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African swine fever (ASF), a highly fatal disease often termed the “number one killer” of pigs, presents clinical symptoms indistinguishable from classical swine fever (CSF), such as fever, diarrhea, and vomiting, complicating on‐site differential diagnosis. As both ASF and CSF are notifiable diseases under the World Organisation for Animal Health (WOAH), rapid and accurate identification is crucial for effective outbreak management. In this study, we developed a multicolor lateral flow immunoassay (LFIA) based on latex microspheres (LMs) for the simultaneous detection of antibodies against ASF virus (ASFV) and CSF virus (CSFV). The assay enables visual differentiation within 15 min, with red indicating ASFV antibodies and blue indicating CSFV antibodies. After optimization, the LFIA demonstrated a sensitivity of 1:256, equivalent to that of a commercial ASFV ELISA kit and four‐fold higher than that for CSFV (1:64). The assay exhibited high specificity, showing no cross‐reactivity with other common swine pathogens and bovine viral diarrhea virus (BVDV). When applied to 180 clinical serum samples and compared with commercial ELISA kits, the LFIA achieved Cohen’s kappa values of 0.986 for ASFV and 0.918 for CSFV, indicating excellent agreement. Additionally, intra and interbatch evaluations confirmed its robust repeatability. Overall, the multicolor LM‐LFIA offers a rapid, sensitive, specific, and cost‐effective tool for point‐of‐care testing (POCT) of ASFV and CSFV antibodies, holding promise for routine field surveillance and disease control.

A wireless magnetoelastic (ME) biosensor immobilized with E2 glycoprotein was first developed to detect classical swine fever virus (CSFV) E2 antibody. The detection principle is that a sandwich complex of CSFV E2 – rabbit anti-CSFV E2 antibody – alkaline phosphatase (AP) conjugated goat anti-rabbit IgG formed on the ME sensor surface, with biocatalytic precipitation used to amplify the mass change of antigen–antibody specific binding reaction, induces a significant change in resonance frequency of the biosensor. Due to its magnetostrictive feature, the resonance vibrations and resonance frequency can be actuated and wirelessly monitored through magnetic fields. The experimental results show that resonance frequency shift increases with the augmentation of the CSFV E2 antibody concentration. Scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS) and fluorescence microscopy analysis proved that the modification and detection process were successful. The biosensor shows a linear response to the logarithm of CSFV E2 antibody concentrations ranging from 5 ng/mL to 10 μg/mL, with a detection limit (LOD) of 2.466 ng/mL and the sensitivity of 56.2 Hz/μg·mL −1 . The study provides a low-cost yet highly-sensitive and wireless method for selective detection of CSFV E2 antibody.

Background Porcine circovirus 2 (PCV2), atypical porcine pestivirus (APPV), and classical swine fever virus (CSFV) are significant pathogens causing congenital tremor in swine, resulting in substantial economic losses worldwide. Methods A TaqMan multiplex real-time PCR method was developed to detect PCV2, APPV, and CSFV, simultaneously. This method was then applied to investigate 476 clinical samples from newborn piglets with congenital tremor symptoms. Results This method exhibited high specificity with no cross-reactivity with other viruses. Furthermore, the assay demonstrated remarkable sensitivity, with a detection limit of 22.6 copies/µL for each pathogen, and excellent repeatability, as evidenced by intra- and inter-assay variability coefficients of less than 2%. The assay was applied to 476 clinical samples, revealing single infection rates of 36.97% for PCV2, 46.43% for APPV, and 14.71% for CSFV. Mixed infection rates were observed at 18.48% for PCV2 + APPV, 5.67% for PCV2 + CSFV, 5.25% for APPV + CSFV, and 3.57% for PCV2 + APPV + CSFV. Conclusions A multiplex real-time PCR assay was created to simultaneously detect PCV2, APPV, and CSFV. Testing on clinical samples showed it to be a quick, sensitive, and specific tool for differentiating viruses linked to congenital tremor, highlighting its strong potential for diagnosing swine diseases.

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-gamma-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-gamma producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence.

The FTA card has emerged as a promising alternative for nucleic acid extraction. The FTA card is a filter paper impregnated with chemicals that preserve and stabilize the genetic material present in the sample, allowing for its storage and transport at room temperature. The aim of this study was to test the card for the detection of RNA and DNA nucleic acids. Two RNA viruses (Senecavirus A and classical swine fever virus) and two DNA viruses (African swine fever virus and suid alphaherpesvirus 1) were tested, and in all cases, there was a decrease in sensitivity. The methods exhibited good repeatability and demonstrated a rapid and practical use for sample transport and nucleic acid extraction.

