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.

Cryptosporidium parvum (Cp), Strongyloides stercoralis (Ss), Trypanosoma cruzi (Tc), Theileria ovis (To) and Echinococcus granulosus (Eg) are significant in causing similar clinical symptoms in both humans and animals. These symptoms include diarrhea, fever, pneumonia, hepatitis, encephalitis, cardiovascular, and gastrointestinal diseases, as well as reproductive system diseases. Therefore, it is urgent to establish a rapid, specific, and sensitive method to simultaneously detect these five pathogens. Here, we develop a TaqMan probe-based pentaplex real-time fluorescence quantitative PCR (qPCR) for simultaneous detection of Cp, Ss, Tc, To, and Eg for the first time. Five sets of specific primers and probes were designed. The 5' end of five probes were labeled with five different fluorescent groups (FAM, VIC, ABY, JUN, CY5) that do not interfere with each other, while the 3' end were labeled with quenching groups (MGB-NFQ, QSY). The instrument collects fluorescence signals at different wavelengths in five independent optical channels in qPCR to achieve simultaneous specific amplification and quantitative detection of five targets within one tube. A five-color qPCR was developed, which can rapidly and quantitatively distinguish and identify Cp, Ss, Tc, To, and Eg in a single tube. This method shows strong specificity and no cross-reaction with other control pathogens. This method has high sensitivity, with the LLOQ of 10 copies and the LOD as low as 1 copy. The standard curve shows a good linear relationship. The amplification efficiency is high. The repeatability and reproducibility are excellent. The interference test shows that high concentrations of nucleic acids do not interfere with low concentrations of nucleic acids, and can ensure excellent detection results even in complex samples for 1.5 hours. A total of 246 clinical samples were detected: 4.88% were positive for Cp, 2.44% for Ss, and 15.85% for To, while 0.81% for co-infections of Cp/To. This TaqMan probe-based pentaplex qPCR enables rapid detection and differential identification of five pathogens from clinical samples, eliminating the need for multiple single-plex qPCR. It can potentially assist clinicians in diagnosis and treatment planning by rapidly detecting pathogens in clinical samples and further quantitative analysis. It is of extremely significant importance for protecting human health.

Pneumocystis is an opportunistic fungal pathogen that causes severe Pneumocystis pneumonia (PCP) in immunocompromised individuals and laboratory animals. Three host-specific species—Pneumocystis murina (P. murina), Pneumocystis carinii (P. carinii), and Pneumocystis jirovecii (P. jirovecii)—are closely associated with infections in humans and laboratory animals. However, the conventional method, microscopic staining, suffers from low sensitivity, operator-dependent subjectivity, and inability to differentiate species, highlighting the urgent need for a multiplex qPCR assay. In this study, we established a multiplex qPCR method targeting the mtLSUrRNA gene of P. murina, the TS gene of P. carinii, and the mtSSUrRNA gene of P. jirovecii. Primers and probes were designed and optimized using a matrix approach. The method was systematically evaluated for sensitivity, specificity, and reproducibility using recombinant plasmid standards and laboratory animal samples. Validation was performed on 260 mouse lung samples, 30 P. murina-positive samples, 25 rat lung samples, 6 rat bronchoalveolar lavage fluid (BALF) samples, and 8 P. carinii-positive samples. Results were compared with single-plex qPCR and staining microscopy (performed on 68 mouse lung samples, 38 Pneumocystis-positive samples). The limits of detection (LOD) were 5 copies/μL for P. murina, 6 copies/μL for P. carinii, and 8 copies/μL for P. jirovecii. Standard curves showed excellent linearity (R2 ≥ 0.999) with amplification efficiencies of 90–110%. No non-specific reactions were observed with 22 common pathogens, and intra-/inter-group coefficients of variation (CV%) were below 1%. Moreover, interference testing revealed minimal matrix effects on the amplification system and no mutual interference among the primers and probes. The multiplex qPCR detected all 38 positive samples (100%), showing 100% concordance with single-plex qPCR, whereas Giemsa staining detected none (0%) and toluidine blue staining only 60% (3/5) of the tested positives, suggesting that the multiplex qPCR achieved higher detection rates than staining microscopy. In conclusion, this novel multiplex qPCR method offers high sensitivity, specificity, and reproducibility, providing a sensitive and specific tool for laboratory animal health monitoring and epidemiological surveillance. Its clinical application for human PCP diagnosis requires further validation with authentic human specimens.

