits of animals. Hainan black goats are the main goat breed in Hainan Island (China), whose development is limited by low reproductive performance. However, the relationship between INHA gene and the reproductive performance of Hainan black goats is still unclear. Therefore, the purpose of this work was to explore the effect of INHA gene polymorphisms on the litter size of Hainan black goats. Methods: Single nucleotide polymorphisms (SNPs) of INHA were detected, and the genetic parameters and haplotype frequency of these SNPs were calculated and association analysis was performed for these SNPs with the litter size. Finally, the SNP with significant correlations to litter size was analyzed by Bioinformatics tools. Results: The results showed that the litter size of individuals with the AC genotype at loci g.28317663A>C of INHA gene was significantly higher than those with the AA genotype. This SNP changed the amino acid sequence, which may affect the function of INHA protein by affecting its structure. Our results suggest that g.28317663A>C loci may serve as a potential molecular marker for improving the reproductive traits in Hainan black goats.

Clearance of bacteria from lymph nodes in sheep immunized with Brucella suis S2 vaccine is associated with M1 macrophage activation.

Ovine brucellosis is a global zoonotic disease of sheep caused by Brucella melitensis, which inflicts a significant burden on human and animal health. Brucella suis strain S2 (B. suis S2) is a smooth live attenuated vaccine for the prevention of ovine brucellosis in China. However, no previous studies have assessed the immunogenicity of B. suis S2 vaccine after oral immunization in sheep. Here, we attempted to evaluate the ovine immune response over the course of B. suis S2 immunization and to identify in vivo predictors for vaccine development. Body temperature, serum Brucella antibodies, serum cytokines (IL-12p70 and interferon [IFN]-γ), and bacterial load in the mandibular lymph nodes (LN), superficial cervical LN, superficial inguinal LN, and spleen were investigated to determine the safety and efficacy of the vaccine. The abnormal body temperature of sheep occurred within 8 days post-infection (dpi). Brucella suis S2 persisted for a short time (< 21 dpi) in the mandibular LN. The highest level of IL-12p70 was observed at 9 dpi, whereas serum IFN-γ levels peaked at 12 dpi. Transcriptome analysis and quantitative reverse transcription PCR were performed to determine gene expression profiles in the mandibular LN of sheep. Antigen processing and presentation pathway was the dominant pathway related to the dataset. Our studies suggest that the immune response in ovine LN resembled type 1 immunity with the secretion of IL-12p70 and IFN-γ after B.suis S2 immunization and the vaccine may eliminate Brucella via stimulation of M1 macrophages through the course of Th cells.

Activation of MyD88-Dependent TLR Signaling Modulates Immune Response of the Mouse Heart during Pasteurella multocida Infection.

Pasteurella multocida (P. multocida) is an important zoonotic pathogen. In addition to lung lesions, necropsies have revealed macroscopic lesions in the heart in clinical cases. However, most previous studies focused on lung lesions while ignoring heart lesions. Therefore, to investigate the immune response of the P. multocida-infected heart, two murine infection models were established by using P. multocida serotype A (Pm HN02) and D (Pm HN01) strains. Histopathological examination revealed heterogeneous inflammatory responses, including immune cell infiltration in the epicardial and myocardial areas of the heart. Transcriptome sequencing was performed on infected cardiac tissues. To explore the traits of immune responses, we performed the functional enrichment analysis of differentially expressed genes, gene set enrichment analysis and gene set variation analysis. The results showed that the innate immune pathways were significantly regulated in both groups, including the NOD-like receptor signaling pathway, the complement and coagulation cascade and cytokine-cytokine receptor interaction. The Toll-like receptor signaling pathway was only significantly activated in the Pm HN02 group. For the Pm HN02 group, immunohistochemistry analysis further verified the significant upregulation of the hub component MyD88 at the protein level. In conclusion, this study reveals critical pathways for host heart recognition and defense against P. multocida serotypes A and D. Moreover, MyD88 was upregulated by P. multocida serotype A in the heart, providing a theoretical basis for future prevention, diagnosis and treatment research.

