The Establishment of a Novel γ-Interferon In Vitro Release Assay for the Differentiation of Mycobacterial Bovis-Infected and BCG-Vaccinated Cattle.

BCG vaccination is increasingly reconsidered in the effective prevention of bovine tuberculosis (bTB). However, the primary challenge in BCG vaccination for cattle is the lack of a technique for differentiating between infected and vaccinated animals (DIVA). This study aimed to establish a novel DIVA diagnostic test based on an interferon-gamma in vitro release assay (IGRA). The plasmid encoding three differential antigens (Rv3872, CFP-10, and ESAT-6) absent in BCG genes but present in virulent M. bovis was previously constructed. Thus, a recombinant protein called RCE (Rv3872, CFP-10, and ESAT-6) was expressed, and an RCE-based DIVA IGRA (RCE-IGRA) was established. The RCE concentration was optimized at 4 µg/mL by evaluating 97 cattle (74 of which were bTB-positive, and 23 were negative) using a commercial IGRA bTB diagnostic kit. Further, 84 cattle were tested in parallel with the RCE-IGRA and commercial PPD-based IGRA (PPD-IGRA), and the results showed a high correlation with a kappa value of 0.83. The study included BCG-vaccinated calves (n = 6), bTB-positive cattle (n = 6), and bTB-negative non-vaccinated calves (n = 6). After 3 months post-vaccination, PPD-IGRA generated positive results in both vaccinated and infected calves. However, RCE-IGRA developed positive results in infected calves but negative results in vaccinated calves. In conclusion, this DIVA method has broad prospects in differentiating BCG vaccination from natural infection to prevent bTB.

A Novel Multiepitope Fusion Antigen as a Vaccine Candidate for the Prevention of Enterotoxigenic E. coli-Induced Calf Diarrhea.

Calf diarrhea caused by enterotoxigenic E. coli (ETEC) poses an enormous economic challenge in the cattle industry. Fimbriae and enterotoxin are crucial virulence factors and vaccine targets of ETEC. Since these proteins have complicated components with large molecular masses, the development of vaccines by directly expressing these potential targets is cumbersome Therefore, this study aimed to develop a multiepitope fusion antigen designated as MEFA by integrating major epitopes of FanC and Fim41a subunits and a toxoid epitope of STa into the F17G framework. The 3D modeling predicted that the MEFA protein displayed the epitopes from these four antigens on its surface, demonstrating the desired structural characteristics. Then, the MEFA protein was subsequently expressed and purified for mouse immunization. Following that, our homemade ELISA showed that the mouse antiserum had a consistent increase in polyclonal antibody levels with the highest titer of 1:217 to MEFA. Furthermore, the western blot assay demonstrated that this anti-MEFA serum could react with all four antigens. Further, this antiserum exhibited inhibition on ETEC adhesion to HCT-8 cells with inhibitory rates of 92.8%, 84.3%, and 87.9% against F17+, F5+, and F41+ ETEC strains, respectively. Additionally, the stimulatory effect of STa toxin on HCT-8 cells was decreased by approximately 75.3% by anti-MEFA serum. This study demonstrates that the MEFA protein would be an antigen candidate for novel subunit vaccines for preventing ETEC-induced diarrhea in cattle.

Identifying the Quantitative Trait Locus and Candidate Genes of Traits Related to Milling Quality in Rice via a Genome-Wide Association Study.

