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Background Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. Methods Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. Results In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. Conclusions Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.

The recent prevalence of coronavirus (CoV) poses a serious threat to animal and human health. Currently, porcine enteric coronaviruses (PECs), including the transmissible gastroenteritis virus (TGEV), the novel emerging swine acute diarrhoea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and re-emerging porcine epidemic diarrhoea virus (PEDV), which infect pigs of different ages, have caused more frequent occurrences of diarrhoea, vomiting, and dehydration with high morbidity and mortality in piglets. PECs have the potential for cross-species transmission and are causing huge economic losses in the pig industry in China and the world, which therefore needs to be urgently addressed. Accordingly, this article summarises the pathogenicity, prevalence, and diagnostic methods of PECs and provides an important reference for their improved diagnosis, prevention, and control.

Rapid and early detection of infectious diseases in pigs is important, especially for the implementation of control measures in suspected cases of African swine fever (ASF), as an effective and safe vaccine is not yet available in most of the affected countries. Additionally, analysis for swine influenza is of significance due to its high morbidity rate (up to 100%) despite a lower mortality rate compared to ASF. The wide distribution of swine influenza A virus (SwIAV) across various countries, the emergence of constantly new recombinant strains, and the danger of human infection underscore the need for rapid and accurate diagnosis. Several diagnostic approaches and commercial methods should be applied depending on the scenario, type of sample and the objective of the studies being implemented. At the early diagnosis of an outbreak, virus genome detection using a variety of PCR assays proves to be the most sensitive and specific technique. As the disease evolves, serology gains diagnostic value, as specific antibodies appear later in the course of the disease (after 7–10 days post-infection (DPI) for ASF and between 10–21 DPI for SwIAV). The ongoing development of commercial kits with enhanced sensitivity and specificity is evident. This review aims to analyse recent advances and current commercial kits utilised for the diagnosis of ASF and SwIAV.

Swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. To develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (TGEV) N, porcine epidemic diarrhea virus (PEDV) M, porcine deltacoronavirus (PDCoV) M, and porcine enteric alphacoronavirus (PEAV) N genes respectively. A TaqMan-probe-based multiplex real-time RT-qPCR assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. The results showed that the limit of detection can reach as low as 10 copies in singular real-time RT-qPCR assays and 100 copies in multiplex real-time RT-qPCR assay, with all correlation coefficients ( R 2 ) at above 0.99, and the amplification efficiency at between 90 and 120%. This multiplex real-time RT-qPCR assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. The multiplex real-time RT-qPCR assay was then employed to detect the swine enteric coronavirus from 354 field diarrheal samples. The results manifested that TGEV and PDCoV were the main pathogens in these samples, accompanied by co-infections. This well-established multiplex real-time RT-qPCR assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses.

Swine influenza causes concern for global veterinary and public health officials. In continuing two previous networks that initiated the surveillance of swine influenza viruses (SIVs) circulating in European pigs between 2001 and 2008, a third European Surveillance Network for Influenza in Pigs (ESNIP3, 2010-2013) aimed to expand widely the knowledge of the epidemiology of European SIVs. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the coordination of appropriate diagnostic tools and subtyping methods. Thus, an extensive virological monitoring, mainly conducted through passive surveillance programs, resulted in the examination of more than 9 000 herds in 17 countries. Influenza A viruses were detected in 31% of herds examined from which 1887 viruses were preliminary characterized. The dominating subtypes were the three European enzootic SIVs: avian-like swine H1N1 (53.6%), human-like reassortant swine H1N2 (13%) and human-like reassortant swine H3N2 (9.1%), as well as pandemic A/H1N1 2009 (H1N1pdm) virus (10.3%). Viruses from these four lineages co-circulated in several countries but with very different relative levels of incidence. For instance, the H3N2 subtype was not detected at all in some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, H3N2-free areas were those that exhibited highest frequencies of circulating H1N2 viruses. H1N1pdm viruses were isolated at an increasing incidence in some countries from 2010 to 2013, indicating that this subtype has become established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these four lineages, especially between previous enzootic SIVs and H1N1pdm. These novel viruses were detected at the same time in several countries, with increasing prevalence. Some of them might become established in pig herds, causing implications for zoonotic infections.