Profiling Chromatin Accessibility Responses in Goat Bronchial Epithelial Cells Infected with Pasteurella multocida.

Pasteurella multocida can cause goat hemorrhagic sepsis and endemic pneumonia. Respiratory epithelial cells are the first line of defense in the lungs during P. multocida infection. These cells act as a mechanical barrier and activate immune response to protect against invading pathogenic microorganisms. Upon infection, P. multocida adheres to the cells and causes changes in cell morphology and transcriptome. ATAC-seq was conducted to determine the changes in the chromatin open region of P. multocida-infected goat bronchial epithelial cells based on transcriptional regulation. A total of 13,079 and 28,722 peaks were identified in the control (CK) and treatment (T) groups (P. multocida infection group), respectively. The peaks significantly increased after P. multocida infection. The specific peaks for the CK and T groups were annotated to 545 and 6632 genes, respectively. KEGG pathway enrichment analysis revealed that the specific peak-related genes in the T group were enriched in immune reaction-related pathways, such as Fc gamma R-mediated phagocytosis, MAPK signaling pathway, bacterial invasion of epithelial cells, endocytosis, and autophagy pathways. Other cellular component pathways were also enriched, including the regulation of actin cytoskeleton, adherent junction, tight junction, and focal adhesion. The differential peaks between the two groups were subsequently analyzed. Compared to those in the CK group, 863 and 11 peaks were upregulated and downregulated, respectively, after the P. multocida infection. Fifty-six known transcription factor motifs were revealed in upregulated peaks in the P. multocida-infected group. By integrating ATAC-seq and RNA-seq, some candidate genes (SETBP1, RASGEF1B, CREB5, IRF5, TNF, CD70) that might be involved in the goat bronchial epithelial cell immune reaction to P. multocida infection were identified. Overall, P. multocida infection changed the structure of the cell and caused chromatin open regions to be upregulated. In addition, P. multocida infection actively mobilized the host immune response with the inflammatory phenotype. The findings provide valuable information for understanding the regulatory mechanisms of P. multocida-infected goat bronchial epithelial cells.

Sheep and goat pathogen database: a pathogen data integration and analysis database of sheep and goat infectious diseases.

The prevalence of infectious diseases in sheep and goats has a significant impact on the development of the sheep and goat industry and public health security. The identification and analysis of pathogens are crucial for infectious disease research; however, existing databases pay little attention to sheep and goat diseases, and pathogen data are relatively scattered. Therefore, the effective integration, analysis and visualization of these data will help us conduct in-depth research on sheep and goat infectious diseases and promote the formulation of disease prevention and control strategies. This article considered the pathogens of 44 infectious diseases in sheep and goats as the main research objects and collected and downloaded relevant scientific literature, pathogen genomes, pathogen transcriptomes, pathogen occurrence records, and other data. The C# programming language and an SQL Server database were used to construct and realize the functions of the Sheep and Goat Pathogen Database (SGPD) within a B/S architecture based on the ASP.NET platform. The SGPD mainly provides an integrated platform for sheep and goat pathogen data retrieval, auxiliary analysis, and user upload, including several functionalities: (1) a Disease Introduction module that queries basic information regarding the 44 recorded sheep and goat infectious diseases, such as epidemiology, clinical characteristics, diagnostic criteria, and prevention and control measures; (2) an Omics Information module that allows users to query and download the genome and transcriptome data related to the pathogens of sheep and goat infectious disease, and provide sequence alignment functionality; (3) a Pathogen Structure module that enables users to view electron micrographs of pathogen structure and tissue sections related to sheep and goat disease from publicly published research; (4) a Literature Search module based on the "Pathogen Dictionary" search strategy that facilitates searches for published research related to pathogens of infectious disease; (5) a Science Popularization module that allows users to view popular science materials related to sheep and goat infectious diseases; and (6) a Public Health module that allows users to query the risk factors of zoonotic disease transmission and the corresponding related literature, and realize the visualization of pathogen distribution. The SGPD is a specialized sheep and goat pathogen information database that provides comprehensive resources and technical support for sheep and goat infectious disease research, prevention, and control.

