绵羊李斯特菌病病原诊断及其生物学特性研究

目的 单核细胞增生性李斯特菌(Lm)是引起人兽共患李斯特菌病的病原菌,该菌感染宿主中枢神经系统是导致高死亡率的主要原因.本研究对农场暴发的呈现神经症状的绵羊李斯特菌病病例进行病原诊断,进而对其病原学特性开展相关研究.方法 利用选择培养基和鉴别培养基进行细菌的分离和鉴定,借助多重PCR方法和Lm诊断血清对分离株的血清型进行研究,并测定了该菌株的溶血活性和药物敏感性,基于毒力基因actA的遗传谱系进行分型,最后对其LD50进行了测定.结果 从绵羊脑实质组织中成功地分离到一株血清型为4b的Lm菌株NTSN,其溶血效价为24,LD50为104.30CFU,是具脑侵袭性的强毒菌株,可用青霉素类药物治疗.遗传谱系分析表明Lm NTSN可能属于高侵袭性的流行克隆.结论 LmNTSN的分离鉴定及其生物学特性的研究,为深入探讨Lm的致病机理和预防控制奠定了基础.     

29种毒力基因在91株食源性单核细胞增生李斯特氏菌中的分布

目的了解河北省食源性单核细胞增生李斯特菌(Listeria monocytogenes,Lm)毒力基因分布特征。方法采用普通PCR方法对Lm的29个毒力基因(包括毒力岛Ⅰ在内的6个位点:prfA、plcA、plcB、hlyA、mpl和actA;内化素家族的10个位点:inlA、inlB、inlC、inlD、inlE、inlF、inlG、inlH/C2、inlI和inlJ;其他13个毒力相关位点:bsh、srtA、iap、sigB、virR、mprF、dltA、dltB、dltC、dltD、srtB、fbpA和hpt)进行检测。结果在91株Lm中,有23个毒力基因的检出率为100%。26株Lm的29种毒力基因全部检出,65株Lm存在inlD、inlF、inlG、inlH/C2、inlJ和mpl等6个毒力基因的不同缺失。缺失最严重的为inlG和inlF,缺失率分别为60.44%和54.95%;其次为mpl基因,缺失率为19.78%。根据毒力基因缺失情况,91株菌可分为10个基因型,优势毒力基因型为具有全部23种毒力基因的Ⅰ型。石家庄地区的毒力菌株缺失率(41/52)高于河北北部地区(11/22)。结论河北省食源性Lm的毒力基因携带率高,毒力基因缺失具有多样性且存在地域差异。     

2011—2021年福建省即食食品中单增李斯特菌的监测及菌株特征分析

目的 了解2011—2021年福建省即食食品中单增李斯特菌(Listeria monocytogenes,Lm)污染状况、毒力基因、耐药及分子分型特征。方法 参照GB 4789.30检测Lm;采用PCR方法对Lm的13种毒力基因进行检测及开展血清学、MLST分型；应用微量肉汤稀释法，选择15种抗生素进行药敏试验。结果 2011—2021年共采集8 166份即食食品，Lm总检出率为1.25%,以寿司、中式凉拌菜及熟肉制品检出率较高，分别为6.26%、5.60%、1.87%;毒力基因plcB、llsX、ptsA的检出率分别为55.42%、16.87%、22.89%,prfA等其余10个毒力基因检出率均为100%;83株Lm对头孢西丁的耐药率为100%,苯唑西林为15.66%、四环素为13.25%;寿司、中式凉拌菜、熟肉制品中检出多重耐药株，多重耐药率达13.25%;83株Lm分4种血清型，1/2a、1/2b为优势血清型；83株Lm MLST分型得到16种序列型(ST),ST87为优势型，其次是ST8,ST101。结论 2011—2021年福建省即食食品存在不同程度的单增李斯特菌污染，寿司、...     

