The respiratory epithelium acts as the airway's first physical barrier against invading pathogens, mainly relying on mucociliary clearance (MCC) and apical junctional complexes (tight junctions/TJs and adherens junctions/AJs) for barrier function. Primary airway epithelial cells cultured in the air-liquid interface (ALI) system can well mimic these characteristics in vitro. This study established and characterized well-differentiated ALI-cultured goat airway epithelial cells (ALI-GAECs), and developed a CPIV3 infection model therein, verifying the apical infection and release of CPIV3 in ALI-GAECs. CPIV3 infection impaired the barrier function of ALI-GAECs, as reflected by decreased trans-epithelial electrical resistance (TEER), increased FITC-Dextran permeability, and reorganization of the ZO-1 and F-actin meshwork. Mechanistically, RT-qPCR and Western blot analyses demonstrated that CPIV3 downregulated major TJ proteins (ZO-1, occludin, claudin-1) while upregulated the expression of proinflammatory cytokines (TNF-α, IL-1β, IL-4, IL-6). The NF-κB signaling pathway, but not the MAPK pathways, was activated upon viral infection. Treatment with the NF-κB inhibitor BAY 11-7082 partially restored the expression of TJ proteins and proinflammatory cytokines. Collectively, the activation of the NF-κB pathway and subsequent production of proinflammatory cytokines is responsible for CPIV3-induced TJ disruption. In addition, the developed ALI-GAECs model provides a valuable in vitro tool for investigating the pathogenesis of CPIV3 and other caprine respiratory pathogens.

Bovine viral diarrhea virus (BVDV) is a key member of the genus Pestivirus, which can cause persistent infections (PI) characterized by immunotolerance, leading to significant economic losses to the cattle industry globally. Among the detection methods for BVDV, conventional gold nanoparticles-based lateral flow immunoassay (Au NPs-based LFIA), which uses gold nanoparticles as signal reporters, often suffers from low sensitivity due to insufficient signal brightness. The development of more sensitive point-of-care testing (POCT) methods is urgently needed. In the present study, we developed a novel signal reporter based on Ag-rich Ag-Au nanoshells (AgR-Au NSs) featuring enhanced visual color brightness. The developed AgR-Au NSs-based LFIA enables one-step BVDV detection within 20 minutes. Under optimized conditions, the visual detection limit reached 1.95 × 103TCID50/mL, a four-fold improvement over conventional Au NPs-based LFIA and consistent with ELISA. The assay showed broad reactivity across different BVDV genotypes, no cross-reactivity with other pathogens, and good stability (4 °C for 180 days). In 215 clinical samples, it achieved 98.14

Respiratory diseases induced by Mycoplasma species, mainly Mycoplasma capricolum subsp. capripneumoniae (Mccp) and Mycoplasma ovipneumonia (Mo), pose a major threat to goat/sheep farming. This study investigated the biological characteristics and antimicrobial susceptibility of Mccp isolates that suddenly spread extensively in China in the first half of 2024. A total of 34 Mccp isolates were obtained from goats with suspected contagious caprine pleuropneumonia (CCPP) across multiple provinces during 2024-2025. All isolates were purified and confirmed via a PCR targeting the arcD gene and exhibited characteristic "fried egg" colony morphology. Phylogenetic analysis based on the arcD gene and multi-locus sequence analysis (MLSA) of eight genetic loci revealed that the circulating strains shared high homology and belonged to Group 1 within Lineage 1, which showed a close genetic relationship with isolates from Qatar and the United Arab Emirates, while differing from previously reported strains in China. Antimicrobial susceptibility testing against nine antimicrobial drugs indicated that the Mccp isolates generally exhibited low resistance levels. However, some strains showed reduced susceptibility to florfenicol and lincomycin. These findings highlight the emergence of a genetically distinct Mccp lineage in China and underscore the importance of ongoing surveillance, strain characterization, and prudent antimicrobial use in CCPP control.

