Vaccination is a critical strategy for controlling H9N2 avian influenza, a subtype with significant implications for poultry health and public safety. Current vaccines hinder serological differentiation between naturally infected and vaccinated animals, complicating disease surveillance and eradication efforts. Here, we developed a novel H9-subtype differentiating-infected-from-vaccinated-animals (DIVA) vaccine using reverse genetics. The recombinant virus, Re-H9-DIVA-J2, was engineered by replacing the neuraminidase (NA) gene of a clinically isolated H9 strain (A/chicken/Guangdong/J2/2016) with the NA ectodomain from a B/Yamagata-lineage influenza virus (B/Massachusetts/2/2012), while retaining six internal genes from the H1N1 PR8 strain. The chimeric virus exhibited low pathogenicity in chicken embryos, high growth titers (HA≥8 log2), and stable genetic inheritance of the B-type NA marker over 10 passages. Three batches of inactivated vaccines were tested in specific-pathogen-free (SPF) chickens, demonstrating robust immunogenicity with hemagglutination inhibition (HI) antibody titers peaking at 10 log2 by 21 days post-vaccination. Challenge experiments confirmed full clinical protection and reduced viral shedding (above 90 % protection). Critically, sera from Re-H9-DIVA-J2-vaccinated chickens showed no cross-reactivity with A-type N2 protein in immunofluorescence (IFA) and ELISA assays, distinguishing them from sera of wild-type-infected or conventional H9N2-vaccinated animals. This study presents a safe, immunogenic H9 marker vaccine compatible with DIVA diagnostics, offering a promising tool for H9N2 control and eradication.

H9N2 subtype avian influenza virus (AIV) facilitates viral adaptative evolution and cross-species transmission, result in inflicting severe economic losses on the poultry industry and posing significant public health threats. Currently, conventional vaccine-based prophylactic strategies exhibit certain limitations, highlighting an urgent need for the development of novel prevention and control measures. In this study, using phage display technology, we screened 37 potential binding sequences from the PBMCs of an alpaca immunized with the H9N2 virus and its HA protein. AlphaFold3 further predicted seven nanobody candidates (Nb1, Nb2, Nb7, Nb13, Nb24, Nb26 and Nb34) with potential neutralizing activity. Subsequently, we successfully expressed these recombinant nanobodies in the Pichia pastoris system and tested the HI activity of the six candidates (excluding Nb24). While all nanobodies exhibited binding activity in ELISA and IFA, several also showed viral neutralizing activity in cells and embryonated chicken eggs. Notably, Nb13 exhibited broad inhibitory activity against the early h9.4.2.1 (JS2002) strain and two temporally distinct h9.4.2.5 strains (DC17 and NJ150), with IC50 values of 14.38 ± 1.01, 1.20 ± 0.20, and 1.14 ± 0.14 μg/mL, respectively. In chick challenge models, intratracheal administration of Nb13 provided robust protection. Both prophylactic and therapeutic regimens dose-dependently reduced the titer and duration of oropharyngeal viral shedding, and effectively alleviated tracheal pathological lesions. Mechanistically, Nb13 neutralizes the virus by blocking viral entry into host cells, which structural predictions suggest is mediated by binding to the RBS. Overall, this study provides a highly promising novel candidate for the clinical prevention of H9N2 infections.

Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), play critical roles in various physiological activities such as cell differentiation, apoptosis, and vascular tension when existing in cells at low levels. However, excessive amounts of free radicals are harmful, causing DNA damage, lipid peroxidation, protein degeneration, and abnormal cell death. Certain viral infections induce cells to produce excessive free radicals, which in multiple ways help the virus to replicate, mature, and exit. Iron is a necessary element for many intracellular enzymes, involved in both cellular activities and viral replication. Ferroptosis, a programmed cell death mode distinct from apoptosis, necrosis, and pyroptosis, is characterized by lipid peroxide accumulation and damage to the antioxidant system, affecting many cellular processes. Viral infection commonly manifests as decreased glutathione (GSH) content and down-regulated glutathione peroxidase 4 (GPX4) activity, similar to ferroptosis. Recent studies have suggested a possible relationship among free radicals, viral infections and ferroptosis. This review aims to elucidate the molecular mechanism linking free radicals and ferroptosis during viral infections and provide a new theoretical basis for studying viral pathogenesis and control.

