一株鸭源新城疫病毒毒力基因分析及其对鸭的致病性研究

为了解中国鸭源新城疫病毒(Newcastle disease virus,NDV)的毒力特点,从2009年广东地区发病鸭群中分离和鉴定出1株新城疫病毒(简称NDV-104),对其生物学特性、致病性和融合蛋白(fusion,F)、血凝素神经氨酸酶(hemagglutinin-neuraminidase,HN)基因进行了研究。结果显示,分离株的MDT、ICPI和IVPI分别为56.4 h、1.95和1.64,结合F蛋白裂解位点(112~117位)的氨基酸序列分析,确定了分离株为新城疫强毒。致病性结果表明,分离病毒对雏鸭具有感染性和致病性。F基因遗传进化结果显示,分离株属于基因Ⅶd亚型。F、HN蛋白的氨基酸同源性结果表明,分离株与2000年以来国内外分离到的基因Ⅶ型NDV同源性较高,分别为96.6%~99.3%和97.0%~99.7%,而其与常用疫苗株B1、V4、Clone30、Mukteswar和LaSota的F、HN蛋白氨基酸序列的同源性较低,且与LaSota株和V4株的同源性最低,分别仅为88.1%和87.6%,说明分离株与经典新城疫病毒毒株存在一定的差异。     

1株鸡新城疫强毒株的分离鉴定及致病性

从山东省一发病率、死亡率高的肉鸡群中分离到1株病毒,经HA、HI试验和动物回归试验确定所分离的毒株为新城疫病毒,命名为新城疫LVCX株。按常规方法测得LVCX株的MDT、ICPI和IVPI分别为47.4h、1.975和2.85,鸡胚半数致死量LD50为10-9.8。F基因分析表明分离株LVCX为基因Ⅶ型,其多肽裂解位点为112-RRQKRF-117,表明LVCX株为新城疫病毒强毒株。该毒株F基因核苷酸同源性与近几年分离的基因Ⅶ型鸡源毒株同源性在93.5%～98.3%之间;与经典强毒株F48E9和Lasota株的核苷酸和氨基酸同源性分别为85.8%、91.3%和83.3%、88.4%;且与近几年分离的几株鹅源、鸭源核苷酸同源性也较高,在95.2%～96.5%之间。     

新城疫LaSota活疫苗对基因Ⅶ型分离株的免疫保护性试验

从山东省发病鸡群分离鉴定了一株新城疫病毒(NDV),命名为SDLY01。经蚀斑纯化后进行毒力测定和序列分析表明分离株SDLY01属于基因Ⅶ型NDV强毒。20只7日龄SPF鸡免疫新城疫活疫苗LaSot a后14 d分别用NDV标准强毒F48E8和分离株SDLY01攻毒,同时设同日龄SPF鸡为对照组,未免疫任何疫苗。攻毒后观察10 d,免疫组在攻毒后食欲、精神均正常;对照组在攻毒后2～4d发病死亡,并表现ND典型的临床症状和病理变化。攻毒后第3、5、7、9 d对免疫组试验鸡取喉头、泄殖腔棉拭进行病毒分离,F48E8攻毒组病毒分离均为NDV阴性,SDLYO1攻毒组第5 d病毒分离NDV阳性,第3、7和9d病毒分离阴性。本研究结果表明LaSot a活疫苗对F48E8和SDLY01均能提供100%免疫保护,但不能完全抑制基因Ⅶ NDV分离株在体内的复制和排毒。     

Experimental infection and cross protection tests in calves with cytopathic strains of bovine rotavirus

Four cytopathic strains (81/32F, 81/36F, 81/40F, 82/80F) of bovine rotavirus were shown to be pathogenic for conventionally reared newborn calves. Calves were infected orally, using 3 calves for each isolate. All became febrile, were depressed and diarrhoeic. Two calves, one of which in the group of those infected with 81/36F isolate, and the other infected with strain 81/40F, were killed when moribund. A 3rd calf from the 81/36F infected group, died. At necropsy localized lesions of the small intestines, which are considered to be typical of rotavirus infection, were found. Virus was consistently isolated from the fecal samples of the inoculated calves up to 13 days post-inoculation. It was speculated that some differences existed in the virulence of the bovine rotaviruses tested. The cross protection tests revealed that 1 strain (81/36F) might be antigenically more complex than the others.     

The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland.