Helicobacter pylori ( H. pylori, Hp ) has been designated a class I carcinogen and is closely associated with severe gastric diseases. During colonization in the gastric mucosa, H. pylori develops immune escape by inducing host immune tolerance. The gastric epithelium acts as the first line of defense against H. pylori , with Toll-like receptors (TLRs) in gastric epithelial cells being sensitive to H. pylori components and subsequently activating the innate immune system. However, the mechanism of immune tolerance induced by H. pylori through the TLR signalling pathway has not been fully elucidated. In this research, we detected the expression of TLRs and inflammatory cytokines in GES-1 cells upon sustained exposure to H. pylori or H. pylori lysate from 1 to 30 generations and in Mongolian gerbils infected with H. pylori for 5 to 90 weeks. We found that the levels of TLR6 and inflammatory cytokines first increased and then dropped during the course of H. pylori treatment in vitro and in vivo. The restoration of TLR6 potentiated the expression of IL-1β and IL-8 in GES-1 cells, which recruited neutrophils and reduced the colonization of H. pylori in the gastric mucosa of gerbils. Mechanistically, we found that persistent infection with H. pylori reduces the sensitivity of TLR6 to bacterial components and regulates the expression of inflammatory cytokines in GES-1 cells through TLR6/JNK signaling. The TLR6 agonist obviously alleviated inflammation in vitro and in vivo. Promising results suggest that TLR6 may be a potential candidate immunotherapy drug for H. pylori infection.

ABSTRACT Helicobacter pylori is a microaerophilic Gram-negative bacterium that resides in the human stomach and is classified as a class I carcinogen for gastric cancer. Numerous studies have demonstrated that H. pylori infection plays a role in regulating the function of host cells, thereby contributing to the malignant transformation of these cells. However, H. pylori infection is a chronic process, and short-term cellular experiments may not provide a comprehensive understanding of the in vivo situation, especially when considering the lower oxygen levels in the human stomach. In this study, we aimed to investigate the mechanisms underlying gastric cell dysfunction after prolonged exposure to H. pylori under hypoxic conditions. We conducted a co-culture experiment using the gastric cell line GES-1 and H. pylori for 30 generations under intermittent hypoxic conditions. By closely monitoring cell proliferation, migration, invasion, autophagy, and apoptosis, we revealed that sustained H. pylori stimulation under hypoxic conditions significantly influences the function of GES-1 cells. This stimulation induces epithelial-mesenchymal transition and contributes to the propensity for malignant transformation of gastric cells. To confirm the in vitro results, we conducted an experiment involving Mongolian gerbils infected with H. pylori for 85 weeks. All the results strongly suggest that the Nod1 receptor signaling pathway plays a crucial role in H. pylori -related apoptosis and autophagy. In summary, continuous stimulation by H. pylori affects the functioning of gastric cells through the Nod1 receptor signaling pathway, increasing the likelihood of cell carcinogenesis. The presence of hypoxic conditions further exacerbates this process. IMPORTANCE Deciphering the collaborative effects of Helicobacter pylori infection on gastric epithelial cell function is key to unraveling the development mechanisms of gastric cancer. Prior research has solely examined the outcomes of short-term H. pylori stimulation on gastric epithelial cells under aerobic conditions, neglecting the bacterium’s nature as a microaerophilic organism that leads to cancer following prolonged stomach colonization. This study mimics a more genuine in vivo infection scenario by repeatedly exposing gastric epithelial cells to H. pylori under hypoxic conditions for up to 30 generations. The results show that chronic exposure to H. pylori in hypoxia substantially increases cell migration, invasion, and epithelial-mesenchymal transition, while suppressing autophagy and apoptosis. This highlights the significance of hypoxic conditions in intensifying the carcinogenic impact of H. pylori infection. By accurately replicating the in vivo gastric environment, this study enhances our comprehension of H. pylori ’s pathogenic mechanisms in gastric cancer.

目的 研究从腹泻实验兔分离到的大肠埃希菌的生物学特性.方法 对一株从因腹泻而死亡的 SPF 级实验兔体内分离的致病性大肠埃希菌的遗传谱系、抗生素敏感性、消毒液敏感性以及细菌携带的耐药基因和毒力基因进行较为全面的研究.结果 该大肠埃希菌属于 B1 型遗传谱系,携带毒力基因 eaeA,属于肠致病性大肠埃希菌.对常用的 5 种消毒液敏感,对于青霉素、氨苄西林、头孢氨苄、头孢拉定、四环素、多西环素、米诺霉素、红霉素、麦迪霉素、万古霉素、复方新诺明等 11 种抗生素耐药,携带 aadA1,gyrA,sul1 和 tetA 等 4 个抗生素耐药基因.结论 从因腹泻而死亡的 SPF级实验兔体内分离的致病性大肠埃希菌为一株 B1 型遗传谱系的肠致病性大肠埃希菌,具有多重耐药性,对常用的消毒剂均敏感.

