动物源产气荚膜梭菌致病因子和耐药性研究进展

产气荚膜梭菌是一种重要的人兽共患病原菌,能够导致气性坏疽、食物中毒和坏死性肠炎等多种疾病,对畜牧生产和公共卫生安全具有较大威胁。近年来,由于抗生素的不合理使用,产气荚膜梭菌耐药性显著升高。本文对影响产气荚膜梭菌致病性的毒力因子,如毒素、芽胞和生物被膜,以及近5年来产气荚膜梭菌耐药性的发展趋势进行归纳总结,以期为其防控提供参考。     

产气荚膜梭菌及其公共卫生危害

产气荚膜梭菌(C. perfringens)在自然界广泛分布,在鸡、鸭、猪、牛等食源动物中均普遍流行。生肉及其制品在加工过程中可能被产气荚膜梭菌污染而引起人的食源性腹泻,而且越来越多的证据表明产气荚膜梭菌出现了多种耐药性,因此控制产气荚膜梭菌污染具有重要的公共卫生意义。本文从该病流行、毒素、耐药基因、屠宰零售及食品污染风险等各环节进行梳理,分析该病引起的公共卫生危害,并提出了相关的控制建议。     

甘肃省牛和羊源产气荚膜梭菌耐药性分析

旨在探究甘肃省牛和羊产气荚膜梭菌的流行情况及对常用抗菌药物的耐药性.对采集自甘肃省不同地区的229份牛、羊源肛拭子进行了产气荚膜梭菌的分离鉴定,并对分离到的产气荚膜梭菌菌株进行毒素基因检测、最小抑菌浓度(MIC)测定及四环素耐药菌株的同源性分析.结果表明,在229份样品中分离到53株A型产气荚膜梭菌,分离率达23.1％...     

产气荚膜梭菌检测方法研究进展

产气荚膜梭菌是革兰氏阳性厌氧菌,广泛存在于环境中,包括污染的水、食物和土壤中,当机体胃肠道菌群紊乱或受到气温骤降等应激反应时,产气荚膜梭菌可大量繁殖并产生毒素,引起人和动物气性坏疽、食物中毒、肠炎、肠毒血症等多种疾病,严重危害人和动物的健康,不仅给养殖户造成严重的经济损失,影响畜牧业的发展,而且对公共卫生安全造成威胁。产气荚膜梭菌分型比较复杂,特异性诊断是防治该病的重要环节,因此需要快捷、高效和准确的鉴定和分型。目前,关于产气荚膜梭菌的主要检测方法有微生物学检测法、免疫学检测法以及分子生物学检测法。本文主要对产气荚膜梭菌的检测法进行总结,为产气荚膜梭菌检测方法的研究方向提供科学依据。     

携带噁唑烷酮耐药基因optrA藏羊源产气荚膜梭菌的检测

对1株携带有噁唑烷酮类耐药基因oprA的藏羊产气荚膜梭菌QHY-2进行了形态学观察、毒素分型、全基因组测序分析和oprA基因环境分析。结果显示，QHY-2为A型产气荚膜梭菌，对磺胺异噁唑、四环素、氟苯尼考、利奈唑胺和氯霉素等药物具有较强抵抗力。除携带有cpa、pfoA、colA、cloSI、nagH等毒素基因外，还携带有tetA(P)、tet(44)、fexA、optrA、ant(6)-Ib、aac(6′)-aph(2″)、erm(A)、erm(B)和erm(Q)等9种耐药基因。QHY-2的optrA基因环境分析显示，其上、下游分别为耐药基因fexA和erm(A),并与多个参考质粒携带的optrA基因高度相似。首次在藏羊产气荚膜梭菌发现噁唑烷酮类耐药基因oprA,对动物源产气荚膜梭菌的耐药性及噁唑烷酮类药物耐药基因传播趋势评估具有参考价值。     

规模牛场产气荚膜梭菌耐药性及其ERIC-PCR指纹图谱分型

[目的 ]了解不同规模牛场分离的产气荚膜梭菌耐药性及其耐药基因分型,为临床治疗和感染控制提供依据。[方法 ]收集2014—2016年来自不同规模牛场分离的产气荚膜梭菌25株,采用K-B法检测其对9种常用抗生素的敏感性;应用基因间重复序列聚合酶链反应(ERIC-PCR),对菌株进行DNA分型及同源性分析。[结果]分离到25株产气荚膜梭菌;在耐药检测的9种药物中,耐药率超过50%的有4种,其中对庆大霉素耐药率最高(92%),其次是对青霉素(84%),而对氨苄青霉素耐药率最低(4%),对其余药物的耐药率在12%~52%之间;ERIC-PCR谱型表现为9个基因型(Ⅰ~Ⅸ),其中大部分来源于不同的克隆株,且有2个克隆株的相似度为100%。[结论 ]奶牛场的产气荚膜梭菌多重耐药现象比较严重,分子多样性复杂;产气荚膜梭菌来源广泛,不同地区、环境和条件下,同一种病原菌的基因型存在一定差异,可能存在多个基因型。     