Mycoplasma ovipneumoniae is a primary causative agent of pneumonia in ruminants, causing chronic non-progressive pneumonia in domestic sheep and goats, but leading to higher morbidity and mortality in bighorn sheep and wild small ruminants. This disease has become a widespread epidemic, resulting in significant losses to the sheep industry. In this study, we evaluated the immunogenicity and initial protective effects of four antigenic proteins of M. ovipneumoniae, namely Eno, EF-Tu, Ulad, and T4SS. These proteins were used to immunize BALB/c mice either individually or in a combination (rProteins group). The mice were intranasally infected with 109 CCU50/mL M. ovipneumoniae strain NJ01 twice, on days 28 and 30 after immunization. Among the four recombinant proteins, rEno demonstrated the most promising results in terms of inducing specific humoral and cellular immune responses. It also resulted in the lowest lung lesion scores and the lowest M. ovipneumoniae loads in the lungs and bronchoalveolar lavage fluid (BALF). Compared to the other three proteins, rEno provided superior protection. Furthermore, the rEno vaccine significantly reduced the inflammatory response in the lungs of mice, as evidenced by the evaluation of pro-inflammatory cytokines. The expression of IL-1β and NF-κB was significantly reduced, while the expression of IL-4 was significantly increased. In conclusion, the rEno vaccine elicited a favorable immunological response and conferred protection against M. ovipneumoniae. This finding presents a novel approach to controlling the global spread of this pathogen.

With the rapid development of cattle industry, bovine viral diarrhea virus (BVDV) is becoming widespread in China, which causes serious economic losses to the industry. Effective vaccination and viral surveillance are critical for the prevent and control of BVDV infection. In the present study, the immunogenic domain of E2 protein of BVDV-1 was expressed by prokaryotic pET-28a vector. Monoclonal antibodies (mAbs) against E2 protein were prepared and systemically examined by western blot, immunofluorescence assay, blocking ELISA (bELISA) and virus neutralization test (VNT). The mAb 1E2B3, which showed good reactivity and neutralizing activity to BVDV-1 strains, was selected for ELISA establishment. After a series of screening and optimization, a novel bELISA for highly sensitive and specific detection of BVDV-1 antibodies was established, using HRP-labeled 1E2B3 and recombinant E2 protein. ROC analysis of 91 positive and 84 negative reference bovine serum samples yielded the area under the curve (AUC) of 0.9903. A diagnostic specificity of 96.43 % and a sensitivity of 95.6 % were achieved when the cutoff value was set at 24.31 %. There was no cross reaction to the positive sera of classical swine fever virus (CSFV), BVDV-2, border disease virus (BDV), bovine parainfluenza virus type 3 (BPIV3), infectious bovine rhinotracheitis virus (IBRV), foot-and-mouth disease virus (FMDV), Mycoplasma bovis (M.bovis) and Brucella. The total agreement rate of bELISA with VNT was 93.96 % (249/265). In addition, the result of bELISA was positively correlated with neutralizing antibody titer, and the bELISA could well distinguish the serum samples before and after BVDV vaccination. These results indicate that the established bELISA in this study is specific, sensitive, simple and convenient, which provides technical support for the vaccine efficacy evaluation, prevention and control of BVD in the future.

为建立一种快速鉴定O8、O9和O89血清型大肠杆菌的三重PCR方法,根据GenBank登录的3种血清型大肠杆菌O抗原合成基因簇序列,设计3对检测引物,通过优化反应条件建立三重PCR方法.结果:经条件优化后的三重PCR方法可有效鉴别O8、O9和O89血清型的大肠杆菌,具有良好的特异性,对其他肉羊养殖场常见血清型的大肠杆菌和致病菌无特异扩增条带;敏感性检测显示,三重PCR方法对O8和O89血清型的最小检出量为10 pg/μL,对09血清型的最小检出量为100pg/μL;采用该方法对临床分离的大肠杆菌进行检测,结果与传统血清凝集方法一致.综上,本研究建立的三重PCR方法可快速、准确、特异地对O8、O9和O89血清型大肠杆菌进行检测,为养殖及肉品加工环节大肠杆菌的流行病学研究提供了更加快捷的检测手段.

