PPNet: Identifying Functional Association Networks by Phylogenetic Profiling of Prokaryotic Genomes.

Identification of microbial functional association networks allows interpretation of biological phenomena and a greater understanding of the molecular basis of pathogenicity and also underpins the formulation of control measures. Here, we describe PPNet, a tool that uses genome information and analysis of phylogenetic profiles with binary similarity and distance measures to derive large-scale bacterial gene association networks of a single species. As an exemplar, we have derived a functional association network in the pig pathogen Streptococcus suis using 81 binary similarity and dissimilarity measures which demonstrates excellent performance based on the area under the receiver operating characteristic (AUROC), the area under the precision-recall (AUPR), and a derived overall scoring method. Selected network associations were validated experimentally by using bacterial two-hybrid experiments. We conclude that PPNet, a publicly available (https://github.com/liyangjie/PPNet), can be used to construct microbial association networks from easily acquired genome-scale data. IMPORTANCE This study developed PPNet, the first tool that can be used to infer large-scale bacterial functional association networks of a single species. PPNet includes a method for assigning the uniqueness of a bacterial strain using the average nucleotide identity and the average nucleotide coverage. PPNet collected 81 binary similarity and distance measures for phylogenetic profiling and then evaluated and divided them into four groups. PPNet can effectively capture gene networks that are functionally related to phenotype from publicly prokaryotic genomes, as well as provide valuable results for downstream analysis and experiment testing.

P38 MAPK pathway regulates the expression of resistin in porcine alveolar macrophages via Ets2 during Haemophilus parasuis stimulation.

Haemophilus parasuis is a widespread bacterial pathogen causing acute systemic inflammation and leading to the sudden death of piglets. Resistin, a multifunctional peptide hormone previously demonstrated to influence the inflammation in porcine, was extremely increased in H. parasuis-infected tissues. However, the mechanism of resistin expression regulation in porcine, especially during pathogen infection, remains unclear. In the present study, we explored for the first time the transcription factor and signaling pathway mediating the expression of pig resistin during H. parasuis stimulation. We found that H. parasuis induced the expression of pig resistin in a time- and dose-dependent manner via the transcription factor Ets2 in porcine alveolar macrophages during H. parasuis stimulation. Moreover, the expression of Ets2 was mediated by the activation of the p38 MAPK pathway induced by H. parasuis, thus promoting resistin production. These results revealed a novel view of the molecular mechanism of pig resistin production during acute inflammation induced by pathogenic bacteria.

Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques.

African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) with clinical symptoms of high fever, hemorrhages and high mortality rate, posing a threat to the global swine industry and food security. Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection. In this study, a recombinase polymerase amplification (RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF. As a highly sensitive method, RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lwCas13a (previously known as C2c2) through quantitative real-time PCR (qPCR) and has the same or even higher sensitivity than the traditional qPCR method. A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of TaqMan qPCR. Likewise, RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity. This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100% for both ASFV positive and negative samples. RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.

Genetic and Molecular Characterization of H9N2 Avian Influenza Viruses Isolated from Live Poultry Markets in Hubei Province, Central China, 2013-2017.

H9N2 subtype avian influenza virus (AIV) is an influenza A virus that is widely spread throughout Asia, where it jeopardizes the poultry industry and provides genetic material for emerging human pathogens. To better understand the epidemicity and genetics of H9 subtype AIVs, we conducted active surveillance in live poultry markets (LPMs) in Hubei Province from 2013 to 2017. A total of 4798 samples were collected from apparent healthy poultry and environment. Real-time RT-PCR revealed that the positivity rate of influenza A was 26.6% (1275/4798), of which the H9 subtype accounted for 50.3% (641/1275) of the positive samples. Of the 132 H9N2 viral strains isolated, 48 representative strains were subjected to evolutionary analysis and genotyping. Phylogenetic analysis revealed that all H9N2 viral genes had 91.1%-100% nucleotide homology, clustered with genotype 57, and had high homology with human H9N2 viruses isolated from 2013 to 2017 in China. Using a nucleotide divergence cutoff of 95%, we identified ten distinct H9N2 genotypes that continued to change over time. Molecular analysis demonstrated that six H9N2 isolates had additional potential glycosylation sites at position 218 in the hemagglutinin protein, and all isolates had I155T and Q226L mutations. Moreover, 44 strains had A558V mutations in the PB2 protein and four had E627V mutations, along with H9N2 human infection strains A/Beijing/1/2016 and A/Beijing/1/2017. These results emphasize that the H9N2 influenza virus in Hubei continues to mutate and undergo mammalian adaptation changes, indicating the necessity of strengthening the surveillance of the AIV H9N2 subtype in LPMs.

