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ABSTRACT: Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium hepatitis syndrome in chickens, which causes severe economic impact to the poultry industry. A simple, swift and reliable detection is crucial for timely identification of FAdV-4 infection, promoting effective viral prevention and control measures. Herein, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system detection platform based on loop-mediated isothermal amplification (LAMP) was studied. The CRISPR RNA (crRNA) and LAMP primers were designed and screened based on the highly conserved region of the FAdV-4 hexon gene. The parameters were then optimized individually to achieve the ideal reaction performance. The platform could lead visual detection of FAdV-4 to achieve as low as 1 copy in less than 40 min without the need for specialized instrumentation or complex equipment. Moreover, it was greatly specific, and did not cross-react with other common avian viruses. Following the validation of 30 clinical samples of suspected FAdV-4 infection, the results LAMP-CRISPR/Cas12a method generated showed fully concordance with which of the gold standard quantitative real-time PCR. To summarize, this study presented a novel, swift, expedient and inexpensive detection platform for FAdV-4, which is beneficial to viral inchoate diagnosis and point-of-care testing.

ABSTRACT: Since 2015, an outbreak of an infectious disease in broilers caused by fowl adenovirus serotype 4 (FAdV-4) has occurred in China, resulting in substantial economic losses. Rapid, accurate, and specific detection are significant in the prevention and control of FAdV-4. In this study, an FAdV-4 detection method combining loop-mediated isothermal amplification (LAMP) and Pyrococcus furiosus Argonaute (PfAgo) was established. Specific primers, guide DNAs (gDNAs), and molecular beacons were designed to target a conserved region of the FAdV-4 hexon gene. After optimizing the reaction conditions, the minimum detection of this assay could reach 5 copies. It only amplified FAdV-4, and there was no cross-reactivity with other pathogens. The assay took about only 50 min, and the results could be visualized with the naked eye under ultraviolet or blue light, getting rid of specialized instruments. This novel LAMP-PfAgo assay was validated by using 20 clinical samples and the results were identical to gold-standard real-time polymerase chain reaction method. In summary, the LAMP-PfAgo assay established in the paper provides a rapid, reliable, convenient, ultra-sensitive and highly specific tool for the on-site detection and clinical diagnosis of FAdV-4.

Abstract Background Avian pathogenic Escherichia coli (APEC) causes tracheal damage and heterophilic granulocytic infiltration and inflammation in infected chicks. In this study, we infected chick tracheal tissue with strain AE17 and produced pathological sections with proteomic sequencing. We compared the results of pathological sections from the APEC-infected group with those from the PBS control group; the pathological sections from the experimental group showed hemorrhage, fibrinization, and infiltration of heterophilic granulocytes in the tracheal tissue. In order to explore the effect on proteomics on inflammation and to further search for the caus. Results The tandem mass tag-based (TMT) sequencing analysis showed 224 upregulated and 140 downregulated proteins after infection with the AE17 strain. Based on the results of KEGG in Complement and coagulation cascades, differential protein expression in the Protein export pathway was upregulated. Conclusions With these results, we found that chemokines produced by the Complement and coagulation cascades pathway may cause infiltration of heterophilic granulocytes involved in inflammation, as well as antimicrobial factors produced by the complement system to fight the infection together.These results suggest that APEC causes the infiltration of heterophilic granulocytes through the involvement of the complement system with serine protease inhibitors.

ABSTRACT: The type VI secretion system (T6SS) of avian pathogenic Escherichia coli (APEC) can affect the functions of eukaryotic cells by secreting or injecting effectors. Hemolysin co-regulatory protein (Hcp), one of the markers of the T6SS, is both a structural protein and an effector protein of the T6SS. According to previous studies, mitochondria in eukaryotic cells are targeted by pathogenic bacteria. However, little is known about the regulation of mitochondria in eukaryotic host cells by the T6SS effector protein Hcp of APEC. In our study, DF-1 cells co-incubated with Hcp2a protein for 6 h showed decreased mitochondrial membrane potential, increased Ca2+ concentration, and increased cellular reactive oxygen species (ROS) levels. We therefore conclude that Hcp2a protein causes dysfunction to mitochondria in DF-1 cells. To explain the mechanism that causes mitochondrial dysfunction, we reanalyzed the Hcp2a interaction protein dataset in DF-1 cells, and the Leucine zipper EF-hand-containing transmembrane protein 1 (LETM1), which is associated with mitochondria, was screened. The protein and molecular docking results showed that Hcp2a protein and LETM1 protein have better binding. Finally, subcellular localization results showed that Hcp2a was localized to mitochondria. In summary, Hcp2a effector proteins caused dysfunction to DF-1 cellular mitochondria, and we hypothesize that the interaction of Hcp2a protein with LETM1 protein induces mitochondrial dysfunction and promotes mitochondrial localization of Hcp2a in DF-1 cells.

