Visual detection of fowl adenovirus serotype 4 via a portable CRISPR/Cas13a-based lateral flow assay.

The widespread occurrence of fowl adenovirus serotype 4 (FAdV-4)-induced hepatitis-hydropericardium syndrome (HHS) has led to significant economic losses for the poultry industry. A sensitive, accurate, and practical FAdV-4 diagnostic approach is urgently required to limit the incidence of the disease. In the present study, a practical method for detecting FAdV-4 was developed using the CRISPR/Cas13a system and recombinase-aided amplification. The approach was based on 37°C isothermal detection with visible results being achieved. The detection limit of the target gene with this approach was only 101 copies/µl, making it very sensitive and specific. Clinical samples fared well when tested with the Cas13a detection method. For identifying FAdV-4, this novel detection approach was found to be sensitive, specific, and effective.RESEARCH HIGHLIGHTS First study using the CRISPR/Cas13a-based lateral flow detection assay for FAdV-4 detection.The results can be observed by the naked eye.The developed assay could provide an alternative tool for detection of FAdV-4 with minimal equipment.

Integrated analysis of noncoding RNAs and mRNAs reveals their potential roles in chicken spleen response to Klebsiella variicola infection.

Klebsiella variicola is an emerging pathogen that has become a threat to human and animal health. There is evidence that long noncoding RNAs (lncRNAs) are involved in a host cell's response to microbial infections. However, no study has defined the link between K. variicola pathogenesis and lncRNAs until now. We used RNA sequencing to comprehensively analyze the lncRNAs and mRNAs in the chicken spleen after K. variicola infection. In total, we identified 2896 differentially expressed mRNAs and 578 differentially expressed lncRNAs. To examine the potential functions of these lncRNAs, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment analyses were performed on the target mRNAs of these differently expressed lncRNAs. The results suggested that lncRNAs play essential roles in modulating mRNA expression and triggering downstream immune signaling pathways to regulate the immune response in the chicken spleen. Using previous microRNA sequencing data, we constructed lncRNA-miRNA-mRNA regulatory networks to clarify the regulatory mechanisms in the chicken immune system. Several potential regulatory pairs related to K. variicola infection were found, involving XR_001467769.2, TCONS_00018386, gga-miR-132a-3p, gga-miR-132b-5p, gga-miR-2954, and novel62_mature. In conclusion, our findings make a significant contribution towards understanding the role of lncRNA in chicken spleen cells during K. variicola infection, thereby establishing a solid foundation for future research in this area.

Altered Proteomic Profile of Exosomes Secreted from Vero Cells Infected with Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) infection causes severe diarrhea in pigs and can be fatal in newborn piglets. Exosomes are extracellular vesicles secreted by cells that transfer biologically active proteins, lipids, and RNA to neighboring or distant cells. Herein, the morphology, particle size, and secretion of exosomes derived from a control and PEDV-infected group are examined, followed by a proteomic analysis of the exosomes. The results show that the exosomes secreted from the Vero cells had a typical cup-shaped structure. The average particle size of the exosomes from the PEDV-infected group was 112.4 nm, whereas that from the control group was 150.8 nm. The exosome density analysis and characteristic protein determination revealed that the content of exosomes in the PEDV-infected group was significantly higher than that in the control group. The quantitative proteomics assays revealed 544 differentially expressed proteins (DEPs) in the PEDV-infected group's exosomes compared with those in the controls, with 236 upregulated and 308 downregulated proteins. The DEPs were closely associated with cellular regulatory pathways, such as the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, extracellular matrix-receptor interaction, focal adhesion, and cytoskeletal regulation. These findings provide the basis for further investigation of the pathogenic mechanisms of PEDV and the discovery of novel antiviral targets.

Genome-scale CRISPR screen identifies TRIM2 and SLC35A1 associated with porcine epidemic diarrhoea virus infection.

