A multiplex liquid array assay based on Luminex xTAG technology was developed for the first time for the simultaneous detection of five respiratory bacterial pathogens in Specific Pathogen-Free (SPF) chickens—Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Pasteurella multocida (Pm), Avibacterium paragallinarum (Apg), and Mycobacterium avium (Mav)—filling the gap that these five pathogens could not be simultaneously detected previously. Specific primers were designed targeting conserved genes of each pathogen, with a TAG sequence attached to the 5′ end of each forward primer and biotin to the 5′ end of each reverse primer. After multiplex PCR amplification, the products were hybridized with MagPlex-TAG microspheres and analyzed using the Luminex system. The method was validated for sensitivity, specificity, and repeatability, and tested with simulated positive samples (a mixture of genomic DNA from the five target pathogens) and SPF chicken samples. The LOD was 10 copies/μL for Pm and 100 copies/μL for the other four pathogens. No cross-reactivity was observed with 18 non-target bacterial species. Intra- and Inter-assay coefficients of variation (CV) ranged from 0.8% to 12.2% and 2.3% to 14.4%, respectively, both below 15%. All simulated mixed positive samples yielded results consistent with expectations, and none of the 114 nasopharyngeal swab samples from SPF chickens tested positive for any of the five targets. The developed assay offers high throughput, high sensitivity, high specificity, and good repeatability, providing a robust tool for routine quality control in SPF chickens.

In the original publication [...].

Science and technological advancements drive human progress,with laboratory animals serving as essential resources for developments in life sciences and medicine.However,the waste generated by these animals presents new challenges for urban management.Issues such as classification,recycling,effective utilization,and biohazard elimination must be addressed,necessitating the development of regulations,standards,and norms to keep pace with advancements.The construction of quality management system is the foundation and framework for the management of inspection and testing organizations.It should have strong operability and inspectability,enabling continuous improvement of the management level and enhancing the stability of basic management.However,current quality management systems often lack clarity in managing laboratory animal waste,including undefined disposal processes for non-medical institutions,inaccurate waste classification,and inadequate disposal methods for different waste categories.This paper addresses these challenges by identifying necessary processes to be added or removed in the quality management system of National Institutes for Food and Drug Control,developing effective SOPs,proposing practical measures to strengthen supervision and management,and integrating 6S management principles into our quality management system.In conclusion,effective management of laboratory animal waste should be centered on improving the quality management system,emphasizing waste classification and management at the source,controlling biological hazards,minimizing environmental pollution and promoting conditions for sustainable development.

兔出血症2型是由兔出血症病毒2型引起的危害家兔和野兔的一种新型高度传染性、急性致死性疫病,该病2010年在法国首次被发现.2020年4月该病传入我国四川省后,迅速传播至我国多个省份,对我国养兔业的健康发展造成了严重危害.目前,国内外关于兔出血症2型的研究比较有限,而且国内没有商品化的疫苗用于该病的预防,因此应该加强对该病的重视程度,积极开展防控技术的研究.论文通过对兔出血症2型的流行情况、病原学及理化特性、流行病学、临床症状、剖检病变、诊断方法和防治措施等多个方面进行阐述,以期为科学预防和控制兔出血症2型提供参考.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains widely pandemic around the world. Animal models that are sensitive to the virus are therefore urgently needed to evaluate potential vaccines and antiviral agents; however, SARS-CoV-2 requires biosafety level 3 containment. To overcome this, we developed an animal model using the intranasal administration of SARS-CoV-2 pseudovirus. As the pseudovirus contains the firefly luciferase reporter gene, infected tissues and the viral load could be monitored by in vivo bioluminescent imaging. We used the model to evaluate the protective efficacy of monoclonal antibodies and the tissue tropism of different variants. The model may also be a useful tool for the safe and convenient preliminary evaluation of the protective efficacy of vaccine candidates against SARS-CoV-2, as well as the treatment efficacy of anti-viral drugs.

详细介绍了某机构A2型Ⅱ级生物安全柜使用现状,从检测人员的角度分析了高效过滤器完整性、照度、噪声、下降气流流速、流入气流流速、气流模式6项性能指标异常原因,并从选择供应商、安装位置、维保、人员培训、规范操作、报废年限等环节为安全柜管理和使用等人员提供了建议,有利于确保此类生物安全柜对实验人员、样品和周围环境的安全.