Isolation, identification and biological characteristics of Mycoplasma bovis in yaks.

Mycoplasma bovis (M. bovis) is one of the important pathogens which may cause bovine respiratory disease syndrome (BRDS), and results in huge economic losses for yaks (Bos gaurus) breeding industry. However, there is limited information about M. bovis in yaks. In our study, 145 nasal mucus samples from yaks with pneumonia were collected to clarify. Bacteriological determination was carried out through biochemical identification and Polymerase Chain Reaction (PCR) detection. And ten strains of Mycoplasma bovis (M. bovis) were found from collected samples. Then, the growth curve of isolated strains was determined by the change of optical density (OD630), pH value and Color Change Cnit (CCU). K-B disk method was also used for antimicrobial susceptibility testing. Results of colony morphology and biochemical testing were consistent with the biological characters of M. bovis. The nucleotide sequences of uvrC specific gene and 16S rRNA gene among the 10 strains were highly homologous. The growth curve assay showed that the isolates cultured in PPLO medium were in lag phase for 24 h, entered stable period in 42 h, and entered decline phase after 78 h. The isolates were found resistant to macrolides, aminoglycosides and lincomycin at various degrees, but they were sensitive or moderately sensitive to doxycycline and kanamycin under antimicrobial susceptibility analysis. In conclusion, the results provided certain reference for the follow-up research and guiding for the treatment of M. bovis in yaks.

P38 mitogen-activated protein kinase promotes Wnt/ß-catenin signaling by impeding Dickkofp-1 expression during Haemophilus parasuis infection.

Haemophilus parasuis induces severe acute systemic infection in pigs, characterized by fibrinous polyserositis, polyarthritis and meningitis. Our previous study demonstrated that H. parasuis induced the activation of p38 mitogen-activated protein kinase (MAPK) pathway, increasing the expression of proinflammatory genes and mediating H. parasuis-induced inflammation. Moreover, Wnt/ß-catenin signaling activation induced by H. parasuis disrupts the adherens junction between epithelial cells and initiates the epithelial-mesenchymal transition (EMT). In the present study, p38 MAPK was found to be involved in the accumulation of nuclear location of ß-catenin during H. parasuis infection in PK-15 and NPTr cells, via modulating the expression of dickkofp-1 (DKK-1), a negative regulator of Wnt/ß-catenin signaling. We generated DKK-1 knockout cell lines by CRISPR/Cas9-mediated genome editing in PK-15 and NPTr cells, and found that knockout of DKK-1 led to the dysfunction of p38 MAPK in regulating Wnt/ß-catenin signaling activity in H. parasuis-infected cells. Furthermore, p38 MAPK activity was independent of the activation of Wnt/ß-catenin signaling during H. parasuis infection. This is the first study to explore the crosstalk between p38 MAPK and Wnt/ß-catenin signaling during H. parasuis infection. It provides a more comprehensive view of intracellular signaling pathways during pathogenic bacteria-induced acute inflammation.

Pathogenicity and Molecular Typing of Fowl Adenovirus-Associated With Hepatitis/Hydropericardium Syndrome in Central China (2015-2018).