3个猪链球菌2型四川分离株4个毒力因子基因序列测定与分析

目的设计了特异性引物对3个致病性猪链球菌2型(Ss2)四川分离株4个主要毒力因子基因mrp、epf、sly和orf2分别进行了扩增,序列测定与分析结果表明3个Ss2四川分离株均存在4个毒力因子基因mrp、epf、sly和orf2,其核苷酸序列与1998年江苏分离株9801及国外参考菌株的同源性均大于99.5%,提示3个四川分离菌株与1998年江苏流行的高毒力菌株可能具有共同的来源。     

陕西省2006年鼠疫菌生物学特性及分析

目的通过对鼠疫菌株的生物学特性分析,探索陕西省动物间鼠疫疫情的流行特点。方法常规方法测定2006年动物间鼠疫疫情中所分离的5株代表性鼠疫菌株的生化、毒力、毒力因子及质粒。结果所有菌株均符合鄂尔多斯高原沙鼠鼠疫菌的生化特性;LD,。在100～12．5亿之间;4种毒力因子n和PstI均存在,3株Pgm＋,2株Pgm-;vw均缺失;检出6、45、65MD3种质粒。结论被鉴定菌株均为鄂尔多斯高原型鼠疫菌,毒力不完全,3株强毒菌,2株弱毒菌。     

不同动物源肠球菌耐药性及毒力基因测定与分析

目的通过测定120株不同来源肠球菌耐药性分布情况,毒力基因cylA、efaA、gelE、esp、ace和AS的携带率,旨在了解肠球菌耐药现状和毒力基因分布情况,为选择临床治疗方案和探索肠球菌致病机制提供参考。方法设计6对特异性引物,应用PCR方法对分离到的120株肠球菌进行6种毒力基因检测,同时用纸片扩散法对11种常用抗生素进行耐药性试验。结果 120个肠球菌分离株对抗生素均具有3重以上的耐药性,且不同来源菌株的多重耐药性有明显差异,其中以病死猪内脏分离株多重耐药性最为严重;总体上,上述分离株对林可霉素耐药率最高,为达96.00%(115/120),对环丙沙星、卡那霉素、四环素和达福普汀的耐药率均达到50%以上,而对替考拉宁的耐药率最低,仅为0.83%(1/120)。6种毒力基因cylA、efaA、gelE、esp、ace和AS在受试菌株中的阳性检出率依次为39.17%、56.67%、68.33%、23.33%、26.67%和13.33%;不同来源的肠球菌毒力基因携带率不同,且携带毒力基因数量也有明显差异,其中,来自病死猪内脏的肠球菌分离株携带毒力基因的比例较高,且有超过50%的菌株同时携带4种毒力基因及以上。结论肠球菌的耐药率和多重耐药性与毒力基因携带阳性率有一致性,且与菌株来源关系密切,因此对这一传统意义上的机会性病原体值得深入研究其中的因果关系。     

河南省某一淡水养殖环节中非O1/O139群霍乱弧菌毒力基因及分子分型分析

目的 了解河南省淡水养殖环节中非O1/O139群霍乱弧菌毒力基因分布及分子分型情况。方法 对河南省淡水养殖环节中50株非O1/O139群霍乱弧菌和3株病人来源菌株进行全基因测序,利用PubMLST-Vc数据库分析其序列分型(ST),利用最小生成树关系图分析进化关系,通过VFDB数据库获得其毒力基因分布。结果 来源于淡水养殖环节和来源于病人的非O1/O139群霍乱弧菌53株菌株均具有黏附、趋化运动、抗吞噬、毒素及酶类等功能的8类毒力相关因子基因,缺失辅助定植、毒素共调、分泌等功能的毒力相关因子基因和副霍乱肠毒素等4个毒素基因。部分毒力相关因子或部分菌株的毒力相关因子毒力基因不全。与3株来源于病人的菌株相比,二者毒力因子基因携带情况相近,除MSHA Ⅳ型菌毛毒力因子mshA基因、荚膜多糖wbuB基因、wbfY基因、rmlB基因、血红素受体hutA基因及Ⅲ型分泌系统vscC2和vcrD2基因外,部分菌株二者携带相同的毒力因子基因。53株非O1/O139群霍乱弧菌分属19个ST型,ST4和ST5是优势ST型,养殖环节来源的菌株与病人来源的菌株分属不同的ST型。19个ST型等位基因位点变异差异数在1~7个,其中分离自淡水养殖环节的非O1/O139群霍乱弧菌菌株17个ST型等位基因位点变异差异数在1~7个,病人来源的非O1/O139群霍乱弧菌菌株2个ST型与分离自淡水养殖环节的非O1/O139群霍乱弧菌菌株ST1、ST2、ST6和ST10属同一簇,与ST1有6个等位基因位点存在差异。结论 河南省淡水养殖环节非O1/O139群霍乱弧菌菌株MLST分型多样化,携带多种毒力相关因子,不同来源菌株携带的毒力基因相同,虽然分属不同的ST型,但还是存在食品安全风险,提醒有关部门采取措施进行防控。     