边界病(border disease)由边界病病毒(border disease virus,BDV)引起,导致绵羊和山羊持续感染和繁殖疾病,2012年在国内首次报道,但目前尚无特异的RT-PCR方法对该病原进行检测.本研究通过比对黄病毒科瘟病毒属病毒的全基因组序列,以3'-UTR基因为靶基因,设计了特异扩增BDV的引物.通过构建重组质粒pMD18-T-BDV,以其作为标准品建立了BDV的RT-PCR检测方法.进一步优化该方法的反应条件,并进行特异性、敏感性及临床样品检测.结果显示,该方法在退火温度48～60℃时均可特异扩增BDV,通过检测牛病毒性腹泻病毒1型(BVDV-1)、牛病毒性腹泻病毒2型(BVDV-2)和猪瘟病毒(CSFV)提取的RNA,该方法可特异扩增BDV而对其他同属病毒检测均呈阴性,表明其特异性良好;同时,该方法具有良好的敏感性,最低检出限可达101拷贝/μL,敏感性极高.利用该方法检测BDV持续感染羊和人工感染羊,发现持续感染羊的心脏、肝脏、脾脏、肺脏、肾脏、淋巴结、卵巢、脑等器官均可检测到BDV,而人工感染羊只能在感染3～7 d的血液和淋巴结中检测到病毒RNA,其他器官中未检测到病毒.本研究建立了特异检测BDV的RT-PCR方法,并证明了BDV在持续感染羊和一过性感染羊中的病毒分布情况,为其可能的排毒途径提供了依据.

Pathogenic Escherichia coli (E. coli) can cause a variety of intestinal and extra-intestinal infections in humans and animals. Similar with the intestinal disease, respiratory disease is also a major threat to the breeding industry of goats. But the reports on respiratory disease associated E. coli are very limited. In this study, E. coli and other pathogens were examined for the 77 submitted respiratory cases. The serotypes, virulence genes, phylogenetic group and antimicrobial resistance characteristics of the E. coli isolates were identified. The results showed that 34 cases (44.16%) were associated with E. coli and 22 cases showed mixed infections of E. coli with Mycoplasma ovipneumoniae, Mannheimia haemolytica or Pasteurella . Among the 49 E. coli isolates, O8 (32.65%), O9 (20.41%) and O89 (10.20%) were the predominant serotypes (31/49, 63.27%). 22 virulence genes were detected and the most prevalent genes were fimH (100%), yijp (100%), mat (97.96%), ompA (95.92%) ibeB (91.84%) and fyuA (77.55%). In addition, ibeA was detected in 6.12% (3/49) of the strains. Markers of extra-intestinal pathogenic E. coli (ExPEC) were also identified and 14 strains were classified as ExPECs. 14 (28.57%), 25 (51.02%), 3 (6.12%) and 7 (14.29%) strains belonged to phylogenetic group A, B1, B2 and D, respectively, and group A and B1 were the predominant ones. The E. coli strains showed high resistant (48.98%-100%) to the 14 selected antimicrobials and all of them were defined as multiple drug resistant (MDR) strains. This is the first systemically study on E. coli of goats respiratory diseases origin in eastern China. The results suggest that E. coli infection may play an important role in goat respiratory diseases and that goats are reservoir hosts of ExPECs, which needs continuous monitoring in the future.

China is the country with the largest number of domestic small ruminants in the world. Recently, the intensive and large-scale sheep/goat raising industry has developed rapidly, especially in nonpastoral regions. Frequent trading, allocation, and transportation result in the introduction and prevalence of new pathogens. Several new viral pathogens (peste des petits ruminants virus, caprine parainfluenza virus type 3, border disease virus, enzootic nasal tumor virus, caprine herpesvirus 1, enterovirus) have been circulating and identified in China, which has attracted extensive attention from both farmers and researchers. During the last decade, studies examining the etiology, epidemiology, pathogenesis, diagnostic methods, and vaccines for these emerging viruses have been conducted. In this review, we focus on the latest findings and research progress related to these newly identified viral pathogens in China, discuss the current situation and problems, and propose research directions and prevention strategies for different diseases in the future. Our aim is to provide comprehensive and valuable information for the prevention and control of these emerging viruses and highlight the importance of surveillance of emerging or re-emerging viruses.

