Porcine deltacoronavirus (PDCoV) is a significant enteric pathogen in swine, necessitating rapid and accessible diagnostic tools for effective control. In this study, we developed a streamlined, extraction-free Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) assay integrated with a TaqMan probe for on-site PDCoV detection. By targeting the conserved N gene and modifying the loop primer (LB) as a fluorescence probe, a highly specific reaction system was established. The assay demonstrated high sensitivity, with a limit of detection (LOD) of 9.3 copies/μL for cDNA and 5.9 copies/μL for RNA within 40 min, while showing no cross-reactivity with common swine viruses. Reproducibility assays yielded a coefficient of variation (CV) of < 2.5%, indicating excellent stability. Furthermore, the method was successfully integrated with nucleic acid extraction-free technology for fecal sample analysis. In clinical validation using 75 samples, the assay showed 100% concordance with column-based RT-LAMP and standard RT-qPCR methods. This extraction-free RT-LAMP-TaqMan assay offers a rapid, reliable, and cost-effective Point-of-Care Testing (POCT) solution for PDCoV surveillance in swine herds.

[This corrects the article DOI: 10.3389/fmicb.2025.1669658.].

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of foodborne infections and represents a significant public health concern. After entering host cells, S. Typhimurium uses effector proteins to support its intracellular survival, but the mechanisms through which these effectors act remain incompletely understood. In this study, we investigated PipB2 as a Salmonella effector associated with increased intracellular bacterial survival and characterized Rab14 as a PipB2-associated host target. We found that recombinant PipB2 directly bound Rab14 and reduced Rab14 GTP hydrolysis in vitro, while the presence of PipB2 in infected cells was associated with increased phosphorylation of AKT and AS160, suggesting that PipB2 may affect Rab14-related host processes and may also be linked to AKT-AS160-associated host signaling. These PipB2-associated changes were accompanied by impaired autophagic flux and increased intracellular bacterial burden, consistent with enhanced intracellular replication. Pharmacological inhibition of AKT with inhibitor VIII partially attenuated PipB2-associated changes in autophagic flux and reduced intracellular bacterial burden both in vitro and in vivo. Our mechanistic investigations provide new insights into how the S. Typhimurium effector PipB2 impairs host autophagic flux and suggest a potential host-directed intervention strategy.

Mycoplasma gallisepticum (MG) is a common and harmful pathogen in the poultry farming industry. Early detection is essential for effective prevention and control of MG. To meet the demand for high-throughput detection in resource-limited testing environments, a colorimetric loop-mediated isothermal amplification (LAMP) assay based on the cresol red indicator was developed. This method enables visual analysis by detecting hydrogen ions produced during the LAMP reaction, causing a color shift from magenta to yellow. The assay successfully detected MG at concentrations as low as 1.07 × 102 copies per µL within 70 min, with no cross-reactivities against other avian respiratory pathogens. It demonstrated stability and reliability, with a coefficient of variation (CV) below 5% across repeated experiments. Additionally, an intelligent software program based on the LAB color space algorithm was developed to complement this assay. The digital analysis function enhances detection precision and facilitates high-throughput analysis by processing up to 64 samples simultaneously. Validation with clinical samples from intensive farms showed a 98% concordance rate with qPCR results (κ > 0.9), confirming the method's accuracy. Consequently, the colorimetric LAMP assay in this study not only accurately identifies MG but also provides an intelligent, high-throughput detection platform for MG detection.

The swine industry faces a substantial economic threat from Lawsonia intracellularis (LI), the bacterium responsible for porcine proliferative enteropathy. Infection commonly presents with diarrhea and increased enterocyte proliferation in the ileum and colon, ultimately resulting in impaired growth performance in affected pigs. Effective management of porcine proliferative enteropathy requires timely and accurate diagnosis. The method provides significant advantages over existing detection techniques, offering enhanced sensitivity and high accuracy. The assay achieves detection within one hour, with no observed cross-reactivity against a panel of common swine pathogens. Results can be visually interpreted under blue light. Clinical validation using 123 samples demonstrated 100% concordance with qPCR results (14 positive and 109 negative). Characterized by simplicity, rapidity, and high sensitivity, the RPA-CRISPR/Cas12a method represents a novel solution for Lawsonia intracellularis detection.