Milling quality directly affects production efficiency in rice, which is closely related to the brown rice recovery (BRR), the milled rice recovery (MRR) and the head milled rice recovery (HMRR). The present study investigated these three traits in 173 germplasms in two environments, finding abundant phenotypic variation. Three QTLs for BRR, two for MRR, and three for HMRR were identified in a genome-wide association study, five of these were identified in previously reported QTLs and three were newly identified. By combining the linkage disequilibrium (LD) analyses, the candidate gene LOC_Os05g08350 was identified. It had two haplotypes with significant differences and Hap 2 increased the BRR by 4.40%. The results of the qRT-PCR showed that the expression of LOC_Os05g08350 in small-BRR accessions was significantly higher than that in large-BRR accessions at Stages 4-5 of young panicle development, reaching the maximum value at Stage 5. The increase in thickness of the spikelet hulls of the accession carrying LOC_Os05g08350TT occurred due to an increase in the cell width and the cell numbers in cross-sections of spikelet hulls. These results help to further clarify the molecular genetic mechanism of milling-quality-related traits and provide genetic germplasm materials for high-quality breeding in rice.

The Identification of a Novel Nucleomodulin MbovP467 of Mycoplasmopsis bovis and Its Potential Contribution in Pathogenesis.

Mycoplasmopsis bovis is a causative agent of crucial diseases in both dairy and beef cattle leading to substantial economic losses. However, limited control measures for M. bovis-related diseases exist due to a lack of understanding about the virulence factors of this pathogen, a common challenge in mycoplasma research. Consequently, this study aimed to characterize a novel nucleomodulin as a virulence-related factor of M. bovis. Employing bioinformatic tools, we initially predicted MbovP467 to be a secreted protein with a nuclear localization signal based on SignalP scores and the cNLS (Nuclear Localization Signal) Mapper, respectively. Subsequently, the MbovP467 gene was synthesized and cloned into a pEGFP plasmid with EGFP labeling to obtain a recombinant plasmid (rpEGFP-MbovP467) and then was also cloned in pET-30a with a consideration for an Escherichia coli codon bias and expressed and purified for the production of polyclonal antibodies against the recombinant MbovP467 protein. Confocal microscopy and a Western blotting assay confirmed the nuclear location of MbovP467 in bovine macrophages (BoMacs). RNA-seq data revealed 220 up-regulated and 20 down-regulated genes in the rpEGFP-MbovP467-treated BoMac group compared to the control group (pEGFP). A GO- and KEGG-enrichment analysis identified associations with inflammatory responses, G protein-coupled receptor signaling pathways, nuclear receptor activity, sequence-specific DNA binding, the regulation of cell proliferation, IL-8, apoptotic processes, cell growth and death, the TNF signaling pathway, the NF-κB signaling pathway, pathways in cancer, and protein families of signaling and cellular processes among the differentially expressed up-regulated mRNAs. Further experiments, investigating cell viability and the inflammatory response, demonstrated that MbovP467 reduces BoMac cell viability and induces the mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, and apoptosis in BoMac cells. Further, MbovP467 increased the promoter activity of TNF-α. In conclusion, this study identified a new nucleomodulin, MbovP467, for M. bovis, which might have an important role in M. bovis pathogenesis.

MbovP0725, a secreted serine/threonine phosphatase, inhibits the host inflammatory response and affects metabolism in Mycoplasma bovis.