规模羊场环境气载产气荚膜梭菌耐药性及其ERIC-PCR分型

为监测不同规模羊场空气中分离的气载产气荚膜梭菌耐药性变迁及不同菌株间的遗传相似性,从而为临床治疗和控制感染提供依据,收集了2016—2017年来自不同规模羊场分离的气载产气荚膜梭菌30株,采用K-B法进行药敏试验,应用基因间重复序列聚合酶链反应(ERIC-PCR),对菌株进行DNA分型及同源性分析。结果显示:30株气载产气荚膜梭菌在监测的9种药物中,对庆大霉素的耐药性最强(86.7%),其次为青霉素(43.3%),对头孢噻肟耐药率最低(1%),对其他药物耐药率在3.3%~36.7%之间;ERIC-PCR谱型表现为14个基因型(Ⅰ~XIV),其中大部分来源于不同的克隆株,且有2个克隆株的相似度为99.0%。本研究结果表明:规模羊场的气载产气荚膜梭菌多重耐药较严重,分子多样性复杂;气载产气荚膜梭菌来源广泛,不同地区、环境和条件下,同一种病原菌的基因型也存在一定差异,可能存在多个基因型;同一个规模羊场的基因型也不同,不同羊场之间也存在相同的基因型。     

陕西省部分地区鸡源A型产气荚膜梭菌分离株的药敏试验

通过应用纸片法对52株分离自陕西部分地区的A型鸡源产气荚膜梭菌分离菌株进行药物敏感性检测,了解各菌株对常见抗菌药物的耐药状况。结果分离菌株对磺胺异恶唑、复方新诺明、强力霉素耐药率较高,均在70%以上;对万古霉素、氯霉素、杆菌肽的敏感率均在80%以上;大部分菌株为多重耐药。结果表明,陕西部分地区的鸡源性产气荚膜梭菌的耐药性较为严重。     

迁徙候鸟产气荚膜梭菌的分离鉴定与特性分析

产气荚膜梭菌是一种人兽共患病原菌,可通过多种途径感染人类。本研究从迁徙候鸟样品中对产气荚膜梭菌进行分离鉴定,并进行毒力基因检测、分型、生物被膜形成能力鉴定及耐药性测定。结果从549份健康候鸟粪便样品中分离细菌42株,35只死亡候鸟病料样品中分离细菌11株,通过PCR方法共鉴定A型菌48株,E型菌5株,所有菌株均未检出β2毒素、肠毒素和NetB毒素基因,30株经测定能够形成生物膜;分离菌株对黏菌素、杆菌肽、克林霉素和红霉素的耐药率在40%以上,所有菌株均对复方新诺明敏感。结论得出迁徙候鸟携带产气荚膜梭菌的比例较低,但是对部分抗生素的高耐药性值得关注。     

一株C型产气荚膜梭菌的分离与鉴定

产气荚膜梭菌是一种重要的人畜共患病原菌,在一定条件下可引起多种严重疾病。从疑似山羊魏氏梭菌病例中分离到一株细菌,通过厌氧培养、显微镜镜检、分子生物学鉴定表明该分离菌为产气荚膜梭菌,使用药敏纸片法进行分离菌株药敏性试验研究,数据分析表明：分离菌对常见抗生素具有不同程度的耐药性,对头孢噻呋敏感性最高,对多西环素、卡那霉素、新霉素、氟苯尼考、环丙沙星、庆大霉素等耐药,以期为山羊健康养殖提供技术参考。     

