Infectious bursal disease virus (IBDV) is a major immunosuppressive pathogen threatening global poultry production. The emergence of very virulent (vvIBDV) and novel variant strains (nVarIBDV) has increased immune escape and reduced vaccine effectiveness, underscoring the need for rapid and reliable diagnostic tools. To promote antigen detection technologies which usually time-consuming and require specialized laboratory facilities, this study developed a simple, rapid, and sensitive fluorescent microsphere immunochromatographic test strip (FM-ICTS) for semi-quantitative detection of IBDV antigen. The FM-ICTS was constructed using lanthanide fluorescent microspheres conjugated to an anti-VP2 monoclonal antibody within a double-antibody sandwich lateral flow format. Reaction conditions were optimized, and a quantitative standard curve was established using serial dilutions of inactivated IBDV (B87 strain). The assay generated stable fluorescence signals within 15 min and showed high analytical sensitivity, detecting IBDV antigen at dilutions up to 1:5120. No cross-reactivity with Chicken Anemia Virus (CAV), Avian Leukosis Virus(ALV), Avian Influenza Virus(AIV), or Marek’s Disease Virus(MDV) was observed, confirming excellent specificity. Repeatability tests demonstrated low coefficients of variation (< 10

ABSTRACT African swine fever virus (ASFV) causes epidemics with high mortality; however, effective vaccines and therapies remain missing. Here, we depict a temporal single-cell landscape of primary porcine alveolar macrophages (PAMs) exposed to three different virulent ASFV strains in vitro . We found that attenuated and low-virulence ASFV strains tend to exhibit higher viral loads than highly virulent strain, which may result from upregulated RNA polymerase subunit genes expression. On the host side, our study highlights the IRF7-mediated positive feedback loop to the activation of the interferon signaling pathway in cells exposed to attenuated and low virulent ASFV strains. Moreover, we unraveled the PAMs populations marked by expressions of the IFI16 and CD163 , respectively, which produce high levels of interferon-stimulated genes (ISGs) and IL18 to regulate the host response to different virulent ASFV strains. Collectively, our data provide insights into the complex host-virus interactions with various ASFV strain infections, which may shed light on the development of effective antiviral strategies. IMPORTANCE There is still no available research on the temporal transcriptional profile of host cells exposed to different virulent ASFV strains at the single-cell level. Here, we first profiled the temporal viral and host transcriptomes in PAMs exposed to high virulent, attenuated virulent, and low virulent ASFV strains. Our analysis revealed that attenuated and low-virulence ASFV strains tend to exhibit higher viral loads than highly virulent strains, which may result from upregulated RNA polymerase subunit genes expression. We also found a positive feedback loop of the interferon signaling pathway mediated through IRF7 and identified the populations of PAMs marked by IFI6 and CD163 , respectively, which produce high levels of ISGs and IL18 to regulate host response to different virulent ASFV strains. Our study delineated a comprehensive single-cell landscape of host-virus dynamics across ASFV strains with different virulences and would provide an important resource for future research.

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs’ eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek’s disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.

This study aimed to optimize the composition of a liquid medium for Mycoplasma gallisepticum growth rate and density through statistical approach. The growth concentration of M. gallisepticum was determined by plotting a standard curve using Real-Time Quantitative PCR. The one-factor-at-a-time method and the Plakett-Burman design were initially applied to identify the factors that influenced the biomass of M. gallisepticum. The steepest ascent experiment and response surface methodology (RSM) with Box-Behnken design was employed to simultaneously assess the effects of multiple factors. Finally, optimization of the initial pH and validation of the model were done. The optimum concentrations of the critical components were obtained as follows: 203.96 mL/L of horse serum, 9.64 g/L of glucose, and 9.49 g/L of PPLO broth. The nucleic acid copy number of M. gallisepticum reached 1010.5147 copies/mL and the viable cell count reached a maximum of 109.8451 CCU/mL. This medium reduced the incubation time by approximately 6 h, and M. gallisepticum nucleic acid concentration and viable cell count were higher than those in the modified Frey medium (9.99 and 7 times, respectively). The new liquid medium is likely to improve productivity and reduce the production costs for vaccine-manufacturing companies in the future by reducing incubation times and increasing the growth rate and concentration of M. gallisepticum.