为明确江苏某羊场外购育肥羊突发呼吸道疾病的病因,本研究结合临床症状、剖检病变观察、病原学检测与分离鉴定等方法对病因进行分析.结果表明:该病是由肠道外致病性大肠杆菌、山羊副流感病毒3型及绵羊肺炎支原体混合感染所致.分离所得的2株大肠杆菌对多黏菌素B、强力霉素敏感,对其他12种常见抗生素均不敏感,检测到其携带有iss、fyuA、iroN、cvaC、fimH、iutA、kpsMT Ⅱ这7种毒力基因,分别属于系统进化群B1群与B2群.通过MDBK细胞成功分离获得1株山羊副流感病毒3型毒株,其M基因片段与流行毒株同源性最高达99.7％.该结果将有助于科学认识和防控山羊呼吸道疾病.

Viruses have evolved diverse strategies to evade the antiviral response of interferons (IFNs). Exogenous IFNs were applied to eliminate the counteracting effect and possess antiviral properties. Caprine parainfluenza virus 3 (CPIV3) and bovine parainfluenza virus type 3 (BPIV3) are important pathogens associated with respiratory diseases in goat and cattle, respectively. To explore the feasibility of type I IFNs for control of CPIV3 and BPIV3 infection, the activated effects of IFN-stimulated genes (ISGs) and the immunomodulation responses of goat IFN-α were detected by transcriptomic analysis. Then, the antiviral efficacy of goat IFN-α and IFN-τ against CPIV3 and BPIV3 infection in MDBK cells was evaluated using different treatment routes at different infection times. The results showed that CPIV3 infection inhibited the production of type I IFNs, whereas exogenous goat IFN-α induced various ISGs, the IFN-τ encoding gene, and a negligible inflammatory response. Consequently, goat IFN-α prophylaxis but not treatment was found to effectively modulate CPIV3 and BPIV3 infection; the protective effect lasted for 1 week, and the antiviral activity was maintained at a concentration of 0.1 μg/mL. Furthermore, the antiviral activity of goat IFN-τ in response to CPIV3 and BPIV3 infection is comparable to that of goat IFN-α. These results corroborate that goat IFN-α and IFN-τ exhibit prophylactic activities in response to ruminant respiratory viral infection in vitro, and should be further investigated for a potential use in vivo.

污蝇杆菌(Wohlfahrtiimonas chitiniclastica)是一种新出现的人畜共患病原菌.2020年从安徽某羊厂1只口腔和舌头溃烂羊的口腔中采样并分离细菌,进行药敏试验.结果显示:分离到1株污蝇杆菌,呈β型溶血,该菌对β-内酰胺类、氨基糖苷类、四环素类耐药、大环内酯类、喹诺酮类、磺胺类、氟苯尼考、多黏菌素类药物均出现耐药情况,对美罗培南、替加环素敏感.这是国内首次报道羊源污蝇杆菌,但其耐药性相比之前报道菌株更严重,这对公共卫生安全具有重大挑战.

随着规模化养羊业的发展,肉羊呼吸道疾病流行普遍、发病严重、防治困难,给养殖业及养殖户造成较大的经济损失.为明确安徽某肉羊养殖场呼吸道疾病的病因,结合流行病学、临床症状、剖检病变观察、细菌学及病毒学检测与分离鉴定以及血清学检测对病因进行分析,结果表明该病是由山羊副流感病毒3型与巴氏杆菌混合感染所致.分离菌株对丁胺卡那霉素、氟苯尼考、阿奇霉素、恩诺沙星和头孢曲松敏感,对青霉素、克林霉素和强力霉素不敏感.通过MDBK细胞成功分离获得山羊副流感病毒3型毒株,其M基因片段与已有毒株同源性达99％.血清学检测也进一步证实病毒的感染.这为临床上科学防控山羊呼吸道疾病提供了指导和借鉴.