Avian influenza virus (AIV) subtype H9N2 is the most widespread AIV in poultry worldwide, causing great economic losses in the global poultry industry. Chickens and ducks are the major hosts and play essential roles in the transmission and evolution of H9N2 AIV. Vaccines are considered an effective strategy for fighting H9N2 infection. However, due to the differences in immune responses to infection, vaccines against H9N2 AIV suitable for use in both chickens and ducks have not been well studied. This study developed an inactivated H9N2 vaccine based on a duck-origin H9N2 AIV and assessed its effectiveness in the laboratory. The results showed that the inactivated H9N2 vaccine elicited significant haemagglutination inhibition (HI) antibodies in both chickens and ducks. Virus challenge experiments revealed that immunization with this vaccine significantly blocked virus shedding after infection by both homogenous and heterologous H9N2 viruses. The vaccine was efficacious in chicken and duck flocks under normal field conditions. We also found that egg-yolk antibodies were produced by laying birds immunized with the inactivated vaccine, and high levels of maternal antibodies were detected in the serum of the offspring. Taken together, our study showed that this inactivated H9N2 vaccine could be extremely favourable for the prevention of H9N2 in both chickens and ducks.

为研究当前鸭疫里氏杆菌1型(RA1)流行株的生物学特性及免疫原性,对从江苏省、山东省等不同地区规模鸭场采集的病料中分离的23个疑似RA的菌株纯化培养后进行细菌形态观察,并利用RA 16S rRNA基因特异性引物进行PCR鉴定及基因序列测定、生化试验、血清学分型鉴定、致病性分析、交叉攻毒保护试验.结果表明,16株分离株符合RA生物学特性,经PCR检测为RA阳性,与GenBank中RA相应基因同源性≥99％,其中11株血清学鉴定为RA1,占分离RA总数的68.7％;选取的5株RA1分离株中JP01、SD02、GY01等3株致病率和致死率达80％～100％;对3株RA1分离株进行免疫原性研究,结果显示JP01株对各毒株的交叉攻毒保护率可达100％,说明JP01株具有良好的免疫原性.

本研究以肝脏出血和脾脏坏死为特征的病死鸭肝脏和脾脏组织接种SPF鸡胚进行病毒分离,并利用PCR方法进行病原鉴定,对分离病毒进行了回归雏鸭试验和S1基因测序分析.结果显示:该分离病毒为新型鸭呼肠孤病毒,并命名为NDRVSY株;将该分离病毒接种SPF鸡胚、BHK-21和Vero细胞进行传代培养,结果表明其能稳定适应鸡胚增殖和致鸡胚死亡,在BHK-21和Vero细胞两种传代细胞上也能良好增殖,并产生明显细胞病变.本研究为NDRV的疫苗和致病机制研究奠定了基础.

利用CD-HIT-EST快速聚类分析和BLASTN同源性比对共鉴定出17个滑液支原体(Mycoplasma synoviae)特有基因,并从中选取保守基因VY93_RS02885作为分子诊断的靶标基因.根据该基因核苷酸序列设计了1对引物,建立了滑液支原体的SYBR green qPCR检测方法.结果表明,该方法对不同浓度的模板和对应的CT值具有较好的线性关系,R2值为0.9988;该方法的特异性较好,BLASTN比对,检测引物对仅能匹配滑液支原体的特有基因VY93_RS02885,结果显示,该方法能特异性地检测滑液支原体,与其他常见的引起禽关节炎的细菌/支原体性病原无交叉反应;该方法的敏感性较好,100％阳性检测的靶标基因最小拷贝数为10;该方法的重复性较好,对3个不同浓度的模板进行组间和组内重复检测,变异系数≤1.1727％.此外,该方法对临床样品的检出率与传统检测方法相符.因此,本研究建立的针对滑液支原体特有基因的SYBR green qPCR检测方法具有用时短、特异性高、准确性高、敏感性高、可重复性强的优点,可为滑液支原体的快速诊断和流行病学调查提供技术手段.