Mycoplasma species are able to produce and release secreted proteins, such as toxins, adhesins, and virulence-related enzymes, involved in bacteria adhesion, invasion, and immune evasion between the pathogen and host. Here, we investigated a novel secreted protein, MbovP0725, from Mycoplasma bovis encoding a putative haloacid dehalogenase (HAD) hydrolase function of a key serine/threonine phosphatase depending on Mg2+ for the dephosphorylation of its substrate pNPP, and it was most active at pH 8 to 9 and temperatures around 40°C. A transposon insertion mutant strain of M. bovis HB0801 that lacked the protein MbovP0725 induced a stronger inflammatory response but with a partial reduction of adhesion ability. Using transcriptome sequencing and quantitative reverse transcription polymerase chain reaction analysis, we found that the mutant was upregulated by the mRNA expression of genes from the glycolysis pathway, while downregulated by the genes enriched in ABC transporters and acetate kinase-phosphate acetyltransferase pathway. Untargeted metabolomics showed that the disruption of the Mbov_0725 gene caused the accumulation of 9-hydroxyoctadecadienoic acids and the consumption of cytidine 5'-monophosphate, uridine monophosphate, and adenosine monophosphate. Both the exogenous and endogenous MbvoP0725 protein created by purification and transfection inhibited lipopolysaccharide (LPS)-induced IL-1ß, IL-6, and TNF-α mRNA production and could also attenuate the activation of MAPK-associated pathways after LPS treatment. A pull-down assay identified MAPK p38 and ERK as potential substrates for MbovP0725. These findings define metabolism- and virulence-related roles for a HAD family phosphatase and reveal its ability to inhibit the host pro-inflammatory response. IMPORTANCE: Mycoplasma bovis (M. bovis) infection is characterized by chronic pneumonia, otitis, arthritis, and mastitis, among others, and tends to involve the suppression of the immune response via multiple strategies to avoid host cell immune clearance. This study found that MbovP0725, a haloacid dehalogenase (HAD) family phosphatase secreted by M. bovis, had the ability to inhibit the host pro-inflammatory response induced by lipopolysaccharide. Transcriptomic and metabolomic analyses were used to identify MbovP0725 as an important phosphatase involved in glycolysis and nucleotide metabolism. The M. bovis transposon mutant strain T8.66 lacking MbovP0725 induced a higher inflammatory response and exhibited weaker adhesion to host cells. Additionally, T8.66 attenuated the phosphorylation of MAPK P38 and ERK and interacted with the two targets. These results suggested that MbovP0725 had the virulence- and metabolism-related role of a HAD family phosphatase, performing an anti-inflammatory response during M. bovis infection.

QTL Detection and Candidate Gene Identification for Eating and Cooking Quality Traits in Rice (Oryza sativa L.) via a Genome-Wide Association Study.

The eating and cooking quality (ECQ) directly affects the taste of rice, being closely related to factors such as gelatinization temperature (GT), gel consistency (GC) and amylose content (AC). Mining the quantitative trait loci (QTLs), and gene loci controlling ECQ-related traits is vital. A genome-wide association study on ECQ-related traits was conducted, combining 1.2 million single nucleotide polymorphisms (SNPs) with the phenotypic data of 173 rice accessions. Two QTLs for GT, one for GC and five for AC were identified, of which two were found in previously reported genes, and six were newly found. There were 28 positional candidate genes in the region of qAC11. Based on a linkage disequilibrium (LD) analysis, three candidate genes were screened within the LD region associated with AC. There were significant differences between the haplotypes of LOC_Os11g10170, but no significant differences were found for the other two genes. The qRT-PCR results showed that the gene expression levels in the accessions with high ACs were significantly larger than those in the accessions with low ACs at 35d and 42d after flowering. Hap 2 and Hap 3 of LOC_Os11g10170 reduced the AC by 13.09% and 10.77%, respectively. These results provide a theoretical and material basis for improving the ECQ of rice.

Identification of Differential Circular RNA Expression Profiles and Functional Networks in Human Macrophages Induced by Virulent and Avirulent Mycobacterium tuberculosis Strains.

Circular RNAs (circRNAs) are noncoding RNAs with diverse functions. However, most Mycobacterium tuberculosis (M.tb)-related circRNAs remain undiscovered. In this study, we infected THP-1 cells with virulent and avirulent M.tb strains and then sequenced the cellular circRNAs. Bioinformatic analysis predicted 58,009 circRNAs in all the cells. In total, 2035 differentially expressed circRNAs were identified between the M.tb-infected and uninfected THP-1 cells and 1258 circRNAs were identified in the virulent and avirulent M.tb strains. Further, the top 10 circRNAs were confirmed by Sanger sequencing, among which four circRNAs, namely circSOD2, circCHSY1, circTNFRSF21, and circDHTKD1, which were highly differentially expressed in infected cells compared with those in uninfected cells, were further confirmed by ring formation, specific primers, and RNase R digestion. Next, circRNA-miRNA-mRNA subnetworks were constructed, such as circDHTKD1/miR-660-3p/IL-12B axis. Some of the individual downstream genes, such as miR-660-3p and IL-12B, were previously reported to be associated with cellular defense against pathological processes induced by M.tb infection. Because macrophages are important immune cells and the major host cells of M.tb, these findings provide novel ideas for exploring the M.tb pathogenesis and host defense by focusing on the regulation of circRNAs during M.tb infection.