Chronic hepatitis E virus (HEV) infection is frequently reported in immunocompromised patients, but has also been increasingly reported in non-immunocompromised individuals. We characterized the course of chronic HEV infection in immunocompetent rabbits. In two independent experiments, 40 specific-pathogen-free rabbits were infected with a rabbit HEV genotype 3 strain in serial diluted titers (108 to 104 copies/mL). Serum and fecal samples were collected weekly and were tested for HEV RNA, antigen, anti-HEV and liver enzymes. Rabbits that spontaneously cleared the infection before 10 weeks post-inoculation (wpi) were kept to the end of the study as recovery control. Liver tissues were collected from HEV-infected rabbits at 5, 10 and 26 wpi for histopathological analysis. Nineteen rabbits (47.5%) developed chronic HEV infection with persistent viraemia and fecal HEV shedding for >6 months. Seroconversion to anti-HEV was observed in 84.2% (16/19) of the chronically infected rabbits. Serum levels of aminotransferase were persistently elevated in most of the rabbits. Characterizations of chronic HEV infection in immunocompetent settings could be recapitulated in rabbits, which can serve as a valuable tool for future studies on pathogenesis.

为了探索纳米孔测序技术在动物传染病病原快速诊断中的应用价值,采集1只发生腹泻实验兔的肝脏和脾脏进行核酸提取、建库和纳米孔测序分析,同时对该腹泻的实验兔分别进行细菌分离鉴定和腹泻相关病毒的血清学检测.结果显示:纳米孔测序方法运行3h后,获得1.9GB的数据量,检出756 521条序列,平均序列长度为2 549.9个碱基.根据对测序数据的生物信息学分析表明,引起实验兔腹泻的病原可能为大肠杆菌.对同批送检的6只实验兔采用常规细菌分离鉴定方法在实验兔的心脏血样品、肝脏、肺脏和回盲部内容物中只分离到了大肠杆菌.对腹泻相关病毒的血清学检测结果显示6只送检的兔均为兔轮状病毒阴性.本研究采用纳米孔测序技术对发生腹泻的实验兔进行诊断,其结果与常规实验室检测方法获得的结果相符合,表明纳米孔技术在动物传染病病原快速检测中具有潜在的应用价值.

Intensive efforts have been made to develop models of hRSV infection or disease using various animals. However, the limitations such as semi-permissiveness and short duration of infection have impeded their applications in both the pathogenesis of hRSV and therapeutics development. Here, we present a mouse model based on a Rag2 gene knockout using CRISPR/Cas9 technology. Rag2−/− mice sustained high viral loads upon intranasal inoculation with hRSV. The average peak titer rapidly reached 1 × 109.8 copies/g and 1c106 TCID50 in nasal cavity, as well as 1 × 108 copies/g and 1 × 105 TCID50 in the lungs up to 5 weeks. Mild interstitial pneumonia, severe bronchopneumonia, elevated cytokines and NK cells were seen in Rag2−/− mice. A humanized monoclonal antibody showed strong antiviral activity in this animal model, implying that Rag2−/− mice that support long-term stable infection are a useful tool for studying the transmission and pathogenesis of human RSV, as well as evaluating therapeutics.

为了比较不同种胶体金试纸条检测牛布鲁氏菌病的效果,本试验测定了 4种胶体金试纸条的敏感性、特异性和重复性,并比较了 4种胶体金试纸条、虎红平板凝集试验、试管凝集试验和竞争酶联免疫吸附试验(cELISA)在检测临床牛血清样品中的符合率.结果显示,4种胶体金试纸条检测牛血清的敏感性均高于虎红平板凝集试验;检测常见牛病的阳性血清时,4种胶体金试纸条均具有良好的特异性,无交叉反应;对已知阴性和阳性牛血清进行5次重复试验结果均一致,说明4种胶体金试纸条均表现良好的重复性;和cELISA相比,4种胶体金试纸条的符合率为85.09％～92.11％.结果表明,本试验中比较的4种胶体金试纸条均具有较高的敏感性、特异性和重复性,与cELISA符合率高,均适用于牛布鲁氏菌病的初筛.

洁净室对于生命科学或电子行业至关重要,其洁净度能否达到设计要求是衡量洁净室能否竣工验收的重要因素之一,也是影响洁净室功能的重要因素之一.现首先对影响洁净室洁净度的因素进行分析,随后针对非单向流生物洁净室检测中发现的共性问题,从设计、施工、管理、物品等方面对导致洁净度不合格的原因进行梳理,并指出洁净度不达标的原因及纠正对策,可以为非单向流生物洁净室合格运行提供一定的借鉴.