E. coli strains isolated from pigs with postweaning diarrhea or edema disease were tested by phenotypic and genotypic methods for the presence of virulence antigens and genes, respectively. The slide agglutination and ELISA analyses were used for determination of F4, F5, F6, F17, and F41 fimbriae whereas the prevalence of fimbrial fedA and toxin eltI, estI, estII, stx1, stx2 and stx2e genes were recorded by the means of PCR. Only F4 antigen (ac variant) was found in strains of the serogroup O149:K91 isolated from pigs with diarrhea. PCR analyses showed that the fedA gene encoding F18 fimbriae was present in 61.9% of strains isolated from pigs with diarrhea and in 84.2% of strains isolated from pigs with edema disease. The eltI genes encoding heat-labile toxin I (LTI) were present only in 9 out of 21 strains recovered from pigs with diarrhea. Shiga toxin 2 variant (stx2e) genes were found in six isolates from edema disease and also in one strain from diarrhea. The PCR test used in the study was a sensitive and valuable method for determination of virulence factors of E. coli strains.     

贵阳地区犬细小病毒VP2基因的克隆及遗传进化分析

为研究贵阳地区犬细小病毒（canine parvovirus,CPV）的流行基因型及其遗传进化情况,本试验对从贵阳市分离的10株CPV的VP2基因进行PCR扩增和克隆并进行序列分析。结果显示,分离的病毒能使F81猫肾细胞产生明显的细胞病变（CPE）,分离的10株病毒中,7株为CPV-2a亚型,3株为CPV-2c亚型,命名为GY-1~GY-10。10株CPV分离株与疫苗株VP2基因的同源性98.4%~99.4%,与其他国内外参考株的同源性为97.7%~99.9%。本试验首次报道贵阳市CPV的流行基因型为CPV-2a亚型并伴随CPV-2c亚型存在,对监测CPV遗传变异趋势及疫苗的研制具有重要意义。     

Cloning and Phylogenetic Analysis of VP2 Gene of Canine Parvovirus in Guiyang Area

This study was aimed to investigate popular genotype and phylogenesis of VP2 gene of canine parvovirus (CPV) in Guiyang area. Ten strains of viruses were isolated from Guiyang and the VP2 gene was amplified by PCR, cloned and sequenced. Results showed that the isolate virus grew could produce typical CPE in F81 cells, seven strains of CPV were CPV type 2a and others were CPV type 2c, named as GY-1 to GY-10. The nucleotide homologies of the isolated strains compared with 3 vaccine strains were 98.4% to 99.4%, with others reference strains nucleotide homologies within 97.7% to 99.9%.This research firstly reported the prevalence of CPV genotype was CPV type 2a in Guiyang area with such CPV type 2c, this was very important for monitoring the trend of CPV genetic variation and the development of vaccine.     

Wintering of neurotropic velogenic Newcastle disease virus and West Nile virus in double-crested cormorants (Phalacrocorax auritus) from the Florida Keys

During November 2002, six double-crested cormorants (DCCs; Phalacrocorax auritus) were found moribund in Big Pine Key, FL, exhibiting clinical signs indicative of neurologic disease. Postmortem diagnostic evaluations were performed on two adult birds. Virulent Newcastle disease virus (NDV) was isolated from a cloacal swab from cormorant 1. West Nile virus (WNV) was isolated from the brain and lung of cormorant 2. Nucleotide sequence analysis of a portion of the fusion (F) protein gene of the NDV cormorant isolate revealed it shared a 100% deduced amino acid identity with only two viruses: the 1992 epizootic cormorant isolate from Minnesota and the 1992 turkey isolate from North Dakota. The epidemiologic significance of the recognition of virulent NDV on cormorant wintering grounds during a nonepizootic period, in addition to the potential implications of the concurrent isolation of NDV and WNV from cormorants, is discussed.     

1株鸽Ⅰ型副黏病毒的分离与鉴定

本试验采集病死鸽临床样品,用SPF鸡胚接种传代分离病毒后,通过血凝（HA）与血凝抑制（HI）试验、RT-PCR及F基因部分片段测序与分析对分离株进行鉴定,并通过测定鸡胚平均死亡时间（MDT）、1日龄雏鸡脑内接种致病指数（ICPI）和动物回归试验评估分离株的毒力和致病性。结果表明,从病鸽组织病料中分离获得1株鸽Ⅰ型副黏病毒（PPMV-1）,命名为GXP120012。分离株GXP120012的F基因裂解位点112－117位的氨基酸组成为R-R-Q-K-R-F,符合强毒株特征;GXP120012与PPMV-1参考毒株pi/CH/LLN/110713的核苷酸序列同源性高达98.9%,并处于同一遗传分支,基因型为Ⅵ型。该分离株的MDT为102 h,ICPI为0。动物回归试验发现,分离株GXP120012对鸽具有较强的致病性,而对鸡没有致病力。本试验结果为今后鸽新城疫的分子流行病学研究和疾病防控提供理论依据。     

