Bovine respiratory disease complex (BRDC), a multifactorial syndrome driven by viral and bacterial co-infections, poses significant challenges to cattle health in northern China. We performed a large-scale epidemiological and phylogenetic investigation (2022-2024) to identify BRDC pathogens in the region. A total of 5052 samples from symptomatic Holstein calves (nasal swabs, sera, tissues) were analysed using virological, bacteriological and molecular methods. Viral pathogens-bovine viral diarrhoea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus-3 (BPIV-3) and bovine herpesvirus-1 (BHV-1)-dominated infections, with pronounced seasonal peaks in colder months. The prevalence of non-cytopathic BVDV highlighted clinical difficulties. Bacterial infections involved Mycoplasma bovis (M. bovis), Mannheimia haemolytica and Pasteurella multocida, with M. bovis prevalent in mixed infections. Phylogenetic analysis revealed biological connections between Chinese isolates and international strains (e.g., BVDV subgenotypes 1a/1d and BPIV-3c), showing global pathogen flow. Transmission electron microscopy revealed the presence of viral popular structure. Meanwhile, seasonality strongly influenced viral dynamics, while bacterial detection remained stable, involving environmental and management factors. Pathogen co-infections demonstrate the complexity of BRDC. This study provides the first comprehensive BRDC pathogen profile for northern China, emphasising the need for region-specific control strategies, including multipathogen vaccines and improved biosecurity.

In recent years, bovine viral diarrhea virus (BVDV) has been associated with increased respiratory and gastrointestinal diseases in cattle. Comprehensive monitoring and investigations into the virus's pathological features are crucial for developing effective prevention strategies. This study investigated BVDV prevalence and pathogenicity in farms undergoing elimination protocols, with a focus on characterizing a novel Cytopathic Bovine Viral Diarrhea Virus (CP-type BVDV) strain (HH839) isolated from a symptomatic calf in Hohhot, Inner Mongolia. During 2021 and 2022, 103 bovine samples were screened for BVDV via nucleic acid detection. Positive cases underwent viral isolation using MDBK cells. The HH839 strain was analyzed for cytopathic effects, ultrastructure (electron microscopy), antigenicity (serum neutralization), and genetic lineage (whole genome sequencing). Pathogenicity of Cytopathic Bovine Viral Diarrhea Virus (CP-type BVDV) infected group, Noncytopathic Bovine Viral Diarrhea Virus (NCP-type BVDV) infected group, and the mixed-infection group of CP-type and NCP-type BVDV was evaluated in New Zealand White rabbits, with viral distribution and histopathological damage assessed in multiple organs. We identified 33 positive BVDV nucleic acid cases, resulting in a positivity rate of 32.04%. Five strains of NCP-type BVDV were isolated, yielding a 15.15% separation rate, alongside one strain of CP-type BVDV with a separation rate of 3.03%. The CP strain HH839 was isolated from a severely symptomatic calf in Hohhot, Inner Mongolia. The HH839 strain demonstrated significant cytopathic effects in MDBK cells, including cellular crumpling and syncytia formation, with a concentration of 5.23 log10TCID50/0.1 mL. Electron microscopy revealed a spherical morphology with a diameter of 40-60 nm. Genetic analysis indicated a close relationship with the BVDV FBS-D8 strain from the BVDV-1d subtype. Pathogenicity trials showed slight fever and minor body weight loss in infected subjects, with BVDV detected in the trachea, lungs, spleen, and small intestines, predominantly in the spleen. The isolation of HH839, a pathogenic CP-type BVDV-1d strain, underscores the coexistence of multiple BVDV biotypes in regional cattle populations. Enhanced pathogenicity observed in mixed infections highlights complex viral interactions. These findings emphasize the necessity for sustained surveillance and biotype-specific control strategies to mitigate BVDV-associated economic losses in livestock industries.

