The devastating impact of porcine epidemic diarrhea virus (PEDV) on the global swine industry underscores an urgent need for effective antiviral therapies. Drug repurposing presents a promising strategy to accelerate the development of such treatments. In this study, we screened a custom‐designed library of 117 cholesterol‐lowering compounds for anti‐PEDV activity using a recombinant PEDV expressing enhanced green fluorescent protein (EGFP). Following two rounds of screening, four compounds exhibiting significant antiviral activity were identified. Among these, cetaben and digitonin displayed superior antiviral efficacy and higher selectivity indices. Subsequent dose–response analyses further confirmed that cetaben effectively suppresses viral replication in both classical and variant strains of PEDV. Time‐of‐addition assays revealed that cetaben exerts potent antiviral effects primarily at the preinfection stage by inhibiting viral internalization and syncytium formation. Notably, supplementation with exogenous cholesterol in infected cells completely abolished cetaben’s antiviral activity and restored viral infectivity, demonstrating that its anti‐PEDV mechanism is mediated through disruption of cellular cholesterol metabolism. Collectively, our findings identify cetaben, a cholesterol‐lowering agent, as a novel broad‐acting antiviral against PEDV with a host‐targeted mechanism of action. This study establishes a foundation for developing antiviral strategies that target cholesterol metabolism to combat PEDV and related coronaviruses.

Accurate diagnosis of bovine tuberculosis (bTB) remains a critical challenge for disease control and eradication programs. While cell‐mediated immune (CMI) response tests such as the comparative cervical intradermal tuberculin test (CCITT) are widely used, their sensitivity is limited, especially in later stages of infection. Antibody‐based assays, such as enzyme‐linked immunosorbent assays (ELISAs), may offer complementary detection and improve case identification. This study aimed to comparatively evaluate two indirect ELISA tests for the detection of antibodies against Mycobacterium bovis in naturally infected cattle from Southern Brazil. One ELISA test is in an experimental phase and uses a chimeric antigen comprising fragments of MPB70, MPB83, and ESAT‐6 proteins. The second is a commercially available ELISA registered with the WOAH, based on MPB70 and MPB83 antigens. Serum samples were collected from 147 cattle across nine herds with known bTB epidemiological histories. All animals underwent CCITT, and a subset was subjected to post‐mortem examination, culture, and nested‐PCR. The experimental ELISA identified 16.6% of animals as seropositive, while the commercial ELISA identified 13.0%, both exceeding the 4.4% apparent prevalence detected by CCITT. Both ELISAs showed low sensitivity (<30%) when compared to culture or PCR‐confirmed cases but were able to detect additional infected animals missed by CCITT. These findings support the use of serological tests as complementary tools to enhance bTB detection in cattle and inform surveillance and eradication strategies in endemic regions.

Cetacean morbillivirus (CeMV) drives recurrent unusual‐mortality events, yet surveillance is uneven where laboratory capacity is limited. We developed a portable reverse transcription‐insulated isothermal PCR (RT‐iiPCR) assay targeting a conserved phosphoprotein (P)‐gene segment and evaluated analytical performance, tissue‐level clinical sensitivity, and concordance with reverse transcription‐quantitative PCR (RT‐qPCR) under low‐copy conditions that resemble challenging strandings. Using synthetic RNA, RT‐iiPCR achieved 100% detection from 62,560 to 513 copies µL−1, and probit analysis estimated a 95% limit of detection (LOD95) of 139 copies µL−1. Clinical sensitivity was assessed with two spiking regimes (RNA added after or before extraction) in cerebrum and lung. Singleplex RT‐iiPCR maintained 100% positivity to approximately cycle threshold (Ct) 33 irrespective of spiking order, indicating that near‐limit failures are governed by template scarcity rather than extraction loss. Duplex RT‐iiPCR co‐amplifying β2‐microglobulin (B2M) provided process control with a slight sensitivity cost, sustaining perfect detection to ~Ct 30–31. Across low‐copy panels, agreement with RT‐qPCR was substantial (overall κ = 0.68–0.76) and very good in cerebrum (singleplex κ = 0.85; duplex κ = 0.87), while duplex lung showed lower concordance (κ = 0.55) driven solely by Ct >33 false‐negative calls, with no false positives. The assay detected five lineages (dolphin morbillivirus [DMV], pilot whale morbillivirus [PWMV], beaked whale morbillivirus [BWMV], Guiana DMV [GDMV], and Fraser’s DMV [FDMV]) in formalin‐fixed, paraffin‐embedded tissues archived up to 28 years, and sequence alignments indicate expected coverage of additional lineages. Lyophilized reagents, compact hardware, and a quick, simple workflow support deployment in hot, humid, resource‐limited settings. A strain‐agnostic, field‐ready RT‐iiPCR can underpin transboundary CeMV surveillance, enable rapid carcass triage and sequencing, and provide early warning where diagnostic gaps currently exist.