In central China, a large number of broiler and layer flocks have suffered from outbreaks of severe hepatitis/hydropericardium syndrome (HHS). This resulted in huge economic losses to the poultry industry, from 2015 to 2018. To identify the specific pathogen and study its pathogenicity, 195 samples from Hubei, Jiangxi, Anhui, Hunan, and Henan provinces in central China were collected. The samples were screened for the adenovirus hexon gene, and neighbor joining was used for the phylogenetic reconstruction of the sequences. Among the collected samples, 122 were found to be positive for fowl adenovirus (FAdV) by PCR, and 73 isolates were obtained. The predominant viral serotype was serotype 4 (FAdV-4), which was found in 48 isolates, while 24 were serotype 10 (FAdV-10), and one was serotype 2 (FAdV-2). The CH/HBTF /1710 isolate was selected for further experiment and inoculated into 33-day-old specific pathogen-free chickens via intramuscular injection or oral administration to evaluate pathogenicity. It was found that the mortality for chickens infected by intramuscular injection or oral administration was 70 and 60%, respectively. Necropsy revealed mild to severe hepatitis and hydropericardium at 5 and 7 days after infection. Ancestor analyses indicated that all of the FAdV-4 strains obtained in this study shared a common Indian precursor and had a close genetic relationship with the JSJ13, SDSX, HN/151025, and SDDM-15 strains common in China.

Identification of an Integrase That Responsible for Precise Integration and Excision of Riemerella anatipestifer Genomic Island.

Riemerella anatipestifer is a Gram-negative, pathogenic bacterium, which is harmful to poultry. However, the genomic islands (GIs) in R. anatipestifer are not well-studied. In this study, a 10K genomic island was predicted by the bioinformatics analysis of R. anatipestifer ATCC 11845, which is widely found in other R. anatipestifer genomes. We had first reported the genomic island integration and excision function in R. anatipestifer. We successfully constructed the integration plasmid by using the integrase and 53 bp attP elements. The 10K GI was found integrated at the 53 bp attB located in the Arg-tRNA of the R. anatipestifer RA-YM chromosome. We identified an integrase that helped in the precise integration and excision in R. anatipestifer and elucidated the molecular mechanism of the 10K genomic island integration and excision. Furthermore, we provided a new method for the gene expression and construction of complementary strain.

Cas9 regulated gene expression and pathogenicity in Riemerella anatipestifer.

Riemerellosis, a Riemerella anatipestifer infection, can cause meningitis, pericarditis, parahepatitis, and airsacculitis in ducks, leading to serious economic losses in the duck meat industry. However, the molecular mechanism of the pathogenesis and virulence factors of this infection are poorly understood. In the present study, we created a mutant strain RA-YMΔCas9 using trans-conjugation. Bacterial virulence tests indicated that the median lethal dose (LD50) of RA-YMΔCas9 was 5.01 × 107 CFU, significantly lower than that of the RA-YM strain, which was 1.58 × 105 CFU. The distribution and blood bacterial load from the infection groups showed no significant difference in the brain between the RA-YMΔCas9 mutant and the wild-type RA-YM strains, however, the number of mutant strains were significantly reduced in the liver, heart, and blood. Animal immunization experiments demonstrated that the intranasal administration of RA-YMΔCas9 in ducklings provided 80% protection after challenge with the wild-type strain, showing potential use as a live mucosal vaccine. RNAseq analysis indicated that Cas9 protein played a regulatory role in gene expression. This study is the first to report on the involvement of Cas9 in the regulation and pathogenesis of R. anatipestifer, and provides a theoretical basis for the development of relevant genetic engineering vaccines.

The Impact of Lactobacillus plantarum on the Gut Microbiota of Mice with DSS-Induced Colitis.

The pathogenesis of inflammatory bowel disease (IBD) is due in part to a loss of equilibrium among the gut microbiota, epithelial cells, and resident immune cells. The gut microbiota contains a large proportion of probiotic commensal Lactobacillus species; some natural microbiota and probiotics confer protection against IBD. In this study, mice with colitis triggered by dextran sodium sulphate (DSS) were given Lactobacillus plantarum orally. We assessed the damage caused by DSS and the therapeutic activity of L. plantarum. The colitis triggered by DSS was less severe in the mice that received the L. plantarum treatment, which also diversified the microbe species in the colon, enhanced the ratio of Firmicutes to Bacteroidetes, and diminished the relative abundance of Lactobacillus. The taxonomic units of greatest diversity in the DSS and L. plantarum groups were identified using a linear discriminant and effect size (LEfSe) analysis. Aliihoeflea was established to be the genus of bacteria that was affected in the L. plantarum group most extensively. In conclusion, gut health was promoted by L. plantarum, as it diversified the microbes in the colon and restricted the activity of pathogenic bacteria in the intestine. Moreover, according to the LEfSe analysis, the DSS group was impacted more significantly by gut microorganisms than the L. plantarum group, suggesting that L. plantarum improved the stability of the intestinal tract.

