蟾蜍壶菌病病原遗传分化研究

壶菌病为近年发现的两栖类动物重要传染病,对野生和养殖种群危害极大,为确定药用经济动物蟾蜍历史上壶菌病感染情况,提高养殖蟾蜍疾病防治水平,选取某博物馆馆藏采集于四川的蟾蜍标本32只,利用Taqman—MGB荧光探针定量PCR技术进行壶菌检测,并对定量PCR产物克隆、测序,通过序列比对和系统发育分析判定其来源。最终得到定量...     

牛支原体TaqMan实时荧光定量PCR检测方法的建立

本研究旨在建立一种快速检测牛支原体(Mycoplasma bovis)的实时荧光定量PCR检测方法。根据GenBank中收录的牛支原体OPPD/F基因序列(登录号:AF130119),利用Primer 5.0软件设计特异性引物与TaqMan探针,以重组质粒作为绝对定量模板,构建检测牛支原体的TaqMan实时荧光定量PCR检测方法。结果显示该检测方法线性关系良好,标准曲线的相关系数R2=0.994,扩增效率E=1.280。荧光定量PCR法检测的敏感性是常规PCR的10倍;特异性良好,检测9种相关细菌和病毒均为阴性;重复性好,组内及组间样本检测Ct值均小于2%。牛支原体TaqMan实时荧光定量PCR检测方法较之于常规PCR方法有快速、特异性强、敏感性高、稳定性好的优点。     

基于牛结节性皮肤病病毒ORF61基因荧光定量检测方法的建立

基于国内流行的牛结节性皮肤病病毒(lumpy skin disease virus, LSDV)的基因组序列,建立了靶向ORF61基因的MGB探针荧光定量PCR检测方法。本研究基于LSDV基因序列设计出带有MGB探针的特异性荧光定量PCR引物,并进行特异性和灵敏性筛选验证。最终,筛选得到靶向于LSDV ORF61基因的一对qPCR引物;利用pCAGGS-ORF61真核表达质粒,获得标准曲线y=-3.287 5x+47.87,线性相关系数R²=0.998 6,扩增效率达101%;荧光定量PCR特异性、重复性和灵敏性试验结果表明,该引物的特异性高、重复性良好和灵敏度高等优点;该引物的最低检测限为6.71拷贝·μL^-1,各批次内与批次间重复性结果的变异系数均小于2%。以上结果表明,作者建立特异性LSDV的荧光定量PCR检测方法,为LSD预防和控制提供有效的检测手段。     

鸡毒支原体TaqMan实时荧光定量PCR检测方法的建立

为快速检测鸡毒支原体,根据鸡毒支原体的保守基因设计特异性引物和TaqMan探针,建立鸡毒支原体TaqMan实时荧光定量PCR方法并分析鸡毒支原体培养过程中颜色单位改变法(color change unit, CCU)和荧光定量PCR检测的相关性。结果显示,标准曲线y=-3.646x+44.208,相关系数R²=0.998,最低检测限度为1 copy/μL;与其他菌株等均无交叉反应;组内和组间变异系数均小于3%,表明该方法具有良好的稳定性和可重复性。用建立的实时荧光定量PCR检测方法对26份临床样品进行检测,该方法较普通PCR方法检出率更高。建立的荧光定量PCR与CCU显示鸡毒支原体在对数生长期时,两者具有一定的对应关系。表明建立了鸡毒支原体TaqMan探针实时荧光定量PCR检测方法,可实现对鸡毒支原体的快速检测,为鸡毒支原体感染的防控提供技术基础。     