Porcine epidemic diarrhoea (PED) caused by the porcine epidemic diarrhoea virus (PEDV) is the most devastating disease in the global pig industry due to its high mortality rate in piglets. The host factors critical for PEDV replication are poorly understood. Here, we designed a pooled African green monkey genome-scale CRISPR/Cas9 knockout (VeroCKO) library containing 75,608 single guide RNAs targeting 18,993 protein-coding genes. Subsequently, we use the VeroCKO library to identify key host factors facilitating PEDV infection in Vero E6 cells. Several previously unreported genes associated with PEDV infection are highly enriched post-PEDV selection. We discovered that knocking out the tripartite motif 2 (TRIM2) and the solute carrier family 35 member A1 (SLC35A1) inhibited PEDV replication. Virtual screening and molecular docking approaches showed that chem-80,048,685 (M2) s ignificantly inhibited PEDV attachment and late replication by impeding SLC35A1. Furthermore, we found that knocking out SLC35A1 in Vero E6 cells upregulated a disintegrin and metalloprotease protein-17 (ADAM17) by splicing porcine aminopeptidase N (pAPN) and angiotensin-converting enzyme 2 (ACE2) ectodomains to reduce PEDV-infection in a CMP-Sialic Acid (CMP-SA) cell entry-independent manner. These findings provide a new perspective for a better understanding of host-pathogen interactions and new therapeutic targets for PEDV infection.

In silico and in vitro evaluation of antiviral activity of wogonin against main protease of porcine epidemic diarrhea virus.

The high mortality rate of weaned piglets infected with porcine epidemic diarrhea virus (PEDV) poses a serious threat to the pig industry worldwide, demanding urgent research efforts related to developing effective antiviral drugs to prevent and treat PEDV infection. Small molecules can possibly prevent the spread of infection by targeting specific vital components of the pathogen's genome. Main protease (Mpro, also named 3CL protease) plays essential roles in PEDV replication and has emerged as a promising target for the inhibition of PEDV. In this study, wogonin exhibited antiviral activity against a PEDV variant isolate, interacting with the PEDV particles and inhibiting the internalization, replication and release of PEDV. The molecular docking model indicated that wogonin was firmly embedded in the groove of the active pocket of Mpro. Furthermore, the interaction between wogonin and Mpro was validated in silico via microscale thermophoresis and surface plasmon resonance analyses. In addition, the results of a fluorescence resonance energy transfer (FRET) assay indicated that wogonin exerted an inhibitory effect on Mpro. These findings provide useful insights into the antiviral activities of wogonin, which could support future research into anti-PEDV drugs.`.

Prevalence and Characterization of Salmonella Isolated from Chickens in Anhui, China.

Salmonella is one of the most important zoonotic pathogens that can cause both acute and chronic illnesses in poultry flocks, and can also be transmitted to humans from infected poultry. The purpose of this study was to investigate the prevalence, antimicrobial resistance, and molecular characteristics of Salmonella isolated from diseased and clinically healthy chickens in Anhui, China. In total, 108 Salmonella isolates (5.66%) were successfully recovered from chicken samples (n = 1908), including pathological tissue (57/408, 13.97%) and cloacal swabs (51/1500, 3.40%), and S. Enteritidis (43.52%), S. Typhimurium (23.15%), and S. Pullorum (10.19%) were the three most prevalent isolates. Salmonella isolates showed high rates of resistance to penicillin (61.11%), tetracyclines (47.22% to tetracycline and 45.37% to doxycycline), and sulfonamides (48.89%), and all isolates were susceptible to imipenem and polymyxin B. In total, 43.52% isolates were multidrug-resistant and had complex antimicrobial resistance patterns. The majority of isolates harbored cat1 (77.78%), blaTEM (61.11%), and blaCMY-2 (63.89%) genes, and the antimicrobial resistance genes in the isolates were significantly positively correlated with their corresponding resistance phenotype. Salmonella isolates carry high rates of virulence genes, with some of these reaching 100% (invA, mgtC, and stn). Fifty-seven isolates (52.78%) were biofilm-producing. The 108 isolates were classified into 12 sequence types (STs), whereby ST11 (43.51%) was the most prevalent, followed by ST19 (20.37%) and ST92 (13.89%). In conclusion, Salmonella infection in chicken flocks is still serious in Anhui Province, and not only causes disease in chickens but might also pose a threat to public health security.

