Rapid and sensitive visual detection of avian leukosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow immunochromatographic strip assay.

Considering that avian leukosis virus (ALV) infection has inflicted massive economic losses on the poultry breeding industry in most countries, its early diagnosis remains an important measure for timely treatment and control of the disease, for which a rapid and sensitive point-of-care test is required. We established a user-friendly, economical, and rapid visualization method for ALV amplification products based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with an immunochromatographic strip in a lateral flow device (LFD). Using the ALVp27 gene as the target, five RT-LAMP primers and one fluorescein-isothiocyanate-labeled probe were designed. After 60 min of RT-LAMP amplification at 64 °C, the products could be visualized directly using the LFD. The detection limit of this assay for ALV detection was 102 RNA copies/µL, and the sensitivity was 100 times that of reverse transcription polymerase chain reaction (RT-PCR), showing high specificity and sensitivity. To verify the clinical practicality of this assay for detecting ALV, the gold standard RT-PCR method was used for comparison, and consistent results were obtained with both assays. Thus, the assay described here can be used for rapid detection of ALV in resource-limited environments.

A multi-epitope subunit vaccine based on CU/ZN-SOD, OMP31 and BP26 against Brucella melitensis infection in BALB/C mice.

Brucellosis, a zoonosis caused by Brucella, is highly detrimental to both humans and animals. Most existing vaccines are live attenuated vaccines with safety flaws for people and animals. Therefore, it is advantageous to design a multi-epitope subunit vaccine (MEV) to prevent Brucella infection. To this end, we applied a reverse vaccinology approach. Six cytotoxic T cell (CTL) epitopes, seven T helper cell (HTL) epitopes, and four linear B cell epitopes from CU/ZN-SOD, Omp31, and BP26 were obtained. We linked the CTL, HTL, B-cell epitopes, the appropriate CTB molecular adjuvant, and the universal T helper lymphocyte epitope, PADRE, with linkers AAY, GPPGG, and KK, respectively. This yielded a 412-amino acid MEV construct, which we named MEVcob. The immunogenicity, stability, safety, and feasibility of the construct were evaluated by bioinformatics tools (including the AlphaFold2 prediction tool, the AlphaFold2 tool, NetMHC-I pan 4.0 server, IEDB MHC-I server, ABCpred service, and C-ImmSim server); the physicochemical properties, secondary and tertiary structures, and binding ability of MEVocb to toll-like receptor 4 (TLR4) was analyzed. Then, codon adaptation and computer cloning studies were performed. MEVocb is highly immunogenic in immunostimulation experiments, The proteins translated by these sequences were relatively stable, exhibiting a high antigenic index. Furthermore, mouse experiments confirmed that the MEVocb construct could raise IFN-γ, IgG, IgG2a, IgG1, IL-2, TNF-α levels in mice, indicating that induced a specific humoral and cellular immune response in BALB/c mice. This vaccine induced a statistically significant level of protection in BALB/c mice when challenged with Brucella melitensis 043 in Xinjiang. Briefly, we utilized immunoinformatic tools to design a novel multi-epitope subunit candidate vaccine against Brucella. This vaccine aims to induce host immune responses and confer specific protective effects. The study results offer a theoretical foundation for the development of a novel Brucella subunit vaccine.

Anaplasma phagocytophilum Ats-1 enhances exosome secretion through Syntenin-1.

Anaplasma phagocytophilum is an intracellular obligate parasite that causes granulocytic anaplasmosis. Effector Ats-1 is an important virulence factor of A. phagocytophilum. Multiomics screening and validation has been used to determine that Ats-1 regulates host cell apoptosis and energy metabolism through the respiratory chain mPTP axis. In this study, a total of 19 potential binding proteins of Ats-1 in host cells were preliminarily screened using a yeast two-hybrid assay, and the interaction between syntenin-1 (SDCBP) and Ats-1 was identified through immunoprecipitation. Bioinformatics analysis showed that SDCBP interacted with SDC1, SDC2, and SDC4 and participated in the host exosome secretion pathway. Further studies confirmed that Ats-1 induced the expression of SDC1, SDC2, and SDC4 in HEK293T cells through SDCBP and increased the exosome secretion of these cells. This indicated that SDCBP played an important role in Ats-1 regulating the exosome secretion of the host cells. These findings expand our understanding of the intracellular regulatory mechanism of A. phagocytophilum, which may enhance its own infection and proliferation by regulating host exosome pathways.