环形泰勒虫SYBR Green I荧光定量PCR方法的建立及应用

为建立一种快速、敏感检测牛环形泰勒虫的方法,本研究根据GenBank中登录的牛环形泰勒虫Tams1基因序列,设计合成1对特异性引物,通过优化反应条件,建立了检测牛环形泰勒虫SYBR Green I荧光定量PCR检测方法。研究结果显示,该方法可以特异地检测牛环形泰勒虫,而对牛巴贝斯虫、反刍动物艾立希体和弓形虫检测均为阴性;该方法的灵敏度可达到180拷贝/μL,比常规PCR敏感10倍;组内和组间变异系数均小于1.0%。对15份临床血液样品和20只璃眼蜱进行检测,SYBR Green I荧光定量PCR和常规PCR的阳性检出率分别为42.86%和28.57%。本研究建立的SYBR Green I荧光定量PCR检测方法具有特异性强、敏感性高的特点,可准确、高效检测牛环形泰勒虫,为牛环形泰勒虫病防控提供技术支持。     

基于鸽新城疫病毒M基因实时定量PCR检测方法的建立

研究通过比对鸽源和鸡源新城疫病毒M基因的序列,找到其差异位点并在保守区设计引物和TaqMan探针,建立了一种可以特异性检测鸽新城疫病毒的实时荧光定量PCR方法。以鸽新城疫病毒M基因阳性质粒为模板制作标准曲线并对检测方法的指标进行系统评价。结果显示,该方法的标准曲线为方程为:y=-3.334logX+37.837,相关系数R2=0.992;重组质粒标准品检测下限为1×101 copies/μL,比普通PCR灵敏100倍;特异性试验显示,该方法与鸡新城疫病毒不同毒株及其他常见禽类病毒无交叉反应;重复性试验的组间及组内的变异系数均小于2%,说明本研究建立的实时定量PCR检测方法敏感性高、特异性强、重复性好,可应用于鸽新城疫病毒的早期检测,为鸽新城疫病毒的防控提供了技术支持。     

非洲猪瘟病毒K196R基因实时荧光定量PCR检测方法的建立

为建立一种准确、特异、高效、快速的非洲猪瘟病毒定量检测方法,本研究根据非洲猪瘟病毒(African swine fever virus, ASFV)早期表达基因K196R的基因序列,设计了TaqMan荧光定量PCR引物及探针,通过优化退火温度、引物及探针浓度,建立了快速检测ASFV的TaqMan荧光定量PCR检测方法。结果表明,该方法选择的引物具有高度灵敏性和特异性,以构建的重组质粒为标准品建立的TaqMan荧光定量PCR方法的标准曲线具有良好的线性关系(R~2=0.998),对ASFV核酸最低检测下限为1.3拷贝,且与猪伪狂犬病病毒、猪细小病毒、猪圆环病毒2型等多种病原不存在交叉反应。本研究建立的K196R基因实时荧光定量PCR检测方法为非洲猪瘟疫情提供了一种新型、灵敏和特异的早期检测方法。     

新疆北疆部分地区鼠类伯氏疏螺旋体携带情况调查

为了解新疆北疆地区鼠类伯氏疏螺旋体携带情况及其基因型分布特征,2021年1—9月在新疆昌吉州吉木萨尔县、伊犁哈萨克自治州新源县、塔城地区乌苏市巴音沟,采集147只野生鼠类的肾脏和膀胱组织样本,采用实时荧光定量PCR和巢式PCR方法,检测鼠类中的伯氏疏螺旋体携带情况;以伯氏疏螺旋体5S—23S基因间隔区进行巢式 PCR检测,对检出的阳性样本进行基因测序及序列分析,确定伯氏疏螺旋体基因型,构建系统发育树。结果显示：在147份鼠类样本中,经实时荧光定量PCR和巢式PCR方法检测,分别检出伯氏疏螺旋体阳性样本11份和13份,阳性率分别为7.48%和8.84%;13份巢式PCR阳性样本中,吉木萨尔县检出1份,新源县检出1份,乌苏市检出11份,通过序列同源性分析确定Borreliella garinii、Borreliella bavariensis为主要致病基因型。结果表明,北疆地区鼠类中存在伯氏疏螺旋体的自然感染,有传播给家畜及人群的风险,应加强该病原体检测和莱姆病防控。     

