鹦鹉热嗜衣原体检测和诊断方法研究进展

鹦鹉热嗜衣原体(Cps)是一种常见的鸟类、禽类、家畜及人类病原体,快速、准确的确定病原体是控制疾病的关键。本文综述了Cps检测和诊断方法的研究进展,这些方法包括抗原检测、抗体检测和核酸检测等。     

鹦鹉热嗜衣原体分型的研究进展

鹦鹉热嗜衣原体广泛分布于世界各地,能感染禽鸟、哺乳动物及人类,严重威胁人类健康,并对世界各国的禽类养殖业造成巨大影响。传统的血清学分型法由于其特异性低、敏感性差等缺点几乎已被淘汰,随着分子生物学技术的发展,基因分型法凭借其高特异性、高敏感性、高分辨率等优点,有效地弥补了血清学分型的不足。本文就Cps分型方法的研究进展进行了综述。     

鹦鹉热嗜衣原体蛋白酶样活性因子(CPAF)原核表达与纯化

应用分子生物学技术,选择鹦鹉热嗜衣原体(Chlamydophila psittaci,C.psittaci,Cps)6BC株的CPAF蛋白的免疫优势区基因,进行构建pGEX6p-2/CPAFm重组质粒与重组菌,使用IPTG诱导重组蛋白的表达并分析诱导温度、诱导剂剂量及诱导时间对蛋白表达的影响.重组蛋白以GST琼脂糖凝胶...     

禽病原性大肠杆菌外膜蛋白的研究进展

大肠埃希氏富是家禽最常见的病原菌之一,可引起家禽胚胎死亡、脐炎、败血症、肉芽肿、卵黄性腹膜炎和全眼球炎等一系列疾病,给养禽业带来重大损失。禽病原性大O杆菌大部分属O1：K1、O2：K1和078：Kwlj以往国内外对禽源性大肠杆菌主要根据O抗原分型［1～3］。最近的研究结果显     

肺炎嗜衣原体基因分型的研究进展

肺炎嗜衣原体是一种严格的专性胞内寄生菌,能引起人和动物多种疾病,主要引起呼吸系统疾病,并且与动脉粥样硬化(Artherosclerosis,AS)、中风等肺外疾病密切相关。不同的Cpn基因型感染宿主范围存在一定的差异,而且有着明显的地理分布特点。基因分型对研究Cpn与疾病的关系、感染的诊断及疫苗的研制等方面具有重要意义。     

鹦鹉嗜衣原体禽鸟株的分离鉴定及小鼠呼吸道感染模型的建立

摘要：【目的】优化鹦鹉热嗜衣原体（Chlamydophila psittaci,C. psittaci,Cps）禽鸟株分离培养技术,建立Cps呼吸道感染的小鼠模型。【方法】从禽鸟肝组织中抽提总DNA,PCR法体外扩增Cps ompA基因,初步鉴定Cps阳性标本;同时将阳性标本的肝组织匀浆液接种到HeLa和Vero细胞中培养,Giemsa和免疫荧光染色法鉴定衣原体包涵体。将临床株扩大培养后,用2×104 、2×105 、2×106 IFU三个剂量鼻内接种小鼠,分别于感染后5d和10d处死小鼠,显微镜观察受染小鼠各脏器病理变化。【结果】采用PCR扩增Cps ompA基因,从100只禽鸟标本中检测到阳性Cps标本6例（6%）,并成功地在HeLa及Vero细胞中培养出3例,且Vero细胞内的衣原体包涵体体积大,结构致密,对衣原体感染引起的宿主细胞溶解耐受性较HeLa细胞强,更适合用于Cps的分离培养及体外研究。成功建立Cps的鼠呼吸道感染模型,105IFU 是建立Cps呼吸道感染小鼠模型的适宜菌量。【结论】优化了Cps临床株的分离培养技术,成功建立了Cps呼吸道感染小鼠模型,为Cps流行病学调查及研究衣原体的致病性和致病机制奠定基础。     