Comparative Proteomic Analysis of Secretory Proteins of Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides Investigates Virulence and Discovers Important Diagnostic Biomarkers.

The most important pathogenic Mycoplasma species in bovines are Mycoplasma bovis (M. bovis) and Mycoplasma mycoides subsp. mycoides (Mmm). Mmm causes contagious bovine pleuropneumonia (CBPP), which is a severe respiratory disease widespread in sub-Saharan Africa but eradicated in several countries, including China. M. bovis is an important cause of the bovine respiratory disease complex (BRD), characterized worldwide by pneumonia, arthritis, and mastitis. Secreted proteins of bacteria are generally considered virulence factors because they can act as toxins, adhesins, and virulent enzymes in infection. Therefore, this study performed a comparative proteomic analysis of the secreted proteins of M. bovis and Mmm in order to find some virulence-related factors as well as discover differential diagnostic biomarkers for these bovine mycoplasmas. The secretome was extracted from both species, and liquid chromatography-tandem mass spectrometry was used, which revealed 55 unique secreted proteins of M. bovis, 44 unique secreted proteins of Mmm, and 4 homologous proteins. In the M. bovis secretome, 19 proteins were predicted to be virulence factors, while 4 putative virulence factors were identified in the Mmm secretome. In addition, five unique secreted proteins of Mmm were expressed and purified, and their antigenicity was confirmed by Western blotting assay and indirect ELISA. Among them, Ts1133 and Ts0085 were verified as potential candidates for distinguishing Mmm infection from M. bovis infection.

An atypical GdpP enzyme linking cyclic nucleotide metabolism to osmotic tolerance and gene regulation in Mycoplasma bovis.

Nucleotide second messengers play an important role in bacterial adaptation to environmental changes. Recent evidence suggests that some of these regulatory molecular pathways were conserved upon the degenerative evolution of the wall-less mycoplasmas. We have recently reported the occurrence of a phosphodiesterase (PDE) in the ruminant pathogen Mycoplasma bovis, which was involved in c-di-AMP metabolism. In the present study, we demonstrate that the genome of this mycoplasma species encodes a PDE of the GdpP family with atypical DHH domains. Characterization of M. bovis GdpP (MbovGdpP) revealed a multifunctional PDE with unusual nanoRNase and single-stranded DNase activities. The alarmone ppGpp was found unable to inhibit c-di-NMP degradation by MbovGdpP but efficiently blocked its nanoRNase activity. Remarkably, MbovGdpP was found critical for the osmotic tolerance of M. bovis under K+ and Na+ conditions. Transcriptomic analyses further revealed the biological importance of MbovGdpP in tRNA biosynthesis, pyruvate metabolism, and several steps in genetic information processing. This study is an important step in understanding the role of PDE and nucleotide second messengers in the biology of a minimal bacterial pathogen.

The Safety and Protective Efficacy Evaluation of an Attenuated M. bovis-BoHV-1 Bivalent Vaccine in Rabbits.