火鸡疱疹病毒荧光定量PCR检测方法的建立及初步应用

本研究针对火鸡疱疹病毒(Herpesvirus of turkey,HVT)FC-126毒株sorf1基因序列,设计特异性的扩增引物和探针,建立了检测HVT的TaqMan荧光定量PCR方法,并对其特异性、敏感性和重复性进行检测。结果表明,建立的荧光定量PCR方法灵敏度高,最低可检测到1×10~2 copies/μL,比普通PCR灵敏10倍;对鸭瘟病毒、传染性喉气管炎病毒、鹅细小病毒、4型禽腺病毒和8型禽腺病毒均无特异性扩增,具有较好的特异性;组间和组内变异系数均小于1%,表明该方法重复性好。用建立的荧光定量PCR方法对82份HVT样品进行检测,结果43份为阳性,普通PCR只检测出30份阳性样品。结果表明本研究建立的HVT TaqMan荧光定量PCR检测方法特异性好,灵敏度高,重要性好,为监测HVT的病毒含量提供了一种有效的手段。     

禽偏肺病毒Taqman荧光定量PCR方法的建立及应用

根据Gen Bank中a MPV-C P基因序列,设计出一对特异性引物和Taqman探针,建立了a MPV-CTaqman探针荧光定量PCR方法。对该反应体系进行优化,进行特异性、敏感性及重复性试验,并且建立标准曲线。结果表明,该方法只对a MPV-C检测为阳性,具有良好的特异性;能够检测到(3.63×102)拷贝数,具有良好的敏感性;建立的标准曲线斜率为-3.312,截距为44.66,相关系数为R2=0.999,循环阈值和模板拷贝数具有良好的相关性,组内及组间重复性好。采用a MPV-C Taqman探针荧光定量方法、普通PCR方法对来自广东、浙江地区25份番鸭疑似阳性a MPV-C的病料进行检测,其中前者23份阳性,后者18份阳性。这表明a MPV-C Taqman荧光定量PCR方法对样品的检测具有特异性和敏感性高的特点,更适合临床样品的检测。     

Chytridiomycosis in amphibians--first report in Europe

Declining of amphibian populations is a worldwide phenomenon. A cutaneous mycosis as a cause of death in free-living amphibians as well as in captive ones due to an chytrid fungus (Batrachochytrium dendrobatidis) was reported at first in 1998. This infections were reported hitherto from Australia, North, Central and South America. This is the first report on chytrid infections in captive anurans from Europe. Dendrobates auratus and D. pumilo imported from Costa Rica and P. vittatus imported from French Guayana died with chytridiomycosis within a week after arrival in Europe. Batrachocytrium was also found on captive bred frogs in Germany and Belgium. Clinical signs, diagnosis and conclusions for protecting free-living amphibian populations and captive frogs are discussed.     

Effect of season and temperature on mortality in amphibians due to chytridiomycosis