Traditional Chinese medicine (TCM) serves as a significant adjunct to chemical treatment for chronic diseases. For instance, the administration of Baitouweng decoction (BTWD) has proven effective in the treatment of ulcerative colitis. However, the limited understanding of its pharmacokinetics (PK) has impeded its widespread use. Chinese Bama miniature pigs possess anatomical and physiological similarities to the human body, making them a valuable model for investigating PK properties. Consequently, the identification of PK properties in Bama miniature pigs can provide valuable insights for guiding the clinical application of BTWD in humans. To facilitate this research, a rapid and sensitive UPLC-MS/MS method has been developed for the simultaneous quantification of eleven active ingredients of BTWD in plasma. Chromatographic separation was conducted using an Acquity UPLC HSS T3 C18 column and a gradient mobile phase comprising acetonitrile and water (containing 0.1% acetic acid). The methodology was validated in accordance with the FDA Bioanalytical Method Validation Guidance for Industry. The lower limit of quantitation fell within the range of 0.60–2.01 ng/mL. Pharmacokinetic studies indicated that coptisine chloride, berberine, columbamine, phellodendrine, and obacunone exhibited low Cmax, while fraxetin, esculin, fraxin, and pulchinenoside B4 were rapidly absorbed and eliminated from the plasma. These findings have implications for the development of effective components in BTWD and the adjustment of clinical dosage regimens.

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.

Avian encephalomyelitis (AE) is a highly infectious disease caused by the avian encephalomyelitis virus (AEV), which primarily affects the central nervous system of 1- to 4-week-old chicks and causes significant economic losses in the worldwide poultry industry. Despite heavy dependency on vaccine immunization, AEV has persisted on farms for extended periods, which increases its virulence and makes quick and accurate detection crucial to preventing and controlling the disease. Classical diagnostic methods have been unable to meet the current requirements for rapid diagnosis of AE cases. To address this issue, this paper reviews the etiological and molecular biological detection techniques of AE, and it seeks to provide a reference for future research and to establish differential diagnostic techniques for AE epidemiological investigation, identification of epidemic strains, and early diagnosis of clinical cases. Through improving our understanding of AE, we can better combat the disease and protect the global poultry industry.

Peste des petits ruminants virus (PPRV) causes a very devastating disease in sheep and goats. Rapid diagnosis and immunisation have been identified as key strategies for successful prevention of the disease. Therefore, a sensitive fluorescent microsphere immunochromatography test strips (FM-ICTS) was developed for rapid detection of special antibodies of PPRV in goats and sheep serum. The FM-ICTS were successfully prepared by fluorescent microspheres (FM) as tracer, which were covalently coupled to PPRV nucleocapsid protein (NP). The NP and monoclonal antibody of NP were separately dispensed onto a nitrocellulose membrane as test and quality control lines, respectively. The critical threshold for determining negative or positive through the ratio of the fluorescent signal of the test line and the control line (T/C) is 0.050. The repeatability of the FM-ICTS was excellent, with an overall average CV of 3.17 %. The detection limit of this assay was 1:5120. Additionally, the FM-ICTS no cross reaction with the sera of other related diseases was observed, only reacting with anti-PPRV serum. 70 serum samples were tested by FM-ICTS and commercial ELISA kit, and the results showed good agreement. Overall, a promising pen-side diagnostic tool was developed for the rapid qualitatively/semi-quantitatively detection of PPRV antibodies within 15 min.

In order to detect chicken infectious anemia virus（CIAV） quickly, accurately and simply, the specific RPA primers and crRNA used in the LwCas13a reaction system were designed with reference to the VP2 gene of CIAV in GenBank, and the CRISPR/Cas13a method for detecting CIAV was established by optimizing the reaction conditions, then the specificity, sensitivity and complexity of the method were verified.The results showed that the detection of other common viruses infecting chickens by this method was negative, with good specificity and no cross-reaction.The sensitivity was 50 copies/μL.It had a good coincidence rate with the detection results of PCR.The CRISPR/Cas13a detection method established in this study, combined with the color development of the test strip, can quickly differentiate the diagnosis of CIAV within 1.5 h, which provides a strong technical support for the diagnosis, prevention and purification of this disease.