Caprine parainfluenza virus type 3 (CPIV3) is one of the most important viral respiratory pathogens of goat. Accumulating evidence demonstrates that apoptosis is a cellular mechanism for the host response to pathogens, and it participates in regulating viral replication. However, there is little study on CPIV3-induced host cells apoptosis. In this study, primary goat tracheal epithelial (GTE) cells were established as a cellular model that is permissive to CPIV3 infection. Then, we showed that CPIV3 infection induced apoptosis in GTE cells, as determined by morphological changes, flow cytometry and TUNEL assay. Moreover, Caspase activity and the expression of pro-apoptotic genes further suggested that CPIV3 induced apoptosis by activating both the intrinsic and extrinsic pathways. Mechanistically, the ability of CPIV3 to induce apoptosis was activated by N protein, and the viral protein increased CPIV3 replication through effecting apoptosis. Overall, our findings showed that GTE cells that will enable further analysis of CPIV3 infection and offers novel insights into the mechanisms of CPIV3induced apoptosis in host cells.

裂谷热(Rift Valley fever,RVF)是由裂谷热病毒(Rift Valley fever virus,RVFV)引起的一种烈性人兽共患传染病.RVFV囊膜蛋白Gn可诱导产生中和抗体,是RVFV检测方法和疫苗研究的重要抗原靶标.本研究通过分析蛋白抗原位点信息,构建包含Gn蛋白两个主要抗原区域的重组表达载体,随后将质粒转化至BL21感受态细胞,以IPTG诱导重组蛋白表达并优化蛋白表达条件,通过Western Blot鉴定重组蛋白;将重组蛋白免疫BALB/c小鼠,制备多克隆抗体,并以ELISA、Western Blot、IFA检测多克隆抗体的反应性.结果显示:诱导表达的Gn重组蛋白分子质量约为45 kDa;蛋白表达条件优化为IPTG终浓度0.25 mmol/L,诱导时间5 h;Western Blot鉴定发现蛋白成功表达.通过ELISA测定小鼠三免后血清抗体,结果发现抗体效价大于1:51200;Western Blot检测显示,制备的多抗血清能与重组蛋白发生反应;进一步的IFA检测结果表明,制备的多克隆抗体可与真核质粒转染细胞中表达的Gn蛋白反应.本研究获得的Gn重组蛋白及制备的多克隆抗体为后续RVFV检测方法的建立奠定了基础.

旨在建立一种检测绵羊肺炎支原体(Mo)血清抗体的间接ELISA方法,笔者构建了 Mo Enolase基因原核表达载体,诱导表达后,纯化的重组蛋白用Western-blot分析其反应原性.以重组蛋白为包被抗原,建立了 Mo间接ELISA抗体检测方法.结果显示,重组蛋白得到可溶性表达,Western-blot证实具有良好的反应原性.间接ELISA反应条件优化结果显示,包被抗原浓度为2mg/L,37℃2h,封闭条件为含10g/L BSA的PBS,4℃过夜,待检血清稀释度为1∶50,37℃1 h,酶标二抗最佳稀释度为1∶4 000,37℃1 h,底物最佳显色条件为37℃避光10min.分别利用38份阴性血清、38份阳性血清确定临界值为S/P=0.365.用该方法与间接血凝法对180份血清进行检测,两者符合率为81.11％.结果表明,本研究建立的间接ELISA方法敏感、特异,具有良好的临床应用前景.

为了解我国主要的规模化养羊区规模化羊场的细菌耐药性情况,2018年8月至2020年11月,从江苏、安徽、浙江、江西、河北等地规模化羊场采集临床发病羊的呼吸道、消化道和其他样品,分离鉴定细菌,用药敏纸片法测定耐药性.结果,共分离到细菌147株;药敏结果显示,革兰阳性菌对大环内酯类耐药严重,对β内酰胺类中度耐药,对氨基糖苷类、喹诺酮类整体耐药率低于40％;革兰阴性菌对大环内酯类耐药率在60％～100％,对青霉素类的耐药率在80％以上,仅对大观霉素和阿米卡星的耐药率低于30％.对24种抗生素药敏试验结果显示,45株细菌耐药20种以上(占30.6％),主要为肠杆菌属菌;50株细菌耐药10～20种(占34％).从2018到2020年总耐药率从80.1％下降到56.9％,江苏、河北等省耐药严重,春夏季耐药率高于秋冬季.这些结果提示,规模化羊场细菌耐药性严重.本研究结果为进一步开展细菌耐药性分析和制定合理的安全用药措施奠定了基础.