Rapid and Easy-Read Porcine Circovirus Type 4 Detection with CRISPR-Cas13a-Based Lateral Flow Strip.

First identified as a new circovirus in Hunan Province in China in 2019, porcine circovirus (PCV4) is now widely detected in other Chinese provinces and South Korea. In recent years, the virus has threatened pig health and operations in the pig industry. Hence, early PCV4 detection and regular surveillance are required to control the spread of infection and prevent collateral damage to the industry. Due to PCV4 being difficult to isolate in vitro, molecular detection methods, such as conventional PCR and real-time PCR, and serological assays are currently the main methods used for the detection of PCV4 infection. However, they are time-consuming, labor-intensive, and complex and require professional personnel. To facilitate rapid pen-side PCV4 diagnoses, we used clustered regularly interspaced short palindromic repeats (CRISPR) and Cas13a technology to develop a quick testing kit. Five recombinase-aided amplification (RPA) primer sets were designed based on the conserved PCV4-Cap gene nucleotide region, which were used to determine several key lateral flow strip (LFD) characteristics (sensitivity, specificity, and accuracy). The results showed that the RPA-Cas13a-LFD reaction could detect PCV4 within 1.5 h in genomic DNA harboring a minimum of a single copy. Furthermore, the assay showed good specificity and absence of cross-reactivity with PCV2, PCV3, or other porcine viruses. When we tested 15 clinical samples, a high accuracy was also recorded. Therefore, we successfully developed a detection assay that was simple, fast, accurate, and suitable for on-site PCV4 testing.

CCR4-NOT Complex 2-A Cofactor in Host Cell for Porcine Epidemic Diarrhea Virus Infection.

The porcine epidemic diarrhea virus (PEDV) has catastrophic impacts on the global pig industry. However, there is no consensus on the primary receptor associated with the PEDV invasion of host cells. An increasing number of studies have reported that PEDV invading host cells may require collaboration between multiple receptors and to better understand the virus-host interaction during PEDV entry, surface plasmon resonance (SPR) assays are performed to investigate relevant host factors interacting with PEDV spike-1 protein (S1) in Vero and IPEC-J2 cell membranes. Subsequently, the rabbit anti-PEDV S1 polyclonal antibody is used as bait to recognize the complexes of IPEC-J2 membrane proteins with or without PEDV infection, followed by detection using liquid chromatography with tandem mass spectrometry (LC-MS-MS). Our results show that 13 and 10 proteins interacting between the S1 protein and plasma membrane protein of Vero or IPEC-J2 can be identified. More specifically, a total of 11 differentially expressed interacting proteins were identified in IPEC-J2 membrane proteins after PEDV infection, compared to the uninfected group. Furthermore, we found that the differentially interacting protein CCR4-NOT complex 2 (CNOT2), identified in PEDV S1 with plasma membrane proteins of Vero cells, is involved in viral infection. The results show that the knockout of CNOT2 significantly inhibits PEDV replication in vitro. These data provide novel insights into the entry mechanism of PEDV.

Visual detection and differentiation of porcine epidemic diarrhea virus wild-type strains and attenuated vaccine strains using CRISPR/Cas13a-based lateral flow strip.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets. Infections result in high mortality and serious economic losses to the swine industry. PEDV attenuated vaccine does not completely protect against all mutant wild-type strains, and PEDV infection can periodically occur. A sensitive, accurate, and simple detection method for PEDV is needed to reduce the occurrence of the disease. In this study, the CRISPR/Cas13a system was combined with recombinase aided amplification to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The method is based on isothermal detection at 37°C. The results are used for visual readout. The assay had high sensitivity and specificity, with a detection limit of 101 copies/µL for the gene of interest, and no cross-reactivity with other pathogens. The Cas13a detection worked well with clinical samples. This visual, sensitive, and specific nucleic acid detection method based on CRISPR/Cas13a should be a powerful tool for detecting PEDV.