C型产气荚膜梭菌实验感染仔猪肠道circRNA的表达特征

circRNAs在病原菌感染宿主的肠道疾病发展中具有重要的调控作用。C型产气荚膜梭菌（C.perfringens type C）是引起仔猪腹泻及相关肠道炎症的主要细菌之一,对产业造成了严重的经济损失。然而,目前有关circRNAs如何调控仔猪C型产气荚膜梭菌性腹泻的全面且系统的研究未见报道。本研究通过RNA高通量测序研究分析了C型产气荚膜梭菌感染的7日龄仔猪回肠组织circRNAs的表达谱,筛选差异表达circRNAs,并进行差异表达基因GO和KEGG功能富集分析,利用miRanda等软件预测circRNA的microRNA靶点,构建circRNA-miRNA-mRNA的互作网络,并进行qPCR验证。结果显示,在C型产气荚膜梭菌感染的处理组（TI组）和对照组（CI组）中共鉴定出3 162个circRNAs,其中差异表达circRNAs有694个,上调表达circRNAs有404个,下调表达circRNAs有290个。功能富集分析结果表明,差异表达circRNAs的亲本基因主要富集在细胞周期、TGF-β信号通路、赖氨酸降解、Wnt信号通路、T细胞受体信号通路、MAPK信号通路等,调节仔猪对产气荚膜梭菌感染的抗性反应。此外,本研究构建了与C型产气荚膜梭菌感染致仔猪腹泻相关的circRNA-miRNA-mRNA互作网络,发现8 circRNAs-5 miRNAs-12 mRNAs组成的ceRNA网络与产气荚膜梭菌感染性疾病密切相关。本试验可为深入研究circRNA调控仔猪C型产气荚膜梭菌性腹泻疾病及猪抗腹泻病品系培育提供参考。     

关中奶山羊源D型产气荚膜梭菌全基因组序列测定及其分子特征分析

本研究自陕西省富平县某规模化关中奶山羊养殖场腹泻奶山羊肛门拭子中分离到5株产气荚膜梭菌,命名为21-D-1~21-D-5。毒素基因检测表明,其均为携带etx的D型产气荚膜梭菌,且21-D-5携带食源性致病毒素基因cpe。全基因组序列测定显示,5株D型产气荚膜梭菌基因组大小、GC含量和基因数量稳定;单核苷酸多态性（SNPs）分析显示,21-D-1和21-D-2以及21-D-3和21-D-4之间的SNPs差异极低（<25）,表明其大概率属于相同的产气荚膜梭菌菌株。分离的D型产气荚膜梭菌基因组中共检测到15种毒素基因,其中,毒素基因etx在分离的D型菌株中高度保守、基因环境相似,且在菌株21-D-5中毒素基因cpe位于etx下游。此外,包括噁唑烷酮类耐药基因optrA在内的9种耐药基因也在分离株中被检测到,并且erm（A）、optrA和fexA具有共同传播的可能性。本研究为国内首次对D型产气荚膜梭菌进行全基因组序列测定分析,结果对产气荚膜梭菌疾病的防治和基因组的进一步研究具有参考价值。     

The effect of hen-egg antibodies on Clostridium perfringens colonization in the gastrointestinal tract of broiler chickens

We evaluated the ability of hen-egg antibodies (HEA) to reduce intestinal colonization by Clostridium perfringens in broiler chickens. Antibodies against C. perfringens or cholera toxin (negative control) were obtained from the eggs of laying hens hyperimmunized using a C. perfringens bacterin or cholera toxin. Eggs were collected, pooled, and egg antibodies were concentrated by polyethylene-glycol precipitation. An initial experiment was conducted to determine the in vivo activity of the administered antibody along the length of the intestine. Thereafter, two feeding trials were performed to assess the efficacy of feed amended with the egg antibodies in reducing the level of colonization of C. perfringens in challenged birds. Antibody activity declined from proximal to distal regions of the intestine but remained detectable in the cecum. In the first experiment there was no significant reduction in the number of C. perfringens in the birds fed the diet amended with the anti-C. perfringens egg antibody, compared to the birds that received the anti-cholera toxin egg antibody (n = 10), at any of the sampling times. In the second experiment there was a significant decrease in C. perfringens intestinal populations 72 h after treatment (n = 15) as assessed by culture-based enumeration, but there was no decrease as measured by quantitative PCR based on the C. perfringens phospholipase C gene. Intestinal-lesion scores were higher in the birds that received the anti-C. perfringens HEA. Our work suggests that administration of HEA did not reduce the level of C. perfringens intestinal colonization and conversely might exacerbate necrotic enteritis.     

Apparition of Clostridium sp. and Bacteroides in the intestine of the newborn delivered by cesarian section

Anaerobic flora plays a key role in preventing intestinal colonization with potential pathogens. Nowadays, the mechanisms involved in the colonization resistance provided by the anaerobic microflora are to be clarified. Numerous factors seem to intervene in the regulation of the intestinal flora.

The purpose of the present study was to correlate the presence or relative absence of Clostridium sp. and Bacteroides with the colonization by C. perfringens, which is involved in lethal infections in an immunologically compromised host.