The porcine epidemic diarrhoea virus (PEDV), a member of the Coronaviridae family, causes acute diarrhoea and dehydration in pigs. Although it was first identified in Europe, it has become increasingly problematic in many Asian countries, including Korea, China, Japan, the Philippines, and Thailand. The economic impacts of the PEDV are substantial, given that it results in significant morbidity and mortality in neonatal piglets and is associated with increased costs related to vaccination and disinfection. Recently, progress has been made in understanding the molecular epidemiology of PEDV, thereby leading to the development of new vaccines. In the current review, we first describe the molecular and genetic characteristics of the PEDV. Then we discuss its molecular epidemiology and diagnosis, what vaccines are available, and how PEDV can be treated.

Porcine circoviruses (PCVs), including porcine circovirus 2 (PCV2) and porcine circovirus 3 (PCV3), have been associated with clinical syndromes in swine, resulting in significant economic losses. To better understand the epidemiology and clinical relevance of PCV2 and PCV3, this study analyzed a dataset comprising diagnostic data from six veterinary diagnostic laboratories (VDLs) in the United States of America. The data comprised of polymerase chain reaction (PCR) test results, sample type, and age group for PCV2 and PCV3 submissions from 2002–2023. Findings indicated a decrease in the percentage of PCV2-positive submissions after introducing a commercial PCV2 vaccine in 2006 and a resurgence in positivity after 2018, particularly in breeding herds, associated with an increased number of submissions using processing fluid samples. After its first report in the U.S. in 2016, PCV3 detection had an upward trend in the percentage of positive cases, peaking in spring 2023. PCV3 detection was more frequent in adult/sow farms, while PCV2 was more frequently detected in the wean-to-market category. An additional analysis used results from tissue diagnostic data from 2019–2023 from one VDL to associate PCR cycle threshold (Ct) values with the probability of confirming a PCV2 or PCV3 disease diagnosis confirmation. An interpretative PCR Ct cutoff for PCV2 and PCV3 diagnoses was assessed based on the logistic regression model associating Ct values with the presence of tissue lesions. The analysis considered only cases tested for PCV2 and PCV3 by PCR with tissue evaluations by diagnosticians. An interpretative Ct cutoff of 22.4 for PCV2 was associated with a high probability of confirming a diagnosis of PCV2 clinical disease through histopathology. For PCV3, the interpretative cutoff with the highest performance was 26.7. These findings contribute to the ongoing efforts to monitor and understand the clinical relevance of PCV2 and PCV3 PCR results, identifying potential disease challenges.

The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93–97% and LOD 95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

Processing fluid is a promising alternative to blood for monitoring porcine diseases, although certain aspects of its routine use remain unclear. This study evaluated serum from females and males, along with corresponding testicular only processing fluid, for antibodies against Actionbacillus pleuropneumonie , hepatitis E virus, porcine epidemic diarrhea virus, influenza A virus, Erysipetothrix rhusiopathie and Mycoplasma hyopneumoniae , using commercial ELISAs (ID Screen APP, Hepatitis E, PEDV, Influenza A from ID Vet, France; Civtest suis SE/MR from Hipra, Spain; and Mycoplasma hyopneumoniae from Idexx, USA). Differences in the proportion of positive results across sample types were analysed to assess the utility of testis-derived processing fluid for litter-level health monitoring. ROC analysis was used to establish optimal cut-offs for processing fluid, followed by evaluation of diagnostic performance using both manufacturer-recommended and ROC-derived thresholds. A pooling simulation was also performed. Results indicate that processing fluid collected exclusively from testes can detect antibodies against selected pathogens effectively. Some ELISA kits validated for serum may be applicable to processing fluid, provided that appropriate cut-off values are determined for this sample type. However, pooling processing fluid samples may reduce sensitivity and increase the risk of false-negative results. These findings highlight the potential of testis-derived processing fluid for large-scale serological surveillance while underscoring the need for test-specific validation.

A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping.