一例猪圆环病毒病的诊断

2007年4月洛阳市某猪场发生一例猪圆环病毒病.根据猪圆环病毒ORF2基因序列设计、合成1对引物,应用PCR技术对病死猪的血液和脾、肺、肾和淋巴结等内脏组织进行PCR扩增,均能扩增出预期大小的目的片段.结合流行病学调查、临床症状和病理剖检变化,确诊为猪群发生猪圆环病毒Ⅱ型感染.     

表达猪2型圆环病毒ORF2基因的重组伪狂犬病病毒的构建及鉴定

【目的】以伪狂犬病病毒为载体，构建表达猪2型圆环病毒ORF2基因的重组病毒，并研究其生物学特性。【方法】将猪2型圆环病毒ORF2基因插入到伪狂犬病病毒gG缺失通用转移载体中，构建猪2型圆环病毒-伪狂犬病病毒重组中间转移质粒，然后将该质粒与伪狂犬病病毒TK-/gG-/LacZ+基因组共转染IBRS-2细胞，待发生细胞病变后收集病毒液进行重组病毒的筛选及生物学特性测定。【结果】利用检测PCV2 ORF2基因和LacZ基因的PCR方法筛选到重组病毒TK-/gG-/ORF2+，同时用Southern blotting证实外源基因PCV2 ORF2已成功插入到TK-/gG-/LacZ+亲本株的基因组中。间接免疫荧光试验（IFA）和Western blotting结果显示重组病毒中PCV2 ORF2基因获得了成功表达。对重组病毒的生物学特性研究表明，重组病毒与其亲本株在不同细胞上的增殖滴度相当，且重组病毒对小鼠无致病性，免疫小鼠后可诱导机体产生抗ORF2蛋白的特异性抗体。【结论】重组伪狂犬病毒构建成功，且表达的ORF2蛋白具有免疫原性。     

一株类猪圆环病毒2型因子P1的全基因组序列测定与分析

【目的】确定猪体内存在与猪圆环病毒2型核苷酸序列高度同源的因子。【方法】首先利用PCR方法对来自临床表现为猪断奶后多系统衰竭综合征的猪血清所抽提的DNA进行扩增,根据获得的序列再设计引物进行反向PCR,拼接得到类猪圆环病毒2型因子P1的全基因组序列,最后根据P1全基因组序列设计引物扩增其全基因组加以验证。【结果】首次完成了类猪圆环病毒2型因子P1的全基因组序列测定。测序结果表明P1因子为环状DNA基因组,全长648个核苷酸,包括3个开放阅读框。除5′端16个核苷酸外,P1因子与国内猪圆环病毒2型BF毒株的核苷酸同源率为98.42%。系统进化分析表明P1与猪圆环病毒有很近的亲缘关系。【结论】猪体内存在类猪圆环病毒2型因子P1。     

猪圆环病毒病原学研究进展

猪圆环病毒(porcine circovirus,PCV)有2种不同的血清型,即猪圆环病毒1型(PCV1)和猪圆环病毒2型(PCV2)。目前研究表明PCV1暂无致病性,但PCV2则被认为是引起猪圆环疾病(PCVDs)的主要病原。很多地区养猪业的经济效益因此而遭受了重大的影响。PCV现已受到全球的关注,引起了许多专家学者的高度重视。本文对PCV的病原学最新研究进展进行了综述。     

百合离体培养与植株再生体系的建立

以香水百合鳞叶为外植体,探讨不同外源激素水平对其小鳞茎分化、增殖与不定根形成的影响。结果表明:以MS+6-BA 0.5~1.0mg/L+IAA 0.1~0.3mg/L培养基的不定芽分化、增殖效果好,分化率达78.5%以上,增殖系数达5.6以上;1/2MS+6-BA 0.1mg/L+IAA 0.5mg/L或1/2MS+6-BA 0.1mg/L+NAA 0.1~0.3mg/L或1/2MS+6-BA 0.1mg/L+IBA 0.8mg/L的生根效果好,生根率可达96.0%以上;在泥炭∶珍珠岩=1∶1的基质中驯苗,成活率可达96.3%。     