The intestinal bacterial colonization of 20 newborns delivered by cesarian section was assessed sequentially over the first 14 days of life. C. perifringens is a strongly reducing microorganism and undoubtedly causes a decrease in the oxidoreduction potential of the newborn feces favouring the subsequent colonization by other putrefactive bacteria. C. perfringens seems to be the precursor for installation of putrefactive bacteria, as Bacteroides and other Clostridium sp.     

Detection and characterization of Clostridium species in soil of Zambia

In the retrospective study of soil-borne diseases of cattle in Zambia, malignant edema and blackquarter were widespread. One hundred and sixty-five cases with malignant edema and 103 cases with blackquarter were reported between 1985 and 1997. It was found that specific soil-conditions associate the emergence of the soil-borne diseases. Soil samples from five areas in Zambia were examined for the presence of genus Clostridium. Direct immuno-fluorescent assay (IFA) examination showed that C. septicum, C. novyi and C. chauvoei were detected in the soil of specific areas in Zambia, respectively. Causal organisms such as C. perfringens were isolated from the soil samples. The information of area-specific distribution of Clositridium species may give an efficient program in protecting cattle and man.     

C型产气荚膜梭菌外毒素致小鼠肠道损伤的转录组分析

旨在探索产气荚膜梭菌外毒素造成机体炎性损伤及免疫调控紊乱的毒性机制,为产气荚膜梭菌病的致病机理研究提供理论基础。将C型产气荚膜梭菌强毒株C59-2培养上清腹腔注射BALB/c小鼠,采集小肠样本进行转录组测序,筛选差异表达基因,并对其进行GO功能注释和KEGG通路富集分析。结果显示,共获得40.99 Gb有效碱基,筛选后共得到795个差异表达基因,其中,229个基因表达上调,566个基因表达下调,对随机选取的10个基因进行q-PCR验证,其相对表达量与转录表达谱一致。GO功能注释主要涉及G蛋白偶联核苷酸受体活性和G蛋白偶联嘌呤核苷酸受体活性等。KEGG通路富集分析发现,主要富集在TNF信号通路、IL-17信号通路、p53信号通路、FOXO信号通路、Toll样受体信号通路、NF-κB信号通路等。产气荚膜梭菌外泌毒素侵入机体,会激活TNF等炎性信号通路,进而造成肠道发生炎性损伤甚至坏死。     

发酵乳杆菌和凝结芽孢杆菌对产气荚膜梭菌感染肉鸡生长性能和肠道健康的影响

试验以产气荚膜梭菌感染肉仔鸡建立坏死性肠炎模型,研究发酵乳杆菌和凝结芽孢杆菌对感染肉鸡生长性能和肠道健康的影响。将336只1日龄爱拔益加肉仔鸡随机分成4个处理组,每个处理6个重复。4个处理组分别为对照组、感染组、感染＋LF组（日粮添加1×10⁹ CFU/kg发酵乳杆菌）、感染＋BC组（日粮添加1×10¹⁰ CFU/kg凝结芽孢杆菌）。所有感染肉鸡在14~21 d经口接种A型产气荚膜梭菌。试验期为28 d。结果显示：与对照组相比,灌服产气荚膜梭菌显著降低22~28 d肉鸡的日均采食量（P<0.05）,显著增加28 d十二指肠和空肠损伤评分（P<0.05）,显著提高21 d肉鸡盲肠的产气荚膜梭菌和大肠杆菌数量,显著降低28 d回肠产气荚膜梭菌数量（P<0.05）。与感染组相比,日粮添加发酵乳杆菌显著提高21 d肉鸡回肠的乳杆菌属数量（P<0.05）,显著降低21 d盲肠大肠杆菌的数量（P<0.05）;日粮添加凝结芽孢杆菌显著降低28 d肉鸡十二指肠损伤评分（P<0.05）,显著降低21 d十二指肠隐窝深度（P<0.05）,显著提高21 d空肠绒毛高度与隐窝深度的比值（P<0.05）,显著降低21 d肉鸡回肠和盲肠的大肠杆菌数量（P<0.05）,显著提高28 d肉鸡回肠乳杆菌属数量（P<0.05）。综上所述,日粮中添加发酵乳杆菌或凝结芽孢杆菌均对感染肉鸡的肠道微生物具有调控作用;其中添加凝结芽孢杆菌有利于肠道绒毛发育,缓解肠道损伤,一定程度上提高了肉鸡的生长性能,而发酵乳杆菌没有缓解肠道损伤的作用。     