Bovine respiratory disease (BRD) is a global prevalent multifactorial infection primarily caused by viral and bacterial coinfections. In China, Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the predominant pathogens associated with BRD. Our previous study involved the development of attenuated M. bovis HB150 and BoHV-1 gG-/tk- vaccine strains, which were thoroughly assessed for their safety profiles and protective efficacy in cattle. In this study, we applied a combination of vaccines in varying ratios and used a rabbit model to determine the safety and protective efficacy. We used PCR/RT-PCR to detect the postimmunization and challenge shedding of M. bovis and BoHV-1. Additionally, we measured antibody titers and the expression of IFN-ß and TNF-α to evaluate the humoral and cellular immune responses, respectively. Furthermore, we performed a histopathological analysis to assess lung damage. Our study provides evidence of the safety and effectiveness of the bivalent M. bovis-BoHV-1 vaccine in rabbits, particularly when applying a combination of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 of the BoHV-1 gG-/tk- strain. The bivalent vaccine significantly enhanced both the long-term antibody immune response and cellular protection against the M. bovis and BoHV-1 challenge. These findings provide a valuable model for the potential application in cattle.

ALKBH5 Stabilized N6-Methyladenosine-Modified LOC4191 to Suppress E. coli-Induced Apoptosis.

E. coli is a ubiquitous pathogen that is responsible for over one million fatalities worldwide on an annual basis. In animals, E. coli can cause a variety of diseases, including mastitis in dairy cattle, which represents a potential public health hazard. However, the pathophysiology of E. coli remains unclear. We found that E. coli could induce global upregulation of m6A methylation and cause serious apoptosis in bovine mammary epithelial cells (MAC-T cells). Furthermore, numerous m6A-modified lncRNAs were identified through MeRIP-seq. Interestingly, we found that the expression of LOC4191 with hypomethylation increased in MAC-T cells upon E. coli-induced apoptosis. Knocking down LOC4191 promoted E. coli-induced apoptosis and ROS levels through the caspase 3-PARP pathway. Meanwhile, knocking down ALKBH5 resulted in the promotion of apoptosis through upregulated ROS and arrested the cell cycle in MAC-T cells. ALKBH5 silencing accelerated LOC4191 decay by upregulating its m6A modification level, and the process was recognized by hnRNP A1. Therefore, this indicates that ALKBH5 stabilizes m6A-modified LOC4191 to suppress E. coli-induced apoptosis. This report discusses an initial investigation into the mechanism of m6A-modified lncRNA in cells under E. coli-induced apoptosis and provides novel insights into infectious diseases.

Mycobacterium tuberculosis Fatty Acyl-CoA Synthetase fadD33 Promotes Bacillus Calmette-Guérin Survival in Hostile Extracellular and Intracellular Microenvironments in the Host.

Tuberculosis, caused by Mycobacterium tuberculosis (M. tb), remains a significant global health challenge. The survival of M. tb in hostile extracellular and intracellular microenvironments is crucial for its pathogenicity. In this study, we discovered a Bacillus Calmette-Guérin (BCG) mutant B1033 that potentially affected mycobacterium pathogenicity. This mutant contained an insertion mutation gene, fadD33, which is involved in lipid metabolism; however, its direct role in regulating M. tb infection is not well understood. Here, we found that the absence of fadD33 reduced BCG adhesion and invasion into human pulmonary alveolar epithelial cells and increased the permeability of the mycobacterial cell wall, allowing M. tb to survive in the low pH and membrane pressure extracellular microenvironment of the host cells. The absence of fadD33 also inhibited the survival of BCG in macrophages by promoting the release of proinflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumors necrosis factor-α, through the mitogen-activated protein kinase p38 signaling pathway. Overall, these findings provide new insights into M. tb mechanisms to evade host defenses and might contribute to identifying potential therapeutic and vaccine targets for tuberculosis prevention.

Evaluation of Antiviral Activity of Ivermectin against Infectious Bovine Rhinotracheitis Virus in Rabbit Model.