First Report and Comparative Genomic Analysis of Mycoplasma capricolum subsp. capricolum HN-B in Hainan Island, China.

Mycoplasma capricolum subsp. Capricolum (Mcc) is an important member of the Mycoplasma mycoides cluster (Mm cluster) and causes caprine contagious agalactia. Mcc can infect goats of all age groups, especially pregnant ewes and kids. It can cause the abortion in pregnant ewes and the death of goat kids, leading to enormous losses in the goat breeding industry. To date, the prevalence of epidemic Mcc strains on Hainan Island, China, remains unclear. This study aimed to isolate and identify Mcc strains endemic to Hainan Island, China. Genome sequencing and comparative genomic analysis were performed to reveal the molecular characteristics and evolutionary relationships of the isolated strain. Mcc HN-B was isolated and identified in Hainan Island, China. The Mcc HN-B genome consists of a 1,117,925 bp circular chromosome with a 23.79% G + C content. It contains 912 encoding genes, 3 gene islands, and 14 potential virulence genes. The core genome with the features of the Mm cluster and the specific genes of Mcc HN-B were identified by comparative genomic analysis. These results revealed the evolutionary relationship between Mcc HN-B and other members of the Mm cluster. Our findings provide a reference for further studies on the pathogenic mechanism and local vaccine development of Mcc.

First Report and Comparative Genomic Analysis of a Mycoplasma mycoides Subspecies capri HN-A in Hainan Island.

Mycoplasma mycoides subspecies capri (Mmc) is one of the six Mycoplasma mycoides cluster (Mm cluster) members, which can cause "MAKePS" (Mastitis, Arthritis, Keratoconjunctivitis, Pneumonia, Septicemia) syndrome in ruminants. These symptoms can occur alone or together in individuals or flocks of goats. However, little is known about the epidemic Mmc strains in Hainan Island, China. We aimed to isolate the endemic Mmc strains in Hainan Island and reveal their molecular characteristics by genomic sequencing and comparative genomics to mitigate the impact of Mmc on local ruminant farming. Here, the Mmc HN-A strain was isolated and identified for the first time in Hainan Island, China. The genome of Mmc HN-A was sequenced. It contains a 1,084,691 bp-long circular chromosome and 848 coding genes. The genomic analysis of Mmc HN-A revealed 16 virulence factors, 2 gene islands, and a bacterial type IV secretion system protein VirD4. Comparative genomics showed that the core genome of the five Mycoplasma mycoides contained 611 genes that could be exploited to develop drugs and endemic vaccines. Additionally, 36 specific genes were included in the Mmc HN-A genome, which could provide the possibility for the further control and prevention of the Mmc effects on local ruminants and enrich the information on Mmc strains.

Whole-Genome Sequencing Reveals the Genomic Characteristics and Selection Signatures of Hainan Black Goat.