In this study, we sought to evaluate the prevalence of bacterial pathogens of mastitis in dairy cattle in the Inner Mongolia Autonomous Region, China. The study was conducted from 2015 to 2024 using a total of 12,053 clinical mastitis (CM) and sub-clinical mastitis (SCM) samples. The pathogens were isolated and identified by standard bacteriological and mycological methods. The most common pathogens isolated were Escherichia coli (13.82%), Staphylococcus aureus (10.28%), Klebsiella spp. (8.96%), Streptococcus agalactiae (7.45%), Streptococcus uberis (6.60%), coagulase-negative staphylococci (5.84%), and Streptococcus dysgalactiae (4.21%). From 2015 to 2017, the primary pathogens responsible for causing mastitis in cows were Staphylococcus aureus and Streptococcus agalactiae. In 2018, the most frequently isolated pathogen was Staphylococcus aureus. Notably, the isolation rate of Escherichia coli increased from 12.31% to 21.72%, and the isolation rate of Klebsiella spp. increased from 7.52% to 14.01% from 2019–2024. Mycoplasma was only detected in clinical mastitis cases, with a separation rate as high as 6.95%. In summary, the isolation rate of environmental pathogens is gradually increasing, while that of contagious pathogens has been continuously declining. This indicates that the current prevention strategies for infectious pathogens are effective. As a next step, it will be important to develop new strategies specifically targeting environmental pathogenic microorganisms.

The neonatal Fc receptor (FcRn) binds to IgG CH2 and CH3 domains (the Fc segment), triggering transendocytosis. Therefore, FcRn transports biological agents across the mucosal barrier. Mucosal administration provides less stimulation to the body than other methods. However, whether FcRn is an effective carrier for antigens across bovine respiratory epithelial cells is unknown. Here, an antigen was fused with the Fc fragment and transferred through the mucosal barrier to antigen-presenting cells via active transport mediated by FcRn. We established a model of FcRn-mediated recombinant IgG Fc protein expression in bovine embryonic tracheal epithelial cells. Western blotting showed that SPA inhibited the relative transport amount of FcRn-mediated IgG Fc fusion protein. Fc fusion protein positively correlated with protein concentration and action time, with the maximum level reached at 1.4 mg/mL (protein concentration) and 18 h (action time). An FcRn-mediated transport model of the IgG Fc recombinant protein in guinea pig lungs was established, and the amount of protein transported at different time points was measured using immunohistochemistry. FcRn mediates vaccine antigen delivery through the mucosal barrier to activate immune cells in the lamina propria, laying a theoretical foundation for the clinical application of nasal mucosal immune vaccines.

绵羊肺炎支原体(Mo)NM2010株的P120蛋白与猪肺炎支原体(Mhp)的黏附素P110高度同源,同属于P110/LppT黏附素N端结构域家族.为了筛选用于研制广谱绵羊支原体肺炎疫苗的保护性抗原,本试验利用生物信息学方法对Mo NM2010株P120蛋白的保守区段、结构、B细胞抗原表位进行预测分析,并对P120基因的N端和C端2个保守区基因片段进行克隆、表达、可溶性分析和抗原特性分析;2个表达产物分别对兔进行免疫试验,用ELISA方法检测免疫兔血清抗体效价和IL-2、IL-4、IL-10和IFN-γ细胞因子浓度.结果显示,P120蛋白N端有信号肽和1个跨膜螺旋,亚细胞定位在外膜,可能是一种外膜蛋白,P120蛋白N端和C端2个保守区段均有较多B细胞线性抗原表位,具有较好的抗原性;经序列测定证实,合成的P120-N和P120-C2个基因片段的基因序列完全正确,长度分别为1 509 bp和741 bp,表达的重组蛋白分子质量分别为58.1 kDa和29.5 kDa;P120蛋白N端以可溶和包涵体2种形式存在,C端以包涵体形式存在,均具有良好的抗原性;新西兰大白兔经4次免疫后,重组蛋白P120-N组和重组蛋白P120-C组兔血清中均产生了特异性抗体IgG,效价分别为1∶512 000和1∶128 000;与阴性对照组相比,重组蛋白P120-N组和重组蛋白P120-C组兔血清中IL-2、IL-4、IL-10和IFN-γ浓度均极显著升高(P＜0.01).结果表明,重组蛋白P120-N和P120-C可以诱导试验动物机体的体液免疫反应,P110/LppT黏附素家族保守区可以作为广谱绵羊支原体肺炎疫苗的候选蛋白.