Deletion of the Riemerella anatipestifer type IX secretion system gene sprA results in differential expression of outer membrane proteins and virulence.

Riemerella anatipestifer (RA), the causative agent of infectious serositis that targets ducklings and other poultry, secretes protein via the type IX secretion system (T9SS). The proteins transported by T9SS are located on the bacterial cell surface or secreted into the extracellular milieu. In this study, a sprA deletion mutant was constructed encoding a core protein of T9SS to investigate its influence on outer membrane protein expression and its role in virulence. Compared with the wild-type RA-YM strain, the deletion mutant ΔsprA failed to digest gelatin, showed the same growth rate in the logarithmic phase and exhibited greater sensitivity to the bactericidal activity of duck sera, whereas the complemented strain restored these phenotypes. The outer membrane proteome of RA-YM and the ΔsprA mutant were analyzed by Tandem Mass Tags, which revealed 198 proteins with predicted localization to the cell envelope. Sixty-three of these proteins were differentially expressed in the outer membrane, with 43 up-regulated and 20 down-regulated. Among the twelve outer membrane proteins which were secreted by T9SS, four proteins were up-regulated and one protein was down-regulated. Animal experiments demonstrated that the median lethal dose of the mutant strain ΔsprA was about 500 times higher than that of the wild-type RA-YM strain, and bacterial loads in blood, brain, heart, liver and spleen of the ΔsprA-infected ducks were significantly reduced. Our results indicate that the SprA is a virulence-associated factor of RA, and its absence results in altered abundance of outer membrane proteins, and secretion disorders associated with some of the T9SS effector proteins.

Functional characterization of duck TBK1 in IFN-ß induction.

TRAF family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) serves as hub molecule at the crossroad of multiple signaling pathways of type I interferon (IFN) induction. The importance of TBK1 in innate immunity has been demonstrated in mammalian, however the characterization and function of TBK1 in avian remains largely unknown. In this study, we cloned duck TBK1 (duTBK1) from duck embryo fibroblasts (DEFs) for the first time, which encoded 729 amino acids and had a high amino acid identity with goose and cormorant TBK1s. The duTBK1 showed a diffuse cytoplasmic localization in DEFs and was extensively expressed in all tested tissues. Overexpression of duTBK1 induced IFN-ß production through the activation of IRF1 and NF-κB in DEFs. The N-terminal kinase domain and the ubiquitin-like domain in middle of duTBK1 played pivotal roles in IFN-ß induction as well as in IRF1 and NF-κB activation. Furthermore, knockdown of duTBK1 by small interfering RNA significantly decreased poly(I:C)- or Sendai virus (SeV)-induced IFN-ß expression. In addition, duTBK1 expression dramatically reduced the replication of both duck reovirus (DRV) and duck Tembusu virus (DTMUV) in DEFs. These results suggested that the duTBK1 played a pivotal role in mediating duck antiviral innate immunity.

Haemophilus parasuis Infection Disrupts Adherens Junctions and Initializes EMT Dependent on Canonical Wnt/ß-Catenin Signaling Pathway.