为建立一种布氏杆菌微滴数字PCR qPCR方法,对布氏杆菌病的定量诊断提供技术支持,在实时荧光PCR (qPCR)检测方法(T/CVMA 20-2020)的基础上,建立了布氏杆菌微滴数字PCR方法,并对方法的反应条件进行了优化,同时对其敏感性、特异性、重复性进行了评估.结果 显示:本方法的最低检测下限为2.6 copies/μL,未发现与常见的5种细菌存在交叉反应,重复性检测的变异系数小于5％;采用该微滴数字PCR和实时荧光PCR检测方法分别对76份流产牛的阴道拭子进行检测,显示该微滴数字PCR比qPCR可以多检出3份阳性样品.本研究建立的微滴数字PCR方法敏感性高、特异性强、重复性好,适用于临床样品的布氏杆菌核酸检测.

Vascular endothelial growth factor receptor 2 (VEGFR2)/KDR plays a critical role in tumor growth, diffusion, and invasion. The amino acid sequence homology of KDR between mouse and human in the VEGF ligand-binding domain was low, thus the WT mice could not be used to evaluate Abs against human KDR, and the lack of a suitable mouse model hindered both basic research and drug developments. Using the CRISPR/Cas9 technique, we successfully inserted different fragments of the human KDR coding sequence into the chromosomal mouse Kdr exon 4 locus to obtain an hKDR humanized mouse that can be used to evaluate the marketed Ab ramucirumab. In addition, the humanized mAb VEGFR-HK19 was developed, and a series of comparative assays with ramucirumab as the benchmark revealed that VEGFR-HK19 has higher affinity and superior antiproliferation activity. Moreover, VEGFR-HK19 selectively inhibited tumor growth in the hKDR mouse model but not in WT mice. The most important binding epitopes of VEGFR2-HK19 are D257, L313, and T315, located in the VEGF binding region. Therefore, the VEGFR2-HK19 Ab inhibits tumor growth by blocking VEGF-induced angiogenesis, inflammation, and promoting apoptosis. To our best knowledge, this novel humanized KDR mouse fills the gaps both in an animal model and the suitable in vivo evaluation method for developing antiangiogenesis therapies in the future, and the newly established humanized Ab is expected to be a drug candidate possibly benefitting tumor patients.

Hand-foot-and-mouth disease is a contagious disease common among children under 5 years old worldwide. It is caused by strains of enterovirus, especially EV-A71, which can lead to severe disease. Vaccines are the only way to fight this disease. Accordingly, it is necessary to establish an efficient and accurate methodology to evaluate vaccine efficacy in vivo. Here, we established a practical method using a hSCARB2 knock-in mouse model, which was susceptible to EV-A71 infection at 5–6 weeks of age, to directly determine the efficacy of vaccines. Unlike traditional approaches, one-week-old hSCARB2 mice were immunized twice with a licensed vaccine, with an interval of a week. The titre of antibodies was measured after 1 week. Mice at 4 weeks of age were challenged with EV-A71 intraperitoneally and intracranially, respectively. The unimmunized hSCARB2 mice displayed systemic clinical symptoms and succumbed to the disease at a rate of approximately 50%. High viral loads were detected in the lungs, brain, and muscles, accompanied by clear pathological changes. The expression of IL-1β, IL-13, IL-17, and TNF-α was significantly upregulated. By contrast, the immunized group was practically normal and indistinguishable from the control mice. These results indicate that the hSCARB2 knock-in mouse is susceptible to infection in adulthood, and the in vivo efficacy of EV-A71 vaccine could be directly evaluated in this mouse model. The method developed here may be used in the development of new vaccines against HFMD or quality control of licensed vaccines.

目的 不确定度是表征被测量的真值所处的量值范围,它是对测量结果受测量误差影响不确定程度的科学描述.本文介绍了实验动物设施噪声测量值的测量不确定度评定方法.方法 使用AWA5636-2声级计测定不同面积、不同采样点数量的噪声.采样点位置按GB 14925—2010要求设置,共测量5次,检测结果以检测区域噪声的最大数值进行报告.结果 不同检测要求下的扩展不确定度均为2.6 dB(A).结论 噪声的不确定度主要源于声级计最大允差和声级计分辨率,检测时应按照声级计和检测标准规定的方法使用声级计,以减小检测结果的测量不确定度.