Infectious bovine rhinotracheitis (IBR) caused by bovine herpes virus 1 (BoHV-1) can lead to enormous economic losses in the cattle industry. Vaccine immunization is preferentially used to decrease its transmission speed and resultant clinical signs, rather than to completely stop viral infection. Therefore, a drug effective in treating IBR is urgently needed. Our previous work demonstrated that ivermectin significantly inhibited viral replication in a cell infection model. This study aimed to investigate its antiviral effects in vivo by using a rabbit infection model. The viral inhibition assay was first used to confirm that ivermectin at low concentrations (6-25 nM) could reduce viral titers (TCID50) significantly (p < 0.001) at 24 h post-infection. In rabbits, ivermectin was administrated with one to three doses, based on the recommended anti-parasite treatment dosage (0.2 mg/kg bodyweight) through subcutaneous injection at different days post-infection in the treated IBRV infection groups, while non-treated infection group was used as the control. The infected rabbits showed hyperthermia and other clinical signs, but the number of high-fever rabbits in the ivermectin treatment groups was significantly lower than that in the non-treated infection group. Furthermore, in ivermectin treatment groups, the cumulative clinical scores correlated negatively with drug doses and positively with delay of administration time post-infection. The overall nasal shedding time in ivermectin-treated groups was two days shorter than the non-treated challenge group. At the same time point, the titer of neutralizing antibodies in the treatment group with triple doses was higher than the other two-dose groups, but the difference between the treatment groups decreased with the delay of drug administration. Correspondingly, the serious extent of lung lesions was negatively related to the dosage, but positively related to the delay of drug administration. The qPCR with tissue homogenates showed that the virus was present in both the lung tissues and trigeminals of the infected rabbits. In conclusion, ivermectin treatment had therapeutic effect by decreasing clinical signs and viral shedding, but could not stop virus proliferation in lung tissues and trigeminals.

Molecular Characterization of UL50 (dUTPase) Gene of Bovine Herpes Virus 1.

Bovine herpes virus -1 (BoHV-1) infection leads to upper respiratory tract infection, conjunctivitis and genital disorders in cattle. To control BoHV-1, it is important to understand the role of viral proteins in viral infection. BoHV-1 has several gene products to help in viral replication in infected cell. One such gene is deoxyuridine triphosphate nucleotidohydrolase (dUTPase) also known as UL50. In this study, we analyzed the amino acid sequence of UL50 (dUTPase) using bioinformatics tools and found that it was highly conserved among herpesvirus family. Then, it was cloned and expressed in Escherichia coli Rosetta (DE3), induced by isopropy1-b-D-thiogalactopyranoside (IPTG) and the recombinant UL50 protein was purified to immunize rabbits for the preparation of polyclonal antiserum. The results indicated that the UL50 gene of BoHV-1 was composed of 978 nucleotides, which encoded 323 amino acids. Western blot analysis revealed that polyclonal sera against UL50 reacted with a band of 34 kDa. Furthermore, immunofluorescence assay showed that UL50 localized in the cytoplasmic area. Taken together, UL50 was successfully cloned, expressed and detected in BoHV-1-infected cells and was localized in the cytoplasm to help in the replication of BoHV-1 in infected cells.

Soluble expression and purification of recombinant bovine ferritin H-chain.

Ferritin is a potential medicine delivery vehicle and vaccine platform, and its efficient expression is a prerequisite for widespread application. This study introduces a soluble expression strategy for recombinant bovine ferritin heavy chain (rFTH) in a prokaryotic system and an improved protein purification method. The amplified rFTH gene was ligated into the prokaryotic expression vector pET30a. The recombinant vectors with the N-terminal His-tag(N-His) or C-terminal His-tag(C-His) were translated and expressed separately. The results showed that the solubility of rFTH with C-His was significantly higher than that with N-His. The expression of rFTH with C-His was attempted at 37 °C and 16 °C, respectively. The results showed that the proportion of soluble protein expressed at 37 °C was more than 90%, higher than that expressed at 16 °C. Then rFTH with C-His was purified successfully using anion exchange chromatography, modified PEG precipitation, and dialysis. The rFTH protein was characterized using SDS-PAGE, Native-PAGE, Western blot, transmission electron microscopy, and dynamic light scattering. The results demonstrated that the purified rFTH protein self-assembled into ferritin nanoparticles with a regular shape and uniform size. This study sheds new light on the soluble expression of ferritin and provides a foundation for the construction of bovine ferritin nanoparticle production platforms.