In this study, animal experimentation verified that the canonical Wnt/ß-catenin signaling pathway was activated under a reduced activity of p-ß-catenin (Ser33/37/Thr41) and an increased accumulation of ß-catenin in the lungs and kidneys of pigs infected with a highly virulent strain of H. parasuis. In PK-15 and NPTr cells, it was also confirmed that infection with a high-virulence strain of H. parasuis induced cytoplasmic accumulation and nuclear translocation of ß-catenin. H. parasuis infection caused a sharp degradation of E-cadherin and an increase of the epithelial cell monolayer permeability, as well as a broken interaction between ß-catenin and E-cadherin dependent on Wnt/ß-catenin signaling pathway. Moreover, Wnt/ß-catenin signaling pathway also contributed to the initiation of epithelial-mesenchymal transition (EMT) during high-virulence strain of H. parasuis infection with expression changes of epithelial/mesenchymal markers, increased migratory capabilities as well as the morphologically spindle-like switch in PK-15 and NPTr cells. Therefore, we originally speculated that H. parasuis infection activates the canonical Wnt/ß-catenin signaling pathway leading to a disruption of the epithelial barrier, altering cell structure and increasing cell migration, which results in severe acute systemic infection characterized by fibrinous polyserositis during H. parasuis infection.

Co-infection with porcine bocavirus and porcine circovirus 2 affects inflammatory cytokine production and tight junctions of IPEC-J2 cells.

Porcine bocavirus (PBoV) has a high prevalence in both healthy and diseased swine around the world. It was recently reported that PBoV and porcine circovirus type 2 (PCV2)-which contribute to porcine diarrheal disease-have a high rate of co-infection. To clarify the pathogenesis of PBoV, we examined the co-infection rate and effects of these two pathogens in IPEC-J2 porcine intestinal enterocytes. Both single and co-infection had cytopathic effects in IPEC-J2 cells. The apoptosis and proliferation rates of cells infected with both viruses did not differ significantly from those of cells infected with either one alone. PBoV and PCV2 induced the upregulation of inflammatory cytokines and the downregulation of the tight junction proteins occludin and claudin 1 in the early stage of infection, leading to destruction of epithelial barrier integrity and enhanced cytotoxicity. These findings provide insight into the pathogenic mechanisms of PBoV and PCV2 and a basis for developing effective strategies to prevent the spread of gastrointestinal diseases in pigs and other livestock.

Relationship between CYP3A29 and pregnane X receptor in landrace pigs: Pig CYP3A29 has a similar mechanism of regulation to human CYP3A4.

The objective of this study was to provide evidence of the validity of utilizing pigs as a model to study the regulation of human CYP3A4, with special emphasis on drug-drug interactions. We determined the mRNA expression and distribution of CYP3A and metabolic nuclear receptors in different tissues isolated from landrace pigs. Our results showed that CYP3A and metabolic nuclear receptor mRNAs were most highly expressed in liver tissues. The expression of the metabolic nuclear receptor pregnane X receptor (PXR) had a significant correlation with expression of CYP3A29, an analog of human CYP3A4. The correlation between their transcriptional levels was further demonstrated using LPS and TNF-α. The mRNA and protein expression of CYP3A29 and PXR in HepLi cells was significantly reduced by LPS and TNF-α treatment. CYP3A29 promoter activity was dramatically elevated by PXR over expression, whereas LPS and TNF-α treatment inhibited the enhanced CYP3A29 promoter activity that was induced by PXR; presumably through inhibition of PXR promoter activity. Furthermore, the inhibition of CYP3A29 promoter activity by LPS and TNF-α treatment was blocked by knockdown of PXR or retinoid X receptor (RXR). These data suggest high similarity in the regulation mechanism of pig CYP3A29 and human CYP3A4. Our research provided a significant evaluation to determine whether pigs are suitable as an experimental animal model.

Development of immunochromatographic test strips for rapid, quantitative detection of H9AIV antibodies.

To improve viral antibody detection and disease control, laboratories need faster and simpler methods for direct detection of H9AIV in clinical samples. In this study, test strips were developed for rapid detection of H9AIV antibodies in poultry serum by a sandwich method with double-line detection. The hemagglutinin protein was labeled with colloidal gold as a detection reagent and was blotted on the test lines, while goat anti-rabbit IgG was utilized on the control line of the nitrocellulose membrane. The test strips have high specificity, sensitivity, and stability, with a correlation coefficient of 0.9656 and coefficients of variation below 10%. Application of the kit to quantitative detection of H9AIV antibodies in 504 samples collected from chickens showed a coincidence rate of 80.56% with previously run HI assays.