Novel Vertical Flow Immunoassay with Au@PtNPs for Rapid, Ultrasensitive, and On-Site Diagnosis of Human Brucellosis.

Brucellosis is an infectious zoonosis caused by Brucella with clinical symptoms of wavy fever, fatigue, and even invasion of tissues and organs in the whole body, posing a serious threat to public health around the world. Herein, a novel vertical flow immunoassay based on Au@Pt nanoparticles (Au@PtNPs-VFIA) was established for detection of Brucella IgG antibody in clinical serum samples. The testing card of Au@PtNPs-VFIA was manufactured by printing the purified Brucella LPS and goat antimouse IgG on the nitrocellulose membrane as the test-spot or control-spot, respectively. Au@PtNPs labeled with protein G (Au@PtNPs-prG) were concurrently employed as detection probes presenting visible spots and catalysts mimicking catalytic enzymes to catalyze the DAB substrate (H2O2 plus O-phenylenediamine) for deepening color development. The testing procedure of Au@PtNPs-VFIA takes 2-3 min, and the limit of detection (LOD) for Brucella antibody is 0.1 IU/mL, which is faster and more sensitive than that of Au@PtNP-based lateral flow immunoassay (Au@PtNPs-LFIA: 15 min and 1.56 IU/mL, respectively). By comparing with vertical flow immunoassay based on classic Au nanoparticles (AuNPs-VFIA), the Au@PtNPs-VFIA is 32 times or 16 times more sensitive with or without further development of DAB substrate catalysis. Au@PtNPs-VFIA did not react with the serum samples of Gram-negative bacterium infections but only weakly cross-reacted with diagnostic serum of Y. enterocolitica O9 infection. In detection of clinical samples, Au@PtNPs-VFIA was validated for possessing 98.33% sensitivity, 100% specificity, and 99.17% accuracy, which were comparable with or even better than those obtained by the Rose-Bengal plate agglutination test, serological agglutination test, AuNPs-VFIA, and Au@PtNPs-LFIA. Therefore, this newly developed Au@PtNPs-VFIA has potential for rapid, ultrasensitive, and on-site diagnosis of human Brucellosis in clinics.

[Preparation and immunogenicity evaluation of mRNA vaccine against porcine epidemic diarrhea].

Porcine epidemic diarrhea (PED) is a highly contagious disease that causes high mortality in suckling piglets. Although several licensed inactivated and live attenuated vaccines were widely used, the infection rate remains high due to unsatisfactory protective efficacy. In this study, mRNA vaccine candidates against PED were prepared, and their immunogenicity was evaluated in mice and pregnant sows. The mRNA PED vaccine based on heterodimer of viral receptor binding region (RBD) showed good immunogenicity. It elicited robust humoral and cellular immune responses in mice, and the neutralizing antibody titer reached 1:300 after a single vaccination. Furthermore, it induced neutralizing antibody level similar to that of the inactivated vaccine in pregnant sows. This study developed a new design of PED vaccine based on the mRNA-RBD strategy and demonstrated the potential for clinical application.

Detection of emerging HoBi-like Pestivirus (BVD-3) during an epidemiological investigation of bovine viral diarrhea virus in Xinjiang: a first-of-its-kind report.