Mycoplasma synoviae is an important pathogen of poultry, causing significant economic losses in this industry. Analysis of the unique genes and shared genes among different M. synoviae strains and among related species is helpful for studying the molecular pathogenesis of M. synoviae and provides valuable molecular diagnostic targets to facilitate the identification of M. synoviae species. We selected a total of 46 strains, including six M. synoviae strains, from 25 major animal (including avian) Mycoplasma species/subspecies that had complete genome sequences and annotation information published in GenBank, and used them for comparative genomic analysis. After analysis, 16 common genes were found in the 46 strains. Thirteen single-copy core genes and the 16s rRNA genes were used for genetic evolutionary analysis. M. synoviae was found to have a distant evolutionary relationship not only with other arthritis-causing mycoplasmas, but also with another major avian pathogen, Mycoplasma gallisepticum, that shares the major virulence factor vlhA with M. synoviae. Subsequently, six unique coding genes were identified as shared among these M. synoviae strains that are absent in other species with published genome sequences. Two of the genes were found to be located in the genetically stable regions of the genomes of M. synoviae and were determined to be present in all M. synoviae isolated strains (n = 20) and M. synoviae-positive clinical samples (n = 48) preserved in our laboratory. These two genes were used as molecular diagnostic targets for which SYBR green quantitative PCR detection methods were designed. The two quantitative PCR methods exhibited good reproducibility and high specificity when tested on positive plasmid controls and genomic DNA extracted from different M. synoviae strains, other major avian pathogenic bacteria/mycoplasmas, and low pathogenic Mycoplasma species. The detection limit for the two genes was 10 copies or less per reaction. The clinical sensitivity and specificity of the quantitative PCR methods were both 100% based on testing chicken hock joint samples with positive or negative M. synoviae infection. This research provides a foundation for the study of species-specific differences and molecular diagnosis of M. synoviae.

大肠杆菌在猪场是一个伺机而动的沉默杀手,得到机会就会毫不犹豫地出手.江苏省农业科学院兽医诊断检测中心在2020年对猪源大肠杆菌分离菌株的药敏试验结果显示:分离菌株对猪场常用药物阿莫西林和氟苯尼考完全耐药,而磷霉素和丁胺卡那的敏感率较高.在"禁抗"时代,对大肠杆菌病的防控是摆在猪场面前的一个重要挑战.

Mycoplasma synoviae (MS) infection causes infectious synovitis and arthritis with hyperplasia of synovial cells in the chicken joint. However, its mechanism is unknown. We used primary chicken synovial fibroblast (CSF) as the research object to study the role of MS in the proliferation of MS-infected CSF and determine the mechanisms involved. Using integrated transcriptomic and proteomic analyses of the interaction between CSF and MS, we screened a proliferation-regulated factor, serum amyloid A (SAA), that may regulate proliferation of MS-infected CSF. SAA appears to be associated with MS-induced CSF proliferation. To study the role of SAA in MS-induced CSF proliferation, a eukaryotic expression vector overexpressing SAA and a small interfering RNA (siRNA) targeting Saa were constructed to manipulate the expression of SAA. Cell proliferation and apoptosis were detected via cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), or terminal deoxyribonucleotidyl transferase-mediated dUTP nick-dnd labeling (TUNEL) assays, respectively. Western blot analysis was used to examine the protein expression level of SAA, cyclin E1, and cyclin-dependent kinase 2 (CDK2). In vitro, MS significantly promoted the proliferation of CSF and increased the production of SAA. Overexpression of SAA accelerated the proliferative ability of CSF, whereas knockdown of SAA depressed the proliferative ability of CSF. A TUNEL assay indicated that MS did not induce apoptosis. Silencing of SAA suppressed the expression of cyclin E1 and CDK2. These results suggest that MS may upregulate the expression of SAA, accelerate the cell cycle, and promote proliferation of CSF.