Correction to: Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review.

The authors would like to call the reader's attention to the fact that unfortunately in the original article Steptococcus suis was introduced as gram-negative bacteria. Steptococcus suis is gram positive. The authors apologize for the mistake.

Haemophilus parasuis infection activates NOD1/2-RIP2 signaling pathway in PK-15 cells.

Haemophilus parasuis, an important swine pathogen, was recently proven able to invade into endothelial or epithelial cell in vitro. NOD1/2 are specialized NLRs that participate in the recognition of pathogens able to invade intracellularly and therefore, we assessed that the contribution of NOD1/2 to inflammation responses during H. parasuis infection. We observed that H. parasuis infection enhanced NOD2 expression and RIP2 phosphorylation in porcine kidney 15 cells. Our results also showed that knock down of NOD1/2 or RIP2 expression respectively significantly decreased H. parasuis-induced NF-κB activity, while the phosphorylation level of p38, JNK or ERK was not changed. Moreover, real-time PCR result showed that NOD1, NOD2 or RIP2 was involved in the expression of CCL4, CCL5 and IL-8. Inhibition of NOD1 and NOD2 significantly reduced CCL5 promoter activity, even in a more effective way compared with inhibition of TLR.

The Role of the Regulator Fur in Gene Regulation and Virulence of Riemerella anatipestifer Assessed Using an Unmarked Gene Deletion System.

Riemerella anatipestifer, an avian pathogen, has resulted in enormous economic losses to the duck industry globally. Notwithstanding, little is known regarding the physiological, pathogenic and virulence mechanisms of Riemerella anatipestifer (RA) infection. However, the role of Ferric uptake regulator (Fur) in the virulence of R. anatipestifer has not, to date, been demonstrated. Using a genetic approach, unmarked gene deletion system, we evaluated the function of fur gene in the virulence of R. anatipestifer. For this purpose, we constructed a suicide vector containing pheS as a counter selectable marker for unmarked deletion of fur gene to investigate its role in the virulence. After successful transformation of the newly constructed vector, a mutant strain was characterized for genes regulated by iron and Fur using RNA-sequencing and a comparison was made between wild type and mutant strains in both iron restricted and enriched conditions. RNA-seq analysis of the mutant strain in a restricted iron environment showed the downregulation and upregulation of genes which were involved in either important metabolic pathways, transport processes, growth or cell membrane synthesis. Electrophoretic mobility shift assay was performed to identify the putative sequences recognized by Fur. The putative Fur-box sequence was 5'-GATAATGATAATCATTATC-3'. Lastly, the median lethal dose and histopathological investigations of animal tissues also illustrated mild pathological lesions produced by the mutant strain as compared to the wild type RA strain, hence showing declined virulence. Conclusively, an unmarked gene deletion system was successfully developed for RA and the role of the fur gene in virulence was explored comprehensively.

A meta-analysis of transcriptomic characterization revealed extracellular matrix pathway involved in the H5N1 and H7N9 infections.

Avian-origin H5N1 and H7N9 influenza A viruses are capable of causing lethal infection in humans, with serious lung pathology and leading to acute respiratory distress syndrome. The contribution of host response associated with the poor prognosis of H5N1 and H7N9 infections remains unclear. The aim of this study was to identify the host factors involved in the high pathogenicity of H5N1 and H7N9 by a systematical meta-analysis. The RNA-seq datasets related to H5N1, H7N9, and H1N1 infections with time series were retrieved from GEO. After merging the data from different series, ComBat was used to adjust the known variances from different batches. The transcription factors binding the genes in each cluster were predicted by PASTAA. We figured out the genes that were differentially expressed at any time point in samples infected with H5N1, H7N9, or H1N1. The analysis of biological function showed that genes related with cytokine were up-regulated in all three viruses. However, genes associated with carbon metabolism were found exclusively down-regulated in H7N9 and the extracellular matrix pathway were only enriched in H5N1 and H7N9. To summary, our study suggested that the extracellular matrix might be associated with the high fatality of H5N1 and H7N9 viruses in humans.