Abstract Background In recent years, outbreaks of respiratory and gastrointestinal diseases in cattle is associated with bovine viral diarrhea virus (BVDV). BVDV infection is a persistent global issue, particularly concerning for calves health. Comprehensive epidemiological surveillance and research into its pathogenic characteristics are essential for effective control and prevention of this virus. Despite limited investigation into the epidemiology of BVDV in Inner Mongolia, there is a lack of comprehensive information in this area. Results From 2021 to 2022, within cattle farms implementing eradication plans for bovine viral diarrhea virus (BVDV), we detected 33 cases of BVDV nucleic acid positivity out of 103 samples (positive rate 32.04%). We isolated five strains of NCP-type BVDV (separation rate 15.15%) and one strain of CP-type BVDV (separation rate 3.03%). This CP BVDV strain was successfully isolated from the nasal swab of a calf with severe clinical symptoms in Hohhot, Inner Mongolia. Subsequently, we designated this strain as HH839 in this study. Non-cytopathic BVDV strains are common and participate in severe clinical acute infections, whereas cytopathic BVDV strains are rare and usually associated with mucosal disease outbreaks. Therefore, this study determined the biological characteristics of CP BVDV 1d strain HH839 using various assays, including cytopathic conditions, electron microscopy, serum neutralization test, whole-genome sequencing, sequence alignment, and replication kinetics. A cytopathic virus strain was isolated, and viral particles were observed within 40–60 nm using transmission electron microscopy. The growth curve showed that the virus propagated effectively in MDBK cells. Phylogenetic analyses based on the 5’untranslated region (5’UTR) and full-length genome sequences indicated that HH839 belonged to the BVDV-1d group and that the strain was in the same branch and had the closest genetic relationship with BJ-1308 (KT951841.1). New Zealand white rabbits were used to study the pathogenicity of different biotypes of BVDV, including the CP-type BVDV-infected group, NCP-type BVDV-infected group, and CP-type NCP-type BVDV mixed-infected group. The animal infection experiments showed that hematology and histopathology showed different degrees of changes, although the infected rabbits did not show typical disease symptoms. Conclusion We isolated a BVDV HH839 strain that can cause severe clinical symptoms in cattle. Genome-wide determination and infection experiments with HH839 provide material to further explore the regional prevalence of BVDV. We propose to reinforce the epidemiological surveillance of this virus.

[目的]验证牛副流感病毒3型(Bovine parainfluenza virus type 3,BOIV3)NP蛋白对BPIV3灭活疫苗的免疫效果是否有增强作用.[方法]利用生物信息学软件对NP基因编码的蛋白进行抗原性分析,筛选出抗原性区域,采用PCR方法扩增BPIV3截短的NP基因序列,连接至pET-32a(+)质粒上,通过大肠杆菌原核表达系统和Ni亲和层析的方法获得了较高纯度的BPIV3 NP蛋白,经Western blotting验证其反应原性.将BPIV3用0.3％甲醛灭活后与弗氏佐剂1 ∶1混合制备灭活疫苗.8只新西兰白兔随机分为4组:灭活疫苗组、NP蛋白组、灭活疫苗和NP蛋白混合组及对照组,每组2只.免疫前及免疫后每7 d采集血液分离血清,采用间接ELISA方法和病毒中和试验测定并比较4组新西兰白兔特异性抗体和中和抗体的水平.[结果]经DNAStar分析,NP蛋白第193-368位氨基酸处的平均抗原指数在0.4～1.7之间,亲水指数为0～1.5,证明该区域抗原性和亲水性较强;通过PCR扩增得到NP基因,并构建重组表达载体,通过基因测序证明该重组表达载体与预期结果一致;SDS-PAGE结果显示,NP蛋白表达量较高,蛋白分子质量为50 ku且以包涵体形式表达;Western blotting结果表明,所表达的蛋白具有较强的反应原性;ELISA结果显示,在免疫后28 d,对照组的特异性抗体效价为0,灭活疫苗组、NP蛋白组及灭活疫苗和NP蛋白混合组的特异性抗体效价分别达到1 ∶211、1 ∶217和1 ∶218.病毒中和试验结果显示,在免疫后28 d,对照组中和抗体效价为0,灭活疫苗组、NP蛋白组及灭活疫苗和NP蛋白混合组的中和抗体效价分别为1∶23.32、1 ∶24.48和1 ∶24.98.[结论]BPIV3 NP蛋白可增强BPIV3灭活疫苗的免疫效果,在灭活疫苗中加入NP蛋白可作为BPIV3灭活疫苗的新型接种方法.