Alterations in bone marrow microRNA expression profiles on infection with avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is one of the most common avian bacterial diseases globally. The bone marrow is a reservoir of immature immune cells. To elucidate the role of bone marrow microRNAs (miRNAs) in regulating the host response to APEC infection, we performed miRNA-seq to investigate alterations in the expression of bone marrow miRNAs in three groups of specific pathogen-free chickens: non-challenged (NC) and challenged with APEC for 12 h (C12) and 24 h (C24). Twenty and 19 differentially expressed miRNAs (fold change >2, P < 0.01) were identified on comparing the NC and C12 and the NC and C24 groups, respectively. On functional annotation analysis of target genes of differentially expressed miRNAs, we found that the gene ontology term "immune system process" was significantly enriched at both 12 h and 24 h; moreover, several important signaling pathways were triggered in response to APEC infection, such as MAPK, cGMP-PKG, Notch, and cAMP pathways. In addition, we performed reverse transcription quantitative real-time PCR (qRT-PCR) to validate the differential expression of miRNAs. qRT-PCR data were similar to the sequencing data. On constructing an miRNA-target gene network, gga-miR-2127, gga-miR-6643-5p, and gga-miR-6567-3p were found to potentially play a vital role in the immune process. Overall, our findings provide deeper insights into miRNA transcriptome changes involved in the immune response of the chicken bone marrow to APEC infection.

Evaluating Salmonella pullorum dissemination and shedding patterns and antibody production in infected chickens.

BACKGROUND: Pullorum disease caused by Salmonella pullorum is one of the most important infectious diseases in the poultry industry, responsible for causing substantial economic losses globally. On farms, the traditional method to detect S. pullorum infection mainly involves the collection of feces and sera to test for antigens and antibodies, respectively, but the regularity of Salmonella pullorum dissemination in internal organs and shedding patterns and antibody production in infected chickens remains unclear. Herein we aimed to investigate the dissemination of S. pullorum to different organs and bacterial shedding patterns in the faeces as well as serum antibody production post-infection in chickens of different ages. RESULT: In this study, the liver and heart of 2-day-old chickens showed the highest copy numbers of S. pullorum at 6.4 × 106 and 1.9 × 106 copies of DNA target sequences/30 mg, respectively. In case of 10-day-old chickens, the percentage of S. pullorum fecal shedding (0%-40%) and antibody production (0%-56.6%) markedly fluctuated during the entire experiment; furthermore, in case of 42-week-old chickens, the percentage of birds showing S. pullorum shedding in the faeces showed a downward trend (from 63.33% to 6.6% in the oral inoculation group and from 43.3% to 10% in the intraperitoneal injection group), while that of birds showing serum antibody production remained at a high level (38.3% and 80% in the oral inoculation and intraperitoneal injection groups, respectively). We also performed cohabitation experiments, showed that 15% 10-day-old and 3.33% 42-week-old chickens were infected via the horizontal transmission in cohabitation with S. pullorum infected chickens, and revealed a high risk of horizontal transmission of S. pullorum. CONCLUSION: This study systematically evaluated the dissemination of S. pullorum in internal organs and bacterial fecal shedding patterns, and antibody production in infected chickens. Collectively, our findings indicate how to effectively screen S. pullorum-negative chickens on livestock farms and should also help in the development of measures to control and eradicate S. pullorum.

Antiviral and Virucidal Activities of Camptothecin on Fowl Adenovirus Serotype 4 by Blocking Virus Replication.