Goats have become one of the most adaptive and important livestock species distributed in developing countries in recent years. The Hainan Black goat is a native goat breed of the Hainan region that is generally well-liked by the local population and is thus raised in large numbers. However, the genomic diversity and selective signals of the Hainan Black goat have not been clearly elucidated yet. Therefore, in this study, we performed whole-genome resequencing of 16 Hainan Black goats and compared the results with those of 71 goats of 6 other breeds from different geographic regions. Principal component analysis (PCA) and phylogenetic analysis identified seven lineages for all goats. Hainan Black goats showed the most similarity with Leizhou goats and the least similarity with Boer goats. Selective sweep analysis identified candidate genes associated with various functions, including immune resistance to disease (TNFAIP2 (TNF alpha induced protein 2) and EXOC3L4 (exocyst complex component 3 like 4)), melanin biosynthetic process (CDH15 (cadherin 15), ASIP (agouti signaling protein), and PARD3 (par-3 family cell polarity regulator)), and light sensitivity (CNGB3 (cyclic nucleotide gated channel subunit beta 3) and CNBD1 (cyclic nucleotide binding domain containing 1)), underlying strong selection signatures in Hainan Black goats. The melanin biosynthetic process, circadian entrainment, regulation of cyclic adenosine 3,5-monophosphate (cAMP)-mediated signaling, and the Rap-1 signaling pathway were significantly enriched in Hainan Black and Alashan Cashmere goats. This result may be important for understanding each trait. Selection signature analysis revealed candidate single nucleotide polymorphisms (SNPs) and genes correlated with the traits of Hainan Black goats. Collectively, our results provide valuable insights into the genetic basis of specific traits correlated with the Hainan island climate, artificial selection in certain local goat breeds, and the importance of protecting breed resources.

Up-Regulation of Interleukin-10 in Splenic Immune Response Induced by Serotype A Pasteurellamultocida.

Pasteurella multocida (P. multocida) is an opportunistic pathogen that is common in livestock and poultry and leads to massive economic losses in the animal husbandry sector. In this study, we challenged mice with P. multocida strain HN02 by intraperitoneal injection and collected spleens to measure bacterial loads. We also performed histopathological analysis by hematoxylin and eosin (H&E) staining. Then we used RNA-sequencing (RNA-seq) to detect the mRNA expression levels in the mouse spleen and quantitative real-time PCR (qRT-PCR) to verify the sequencing data. Finally, we examined the effect of HN02 on anti-inflammatory cytokine interleukin-10 (IL-10) protein expression in the spleen through immunohistochemical analysis. The results showed that compared to those in the control group, the mouse spleens in the challenge group had lesions, and the average bacteria loads was (3.07 ± 1.09) × 106 CFU (colony-forming unit)/g. The RNA-seq results determined 3653 differentially expressed genes (DEGs), and the qRT-PCR analysis revealed immune-related genes consistent with the expression trend in the sequencing data. The number and area of IL-10 positive cells substantially increased to resist inflammation in the challenge group. In conclusion, we analyzed the spleens of mice infected with P. multocida from multiple perspectives, and our findings lay a foundation for subsequent studies on the mechanism of pathogen-host interactions.

Microbiological Characteristics and Pathogenesis of Klebsiella pneumoniae Isolated from Hainan Black Goat.

K. pneumoniae is an opportunistic pathogen that leads to widespread infection in humans and animals, seriously threatening human health and animal husbandry development. In our research, we investigated the biological characteristics of the isolate by using a 16S rRNA gene sequencing, biochemical assay, and drug sensitivity test. Moreover, the pathogenicity study, including the bacteria load determination, the histopathology examination, and the RNA sequencing was carried out to explore whether the isolate could cause lung injury in mice through intraperitoneal injection. The results indicated that the isolate was identified as K. pneumoniae and named as KPHN001. The drug susceptibility test showed that KPHN001 was only sensitive to polymyxin B and furazolidone, and was resistant to other 28 antibiotics. In the bacteria load determination, the highest bacterial load of the organs was found in the spleen, and abundant bacterial colonization was also found in the lung. The histopathology showed the mainly acute inflammations in the lung were due to congestion, edema, and exudation. RNA-seq analysis revealed that the differentially expressed genes (DEGs) of inflammatory cytokines and chemokines were expressed massively in mice. In the present research, the biological characteristics and pathogenesis of clinically isolated K. pneumoniae were systematically studied, revealing the pathogenic mechanism of K. pneumoniae to animals, and providing a theoretical basis for the following prevention, control, and diagnosis research.

Bovine skin fibroblasts mediated immune responses to defend against bovine Acinetobacter baumannii infection.