Objective To analyze the status of proficiency testing samples in the field of laboratory animal testing, and to evaluate the role of the testing samples in the laboratory animal laboratory proficiency testing program.MethodsA retrospective study was conducted to compare the 30 proficiency testing plans and reports in the field of laboratory animal organized by National Institute for Food and Drug Control (NIFDC) from 2013 to 2022. The type, quantity, preparation method, homogeneity and stability of the proficiency testing samples, as well as the satisfaction of the participating units to the samples were analyzed. Then, the supply of new samples, the addition of quantitative projects, the improvement of stability research and the attention to customer needs were discussed.ResultsThe 30 proficiency testing programs included 10 items in the direction of bacteria, 10 items in the direction of virus and 10 items in the direction of genetics in the field of experimental animals. A total of 80 non-nucleic acid samples (including serums, feces, tracheal flushing fluid and proteins) and 4 nucleic acid samples were provided, all of which were qualitative samples. Compared with nucleic acid samples, non-nucleic acid samples always required large numbers of animals for sample preparation. The pass rate of homogeneity verification of the sample was 100%, but there is no quantitative study at present. Stability verification can be conducted for the short-term storage, transportation, freeze-thaw, different concentrations and terminal stability of different samples, but it lacks long-term stability verification.ConclusionIn the past ten years, the proficiency testing samples have basically met the basic requirements of the proficiency testing plan of laboratory animal testing area. However, the provision of nucleic acid samples should be reasonably increased; quantitative research samples and projects should be added; the influence of proficiency testing in the field of laboratory animal testing should be continuously expanded and maintain high-quality services.

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.

Identifying Helicobacter pylori (H. pylori, Hp) infection in animals before and after artificial infection influences the subsequent experiment. We established effective and noninvasive detection methods, including the gastric fluid nested polymerase chain reaction (PCR) method and the 13C-urea breath test, which can detect Hp before modeling Hp infection in Mongolian gerbils. We designed a gas collection equipment for gerbils. Hp nested PCR was also performed on gastric fluid, gastric mucosa, duodenal contents, and faeces of gerbils challenged with Hp. Conventional Hp detection methods, including rapid urease assay and immunohistochemistry, were compared. Moreover, we assessed the natural infection of Hp in 135 gerbils that had never been exposed to Hp artificially from the major laboratory gerbil groups in China. In 10 Hp infected gerbils, the positive detection results were 100%, 100%, 90%, and 10% in gastric fluid, gastric mucosa, duodenal contents, and faeces with nested PCR, respectively. A rapid urease test performed on gastric mucosa showed that all animals were infected with Hp. Immunohistochemical detection and bacteria culture of gastric mucosa samples that were positive by the nested PCR method also confirmed the presence of Hp. 9% (3/35) and 6% (2/31) natural infection rates were found in conventional gerbil groups from the Capital Medical University and Zhejiang Laboratory Animal Center. In conclusion, we established two noninvasive Hp detection methods that can be performed before modelingHp infection, including the gastric fluid nested PCR method and the 13C-urea breath test.

目的 通过实验动物小肠结肠炎耶尔森菌(Yersinia enterocolitica)检出能力验证,了解实验动物检测机构的检验能力,提高实验动物质量检测水平.方法 按照中国合格评定国家认可委员会(China National Accreditation Service for Conformity,CNAS)批准的能力验证方案,低温冷冻干燥制备样品;稳定性和均匀性检验合格后,通过CNAS平台随机分组编号,发放给参加单位,并附作业指导书.要求在规定时限提交检验报告和原始记录,其结果与样品设置一致的判为满意结果,不一致或未按时提交的判为不满意结果.结果 共有24个实验室参加本项能力验证;其中获得满意结果的实验室为22个,占总参加机构的91.7％;得到不满意结果的实验室有2个,占8.3％.结论 实验动物质量检测机构对小肠结肠炎耶尔森菌的检测能力较高.