Infectious bronchitis (IB) virus (IBV) remains a major pathogen threatening the poultry industry. Its rapid mutation and recombination continuously generate variants that disseminate worldwide. Between May 2024 and February 2025, 49 field strains were isolated from chickens vaccinated with live‐attenuated IBV vaccines (H120, 4/91, or QXL87) in four Chinese provinces (Jiangsu, Anhui, Shandong, and Guangdong). Based on full‐length S1 gene sequencing, all isolates were classified into genotype GI, including lineages GI‐13 and GI‐19. Phylogenetic analysis revealed that GI‐19 (QX‐type) comprised as much as 83.67%, with the nucleotide homology of the S1 gene to QXL87 varying from 93.4% to 99.8%. Recombination analysis indicated that the S1 genes of three isolates incorporate QXL87 and 4/91 genetic material, possibly arising from recombination between the QX‐type and 4/91 vaccine strains. Virulence assessment in 1‐day‐old specific‐pathogen‐free (SPF) chickens demonstrated that four phylogenetically distant QX‐type strains and one recombinant strain (with QXL87 as the major parent) induced varying degrees of tissue damage and mortality. Cross‐neutralization assays demonstrated reduced antigenic relatedness between the circulating isolates and QXL87 vaccine strain. Structural mapping analysis further indicated that three amino acid mutations within the N‐terminal domain (NTD) and two amino acid mutations in the C‐terminal domain (CTD) of the S1 subunit alter its overall conformation, potentially leading to antigenic variation and facilitating immune evasion. Overall, these findings offer timely insights into the epidemiology and virulence heterogeneity of QX‐IBV, providing valuable references for optimizing vaccine selection and development, as well as for preventing and controlling the disease.

Larvae of ascaridoid nematodes, particularly Anisakis spp., are common parasites of commercially important marine fishes and may represent a zoonotic hazard following ingestion of raw or undercooked seafood. We investigated the ascaridoid fauna of the sympatric European hake (Merluccius merluccius) and greater forkbeard (Phycis blennoides) from the Ionian Sea (Eastern Mediterranean), integrating host biometric and seasonal drivers with molecular identification and quantitative risk assessment (QRA) for the zoonotic Anisakis pegreffii. Anisakis pegreffii was the dominant species in both hosts, followed by Hysterothylacium aduncum; other detected taxa included A. typica, A. ziphidarum, Skrjabinisakis physeteris, and H. fabri. In both hosts, the larval abundance exhibited marked seasonal peaks in summer and correlated more strongly with host liver and gonad condition indices, suggesting that seasonality, togheter with host physiological state, rather than size alone, modulates infection levels. Most larvae were found in the visceral non edible parts of the fish, while only a small proportion of these were detected in skeletal muscles (2.6% in hake and 0.6% in forkbeard), primarily in the anterior ventral fillet portion. QRA indicated a low per‐meal probability of anisakiasis from untreated hake (~1 case per 52,609 meals). These findings highlight species‐specific, trophically mediated infection patterns and reinforce that European hake and greater forkbeard represent minor but nonnegligible sources of zoonotic risk. Preventive measures, including immediate evisceration, proper freezing or cooking, and selective fillet trimming, are recommended to minimize human exposure.