OBJECTIVE: To investigate the distribution and incidence of chytridiomycosis in eastern Australian frogs and to examine the effects of temperature on this disease. DESIGN: A pathological survey and a transmission experiment were conducted. PROCEDURE: Diagnostic pathology examinations were performed on free-living and captive, ill and dead amphibians collected opportunistically from eastern Australia between October 1993 and December 2000. We conducted a transmission experiment in the laboratory to investigate the effects of temperature: eight great barred frogs (Mixophyes fasciolatus) exposed to zoospores of Batrachochytrium dendrobatidis and six unexposed frogs were housed individually in each of three rooms held at 17 degrees C, 23 degrees C and 27 degrees C. RESULTS: Chytridiomycosis was the cause of death or morbidity for 133 (55.2%) of 241 free-living amphibians and for 66 (58.4%) of 113 captive amphibians. This disease occurred in 34 amphibian species, was widespread around the eastern seaboard of Australia and affected amphibians in a variety of habitats at high and low altitudes on or between the Great Dividing Range and the coast. The incidence of chytridiomycosis was higher in winter, with 53% of wild frogs from Queensland and New South Wales dying in July and August. Other diseases were much less common and were detected mostly in spring and summer. In experimental infections, lower temperatures enhanced the pathogenicity of B. dendrobatidis in M. fasciolatus. All 16 frogs exposed to B. dendrobatidis at 17 degrees C and 23 degrees C died, whereas 4 of 8 frogs exposed at 27 degrees C survived. However, the time until death for the frogs that died at 27 degrees C was shorter than at the lower temperatures. Infections in survivors were eliminated by 98 days. CONCLUSION: Chytridiomycosis is a major cause of mortality in free-living and captive amphibians in Australia and mortality rate increases at lower temperatures.     

A novel subgroup among genotypes of equine arteritis virus: genetic comparison of 40 strains

The authors determined partial nucleic sequences of the variable regions of open-reading frame (ORF5) from 151 nucleotide to 668 nucleotide and deduced amino acid sequences of 518 nucleotide respectively of 20 equine arteritis virus (EAV) isolates. About 19 Hungarian and one Austrian EAV strains were subjected to sequence analysis, the further data of 20 EAV strains: six North American and 14 European were obtained from the GenBank. Comparative sequence analysis of the Hungarian EAV strains indicated that among the three variable regions the first has been affected mostly by point mutations. Genetic comparison of the Hungarian strains with other EAV isolates from western Europe and North America (including the Bucyrus reference strain) has been performed on the aforementioned genome region. Besides the already known genetic subgroups of EAV; phylogenetic analysis revealed a novel subgroup comprising mainly Hungarian strains. Compared with the Bucyrus virus, the overall sequence divergencies of the examined Hungarian strains ranged from 81.47 to 90.73% at nucleotide and from 84.88 to 91.86% at amino acid level. Epizootiological studies have shown that the significant part of the EAV strains having been existed in Hungary before and in 2000 belong to this unique cluster (II.D) which was not indicated in former phylogenetic studies. After 2000 new EAV strains emerged in Hungary, one of them causing abortions or neonatal death. The previously dominant 'Hungarian' EAV genotypes were replaced by these new strains belonging to North American and European subgroups (I.A, I.B, II.A, II.B). The anamnesis of these cases revealed connections with persistent virus shedder stallions, those were imported to the country after 2000 or have been infected abroad. One of these Hungarian stallions became the source of abortion storms in Hungarian studs.     

Etiological Investigation of Diarrhea in Newborn Piglets and Molecular Characterization and Genetic Variation Analysis of ORF3 Gene of PEDV in North Guangdong

In order to understand the main pathogen of newborn piglets diarrhea in North Guangdong region, 31 diarrhea samples were collected from six pig farms in North Guangdong, the pathogen of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) were detected by Real-time RT-PCR, meanwhile, ORF3 gene of PEDV amplified from positive samples were sequenced and analyzed. Pathogen detection results showed that 83.87%(26/31) samples were positive for PEDV, all herds and samples were negative for TGEV and PoRV. The sequence analysis revealed that the ORF3 gene of 5 epidemic strains of PEDV were all 675 bp, homologies of nucleotides were 98.7% to 100.0%, and homologies with reference sequences of nucleotides were 94.5% to 100.0%, and some gene mutation in common nucleotides site. The results of gene phylogenetic trees showed that PEDV could be divided into two groups, PEDV genetic relationship between field strains in North Guangdong and some regions in China, Southeast Asia, North America, Europe from 2013 to 2015 was closer, classed as a gene subgroup, from 2011 to 2012 main epidemic strains in our country and vaccine strains classed as other two gene subgroups. These results indicated that PEDV infection was the main pathogen of newborn piglets diarrhea in North Guangdong region, as time passed, the PEDV epidemic strains gene presented a tendency of evolution and variation.     