ObjectiveTo promote the development of domestic laboratory animal detection proficiency testing activities and testing levels by assessing self testing capabilities.MethodsThe National Institutes for Food and Drug Control participated in the International Council for Laboratory Animal Science (ICLAS) laboratory animal detection proficiency test (i.e. Performance Evaluation Program for Diagnostic Laboratories, PEP) in 2013—2020. PEP pathogenic bacteria samples were tested by culture, biochemical identification, PCR, and sequencing. Finally, the results were analyzed and summarized.ResultsThere were 40 samples in seven international PEP activities, involving 27 kinds of pathogenic bacteria. The coincidence rates of the seven results were 8/8, 2/2, 5/8, 9/9, 5/5, 6/7, and 1/1, respectively, including 33 pathogen detection items and 7 antibody detection items. Nine pathogens were missing from the national standards: Bordetella hinzii, Serratia marcescens, Corynebacterium mastitis, CAR (Cilia-associated respiratory) bacillus, Actinobacillus muris, Citrobacter freundii, Klebsiella oxytoca, Streptococcus agalactiae, and pathogenic Escherichia coli.ConclusionThrough participating in PEP, we can identify the differences between domestic and foreign standards in laboratory animal detection and the testing capabilities that we need to improve, expecting to be in line with international standards as soon as possible.

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.

目的 评价基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)检测实验动物病原菌的效果.方法 使用德国Bruker Biotyper系统对40株标准菌株及384株实验动物分离菌进行鉴定.结果 Bruker Biotyper检测标准菌株和分离株的准确率分别为90.0％和80.0％.对鼠伤寒沙门氏菌(Salmonella typhimurium)、假结核耶尔森氏菌(Yersinia pseudotuberculosis)、志贺菌(Shigella spp.)、肺炎克雷伯杆菌(Klebsiella pneumoniae)、嗜肺巴斯德杆菌Heyl生物型(Rodentibacter heylii)和支气管鲍特杆菌(Bordetella bronchiseptica)的鉴定存在偏差.结论MALDI-TOF MS方法高效、准确率高,可用于实验动物病原菌的快速鉴定.

Helicobacter pylori is designated as a class I carcinogen of human gastric cancer following long-term infection. During this process, H. pylori bacteria persist in proliferation and death, and release bacterial components that come into contact with gastric epithelial cells and regulate host cell function. However, the impact of long-term exposure to H. pylori lysate on the pathological changes of gastric cells is not clear. In this study, we aimed to investigate the regulation and mechanisms involved in gastric cell dysfunction following continuous exposure to H. pylori lysate. We co-cultured gastric cell lines GES-1 and MKN-45 with H. pylori lysate for 30 generations, and we found that sustained exposure to H. pylori lysate inhibited GES-1 cell invasion, migration, autophagy, and apoptosis, while it did not inhibit MKN-45 cell invasion or migration. Furthermore, Mongolian gerbils infected with H. pylori ATCC 43504 strains for 90 weeks confirmed the in vitro results. The clinical and in vitro data indicated that sustained exposure to H. pylori lysate inhibited cell apoptosis and autophagy through the Nod1-NF-κB/MAPK-ERK/FOXO4 signaling pathway. In conclusion, sustained exposure to H. pylori lysate promoted proliferation of gastric epithelial cells and inhibited autophagy and apoptosis via Nod1-NF-κB/MAPK-ERK/FOXO4 signaling pathway. In the process of H. pylori-induced gastric lesions, H. pylori lysate plays as an "accomplice" to carcinogenesis.

目的 了解北京地区实验大鼠金黄色葡萄球菌的携带情况及分子特征,为实验大鼠金黄色葡萄球菌的防控提供基础信息.方法 按照GB/T 14926.14—2001实验动物金黄色葡萄球菌检测方法,对2008—2017年北京地区实验大鼠进行检测,并按年份和季节对检出情况进行分析;然后,对其中主要菌株进行MLST分型及分析.结果 2008—2017年在北京地区实验大鼠中检测到金黄色葡萄球菌165株,阳性率为8.44％;2013年达到金葡菌阳性峰值(39株,18.48％),而阳性率在季节间的差异不明显.MLST分型将148株金黄色葡萄球菌分为7个型别(ST8、ST3、ST1、ST6、ST2871、ST88及ST641),其中ST1占比最大(51.35％),其次是ST2871(30.41％),其余ST型别占比都低于10％;进一步分析发现其具有显著的时空分布特点,并存在进化相关性.结论 本研究为实验大鼠金黄色葡萄球菌的防控提供了基础信息.