Abstract APEC encodes multiple virulence factors that have complex pathogenic mechanisms. In this study, we report a virulence factor named EspE3, which can be secreted from APEC. This protein was predicted to have a leucine-rich repeat domain (LRR) and may have a similar function to IpaH class effectors of the type III secretion system (T3SS). For further exploration, the regulatory correlation between the espE3 and ETT2 genes in APEC was analysed. We then assessed the pathogenicity of EspE3, detected it in APEC secretion proteins and screened the proteins of EspE3 that interact with chicken trachea epithelial cells. This study provides data on a new virulence factor for further exploring the pathogenic mechanism of APEC.

Abstract The type VI secretion system (T6SS) is a secretion apparatus widely found in pathogenic Gram-negative bacteria and is important for competition among various bacteria and host cell pathogenesis. Hcp is a core component of functional T6SS and transports toxic effectors into target cells by assembling to form tube-like structures. Studies have shown that Hcp simultaneously acts as an effector to influence cellular physiological activities; however, the mechanism of its activity in host cells remains unclear. To investigate the target of effector protein Hcp2a in a chicken fibroblast cell line, we first detected the subcellular localization of Hcp2a in DF-1 cells by indirect immunofluorescence assay. The results showed that Hcp2a protein was localized in the endoplasmic reticulum of DF-1 cells. We also used a streptavidin–biotin affinity pull-down assay combined with LC–MS/MS to screen DF-1 cell lysates for proteins that interact with Hcp2a and analyze the cellular functional pathways affected by them. The results showed that Hcp2a interacted with 52 DF-1 cellular proteins that are involved in multiple intracellular pathways. To further explore the mechanism of Hcp2a protein targeting the endoplasmic reticulum of DF-1 cells, we screened three endoplasmic reticulum-associated proteins (RSL1D1, RPS3A, and RPL23) from 52 prey proteins of Hcp2a for protein–protein molecular docking analysis. The docking analysis showed that the effector protein Hcp2a and the RPL23 protein had good complementarity. Overall, we propose that Hcp2a has strong binding activity to the RPL23 protein in DF-1 cells and this may help Hcp2a anchor to the endoplasmic reticulum in DF-1 cells.

Abstract Background Bacteria deliver effector proteins into the host cell via a secretory system that can directly act on the target to cause disease. As an important pipeline structural protein of the type VI secretion system (T6SS) complex, Hcp acts together with other virulence factors in the target cell. There is growing evidence that T6SS plays a key role in the pathogenic mechanism of APEC. However, the regulatory function played by the effector protein Hcp during its interaction with host cells is not clear. Here, tandem mass tag (TMT) analysis was used to quantify the proteins affected by increased expression of Hcp2a in DF-1 cells. Results The host response was significantly different between the overexpression and null groups at the protein level. A total of 195 differentially expressed proteins (DEPs) were detected in the overexpression group (upregulated, n = 144, downregulated, n = 51). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the biological functions and pathways of differentially expressed proteins. The results showed that these DEPs were mainly enriched in RNA degradation, spliceosome, and mRNA surveillance pathways. Conclusions This study suggests that Hcp2a, the effector protein of APEC, plays an important role in regulating mRNA splicing and protein quality control in DF-1 cells. These findings provide useful clues to elucidate the pathogenic mechanism of effector protein Hcp2a on host target cells.

