Foot-and-mouth disease (FMD) is a highly contagious animal disease caused by foot-and-mouth disease virus (FMDV), primarily affecting cloven-hoofed animals such as swine, cattle, and sheep. As a core metabolic pathway for maintaining cellular homeostasis, lipid metabolism is frequently hijacked by viruses via metabolic reprogramming mechanisms to support their infection cycle. Studies have demonstrated that positive-sense single-stranded RNA viruses can reshape the host cell membrane system and modulate the lipid metabolic network, thereby constructing a favorable microenvironment for their invasion and replication. However, the molecular mechanisms by which FMDV-also a positive-sense single-stranded RNA virus-promotes viral replication through the regulation of lipid metabolism remain incompletely elucidated. In this study, we found that inhibiting the key enzymes involved in the lipid metabolic pathway could significantly suppress FMDV proliferation. Exogenous supplementation of the downstream products catalyzed by these key enzymes notably restored FMDV replication, indicating that FMDV replication is dependent on lipids. Furthermore, we observed a significant upregulation in the protein expression of carnitine palmitoyltransferase 1A (CPT1A) in host cells following FMDV infection. Inhibition of this enzyme led to a marked reduction in FMDV replication, suggesting that FMDV may enhance the fatty acid β-oxidation pathway to supply energy for its replication. In conclusion, this study comprehensively verified the critical role of lipid metabolism in FMDV replication through multidimensional assays involving the administration of inhibitors targeting key enzymes in the lipid metabolic pathway. These findings provide novel theoretical insights for the development of antiviral drugs and the prevention and control of FMD.

The spread of the tet(X4) gene threatens the efficacy of tigecycline, a last-resort antibiotic. To understand this threat, we studied its prevalence and spread in Escherichia coli from pigs in China. Our investigation of 190 swine E. coli from Henan Province identified 26 (13.68%) as tet(X4)-positive. Whole-genome sequencing and phylogenetic analysis revealed considerable genetic diversity across multiple sequence types. Among these, we identified a globally epidemic O11:H25-ST457 clone. Conjugation assays confirmed high transferability, with 76.92% (20/26) of tet(X4)-harboring plasmids being successfully transferred, predominantly through independent transfer or cotransfer with other plasmids. We further elucidated, for the first time, the molecular mechanism of an IS15DI-mediated fusion between a tet(X4)-bearing IncHI1A/IncHI1B/IncFIA plasmid and an IncFIC(FII)/IncR plasmid. However, the resulting fusion plasmid was highly unstable without antimicrobial selection. Additionally, we report a tet(X4)-positive IncHI2/IncHI2A plasmid whose flexible multidrug resistance region readily underwent rearrangements during conjugation, generating heterogeneous plasmids in transconjugants. Our findings demonstrate that tet(X4) dissemination is primarily driven by the flexible cotransfer of plasmids rather than stable fusion events, highlighting an ongoing and dynamic threat of tigecycline resistance dissemination from livestock to the global health landscape.

Equine herpesvirus type 1 (EHV-1) is a high morbidity and mortality virus that impacts horse populations worldwide. As a respiratory virus, it enters through the upper respiratory tract (URT), where mucosal immunity plays a crucial role in preventing severe disease. In this study, flow cytometry was used to characterize the nasal leukocyte population during EHV-1 infection, and RNA sequencing of nasal secretions was employed to assess transcriptional markers of the mucosal immune response. Horses with distinct immune statuses were compared at four stages: pre-infection, early (day 1 and 3 post-infection [pi]), mid (days 8 and 10pi) and late (day 18pi) infection. Non-immune horses (n = 4), developed clinical signs, shed virus, and established viremia, whereas, immune horses (n = 4), possessing pre-existing immunity, showed no disease or detectable virus after EHV-1 infection. This comparison revealed differential expression of various T cell associated markers based on pre-existing immunity. In immune horses, upregulation of fewer genes occurred during early infection only, while in non-immune horses, a large number of genes were upregulated during mid and late infection. Collectively, these findings highlight a key role for mucosal T cells in mediating viral clearance and restoring homeostasis.

Lawsonia intracellularis, the etiological agent for proliferative enteropathy, remains a major cause of productivity loss and economic burden in the global swine industry. Currently available vaccines provide suboptimal protection and raise safety concerns, highlighting the need for improved immunization strategies. In this study, we developed Salmonella Typhimurium-based vaccine candidates against L. intracellularis in a murine model, incorporating immunoprotective epitopes from the surface autotransporter protein LatA, the flagellar protein FliC, and the heat shock protein 60 (HSP60). These antigens were expressed using dual bacterial-host plasmids, pJHL270 and pJHL305, containing prokaryotic (Ptrc) and eukaryotic (CMV) promoters to ensure both extracellular and intracellular antigen presentation. The attenuated Salmonella delivery strain JOL3086 (Δlon ΔpagL) exhibited a high safety profile with markedly reduced endotoxicity. Western blot analysis confirmed antigen expression in both bacterial and mammalian systems in vitro. Immunization with the recombinant Salmonella strains elicited significant humoral (systemic IgG and mucosal IgA) and cell-mediated (CD4+, CD8+ T cells) immune responses, accompanied by significantly increased levels of Ifng, Il6, Il4, and Il10 cytokines. Among the three vaccine strains, JOL3149 (pJHL305 expressing FliC-HSP60) induced the most pronounced immunological responses and conferred superior protection in C57BL/6 mice challenged with L. intracellularis, as evidenced by decreased fecal bacterial shedding, reduced intestinal bacterial load, and alleviated intestinal lesions postchallenge. Collectively, these findings demonstrate the potential of the Salmonella-mediated dual expression platform as a safe and effective delivery system forL. intracellularisantigens, offering a promising foundation for next-generation vaccines against L. intracellularis.