目的 针对嗜肺巴斯德杆菌分类变更及其在实验动物中感染率高的问题,建立该菌的快速基因分型方法,为其检测方法的修订及有效控制提供支撑.方法 利用扩增片段长度多态性方法(AFLP),对实验动物中分离的314株嗜肺巴斯德杆菌及2株标准菌株进行遗传多态性及分子流行病学分析.结果 AFLP方法将受试分离株共分为了11个基因簇、190个基因型,多态性条带主要有31～36条,辛普森多样性指数0.992.基因簇AC7为北京地区的主要流行群.结论 本研究表明北京地区实验动物中的嗜肺巴斯德杆菌基因型丰富,2株标准菌株所在的基因簇在多态性上有明显区别.某些不同来源动物存在交叉污染、污染源多,以及长期污染的情况.

实验动物科学作为生命科学研究的支撑要素,需要有良好的动物质量做保证.动物质量的检测依靠完善、科学的检测标准.牛棒状杆菌作为引起裸鼠感染的一种重要病原菌,仍然缺少相关检测标准.本标准的制定,提供和规范了牛棒状杆菌的检测方法,有助于对此菌的及时发现与控制.

布鲁杆菌病是一种人畜共患的传染病,病原为布鲁杆菌,该菌是实验动物必需排除的微生物之一.随着时间的推移、条件的变化及技术的进步,原有检测方法和标准已明显不适用于检测现状,需要制定新的实验动物布鲁杆菌检测方法和标准.本文介绍实验动物布鲁杆菌PCR检测方法团体标准的编制背景、法律依据、内容编制、及未来展望等内容,以便于相关人员更好的理解该标准,更好地进行实验动物布鲁杆菌的检测.本团体标准的编制完成进一步完善了实验动物相关标准,为保障实验动物质量,适应我国实验动物国际化发展的需求提供了技术支持.

目的 开展2017年能力验证活动,评价国内实验动物检测实验室对支气管鲍特杆菌的检测能力.方法 依据CNAS相关文件,中国食品药品检定研究院作为能力验证提供者,制备含有支气管鲍特杆菌及产气巴斯德杆菌和大肠杆菌为干扰菌的比对样品,提供给参加实验室,要求在规定时限内反馈检测结果及报告.结果 所制备三种样品的平均含菌量均大于1× 108 CFU/mL,37℃加速稳定性时间不少于20 d,符合比对要求.全国共有18个省市的25个实验动物检测实验室参加,均按时反馈结果和报告,满意率88％.结论 本次能力验证为实验动物呼吸道细菌比对项目的进一步开展奠定了基础.大部分参加实验室具备一致可靠的支气管鲍特杆菌检测能力.

The natural antimicrobial resistance of Pseudomonas aeruginosa (P. aeruginosa) represents a serious therapeutic challenge to antibiotic therapy approaches in clinical caring. As a new non-antibiotic therapy approach, the bactericidal effect of 470 nm blue light on P. aeruginosa has been reported and investigated. However, the relationships between the bactericidal ratio and the irradiation doses have not been reported, and the bactericidal mechanism has not been explicated. In this paper, the bactericidal effects and the bactericidal mechanism of 470 nm blue LED irradiation on P. aeruginosa are investigated. P. aeruginosa suspensions are irradiated with blue LED (100 mW cm(-2)) with a different irradiation time, then the number of the surviving P. aeruginosa is measured and the bactericidal effect is calculated. The fluorescence and ultrastructure of P. aeruginosa are measured and compared after 470 nm blue light irradiation. The survival ratio of the animal model of a P. aeruginosa infection with 470 nm blue light irradiation is researched. The experiment results show that a bactericidal rate of 94% is achieved for P. aeruginosa suspension with an irradiation time of 240 min and an irradiation intensity of 100 mW cm(-2), and the bactericidal rate is higher for P. aeruginosa streaked on petri dish. The bactericidal effect of 470 nm blue light on P. aeruginosa is a photochemical reaction process, and the bactericidal effect increases with the increase of irradiation intensity and/or irradiation time. The survival rate of mice infected by P. aeruginosa can be effectively improved by using 470 nm blue light irradiation. The bactericidal rate of 470 nm blue light on P. aeruginosa depends on the irradiation doses, and the bactericidal effect of 470 nm blue light on P. aeruginosa is a photochemical reaction process. These results provide good reference values for the non-antibiotic bactericidal effect research of P. aeruginosa.