Recombinant production of SAG1 fused with xylanase in Pichia pastoris induced higher protective immunity against Eimeria tenella infection in chicken.

Chicken coccidiosis is an intestinal disease caused by the parasite Eimeria, which severely damages the growth of chickens and causes significant economic losses in the poultry industry. Improvement of the immune protective effect of antigens to develop high efficiency subunit vaccines is one of the hotspots in coccidiosis research. Sporozoite-specific surface antigen 1 (SAG1) of Eimeria tenella (E. tenella) is a well-known protective antigen and is one of the main target antigens for the development of subunit, DNA and vector vaccines. However, the production and immunoprotective effects of SAG1 need to be further improved. Here, we report that both SAG1 from E. tenella and its fusion protein with the xylanase XynCDBFV-SAG1 are recombinant expressed and produced in Pichia pastoris (P. pastoris). The substantial expression quantity of fusion protein XynCDBFV-SAG1 is achieved through fermentation in a 15-L bioreactor, reaching up to about 2 g/L. Moreover, chickens immunized with the fusion protein induced higher protective immunity as evidenced by a significant reduction in the shedding of oocysts after E. tenella challenge infection compared with immunized with recombinant SAG1. Our results indicate that the xylanase enhances the immunogenicity of subunit antigens and has the potential for developing novel molecular adjuvants. The high expression level of fusion protein XynCDBFV-SAG1 in P. pastoris holds promise for the development of effective recombinant anti-coccidial subunit vaccine.

Impact of MgtC on the Fitness of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis is an extracellular foodborne pathogen and usually causes self-limiting diarrhea in healthy humans. MgtC is known as a key subversion factor that contributes to intramacrophage adaptation and intracellular survival in certain important pathogens. Whether MgtC influences the fitness of Y. pseudotuberculosis is unclear. According to in silico analysis, MgtC in Y. pseudotuberculosis might share similar functions with other bacterial pathogens, such as Salmonella. Studies indicated that MgtC was clearly required for Y. pseudotuberculosis growth in vitro and bacterial survival in macrophages under Mg2+ starvation. Transcriptome analysis by RNA-seq indicated that 127 differentially expressed genes (DEGs) (fold change > 2 and p < 0.001) were discovered between wild-type PB1+ and mgtC mutant inside macrophages. However, a lack of MgtC only moderately, albeit significantly, reduced the virulence of Y. pseudotuberculosis in mice. Overall, this study provides additional insights for the role of MgtC in Y. pseudotuberculosis.

ClpP protease modulates bacterial growth, stress response, and bacterial virulence in Brucella abortus.

The process of intracellular proteolysis through ATP-dependent proteases is a biologically conserved phenomenon. The stress responses and bacterial virulence of various pathogenic bacteria are associated with the ATP-dependent Clp protease. In this study, a Brucella abortus 2308 strain, ΔclpP, was constructed to characterize the function of ClpP peptidase. The growth of the ΔclpP mutant strain was significantly impaired in the TSB medium. The results showed that the ΔclpP mutant was sensitive to acidic pH stress, oxidative stress, high temperature, detergents, high osmotic environment, and iron deficient environment. Additionally, the deletion of clpP significantly affected Brucella virulence in macrophage and mouse infection models. Integrated transcriptomic and proteomic analyses of the ΔclpP strain showed that 1965 genes were significantly affected at the mRNA and/or protein levels. The RNA-seq analysis indicated that the ΔclpP strain exhibited distinct gene expression patterns related to energy production and conversion, cell wall/membrane/envelope biogenesis, carbohydrate transport, and metabolism. The iTRAQ analysis revealed that the differentially expressed proteins primarily participated in amino acid transport and metabolism, energy production and conversion, and secondary metabolites biosynthesis, transport and catabolism. This study provided insights into the preliminary molecular mechanism between Clp protease to bacterial growth, stress response, and bacterial virulence in Brucella strains.