Glaesserlla parasuis (G. parasuis), a Gram-negative pathogen responsible for Glässer's disease, employs outer membrane vesicles (OMVs) as sophisticated nanoscale effectors to modulate host‒pathogen interplay. While bacterial OMVs are recognized as critical mediators of virulence dissemination, their functional orchestration in G. parasuis immunopathogenesis remains unclear. To date, few reports have focused on the relationships among G. parasuis, OMVs and host-susceptible cells; thus, more evidence is urgently needed to explore their crosstalk further. This study revealed a novel immune activation paradigm: both G. parasuis and its OMVs trigger robust type I interferon (IFN) responses via a DNA-sensing cascade. G. parasuis OMVs-Dio were internalized by macrophages in a time-dependent manner, partially via clathrin-mediated endocytosis but mainly via dynamin-dependent endocytosis. Studies have shown that IFNs play key antiviral roles in viral infections and important roles in bacterial infections. Our results suggested that IFNs inhibited G. parasuis adhesion and invasion of pulmonary alveolar macrophage (PAM) cells. Furthermore, by assessing the major components of OMVs, we confirmed that the DNA of G. parasuis, which is carried by OMVs, is the key component that induces the production of IFN in macrophages. The cGAS–STING–IRF3 pathway links the host’s recognition of G. parasuis OMVs to IFN production. Taken together, our data reveal that G. parasuis OMVs activate cGAS/STING/IRF3 signaling and induce IFN production, which then affects the adhesion and invasion of G. parasuis. The discovery of this vesicle-mediated nucleic acid delivery system redefines the pathogenesis framework for G. parasuis and provides a trans-species conceptual advance in understanding how Gram-negative pathogens exploit vesicular trafficking to manipulate host immunity.

Despite decades of research on effective methods to resist Salmonella enterica serovar Typhimurium (S. Typhimurium) pathogenicity, the mechanisms of S. Typhimurium-host interactions have not been fully determined. S. Typhimurium is characterized as an important zoonosis in public health worldwide because of its endemicity, high morbidity, and difficulty in applying control and prevention measures. Herein, we introduce a novel bacterial factor, secretion system effector J (SseJ), and its interactive host protein, PHB2 (prohibitin 2). We explored whether SseJ affected S. Typhimurium replication and survival in the host. S. Typhimurium infection caused severe mitochondrial damage and mitophagy, which facilitated S. Typhimurium proliferation in cells. S. Typhimurium SseJ activated the PINK1 (PTEN induced kinase 1)-PRKN (parkin RBR E3 ubiquitin protein ligase)-autophagosome-dependent mitophagy pathway, aided by the mitophagy receptor PHB2, for bacterial survival and persistent infection. Moreover, suppression of mitophagy alleviated the pathogenicity of S. Typhimurium. In conclusion, S. Typhimurium infection could be antagonized by targeting the SseJ-PHB2-mediated host mitochondrial autophagy pathway.Abbreviation: ACTB: actin beta; BafA1: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; co-IP: co-immunoprecipitation; CFU: colony-forming units; COX4/COXIV: cytochrome c oxidase subunit 4; CQ: chloroquine; hpi: h post-bacterial infection; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Mdivi-1:mitophagy inhibitor mitochondrial division inhibitor 1; MFN2: mitofusin 2; MG132: z-leu-leu-leucinal; MOI: multiplicity of infection; mtDNA: mitochondrial DNA; PBS: phosphate-buffered saline; PGAM5: PGAM family member 5, mitochondrial serine/threonine protein phosphatase; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; qPCR: quantitative real-time reverse transcription PCR; Roc-A: Rocaglamide A; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; SCVs: Salmonella-containing vacuoles; siRNA: small interfering RNA; SPI-2: Salmonella pathogenicity island 2; SseJ: secretion system effector J; S. Typhimurium: Salmonella enterica serovar Typhimurium; S.T-ΔSseJ: SseJ gene-deleted Salmonella Typhimurium strains; S.T-CΔSseJ: SseJ-complemented Salmonella Typhimurium strains; WT: wild-type.

As the only member of the genus Senecavirus within the family Picornaviridae, Senecavirus A (SVA) has posed an enormous challenge for the pig industry worldwide. In our previous study, a SVA strain was isolated from a buffalo with mouth ulcers. To systematically assess its pathogenicity, this study compared the outcome of piglets and buffaloes artificially infected by the different viral dose of the buffalo-origin SVA strain (SVA/GD/China/2018). These results indicated that vesicular diseases can occur in infected piglets and buffaloes. Severe clinical symptoms were observed in the piglets and buffaloes with the inoculation of 105.0 50% tissue culture infective dose (TCID50/mL). The SVA antigen expression was also detected in the lung tissue, chin blister lesion tissue, nasolabial tissue of the piglets, and the upper lip blister tissue of the buffaloes. This study demonstrated that the buffalo-origin SVA strain was pathogenic to piglets and buffaloes, revealing the possibility of cross-species transmission of SVA between pigs and buffaloes. In the future, it is necessary to strengthen the surveillance of SVA in cattle herds.