为探究奶牛乳房炎源性肺炎克雷伯菌(K.pn)黏附素MrkD蛋白对K.pn黏附奶牛乳腺上皮细胞的影响以及黏附素抗血清对阻断K.pn黏附奶牛乳腺上皮细胞的效果,本研究利用PCR扩增了K.pn MrkD基因,构建重组质粒pColdI-MrkD,将其转化至大肠杆菌后诱导表达,采用SDS-PAGE和western blot鉴定.结果显示,MrkD重组蛋白以可溶性形式表达,并具有较好的反应原性.将牛乳腺上皮细胞与1×108 cfu/mL K.pn稀释液共同孵育后,利用间接免疫荧光试验检测K.pn对奶牛乳腺上皮细胞的黏附,结果显示K.pn可以黏附奶牛乳腺上皮细胞.利用K.pn稀释液孵育经不同浓度MrkD重组蛋白作用的奶牛乳腺上皮细胞后,通过对黏附细菌数的统计和分析,结果表明MrkD重组蛋白可以显著抑制K.pn黏附奶牛乳腺上皮细胞.利用MrkD重组蛋白抗血清和K.pn抗血清分别与K.pn作用后计数黏附的奶牛乳腺上皮细胞,结果显示,MrkD重组蛋白抗血清与K.pn抗血清均能有效阻断K.pn的黏附,且重组蛋白抗血清效果较好.本研究获得了MrkD重组蛋白,首次检测了其在K.pn黏附奶牛乳腺上皮细胞中的影响以及该重组蛋白抗血清在阻断K.pn黏附细胞中的作用,为探究阻断细菌黏附以及奶牛乳房炎亚单位疫苗的研究奠定基础.

[目的]研究牛病毒性腹泻病毒(BVDV)感染对新西兰白兔的致病性以及BVDV E2重组蛋白的免疫效果.[方法]将实验室培养保存的BVDV病毒纯化并按照Reed-Muench法测定其病毒滴度.在致病性试验中,将10只新西兰白兔随机分为感染组和对照组,每组5只.感染组用1 mL纯化的BVDV病毒攻毒(滴鼻500 μL、耳缘静脉注射500 4L),对照组用等体积的生理盐水处理,连续3 d,每天1次,每天观察各组兔的临床症状并测量体温;分别于接种病毒后第6、9、12、15、17天通过耳缘静脉采集血液检测血常规;感染病毒第17天采集鼻拭子进行RT-PCR鉴定,采集后剖杀并采集气管、肺脏、脾脏和小肠组织,制备病理切片观察病理变化.在免疫效果评价试验中,将10只新西兰白兔随机分为免疫组和对照组,每组5只,免疫组用E2重组蛋白(1 mg/只)与佐剂混合后经肌内多点注射免疫新西兰白兔,对照组接种等体积生理盐水;共免疫2次,2次免疫间隔为14 d.在一免后0、7、14、21、28 d采集血清,通过间接ELISA方法检测血清中抗重组蛋白特异性抗体水平;在一免后第28天按致病性试验中方法攻毒,在攻毒第17天采集鼻拭子进行RT-PCR鉴定,采集气管、肺脏、脾脏和小肠组织制备病理切片观察病理变化及免疫组织化学检测.[结果]纯化后BVDV的病毒滴度为4.16×106 TCID50/mL.与对照组相比,感染组部分新西兰白兔6 d内活动减少,采食略微减少,6 d后逐渐恢复正常,在感染第13天出现腹泻症状,从第5天开始体温略微升高,但均在正常范围内波动.与对照组相比,在攻毒第6和9天,感染组白细胞和血小板分别显著和极显著降低(P＜0.05;P＜0.01);在攻毒第12、15和17天,感染组白细胞、血小板和淋巴细胞均极显著降低(P＜0.01).鼻拭子RT-PCR检测为阳性,气管、肺脏、脾脏及小肠组织表现出轻度至重度的组织病理学变化.间接ELISA检测结果表明,在一免后7 d时,血清抗体滴度为1:16～1 ∶ 32;在一免后28 d时,血清抗体滴度为1 ∶ 256～1 ∶ 512;免疫攻毒组新西兰白兔鼻拭子经RT-PCR检测为阴性;组织病理学观察显示,免疫攻毒组气管及肺脏表现出轻微的组织病理学变化.免疫组化检测结果显示,免疫组结果均呈阴性,对照组结果均为阳性.[结论]通过滴鼻及耳缘静脉注射BVDV的方式可以构建新西兰白兔致病模型,BVDV E2亚单位疫苗能够刺激机体产生特异性抗体,起到免疫防御的作用.