Several animal species, including horses and pigs, can be infected with Japanese encephalitis virus (JEV). Feral pigs (Sus scrofa) are considered an effective amplifying host for JEV in natural habitats. In this study, 265 dried blood spots were collected from feral pigs in four selected North Queensland local government areas, and antibodies against JEV were detected using a competitive enzyme-linked immunosorbent assay (cELISA). The results showed that 26.4% (70/265) of the feral pigs had antibodies specific to JEV. We found a significant difference in the seroprevalence of JEV in the Wet Tropics (p < 0.05), compared to the Dry Tropics region of North Queensland. The results indicate that feral pigs in North Queensland have been exposed to JEV, suggesting that they may play an important role as an amplifying host. These findings may inform the planning of mitigation measures through a multisectoral One Health approach.

In summer 2024, Western Europe experienced extensive outbreaks of bluetongue virus Serotype 3 (BTV‐3), a Culicoides‐borne orbivirus. Clinical disease was first detected in August 2024 in one of two Belgian zoos, predominantly affecting European bison (Bison bonasus) and American bison (Bison bison). These species showed high morbidity, two fatalities, and severe multisystemic lesions at necropsy. This outbreak coincided with reemergence of BTV‐8 and increasing reports of emerging BTV‐12 and epizootic hemorrhagic disease virus (EHDV) circulation in Europe. Following the outbreak, samples from 116 animals representing 33 species were analyzed across two Belgian zoos (urban and rural), spanning a 20‐year period (2005–2025). Species belonged to Artiodactyla, Perissodactyla, Proboscidea, Rodentia, Diprodontia, and Carnivora. Archived sera predating 2024 (retrospective), samples collected during the 2024 outbreak (outbreak), and follow‐up samples obtained through June 2025 (follow‐up) were tested at the Belgian National Reference Laboratory (NRL). Antibodies were screened using a pan‐BTV ELISA, and ELISA‐positive samples were further analyzed by virus neutralization tests (VNTs) to differentiate BTV‐3 and BTV‐8. Viral RNA detection was performed using RT‐qPCR assays targeting pan‐BTV, BTV‐3, BTV‐12, and EHDV. Serological reactivity was confined to Artiodactyla and Perissodactyla. In total, 46 ruminants (67% of tested ruminants) and one greater one‐horned (GOH) rhinoceros (Rhinoceros unicornis) were ELISA positive. VNT confirmed BTV‐3 infection in 62% and BTV‐8 in 9% of seropositive ruminants. Viral RNA was detected in whole blood from 28 individuals, representing 42% of the tested ruminants. Marked epidemiological differences were observed between zoos, with limited detection in the urban collection and widespread infection in the rural collection, likely reflecting differences in vector exposure. No evidence of BTV‐12 or EHDV was detected. This study provides comprehensive documentation of BTV‐3 in zoo‐housed species, integrating clinical, pathological, and longitudinal surveillance data. It underscores pronounced interspecies variability in susceptibility and immune response, providing critical insights for outbreak preparedness.