Transcriptome and the gut microbiome analysis of the impacts of Brucella abortus oral infection in BALB/c mice.

Brucellosis is a zoonotic infectious disease caused by Brucella spp, which could cause serious economic losses to animal husbandry and threaten human public health. Ingestion of contaminated animal products is a common way to acquire Brucella infection in humans, while research on effect of oral Brucella infection on host gut microbiota and the gene expression in intestinal tissues is limited. In the present study, 16S rRNA sequencing and RNA sequencing were conducted to explore gut microbiota and expression profiles of mRNAs in the colon of BALB/c mice, which were infected by Brucella abortus 2308. The fecal samples were collected at 7 and 28 days post infection to observe changes in the gut microbiota during Brucella infection. In the alpha diversity analysis, significantly increased Chao 1 index was observed at 28 days after Brucella infection. The Bray-Curtis distancebased principal coordinate analysis indicated that the WT group showed a separation from the Brucella infection groups. In addition, analysis of composition of microbes revealed that Prevotellaceae_NK3B31_group were more abundant in 1 week and 4 week infection groups, while Turicibacter was only more abundant in 4 week infection group. Based on the RNA-seq assay, a total of 45 differentially expressed genes were detected between Brucella abortus infection group and control group. Furthermore, KEGG pathway enrichment analysis showed that protein processing in endoplasmic reticulum, Legionellosis, Spliceosome, Hippo signaling pathway and Influenza A were significantly enriched in response to Brucella abortus infection. Our finding will help to improve the knowledge of the mechanisms underlying Brucella infection and may provide novel targets for future treatment of this pathogen infection.

Molecular Characteristics of Bovine Viral Diarrhea Virus Strains Isolated from Persistently Infected Cattle.

In this study, we reported the isolation, identification, and molecular characteristics of nine BVDV strains that were isolated from the serum of persistently infected cattle. The new strains were designated as BVDV TJ2101, TJ2102, TJ2103, TJ2104, TJ2105, TJ2106, TJ2107, TJ2108 and TJ2109. The TJ2102 and TJ2104 strains were found to be cytopathic BVDV, and the other strains were non-cytopathic BVDV. An alignment and phylogenetic analysis showed that the new isolates share 92.2-96.3% homology with the CP7 strain and, thus, were classified as the BVDV-1b subgenotype. A recombination analysis of the genome sequences showed that the new strains could be recombined by the major parent BVDV-1a NADL strain and the minor parent BVDV-1m SD-15 strain. Some genome variations or unique amino acid mutations were found in 5'-UTR, E0 and E2 of these new isolates. In addition, a potential linear B cell epitopes prediction showed that the potential linear B cell epitope at positions 56-61 is highly variable in BVDV-1b. In conclusion, the present study has identified nine strains of BVDV from persistently infected cattle in China. Further studies on the virulence and pathogenesis of these new strains are recommended.

A homolog of low molecular weight protein tyrosine phosphatase isolated from Brucella melitensis displays an acidic dual specific phosphatase activity, nonessential for bacterial resistance to bactericidal factors and virulence.

Brucellosis is a bacterial infectious zoonosis which is spread worldwide, caused by Brucella, with infertility and abortion in domestic animals. Protein-tyrosine phosphatase (PTPs) have been discovered in many kinds of bacterial species, which play crucial roles in many aspects, such as bacterial physiology and virulence. However, no PTPs have been identified in Brucella to date. Here, we identified a novel gene BM28_RS15985 in Brucella melitensis that encodes a homolog of a low weight molecular PTP. Enzyme activity analysis showed that this PTP is a dual specific phosphatase, removing phosphate group from phosphotyrosine and phosphoserine/phosphothreonine peptides, which was designated as Dsp1. The optimal pH of the Dsp1 enzyme activity were 5.5, suggesting that the Dsp1 is an acidic phosphatase, and the optimal reaction temperature of the Dsp1 was 35.0 °C. Besides, the Michaelis constant and maximum reaction velocity of the Dsp1 were 40.17 mM and 24.33 nM/min/mg, respectively. In further study, we investigated the role of Dsp1 in B. melitensis phenotype and virulence. Growth curve and resistance test exhibited that the dsp1 had no role in Brucella growth and resisting bactericidal factors. Cell and animal infection experiment showed that the dsp1 deletion did not affect the intracellular survival and virulence of B. melitensis. In summary, we identified a novel acidic dual specific phosphatase in B. melitensis and evaluated its characteristics of the enzyme activity, this study will expand the understanding of Brucella phosphatase.