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (> or =80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick-pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick-pathogen interactions.     

The prevalent genotypes of bovine viral diarrhea virus in Japan, Germany and the United States of America

Genotypes and subgenotypes of bovine viral diarrhea virus (BVDV) field isolates from Japan, Germany and the United States of America (USA) were identified, and the prevalent pattern of BVDV in individual countries was estimated genetically. Subgenotypes were determined based on phylogenetic analyses of nucleotide sequences of a part of the E2-coding gene of BVDV. Forty-five, 61 and 56 BVDV strains were isolated from naturally infected cattle in Japan, Germany and USA, respectively, between 1980 and 2003. The most prevalent BVDV in these three countries was BVDV-1b. The second most prevalent BVDV strains were 1a, 1d and BVDV-2 in Japan, Germany and USA, respectively. The most prevalent subgenotype 1b in each country constructed individual small clusters in the subgenotype 1b branch in the phylogenetic tree. Although cattle and/or cattle products were moving among the three countries as part of international trade, the distribution of BVDV in the field in each country showed long-standing individual patterns.     

牛支原体生物学特性及其膜蛋白的研究进展

牛支原体(Mycoplasma bovis,Mb)是引起牛肺炎、关节炎、乳房炎和中耳炎等众多疾病的主要病原之一,牛支原体引起的疾病在欧美地区广泛蔓延并造成巨大的经济损失。中国2008年首次报道在犊牛肺炎中分离到牛支原体,目前国内对牛支原体的认识还很欠缺,现对牛支原体生物学特性及其膜蛋白进行简要综述,以期为牛支原体病的防控与治疗提供一定的参考依据。     

用CPN60基因序列研究隐孢子虫种系发育关系

本试验以编码线粒体功能性蛋白Chaperonin 60(CPN60)的核基因作为研究对象,对隐孢子虫分离株CPN60基因进行扩增测序,用Clustal X1.81对扩增序列与GenBank相关参考序列进行比对,然后用PAUP4.0程序中邻接法(Neighbor-joining,NJ)、最大简约法(Parsimony,MP)构建基因树,同时用TREEPUZZLE程序Version4.1构建最大似然树(Maximum likelihood,ML),以确定不同隐孢子虫虫株之间的进化关系,并以18S rRNA和HSP70基因构建的进化树作参照,评价CPN60是否更适合作为隐孢子虫基因分型和进化关系的分子标记。结果显示:基于CPN60构建的进化树将隐孢子虫分为两大类:C.baileyi和C.meleagridis处于一个分枝,C.hominis、C.suis、C.parvum牛基因型和C.parvum鼠基因型处于另一个分枝上。不同隐孢子虫之间的同源性介于96%~100%,能有效区分隐孢子虫不同基因型。因此,CPN60基因序列也可作为隐孢子虫分离株种系发育的遗传标记。     

Genetic typing of pestiviruses from New Zealand

AIM: To investigate the genetic type of 20 pestiviruses collected from New Zealand over the period 1967-97. METHODS: The pestiviruses were genetically typed by the sequencing of polymerase chain reaction (PCR) products. The primers selected were from the 5'-untranslated region (5'-UTR) of the pestivirus genome and consistently amplified a 288 bp fragment from all samples tested. RESULTS: Sequencing and phylogenetic analysis of PCR products revealed that all samples obtained from cattle represented bovine viral diarrhoea (BVDV) type I. Two sheep isolates were characterised as border disease virus (BDV). A pestivirus isolated from foetal calf serum of USA origin was typed as BVDV type II. CONCLUSIONS: The findings show that the evolution of pestiviruses in New Zealand has been similar to Europe and North America, indicating the occurrence of a conservative phylogenetic branch of BVDV type I in cattle and the presence of BDV in the sheep population.