We analyzed metagenome data of feces from sows at different physiological periods reared on large-scale farms in Anhui Province, China, to provide a better understanding of the microbial diversity of the sow intestinal microbiome and the structure of antibiotic-resistance genes (ARGs) and virulence genes it carries. Species annotation of the metagenome showed that in the porcine intestinal microbiome, bacteria were dominant, representing >97% of the microorganisms at each physiological period. Firmicutes and Proteobacteria dominated the bacterial community. In the porcine gut microbiome, the viral component accounted for an average of 0.65%, and the species annotation results indicated that most viruses were phages. In addition, we analyzed the microbiome for ARGs and virulence genes. Multidrug-like, MLS-like, and tetracycline-like ARGs were most abundant in all samples. Evaluation of the resistance mechanisms indicated that antibiotic inactivation was the main mechanism of action in the samples. It is noteworthy that there was a significant positive correlation between ARGs and the total microbiome. Moreover, comparative analysis with the Virulence Factor Database showed that adhesion virulence factors were most abundant.

With the widespread promotion of the green feeding concept of “substitution and resistance”, there is a pressing need for alternative products in feed and breeding industries. Employing lactic acid bacteria represents one of the most promising antimicrobial strategies to combat infections caused by pathogenic bacteria. As such, we analyzed the intestinal tract of Anhui local pig breeds, including LiuBai Pig, YueHei Pig, and HuoShou Pig, to determine the composition and diversity of intestinal microbiota using 16S rRNA. Further, the functionality of the pigs’ intestinal microbiota was studied through metagenomic sequencing. This study revealed that lactic acid bacteria were the primary contributors to the functional composition, as determined through a species functional contribution analysis. More specifically, the functional contribution of lactic acid bacteria in the HuoShou Pig group was higher than that of the LiuBai Pig and YueHei Pig. Subsequently, the intestinal contents of the HuoShou Pig group were selected for the screening of the dominant lactic acid bacteria strains. Out of eight strains of lactic acid bacteria, the acid-production capacity, growth curve, and tolerance to a simulated intestinal environment were assessed. Additional assessments included surface hydrophobicity, the self-aggregation capability, co-agglutination of lactic acid bacteria with pathogenic bacteria, and an in vitro bacteriostatic activity assay. Lactobacillus johnsonii L5 and Lactobacillus reuteri L8 were identified as having a strong overall performance. These findings serve as a theoretical basis for the further development of pig-derived probiotics, thereby promoting the application of lactic acid bacteria to livestock production.

Abstract Avian pathogenic Escherichia coli (APEC) is a pathotype of extraintestinal pathogenic E. coli and one of the most serious infectious diseases of poultry. It not only causes great economic losses to the poultry industry, but also poses a serious threat to public health worldwide. Here, we examined the role of YqeH, a transcriptional regulator located at E. coli type III secretion system 2 (ETT2), in APEC pathogenesis. To investigate the effects of YqeH on APEC phenotype and virulence, we constructed a yqeH deletion mutant (APEC40-ΔyqeH) and a complemented strain (APEC40-CΔyqeH) of APEC40. Compared with the wild type (WT), the motility and biofilm formation of APEC40-ΔyqeH were significantly reduced. The yqeH mutant was highly attenuated in a chick infection model compared with WT, and showed severe defects in its adherence to and invasion of chicken embryo fibroblast DF-1 cells. However, the mechanisms underlying these phenomena were unclear. Therefore, we analyzed the transcriptional effects of the yqeH deletion to clarify the regulatory mechanisms of YqeH, and the role of YqeH in APEC virulence. The deletion of yqeH downregulated the transcript levels of several flagellum-, biofilm-, and virulence-related genes. Our results demonstrate that YqeH is involved in APEC pathogenesis, and the reduced virulence of APEC40-ΔyqeH may be related to its reduced motility and biofilm formation.

ABSTRACT: Avian pathogenic Escherichia coli (APEC) is a serious systemic infectious disease in poultry infections, causing severe economic losses to the poultry industry. Previous studies have shown that secretion of virulence proteins was required for the pathogenicity of APEC through the secretion system. Outer membrane vesicles (OMVs) are a generalized secretion system of Gram-negative bacteria that play a key role in the long-distance delivery of virulence factors, but whether they are associated with the pathogenic mechanism of APEC has not been determined. In this study, OMVs were purified and characterized from AE17 (O2 serotype) by ultracentrifugation and density gradient centrifugation and their protein cargo was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, 89Zr was labeled after chelating AE17 OMVs by DFO and positron emission tomography PET imaging was used to track 89Zr-DFO-OMVs in chickens and to pathologically analyze the distribution sites. This study showed that AE17 OMVs were membrane vesicles ranging in size from 20 to 200 nm and proteomic analysis revealed the presence of virulence proteins, including adhesion proteins OmpA, OmpC, OmpF, OmpX, FimH, FimC and FigE, and serum resistance proteins OmpT and MliC and immune response regulator proteins (FliC). In addition, in vivo PET imaging to track the biodistribution of AE17 OMVs showed that AE17 OMVs were taken up by the lung region and the gastrointestinal and renal regions but were not detected in other areas. Pathological analysis of the tissue sites where AE17 OMVs were ingested showed inflammatory responses and damage. These findings suggested that AE17 OMVs not only contained a group of virulence proteins associated with AE17 infection but can also deliver these virulence proteins over long distances and caused tissue inflammatory damage. Our study revealed a previously unidentified causative microbial signal in the pathogenesis of APEC that could aid in the development of vaccines and antibiotics effective against APEC.

ABSTRACT: EnvZ, the histidine kinase (HK) of OmpR/EnvZ, transduces osmotic signals in Escherichia coli K12 and affects the pathogenicity of Shigella flexneri and Vibrio cholera. Avian pathogenic E. coli (APEC) is an extra-intestinal pathogenic E. coli (ExPEC), causing acute and sudden death in poultry and leading to severe economic losses to the global poultry industry. How the functions of EnvZ correlate with APEC pathogenicity was still unknown. In this study, we successfully constructed the envZ mutant strain AE17ΔenvZ and the inactivation of envZ significantly reduced biofilms and altered red, dry, and rough (rdar) morphology. In addition, AE17ΔenvZ was significantly less resistant to acid, alkali, osmotic, and oxidative stress conditions. Deletion of envZ significantly enhanced sensitivity to specific pathogen-free (SPF) chicken serum and increased adhesion to chicken embryonic fibroblast DF-1 cells and elevated inflammatory cytokine IL-1β, IL6, and IL8 expression levels. Also, when compared with the WT strain, AE17ΔenvZ attenuated APEC pathogenicity in chickens. To explore the molecular mechanisms underpinning envZ in APEC17, we compared the WT and envZ-deletion strains using transcriptome analyses. RNA-Seq results identified 711 differentially expressed genes (DEGs) in the envZ mutant strain and DEGs were mainly enriched in outer membrane proteins, stress response systems, and TCSs. Quantitative real-time reverse transcription PCR (RT-qPCR) showed that EnvZ influenced the expression of biofilms and stress responses genes, including ompC, ompT, mlrA, basR, hdeA, hdeB, adiY, and uspB. We provided compelling evidence showing EnvZ contributed to APEC pathogenicity by regulating biofilms and stress response expression.

ABSTRACT: Fatal gout in geese caused by goose astrovirus (GAstV) has been spreading rapidly in China since 2018, causing serious economic losses in the goose breeding industry. To achieve simple, convenient and sensitive detection of GAstV, a novel diagnostic test was developed by combining reverse transcription-enzymatic recombinase amplification (RT-ERA) and CRISPR-Cas12a technologies. RT-ERA primers were designed to pre-amplify the conserved region of the ORF2 gene of GAstV and the predefined target sequence detected using the Cas12a/crRNA complex at 37℃ for 30 min. Specific detection of GAstV was achieved with no cross-reaction with non-GAstV templates and a sensitivity detection limit of 2 copies. The experimental procedure could be completed within 1 h, including RNA extraction (15 min), RT-ERA reaction (20 min), CRISPR-Cas12a/crRNA detection (5 min) and result readout (within 2 min) steps. In conclusion, the combination of RT-ETA and CRISPR-Cas12a provides a rapid and specific method that should be effective for the control and surveillance of GAstV infections in farms from remote locations.