为了快速、准确和简便地检测K亚群禽白血病病毒(ALV-K),本研究参照GenBank中ALV-K的gp85基因设计了特异的RPA引物和用于LwCas 13a反应系统的crRNA,通过优化反应条件建立了检测ALV-K的CRISPR/Cas 13a方法,并验证了该方法的特异性、敏感性和符合性.结果显示,该法对感染鸡的其他常见病毒的检测均为阴性,特异性良好无交叉反应;灵敏度为50 copies/μL;与PCR的检测结果有很好的符合性.本研究建立的CRISPR/Cas13a检测方法配合试纸条显色在1.5 h左右可对ALV-K进行快速的鉴别诊断,为该病的诊断及防控净化提供了强有力的技术支撑.

为了建立快速检测猪繁殖与呼吸综合征病毒(PRRSV)和猪肺炎支原体(Mhp)的双重荧光PCR检测方法,本试验根据NCBI数据库中的PRRSV ORF7和MhpP36基因,分别设计引物和TaqMan探针,通过优化扩增反应条件,建立PRRSV和Mhp的双重荧光PCR检测方法;分析所建立PCR方法的特异性、敏感性和重复性,并初步应用于临床样品检测.结果显示,25 μL PCR 反应体系中各引物最佳添加量:PRRSV-F 1.5 μL、PRRSV-R 1.0μL、PRRSV-P 0.8 μL、Mhp-F 1.2 μL、Mhp-R 1.3 μL、Mhp-P0.7μL.优化后的PCR反应程序:50℃反转录20min,95℃预变性2 min,95℃变性5 s,55℃退火10 s,72℃延伸20 s(此处收集荧光),40个循环.建立的双重荧光PCR方法特异性较好,与其他猪常见病原体无交叉反应;检测PRRSV和Mhp的敏感性分别为4.2拷贝/μL和9.6拷贝/μL;批内和批间变异系数均不高于2％,具有很好的重复性.因此,本试验建立的双重荧光PCR方法可同时检测PRRSV和Mhp,为猪繁殖与呼吸综合征(PRRS)和猪支原体肺炎(MPS)的诊断和防控工作提供支持.

The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) are two major protein degradation pathways in eukaryotic cells. In the present study, we investigated the role of two systems and their interaction after Brucella.suis (B.suis) infected RAW264.7 murine macrophage. We demonstrated that B.suis activated ALP by upregulating LC3-Ⅱlevels as well as incomplete inhibition of P62 expression in RAW264.7 cells. On the other hand, we used pharmacological agents to confirm that ALP contributed the intracellular proliferation of B.suis. At present, the studies on the relationship between UPS and Brucella remain less understanding. In the study, we demonstrated that UPS machinery was also activated by promoting expression of 20 s proteasome after B.suis infected RAW264.7 cells, and that, the UPS could also promote intracellular proliferation of B.suis. Many recent studies propose the close link and dynamic interconversion between UPS and ALP. Currently, the experiments demonstrated that after RAW264.7 cells infected B.suis, ALP was activated following UPS inhibition, while the UPS was not effectively activated after ALP inhibition. Last, we compared the ability to promote intracellular proliferation of B.suis between UPS and ALP. The results displayed that the ability of UPS to promote intracellular proliferation of B.suis was stronger than that of ALP, and simultaneous inhibition of UPS and ALP led to seriously affection on intracellular proliferation of B.suis. All above, our research provides a better understanding on the interaction between Brucella and both systems.