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen which can cause pneumonia, sepsis and infections of skin and soft tissue. The host mostly relies on innate immune responses to defend against the infection of A. baumannii. Currently, it has been confirmed that fibroblasts involved in innate immune responses. Therefore, to explore how bovine skin fibroblasts mediated immune responses to defend against A. baumannii infection, we analyzed the differential transcripts data of bovine skin fibroblasts infected with bovine A. baumannii by RNA-sequencing (RNA-seq). We found that there were 3014 differentially expressed genes (DEGs) at 14h with bovine A. baumannii infection, including 1940 up-regulated genes and 1074 down-regulated genes. Gene Ontology (GO) enrichment showed that ubiquitin protein ligase binding, IL-6 receptor complex, ERK1 and ERK2 cascade terms were mainly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that innate immune pathways were significantly enriched, such as TNF, IL-17, NLR, MAPK, NF-κB, endocytosis, apoptosis and HIF-1 signaling pathways. Furthermore, Gene Set Enrichment Analysis (GSEA) revealed that GO terms such as chemokine receptor binding and Th17 cell differentiation and KEGG pathways such as TLR and cytokine-cytokine receptor interaction pathways were up-regulated. In addition, CASP3 and JUN were the core functional genes of apoptosis, while IL-6, ERBB2, EGFR, CHUK and MAPK8 were the core functional genes of immunity by Protein-Protein Interaction (PPI) analysis. Our study provided an in-depth understanding of the molecular mechanisms of fibroblasts against A. baumannii infection. It also lays the foundation for the development of new therapeutic targets for the diseases caused by A. baumannii infection and formulates effective therapeutic strategies for the prevention and control of the diseases caused by A. baumannii.

Effects of Pasteurella multocida on Histopathology, miRNA and mRNA Expression Dynamics in Lung of Goats.

Pasteurella multocida (Pm) infection causes severe respiratory disease in goats. We investigated the effects of the Pm infection intratracheally on the histopathology, miRNA and mRNA expression dynamics in the lung of goats infected for 1, 2, 5 and 7 days. Pm infection caused fever, which significantly (p < 0.05) increased the body temperature of the goats from day 1 to 5. Haemotoxylin−eosin staining of the infected lung tissue showed characteristics of suppurative pneumonia with inflammatory cells infiltration and the lung structure destruction. During the Pm infection of the goats, compared with the control group, there were 3080, 3508, 2716 and 2675 differentially expressed genes and 42, 69, 91 and 108 significantly expressed miRNAs (|log2Fold Change| > 1, p < 0.05) in the Pm_d1, Pm_d2, Pm_d5 and Pm_d7 groups, respectively. Five miRNAs and nine immune-related genes were selected for confirmation by reverse transcription−polymerase chain reaction. The results indicated that the expression patterns of the miRNAs and genes were consistent with those determined by next-generation sequencing. The differentially expressed genes were enriched in cytokine−cytokine receptor interaction, cell adhesion molecules, complement and coagulation cascades, tight junction and phagosome Kyoto Encyclopedia of Genes and Genomes pathways and cytokine production, leukocyte migration, myeloid leukocyte migration, cell periphery, plasma membrane, extracellular region part, extracellular region and other Gene Ontology terms. The differentially expressed genes were mapped to marker genes in human and mouse lung cells. The results showed the presence of some marker genes of the immune cells. Compared with the CK group, five miRNAs and 892 common genes were differentially expressed in the Pm_d1, Pm_d2, Pm_d5 and Pm_d7 groups. The target relationships between the common 5 miRNAs and 892 differentially expressed genes were explored and the miRNAs involved in the host immune reaction may act through the target genes. Our study characterized goats' reaction in the lung from histopathological and molecular changes upon Pm infection, which will provide valuable information for understanding the responses in goats during Pm infection.

Downregulation of Three Novel miRNAs in the Lymph Nodes of Sheep Immunized With the Brucella suis Strain 2 Vaccine.