陕西省两起鼠间鼠疫流行鼠疫菌生物学特性比较

目的通过对两起鼠间鼠疫流行鼠疫菌生物学特性进行比较,了解陕西省动物间鼠疫流行的病原特点。方法对2000～2001年、2006年动物鼠疫流行期间所分离菌株的生化、毒力、毒力因子及质粒进行比较分析。结果被鉴定菌株发酵阿胶糖,分解甘油,不发酵鼠李糖、麦芽糖,脱氮阴性。所有测试菌株含有F1和Pst1因子,2000～2001年19株鼠疫菌除1株Pgm±外,均含有4种毒力因子,2006年5株测试鼠疫菌均不含VW因子,2株不含Pgm因子。2000～2001年5株测试菌LD50在41～180个菌之间,2006年5株测试菌LD50在100～12.5亿个菌之间;2000～2001年未进行质粒检出工作,2006年鼠疫菌检出6、45、65 MD 3种质粒。结论所有鉴定菌株生化特点符合鄂尔多斯高原沙鼠鼠疫菌生化特性,引起两起鼠间鼠疫的鼠疫菌毒力因子及毒力不完全相同,2000～2001年鼠疫菌为强毒鼠疫菌,2006年鼠疫菌毒力有强弱之分,5株测试菌3株强毒菌,2株弱毒菌。     

陕西省2000～2001年分离鼠疫菌株特性及分析

目的 为了解陕西省动物鼠疫流行的病原特点。方法 对2000～2001年定边县分离于不同宿主、媒介动物的19株鼠疫菌进行了生化、毒力因子和毒力测定。结果 所有菌株生化特点一致,4种毒力因子除1株菌为Pgm±外,均齐备。对5株菌的毒力测定LD_(50)介于41～180之间。结论 分离菌株为鄂尔多斯高原生态型强毒鼠疫菌。     

东北地区恙虫病立克次体分离株的生物学及免疫学特性的研究

目的：本文对从黑龙江省密山、吉林省珲春及辽宁省宽甸三个地区分离到的恙虫病立克次体每株的生物学及免疫学性质进行了比较分析。方法：采用间接免疫荧光法、小鼠保护试验以及SDS-聚丙烯胺电泳法，分别测定分离株的血清类型、毒力和蛋白图谱。结果：三个地区的分离株除二株外均能引起小白鼠典型的发病过程。密山和珲春分离株均以Gilliam型为主；宽甸分离株则以Karp型为主。毒力以密山分离株最强（LD50=6．5-8．1）；珲春分离株次之（LD50=4．6-4．8）；宽甸分离株较弱（LD50=2．0-3．2）。结论：东北地区恙虫病立克次体分离株存在着3种血清型，其毒力由北向南逐渐减弱的趋势。三个地区的分离株都具有较好的免疫力，SDS-聚丙烯酰胺电泳所显示的各分离株图谱未见差异。     

Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8(+) effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8(+) effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens.     

Reductions in cross-neutralizing antibody responses in infants after attenuation of the human rotavirus vaccine candidate 89-12

The G1P1A[8] rotavirus vaccine candidate 89-12, the precursor to Rotarix, stimulated high titers of neutralizing antibodies to non-G1/P1A[8] serotypes of human rotavirus in naturally infected subjects before attenuation by cell-culture passages. These responses were greatly diminished in young infants (median age, 11 weeks) administered the attenuated vaccine. Because of the possibility of improved responses in older infants, the immunogenicity of the 89-12 vaccine candidate was evaluated after administration of 2 doses beginning at either 4 or 6 months of age. As was found in young infants, neutralizing antibody responses to non-G1/P1A[8] rotaviruses were considerably lower than those observed after natural infection. The reasons identified were overall (P<.0001) lower neutralizing antibody responses stimulated by the attenuated 89-12 strain, compared with those stimulated by its virulent precursor, and 5 mutations selected in the gene encoding the immunodominant VP4 (P) neutralization protein. Even so, the Rotarix vaccine developed from attenuated 89-12 was found to elicit excellent protection against non-G1 rotaviruses.     