The Pseudogene BMEA_B0173 Deficiency in Brucella melitensis Contributes to M-epitope Formation and Potentiates Virulence in a Mice Infection Model.

It is widely acknowledged that pseudogenes play important roles in bacterial diversification and evolution and participate in gene regulation and RNA interference (RNAi). However, the function of most pseudogenes in Brucella spp remains poorly understood, warranting further studies.To comprehensively analyze the function of the pseudogenes BMEA_B0173 in Brucella melitensis strain 63/9, a BMEA_B0173 in-frame deleted mutant strain was constructed. Then, the phenotypes of the mutant strain, such as growth characteristics and bacterial virulence, were assessed in mice infection models. Finally, iTRAQ analysis was performed to investigate the gene expression profile affected by the pseudogenes BMEA_B0173. In this study, we found that BMEA_B0173 deletion exhibited increased agglutination with M monospecific sera. In a mouse model of chronic infection, the BMEA_B0173 deletion strain displayed increased colonization in the spleen compared to the wild-type pathogen. The iTRAQ assay revealed that 252 proteins were differentially expressed between the BMEA_B0173 deletion and the wild-type strains. In addition, deletion of BMEA_B0173 significantly increased the expression of proteins involved in the denitrification pathway, iron metabolism, and several transcriptional regulators, which might cause increased virulence of the mutant strain. In conclusion, this study preliminary uncovered the function of the pseudogene BMEA_B0173 in Brucella melitensis 63/9 and provided novel insights for studying the pathogenesis of Brucella strains.

Regulated necrosis, a proinflammatory cell death, potentially counteracts pathogenic infections.

Since the discovery of cell apoptosis, other gene-regulated cell deaths are gradually appreciated, including pyroptosis, ferroptosis, and necroptosis. Necroptosis is, so far, one of the best-characterized regulated necrosis. In response to diverse stimuli (death receptor or toll-like receptor stimulation, pathogenic infection, or other factors), necroptosis is initiated and precisely regulated by the receptor-interacting protein kinase 3 (RIPK3) with the involvement of its partners (RIPK1, TRIF, DAI, or others), ultimately leading to the activation of its downstream substrate, mixed lineage kinase domain-like (MLKL). Necroptosis plays a significant role in the host's defense against pathogenic infections. Although much has been recognized regarding modulatory mechanisms of necroptosis during pathogenic infection, the exact role of necroptosis at different stages of infectious diseases is still being unveiled, e.g., how and when pathogens utilize or evade necroptosis to facilitate their invasion and how hosts manipulate necroptosis to counteract these detrimental effects brought by pathogenic infections and further eliminate the encroaching pathogens. In this review, we summarize and discuss the recent progress in the role of necroptosis during a series of viral, bacterial, and parasitic infections with zoonotic potentials, aiming to provide references and directions for the prevention and control of infectious diseases of both human and animals.

Bacteriological and molecular typing of Clostridium perfringens strains isolated in retail beef in Beijing, China.