Isolation and Identification of One Strain of Pigeon Paramyxovirus SerotypeⅠ

A pigeon paramyxovirus serotypeⅠ(PPMV-1) isolate was recovered from samples collected from dead pigeons and initial isolation of the virus was performed in 9-day-old specific-pathogen-free (SPF) chicken embryos.The isolate was identified by the hemagglutination (HA) test,hemagglutination-inhibition (HI) assays,RT-PCR,sequencing and analysis of partial of F gene of the virus.The virulence and pathogenicity of the isolate were determined by mean death time (MDT),intracerebral pathogenicity index (ICPI) in 1-day-old chicks and animal regression experiment.The results indicated that one PPMV-1 was isolated from dead pigeon and named as GXP120012.The F gene sequence analysis of PPMV-1 GXP120012 strain showed that the cleavage sites of the virus was ¹¹²R-R-Q-K-R-F¹¹⁷ motif in accordance with characteristic of velogenic strains;The nucleotide sequence homologies of F gene between strain GXP120012 and PPMV-1 reference strain pi/CH/LLN/110713 were high and reached 98.9%.They located the same branch on phylogenetic tree and fell into genotype Ⅵ.The MDT and ICPI values of isolate were 102 h and 0,respectively.The results of animal regression experiment showed that GXP120012 isolate was virulent for pigeons and avirulent for chickens.The present study provided useful material and theoretical basis for the study of molecular epidemiology and prevention and control of Newcastle disease in pigeon in future.     

一株Class Ⅰ新城疫病毒中国分离株分子特性的研究

对从健康家鸭中分离到的一株Class I新城疫病毒分离株Duck/China/08-004/2008进行了遗传进化特性研究。利用RT-PCR扩增了该分离株F基因的主要功能区片段,并进行了克隆与序列分析。序列测定结果已经登录到GenBank,登录号为EU589149。该分离株F蛋白裂解位点的组成为112E-Q-Q-E-R-L117,具有典型新城疫弱毒株的分子特征。同源性分析表明其与国内普遍使用的弱毒疫苗株LaSota和V4核苷酸的同源性较低(分别为55.4%和57.7%)。通过构建F基因的遗传进化树,结果表明该分离株在分类地位上属于ClassI,与我国目前普遍使用的弱毒疫苗株LaSota(Class II中的基因II型)和V4(Class II中的基因I型)处在不同的进化分支。通过构建57株ClassI新城疫病毒的遗传进化树,表明本分离株与近年来香港活禽市场分离株较为类似,同属于基因3型。     

一株基因Ⅸ型鸭源新城疫病毒F基因的克隆及序列分析

从广东地区疑似患有新城疫的病死雏番鸭群中分离到1株新城疫病毒,命名为GD1002株。毒力测定结果表明,致死鸡胚平均死亡时间(MDT)、1日龄雏鸡脑内接种致病指数(ICPI)、6周龄鸡静脉接种致病指数(IVPI)分别为56.0 h、1.64、2.29,符合强毒株的毒力判断标准。利用RT-PCR扩增了该分离株F基因的主要功能区片段,并进行了克隆与序列分析。该分离株F蛋白裂解位点的组成为¹¹²R-R-Q-R-R-F¹¹⁷,具有典型新城疫强毒株的分子特征。F基因第47-420 nt片段基因分型分析表明,该流行株属于基因Ⅸ型。相似性及遗传进化分析结果表明,该分离株与基因Ⅸ型毒株F₄₈E₉、JS/1/97/Ch和FJ/1/85/Ch株有较高同源性和较近的亲缘关系。     

伪狂犬病弱毒株的分离鉴定及生物学特性的研究

在流行病学调查中分离到1株病毒,经鉴定为伪狂犬病弱毒株,定名为F971株。分离病毒经克隆纯化后测得其毒价为10^7.59TCID50/ml,通过细胞中和试验表明分离病毒能也有效地被猪伪狂犬病毒闽A株阳性血清中和。病毒在电镜下可以清楚地观察到囊膜及外周纤突。分离株对3日龄乳鼠有一定的致病力,但对家兔、3日龄乳猪及妊娠母猪都有很高的安全性。用不同的剂量10^0、10^-1、10^-2肌肉注射3日龄乳猪后14天用10^5.7TCID50伪狂犬病强毒攻击,所有试验仔猪均得到保护。用分离株免疫母猪,其后代可获高滴度的母源抗体,15日龄的仔猪能抵御10^5.7TCID50强毒的攻击。用ELISA普查试剂盒测定免疫猪抗体,结果均为阳性,而用g^1-ELISA试剂盒测定抗体时,结果均为阴性。证明分离株具有缺损g^1糖蛋白的特性。综合上述特性,确定F971为1株g^1糖蛋白缺损的猪伪狂犬病弱毒株。     