A new allele PEL9 GG identified by genome-wide association study increases panicle elongation length in rice (Oryza sativa L.).

Considering the male sterile line has the phenomenon of panicle enclosure, panicle elongation length (PEL) plays an important role in hybrid rice seed production. However, the molecular mechanism underlying this process is poorly understood. In this study, we investigated the PEL phenotypic values of 353 rice accessions across six environments, which shows abundant phenotypic variation. Combining the 1.3 million single-nucleotide polymorphisms, we performed a genome-wide association study on PEL. Three quantitative trait loci (QTL) qPEL4, qPEL6, and qPEL9 were identified as significantly associated with PEL, of which qPEL4 and qPEL6 were previously reported QTLs and qPEL9 was novel. One causal gene locus, PEL9, was identified and validated. The PEL of accessions carrying allele PEL9 GG was significantly longer than that of those carrying allele PEL9 TT. We also demonstrated that the outcrossing rate of female parents carrying allele PEL9 GG increased by 14.81% compared with that of the isogenic line carrying allele PEL9 TT in an F1 hybrid seed production field. The allele frequency of PEL9GG increased gradually with an increase in latitude in the Northern Hemisphere. Our results should facilitate the improvement of the PEL of the female parent of hybrid rice.

Epidemiological Survey and Risk Factor Analysis of 14 Potential Pathogens in Golden Snub-Nosed Monkeys at Shennongjia National Nature Reserve, China.

Golden snub-nosed monkeys (Rhinopithecus roxellanae) belong to Class A, the highest level of endangered primate species. Exploring the infection status of potential pathogens in golden snub-nosed monkeys is important for controlling associated diseases and protecting this species. The objective of this study was to investigate the seroprevalence for a number of potential pathogens and the prevalence of fecal adenovirus and rotavirus. A total of 283 fecal samples were collected from 100 golden snub-nosed monkeys in December 2014, June 2015, and January 2016; 26 blood samples were collected from 26 monkeys in June 2014, June 2015, January 2016 and November 2016 at Shennongjia National Reserve in Hubei, China. The infection of 11 potential viral diseases was examined serologically using an Indirect Enzyme-linked Immunosorbent Assay (iELISA) and Dot Immunobinding Assays (DIA), while the whole blood IFN-γ in vitro release assay was used to test tuberculosis (TB). In addition, fecal Adenovirus and Rotavirus were detected using Polymerase Chain Reaction (PCR). As a result, the Macacine herpesvirus-1 (MaHV-1), Golden snub-nosed monkey cytomegalovirus (GsmCMV), Simian foamy virus (SFV) and Hepatitis A virus (HAV) were detected with the seroprevalence of 57.7% (95% CI: 36.9, 76.6), 38.5% (95% CI: 20.2, 59.4), 26.9% (95% CI: 11.6, 47.8), and 7.7% (95% CI: 0.0, 84.2), respectively. Two fecal samples tested positive for Adenovirus (ADV) by PCR, with a prevalence of 0.7% (95% CI: 0.2, 2.5), and further, the amplification products were sequenced. Phylogenetic analysis revealed that they belonged to the HADV-G group. However, other pathogens, such as Coxsackievirus (CV), Measles virus (MeV), Rotavirus (RV), Simian immunodeficiency virus (SIV), Simian type D retroviruses (SRV), Simian-T-cell lymphotropic virus type 1 (STLV-1), Simian varicella virus (SVV), Simian virus 40 (SV40) and Mycobacterium tuberculosis complex (TB) were negative in all samples. In addition, a risk factor analysis indicated that the seroprevalence of MaHV-1 infection was significantly associated with old age (≥4 years). These results have important implications for understanding the health status and conservation of the endangered golden snub-nosed monkey population at Shennongjia Nature Reserve.