Dominant-negative mutants as antiviral agents: simultaneous infection with the cold-adapted live-virus vaccine for influenza A protects ferrets from disease produced by wild-type influenza A

The attenuated cold-adapted strain of influenza A virus that is a candidate live-virus vaccine suppressed clinical disease in ferrets when given simultaneously with a virulent epidemic strain of influenza A virus. The cold-adapted virus effectively prevented disease, even when the epidemic strain was of a different subtype than the attenuated virus. In this case, ferrets given a mixed inoculum produced antibody to both subtypes in the absence of clinical disease, indicating that both viruses are replicating in the respiratory tract. These findings suggest the possibility of the development of a novel class of antivirals for influenza, namely a live virus that is a dominant-negative attenuated mutant that interferes with the replication of epidemic strains of virus.     

Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Objective:To develop allenualed slrains of Salmonella enterica serorar Typhi(S.typhi) for the candidate vaccine by osmolar stress.Mothods:S.typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Mamakkal,Tamil Nadu.India.Both strains were grown in LB(Luria Bertani) medium supplemented with various concentration of NaCl(0.1- 0.7M) respectively.The effecl of osmolar stress was determined at molecular level by PCR using MCR 06 and MCR07 primers corresponding to ompR with chromosomal DNA of S.typhi SS3 and SS5 strains.Attenuation by osmolar stress results in deletion mutation of the.S.typhi slrains was determined by agglutination assays,precipitation method.SDS PAGE analysis and by animal models.Results:The 799 bp amplified ompR gene product from wild type S.typhi SS3 and SS5 illustrate the presence of virulent gene.Interestingly,there was only a 282 bp amplified product from S.typhi SS3 and SS5 grown in the presence of 0.5.0.6 and 0.7 M NaCl.This illustrates the occurrence of deletion mutation in ompR gene al high concentration of NaCl.Furthermore,both the wildtype and mutant S.typhi outer membrane SDS-PAGF.profile reveals the differences in the expression of ompF.ompC and ompA proteins.In mice,wild type and mutant strains lethal dose(LD_(50)) were determined.The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU-mL~(-1).When the mice were injected with the mutants in same dosage,no clinical symptoms were observed;whereas the serum antibodv litre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response.These results suggest that S.typhi SS3 and SS5 may bo used as good candidate strains for the development of live attenuated vaccine against salmonellosis.Conclusions:This study demonstrates that the S.typhi strains were allenualed and could be good vaccine candidates in future.     

Recombinant ASF Live Attenuated Virus Strains as Experimental Vaccine Candidates

African swine fever (ASF) is causing a pandemic affecting swine in a large geographical area of the Eastern Hemisphere, from Central Europe to East and Southeast Asia, and recently in the Americas, the Dominican Republic and Haiti. The etiological agent, ASF virus (ASFV), infects both domestic and wild swine and produces a variety of clinical presentations depending on the virus strain and the genetics of the pigs infected. No commercial vaccines are currently available, although experimental recombinant live attenuated vaccine candidates have been shown to be efficacious in protecting animals against disease when challenged with homologous virulent strains. This review attempts to systematically provide an overview of all the live attenuated strains that have been shown to be experimental vaccine candidates. Moreover, it aims to analyze the development of these vaccine candidates, obtained by deleting specific genes or group of genes, and their efficacy in preventing virus infection and clinical disease after being challenged with virulent isolates. This report summarizes all the experimental vaccine strains that have shown promise against the contemporary pandemic strain of African swine fever.     

单增李斯特菌inl A/ inl B双基因缺失株生长特性观察及对小鼠致病性研究

目的探究单增李斯特菌inl A/inl B双基因缺失株的生物学特性。方法通过体外胁迫培养条件下OD600测定以及小鼠感染试验,对Lm 90-△inlAB和Lm 90的抗胁迫能力及细菌致病力进行研究。结果低温(4 ℃、30 ℃)环境下,Lm 90和Lm 90-△inlAB生长差异无统计学意义(t4 ℃=0.057,P>0.05;t30 ℃=0.441,P>0.05),适温37 ℃及42 ℃高温环境下,Lm 90-△inlAB较Lm 90生长受到抑制,生长差异具有统计学意义(t37 ℃=0.763,P<0.05;t42 ℃=1.147, P<0.05);体外耐酸碱试验中,Lm 90-△inlAB耐酸性未明显改变,而耐碱能力强于亲本株Lm 90,生长差异具有统计学意义(pH=9时t=2.837,P<0.05);在含有3.5%酒精的BHI培养基及含5% NaCl高渗BHI培养基中,Lm 90-△inlAB增长趋势明显低于Lm 90,生长差异具有统计学意义(t酒精=1.422,P<0.05;tNaCl=1.382,P<0.05),Lm 90-△inlAB对酒精及高盐的耐受力显著低于Lm 90;小鼠毒力测定结果表明,Lm 90-△inlAB与Lm 90半数致死量分别为106.94CFU、105.68CFU,Lm 90-△inlAB毒力下降明显,在不同检测时间点Lm 90-△inlAB在小鼠肝脏和脾脏内的载菌量均少于Lm 90,载菌量差异具有统计学意义(t肝=2.454,P<0.05;t脾=2.443,P<0.05)。结论　Lm的抗胁迫能力可能随着基因的缺失发生显著改变,内化素InlA/InlB与单增李斯特菌侵染宿主的能力有一定的调控作用。缺失株生物学特性测定对研究Lm胁迫环境生存及毒力因子的致病机理提供了科学依据。     