Clostridium perfringens is an important zoonotic pathogen. This study was designed to explore the prevalence and toxin types of C. perfringens in retail beef collected from Beijing, China. Among 221 beef samples collected, 53 samples were positive for C. perfringens, resulting in the average prevalence as 23.98%. By toxin gene-based typing, the most C. perfringens strains belong to type A (96.23%, 51/53), only 2 strains were identified as type D. By a multi-locus sequence typing (MLST)-based analysis, a total of 36 sequence types (STs) were detected, and the most STs (n=30) represented just a single strain. These finding suggested that the prevalence of C. perfringens in retail beef in Beijing was considerably high and these bacteria displayed extreme diversity in genetics.

Deletion of the Transcriptional Regulator MucR in Brucella canis Affects Stress Responses and Bacterial Virulence.

The transcriptional regulator MucR is related to normal growth, stress responses and Brucella virulence, and affects the expression of various virulence-related genes in smooth-type Brucella strains. However, the function of MucR in the rough-type Brucella canis remains unknown. In this study, we discovered that MucR protein was involved in resistance to heat stress, iron-limitation, and various antibiotics in B. canis. In addition, the expression level of various bacterial flagellum-related genes was altered in mucR mutant strain. Deletion of this transcriptional regulator in B. canis significantly affected Brucella virulence in RAW264.7 macrophage and mice infection model. To gain insight into the genetic basis for distinctive phenotypic properties exhibited by mucR mutant strain, RNA-seq was performed and the result showed that various genes involved in translation, ribosomal structure and biogenesis, signal transduction mechanisms, energy production, and conversion were significantly differently expressed in ΔmucR strain. Overall, these studies have not only discovered the phenotype of mucR mutant strain but also preliminarily uncovered the molecular mechanism between the transcriptional regulator MucR, stress response and bacterial virulence in B. canis.

Safety and Transcriptome Analysis of Live Attenuated Brucella Vaccine Strain S2 on Non-pregnant Cynomolgus Monkeys Without Abortive Effect on Pregnant Cynomolgus Monkeys.

Brucellosis, caused by Brucella spp., is an important zoonotic disease leading to enormous economic losses in livestock, posing a great threat to public health worldwide. The live attenuated Brucella suis (B. suis) strain S2, a safe and effective vaccine, is widely used in animals in China. However, S2 vaccination in animals may raise debates and concerns in terms of safety to primates, particularly humans. In this study, we used cynomolgus monkey as an animal model to evaluate the safety of the S2 vaccine strain on primates. In addition, we performed transcriptome analysis to determine gene expression profiling on cynomolgus monkeys immunized with the S2 vaccine. Our results suggested that the S2 vaccine was safe for cynomolgus monkeys. The transcriptome analysis identified 663 differentially expressed genes (DEGs), of which 348 were significantly upregulated and 315 were remarkably downregulated. The Gene Ontology (GO) classification and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these DEGs were involved in various biological processes (BPs), including the chemokine signaling pathway, actin cytoskeleton regulation, the defense response, immune system processing, and the type-I interferon signaling pathway. The molecular functions of the DEGs were mainly comprised of 2'-5'-oligoadenylate synthetase activity, double-stranded RNA binding, and actin-binding. Moreover, the cellular components of these DEGs included integrin complex, myosin II complex, and blood microparticle. Our findings alleviate the concerns over the safety of the S2 vaccine on primates and provide a genetic basis for the response from a mammalian host following vaccination with the S2 vaccine.

Phylogenetic and genomic analysis reveals high genomic openness and genetic diversity of Clostridium perfringens.