根据新型鸭呼肠孤病毒(Novel duck reovirus,NDRV)特有的非结构蛋白P18基因保守序列设计1对特异性引物,建立了NDRV荧光定量RT-PCR检测方法.用该方法和普通RT-PCR方法对239份临床样品进行检测,NDRV阳性检出率分别为23.85％(57/239)和17.57％(42/239),荧光定量RT-PCR检测方法的检出率明显高于普通RT-PCR检测方法.随机取5份荧光定量RT-PCR检测方法检测的阳性的样品用鸭胚进行病毒分离培养,经普通RT-PCR扩增和基因测序证明均为NDRV.用103.0ELD50剂量的NDRV-SY株人工感染14日龄雏鸭,不同时间采集泄殖腔肛拭子样品,通过荧光定量RT-PCR检测方法进行排毒情况监测,结果显示,雏鸭人工感染NDRV 48 h时可以从泄殖腔检测到病毒排出,持续时间可达20 d,并发现在病毒感染后的第5 d和第10 d出现2次排毒高峰.试验结果表明,建立的NDRV荧光定量RT-PCR检测方法具有良好的特异性和较高的敏感性,可用于NDRV感染的排毒监测以及临床感染的早期诊断和流行病学调查.

Here, we report the complete genome sequence of Mycoplasma synoviae HN01, a virulent epidemic strain isolated from a sick chicken with synovitis in Henan Province, China. HN01 is the Asian source of an M. synoviae strain that is completely sequenced, genome annotated, and published with relevant data.

Mycoplasma synoviae (MS) is an important avian pathogen causing considerable economic hardship in the poultry industry. A major inflammation caused by MS is synovitis that occurs in the synovial tendon sheath and joint synovium. However, the overall appearance of pathological changes in the tendon sheath and surrounding tissues caused by MS infection at the level of pathological tissue sections was poor. Studies on the role of MS and synovial sheath cells (SSCs) interaction in the development of synovitis have not been carried out. Through histopathological observation, our study found that a major MS-induced pathological change of the tendon sheath synovium was extensive scattered and focal inflammatory cell infiltration of the tendon sheath synovial layer. In vitro research experiments revealed that the CFU numbers of MS adherent and invading SSC, the levels of expression of various pattern recognition receptors, inflammatory cytokines, and chemokines coding genes, such as IL-1β, IL-6, IL-8, CCL-20, RANTES, MIP-1β, TLR7, and TLR15 in SSCs, and chemotaxis of macrophages were significantly increased when the multiplicity of infection (MOI) of MS to SSC were increased tenfold. The expression level of IL-12p40 in SSC was significantly higher when the MOIs of MS to SSC were increased by a factor of 100. The interaction between MS and SSC can activate macrophages, which was manifested by a significant increase in the expression of IL-1β, IL-6, IL-8, CCL-20, RANTES, MIP-1β, and CXCL-13. This study systematically demonstrated that the interaction of MS with chicken SSC contributes to the inflammatory response caused by the robust expression of related cytokines and macrophage chemotaxis. These findings are helpful in elucidating the molecular mechanism of MS-induced synovitis in chickens.

数字PCR是基于反应体系有限分割的原理,突破了荧光定量PCR以"荧光信号"的变化进行检测的方式,实现了"数字式"检测.在特异性、可重复性等方面实现了里程碑式的跨越,在生物研究、医学检测、环境食品安全监测等科技领域得到广泛认可和推广.在国家政策大力扶持下,中国数字PCR行业保持稳定增长,数据显示,2018年中国数字PCR行业市场规模达到26.3亿元,同比增长17.9％.但目前在兽医检测领域,数字PCR在国内仍处于萌芽阶段,从生物科技的发展经验看,数字PCR预计将成为兽医检测领域未来的主要发展方向,非洲猪瘟的肆虐将催化这一进程.

2019年,我国的养猪业虽然受到重创,但部分养猪科技新技术仍得到了一定的发展.如:养猪"智能+"、"铁桶"楼房养猪技术、无人养猪技术、非洲猪瘟感染猪场复养技术和数字PCR检测技术等.防控非洲猪瘟疫情告诉我们:在充满不确定的世界,杀不死你的,必使你更加强大.面对突如其来的意外,与其逃避,不如正面面对,把意外当做锻炼自己的机会,主动拥抱变化,适应变化,反而能茁壮成长和壮大.

2020年突如起来的新冠肺炎疫情使"核酸检测"进入了公众的视野,但在核酸检测中,部分病例在不同时间经2次以上的检测后结果由阴性转为阳性,这种现象的出现给临床诊断和疾病控制带来了极大的困扰.同样,在猪病的核酸检测中,假阴性问题亦普遍存在.新病毒的确认时间周期长、采集的待检样品不合格、核酸提取效果差是出现上述现象的主要因素.因地制宜地制定样品采集方案,试剂盒质量可靠,监督环节不缺失等,是输出合格检测结果的重要保障.

