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Dietary fatty acids, particularly arachidonic acid (ARA), affect cortisol and may influence the expression of genes involved in stress response in fish. The involvement of ARA on stress, lipid, and eicosanoid metabolism genes, in Senegalese sole, was tested. Post-larvae were fed Artemia presenting graded ARA levels (0.1, 0.4, 0.8, 1.7, and 2.3 %, dry matter basis), from 22 to 35 days after hatch. Whole-body cortisol levels were determined, before and 3 h after a 2 min air exposure, as well as the expression of phospholipase A₂ (PLA ₂ ), cyclooxygenase-2 (COX-2), steroidogenic acute regulatory protein (StAR), glucocorticoid receptors (GRs), phosphoenolpyruvate carboxykinase (PEPCK), and peroxisome proliferator-activated receptor alpha (PPARα). Relative growth rate (6.0–7.8 % day⁻¹) and survival at the end of the experiment (91–96 %) and after stress (100 %) were unaffected. Fish reflected dietary ARA content and post-stress cortisol increased with ARA supply up to 1.7 %, whereas 2.3 % ARA seemed to enhance basal cortisol slightly and alter the response to stress. Results suggested that elevating StAR transcription might not be necessary for a short-term response to acute stress. Basal cortisol and PLA ₂ expression were strongly correlated, indicating a potential role for this enzyme in steroidogenesis. Under basal conditions, larval ARA was associated with GR1 expression, whereas the glucocorticoid responsive gene PEPCK was strongly related with cortisol but not GR1 mRNA levels, suggesting the latter might not reflect the amount of GR1 protein in sole. Furthermore, a possible role for PPARα in the expression of PEPCK following acute stress is proposed.

Background Nipah virus (NiV) is a zoonotic pathogen that poses a significant threat because of its wide host range, multiple transmission modes, high transmissibility, and high mortality rates, affecting both human health and animal husbandry. In this study, we developed a one-step reverse transcription droplet digital PCR (RT-ddPCR) assay that targets the N  gene of NiV. Results Our RT-ddPCR assay exhibited remarkable sensitivity, with a lower limit of detection of 6.91 copies/reaction. Importantly, it displayed no cross-reactivity with the other 13 common viruses and consistently delivered reliable results with a coefficient of variation below 10% across different concentrations. To validate the effectiveness of our RT-ddPCR assay, we detected 75 NiV armored RNA virus samples, mimicking real-world conditions, and negative control samples, and the RT-ddPCR results perfectly matched the simulated results. Furthermore, compared with a standard quantitative real-time PCR (qPCR) assay, our RT-ddPCR assay demonstrated greater stability when handling complex matrices with low viral loads. Conclusions These findings show that our NiV RT-ddPCR assay is exceptionally sensitive and provides a robust tool for quantitatively detecting NiV, particularly in stimulated field samples with low viral loads or complex matrices. This advancement has significant implications for early NiV monitoring, safeguarding human health and safety, and advancing animal husbandry practices.

Background Pseudorabies virus (PRV), porcine parvovirus (PPV) and porcine circovirus 3 (PCV3) are common in swine farms in China. Single infection or co-infection with PRV, PPV and/or PCV3 was difficult to distinguish between their clinical symptoms and pathological changes. Therefore, a quick and accurate detection method is needed for epidemiological surveillance, disease management, import and export control. Methods In the present study, we established a multiplex real-time PCR assay based on SYBR Green I for the simultaneous detection of PRV, PPV and PCV3 genomes. Results PRV, PPV and PCV3 were distinguished in the same sample by their different melting temperatures (Tm), with melting peaks at 90 °C for PRV, 84 °C for PPV and 80 °C for PCV3, respectively, and other non-targeted swine pathogens did not exhibit specific melting peaks. The assay showed a high degree of linearity (R 2 ≧0.995), and the detection limits were 4.76 copies/μL for PRV, 3.67 copies/μL for PPV, 3.07 copies/μL for PCV3 and 1.87 × 10 2 copies/μL for the three mixed plasmids, respectively. In this research, 81 clinical samples from pig farms in nine different regions of Guangdong Province were used to evaluate this new method. The detection rate of the multiplex real-time PCR assay was higher than that of the conventional PCR assay. Conclusions This multiplex real-time PCR assay could be used as a diagnostic tool that is rapid, sensitive and reliable for the detection of co-infection of PRV, PPV and PCV3 as well as for molecular epidemiological surveillance.

Background Porcine respiratory disease complex poses a substantial economic challenge for the swine farms due to its prevalence in pig farming environments. The disease is primarily caused by viral pathogens such as swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 (PCV2). Given the high incidence and morbidity associated with these viruses, effective control strategies rely on rapid and accurate diagnosis. Results To this end, we developed and validated a triplex TaqMan probe-based real-time PCR assay for the simultaneous detection and differentiation of SIV, PRRSV, and PCV2. This assay demonstrated high specificity, with no observed cross-reactivity between the target viruses. The assay’s sensitivity was determined to be 100 copies/µL for SIV and 10 copies/µL for PRRSV and PCV2. Comparison with a national standard detection method using 110 clinical samples revealed 100% agreement, confirming the reliability of the newly developed assay for clinical diagnostic applications. Conclusions Given the zoonotic potential of SIV, this assay not only provides a valuable diagnostic tool for veterinary medicine but also contributes to enhanced public health surveillance efforts.