Clostridium perfringens is associated with a variety of diseases in both humans and animals. Recent advances in genomic sequencing make it timely to re-visit this important pathogen. Although the genome sequence of C. perfringens was first determined in 2002, large-scale comparative genomics with isolates of different origins is still lacking. In this study, we used whole-genome sequencing of 45 C. perfringens isolates with isolation time spanning an 80-year period and performed comparative analysis of 173 genomes from worldwide strains. We also conducted phylogenetic lineage analysis and introduced an openness index (OI) to evaluate the openness of bacterial genomes. We classified all these genomes into five lineages and hypothesized that the origin of C. perfringens dates back to ~80 000 years ago. We showed that the pangenome of the 173 C. perfringens strains contained a total of 26 954 genes, while the core genome comprised 1020 genes, accounting for about a third of the genome of each isolate. We demonstrated that C. perfringens had the highest OI compared with 51 other bacterial species. Intact prophage sequences were found in nearly 70.0 % of C. perfringens genomes, while CRISPR sequences were found only in ~40.0 %. Plasmids were prevalent in C. perfringens isolates, and half of the virulence genes and antibiotic resistance genes (ARGs) identified in all the isolates could be found in plasmids. ARG-sharing network analysis showed that C. perfringens shared its 11 ARGs with 55 different bacterial species, and a high frequency of ARG transfer may have occurred between C. perfringens and species in the genera Streptococcus and Staphylococcus. Correlation analysis showed that the ARG number in C. perfringens strains increased with time, while the virulence gene number was relative stable. Our results, taken together with previous studies, revealed the high genome openness and genetic diversity of C. perfringens and provide a comprehensive view of the phylogeny, genomic features, virulence gene and ARG profiles of worldwide strains.

First identification of a Brucella abortus biovar 4 strain from yak in Tibet, China.

Brucellosis is one of the major zoonotic diseases in the world. In China, understanding on its causative agent Brucella is still limited. Recently, we isolated a Brucella strain XZ19-1 from yak in Lhasa, Tibet. Phenotypical characterization proved that it belongs to B. abortus biovar 4, a biotype that has never been reported in China. MLVA-16 genotyping revealed a novel profile (4-5-3-12-2-2-3-3-8-32-8-5-4-3-3-3) in this strain, while MLST sequence typing demonstrated that it belongs to ST 71. Furthermore, the whole genome of XZ19-1 strain was sequenced. Subsequent phylogenetic analysis demonstrated that XZ19-1was genetically more closely related to B. abortus strains originated from European countries rather than to those collected from China previously. Isolation and identification of XZ19-1 strain may thus indicate a unique Brucella lineage existing in Qing-Tibet plateau. These findings will help to improve the diagnosis and epidemiological studies of brucellosis in animals and human in this part of China.

Potential Diagnostic Value of the Peripheral Blood Mononuclear Cell Transcriptome From Cattle With Bovine Tuberculosis.

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis. During early-stage infection, M. bovis-infected cattle shed mycobacteria through nasal secretions, which can be detected via nested-polymerase chain reaction (PCR) experiments. Little research has focused on immune responses in nested PCR-positive (bTB PCR-P) or nested PCR-negative (bTB PCR-N) M. bovis-infected cattle. Here, we investigated the transcriptomes of peripheral blood mononuclear cells (PBMCs), with or without stimulation by purified protein derivative of bovine tuberculin (PPD-B), among bTB PCR-P, bTB PCR-N, and healthy cattle using RNA-Seq. We also explored the potential value of PBMC transcripts as novel biomarkers for diagnosing bTB. Numerous differentially expressed genes were identified following pair-wise comparison of different groups, with or without PPD-B stimulation (adjusted p < 0.05). Compared with healthy cattle, bTB PCR-P, and bTB PCR-N cattle shared 5 significantly dysregulated biological pathways, including Cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, Hematopoietic cell lineage, Osteoclast differentiation and HTLV-I infection. Notably, dysregulated biological pathways of bTB PCR-P and bTB PCR-N cattle were associated with cell death and phagocytosis, respectively. Lymphotoxin alpha and interleukin-8 could potentially differentiate M. bovis-infected and healthy cattle upon stimulation with PPD-B, with area-under-the-curve (AUC) values of 0.9991 and 0.9343, respectively. B cell lymphoma 2 and chitinase 3-like 1 might enable differentiation between bTB PCR-P and bTB PCR-N upon stimulation with PPD-B, with AUC values of 0.9100 and 0.8893, respectively. Thus, the PBMC transcriptome revealed the immune responses in M. bovis-infected cattle (bTB PCR-P and bTB PCR-N) and may provide a novel sight in bTB diagnosis.