本研究以牛传染性鼻气管炎gE基因的原核表达产物为抗原建立检测IBRV抗体间接ELISA检测方法.通过DANStar比对分析牛传染性鼻气管炎病毒gE蛋白序列,通过亲和层析对重组表达产物进行纯化,经Western blot检测证明重组表达产物能被IBRV标准性鼻气管炎gE蛋白长度为500 bp的特异性肽序列,随后构建了pCold-TF-gE原核表达体系,并用Ni-IDA进行纯化.以纯化鉴定后的表达产物为抗原(1μg/mL)包被酶标板制备多克隆抗体血清,并建立检测牛传染性鼻气管炎血清抗体间接gE-ELISA方法.结果 显示:阳性OD450值为0.369,组内组间重复性小于5％,敏感性强,与口蹄疫等阳性血清均无交叉反应且特异性达95％.利用建立的gE-ELISA方法,同时和IDEXX (IBR)抗体检测试剂盒对200份临床阳性血清进行检测比较,符合率均达到95％.该方法特异敏感高效,可用于牛传染性鼻气管炎血清抗体的检测.

为验证绵羊肺炎支原体(M.ovi)或脂质相关膜蛋白(LAMPs)是否通过Toll样受体2(TLR2)诱导小鼠腹腔巨噬细胞TNF-α的表达,本研究利用不同浓度的M.ovi或LAMPs刺激C57BL/6J WT小鼠及Tlr2-/-基因缺失小鼠的腹腔巨噬细胞12h后,利用荧光定量PCR(qPCR)检测Tlr2和TNF-α基因的转录水平.结果显示,C57BL/6J WT小鼠巨噬细胞中Tlr2和TNF-α基因的转录水平均不同程度上升,而Tlr2-/-基因缺失小鼠巨噬细胞TNF-α基因的转录水平明显受到抑制.进一步以终浓度为1×107cfu/mL M.ovi或8 μg/mL LAMPs分别刺激C57BL/6J WT小鼠和Tlr2-/-基因缺失小鼠的巨噬细胞,不同时间后利用qPCR检测Tlr2和TNF-α基因的转录水平;利用流式细胞术检测C57BL/6J WT小 鼠巨噬细胞中TLR2蛋白的表达水平;采用ELISA方法检测两种小鼠巨噬细胞上清液中TNF-α 的分泌水平;利用western blot检测细胞中MAPK信号通路的激活.qPCR结果显示,以M.ovi或LAMPs经不同时间刺激的C57BL/6J WT小鼠巨噬细胞中Tlr2和TNF-α基因的转录水平均不同程度上升,而Tlr2-/-基因缺失小鼠巨噬细胞中TNF-α基因的转录水平均明显受到抑制;流式细胞术检测结果显示,C57BL/6J WT小鼠巨噬细胞在受到M.ovi或LAMPs刺激后,TLR2蛋白的表达水平均不同程度上升;ELISA结果显示,C57BL/6J WT小鼠巨噬细胞在受到M.ovi或LAMPs刺激后,TNF-α蛋白的表达水平均不同程度上升,而Tlr2-/-基因缺失小鼠巨噬细胞中TLR2蛋白的表达则明显受到抑制;Western blot结果显示,C57BL/6J WT小鼠巨噬细胞经M.ovi或LAMPs刺激后MAPK信号通路中各信号蛋白(Erk、p38、JNK)均发生磷酸化反应,而Tlr2-/-基因缺失小鼠巨噬细胞在受到这两种物质刺激后MAPK信号通路明显受到抑制.上述结果表明,M.ovi及其LAMPs可通过小鼠腹腔巨噬细胞的TLR2激活MAPK信号通路,继而引起细胞因子TNF-α的分泌增多.本研究首次证实M.ovi及其LAMPs是通过TLR2及MAPK信号通路引起小鼠腹腔巨噬细胞TNF-α的分泌水平发生变化,为进一步明确巨噬细胞参与抗M.ovi天然免疫应答的机制奠定基础.