不同践踏强度对3种暖季型草坪草的影响北大核心CSCD

开放的公园绿地草坪可满足居民游憩健身、防灾避险需求,保障城市居民的公共安全和健康,但过度践踏会对草坪产生直接的机械磨损和土壤紧实造成的间接损伤,因此草坪的耐践踏性成为衡量公园绿地质量的关键因素之一。为了探究不同草坪草在不同践踏强度下耐践踏能力,以上海常用的结缕草属、雀稗属、狗牙根属(Cynodon)3个暖季型草坪草品种为试验材料,设置不同客流量大小的践踏强度和践踏频率,在草坪生长高峰的5-9月分散式模拟践踏后,从草坪草的耐磨损性、耐土壤紧实性及恢复能力3个方面进行多指标隶属函数综合评价。结果表明:在轻度践踏水平时,3个草种的耐践踏性为:‘Belair’结缕草>‘SeaIsle2000’海滨雀稗>‘Tifdwarf’杂交狗牙根,40人·次^(-1)的轻度践踏可促进3个草种生长;在中度践踏时3个草种的耐践踏性为:杂交狗牙根>海滨雀稗>结缕草,杂交狗牙根在5-9月生长期内受80人·次^(-1)的践踏时表现出最强的耐践踏能力;重度践踏下,3个草种的耐践踏综值为:杂交狗牙根>海滨雀稗>结缕草,杂交狗牙根的耐践踏能力与其本身较强的生长量、恢复能力相关。上述试验结果,不仅可为公园绿地规划建设中所需耐践踏草坪草资源的推广利用提供理论依据,也可为公园绿地承载、绿地养护及客流导引间形成动态反馈机制的智能化管理系统提供基础数据,保障游客的安全和舒适度。     

重组产气荚膜梭菌β毒素突变体的表达与免疫保护性分析

旨在获得产气荚膜梭菌β毒素（CPB）的重组突变体,并评价其毒力及免疫保护性。对已知的产气荚膜梭菌CPB编码基因进行优化设计,同时引入4个氨基酸突变位点,分别是第212位的精氨酸突变为谷氨酸,第268位的亮氨酸突变为甘氨酸,266位的酪氨酸和275位的色氨酸突变为丙氨酸。此外,在该基因5'端添加Th细胞表位（T）和鞭毛蛋白（flagellin）N末端的编码序列,经人工合成获得重组基因片段（GTF_NCPB_m4）。将GTF_NCPB_m4克隆至原核表达载体pET-30a（＋）中进行表达与纯化,获得重组蛋白rTF_NCPB_m4。利用Western blot方法检测rTF_NCPB_m4与C型产气荚膜梭菌毒素抗血清的反应性,并检测rTF_NCPB_m4对小鼠的毒力。随后,以rTF_NCPB_m4免疫家兔,按照《中华人民共和国兽药典》（2015年版）规定的方法检测家兔血清对C型产气荚膜梭菌毒素的中和抗体效价。结果表明,rTF_NCPB_m4主要以包涵体的形式表达且能与C型产气荚膜梭菌毒素抗血清反应。小鼠安全性试验显示,50 μg的rTF_NCPB_m4对小鼠仍无致死性;免疫rTF_NCPB_m4后,每毫升家兔二免抗血清可中和10~20个小鼠最小致死量（MLD）的C型产气荚膜梭菌毒素;1个家兔MLD的C型产气荚膜梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔得到了100%（4/4）的保护。以上结果说明,rTF_NCPB_m4在丧失毒力的同时保留了良好的免疫原性,从而为C型产气荚膜梭菌病基因工程疫苗的研制提供了重要的数据。     

Differential Proteomic Analysis between Sporulated Oocyst and Unsporulated Oocyst of Cystoisospora suis

The differentially expressed proteins between sporulated oocyst and unsporulated oocyst of Cystoisospora suis strains were screened and compared. In this study, the C. suis infection model of piglets were established and a quantitative proteomic analysis between sporulated oocyst and unsporulated oocyst of C. suis were conducted by iTRAQ labeling and LC-MS/MS. Part of the iTRAQ results were validated by qRT-PCR. Total 174 proteins were identified, among which 40 proteins were differentially expressed, 38 proteins were upregulated and 2 proteins were downregulated. The differentially expressed proteins were mainly involved in metabolism, antibiotics biosynthesis, secondary metabolites biosynthesis and glycolysis/gluconeogenesis pathways. The results of this study provide reference for elucidating the developmental mechanism of C. suis sporulated oocyst and facilitated discovering a new biomarker for C. suis.