Brucellosis in China: history, progress and challenge.

Brucellosis is a neglected zoonosis. It causes acute febrile illness and a potentially debilitating chronic infection in humans, and livestock infection has substantial socioeconomic impact. Over the past two decades, improvements have been made to better understand the various aspects of human and animal brucellosis. Meanwhile, especially in the developing world, immense challenges that remain in controlling and eradicating brucellosis are novel diagnostics tools and efficacious vaccines. Here, we will focus on the remarkable issues on epidemiological survey, as well as the priority and challenge of brucellosis in China. Brucellosis will be controlled with meaningful collaboration between local and public partnerships effectively applying a One Health framework.

Complete Genome Sequence of Mycoplasma bovis Strain XBY01, Isolated from Henan Province, China.

We report the complete genome sequence of Mycoplasma bovis strain XBY01, which was isolated from a severely diseased young calf in Henan Province, China, in 2019. The genome of XBY01 contains a single circular chromosome of 986,067 bp, with a GC content of 29.30%.

Mutations in PB2 and HA enhanced pathogenicity of H4N6 avian influenza virus in mice.

The H4 subtype avian influenza virus (AIV) continues to circulate in both wild birds and poultry, and occasionally infects mammals (e.g. pigs). H4-specific antibodies have also been detected in poultry farm workers, which suggests that H4 AIV poses a potential threat to public health. However, the molecular mechanism by which H4 AIVs could gain adaptation to mammals and whether this has occurred remain largely unknown. To better understand this mechanism, an avirulent H4N6 strain (A/mallard/Beijing/21/2011, BJ21) was serially passaged in mice and mutations were characterized after passaging. A virulent mouse-adapted strain was generated after 12 passages, which was tentatively designated BJ21-MA. The BJ21-MA strain replicated more efficiently than the parental BJ21, both in vivo and in vitro. Molecular analysis of BJ21-MA identified four mutations, located in proteins PB2 (E158K and E627K) and HA (L331I and G453R, H3 numbering). Further studies showed that the introduction of E158K and/or E627K substitutions into PB2 significantly increased polymerase activity, which led to the enhanced replication and virulence of BJ21-MA. Although individual L331I or G453R substitutions in HA did not change the pathogenicity of BJ21 in mice, both mutations significantly enhanced virulence. In conclusion, our data presented in this study demonstrate that avian H4 virus can adapt to mammals by point mutations in PB2 or HA, which consequently poses a potential threat to public health.

Deletion of the LuxR-type regulator VjbR in Brucella canis affects expression of type IV secretion system and bacterial virulence, and the mutant strain confers protection against Brucella canis challenge in mice.

Brucella spp. are facultative intracellular pathogens and zoonotic agents which pose a huge threat to human health and animal husbandry. The B. melitensis, B. abortus, and B. suis cause undulant fever and influenza-like symptoms in humans. However, the effects of B. canis have not been extensively studied. The quorum sensing-dependent transcriptional regulator VjbR influences the Brucella virulence in smooth type Brucella strains, such as B. melitensis, B. abortus and rough type Brucella ovis. However, the function of VjbR in the rough-type B. canis is unknown. In the present study, we discovered that deletion of this regulator significantly affected Brucella virulence in macrophage and mice infection models. The expression levels of virB operon and the ftcR gene were significantly altered in the vjbR mutant strain. We further investigated the protective effect of different doses of the vjbR mutant in mice and the results indicated that VjbR conferred protection against the virulent B. canis strain. This study presents the first evidence that the transcriptional regulator VjbR has important function in B. canis. In addition, according to its reduced virulence and the protective immunity it induces in mice, it can be a potential live attenuated vaccine against B. canis.