禽冠状病毒组织嗜性和固有免疫应答差异研究

鸡传染性支气管炎(Infectious Bronchitis,IB)一直是危害世界养禽业的最重要的疫病之一.IBV变异频繁,导致出现多种不同致病性毒株,不同致病性IBV的出现可能与其感染后免疫系统的激活有关.对IBV组织嗜性和感染后的免疫机制的研究对疫病防控具有重要意义.本研究为了探究IBV不同毒株组织嗜性和固有免疫应答之间的关系.将不同IBV毒株接种原代细胞、鸡胚、SPF鸡,进行各器官的组织嗜性鉴定,应用荧光定量的方法测定TLR3、TLR7、MAVS、MDA5、IFN-β和IFN-γ等细胞因子在SPF鸡的不同组织器官包括气管、肾、胃、肺、脾和法氏囊中mRNA的表达差异.结果表明,除-H52为已知疫苗毒株外,AH毒株对泌尿系统致病性强,TM毒株对消化系统有致病性.感染实验后显示,在气管中,AH感染组MDA5的mRNA表达显著下调,TM组IFN-β和IFN-γ的mRNA表达显著性上调,H52组MAVS与IFN-β表达上调;在肺脏中,AH组TLR3、TLR7、IFN-β和IFN-γ显著性上调,在TM组能看到MDA5和IFN-γ表达显著升高,H52组的TLR3、TLR7、MDA5、IFN-β和IFN-γ上调;在肾中,AH组和TM组IFNβ和TLR7表达下调,H52组TLR7、MAVS、IFN-β和IFN-γ表达上调;在腺胃中,AH组TLR7、MAVS﹑MDA5、IFN-β和IFN-γ表达上调,TM株IFN-γ表达上调,H52组TLR7表达显著性下调,MAVS、IFN-β和IFN-γ表达上调;在脾脏中,AH组MAVS表达上调,TM组TLR3、TLR7和MDA5表达显著性上调.H52组TLR7和IFN-γ的表达也显著提高;在法氏囊中,AH组各因子表达无显著性差异,TM组TLR7、MDA5和IFN-y表达上调,H52组中TLR3、TLR7、MAVS、MD)A5、IFN-β和IFN-γ表达均显著上调.不同组织嗜性的IBV毒株在各组织器官中各细胞因子表达量不同,不同组织嗜性毒株在固有免疫应答上存在差异且与免疫分子表达水平有相关性.本实验为不同IBV毒株感染机制、疫苗开发、病原与细胞相互作用的研究应用奠定理论基础.     

禽致病性大肠杆菌毒力基因多重PCR方法的建立和应用

【目的】建立禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)黏附相关基因、侵袭及毒素相关基因、抗血清存活相关基因及铁转运相关基因的多重PCR方法,实现禽致病性大肠杆菌毒力基因的简便、快速检测。【方法】根据GenBank公布的基因序列,设计合成18对特异性引物,通过条件优化,建立四组多重PCR体系,并通过模板倍比稀释检测各组多重PCR的灵敏性。利用多重PCR检测100株APEC毒力基因的分布,验证多重PCR方法的可行性。【结果】根据PCR扩增片段大小判定,上述四组多重PCR体系均能同时扩增出该组中的各个毒力基因,且灵敏度分别为:103CFU、103CFU、105CFU、105CFU细菌和1ng、1ng、10ng、10ng DNA。100株APEC的毒力因子检测结果显示,多重PCR和单基因PCR结果一致。【结论】建立的四组多重PCR方法能够简便、快速地检测禽致病性大肠杆菌的毒力基因,可用于毒力基因的鉴定以及流行病学调查。     

三种检验方法在诊断女性生殖道沙眼衣原体感染的比较研究

目的:比较不同方法对宫颈拭子样本沙眼衣原体的检测效果.方法:采集300例20-50岁无感染症状女性宫颈分泌物,分别采用细胞培养、聚合酶链式反应及胶体金法进行沙眼衣原体检测,培养阳性或两个非细胞培养方法检测为阳性,定为真阳性,称为"扩大金标准".结果:300例受检查者.按"扩大金标准"CT感染者共36例,发病率为12%(36/300),细胞培养、聚合酶链式反应、胶体金法敏感性分别为72.22%、91.67%、52.78%.特异性分别为100%、98.11%、98.86%.阳性预测值分别为100%、86.84%、86.36%.阴性预测值分别为96.35%、98.85%、93.88%.结论:三种检测方法均可用于CT感染的诊断.     

