Nocardia is a Gram-positive pathogen responsible for causing dairy mastitis,which leads to purulent granulomatous lesions in mammary tissue and can significantly impact the dairy indus-try,resulting in substantial economic losses.To develop a rapid and accurate diagnostic method for detecting Nocardia of bovine origin,a conserved sequence of the 16S rRNA gene from Nocardia was selected from the NCBI database.Based on this sequence,a pair of primers and a TaqMan fluo-rescent quantitative probe were designed.The validation of the TaqMan fluorescence quantitative PCR(qPCR)method found in this study showed that the Ctvalue had a good linear relationship with recombinant plasmid concentrations ranging from 1×1010 to 1×102 copies/μL,with a regres-sion equation of y=-3.536x+43.78,a correlation coefficient(R2)of 0.997 5,a slope of-3.536,and an amplification efficiency(E)of 91％(where 90％＜E＜110％).The specificity was strong,with no cross-reactions with other pathogens.The standard curve had a high sensitivity with a low-er detection limit of 1 × 102 copies/μL,it was 100-fold higher than conventional PCR.The repeatability of the standard curve was also good.Both intra-and inter-group coefficients of varia-tion were below 2％.Using this method,234 milk samples and 80 environmental samples were tested using this method,respectively,with a positive detection rate of 27.07％,whereas conven-tional PCR had a positive detection rate of 19.43％,indicating that this method was more sensitive compared to conventional PCR.The fluorescent quantitative PCR detection method established in this study provides an effective means for the clinical detection of Nocardia in dairy cows.

To identify clinical viral diseases characterized by reproductive disorders and abortion,a quadruple qPCR method was established for simultaneous detection of PRV,PPV,PCV2 and AS-FV.Four pairs of specific primers and probes were designed according to the conserved genes of four viruses in the NCBI gene bank.The annealing temperature,primer concentration and probe concentration of the reaction were optimized,and the specificity,sensitivity and repeatability of the method were tested.The results showed that the method could not detect other pathogens except the target ones.The minimum detection limit of PRV,PPV,PCV2 and ASFV was 10 copies.Intra-group and inter-group repeatability tests showed that the coefficient of variation of C,values be-tween different batches was less than 3％,indicating that the method was highly specific,sensitive and stable.Establishment of an efficient and sensitive quadruple qPCR method provides technical reference for the clinical prevention and control of porcine pseudorabies virus disease,porcine circo-virus disease,porcine parvovirus disease and African swine fever.

This study investigates the effects of pseudorabies virus(PRV)infection on the antiviral immune signaling pathways and type Ⅰ interferon factors in mouse trigeminal ganglion(TG)cells.In this experiment,primary TG cells were infected with PRV at a multiplicity of infection(MOI)of 1,while mice were infected via a drop-nose method using 106,29 TCID50 of PRV.Real-time fluorescence quantitative PCR(qPCR),Western blot and ELISA were used to assess gene tran-scription,protein expression,and the secretion of IFN-α and IFN-β.The results indicated that PRV infection of mouse TG primary cells led to alterations in the gene and protein expression of RIG-Ⅰ,MAVS,and IRF3,as well as the phosphorylation of IRF3 and IKBα both in vivo and ex vivo.ELISA results showed that PRV infection could regulate the secretion of IFN-α and IFN-β in mouse primary TG cells and mouse TGs.The results of RIG-Ⅰ signaling pathway-related proteins and the secretion of IFN-a and IFN-β were analyzed using Western blot after using siRNA to interfere with RIG-Ⅰ expression in TG cells.The results showed that siRIG-Ⅰ successfully inter-fered with RIG-Ⅰ protein expression in TG cells and caused changes in the expression of down-stream proteins such as MAVS and IRF3,and also regulated the secretion of IFN-α and IFN-β in TG cells.Furthermore,the results indicated that PRV infection induced the expression of RIG-Ⅰ in mouse TG progenitor cells,regulating the antiviral immune response of type Ⅰ interferon factors in TG cells through the RIG-Ⅰ-MAVS-IRF3 signaling axis.Notably,PRV inhibited the expression of IRF3 in TG cells while significantly upregulating the expression of IFN-β during the later stages of infection,which may be an important factor in the important reason for the rapid mortality ob-served in mice during the late stages of PRV infection.This experiment elucidates part of the anti-viral immune mechanism mediated by the RIG-Ⅰ-MAVS-IRF3 signaling pathway in regulating type Ⅰ interferon factor after PRV infection of mouse TG cells,as well as the discovery of differ-ent trends of IRF3 protein changes in vivo and ex vivo,laying the groundwork for future in-depth studies.