Xinjiang pastoral area is the second largest pastoral area in China, accounting for 26.8% of the available grassland area in the country, and the geographical advantage of cattle breeding industry is very obvious. Bovine viral diarrhea virus (BVDV) has always been one of the important viral diseases that have plagued the development of cattle farming industry in the world. As one of the main pastoral areas of China's cattle farming industry, the Xinjiang pastoral area has also been deeply affected. In this study, 6,153 bovine serum samples were collected from 18 large-scale cattle farms in 13 cities in Xinjiang. The antibodies and antigens of 6,153 and 588 serum samples were detected by serological detection methods, respectively. Ten serum samples, which were antigen-positive by ELISA, were randomly selected for RT-PCR detection, sequencing, and phylogenetic analysis of suspected HoBi-like Pestivirus (HoBiPeV) strains. The results showed that the positive rates of BVDV antibodies and antigens were 53.68% (3,303/6,153) and 6.12% (36/588), respectively. One of the 10 randomly selected seropositive samples was infected with the HoBiPeV strain. HoBiPeV, also referred to as BVDV-3, is an emerging atypical Pestivirus that occurs in cattle and small ruminants, and its clinical signs are similar to those of BVDV infection. Based on the whole genome of the BVDV-3 reference strain (JS12/01) on the GenBank, the homology of the detected strain was 96.02%. The whole genome nucleotide sequence was submitted to the GenBank database, and the gene accession number was obtained: OP210314. The whole genome of isolate OP210314 was 12.239 nucleotides and contained a 5'-UTR of 340 nucleotides, a 3'-UTR of 199 nucleotides, and a large open reading frame (ORF) encoding a polyprotein consisting of 3,899 amino acids. In conclusion, the prevalence rate of BVDV infection in Xinjiang dairy cows is high, and the genetic diversity is increasing. This study successfully identified and isolated HoBiPeV in Xinjiang for the first time, posing a potential threat to the cattle industry in Xinjiang.

Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Previous studies suggested that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, in the present study, a Dap-seq approach was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes was identified using qRT-PCR. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), ftrA (BME_RS14525), cation-transporting P-type ATPase (zntA) (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. Furthermore, the iron utilization and growth assay experiments confirmed that rirA was involve in iron metabolism and growth of Brucella. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism, and the rirA was identified as a regulon of Irr and it also plays a role in iron metabolism of Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism.

Exosomes released by Brucella-infected macrophages inhibit the intracellular survival of Brucella by promoting the polarization of M1 macrophages.

Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.

LDH as an adjuvant makes Brucella outer-membrane vesicles and outer-membrane vesicle-associated proteins highly protective in mice.

Objectives: Existing Brucella vaccines are attenuated and can cause vaccine-associated brucellosis; and these safety concerns have affected their application. Although subunit vaccines have the advantages of safety, efficacy, low cost, and rapid production, they are usually poorly immunogenic and insufficient to trigger persistent immunity. Therefore, we added layered double hydroxide (LDH) as an adjuvant to Brucella subunit vaccine formulations to enhance the immune response to the antigen. Materials and Methods: LDH and Freund's adjuvant were combined with Brucella outer-membrane vesicles (OMVs) and OMV-associated proteins to form a subunit vaccine, respectively. The immunogenicity of LDH as an adjuvant was assessed in BALB/c mice. We examined levels of immunoglobulin G, G1, and G2a (IgG, IgG1, and IgG2a) antibodies (aBs); percentages of Cluster of Differentiation 4-positive (CD4+) and CD8+ T cells in peripheral-blood lymphocytes; and secretion of cytokines in mouse spleen lymphocytes. Finally, splenic index and splenic bacterial load were assessed via Brucella challenge experiments on mice. Results: The LDH subunit vaccine also produced high levels of specific aBs in mice compared with Freund's adjuvant subunit vaccine and induced mainly T-helper 1 cell (Th1)-type immune responses. In addition, mice in the LDH subunit vaccine group had significantly lower bacterial loads in their spleens than those in the Freund's adjuvant subunit vaccine group, and the LDH-OMV vaccine offered a higher level of protection against Brucella attack. Conclusion: LDH as an adjuvant-paired vaccine provided a high level of protection against Brucella infection.