Duck hepatitis A virus type 3 (DHAV-3) is an important pathogen that causes substantial losses in the Chinese duck industry. DHAV-3 is highly fatal to ducklings and there is no licensed vaccine in China available to reduce DHAV-3 infection. Our goal was to develop a live attenuated vaccine candidate against DHAV-3. A field isolated strain, SD, was attenuated by serially passaging in specific-pathogen-free (SPF) chicken embryos, and it lost its pathogenicity after 40 passages. The 70th passaged strain (SD70), which achieved good growth capacity in chicken embryos with a viral titer of 107.5 ELD50/mL, was chosen to be the live attenuated vaccine candidate. The SD70 strain did not cause clinical signs of disease or mortality in 1-day-old ducklings and showed no virulence reversion after seven rounds of in vivo back passages. The minimum effective dose of SD70 was determined to be 102.5 ELD50 via the vaccination route of subcutaneous inoculation. A single dose of the SD70 provided good protection to susceptible ducklings against the lethal DHAV-3 strain. Compared with the genomic sequence of the parent SD strain, the SD70 had 12 amino acid substitutions, some of which may play a role in virulence attenuation. This study demonstrated that the attenuated SD70 strain is a promising vaccine candidate for the prevention of DHAV-3 infection in China. It exhibited safety, good stability and excellent protection.

Mycoplasma synoviae (MS), which causes respiratory disease, eggshell apex abnormalities, infectious synovitis, and arthritis in avian species, has become an economically detrimental poultry pathogen in recent years. In China, the disease is characterized by infectious synovitis and arthritis. However, the mechanism by which MS causes infectious synovitis and arthritis remains unknown. Increasing evidence suggests that synovial fibroblasts (SF) play a key role in the pathogenesis of arthritis. Here, both RNA sequencing and tandem mass tag analyses are utilized to compare the response of primary chicken SF (CSF) following infection with and without MS. The host response between non-infected and infected cells was remarkably different at both the mRNA and protein levels. In total, 2,347 differentially expressed genes (DEGs) (upregulated, n = 1,137; downregulated, n = 1,210) and 221 differentially expressed proteins (DEPs) (upregulated, n = 129; downregulated, n = 92) were detected in the infected group. A correlation analysis indicated a moderate positive correlation between the mRNA and protein level changes in MS-infected CSF. At both the transcriptomic and proteomic levels, 149 DEGs were identified; 88 genes were upregulated and 61 genes were downregulated in CSF. Additionally, part of these regulated genes and their protein products were grouped into seven categories: proliferation-related and apoptosis-related factors, inflammatory mediators, proangiogenic factors, antiangiogenic factors, matrix metalloproteinases, and other arthritis-related proteins. These proteins may be involved in the pathogenesis of MS-induced arthritis in chickens. To our knowledge, this is the first integrated analysis on the mechanism of CSF-MS interactions that combined transcriptomic and proteomic technologies. In this study, many key candidate genes and their protein products related to MS-induced infectious synovitis and arthritis were identified.

正确"归因"意义非凡,不少猪场在寻找事物发生的原因时,往往注重形式逻辑,而忽略实质逻辑;那些更深层,更隐蔽,更随机的因素很难被发现.很多时候"归因谬误"会出现与真实情况相距甚远的偏差.这种偏差的存在,是猪场管理中的最大障碍,也是很多猪场多走弯路的重要因素.

2019年7月至2020年6月,从江苏不同地区38个规模水禽(鸭、鹅)养殖场采集各类样品共1671份,分别进行了鸭疫里氏杆菌、巴氏杆菌、大肠杆菌等当前危害水禽生产的主要细菌性疾病病原的监测.结果表明,鸭疫里氏杆菌阳性率为36.01％,巴氏杆菌阳性率为24.43％,大肠杆菌阳性率为31.17％,沙门氏菌阳性率为12.82％,葡萄球菌阳性率为19.05％.根据监测结果结合江苏省内水禽养殖现状进行分析,为水禽细菌性疾病防控技术推广实施提供依据.