本试验克隆、表达了牛病毒性腹泻病毒(BVDV)的E2蛋白,以表达纯化后的E2蛋白作为包被抗原,通过优化反应条件建立了间接ELISA检测法.确定该方法的阴阳判定临界值为0.33,抗原包被浓度为8μg/mL,一抗的最佳稀释度为1:100,10％PEG4000为最佳封闭液,酶标二抗最佳稀释度为1:6000.用建立的ELISA检测方法和IDEXX ELISA抗体检测试剂盒检测180份血清临床样品,总符合率为95％.该方法的建立为进一步研制BVDV ELISA检测试剂盒奠定了基础.

本研究旨在获得重组牛呼吸道合胞体病毒(BRSV)G蛋白并对其进行反应原性鉴定.从NCBI上找出G基因的核苷酸序列,分析其抗原区域,利用Primer Premier 5.0软件设计1对特异性引物,利用RT-PCR方法扩增BRSVG基因片段,将目的片段连接到克隆载体pMD19-T后,对其进行双酶切鉴定和测序.将双酶切后的目的片段进行胶回收,构建重组质粒pET-32a-G,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,用Ni-IDA亲和层析法纯化经IPTG诱导表达的蛋白,并测定其浓度;通过SDS-PAGE和Western blotting方法对该蛋白进行分析与鉴定.结果显示,本试验成功克隆出大小约为567 bp的G基因,获得分子质量约为40 ku的可溶性重组蛋白,优化诱导条件后用SDS-PAGE对表达产物进行鉴定,在16℃、IPTG浓度1.2 mmol/L、诱导4 h时蛋白表达量最大;通过Western blotting检测发现,重组蛋白可与山羊源BRSV标准阳性血清发生特异性反应,说明该蛋白具有反应原性.综上,本研究成功表达并纯化了BRSV G蛋白,为建立BRSV抗体间接ELISA检测方法和BRSV亚单位疫苗的研制奠定了基础.