Integrated bioinformatic analyses investigate macrophage-M1-related biomarkers and tuberculosis therapeutic drugs.

Tuberculosis (TB) is a common infectious disease linked to host genetics and the innate immune response. It is vital to investigate new molecular mechanisms and efficient biomarkers for Tuberculosis because the pathophysiology of the disease is still unclear, and there aren't any precise diagnostic tools. This study downloaded three blood datasets from the GEO database, two of which (GSE19435 and 83456) were used to build a weighted gene co-expression network for searching hub genes associated with macrophage M1 by the CIBERSORT and WGCNA algorithms. Furthermore, 994 differentially expressed genes (DEGs) were extracted from healthy and TB samples, four of which were associated with macrophage M1, naming RTP4, CXCL10, CD38, and IFI44. They were confirmed as upregulation in TB samples by external dataset validation (GSE34608) and quantitative real-time PCR analysis (qRT-PCR). CMap was used to predict potential therapeutic compounds for tuberculosis using 300 differentially expressed genes (150 downregulated and 150 upregulated genes), and six small molecules (RWJ-21757, phenamil, benzanthrone, TG-101348, metyrapone, and WT-161) with a higher confidence value were extracted. We used in-depth bioinformatics analysis to investigate significant macrophage M1-related genes and promising anti-Tuberculosis therapeutic compounds. However, more clinical trials were necessary to determine their effect on Tuberculosis.

Expanded catalogue of metagenome-assembled genomes reveals resistome characteristics and athletic performance-associated microbes in horse.

BACKGROUND: As a domesticated species vital to humans, horses are raised worldwide as a source of mechanical energy for sports, leisure, food production, and transportation. The gut microbiota plays an important role in the health, diseases, athletic performance, and behaviour of horses. RESULTS: Here, using approximately 2.2 Tb of metagenomic sequencing data from gut samples from 242 horses, including 110 samples from the caecum and 132 samples from the rectum (faeces), we assembled 4142 microbial metagenome-assembled genomes (MAG), 4015 (96.93%) of which appear to correspond to new species. From long-read data, we successfully assembled 13 circular whole-chromosome bacterial genomes representing novel species. The MAG contained over 313,568 predicted carbohydrate-active enzymes (CAZy), over 59.77% of which had low similarity match in CAZy public databases. High abundance and diversity of antibiotic resistance genes (ARG) were identified in the MAG, likely showing the wide use of antibiotics in the management of horse. The abundances of at least 36 MAG (e.g. MAG belonging to Lachnospiraceae, Oscillospiraceae, and Ruminococcus) were higher in racehorses than in nonracehorses. These MAG enriched in racehorses contained every gene in a major pathway for producing acetate and butyrate by fibre fermentation, presenting potential for greater amount of short-chain fatty acids available to fuel athletic performance. CONCLUSION: Overall, we assembled 4142 MAG from short- and long-read sequence data in the horse gut. Our dataset represents an exhaustive microbial genome catalogue for the horse gut microbiome and provides a valuable resource for discovery of performance-enhancing microbes and studies of horse gut microbiome. Video Abstract.

bta-miR-2904 inhibits bovine viral diarrhea virus replication by targeting viral-infection-induced autophagy via ATG13.

MicroRNAs (miRNAs) are endogenous small and noncoding RNA molecules (18-25 nt) that can regulate expression of their target genes post-transcriptionally. Previously, using high-throughput sequencing data obtained on a Solexa platform, we found that Bos taurus bta-miR-2904 (miR-2904) was significantly upregulated in Madin-Darby bovine kidney (MDBK) cells infected with bovine viral diarrhea virus (BVDV) strain NADL at 2, 6, and 18 h postinfection (hpi) compared to uninfected MDBK cells. Moreover, miR-2904 overexpression significantly reduced BVDV replication. However, the mechanism by which miR-2904 inhibits viral replication remains unclear. In this study, we used electron microscopy, laser confocal microscopy, dual-luciferase reporter analysis, real-time PCR, and Western blot assays to investigate the effect of the miR-2904 expression on BVDV NADL replication and virus-infection-induced autophagy. The results indicate that miR-2904 inhibits autophagy of MDBK cells by targeting autophagy-related gene 13 (ATG13), and overexpression of miR-2904 inhibited the replication of BVDV NADL.