禽病原性大肠杆菌iro和tsh基因缺失株的构建

通过SSH和SCOTS研究, 铁系统(Iro)和温度敏感性血凝素(Tsh)在禽病原性大肠杆菌(APEC)的感染中可能发挥重要作用。基因检测发现, 在243个禽源大肠杆菌分离株中, 有205株为iro+菌株, 其中高、中度和低致病株分别为89.8%(184/205)、8.8%(18/205)和1.5%(3/205); 有167株为tsh+菌株, 高、中度、低致病株分别为87.4%(146/167)、12.6%(21/167)和0%(0/167), 结果显示iro+或tsh+株大多数为高致病株。为了确定iro和tsh基因在APEC致病力中的作用, 以APEC E037株为基础, 通过自杀性载体分别构建了iro和tsh基因缺失突变株E037(Δiro)、E037(Δtsh)和E037(ΔiroΔtsh)。动物感染性试验表明, 突变株在鸡体内的繁殖能力和致病性均明显下降, 但两个基因的协同致病作用不显著。进一步证实Iro和Tsh为APEC重要的致病因子。     

Polymerase Chain Reaction for the Detection of Chlamydia psittaci in the Feces of Budgerigars

The polymerase chain reaction (PCR) targeting the ompA gene of Chlamydia psittaci was evaluated for its ability to detect chlamydiae in fecal specimens of budgerigars as compared with isolation procedures using cell culture and embryonated egg inoculations. Several procedures for PCR template DNA preparation were compared so as to determine their detection levels for chlamydiae propagated in cell culture in the presence of fecal materials. Tween-20 and proteinase K treatments followed by centrifugation of the template DNA were found to be an appropriate procedure for DNA preparation for primary PCR. Subsequent nested PCR was shown to detect 4.8 IFU/ml or 84 particles/ml of chlamydiae. Chlamydiae in 50 fecal specimens from apparently healthy budgerigars were examined by nested PCR and several other methods. Nested PCR detected chlamydiae at a higher rate (12/50, 24%) than the isolation procedure in embryonated eggs (6/50, 12%). Primary PCR combined with the isolation procedure in cell culture gave a detection rate (5/50, 10%) similar to that of isolation from embryonated eggs. Detection rates by primary PCR (1/50, 2%) and in cell culture (0%) were inferior to the other procedures.     

Genetic diversity and epizootiology of Chlamydophila psittaci prevalent among the captive and feral avian species based on VD2 region of ompA gene

To study genetic diversity and occurrence of Chlamydophila psittaci, a total of 1,147 samples from 11 avian orders including 53 genera and 113 species of feral and captive birds were examined using ompA gene based nested PCR. Three types of chlamydiae: C. psittaci (94.12%), C. abortus (4.41%) and unknown Chlamydophila sp. (1.47%) were identified among 68 (5.93%) positive samples (Psittaciformes-59, Ciconiiformes-8 and Passeriformes-1). Based on nucleotide sequence variations in the VD2 region of ompA gene, all 64 detected C. psittaci strains were grouped into 4 genetic clusters. Clusters I, II, III and IV were detected from 57.35%, 19.12%, 10.29% and 7.35% samples respectively. A single strain of unknown Chlamydophila sp. was found phylogenetically intermediate between Chlamydophila species infecting avian and mammalian hosts. Among Psittaciformes, 28 out of 81 tested species including 10 species previously unreported were found to be chlamydiae positive. Chlamydiosis was detected among 8.97% sick and 48.39% dead birds as well 4.43% clinically normal birds. Therefore, it was observed that though various genetically diverse chlamydiae may cause avian chlamydiosis, only a few C. psittaci strains are highly prevalent and frequently associated with clinical/subclinical infections.     

Prevalence of Chlamydophila psittaci in Fecal Droppings from Feral Pigeons in Amsterdam, The Netherlands

In many cities, the feral rock dove is an abundant bird species that can harbor Chlamydophila psittaci. We determined the prevalence and genotype of C. psittaci in fresh fecal samples from feral pigeons in Amsterdam, The Netherlands. The prevalence was 7.9% overall (26/331; 95% confidence interval, 5 to 11). Ten genotyped PCR-positive samples were all genotype B.     