为探究由IgG FcRn介导奶牛乳房炎无乳链球菌（S. agalactiae,Sgc）和金黄色葡萄球菌（Staphylococcus aureus,Sau）保护性抗原亚单位疫苗的免疫效果,在本实验室前期研究基础上,本研究按体积11的比例将Sgc的Fc-Sip和Sau的Fc-FnBPB-ClfA两种蛋白分别与1%的盐酸左旋咪唑（1%LH）充分混合,制备两种亚单位疫苗。将即将干奶（妊娠220 d）、CMT试验为"++"以上的奶牛随机分为4组,分别将Fc-Sip+Fc-FnBPB-ClfA二联亚单位疫苗（A组）、Fc-Sip亚单位疫苗（B组）、Fc-FnBPB-ClfA亚单位疫苗（C组）通过首次肌肉免疫和二次乳头管灌注免疫方式进行免疫试验。通过免疫前后对乳样细菌分离、体细胞数测定及免疫血清抗体滴度测定,评价上述亚单位疫苗的免疫效果。结果显示,免疫前免疫组（A、B、C）奶牛的乳样细菌分离率分别为80%、90%、80%,其中Sgc检出率分别为20%、30%、10%,Sau检出率分别为30%、20%、10%;首免90 d后3个免疫组奶牛乳样细菌分离率分别降至20%、20%、40%,其中Sgc的检出率分别降至0、0、10%,Sau的检出率分别降至10%、10%、0,对照组免疫前后以上各项检测无明显变化。使用利拉伐牛奶体细胞检测仪检测免疫前后试验奶牛乳样中的体细胞数,免疫前免疫组（A、B、C）奶牛体细胞数均值分别为6.19×105个/mL、5.16×105个/mL、5.49×105个/mL,首免90 d后3个免疫组奶牛乳样中体细胞数均值分别为2.05×105个/mL、2.04×105个/mL、2. 52×105个/mL,而对照组奶牛乳样体细胞数与免疫前无明显变化。检测首免后0、7 d、14 d、28 d、60 d、90 d的血清抗体滴度,结果显示,首免后7 d,免疫组（A、B、C）80%的奶牛血清抗体滴度为11 024;第14 d后,免疫组（A、B、C）90%奶牛血清抗体滴度达到12 048;第28 d后,免疫组（A、B、C）80%奶牛血清抗体滴度在14 096～18 192,比对照组平均上升3～4个抗体滴度,对照组奶牛血清抗体滴度一直保持在1256～1512。综合分析以上试验结果显示免疫A组（Fc-Sip+Fc-FnBPB-ClfA）综合免疫效果在各免疫组中最佳。本研究结果表明Fc-Sip+Fc-FnBPB-ClfA二联亚单位疫苗对无乳链球菌和金黄色葡萄球菌性奶牛乳房炎具有较好的治疗和预防作用,为奶牛隐形乳房炎的防治提供了实验依据。

Mycoplasma bovis is an important pathogenic bacterium affecting cows and cattle. Clinically, an inactivated vaccine of M. bovis is mainly used to prevent infection by this bacterium. The changes that occur in the antigen when M. bovis is continuously passaged in vitro remain unknown. Therefore, we performed an in vitro serial passage of the M. bovis NM-28 strain, which was isolated and identified in our laboratory. An isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics method was used to analyse the differences between generations 3 and 60. Many major membrane proteins or protective antigens reported in the literature did not exhibit changes between these generations. We found an imbalance between growth rate and nutrition in the 60th generation. The proteomics results were verified by western blotting and real-time PCR. Growth curves were also prepared based on colony-forming units (CFUs) between the 3rd and 60th generations. The number of colonies in the 60th generation in the stationary phase was 5 x 10(9) CFU mL(-1), which was 10-fold higher than that in the 3rd generation. The 60th generation of the NM-28 strain can be used as an inactivated vaccine strain of M. bovis to lower production costs compared to use of the 3rd generation. (C) 2019 Elsevier Ltd. All rights reserved.

为证明TLR2是否参与绵羊肺炎支原体(Mo)或LAMPs刺激肺泡巨噬细胞时细胞因子的表达,使用Mo或LAMPs刺激绵羊肺泡巨噬细胞后,利用荧光定量PCR、流式细胞术、Western blot以及ELISA等方法分别对TLR2转录和翻译水平、MAPK信号通路激活情况和IL-1β分泌情况进行检测.同时,使用TLR2抑制抗体及MAPK抑制剂后,利用荧光定量PCR、Western blot以及ELISA检测MAPK信号通路激活情况和IL-1β分泌情况.结果 显示,绵羊肺泡巨噬细胞受Mo或LAMPs刺激后,TLR2在基因和蛋白表达水平均有不同程度上升,MAPK信号通路中各信号蛋白均发生磷酸化反应,IL-1β在基因和蛋白表达水平均有不同程度上升.对TLR2及MAPK功能抑制后,MAPK信号通路的激活及IL-1β的表达均受到明显抑制.结果 提示,Mo及其LAMPs可通过绵羊肺泡巨噬细胞的TLR2受体激活MAPK信号通路,继而引起细胞因子IL-1β的分泌增多.