目的 建立输血感染戊型肝炎病毒(hepatitis E virus,HEV)兔模型,了解HEV全血输注传播的可能性,并观察感染后的指标动态.方法 以兔HEV接种4只SPF兔,经腹腔静脉窦采集抗凝血,然后立即以约10 mL/kg经耳缘静脉注射输血方式分别给予正常SPF受体兔5只,每周监测受体兔粪便和血清抗原、抗体和核酸,连续13周.结果 静脉输血后,5只受体兔均出现粪便排毒,其中3只受体兔引起慢性戊肝,排毒时间超过13周,同时出现病毒血症和HEV抗原阳性,HEV抗体阴性或弱阳性;2只受体兔引起急性戊肝,排毒时间均小于6周,同时HEV抗体检测Sample/Cut-off(S/CO)值均大于13、HEV抗原阴性、无病毒血症.结论 通过输全血方式可以感染兔,并引起急性或慢性HEV感染.

为了建立一种鉴别布鲁菌S2疫苗株和其他种类布鲁菌的双重实时荧光PCR检测方法,通过查阅文献,分别设计了特异性扩增布鲁菌IS711插入序列和S2疫苗株缺失序列的2套引物探针,并对引物和探针浓度进行了优化,对其敏感性、特异性和重复性进行了评价,同时,使用该方法对不同类型的临床样品进行了检测.结果 表明,用该方法对2种阳性质粒标准品的最低检测限均为10 copies/μL;该方法能特异性鉴别S2疫苗株,其他非布鲁菌的细菌核酸均未有特异性的扩增曲线;批内和批间重复变异系数均小于3％,重复性良好.对112份临床样品的检测结果显示该方法的阳性样品检出率高于套式PCR检测方法,并可以成功对S2疫苗株的核酸进行鉴别.该研究建立的鉴别布鲁菌S2疫苗株的双重实时荧光PCR方法特异性好、灵敏度高、重复性好,具有较好的临床应用价值.

Hepatitis E virus (HEV) is zoonotic and the leading cause of acute viral hepatitis worldwide. Rabbit HEV can infect humans and is prevalent globally. It is reported that laboratory rabbits are also naturally infected with HEV. Therefore, it is important to investigate in a large scale the prevalence of HEV in laboratory rabbits. Serum samples were collected from 649 laboratory rabbits of 13 different commercial vendors in Beijing, China, from 2017 to 2019, and anti-HEV and HEV antigen (Ag) were tested. Fecal samples were collected from 50 laboratory rabbits from one of the vendors for HEV RNA detection. Six laboratory rabbits with natural HEV infection were euthanized and their liver, kidney, bile and urine samples were collected for HEV RNA quantification. Liver tissues were subjected to histopathology analysis. The overall positive rates of anti-HEV antibodies and HEV-Ag are 2.6% (15/588) and 7.9% (51/649), respectively. HEV RNA was detected in 12.0% (6/50) of the rabbits. High viral load of HEV RNA was detected in liver and bile samples. Liver inflammation was observed. HEV is circulating in laboratory rabbit population in China. Strict screening is crucial to ensure experimental accuracy and prevent zoonotic transmission to research personnel.

Inflammatory bowel disease (IBD), consisting of ulcerative colitis (UC) and Crohn's disease (CD), is a chronic relapsing and life-threatening inflammatory disorder that mainly affect the intestinal tract. The mainstream therapies for moderate to severe IBD lie in the use of immunosuppressive agents. However, it encountered the problem of drug tolerance and significant adverse events. Therefore, identifying novel signal pathways involved in IBD is necessary to satisfy the unmet treatment needs of IBD patients. There existed some hints between iron and IBD, and was reported that ferroptosis induced in UC. However, as another important subtype of IBD, whether ferroptosis also occurred in CD remains unclear. In this study, we found that the dysregulation of iron, lipid peroxidation and redox homeostasis were involved in CD; the administration of ferroptosis inhibitor Ferrostatin-1 could alleviate pathological phenotypes of TNBS induced CD-like colitis in mice. Our results provide a new hopeful therapeutic strategy in treating CD, especially for those who suffered from the tolerance of existing immunosuppressive agent drugs.

实验动物设施环境检测是设施建设验收及实验动物行政许可制度实施的组成部分,也是设施使用单位监测了解设施运行状况的重要手段.中国食品药品检定研究院作为实验动物设施环境检测业务提供单位,在获得大量的现场检测数据后,有必要尽快完成数据的计算和报告制作,以实现对客户在质量和效率上的承诺.本文介绍了一款由中检院设施环境检测团队基于VB编程设计的实验动物设施环境参数快速计算器的主要功能和关编程逻辑图.