Xiao Han

Foodborne bacterial pathogens, including S. aureus, L. monocytogenes, C. sakazakii, and M. bovis, pose significant threats to dairy safety and public health. Current detection methods, such as culture-based techniques and real-time quantitative PCR (qPCR), are either time-consuming or limited in absolute quantification accuracy. Herein, we developed and validated a novel quadruplex droplet digital PCR (ddPCR) assay for simultaneous detection and absolute quantification of these four pathogens in dairy products. The assay targets the femA, hly, ompA, and esxA genes, respectively, with optimized primer/probe concentrations of 500 nM/400 nM and an annealing temperature of 58 °C. The established method demonstrated high specificity, with no cross-reactivity against common dairy-associated bacteria. The limits of detection (LoDs) ranged from 7.04 to 10.31 copies/reaction, with coefficients of variation (CVs) below 14% for intra-assay and 10% for inter-assay repeatability. Notably, the ddPCR assay detected a 25% co-contamination rate compared to 13% by qPCR among 120 dairy samples, suggesting higher sensitivity for low-abundance targets. This quadruplex ddPCR platform offers a rapid, sensitive, and high-throughput solution for food safety surveillance, particularly in high-risk dairy matrices such as infant formula.

Porcine diarrheal diseases caused by mixed viral and bacterial infections pose significant challenges to swine health and production. Rapid and accurate identification of key pathogens, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine rotavirus (PoRV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine bocavirus (PBoV), hepatitis E virus (HEV), and Salmonella , remains a challenge due to overlapping clinical symptoms and co-infections. The zoonotic potential of HEV and PBoV also highlights the need for reliable, high-throughput detection methods. We developed a multiplex Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Nucleic Acid Mass Spectrometry (MALDI-TOF NAMS) assay for the simultaneous detection of eight major porcine gastrointestinal pathogens. The method utilizes a multiplex PCR approach and a single-base extension strategy to improve sensitivity and specificity, and the extension products are displayed by mass spectrometry. The analytical performance of the assay was assessed by evaluating the limits of detection (LoD), analytical specificity, and repeatability. Additionally, we validated its diagnostic accuracy using 242 clinical samples, comparing the results with real-time quantitative PCR (qPCR) as the reference method. The MALDI-TOF NAMS assay demonstrated high analytical specificity, with no cross-reactivity to non-target pathogens. The limit of detection (LoD) ranged from 12.20 to 33.59 copies/μL. The intra- and inter-batch reproducibility assessments showed a 100% detection rate across high, medium, and low concentrations (20/20 per concentration, 60/60 total). Validation using 242 clinical samples demonstrated a 98.3% sensitivity and 99.5% specificity compared to qPCR, with an overall concordance rate of 96.2%. The MALDI-TOF NAMS assay provides a sensitive and high-throughput method for the detection and genotyping of major swine diarrhea pathogens. This method performs well in mixed infections and allows for expansion of pathogen species, making it an important addition to traditional diagnostic methods. IMPORTANCE: Porcine diarrheal diseases are a major threat to the swine industry, often caused by multiple viruses and bacteria infecting animals at the same time. Fast and accurate detection of these pathogens is crucial to prevent outbreaks, reduce economic losses, and protect public health, especially given the potential of some pathogens to infect humans. This study introduces a new method that uses nucleic acid mass spectrometry technology to quickly and accurately detect eight major pathogens from pig samples, such as feces, blood, or tissue. Unlike traditional tests that often detect one pathogen at a time, this method can screen for many at once, even in complex cases of mixed infections. It is sensitive, reliable, and can handle large numbers of samples efficiently. This tool offers farmers, veterinarians, and disease control agencies a faster and more effective way to monitor pig health and respond to outbreaks before they spread.

Porcine enteric coronaviruses (CoVs), including swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine transmissible gastroenteritis virus (TGEV), are major pathogens causing porcine viral diarrhea syndrome (VDS), which brings significant economic losses to the swine industry; distinguishing between these clinically similar viruses has become a serious challenge. We developed a highly specific and interference-resistant porcine CoV multiplex digital PCR (dPCR) assay. The assay exhibited robust anti-interference capabilities, as the concentrations of the four viruses did not affect their accurate quantification. The coefficients of variation (CV%) of intra-batch and inter-batch repeatability for all target viruses were less than 11%. The limit of quantification (LoQ) of this dPCR assay reached 7.5 copies/reaction for each target, and it was one order of magnitude more sensitive than qPCR. The limits of detection (LoD) for SADS-CoV, PEDV, PDCoV, and TGEV were 2.72, 3.00, 3.56, and 3.19 copies/reaction, respectively. A total of 408 known samples were used for validation tests, and the results were highly consistent with the known conditions, showing a compliance rate of 97–100%. The diagnostic specificity (Dsp) of the method was 99–100%. In conclusion, the developed multiplex dPCR assay is highly suitable for early detection and quarantine in four porcine CoVs. The results indicate that this dPCR method is characterized by high specificity, anti-interference capabilities, repeatability, and high sensitivity. It also demonstrates a high compliance rate and diagnostic specificity in sample detection. This multiplex dPCR will contribute to the control of porcine enteric CoV-caused VDS and provide clues for subsequent research.

Classical swine fever virus (CSFV) poses a major threat to the pig industry. The mechanisms that CSFV uses to evade host innate immunity are not fully understood. Acetylation of histones and non-histone proteins is involved in modulating innate immune responses. Histone deacetylase 1 (HDAC1) could be proviral or antiviral by modulating the acetylation status of histones, viral proteins or non-histone host proteins, depending on the type of viruses involved. First, we found that CSFV infection in IPEC-J2 cells resulted in reduced expression of HDAC1. By chemical inhibition, gene silencing, and overexpression, we revealed that HDAC1 acts as a negative regulator of CSFV replication in IPEC-J2 cells probably through activation of poly(I:C) and IFN-λ3-induced IFN-I/III innate immunity. Mechanistically, CSFV N ^pro downregulated HDAC1 and its transcriptional regulator specificity protein 1 (Sp1). N ^pro interacted with Sp1 to facilitate its degradation through the ubiquitin-proteasome pathway via its N-terminal domain, a region that does not have significant effect on IRF3 stability. Thus, it is clear that CSFV deploys the two domains of its N ^pro to counteract the innate immune responses, the C-terminal one targeting the IRF3 pathway as previously reported, and the N-terminal one targeting the Sp1-HDAC1 axis. IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express N ^pro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its N ^pro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving N ^pro -mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.

PEDV has developed sophisticated evasion mechanisms to escape host IFN signaling via its structural and nonstructural proteins. STAT1 is one of the key transcription factors in regulating expression of ISGs.

Considering the fact that clinical porcine circovirus-associated diseases (PCVAD) mostly results from activation of latent PCV2 infection by confounding factors such as coinfection or environmental stresses, we propose that such confounding factors might impose oxidative stress to the animals, where PCV2 in infected cells might utilize the elevated reactive oxygen species (ROS) to promote HMGB1 migration out of nuclei in favor of its replication. An animal infection model with a particular stressor could be approached with or without antioxidant treatment to examine the relationship among the stressor, ROS level, HMGB1 distribution in target tissues, virus replication, and severity of PCVAD.