The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells.

Mycobacterium tuberculosis (M. tb) is the causative agent of tuberculosis (TB) that leads to millions of deaths each year. Extensive evidence has explored the involvement of microRNAs (miRNAs) in M. tb infection. Limitedly, the concrete function of microRNA-100-5p (miR-100-5p) in M. tb remains unexplored and largely elusive. In this study, using Bacillus Calmette-Guérin (BCG) as the model strain, we validated that miR-100-5p was significantly decreased in BCG-infected THP-1 cells. miR-100-5p inhibition effectively facilitated the apoptosis of infected THP-1 cells and reduced BCG survival by regulating the phosphatidylinositol 3-kinase/AKT pathway. Further, SMARCA5 was the target of miR-100-5p and reduced after miR-100-5p overexpression. Since BCG infection down-regulated miR-100-5p in THP-1 cells, the SMARCA5 expression was up-regulated, which in turn increased apoptosis through caspase-3 and Bcl-2 and, thereby, reducing BCG intracellular survival. Collectively, the study uncovered a new molecular mechanism of macrophage to suppress mycobacterial infection through miR-100-5p and SMARCA5 pathway.

Gut microbiome variations in Rhinopithecus roxellanae caused by changes in the environment.

BACKGROUND: The snub-nosed monkey (Rhinopithecus roxellanae) is an endangered animal species mainly distributed in China and needs to be protected. Gut microbiome is an important determinant of animal health and population survival as it affects the adaptation of the animals to different foods and environments under kinetic changes of intrinsic and extrinsic factors. Therefore, this study aimed to elucidate gut fecal microbiome profiles of snub-nosed monkeys affected by several extrinsic and intrinsic factors, including raising patterns (captive vs. wild), age, sex, and diarrheal status to provide a reference for making protection strategies. RESULTS: The 16S rRNA gene sequencing was firstly used to pre-check clustering of 38 fecal samples from the monkeys including 30 wild and 8 captive (5 healthy and 3 diarrheal) from three Regions of Shennongjia Nature Reserve, Hubei Province, China. Then the 24 samples with high-quality DNA from 18 wild and 6 captive (4 healthy and 2 diarrheal) monkeys were subjected to shotgun metagenomic sequencing to characterize bacterial gut microbial communities. We discovered that the raising pattern (captive and wild) rather than age and sex was the predominant factor attributed to gut microbiome structure and proportionality. Wild monkeys had significantly higher bacterial diversity and lower Bacteroidetes/Firmicutes ratios than captive animals. Moreover, the gut microbiomes in wild healthy monkeys were enriched for the genes involved in fatty acid production, while in captive animals, genes were enriched for vitamin biosynthesis and metabolism and amino acid biosynthesis from carbohydrate intermediates. Additionally, a total of 37 antibiotic resistant genes (ARG) types were detected. Unlike the microbiome diversity, the captive monkeys have a higher diversity of ARG than the wild animals. CONCLUSION: Taken together, we highlight the importance of self-reprogramed metabolism in the snub-nosed monkey gut microbiome to help captive and wild monkeys adapt to different intrinsic and extrinsic environmental change.

The Role of N6-Methyladenosine in Inflammatory Diseases.

N6-Methyladenosine (m6A) is the most abundant epigenetic RNA modification in eukaryotes, regulating RNA metabolism (export, stability, translation, and decay) in cells through changes in the activity of writers, erasers, and readers and ultimately affecting human life or disease processes. Inflammation is a response to infection and injury in various diseases and has therefore attracted significant attention. Currently, extensive evidence indicates that m6A plays an essential role in inflammation. In this review, we focus on the mechanisms of m6A in inflammatory autoimmune diseases, metabolic disorder, cardio-cerebrovascular diseases, cancer, and pathogen-induced inflammation, as well as its possible role as targets for clinical diagnosis and treatment.