Pasteurella multocida (Pm) is an important veterinary and zoonotic pathogen that causes significant economic losses in poultry production. However, long‐term surveillance studies integrating antimicrobial resistance (AMR), biocide tolerance, and genomic epidemiology of Pm remain scarce. In this study, we investigated the antimicrobial susceptibility, biocide tolerance, and the phenotypic associations of 136 avian Pm isolates collected from six provinces in China between 2002 and 2024. Whole‐genome sequencing was performed to characterize population structure, identify antimicrobial resistance genes (ARGs), and assess genotype–phenotype concordance. The A:L1:ST129 lineage remained the predominant clone throughout the 23‐year surveillance period, with a high prevalence of AMR‐associated traits observed within this lineage. Although resistance to several commonly used antimicrobial classes declined significantly after 2021, florfenicol resistance continued to increase, suggesting an emerging challenge for the clinical management of pasteurellosis. While the isolates generally exhibited low tolerance to the four representative biocides tested, phenotypic correlations were observed between AMR profiles and biocide tolerance patterns. Furthermore, substantial phenotype–genotype discordance was observed, indicating that the presence of ARGs alone may not be sufficient to accurately predict antimicrobial susceptibility. Overall, this study provides a longitudinal assessment of long‐term AMR trends, biocide tolerance, and genomic epidemiology of avian Pm in China, offering epidemiological evidence for monitoring AMR trends and improving antimicrobial management strategies in poultry production.

African horse sickness (AHS) is a vector-borne and noncontagious disease caused by African horse sickness virus (AHSV) that poses a severe threat to the global equine industry. As a country historically free from AHS, China faces elevated risks due to recent outbreaks in Southeast Asia in 2020 and its ecological suitability of its border regions for AHSV vectors. Therefore, we conducted consecutive molecular and serological surveillance for AHSV in high-risk border regions from 2020 to 2024. A total of 3203 equine serum samples, 2909 whole-blood samples, and 1037 collections of blood-sucking insects were collected from 103 counties across five border provinces from June to October (peak Culicoides season). Samples were analyzed using World Organization for Animal Health (WOAH)-recommended RT-qPCR (targeting VP7 gene) and blocking ELISA. Morphological identification confirmed that the collected vectors were primarily Culicoides midges, with some mosquitoes. No AHSV nucleic acid or specific antibodies were detected, which is consistent with China's AHS-free status. Combined with predictive models and regional risk factors, the likelihood of AHSV occurrence in southern China is "likely." The study highlights the urgent need for continuous surveillance and risk assessment to safeguard China's equine industry. Further regional and ecological studies on vectors and animal transportation are also essential for understanding and mitigating the risks associated with AHSV introduction.

Colistin resistance genes have attracted increasing global attention due to their plasmid‐mediated transmissibility. To clarify the transmission dynamics of colistin resistance genes in the endangered crested ibis (Nipponia nippon) and its environment, surveillance for mcr-1 was conducted at a captive breeding center in Deqing, Zhejiang, China, from 2017 to 2024. Genomic sequencing was used to analyze the phylogenetic relationships among the Enterobacteriaceae isolates harboring these resistance genes. A total of 2660 fecal samples of crested ibises were collected seasonally. There was only one episode of mcr-1 occurrence with a total of 144 mcr-1‐positive strains in crested ibises. The prevalence was first detected at 8% in April 2021, surged to 25% in July, and then declined to 10.6% in October. Additionally, mcr-1‐harboring Escherichia coli isolates were also found in feces from wild egrets (33%), the staff member’s dog (8.3%), and effluent sewage, but not in the staff member, loach samples, or water sources. Phylogenetic analysis showed that the egret‐derived isolate BLM86 and the crested ibis‐derived isolate ZHM12 belonged to the same ST58‐cgST28300 type; no core‐genome SNP differences were detected. All 36‐deeper analyzed mcr-1‐positive isolates displayed multidrug resistance, with four distinct mcr-1‐containing plasmid types identified: IncI2 (20/36, 55.6%), IncX4 (19.4%), IncP1 (11.1%), and IncHI2 (13.9%). Phylogenetic analysis indicated that several of these crested ibis‐derived isolates were closely related to those from egrets, implying potential environmental transmission routes to both the crested ibis and egrets. Mcr-1 was no longer detected in the crested ibis population after implementation of biosecurity interventions. Our findings demonstrate that wild egrets could act as potential vectors for the environmental transmission of mcr-1 to captive crested ibises, highlighting the importance of interspecies barriers in antimicrobial resistance (AMR) containment.