禽白血病病毒J亚群（ALV—J）的血清学调查及PCR诊断

禽骨髓细胞性白血病 (ｍｙｅｌｏｉｄｌｅｕｃｏｓｉｓ) (或称禽骨髓细胞瘤 ,ｍｙｅｌｃｙｔｏｍａｔｏｍａｔｏｓｉｓ) (ＭＬ)是由禽白血病病毒 (Ａｖｉａｎｌｅｕｋｏｓｉｓｖｉｒｕｓ)Ｊ亚群 (ＡＬＶ Ｊ)引起的禽的一种肿瘤性传染病[1] ,ＡＬＶ -Ｊ是英国的Ｐａｙｎｅ于 199     

Prevalence of Chlamydophila psittaci in fecal droppings from feral pigeons in Amsterdam, The Netherlands

In many cities, the feral rock dove is an abundant bird species that can harbor Chlamydophila psittaci. We determined the prevalence and genotype of C. psittaci in fresh fecal samples from feral pigeons in Amsterdam, The Netherlands. The prevalence was 7.9% overall (26/331; 95% confidence interval, 5 to 11). Ten genotyped PCR-positive samples were all genotype B.     

禽白血病病毒J亚群(ALV-J)的血清学调查及PCR诊断

禽骨髓细胞性白血病(myeloid leucosis)(或称禽骨髓细胞瘤,myelcytomatomatosis)(ML)是由禽白血病病毒(Avian leukosis virus)J亚群(ALV-J)引起的禽的一种肿瘤性传染病[1],ALV-J是英国的Payne于1991年从肉鸡中分离出来的一个新的囊膜亚群[2,3].对ALV-J的原型株,HPRS-103的致病性和传播的研究中发现,本病毒能诱导肉鸡产生骨髓细胞瘤病(ML)、肾瘤和其它多种肿瘤,死亡率为1%～2%,偶尔可高达20%.由于本病毒为ALV和禽内源性反转录病毒囊膜(E51)的重组体[4,5],因此其可通过水平传播和垂直传播迅速地感染整个鸡群,使鸡群在短时间内遭受灭顶之灾.近十年来,在许多国家,包括美国在内的肉鸡中,ML已经是引起死亡和其它生产性问题的严重原因.     

Characterization of a hypervariable region in the genome of Chlamydophila pneumoniae

Chlamydophila pneumoniae displays surprisingly little genomic variation, as seen by comparisons of the published genomes from three different isolates and sequencing of four different genes from different isolates. We have in the present study, however, demonstrated genomic variation between 10 C. pneumoniae isolates in the 11690-bp region between the two outer membrane protein genes pmp1 and pmp2. This region of the C. pneumoniae CWL-029 isolate contains seven C. pneumoniae-specific open reading frames (hb1-7, encoding hydrophobic beta-sheet-containing proteins). We identified additionally 12 open reading frames in the C. pneumoniae CWL-029 genome encoding hypothetical proteins with similarity to the seven hypothetical Hb-proteins. Compared to other isolates, genomic variation is seen to cause frame-shifting of three of the 19 hb-open reading frames, which are proposed to be three full-length genes and eight frame-shifted pseudogenes. The hypothetical proteins encoded by these proposed genes contain an N-terminally located highly hydrophobic stretch of 50-60 residues. A similar motif is found in all identified Chlamydia inclusion membrane proteins and therefore the Hb-proteins are candidate inclusion proteins.     

牡丹江城市鸟类调查

2004年5月至2009年4月对牡丹江城市鸟类进行调查,共记录鸟类91种,隶属14目31科,占全国鸟类总种数的7.69%.说明牡丹江地区鸟类资源比较丰富,其丰富度指数1.967,均匀度指数0.653 8,Shannon-Wiener指数2.949.记录的91种鸟中留鸟26种,夏候鸟53种,冬候鸟3种,旅鸟9种,分别占总数28.6%、58.2%、3.3%、和9.9%.在居留型上,主要以夏候鸟为主.在区系组成上,东洋种5种,占5.5%,古北种54种,占59.3%,广布种32种,占35.2%,以古北种为主.记录到的91种鸟类中包括松雀鹰(Accipiter virgatus)、苍鹰(A.gentilis)等8种国家Ⅱ级保护野生动物.在所调查的鸟类中雀形目鸟类最多,15科52种.