Mycoplasma ovipneumoniae (Mo) is difficult to culture, resulting in many difficulties in related research and application. Since nucleotide metabolism is a basic metabolism affects growth, this study conducted a "point-to-point" comparison of the corresponding growth phases between the Mo NM151 strain and the Mycoplasma mycoides subsp. capri (Mmc) PG3 strain. The results showed that the largest difference in nucleotide metabolism was found in the stationary phase. Nucleotide synthesis in PG3 was mostly de novo, while nucleotide synthesis in NM151 was primarily based on salvage synthesis. Compared with PG3, the missing reactions of NM151 referred to the synthesis of deoxythymine monophosphate. We proposed and validated a culture medium with added serine to fill this gap and prolong the stationary phase of NM151. This solved the problem of the fast death of Mo, which is significant for related research and application.

为了探讨绵羊肺炎支原体NM2010株假定P60样脂蛋白用于制备基因工程亚单位疫苗的可能性,试验运用ExPASy、SignalP 4.0、TMHMM 2.0、SOPMA、PSORT 6.4、IEDB、BlastP、Hcluster_sg、Muscle等生物信息学软件,对绵羊肺炎支原体NM2010株P60基因编码蛋白的理化性质、跨膜区、信号肽、二级结构、亚细胞定位、B细胞抗原表位、蛋白功能和基因家族进行预测分析.利用人工合成方法获得P60成熟肽编码区基因序列,并克隆到表达载体pET-32a(+)上进行原核表达及Western-blot分析.结果 表明:绵羊肺炎支原体NM2010株P60基因编码蛋白的二级结构以α-螺旋和随机卷曲为主,其结构稳定,可溶性较高;该编码蛋白有信号肽,亚细胞定位在外膜,可能是一种外膜蛋白,具有较好的抗原性;参与绵羊肺炎支原体烟酸和烟酰胺代谢途径,与绵羊肺炎支原体SC01株的GL000100(WP_010321430.1)、猪肺炎支原体7448株的0353(WP_011290189)属于同一家族,均为P60样脂蛋白.P60基因序列中的TGA成功突变为同义密码子TGG,重组蛋白的分子质量为74.0 ku,以可溶形式存在,具有良好的反应原性.说明P60重组蛋白可以作为绵羊支原体性肺炎基因工程亚单位疫苗的候选蛋白.

BACKGROUND:Mycoplasma ovipneumoniae (M. ovi) is the causative agent of chronic non-progressive pneumonia in sheep, goats, bighorn, and wild small ruminants. However, the mechanism of infection and immune response to M. ovi remain unclear. Invading microbes express lipid-associated membrane proteins (LAMPs) on the cell surface that interact with host cells to facilitate infection, and are thus the major molecules recognised by the host immune system. Upon LAMP recognition, Toll-like receptor 2 (TLR2) and NLRP3 inflammasome sense the pathogens and signalling pathways for cytokine secretion. In this study, we investigated whether M. ovi and M. ovi-derived LAMPs are immuno-biologically active compounds capable of activating mouse peritoneal macrophages and explored the underlying mechanism.RESULTS:After infection of wild-type mice with M. ovi, the expression of TLR2 and NLRP3 at the transcriptional and translational levels was determined with reverse transcription-polymerase chain reaction and flow cytometry. In addition, the cytokine levels and associated pathways were detected in infected wild-type, Tlr2-/-, and Nlrp3-/- mice via enzyme-linked immunosorbent assays and western blotting. The nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signalling pathways were found to mediate the expression of inflammatory cytokines in M. ovi or M. ovi-derived LAMP-infected peritoneal macrophages, and cytokines were not induced in Tlr2-/- and/or Nlrp3-/- macrophages.CONCLUSION:Host cytokine production is activated in response to M. ovi-derived LAMPs through the NF-κB and MAPK signalling pathway via TLR2.