Ovine and caprine brucellosis, both caused by Brucella melitensis, lead to substantial economic losses in the animal industry and health problems in human populations. Brucella suis strain 2 (B.suis S2), as a live attenuated vaccine, is used extensively in China to prevent brucellosis. It has been proven that microRNA (miRNAs) are involved in the immunopathogenesis of brucellosis; however, the miRNA-driven mechanism of immune response to B.suis S2 in vivo remains unknown. To determine which new miRNAs are involved in the host immune response to B.suis S2 and elucidate the function of these miRNAs, we performed a comprehensive analysis of miRNA expression profiles in sheep immunized with B.suis S2 using the high-throughput sequencing approach. The submandibular lymphatic nodes from sheep seropositive for Brucella were collected at 7, 14, 21, 30, 60 and 90 days post-immunization. MiRNA sequencing analysis revealed that 282 differentially expressed miRNAs (|log2 fold-change |>0.5 and p < 0.05) were significantly enriched in the immune pathways, including the NF-kappa B signaling pathway, B cell receptor signaling pathway, p53 signaling pathway and complement and coagulation cascades. Increasing the threshold to |log2 fold change|>1 and p < 0.01 revealed 48 differentially expressed miRNAs, 31 of which were novel miRNAs. Thirteen of these novel miRNAs, which were differentially expressed for at least two time points, were detected via RT-qPCR assays. The novel_229, novel_609, novel_973 and oar-miR-181a assessed by RT-qPCR were detectable and consistent with the expression patterns obtained by miRNA sequencing. Functional analyses of these miRNAs demonstrated that their target genes participated in the immune response pathways, including the innate and adaptive immunity pathways. The immune-related target genes of novel_229 included ENSOARG00000000649 and TMED1, as well as LCN2, PDPK1 and LPO were novel_609 target genes. The immune-related target genes of novel_973 included C6orf58, SPPL3, BPIFB1, ENSOARG00000021083, MPTX1, CCL28, FGB, IDO1, OLR1 and ENSOARG00000020393. The immune-related target genes of oar-miR-181a included ENSOARG00000002722, ARHGEF2, MFAP4 and DOK2. These results will deepen our understanding of the host miRNA-driven defense mechanism in sheep immunized with B.suis S2 vaccine, and provide the valuable information for optimizing vaccines and developing molecular diagnostic targets.

The mRNA and miRNA profiles of goat bronchial epithelial cells stimulated by Pasteurella multocida strains of serotype A and D.

Pasteurella multocida (P. multocida) is a zoonotic bacterium that predominantly colonizes the respiratory tract and lungs of a variety of farmed and wild animals, and causes severe respiratory disease. To investigate the characteristics of the host immune response induced by P. multocida strains of serotype A and D, high-throughput mRNA-Seq and miRNA-Seq were performed to analyze the changes in goat bronchial epithelial cells stimulated by these two serotypes of P. multocida for 4 h. Quantitative RT-PCR was used to validate the randomly selected genes and miRNAs. The results revealed 204 and 117 differentially expressed mRNAs (|log2(Fold-change)| ≥ 1, p-value < 0.05) in the P. multocida serotype A and D stimulated groups, respectively. Meanwhile, the number of differentially expressed miRNAs (|log2(Fold-change)| > 0.1, p-value < 0.05) were 269 and 290, respectively. GO and KEGG enrichment analyses revealed 13 GO terms (p-value < 0.05) and four KEGG pathways (p-value < 0.05) associated with immunity. In the serotype A-stimulated group, the immune-related pathways were the GABAergic synapse and Toll-like receptor signaling pathways, while in the serotype D-stimulated group, the immune-related pathways were the phagosome and B cell receptor signaling pathways. Based on the predicted results of TargetScan and miRanda, the differentially expressed mRNA-miRNA network of immune-related GO terms and KEGG pathways was constructed. According to the cell morphological changes and the significant immune-related KEGG pathways, it was speculated that the P. multocida serotype D strain-stimulated goat bronchial epithelial cells may induce a cellular immune response earlier than serotype A-stimulated cells. Our study provides valuable insight into the host immune response mechanism induced by P. multocida strains of serotype A and D.