Porcine deltacoronavirus(PDCoV)is the main pathogen of porcine deltacoronavirus dis-ease.After infection,pigsmanifest a series of main symptoms,such as persistent vomiting,watery diarrhea and severe dehydration.Pigs at almost all growth stages are likely to be infected with the virus,especially suckling piglets are much sensitive to the virus.Once PDCoV infects the host,it u-sually causes significant immunosuppression.In recent years,studies on the immunosuppressive mechanism of PDCoV have gradually attracted widespread attention.The results showed that mul-tiple proteins of PDCoV were involved in the regulation of host innate immunity,revealing the mechanism of these proteins in regulating host innate immunity.In this paper,the interaction mechanism between PDCoV protein and host innate immunity were rsummarized,which will pro-vide a theoretical basis for further understanding the pathogenesis of PDCoV and effective preven-tion and control of porcine delta coronavirus disease.

To establish a TaqMan-based multiplex RT-qPCR method for the identification of Japa-nese encephalitis virus(JEV),Porcine reproductive and respiratory syndrome virus(PRRSV),and Classical swine fever virus(CSFV),this study designed and synthesized three pairs of specific primers and probes based on the conserved sequences of JEV E,PRRSV ORF6,and CSFV E2 a-vailable in the NCBI GenBank.By optimizing the reaction system and protocol,a multiplex RT-qPCR method for detecting these three viruses was developed and applied to the detection of clini-cal samples.The results showed that the established TaqMan multiplex RT-qPCR specifically am-plified the gene fragments of JEV,PRRSV,and CSFV,and did not amplify other non-target genes,indicating good specificity of the method.Intra-assay and inter-assay repeatability tests showed that the coefficient of variation(Cv)values were all below 3％,demonstrating that the method has ex-cellent repeatability.Sensitivity tests revealed that the minimum detectable amount for the recom-binant plasmids of the three viruses was 100 copies/pL.Using the established method,a total of 969 samples,including blood,aborted fetuses,semen,and deceased pigs,from 26 pig farms in Guizhou Province were tested.The detection rates were 34.3％(332/969)for JEV,28.3％(274/969)for PRRSV,and 19.8％(192/969)for CSFV.The co-infection rates were 10.1％(98/969)for JEV and PRRSV,12.1％(117/969)for JEV and CSFV,and 14.6％(141/969)for CSFV and PRRSV.Additionally,the triple co-infection rate of JEV,PRRSV,and CSFV was 7.9％(77/969).These results indicate that the TaqMan multiplex RT-qPCR method developed in this study is ef-fective for detecting these three viruses in pig farms,providing technical support for identifying vi-ral causes of reproductive disorders.

Japanese encephalitis virus(JEV)belongs to the genus Flavivirus and the family Flavi-viridae,and is classified as a single-stranded,positive-sense RNA virus.The disease known as Japa-nese encephalitis(JE),which results from JEV infection,is a viral zoonosis that is prevalent worldwide and poses a significant public health concern.JEV infection activates a variety of signa-ling pathways,leading to a series of changes that are crucial to the virus's pathogenesis.Among these pathways,Toll-like receptors(TLRs)are particularly significant,and their diverse range and complex signal transduction mechanisms present substantial challenges for the prevention and con-trol of JEV.Currently,there is no specific treatment for JEV.Although some vaccines have been developed to prevent JE,eradicating JEV remains difficult due to its zoonotic transmission cycle and the limited efficacy of the available vaccines.This article reviews the alterations in various TLR signaling pathways induced by JEV infection in the host,aiming to provide insights into the patho-genic mechanisms of JEV and to identify potential new antiviral targets.

Porcine reproductive and respiratory syndrome virus(PRRSV)is the pathogen of porcine reproductive and respiratory syndrome(PRRS),and its infection mainly causes abortion,stillbirth in sows and piglet respiratory infections,which are widely prevalent in the world and seriously jeopardize the development of the world's animal husbandry industry.PRRSV infection of the host is capable of inducing significant immunosuppression,and in recent years,the study of the mecha-nism of PRRSV immunosuppression has become a hot topic,with studies showing that numerous PRRSV proteins are involved in the regulation of host innate immunity and elucidating the mecha-nism by which PRRSV proteins modulate host innate immunity.In this paper,we reviewed the progress of research on the interaction mechanism between PRRSV proteins and host innate im-munity to provide a theoretical basis for a comprehensive understanding of the pathogenesis of PRRSV and the prevention and control of PRRS.