1株基因Ⅶ型新城疫病毒的生物学特性研究及全基因组序列分析

为研究当前流行的新城疫病毒（Newcastle disease virus,NDV）基因型、致病性及其与传统新城疫病毒疫苗株（La Sota等）的核苷酸差异,试验从某发病鸡场病死鸡体内分离到1株疑似NDV毒株,经红细胞凝集试验（HA）和红细胞凝集抑制试验（HI）初步确定为鸡源NDV。参照GenBank公布的NDV F基因部分片段（登录号：JF950510.1）设计1对引物,通过RT-PCR技术扩增分离株的F基因并克隆、测序,测序结果与NCBI中NDV F基因序列进行比对,构建系统进化树并分析其基因型;通过测定鸡胚平均致死时间（MDT）、1日龄鸡脑内致病指数（ICPI）和6周龄鸡静脉致病指数（IVPI）判断病毒致病性;参照GenBank公布的NDV全基因组序列（登录号：JF950510.1）设计9对引物对分离株进行全基因组序列测定,并分析其基因组结构。结果表明,RT-PCR扩增得到F基因长约500 bp,基于F基因构建的系统进化树显示分离株为基因Ⅶ型NDV;MDT、ICPI和IVPI分别为52.8 h、1.675和2.46,表明分离株属于强毒株。全基因组序列分析显示,分离株全基因组全长15 192 bp,与传统La Sota株基因组相比,序列多出6个碱基,核苷酸序列同源性82.8%。本研究成功分离到1株基因Ⅶ型NDV强毒株,且与传统疫苗毒株La Sota的核苷酸序列同源性差异较大。     

从自然发病鸡中分离出一株新城疫病毒

应用电镜技术、血清学检查、生物学试验等方法,从未注射过疫苗的某自然发病鸡群分离出一株新城疫病毒。该分离株对１０日龄鸡胚的平均致死时间为１１６小时,静脉接种致病指数为０．０６,其毒力介于新城疫病毒的ＬａＳｏｔａ株与Ｖ４株之间。免疫原性试验结果表明,该分离株具有免疫后无不良反应、免疫后６天开始产生抗体、产生的抗体效价高、对新城疫强毒的攻击能１００％保护等特点,是一株良好的新城疫候选疫苗株     

1株鸭源新城疫病毒的分离鉴定及生物学特性研究

从广东地区发病鸭中分离到1株新城疫病毒,血凝试验、血凝抑制试验结果表明此分离株具有血凝活性,且这种血凝性可被NDV标准阳性血清抑制,而不能被AIV-H5、AIV-H7、AIV-H9、EDSV阳性血清抑制;用针对NDV F基因特异性鉴定引物对该分离株进行聚合酶链反应扩增,可扩增出相应的目的片段。由此,初步鉴定该分离株为鸭源新城疫病毒,并命名为NDV/DUCK/SD/2006。另外,通过毒力测定,该毒株的MDT、IVPI分别为51.75 h、2.44,判定为强毒力株。     

新城疫病毒ShD-5-04株F基因的克隆与毒力分析

通过RT-PCR特异性扩增出新城疫病毒(NDV)山东分离株(ShD-5-04)的F基因序列,对其进行核苷酸序列测定和分析.结果表明,ShD-5-04株的F基因开放性阅读框为1 662 bp,编码489个氨基酸.与国外发表的部分新城疫病毒强毒株和弱毒株之间相同序列进行比较,F基因核苷酸序列的同源性在84.1%～88.7%之间,氨基酸同源性在88.1%～93.3%之间;F蛋白裂解位点区(112位～117位)氨基酸组成与强毒株一致,从基因水平上说明NDV ShD-5-04株为新城疫强毒株.     

Isolation of an atypical pigeon paramyxovirus type 1 in Poland

In this study, a pigeon paramyxovirus type 1 (PPMV-1) isolated from a flock of ornamental pigeons in Poland in 2010 is described. The PPMV-1/Poland/H2/10 isolate showed the amino acid sequence at the cleavage site of F2/F1 112KRQKRF117 i.e. typical of virulent strains. Despite having the monoclonal antibody binding pattern typical of pigeon variants PPMV-1 (antigenic group "P"), the Polish isolate clustered into genetic sublineage 4a, which is usually associated with PMV-1 isolated from poultry.     

Mycobacterium fortuitum isolated from three species of fish in South Africa

Mycobacterium fortuitum was isolated from 3 species of freshwater fish in South Africa for the first time. The bacterium was isolated from oscars, guppies and discus fish from different sources. Heavy mortalities as a result of infection with this bacterium were reported from a guppy farm and the multi-resistance of this isolate to antimicrobials rules out the treatment of infected fish.