4b型单核细胞增生李斯特菌内化素基因NTSN_0462的功能初探

目的单核细胞增生性李斯特菌(Listeria monocytogenes,Lm)对宿主的粘附、侵袭作用与其多种内化素蛋白密切相关。本研究以4b型菌株LmNTSN的內化素基因NTSN_0462为研究对象,初步探究其致病作用。方法利用同源重组技术构建该基因缺失的突变株,研究0462基因对NTSN在生长、细胞侵袭和体内定植中的作用。人结肠腺癌细胞Caco-2、人肝癌细胞HepG2的侵袭率,以及BALB/c小鼠体内肝、脾脏中定植能力的差异。结果缺失株的生长曲线结果显示,NTSN_0462基因对Lm在BHI培养基中的生长及代谢未造成明显影响。以人结肠腺癌细胞Caco-2、人肝癌细胞HepG2进行的体外试验表明,缺失株的侵袭能力低于野生株(P0.001)。以BALB/c小鼠进行的体内试验显示,缺失株在肝脏中定殖的能力显著低于野生株(P0.001),在脾脏中无统计学差异。结论 NTSN_0462基因在LmNTSN入侵肝脏和定植中发挥重要作用,是侵袭相关的重要毒力因子。     

Immunobiological Characteristics of the Attenuated African Swine Fever Virus Strain Katanga-350

The African swine fever virus (ASFV) is the cause of a recent pandemic that is threatening the global pig industry. The virus infects domestic and wild pigs and manifests with a variety of clinical symptoms, depending on the strain. No commercial vaccine is currently available to protect animals from this virus, but some attenuated and recombinant live vaccine candidates might be effective against the disease. This article describes the immunobiological characteristics of one such candidate—the laboratory-attenuated ASFV strain, Katanga-350—which belongs to genotype I. In this study, we assessed clinical signs and post-mortem changes, the levels of viremia and the presence of viral DNA caused by injection of ASF virus strains Katanga-350, Lisbon-57, and Stavropol 08/01. Intramuscular injection of this strain protected 80% of pigs from a virulent strain of the same genotype and seroimmunotype (Lisbon-57). At least 50% of the surviving pigs received protection from subsequent intramuscular infection with a heterologous (genotype II, seroimmunotype VIII) virulent strain (Stavropol 08/01). Virus-specific antibodies were detectable in serum and saliva samples between 8–78 days after the first inoculation of the Katanga-350 strain (the observational period). The results suggested that this strain could serve as a basis for the development of a recombinant vaccine against ASF viruses belonging to seroimmunotype I.     

马链球菌兽疫亚种10株国内猪源分离株对小鼠免疫效力的评价

目的近年来马链球菌兽疫亚种引起的猪链球菌病在我国造成重大损失。为评价猪源兽疫亚种分离株的抗原变异,取国内10省市分离的10株猪源兽疫亚种分离株,分别制备灭活抗原,加入弗氏完全佐剂免疫12 w龄ICR小鼠,4 w后加入弗氏不完全佐剂进行再次免疫,再次免疫2 w后分别用5 LD50同源菌株和1.6×105CFU ATCC35246株攻击。结果表明,同源菌株攻击,免疫保护率均在90%以上;而以ATCC35246攻击的异源菌免疫小鼠,免疫保护率为80%-100%。可见我国不同地区的猪源马链球菌兽疫亚种分离株的抗原性差异不大,单一菌株制备的疫苗具有应用前景。