The increasing spread of African swine fever (ASF) in recent years and the presence of classical swine fever (CSF) subclinical forms in endemic countries suggests that the possibility of coinfection with ASF virus (ASFV) and CSF virus (CSFV) in pigs cannot be ruled out in areas where both diseases are prevalent. Thus, rapid and reliable diagnosis through molecular testing is essential for the timely implementation of control measures to prevent the spread of these devastating swine diseases. Here, we have coupled two of the most validated PCR assays for the detection of CSFV and ASFV in a single reaction tube. The combination of the two tests for the detection of two target nucleic acids did not affect the analytical sensitivity, and the duplex RT-qPCR assay was comparable with the standard molecular techniques. The detection limits for CSFV RNA and ASFV DNA were 0.12 TCID50/reaction and 0.25 TCID50/reaction, respectively. The test showed high repeatability and reproducibility, the coefficient of variation was below 2%, and excellent performance was demonstrated in clinical samples. The duplex assay shows great potential to become a robust diagnostic tool for the rapid and reliable detection and differentiation of CSFV and ASFV in areas where both viruses may be circulating.

Classical swine fever (CSF) is a highly contagious disease affecting domestic pigs and wild boars, posing a serious threat to the global swine industry. In Japan, CSF re‐emerged on a pig farm in Gifu Prefecture in 2018, just 3 years after the country was declared CSF‐free. The CSF virus (CSFV) was soon detected in neighboring wild boars and subsequently spread to adjacent areas, leading to further farm outbreaks. Given that long‐distance transmission accelerates both spatial expansion and epidemic persistence, we aimed to identify such events during the current Japanese epidemic. Whole‐genome sequences were generated for 100 farm isolates and 585 wild boar isolates collected through national surveillance. Putative ancestral strains were inferred for each isolate by comparing single‐nucleotide variants (SNVs), and the great‐circle distance to the nearest ancestral strain was considered the transmission distance. Six routes exceeding the 99th percentile of the distance distribution (182.2 km) were classified as long‐distance transmission events: three involving farms and three involving wild boars. The sources of all these transmission events were identified as infected wild boars. The route to a farm in Okinawa Prefecture (January 2020) was linked to the illegal feeding of unheated food waste containing meat products. No specific sources were identified in the remaining two farm outbreaks. The three introductions into wild boar populations were most plausibly associated with anthropogenic activities, such as the movement of people or vehicles through infected habitats. To the best of our knowledge, this is the first study to comprehensively quantify long‐distance CSFV spread across the entire course of the Japanese epidemic (2018–2024). Our findings will inform targeted control measures to prevent farm infections and the inadvertent spread of contaminated material to remote areas.

Emergency surveillance following an outbreak of transboundary animal diseases such as classical swine fever (CSF), is conducted to find another new infection as early as possible. Although larger sample sizes can help achieve higher disease surveillance sensitivity, the sample size is limited by the availability of resources in an emergency situation. Moreover, the recent CSF outbreak reported in Japan was associated with fewer clinical signs; this emphasizes the importance of detecting infected farms by surveillance. In this study, we aimed to identify effective and labor-efficient sampling methods showing high probabilities of detecting infection, by simulating infection and sampling in pigsties. We found that impartial sampling, which involves selection of pigs to be sampled from the four corners and the center of the pigsty, and random sampling showed comparable probabilities of detection. Impartial sampling involves sample collection without pig identification and random selection. Owing to its simplicity, impartial sampling is labor-efficient and thus a possible substitute for random sampling. In a group-housing pigsty, testing five pigs from five pens showed a higher detection probability than testing five pigs from one pen. These results suggest preferable surveillance methods for conducting emergency surveillance of infectious diseases.

Background: Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. Methods: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Results: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have crossreaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. Conclusion: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Wild boars are recognized reservoirs of numerous viral pathogens, posing a significant risk to domestic pig populations, particularly in areas with poor biosecurity. This study assessed the prevalence and co-infection patterns of porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine cytomegalovirus (PCMV), African swine fever virus (ASFV), classical swine fever virus (CSFV), and pseudorabies virus (PRV) in wild boars from western Serbia and the Republic of Srpska (Bosnia and Herzegovina). Sixty-six spleen samples from legally hunted wild boars were analyzed by qPCR. All animals were negative for ASFV, CSFV, and PRV. The cumulative prevalence of infection with at least one of the other three viruses was 86.4% (95% CI: 76.2–92.8%). PCMV was detected in 74.2% of samples, PCV2 in 50%, and PPV in 28.8%. Co-infections were common: 42.4% of animals were positive for two viruses, and 12.1% for all three. A statistically significant association was observed between triple co-infection and sex, with higher rates in males. Subadult wild boars showed the highest PCV2 + PCMV co-infection rate (p = 0.0547). These findings highlight the need to expand molecular surveillance, particularly for PCMV, in both wild and domestic pigs, especially in regions reliant on low-biosecurity backyard farming.