[目的]研究熟地黄多糖对环磷酰胺诱导的免疫抑制小鼠的免疫调节作用,为熟地黄多糖用于临床治疗免疫低下疾病或开发相关保健食品提供科学依据.[方法]选用36只昆明系小鼠,随机分为6组:空白对照组、免疫抑制模型组、黄茂多糖组及熟地黄多糖低、中、高(分别灌胃熟地黄多糖50、100、200 mg/kg)剂量组(PRRPL、PRRPM、PRRPH),每组6只.除空白对照组外,其余各组小鼠均腹腔注射80 mg/kg环磷酰胺,建立免疫抑制模型.造模后各组小鼠分别灌胃相应药物,1次/d,连续15 d,测定小鼠体重、腹腔巨噬细胞吞噬能力、免疫器官系数、脾淋巴细胞增殖及细胞周期、血清细胞因子含量,观察脾脏和胸腺组织形态学、肠道派氏节数目等免疫指标.[结果]与空白对照组相比,模型组脾脏指数极显著升高(P＜0.01),脾脏红髓、白髓界限不清,脾淋巴细胞数量减少;胸腺指数降低,皮质、髓质不清,结构破坏;脾淋巴细胞增殖能力、小鼠腹腔巨噬细胞吞噬能力及血清白细胞介素1β(IL-1β)、IL-6、γ-干扰素(INF-γ)水平均显著或极显著降低(P＜0.05;P＜0.01);脾淋巴细胞滞留在G0/G1期.与模型组相比,熟地黄多糖能极显著降低小鼠的脾脏指数(P＜0.01),缓解环磷酰胺造成的脾脏组织损坏,熟地黄多糖各剂量组对脂多糖(LPS)诱导的脾淋巴细胞增殖具有极显著的促进作用(P＜0.01),熟地黄多糖各剂量组能显著或极显著提高免疫抑制小鼠血清IL-1β、IL-6、肿瘤坏死因子α(TNF-α)、INF-γ水平(P＜0.05;P＜0.01),缓解淋巴细胞周期滞留,且高剂量组熟地黄多糖能显著提高小鼠腹腔巨噬细胞吞噬功能(P＜0.05).造模前后及熟地黄多糖对肠道派氏结数量均无显著影响(P＞0.05).[结论]熟地黄多糖能提高免疫抑制小鼠的免疫功能,可用于治疗免疫低下类疾病药物和保健品的开发.

Using complex plant protein sources as raw materials， the effects of mixed fermentation on anti-nutritive factors and nutrients of complex plant protein sources were studied through the orthogonal optimization experiments of mixed bacteria inoculation ratio and mixed bacteria fermentation. The results showed that the maximum degradation rate of free gossypol was 61.67%， which was significantly higher than that of the other groups when the inoculation ratio of Cyberlindnera jadinii and Bacillus subtilis was 1:3 （P＜0.05）. The optimum fermentation conditions of compound plant protein source were as follows: temperature 32℃， time 24h， ratio of material to water 1:0.6， inoculum 8%. Under the optimum conditions， the degradation rate of free gosseol and glucosinolate in fermented compound plant protein source was 77.38%， 66.98%， 12.98% and 4.87%， respectively. The tannin content decreased from 5.00 g/kg to 4.49 g/kg， the contents of calcium and total phosphorus increased from 0.61% and 1.52% to 0.67% and 1.54%， respectively， and threonine， valine， isoleucine， leucine and lysine increased by 9.56%， 7.64%， 6.70%， 7.10% and 10.37%， respectively.

An emerging infectious disease (EID) in foxes called "large kidney disease", characterized by enlarged kidneys, has been breaking out in fox farms in China, although its pathogenesis has not yet been elucidated. Here, we performed viral metagenomics sequencing on diseased fox tissue samples that identified a virus with 82.6% homology to the xenotropic murine leukemia virus-related virus (XMRV) PreXMRV-1 provirus strain (GenBank accession number NC_007815.2) in sick fox tissue. It was named PreXMRV-20, and its genome was verified by reverse transcription-polymerase chain reaction (PCR) and PCR product sequencing. Nonenveloped and polygonal virus-like particles consistent with the shape and size of XMRV were observed by negative staining electron microscopy. Administered subcutaneously, PreXMRV-20 infected weaned foxes, leading to growth retardation. The discovery of the PreXMRV-20 strain (the first isolation of an XMRV homolog in blue foxes) identifies a potential public health issue in blue fox breeding since XMRV has been confirmed to be a zoonotic virus.

African swine fever virus (ASFV) causes a lethal swine hemorrhagic disease and is currently responsible for widespread damage to the pig industry. The pathogenesis of ASFV infection and its interaction with host responses remain poorly understood. In this study, we profiled the temporal viral and host transcriptomes in porcine alveolar macrophages (PAMs) with virulent and attenuated ASFV strains. We identified profound differences in the virus expression programs between SY18 and HuB20, which shed light on the pathogenic functions of several ASFV genes. Through integrated computational analysis and experimental validation, we demonstrated that compared to the virulent SY18 strain, the attenuated HuB20 quickly activates expression of receptors, sensors, regulators, as well as downstream effectors, including cGAS, STAT1/2, IRF9, MX1/2, suggesting rapid induction of a strong antiviral immune response in HuB20. Surprisingly, in addition to the pivotal DNA sensing mechanism mediated by cGAS-STING pathway, infection of the DNA virus ASFV activates genes associated with RNA virus response, with stronger induction by HuB20 infection. Taken together, this study reveals novel insights into the host-virus interaction dynamics, and provides reference for future mechanistic studies of ASFV pathogenicity.