Integrated mRNA-seq and miRNA-seq analysis of goat fibroblasts response to Brucella Melitensis strain M5-90.

Brucellosis is a globally zoonotic bacterial disease of humans and various animals including goats, sheep, and cattle. Brucella melitensis M5-90, a live attenuated vaccine strain, has been widely used to prevent brucellosis in goats and sheep. However, the molecular mechanisms governing protective immunity response in non-professional phagocytes infected with B. melitensis M5-90 have not been fully investigated, especially in goats. In our research, goat fibroblasts were used as in vitro models to determine these mechanisms by transcriptome analysis. After incubating with B. melitensis M5-90 3 h, the infected goat fibroblasts were collected at 0 h, 4 h, 24 h, 48 h and 72 h for RNA-seq. The results indicated that there were totally 11,819 differentially expressed genes (DEGs) and 777 differentially expressed (DE) miRNAs found in experiment groups compared with the control groups (|log2(Foldchange)|≥1, FDR<0.05). GO and KEGG enrichment analyses revealed that down-regulated genes were involved in the riboflavin metabolism and positive regulation of IL-8 secretion pathway. The up-regulated genes were mainly involved in adaptive immunity, including TNF signaling pathway, MAPK signaling pathway and JAK/STAT pathway. Additionally, cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity and toll-like receptor signaling pathway, which associated with innate immunity pathways, were also induced. Based on the Pearson correlation coefficients and prediction results of TargetScan and miRanda, the miRNA-mRNA networks of NFKB1, IFNAR2 and IL10RB were constructed and verified in goat fibroblasts by qPCR, which demonstrated that goat fibroblasts displayed immunomodulatory properties. Our findings provide a deeper insight into the host miRNA-driven B. melitensis defense mechanism and reveal the transcriptome changes involved in the innate and adaptive immune response of the goats to B. melitensis infection.

siRNA targeting mCD14 inhibits TNF-α, MIP-2, and IL-6 secretion and NO production from LPS-induced RAW264.7 cells.

Innate immunity plays a key role in protecting a host against invading microorganism, including Gram-negative bacteria. Cluster of differentiation antigen 14 (CD14) is an important innate immunity molecule, existing as a soluble (sCD14) and membrane-associated (mCD14) protein. Endotoxin [lipopolysaccharide (LPS)] is recognized as a key molecule in the pathogenesis of sepsis and septic shock caused by Gram negative bacteria. Emerging evidences indicate that upstream inhibition of bacterial LPS/Toll-like receptor 4(TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. RNA interference (RNAi) provides a promising approach to down-regulate gene expression specifically. To explore the possibility of using RNAi against mCD14 as a strategy for inhibiting the secretion of cytokines and the nitric oxide (NO) production from LPS-activated RAW264.7 cells, four different short interfering RNA (siRNA) molecules corresponding to the sequence of mCD14 gene were designed and synthesized. We then tested the inhibition effects of these siRNA molecules on mCD14 expression by real-time quantitative RT-PCR and Western blot. After effective siRNA molecule (mCD14-siRNA-224), which is capable of reducing messenger RNA (mRNA) accumulation and protein expression of mCD14 specifically, was identified, RAW264.7 cells pretreated with mCD14-siRNA-224 were stimulated with LPS, and the secretion of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) and the NO production were evaluated. The results indicated that mCD14-siRNA-224 effectively inhibited TNF-α, MIP-2, and IL-6 release and NO production from LPS-stimulated RAW 264.7 cells by down-regulating mRNA accumulation and protein expression of mCD14 specifically. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for endotoxin-related diseases.