Virus-like particles vaccines based on glycoprotein E0 and E2 of bovine viral diarrhea virus induce Humoral responses.

Bovine viral diarrhea/mucosal disease (BVD/MD) is a viral infectious disease that seriously endangers the health of cattle herds and brings serious economic losses to the global cattle industry. Virus-like particles (VLPs) are empty shell structures without viral nucleic acid, which are similar to natural virus particles in morphology and structure. Because of their strong immunogenicity and biological activity, some of them have been used as vaccines in clinical trials. In this study, we developed a strategy to generate BVDV (E0 + E2, E2 + E2) VLPs using an insect baculovirus expression vector system (BEVS). The VLPs obtained were detected by immunofluorescence assay (IFA), western blotting analyses and transmission electron microscope (TEM), and the results showed that VLPs of high purity were obtained. Mice immunized with VLPs (15 µg) and Freund's adjuvant (100 µl) elicited higher BVDV-neutralizing antibody in comparison with Freund's adjuvant control (p < 0.0001), and even on day 21 or 35 post-prime immunization, the neutralizing antibody levels of mice immunized with E0 + E2 or E2 + E2 VLPs were significantly higher compared with inactivated vaccine (p < 0.05). A subsequent challenge reveals that the viral loads of livers, kidneys, spleens, lungs and small intestines were significantly lower compared with control (p < 0.0001), and the viral loads of mice immunized with E0 + E2 or E2 + E2 VLPs in the small intestines were significantly lower compared with inactivated vaccine (p < 0.05). Thus, VLPs are a promising candidate vaccine and warrants further clinical evaluation.

Multiomics analyses reveals Anaplasma phagocytophilum Ats-1 induces anti-apoptosis and energy metabolism by upregulating the respiratory chain-mPTP axis in eukaryotic mitochondria.

BACKGROUND: Anaplasma translocated substrate 1 (Ats-1) is an effector of type 4 secretory systems (T4SS) and the main virulence factor of Anaplasma phagocytophilum. Ats-1 is involved in the regulation of host cell biological processes, but the specific molecular mechanism of its action is unclear. RESULTS: In this study, we identified Ats-1 as involved in mitochondrial respiratory regulation of HEK293T cells by multi-omics analysis. After intracellular expression of Ats-1, adenosine triphosphate levels and the proliferation of HEK293T cells were both up-regulated, while HEK293T cells apoptosis was inhibited. Ats-1 targeted translocation to the mitochondria where it up-regulated the expression of NDUFB5, NDUFB3, NDUFS7, COX6C, and SLC25A5, thereby enhancing energy production and inhibiting HEK293T cells apoptosis while enhancing HEK293T cells proliferation, and ultimately facilitating Anaplasma phagocytophilum replication in HEK293T cells. CONCLUSIONS: This study demonstrated that Anaplasma phagocytophilum Ats-1 induces anti-apoptosis and energy metabolism by upregulating the respiratory chain-mPTP axis in eukaryotic mitochondria. These results provide a better understanding of the pathogenic mechanism of Anaplasma phagocytophilum within host cells.

Survival characteristics and transcriptome profiling reveal the adaptive response of the Brucella melitensis 16M biofilm to osmotic stress.