Upon RNA virus infection, nuclear translocation of activated transcriptional factors via the RNA-sensing signal pathway is a key event in the interferon (IFN)-mediated antiviral response, and a specific target of viral immune evasion. Foot-and-mouth disease virus (FMDV) causes an acute vesicular disease in cloven-hoofed animals and poses a serious economic risk to the dairy industry. FMDV VP4, one of the structural proteins, is an internal protein of the viral capsid and is known to play an important role in cell entry. Here, we demonstrate a novel molecular mechanism by which VP4 inhibits karyopherin (KPNA)-mediated antiviral immune responses. VP4 and IRF3 specifically interacted with the nuclear localization signal (NLS) binding site on the KPNA4 molecule, and VP4 inhibited the interaction between KPNA4 and IRF3 via competitive binding with higher affinity. Thus, VP4 inhibited nuclear translocation of IRF3 without affecting dimerization and phosphorylation of IRF3. Consequently, VP4 significantly enhanced the replication of RNA and DNA viruses by suppressing IFN production through inhibition of the IRF3-mediated type I IFN signaling pathway. Taken together, these results suggest that VP4 negatively regulates host type I IFN signaling by inhibiting the nuclear translocation of IRF3 and provide a critical implication for better understanding the pathogenesis of FMDV.

Cathelicidin CATH-2 has been reported to exert potent anti-inflammatory activity in different species though neutralizing stimuli such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA). CATH-2 has been shown to inhibit Streptococcus suis(S. suis)-induced activation of dendritic cells and macrophages by binding to LTA. However, the exact mechanism of this prophylactically anti-inflammatory activity remains unclear. Therefore, we investigated the anti-inflammatory activity and mechanism of CATH-2 in mice peritoneal macrophages pretreated with CATH-2 followed by S. suis infection. The results showed that CATH-2 pretreatment significantly reduced S. suis-induced transcription and secretion of interleukin (IL)-1β, IL-6, and IL-12. CATH-2 also downregulated NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) expression and apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, and inhibited the maturation of IL-1β, suggesting that CATH-2 inhibits NLRP3 activation. In addition, CATH-2 significantly inhibited S. suis-induced phosphorylation of p65 and extracellular signal-regulated kinase (ERK). Further study showed that CATH-2 inhibited S. suis-induced reactive oxygen species (ROS) by upregulating the expression of ROS scavenging genes including catalase (CAT) and superoxide dismutase 1 (SOD1). Mechanistically, transcriptome analysis revealed that CATH-2 regulated the protein kinase B (ATK)/mammalian target of rapamycin (mTOR) pathway, which was evident by the downregulation of phosphorylated (p)-ATK and p-mTOR induced by CATH-2. Notably, CATH-2 induced autophagy and autophagic flux. Inhibition of mTOR using rapamycin enhanced the CATH-2-induced autophagic efficacy, demonstrating that CATH-2 induces mTOR-dependent autophagy. However, inhibition of autophagy using 3-methyladenine (3-MA) reversed the reduction in the expression of p-p65, p-ERK, and IL-1β induced by CATH-2. Our study reveals that CATH-2 inhibits the nuclear factor kappa-B (NF-κB)/NLRP3-mediated inflammatory response through the induction of mTOR-dependent autophagy during S. suis infection, which provides new insight into the anti-inflammatory pathways of antimicrobial peptides.

Circoviruses (CVs) infect a wide range of avian hosts, including domestic, ornamental and wild birds. The immunosuppressive effect of these viruses could make the host more susceptible to other pathogens. This study aimed to assess the potential hosts and genetic features of CVs or other related viruses in wild birds. Cloacal swab samples of 588 birds were processed for screening of circoviral sequences using nested PCR. Altogether, 19 complete genome and 21 partial sequences of small, circular, rep-encoding DNA viruses were determined, 32 of which belonged to CVs. Along with some newly established CV species (virus name long-eared owl-associated CV1 and barn owl-associated CV1), genomic sequences of previously characterized avian CVs (pigeon CV, duck CV, goose CV, gull CV, swan CV and little bittern CV) were identified. Pathogenic CVs and CVs of unknown aetiology occur in wild birds taxonomically distant from the originally described host species, such as the duck CV and pigeon CV in white stork (Ciconia ciconia), as well as pigeon CV in peregrine falcon (Falco peregrinus) and black-headed gull (Chroicocephalus ridibundus). The results draw attention to the widespread distribution of these viruses among wild birds which, by hiding in the host and reducing defensibility, could pose a threat to both poultry farming and efforts to wild bird conservation.