Background Aleutian mink disease, mink viral enteritis and canine distemper are known as the three most serious diseases that cause great economic loss in the mink industry. In clinical practice, aleutian mink disease virus (AMDV), mink enteritis virus (MEV) and canine distemper virus (CDV) are common mixed infections, and they have similar clinical clinical signs, such as diarrhoea. Therefore, a rapid and accurate differential diagnosis method for use on mink ranches is essential for the control of these three pathogens. Here, we developed multiplex one-step real-time quantitative PCR (RT‒qPCR) assays for the simultaneous detection and quantification of AMDV, MEV and CDV by using three primers and probes based on the conserved NS1, VP2 and N genes, respectively. Results The results showed that the established method can not cross-react with other mink pathogens, with a detection sensitivity of 25 copies/µL and a coefficient of variation less than 3.51%. Moreover, the interference experiment showed that the presence of AMDV, MEV and CDV templates at different concentrations would not interfere with the detection results. Furthermore, two hundred clinical samples of mink with diarrhoea were simultaneously analysed using multiplex RT‒qPCR and single RT‒qPCR, the Kappa values were all greater than 0.921, indicating that there was a high degree of coincidence between the two detection methods. Conclusions In conclusion, multiplex RT‒qPCR exhibited high specificity, sensitivity, and reproducibility, indicating that this method can be used as a reliable and specific tool for the differential detection and quantification of AMDV, MEV and CDV.

Background Canine respiratory coronavirus (CRCoV) is a major contributor to the canine infectious respiratory disease complex (CIRDC). Despite its widespread prevalence, molecular assays for CRCoV detection remain limited. Additionally, the efficiency and accuracy of detection can vary depending on the type of clinical sample used, such as nasal swabs (NS), oropharyngeal swabs (OS), and rectal swabs (RS). To address these challenges, we developed a nanoplate-based reverse transcription digital polymerase chain reaction (RT-dPCR) method for detecting the spike gene of CRCoV in various clinical samples. Results The RT-dPCR assay demonstrated consistent repeatability and reproducibility, ensuring reliable results. With a detection limit of 1.83 copies/µL, the RT-dPCR assay exhibited 100-fold greater sensitivity than probe-based reverse transcription quantitative polymerase chain reaction (RT-qPCR). It showed no cross-reactivity with other common CIRDC-associated viruses or coronaviruses, confirming its high specificity for CRCoV. The assay was further validated using 162 clinical swab samples (NS, OS, and RS) collected from both healthy dogs and those with respiratory distress. The RT-dPCR assay showed a higher overall positivity rate for CRCoV compared to RT-qPCR, with the most notable difference observed in rectal swabs ( P  < 0.05), where RT-dPCR detected CRCoV in 53.7% of samples compared to 22.22% by RT-qPCR. Conclusions This study demonstrated that the RT-dPCR assay provided high sensitivity for detecting low viral loads across various sample types, making it a valuable tool for precise CRCoV detection. In contrast, RT-qPCR remains valuable for its broader detection range and suitability in initial screening. Both techniques proved to be versatile tools that can contribute to advancing CRCoV research and improving clinical diagnostics.

Background As crucial pollinators sustaining agricultural ecosystem services and biodiversity, bees mediate pollination for approximately 35% of global insect-pollinated crops and generate multidimensional ecological value through apicultural products in the pharmaceutical and food industries. However, emerging viral pathogens pose escalating threats to bee health. Results To address the technical bottlenecks in pathogen detection for viral paralysis disease in bees, this study innovatively integrated multiplex RT-PCR amplification, lateral flow dipstick (LFD), and centrifugal microfluidic chip technology (MFCT) to develop an on-site quadruple detection platform capable of simultaneously identifying four viruses: Chronic Bee Paralysis Virus (CBPV), Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), and Israeli Acute Paralysis Virus (IAPV). Through multiple sequence alignment, conserved genomic regions of the four viruses were identified, and systematic screening was performed to optimize primer combinations, with critical parameters such as primer concentration (10 µM) and annealing temperature (55 °C) determined. Building on this, a RT-PCR-LFD-MFCT integrated detection system was established by incorporating chemically modified downstream primers/probes and MFCT. Experimental results demonstrated a sensitivity of 10² copies/µL for single-virus detection, enabling precise identification of low viral loads. The method exhibited exceptional specificity with no cross-reactivity, and clinical sample validation achieved 100% concordance with conventional RT-qRT-PCR. Conclusions This system features simultaneous multi-target detection, high specificity, rapid processing, minimal instrumentation requirements, portability, and field applicability. It provides a robust tool for precise diagnosis and control of bee paralysis diseases, particularly suitable for resource-limited apiaries and outbreak scenarios, demonstrating significant practical value for safeguarding apicultural health.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

Trichuriasis is a serious threat to the economic development of animal husbandry. This research aimed to establish a droplet digital PCR (ddPCR) method to detect Trichuris spp. for the early diagnosis and prevention of trichuriasis in sheep. The real-time quantitative PCR (qPCR) and ddPCR methods were used for the detection of nematodes by targeted amplification of the ITS gene. Each means was evaluated to optimize the limit of detection and reproducibility. For a recombinant plasmid, the qPCR results showed that the detection limit was 31.7 copies per reaction. In contrast to qPCR, ddPCR was able to detect concentrations below 3.17 copies per reaction. Both assays exhibited good reproducibility. However, the ddPCR method was more stable for low-copy-number detection. This new assay was specific for Trichuris spp. and did not cross-react with other relevant gastrointestinal nematodes. A total of 98 clinical samples were tested with both assays. The results showed that the positive rate of ddPCR (80.6%) was higher than that of qPCR (72.4%). This method could be used as an efficient molecular biology tool to test for Trichuris spp. and could be a new valuable tool for the clinical diagnosis and prevention of trichuriasis.