Campylobacter represents a major zoonotic threat to both public health and the livestock industry. To address the limitations of current detection approaches, a smartphone-assisted, portable, on-site fast-extraction testing (POET) platform was developed. This system integrates a highly specific EP-LAMP assay, a nucleic acid fast-extraction reagent, a portable reaction device, and a smartphone app for colorimetric analysis. The EP-LAMP method shows high specificity and sensitivity, with detection limits of 2.1×10² copies/μL for plasmid templates and 250 CFU/mL for genomic templates. The entire detection process can be completed on-site within one hour. The detection capability of POET platform for real samples was validated using 40 pork samples, demonstrating 100% concordance with TaqMan qPCR results. This platform enables visual detection without external power or specialized equipment, fulfilling the requirements for rapid, simple, and field-deployable Campylobacter coli diagnostics.

BackgroundPorcine epidemic diarrhea virus (PEDV) causes severe diarrhea, vomiting, and high mortality in neonatal piglets, but no fully effective treatments or vaccines are currently available. Although gut microbiota transplantation can alleviate post infection symptoms, the specific protective bacterial strains or metabolites involved, along with their underlying mechanisms of action against PEDV, remain unclear.ResultsOral administration of L. reuteri GZ-1 or its metabolite hyodeoxycholic acid (HDCA) to three-day-old piglets significantly mitigated clinical symptoms and improved survival outcomes following PEDV challenge. This protection was achieved through five-day pretreatment preceding viral exposure. Both interventions substantially preserved the intestinal architecture, maintaining normal villus height and goblet cell density while markedly reducing PEDV loads in jejunal tissue. Metabolomic profiling established HDCA—a secondary bile acid derivative of L. reuteri metabolism—as the core protective mediator. The direct antiviral activity of HDCA against PEDV was subsequently confirmed through complementary in vitro and in vivo experimental validation. Integrated transcriptomic and proteomic analyses revealed a dual mechanistic pathway underlying HDCA efficacy: (1) suppression of NF-κB-driven inflammatory cascades and (2) activation of interferon-stimulated gene 15 (ISG15)-dependent antiviral pathways.ConclusionThis study establishes the L. reuteri-HDCA-TGR5-IFNβ-ISG15 metabolic axis as a novel antiviral pathway. This study identified microbial-derived HDCA as a key effector metabolite that mediates protection against PEDV through the coordinated suppression of inflammation and enhancement of antiviral defenses. These findings highlight microbial-metabolic crosstalk as a promising therapeutic strategy against enteric coronaviruses and provide foundational evidence for commensal-derived interventions to manage porcine epidemic diarrhea.

Rapid on-site typing methods for SARS-CoV-2 variants of concern are crucial for its effective surveillance and control. Herein, a smart single-loop-mediated isothermal amplification (ssLAMP) method with the absence of an inner primer but the addition of a swarm primer for differentiation of SARS-CoV-2 Omicron variants is developed. This unique primer design strategy offers greater flexibility in introducing single nucleotide polymorphism (SNP) identification probes and enables multiple detection assays for SARS-CoV-2 Omicron variants including BA.1, BA.2, BA.3, BA.4, and BA.5. A 3D-printed portable dual fluorescence visualization device and smartphone app are developed to enable point-of-care testing. This assay is rapid (within 90 min), highly sensitive (100 copies/reaction), and specific (identification of SNP) for SARA-CoV-2 Omicron variants. The ssLAMP method identifies five BA.5-positive samples among 97 nasopharyngeal swab samples from the clinic, with a 100% concordance rate with Sanger sequencing. The ssLAMP assay system is expected to be utilized for on-site, highly specific, and rapid visualization detection of SARS-CoV-2 and its variants, with great application potential in pathogen genotyping, early cancer screening, and other areas of SNP mutation detection.

Porcine epidemic diarrhea virus (PEDV) is responsible for causing fatal watery diarrhea in piglets, resulting in significant economic losses within the pig farming industry. Although vaccination is currently employed as a preventive measure, certain vaccines do not provide complete protection against PEDV field strains. Probiotics present a promising alternative due to their ability to regulate intestinal flora, enhance host immunity, and improve resistance against pathogenic microorganisms. We isolated six lactic acid bacteria (LAB) from the fecal microorganisms of Bama pigs, compared to Limosilactobacillus mucosae DSM13345 of the same genus in which Limosilactobacillus mucosae G01 (L. mucosae G01) proved to have a potent anti-PEDV effect. In a comprehensive manner, L. mucosae G01 significantly augmented the phosphorylation of IRF3 in IPEC-J2 cells, resulting in the induction of interferons (IFN α, IFN β, IFN λ1, and IFN λ3) and subsequent upregulation of interferon-stimulated genes (ISGs) (MX1, MX2, OAS1, and ZAP) in a dose-dependent fashion, consequently leading to the mitigation of PEDV replication. These findings underscore the promising prospects of L. mucosae G01 as a naturally derived substitute for combating PEDV and other enteric coronavirus infections.

Streptococcus suis serotypes 2 and 14 are the most common zoonotic strains, but previous identification methods made distinguish these two serotypes from other S. suis serotypes difficult. To effectively prevent and control them, there is an urgent need for a highly sensitive and specific method to identify these two serotypes. In this study, a fluorescent probe was designed for the single nucleotide polymorphism site at cpsK 483 of Streptococcus suis type 2 and type 14 compared with other serotypes, and an enzyme-activated probe quantitative PCR (EA-probe qPCR) method was established for the detection of Streptococcus suis type 2 and type 14 by combining with the specific hydrolysis characteristics of the RNase H2 enzyme. The results showed that the optimal probe concentration for this method was 0.5 µM and the optimal RNase H2 enzyme concentration was 25 mU.This method showed no reactivity with genomic DNA from Streptococcus suis strains 1/2, 5, 7, 9, 23, 28, 29, and 31, confirming its high specificity. And its sensitivity can reach 18.4 CFU. In addition, 19 clinical strains of Streptococcus suis type 2 or type 1/2 were tested. The results showed 100

Actinobacillus pleuropneumoniae, a significant respiratory pig pathogen, is causing substantial losses in the global swine industry. The resistance spectrum of A. pleuropneumoniae is expanding, and multidrug resistance is a severe issue. Horizontal gene transfer (HGT) plays a crucial role in the development of the bacterial genome by facilitating the dissemination of resistance determinants. However, the horizontal transfer of resistance genes via A. pleuropneumoniae-derived outer membrane vesicles (OMVs) has not been previously reported. In this study, we used Illumina NovaSeq and PacBio SequeI sequencing platforms to determine the whole genome sequence of A. pleuropneumoniae GD2107, a multidrug-resistant (MDR) isolate from China. We detected a plasmid in the isolate named pGD2107-1; the plasmid was 5,027 bp in size with 7 putative open reading frames (ORF) and included the floR resistance genes. The carriage of resistance genes in A. pleuropneumoniae OMVs was identified using a polymerase chain reaction (PCR) assay, and then we thoroughly evaluated the influence of OMVs on the horizontal transfer of drug-resistant plasmids. The transfer of the plasmid to recipient bacteria via OMVs was confirmed by PCR. In growth competition experiments, all recipients carrying the pGD2107-1 plasmid exhibited a fitness cost compared to the corresponding original recipients. This study revealed that OMVs could mediate interspecific horizontal transfer of the resistance plasmid pGD2107-1 into Escherichia coli recipient strains and significantly enhance the resistance of the transformants. In summary, A. pleuropneumoniae-OMVs play the pivotal role of vectors for dissemination of the floR gene spread and may contribute to more antimicrobial resistance gene transfer in other Enterobacteriaceae.

Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that clinically causes severe swine and human infections (such as meningitis, endocarditis, and septicemia). In order to cause widespread diseases in different organs, S. suis 2 must colonize the host, break the blood barrier, and cause exaggerated inflammation. In the last few years, most studies have focused on a single virulence factor and its influences on the host. Membrane vesicles (MVs) can be actively secreted into the extracellular environment contributing to bacteria-host interactions. Gram-negative bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide (LPS), causing host cell pyroptosis. However, little is known about the effect of the MVs from S. suis 2 (Gram-positive bacteria without LPS) on cell pyroptosis. Thus, we investigated the molecular mechanism by which S. suis 2 MVs participate in endothelial cell pyroptosis. In this study, we used proteomics, electron scanning microscopy, fluorescence microscope, Western blotting, and bioassays, to investigate the MVs secreted by S. suis 2. First, we demonstrated that S. suis 2 secreted MVs with an average diameter of 72.04 nm, and 200 proteins in MVs were identified. Then, we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis. The S. suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway, resulting in cell pyroptosis, but it did not activate the Caspase-4/-5 pathway. More importantly, endothelial cells produce large amounts of reactive oxygen species (ROS) and lost their mitochondrial membrane potential under induction by S. suis 2 MVs. The results in this study suggest for the first time that MVs from S. suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage, which produced mtDNA and ROS under induction, leading to the pyroptosis of endothelial cells.