Porcine circovirus type 4 (PCV4) is a newly emerging pathogen that was first detected in 2019 and is associated with diverse clinical signs, including respiratory and gastrointestinal distress, dermatitis and various systemic inflammations. It was necessary to develop a sensitive and specific diagnostic method to detect PCV4 in clinical samples, so in this study, a multienzyme isothermal rapid amplification (MIRA) assay was developed for the rapid detection of PCV4 and evaluated for sensitivity, specificity and applicability. It was used to detect the conserved Cap gene of PCV4, operated at 41°C and completed in 20 min. With the screening of MIRA primer-probe combination, it could detect as low as 101 copies of PCV4 DNA per reaction and was highly specific, with no cross-reaction with other pathogens. Further assessment with clinical samples showed that the developed MIRA assay had good correlation with real-time polymerase chain reaction assay for the detection of PCV4. The developed MIRA assay will be a valuable tool for the detection of the novel PCV4 in clinical samples due to its high sensitivity and specificity, simplicity of operation and short testing time.

Porcine parvovirus (PPV) is one of the important causes of pig reproductive diseases. The most prevalent methods for PPV authentication are the polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and quantitative real-time PCR. However, these procedures have downsides, such as the fact that they take a long time and require expensive equipment. As a result, a rapid, visible, and economical clinical diagnostic strategy to detect PPV is necessary. In this study, three pairs of crRNA primers were designed to recognize the VP2 gene, and an ERA-CRISPR/Cas12a system for PPV detection was successfully developed. The approach involved isothermal detection at 37°C, and the method can be used for visual inspection. The detection limit of the ERA-CRISPR/Cas12a system was 3.75 × 102 copies/μL, and no cross reactions with other porcine viruses were found. In view of the preceding, a rapid, visible, and low-cost nucleic acid testing approach for PPV has been developed using the ERA-CRISPR/Cas12a system.

ABSTRACT: Avian colibacillosis is a serious systemic infectious disease in poultry and caused by avian pathogenic Escherichia coli (APEC). Previous studies have shown that 2-component systems (TCSs) are involved in the pathogenicity of APEC. OmpR, a response regulator of OmpR/EnvZ TCS, plays an important role in E. coli K-12. However, whether OmpR correlates with APEC pathogenesis has not been established. In this study, we constructed an ompR gene mutant and complement strains by using the CRISPR-Cas9 system and found that the inactivation of the ompR gene attenuated bacterial motility, biofilm formation, and the production of curli. The resistance to environmental stress, serum sensitivity, adhesion, and invasion of DF-1 cells, and pathogenicity in chicks were all significantly reduced in the mutant strain AE17ΔompR. These phenotypes were restored in the complement strain AE17C-ompR. The qRT-PCR results showed that OmpR influences the expression of genes associated with the flagellum, biofilm formation, and virulence. These findings indicate that the regulator OmpR contributes to APEC pathogenicity by affecting the expression and function of virulence factors.

Avian pathogenic Escherichia coli (APEC) is one of the most common pathogens in poultry and a potential gene source of human extraintestinal pathogenic E. coli (ExPEC), leading to serious economic losses in the poultry industry and public health concerns. Exploring the pathogenic mechanisms underpinning APEC and the identification of new targets for disease prevention and treatment are warranted. YgeK is a transcriptional regulator in APEC and is localized to the type III secretion system 2 of E. coli. In our previous work, the transcription factor ygeK significantly affected APEC flagella formation, bacterial motility, serum sensitivity, adhesion, and virulence. To further explore ygeK functions, we evaluated its influence on APEC biofilm formation and resistance to environmental stress. Our results showed that ygeK inactivation decreased biofilm formation and reduced bacterial resistance to environmental stresses, including acid and oxidative stress. In addition, the multi-level regulation of ygeK in APEC was analyzed using proteomics, and associations between differentially expressed proteins and the key targets of ygeK were investigated. Overall, we identified ygeK’s new function in APEC. These have led us to better understand the transcriptional regulatory ygeK and provide new clues about the pathogenicity of APEC.