旨在研究羊感染肺炎支原体后胸部C T影像学特征.将20只山羊分为2组,对照组5只、试验组15只,试验组通过气管注射感染5 mL绵羊肺炎支原体(1×107 CCU·mL-1)人工诱发山羊支原体肺炎,对照组注射等体积生理盐水.感染后观察两组羊的临床症状,在感染后第0、7、14、21、28天对两组羊胸部进行C T扫查,分析支原体肺炎的C T影像学特征.感染后第29天剖杀羊,观察肺部病理解剖变化.取病变组织制备石蜡切片,观察组织病理学变化.结果显示:试验组山羊感染肺炎支原体后表现出呼吸道感染症状,体温升高,咳嗽,流浆液性或脓性鼻液.胸部CT影像主要表现为片状磨玻璃密度影,网格状阴影,多见双侧肺叶病变,好发于右前叶,以间质性肺炎伴发支气管肺炎为主,其中,重症羊多见空气支气管征,个别羊见胸膜增厚影.对照组临床症状和胸部CT影像在感染前后未见明显差异和异常.综上表明,CT影像诊断技术有利于羊支原体肺炎的早期诊断,并有助于疾病转归的判断.

裂谷热(RVF)是由裂谷热病毒(RVFV)引起的一种人兽共患传染病,主要由蚊媒传播.该病主要感染反刍动物,可引起流产和新生胎儿死亡,人类对该病也易感,严重者可导致死亡.对于无RVF的国家,建立相应的病原学与血清学检测方法对于防止该病传入至关重要.为了建立基于病毒N蛋白的诊断技术,本研究构建了裂谷热病毒N基因原核表达质粒pET-28a-N,转化BL21(DE3),IPTG诱导表达,通过SDS-PAGE和Western blot检测蛋白的表达,重组蛋白分子量约为29 kDa,主要以上清液形式存在.大量表达并纯化重组蛋白,用纯化后的重组蛋白免疫BALB/c小鼠,制备单克隆抗体,并对其进行鉴定.采用间接ELISA和ID Vet公司阻断ELISA试剂盒检测免疫小鼠抗体效价及特异性,并用Western blot检测多克隆抗体反应性.结果表明,免疫后的抗体效价迅速升高,最终可达51 200以上;阻断ELISA和Western blot检测结果表明,制备的多克隆抗体具有良好的反应性与特异性.通过3次亚克隆最终获得2株分泌单克隆抗体的杂交瘤细胞株,分泌的单克隆抗体均可与重组蛋白反应,重链均为IgG2a型,轻链为κ型.本研究为RVF诊断方法的建立奠定了基础.

The Caprine parainfluenza virus 3 (CPIV3) is a novel Paramyxovirus that is isolated from goats suffering from respiratory diseases. Presently, the pathogenesis of CPIV3 infection has not yet been fully characterized. The Type I interferon (IFN) is a key mediator of innate antiviral responses, as many viruses have developed strategies to circumvent IFN response, whether or how CPIV3 antagonizes type I IFN antiviral effects have not yet been characterized. This study observed that CPIV3 was resistant to IFN-α treatment and antagonized IFN-α antiviral responses on MDBK and goat tracheal epithelial (GTE) cell models. Western blot analysis showed that CPIV3 infection reduced STAT1 expression and phosphorylation, which inhibited IFN-α signal transduction on GTE cells. By screening and utilizing specific monoclonal antibodies (mAbs), three CPIV3 accessory proteins C, V and D were identified during the virus infection process on the GTE cell models. Accessory proteins C and V, but not protein D, was identified to antagonize IFN-α antiviral signaling. Furthermore, accessory protein C, but not protein V, reduced the level of IFN-α driven phosphorylated STAT1 (pSTAT1), and then inhibit STAT1 signaling. Genetic variation analysis to the PIV3 accessory protein C has found two highly variable regions (VR), with VR2 (31-70th aa) being involved in for the CPIV3 accessory protein C to hijack the STAT1 signaling activation. The above data indicated that CPIV3 is capable of inhibiting IFN-α signal transduction by reducing STAT1 expression and activation, and that the accessory protein C, plays vital roles in the immune escape process.