Fowl adenovirus serotype 4 (FAdV-4) caused hepatitis-hydropericardium syndrome in poultry and caused huge economic losses to the poultry industry. At present, antiviral drugs have not been reported to be effective against this virus, and new treatment methods are urgently needed to treat FAdV-4. Camptothecin has been shown to have antiviral activity against various viruses; however, whether it can inhibit FAdV-4 infection remains unclear. This study aimed to explore the anti-FAdV-4 effects and mechanisms of camptothecin in vitro and in vivo. Several camptothecin treatments were used to study the antiviral activity of camptothecin on FAdV-4-infected Leghorn male hepatocellular (LMH) cells. The FAdV-4 titers of mock and camptothecin-treated infected cell cultures were determined using tissue culture infective dose assay, and the FAdV-4 copy number was determined using quantitative real-time polymerase chain reaction. In addition, the therapeutic effect of camptothecin on FAdV-4-infected chickens was also evaluated. The results showed that camptothecin significantly reduced the viral replication in LMH cells in a dose-dependent manner, resulting in a reduction in viral titer, viral copy number, and viral Hexon protein expression. Camptothecin was also found to have a significant inhibitory effect on the viral replication step. Finally, camptothecin showed anti-FAdV-4 efficacy in the chicken infection model, and the survival rate was improved. This study was novel in proving that camptothecin had a protective effect against FAdV-4, indicating its potential as an antiviral drug against FAdV-4 infection.

Characteristics of the MicroRNA Expression Profile of Exosomes Released by Vero Cells Infected with Porcine Epidemic Diarrhea Virus.

Exosomes are nanoscale vesicles actively secreted by a variety of cells. They contain regulated microRNA (miRNA), allowing them to function in intercellular communication. In the present study, the role of exosomal miRNAs in porcine epidemic diarrhea virus (PEDV) infection was investigated using exosomes isolated from Vero cells infected with PEDV. The results of transmission electron microscopy observation showed that the exosomes are spherical in shape, uniform in size, and negatively stained in the membrane. Nanoparticle tracking analysis showed that the average exosome particle size is 130.5 nm. The results of miRNA sequencing showed that, compared with the control group, a total of 115 miRNAs are abnormally expressed in the exosomes of infected cells. Of these, 80 miRNAs are significantly upregulated and 35 miRNAs are significantly downregulated. Functional annotation analysis showed that the differentially expressed miRNAs are associated with PEDV infection through interaction with the cAMP, Hippo, TGF-beta, HIF-1, FoxO, MAPK, and Ras signaling pathways. Thus, our findings provide important information about the effects of PEDV infection on exosomal miRNA expression and will aid the search for potential anti-PEDV drug candidates.

Visual Detection of Duck Tembusu Virus With CRISPR/Cas13: A Sensitive and Specific Point-of-Care Detection.

Duck tembusu virus (DTMUV), which causes huge economic losses for the poultry industries in Southeast Asia and China, was first identified in 2010. DTMUV disease has become an important disease that endangers the duck industry. A sensitive, accurate, and convenient DTMUV detection method is an important means to reduce the occurrence of the disease. In this study, a CRISPR/Cas13a system was combined with recombinase polymerase amplification to develop a convenient diagnostic method to detect DTMUV. The novel method was based on isothermal detection at 37°C, and the detection was used for visual readout or real-time analysis. The assay was highly sensitive and specific, with a detection limit of 1 copy/µL of the target gene and showed no cross-reactivity with other pathogens. The enhanced Cas13a detection worked well with clinical samples. Overall, a visual, sensitive, and specific nucleic acid detection method based on CRISPR/Cas13a proved to be a powerful tool for detecting DTMUV.

Transcriptome response of a new serotype of avian type Klebsiella varicella strain to chicken sera.