The Porcine epidemic diarrhea virus (PEDV) presents a substantial risk to the domestic pig industry, resulting in extensive and fatal viral diarrhea among piglets. Recognizing the mucosal stimulation triggered by PEDV and harnessing the regulatory impact of lactobacilli on intestinal function, we have developed a lactobacillus based vaccine that is carefully designed to elicit a strong mucosal immune response. Through bioinformatics analysis, we examined PEDV S proteins to identify B-cell linear epitopes that meet the criteria of being non-toxic, soluble, antigenic, and capable of neutralizing the virus. In this study, a genetically modified strain of Lactobacillus mucosae G01 (L.mucosae G01) was created by utilizing the S layer protein (SLP) as a scaffold for surface presentation. Chimeric immunodominant epitopes with neutralizing activity were incorporated at various sites on SLP. The successful expression of SLP chimeric immunodominant epitope 1 on the surface of L.mucosae G01 was confirmed through indirect immunofluorescence and transmission electron microscopy, revealing the formation of a transparent membrane. The findings demonstrate that the oral administration of L.mucosae G01, which expresses the SLP chimeric immunodominant gene epitope1, induces the production of secreted IgA in the intestine and feces of mice. Additionally, there is an elevation in IgG levels in the serum. Moreover, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 are significantly increased compared to the negative control group. These results suggest that L.mucosae G01 has the ability to deliver exogenous antigens and elicit a specific mucosal immune response against PEDV. This investigation presents new possibilities for immunoprophylaxis against PEDV-induced diarrhea.

EDITORIAL article Front. Vet. Sci., 19 December 2023Sec. Veterinary Infectious Diseases Volume 10 - 2023 | https://doi.org/10.3389/fvets.2023.1349844

Haemophilus parasuis (H. parasuis, HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. Therefore, the rapid diagnosis of this pathogen is of utmost importance. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for H. parasuis that is fast to detect, easy to implement, and accurate to analyze, known as RPA-CRISPR/Cas12a analysis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/μL of DNA template, p < 0.05), which is 104 -fold higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of H. parasuis did not react with other common pig pathogens, including Streptococcus suis type II and IX, Actinobacillus pleuropneumoniae, Escherichia coli, Salmonella, Streptococcus suis, and Staphylococcus aureus (p < 0.0001). The RPA-CRISPR/Cas12a assay was applied to 15 serotypes of H. parasuis clinical samples through crude extraction of nucleic acid by boiling method, and all of the samples were successfully identified. It greatly reduces the time and cost of nucleic acid extraction. Moreover, the method allows results to be visualized with blue light. The accurate and convenient detection method could be incorporated into a portable format as point-of-care (POC) diagnostics detection for H. parasuis at the field level.

EDITORIAL article Front. Cell. Infect. Microbiol., 26 June 2023Sec. Clinical Microbiology Volume 13 - 2023 | https://doi.org/10.3389/fcimb.2023.1219506

Currently, porcine coronaviruses are prevalent in pigs, and due to the outbreak of COVID-19, porcine coronaviruses have become a research hotspot. porcine epidemic diarrhea virus (PEDV), Transmissible Gastroenteritis Virus (TGEV), and Porcine Deltacoronavirus (PDCoV) mentioned in this study mainly cause diarrhea in pigs. These viruses cause significant economic losses and pose a potential public health threat. In this study, specific primers and probes were designed according to the M gene of PEDV, the S gene of TGEV, and the M gene of PDCoV, respectively, and TaqMan probe-based multiplex real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was developed for the simultaneous detection of PEDV, TGEV, and PDCoV. This method has high sensitivity and specificity, and the detection limit of each virus can reach 2.95 × 100 copies/μl. An assay of 160 clinical samples from pigs with diarrhea showed that the positive rates of PEDV, TGEV, and PDCoV were 38.13, 1.88, and 5.00%; the coinfection rates of PEDV+TGEV, PEDV+PDCoV, TGEV+PDCoV, PEDV+TGEV+PDCoV were 1.25, 1.25, 0, 0.63%, respectively. The positive coincidence rates of the multiplex qRT-PCR and single-reaction qRT-PCR were 100%. This method is of great significance for clinical monitoring of the porcine enteric diarrhea virus and helps reduce the loss of the breeding industry and control the spread of the disease.