Objective To explore the efficacy of arthroscopic reduction combined with percutaneous screw fixation in the treatment of Sanders types Ⅱ and Ⅲ calcaneal fractures.Methods Forty-eight patients with Sanders types Ⅱ and Ⅲcalcaneal fractures were divided into the arthroscopic reduction combined with percutaneous screw fixation(arthroscopy)group(n=23)and the tarsal sinus approach open reduction combined with screw internal fixation(tarsal sinus approach)group(n=25)according to different surgical methods and plans.The operative time,incision length,postoperative complications and secondary removal of internal fixation were compared between the two groups.Changes in B?hler's angle and Gissane's angle were evaluated and compared between the two groups at 3 months,6 months after operation and at the last follow-up.Additionally,the American Orthopaedic Foot&Ankle Society(AOFAS)ankle-hindfoot score and the visual analog scale(VAS)for pain were used to assess outcomes at the last follow-up.Results Compared with the tarsal sinus approach group,the incision length was reduced,the operation time was prolonged and the incidence of complications was decreased in the arthroscopic group(P＜0.05).At 3 months,6 months after the operation and the last follow-up,there were no significant differences in the B?hler Angle,Gissanes Angle,AOFAS and VAS scores between the arthroscopic group and the tarsal sinus approach group(P＞0.05).Conclusion Both groups of regimens can enable patients to achieve good postoperative therapeutic efficacy.However,under arthroscopic assistance,surgical incisions inherently possess minimally invasive advantages,while the incidence of postoperative complications is lower compared to the sinus tarsi approach.

Porcine deltacoronavirus(PDCoV)is the main pathogen of porcine deltacoronavirus dis-ease.After infection,pigsmanifest a series of main symptoms,such as persistent vomiting,watery diarrhea and severe dehydration.Pigs at almost all growth stages are likely to be infected with the virus,especially suckling piglets are much sensitive to the virus.Once PDCoV infects the host,it u-sually causes significant immunosuppression.In recent years,studies on the immunosuppressive mechanism of PDCoV have gradually attracted widespread attention.The results showed that mul-tiple proteins of PDCoV were involved in the regulation of host innate immunity,revealing the mechanism of these proteins in regulating host innate immunity.In this paper,the interaction mechanism between PDCoV protein and host innate immunity were rsummarized,which will pro-vide a theoretical basis for further understanding the pathogenesis of PDCoV and effective preven-tion and control of porcine delta coronavirus disease.

To establish a TaqMan-based multiplex RT-qPCR method for the identification of Japa-nese encephalitis virus(JEV),Porcine reproductive and respiratory syndrome virus(PRRSV),and Classical swine fever virus(CSFV),this study designed and synthesized three pairs of specific primers and probes based on the conserved sequences of JEV E,PRRSV ORF6,and CSFV E2 a-vailable in the NCBI GenBank.By optimizing the reaction system and protocol,a multiplex RT-qPCR method for detecting these three viruses was developed and applied to the detection of clini-cal samples.The results showed that the established TaqMan multiplex RT-qPCR specifically am-plified the gene fragments of JEV,PRRSV,and CSFV,and did not amplify other non-target genes,indicating good specificity of the method.Intra-assay and inter-assay repeatability tests showed that the coefficient of variation(Cv)values were all below 3％,demonstrating that the method has ex-cellent repeatability.Sensitivity tests revealed that the minimum detectable amount for the recom-binant plasmids of the three viruses was 100 copies/pL.Using the established method,a total of 969 samples,including blood,aborted fetuses,semen,and deceased pigs,from 26 pig farms in Guizhou Province were tested.The detection rates were 34.3％(332/969)for JEV,28.3％(274/969)for PRRSV,and 19.8％(192/969)for CSFV.The co-infection rates were 10.1％(98/969)for JEV and PRRSV,12.1％(117/969)for JEV and CSFV,and 14.6％(141/969)for CSFV and PRRSV.Additionally,the triple co-infection rate of JEV,PRRSV,and CSFV was 7.9％(77/969).These results indicate that the TaqMan multiplex RT-qPCR method developed in this study is ef-fective for detecting these three viruses in pig farms,providing technical support for identifying vi-ral causes of reproductive disorders.