Brucella can inhabit hostile environments, including osmotic stress. How Brucella responds collectively to osmotic stress is largely unexplored, particularly in spatially structured communities such as a biofilm. To gain insight into this growth mode, we set out to characterize the Brucella melitensis 16M biofilm, describe its phenotype, and carry out a comparative transcriptomic analysis between biofilms under osmotic stress and control conditions. We determined that the bacteria challenged with 1.5 M NaCl had a reduced ability to aggregate and form clumps and develop a biofilm; however, the salt stress promoted the release of the outer membrane vesicles from the biofilm. Together with the genotypical response to osmotic stress, we identified 279 differentially expressed genes in B. melitensis 16M grown under osmotic conditions compared with control conditions; 69 genes were upregulated and 210 downregulated. Under osmotic stress, the main changed genes of biofilm were predicted to be involved in flagellar assembly, cell envelope, translation, small RNA regulation, transport and binding proteins, and energy metabolism. In addition, the ABC transporter was enriched in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We highlight 12 essential ABC transporter genes associated with a bacterial response to osmotic stress at the biofilm stage, including one specific locus, BME_RS12880, mediating betaine accumulation in biofilms to eliminate osmotic stress. The current study results can help researchers gain insights into B. melitensis 16M biofilm adaptation to osmotic stress and provide information for developing intervention strategies to control Brucella.

The Flagellar Transcriptional Regulator FtcR Controls Brucella melitensis 16M Biofilm Formation via a betI-Mediated Pathway in Response to Hyperosmotic Stress.

The expression of flagellar proteins in Brucella species likely evolved through genetic transference from other microorganisms, and contributed to virulence, adaptability, and biofilm formation. Despite significant progress in defining the molecular mechanisms behind flagellar gene expression, the genetic program controlling biofilm formation remains unclear. The flagellar transcriptional factor (FtcR) is a master regulator of the flagellar system's expression, and is critical for B. melitensis 16M's flagellar biogenesis and virulence. Here, we demonstrate that FtcR mediates biofilm formation under hyperosmotic stress. Chromatin immunoprecipitation with next-generation sequencing for FtcR and RNA sequencing of ftcR-mutant and wild-type strains revealed a core set of FtcR target genes. We identified a novel FtcR-binding site in the promoter region of the osmotic-stress-response regulator gene betI, which is important for the survival of B. melitensis 16M under hyperosmotic stress. Strikingly, this site autoregulates its expression to benefit biofilm bacteria's survival under hyperosmotic stress. Moreover, biofilm reduction in ftcR mutants is independent of the flagellar target gene fliF. Collectively, our study provides new insights into the extent and functionality of flagellar-related transcriptional networks in biofilm formation, and presents phenotypic and evolutionary adaptations that alter the regulation of B. melitensis 16M to confer increased tolerance to hyperosmotic stress.

Using a Relative Quantitative Proteomic Method to Identify Differentially Abundant Proteins in Brucella melitensis Biovar 3 and Brucella melitensis M5-90.

Brucellosis, caused by Brucella spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, B. melitensis biovar 3 and compare it with those expressed by B. melitensis M5-90. The higher level of virulence for B. melitensis biovar 3 compared to B. melitensis M5-90 was validated in vitro and in vivo. A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the B. melitensis proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to B. melitensis M5-90 and B. melitensis biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in B. melitensis biovar 3 was 8-fold and 2-fold higher than in B. melitensis M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of B. melitensis M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified Brucella virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.

Molecular Detection of Zoonotic and Veterinary Pathogenic Bacteria in Pet Dogs and Their Parasitizing Ticks in Junggar Basin, North-Western China.