为了建立简便、快速检测猪繁殖与呼吸综合征病毒(PRRSV)类NADC30毒株免提取核酸荧光定量RT-PCR方法,试验根据GenBank中PRRSV类NADC30毒株NSP2基因设计引物和探针,建立了直接对样品中PRRSV类NADC30毒株检测的荧光定量RT-PCR方法.对该方法的敏感性、特异性和重复性进行评估,并对临床样本进行检测.结果表明:建立的PRRSV类NADC30毒株免提取核酸荧光定量 RT-PCR 方法的扩增体系(20 μL)为 2×PrimeDirect Probe RT-qPCR Mix 10 μL,PRRSV类 NADC30 毒株液 1 μL,NADC30-F(15 μmol/μL)0.8 μL,NADC30-R(15 μmol/μL)0.8 μL,NADC30-P(15 μmol/μL)0.4 μL,灭菌纯化水7 μL.扩增程序为 90℃3 min;60℃5 min;95℃5 s,54℃20 s(收集荧光信号,选择FAM通道),40个循环.该法特异性好,对其他猪常见病原无交叉反应.对PRRSV类NADC30毒株液的最低检测限度为3.98 TCID50/100μL,与传统提取核酸的荧光定量RT-PCR检测方法敏感性一致.不同病毒含量样本组内和组间变异系数均不高于2％,具有良好的重复性和稳定性.临床样本检测结果与传统荧光定量RT-PCR方法检测结果一致.说明建立的PRRSV类NADC30毒株免提取核酸荧光定量RT-PCR检测方法特异性好、敏感性高,具有良好的重复性和稳定性,可用于PRRSV类NADC30毒株的临床检测.

Imidacloprid (IMI) is a widely used neonicotinoid insecticide that has toxic effects on nontarget organisms. 1,8Cineole (eucalyptol) is purified from essential oils in several aromatic plants and can prevent xenobiotic toxicity. The kidney is a major organ for xenobiotic elimination and thus has high risk of exposure. The purpose of this research was to clarify the effect of IMI exposure on autophagy in fish kidney cells, determine the potential of eucalyptol to provide cytoprotection from the toxicity of the neonicotinoid pesticide IMI, and identify its mechanism of action. Therefore, the Ctenopharyngodon idellus kidney cell line (CIK cell) was treated with 20 mg/L IMI and/or 20 mu M eucalyptol for 48 h as the research objective. The results showed that IMI exposure induced autophagy accompanied by advanced autophagy markers BNIP3, Beclin1 and LC3II/I in CIK cells, reduced the levels of miR-451, increased the expression of Cab39 and AMPK, inhibited AKT/mTOR signaling, and activated the JNK pathway. Eucalyptol treatment alleviated IMI-induced autophagy and relieved the activation of autophagy-associated signals. These results indicate that eucalyptol could alleviate IMI-induced autophagy through the miR-451/Cab39/AMPK axis in fish kidney cells. These results partly explained the mechanism of biological threat on fish under IMI exposure and the potential application value of EUC in aquaculture.

为建立一种山羊痘病毒(GTPV)抗体快速检测方法,通过PCR技术扩增了P32蛋白优势抗原区(2-141AA)基因序列,将其连接至原核表达载体pET-28a(+)上,转化至E.coli BL21(DE3)中,经IPTG诱导后获得了重组蛋白,纯化后经Western blot鉴定表明该蛋白具有良好的特异性和抗原反应性.以重组蛋白作为包被抗原,通过优化反应条件建立了快速检测GTPV抗体的间接ELISA方法,该方法具有良好的特异性、敏感性与可重复性,为GTPV的免疫抗体检测及流行病学调查提供了一种血清学诊断方法.