Cyclic dimeric adenosine monophosphate (c-di-AMP) is a second messenger commonly found in bacteria, particularly within the Firmicutes and Actinobacteria phyla, as well as in Archaea. Phosphodiesterase (PDE) is an enzyme that plays a crucial role in regulating the homeostasis of bacterial c-di-AMP and is capable of hydrolysing this molecule both intracellularly and extracellularly. This enzymatic activity influences various physiological functions in bacteria, modulates the host immune response, and impacts the pathogenic processes of infectious agents. Mesomycoplasma ovipneumoniae (M. ovipneumoniae) is the primary pathogen responsible for mycoplasma pneumonia in sheep, but its pathogenic mechanisms remain poorly understood. This study aimed to investigate the potential role of c-di-AMP and its binding proteins in M. ovipneumoniae in affecting host immune responses. Our findings confirmed that M. ovipneumoniae produced c-di-AMP in both the intracellular extracts and culture supernatants and that it generated c-di-AMP during the infection of sheep primary alveolar macrophages. Transcriptome analysis revealed that c-di-AMP can activate the immune response in macrophages. Furthermore, we identified and characterized the c-di-AMP binding protein nicotinate-nucleotide adenylyltransferase (NadD), which represents a novel class of PDE containing His-Asp (HD) domains in Mycoplasmas. Notably, our study demonstrated that NadD suppresses inflammatory responses in host cells infected with M. ovipneumoniae. In conclusion, this study provides new insights into the pathogenic mechanisms of M. ovipneumoniae and develops potential targets for the prevention and control of ovine mycoplasma pneumonia, as well as for the development of therapeutic agents.

African horse sickness (AHS) is a highly severe, midge-borne disease of equids showing a mortality rate of > 90% in naive domestic horses. Outbreaks are caused by AHS virus (AHSV) and result in devastating economic losses in developing countries as equids play a crucial role in the agricultural industry and many households. The AHSV species encompasses nine serotypes showing no or limited cross protection, which is challenging regarding AHS vaccine development. Recently, and like bluetongue virus (BTV), the AHS Disabled Infectious Single Animal (DISA)-DIVA vaccine platform was developed and applied for all nine AHSV serotypes. The vaccine platform is based on a single deletion crucial for vaccine safety and affording differentiation of infected from vaccinated individuals (DIVA) testing. Before considering ethically debatable and expensive experiments in equids, DISA-DIVA vaccine candidates were evaluated here in the previously validated IFNAR (-/-) mouse model for AHS. A multivalent AHS DISA-DIVA vaccine containing DISA constructs for each of the nine serotypes was studied. This "DISA cocktail" was safe and protected mice against two virulent AHSV serotypes used in challenge infection studies. DISA multivalent vaccine induced serotype-specific neutralizing antibodies (nAbs) against all AHSV serotypes suggesting broad protection, as correlates of protection (CoP) based on nAb titers for studied serotypes can be extrapolated to others. These results now justify further experiments in horses to study safety, efficacy and DIVA by DISA cocktail vaccine. Importantly, the presented results for IFNAR (-/-) mice indicate that broad protection of equines by vaccination with DISA cocktail vaccine can be feasible.

Porcine epidemic diarrhea (PED), a highly contagious intestinal disease caused by PEDV, threatens newborn piglets and causes enormous losses in the swine industry. Intestinal mucosal immunity is vital for defense against PEDV. Chitosan (CS), a mucin-adhesive biodegradable polysaccharide, has emerged as a promising mucosal vaccine delivery vector and antigen adjuvant. To develop a PEDV mucosal vaccine, we prepared S1-CS nanoparticles (NPs) loaded with the S1 domain of the PEDV spike (S) protein. We evaluated their ability to induce mucosal immune responses in mice via different inoculation routes and to confer immune protection in piglets after oral inoculation. The results showed that S1-CS NPs exhibited no cytotoxicity and remained stable in simulated gastric fluid in vitro. Compared with oral administration, intramuscular inoculation of S1-CS NPs induced higher levels of PEDV-specific serum IgG in mice. However, PEDV-specific IgA was detected only in the serum and intestinal lavage fluid of orally vaccinated mice. Oral administration also elicited higher IFN-γ levels than intramuscular injection, suggesting stronger activation of cellular immunity. In piglets, oral S1-CS NPs induced serum neutralizing antibodies and IgA responses in both serum and intestinal mucosa, increased the number of IgA-secreting cells in the intestine, reduced viremia and virus shedding, and improved intestinal villus morphology. These findings indicate that the S1-CS NPs developed in this study represent promising candidates for an oral mucosal PEDV vaccine.