Despite the recognized epidemiological importance of ticks as vectors for pathogens that cause numerous zoonotic and veterinary diseases, data regarding the pathogens of pet dogs and their parasitic ticks in the Junggar Basin are scarce. In this study, a total of 178 blood samples and 436 parasitic ticks were collected from pet dogs in Junggar Basin, Xinjiang Uygur Autonomous Region (XUAR), north-western China. All ticks were identified as Rhipicephalus turanicus sensu stricto (s.s.) according to morphological and molecular characteristics. Rh. turanicus s.s. ticks were collected from pet dogs in China for the first time. Seven tick-borne pathogens, such as Ehrlichia chaffeensis, Anaplasma phagocytophilum, Rickettsia massiliae, Candidatus R. barbariae, Brucella spp., Rickettsia sibirica, and Anaplasma ovis, were detected from ticks, whereas the first five bacteria were detected from blood samples of dogs. Brucella spp. was the most predominant pathogen in both blood samples and ticks of pet dogs, with the detection rates of 16.29 and 16.74%, respectively. Moreover, 17 ticks and 1 blood sample were co-infected with two pathogens, and 1 tick was co-infected with three pathogens. This study provided molecular evidence for the occurrence of Anaplasma spp., Ehrlichia spp., Rickettsia spp., and Brucella spp. circulating in pet dogs and their parasitic ticks in Junggar Basin, north-western China. These findings extend our knowledge of the tick-borne pathogens in pet dogs and their parasitic ticks in Central Asia; therefore, further research on these pathogens and their role in human and animal diseases is required.

A combined application of molecular docking technology and indirect ELISA for the serodiagnosis of bovine tuberculosis.

BACKGROUND: There is an urgent need to find reliable and rapid bovine tuberculosis (bTB) diagnostics in response to the rising prevalence of bTB worldwide. Toll-like receptor 2 (TLR2) recognizes components of bTB and initiates antigen-presenting cells to mediate humoral immunity. Evaluating the affinity of antigens with TLR2 can form the basis of a new method for the diagnosis of bTB based on humoral immunity. OBJECTIVES: To develop a reliable and rapid strategy to improve diagnostic tools for bTB. METHODS: In this study, we expressed and purified the sixteen bTB-specific recombinant proteins in Escherichia coli. The two antigenic proteins, MPT70 and MPT83, which were most valuable for serological diagnosis of bTB were screened. Molecular docking technology was used to analyze the affinity of MPT70, MPT83, dominant epitope peptide of MPT70 (M1), and dominant epitope peptide MPT83 (M2) with TLR2, combined with the detection results of enzyme-linked immunosorbent assay to evaluate the molecular docking effect. RESULTS: The results showed that interaction surface Cα-atom root mean square deviation of proteins (M1, M2, MPT70, MPT83)-TLR2 protein are less than 2.5 A, showing a high affinity. It is verified by clinical serum samples that MPT70, MPT83, MPT70-MPT83 showed good diagnostic potential for the detection of anti-bTB IgG and M1, M2 can replace the whole protein as the detection antigen. CONCLUSIONS: Molecular docking to evaluate the affinity of bTB protein and TLR2 combined with ELISA provides new insights for the diagnosis of bTB.

Brucella melitensis outer membrane protein 25 interacts with ferritin heavy polypeptide 1 in human trophoblast cells.

Outer membrane protein 25 (OMP25) is involved in Brucella virulence and serves an important role in Brucella pathogenesis during infection. However, the protein interacting with OMP25 in host cells remains to be elucidated. The present study constructed a cDNA library from Brucella melitensis 16M­infected human placenta trophoblastic cells (HPT­8) and identified and confirmed the interaction between Brucella OMP25 and ferritin heavy polypeptide 1 (FTH1) of HPT­8 using yeast two­hybrid and co­immunoprecipitation technologies. The expression of Toll­like receptor (TLR)4, myeloid differentiation primary response protein MyD88 (MyD88) mRNA and inflammatory factors was detected by RNA interference. The results showed that OMP25 interacted with FTH1. Subsequently, TLR4 and MyD88 mRNA expression levels and inflammatory factors, such as nitric oxide, lactate dehydrogenase and TNF­α, significantly increased after inserting OMP25 into the HPT­8 cells. Notably, a low dose of OMP25 resulted in immunological protection, whereas high dose of OMP25 resulted in a cytotoxic effect on the HPT­8 cells. It is suggested that OMP25 and FTH1 serve important roles in intracellular parasitism of Brucella and inhibition of expression.