Klebsiella variicola is a newly discovered pathogen of zoonotic importance, commonly causing serious systemic infection via the bloodstream route. However, the mechanism by which K. variicola survives and grows in the bloodstream is poorly understood. In a previous study, a strain of Klebsiella causing chicken bloodstream infection was obtained, and whole genome sequencing showed that it was a new ST174 type K. variicola. Therefore, the present study aimed to determine the molecular mechanism underlying the survival and development of K. variicola in host serum. First, we compared the transcriptomes of K. variicola grown in Luria-Bertani broth and chicken serum. We sequenced six RNA libraries from the two groups, each library had three repeats. A total of 1046 differentially expressed genes were identified. Functional annotation analysis showed that the differentially expressed genes are mainly involved in adaptive metabolism, biosynthesis pathways (including biosynthesis of siderophore group nonribosomal peptides and lipopolysaccharide (LPS) biosynthesis), stress resistance, and several known virulence regulatory systems (including the ABC transporter system, the two-component signal transduction system and the quorum sensing system). These genes are expected to contribute to the adaptation and growth of K. variicola in host birds. This analysis provides a new insight into the pathogenesis of K. variicola.

ß-Glucans from Trametes versicolor (L.) Lloyd Is Effective for Prevention of Influenza Virus Infection.

Coriolus versicolor (C. versicolor) is a higher fungi or mushroom which is now known by its accepted scientific names as Trametes versicolor (L.) Lloyd. Many studies have shown that ß-glucans from C. versicolor have various physiological activities, including activating macrophages to protect against Salmonella infection. However, whether ß-glucans have antiviral effects has not been reported. Hence, the objective of this study was to confirm whether ß-glucans could boost the immune response to combat influenza virus in mouse and chick models. The results show that ß-glucans induced the expression of Dectin-1, costimulatory molecules (CD80/86) and cytokines IL-6, IL-1ß, IFN-ß and IL-10 in murine bone marrow dendritic cells (BMDCs). In addition, orally administered ß-glucans reduced weight loss, mortality and viral titers in the lungs of mice infected with influenza virus and attenuated pathological lung damage caused by the virus in the mice. Orally administered ß-glucans improved survival and reduced lung viral titers in chickens infected with H9N2 avian influenza virus. These results suggest that ß-glucans have a significant antiviral effect. Therefore, ß-glucans could become a potential immunomodulator against influenza virus.

Tofu Whey Wastewater as a Beneficial Supplement to Poultry Farming: Improving Production Performance and Protecting against Salmonella Infection.

Tofu whey wastewater (TWW) is a by-product of the tofu production process, and contains high amounts of organic products and Lactobacillus ap. However, no studies have been reported on whether naturally fermented TWW can be used as a beneficial additive for poultry production. This study analyzed the main nutritional components and microbial flora of naturally fermented TWW from rural tofu processing plants and their effect on chick production performance, role in modulating the biochemical and immune parameters, and protection against Salmonella enteritidis (S. enteritidis) infection. It was observed that the average pH of TWW was 4.08; therefore, the total viable count was 3.00 × 109 CFU/mL and the abundance of Lactobacillus was 92.50%. Moreover, TWW supplementation increased the total weight gain and feed intake, reduced the feed/gain ratio, increased the length and relative weight of the gut, and reduced the colonization and excretion of S. enteritidis in chickens. Additionally, TWW decreased oxidative damage and pro-inflammatory cytokine secretion caused by S. enteritidis infection. In addition, TWW supplementation ensured the structure of the intestine remained relatively intact in S. enteritidis-infected chicken. Furthermore, TWW markedly promoted the intestinal barrier integrity and up-regulated the relative abundance of Lactobacillus, counteracting the changes in gut microbiota caused by S. enteritidis infection in chicken. In conclusion, our data demonstrated that TWW could be used as a beneficial addition to poultry production, providing a research basis for the further development of TWW as a health care application in in food-producing animal.

The Escherichia coli type III secretion system 2 Is involved in the biofilm formation and virulence of avian Pathogenic Escherichia coli.

The Escherichia coli type III secretion system 2 (ETT2) is found in most pathogenic E. coli strains. Although many ETT2 gene clusters carry multiple genetic mutations or deletions, ETT2 is known to be involved in bacterial virulence. To date, no studies have been conducted on the role of ETT2 in the virulence of avian pathogenic Escherichia coli (APEC), which harbours ETT2. Thus, we deleted the ETT2 of APEC strain and evaluated the phenotypes and pathogenicities of the mutant. The results showed that deletion of ETT2 had no effect on APEC growth, but significantly promoted biofilm formation. In addition, as compared to the wild-type （WT） strain, the ETT2 deletion significantly promoted adherence to and invasion of DF-1 chicken fibroblasts and facilitated survival in the sera of specific-pathogen-free chickens. Analysis of the role of ETT2 in animal infection models demonstrated that the distribution of viable bacteria in the blood and organs of chicks infected with the ΔETT2 was significantly higher than those infected with WT. The results of RNA sequencing indicated that multiple genes involved in biofilm formation, lipopolysaccharide components, fimbrial genes and virulence effector proteins are regulated by ETT2. Collectively, these results implicated ETT2 is involved in the biofilm formation and pathogenicity of APEC.

Flagellar rotor protein FliG is involved in the virulence of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC), a type of extraintestinal pathogenic E. coli, causes avian colibacillosis, a disease of significant economic importance to poultry producers worldwide, which is characterized by systemic infection. However, the pathogenesis of avian pathogenic E. coli strains is not well defined. Here, the role of a flagellar rotor protein encoded by the fliG gene of avian pathogenic E. coli strain AE17 was investigated. To study the role of FliG in the pathogenicity of APEC, fliG mutant and complemented strains were constructed and characterized. The inactivation of fliG had no effect on APEC growth, but significantly reduced bacterial motility. Compared with the wild type, the fliG mutant was highly attenuated in a chick infection model and showed severe defects in its adherence to and invasion of chicken embryo fibroblast DF-1 cells. The fliG mutant also showed reduced resistance to serum in chicks. The expression of the inflammatory cytokines interleukin 1ß (IL1ß), IL6, and IL8 was reduced in HD-11 macrophages infected with the fliG mutant strain compared with their expression in the wild-type strain. These results demonstrate that the FliG contributes to the virulence of APEC.

RNA-seq and nuclear proteomics provide insights into the lactation regulation mechanism of goat transfected IGF-I and GH recombinant vectors.

There exists little available information on the mechanisms of lactation regulation until now. In order to explore the underlying mechanism, we injected IGF-I and GH recombinant vectors into the mammary gland, then RNA-seq analysis and nuclear proteomics were used for rapid high-throughput screening of DEGs and DEPs in the two groups linked to lactation regulation. KEGG analysis of 206 DEGs showed that the same 4 of top 10 enrichment pathways (ECM receptor interaction, protein digestion and absorption, focal adhesion and phagosome) involved in 4 co-expressed genes (IDO, BTG1, ITGB6 and keratin 83), the two groups enriched different metabolic pathways yet. Nuclear proteomics analysis showed 75 and 36 DEPs in the IGF-I and GH group respectively; Sixteen common proteins were identified between the IGF-I group and GH group, four of which (ALB, TPT1, CXXC-5 and ACTR2) significantly decreased and three of which (PRP1, PAG-9 and Hsp70) significantly increased. Similarly, DEPs in the two groups were enriched in same one of top 10 enrichment pathways (PI3K-Akt signaling pathway). Protein-protein interaction networks highlighted the contribution of glycosphingolipid biosynthesis, porphyrin and chlorophyll metabolism and the Jak-STAT signaling pathway to lactation regulation of GH and IGFI. GH and IGF-I improve milk yield, which may be linked to important nodal proteins (ALB and ACTB). Our research advances the understanding of the mammary gland transcriptome and nuclear proteomics during GH and IGF-I overexpression. Individual genes, proteins and pathways in this study point towards potential targets for lactation regulation.