南疆地区某猪场猪圆环病毒Ⅱ型的PCR检测

应用PCR技术对来自南疆某猪场的24例疑似病例进行猪圆环病毒Ⅱ检测,共检出PCV2阳性10份,阳性率为41.6%（10/24）。结果表明,该猪场存在PCV2的感染,且感染较为严重。     

Virus Location and Lymphocyte Apoptosis in Lymph Nodes of Piglets Infected with PCV-2

The present study has been performed to understand the location of the virus, type of apoptotic cells, and their relation to lymph nodes of piglets infected with porcine circovirus type Ⅱ （PCV-2）. Nine 32-day-old conventional piglets free of infection with PCV-2 were used, and distributed into three groups： control group （n = 3）, piglets inoculated with PCV-2 alone （PCV-2, n = 3）, and PCV-2 inoculated and KLH immunostimulated group （PCV-2 ＋ KLH, n = 3）. Superficial inguinal lymph nodes from all piglets were collected for histological examination after 32 days postinoculation, and immunohistochemistry for PCV-2 detection. Location of apoptotic cells was detected with TdT-mediated dUTP nick end labeling （TUNEL） and cell cycle, and the apoptotic rates were measured by flow cytometry. The characteristic histopathological lesions of the piglets in PCV-2 and PCV-2 ＋ KLH were lymphocyte depletions in the cortex and paracortex of the lymph nodes, epithelioid-like macrophage infiltration, and intracytoplasmic inclusion bodies presented in epithelioid-like macrophages. PCV-2 was mainly found in epithelioid-like macrophages by immunohistochemistry. In the lymph nodes, lymphocytes presented higher apoptotic rates in the cortex by TUNEL, special B-cell areas, and similar apoptotic cells were found in this compartment in the control. The apoptotic rates of the lymph nodes were 0.41, 3.34, and 4.88% in the control, PCV-2, and PCV-2 ＋ KLH groups by flow cytometry, respectively. The apoptotic rates of lymph nodes for PCV-2 and PCV-2 ＋ KLH piglets were significantly higher than those for the control group （P〈0.05 and P〈0.01）. The proliferation index （PI） was 0.17_＋0.01, 0.12_＋0.01 and 0.12_＋0.04 in the control, PCV-2, and PCV-2 ＋ KLH group, the PI of the control group was higher than that of the other groups, but without the statistical difference. PCV-2 can induce lymphocyte depletion in lymph nodes of piglets by blocking cell proliferation and promoting apoptosis. This is one o     

猪圆环病毒2型SYBR Green Ⅰ实时荧光定量PCR方法的建立

参考GenBank上的猪圆环病毒2型(PCV2)全基因序列,根据其开放阅读框ORF1保守序列设计引物,建立基于SYBR Green Ⅰ荧光定量PCR方法来检测PCV2.所建立的荧光定量PCR方法最低检测限为82.2拷贝·反应-1,与常规PCR相比,灵敏度高出100倍.扩增产物的融解曲线只出现1个单特异峰,无引物二聚体,...     

In vitro Drug Sensitivity of Eperythrozoon from Mink

[Objective] This study aimed to test the sensitivity of Eperythrozoon from mink to various drugs in vitro. [Method] The red blood cells isolated from Eperythro- zoon positive mink was cultured in complete medium （70% RPMI-1640 medium and 30% calf serum）, supplemented with bernier, oxytetracycline, trichlorfon, tylosin, imi- docarb, florfenicol, Fuhongjuesha or primaquine phosphate at the working concentra- tions of 12, 24, 36, 48 and 96 μL/ml, incubated at 37.3 ℃, 5% CO2. [Result] Fuhongjuesha was most efficient for killing Eperythrozoon, followed by trichiorfon and primaquine phosphate, but trichlorfon is toxic. Bernier, imidocarb and florfenicol are efficient. [Conclusion] The study provides a scientific reference for clinical treatment of eperythrozoonosis.     

溴氰菊酯对草鱼鱼种脑AchE及ATP酶活性的影响

本试验研究了溴氰菊酯对草鱼鱼种的急性效应以及溴氰菊酯对草鱼鱼种脑ＡｃｈＥ及ＡＴＰ酶活性的影响。结果表明,溴氰菊酯对规格Ⅰ（平均体重８．２１ｇ）、规格Ⅱ（平均 重６６．６ｇ）草鱼鱼种９６ｈ半数致死浓度（９６ｈＬＣ５０）分别为２．１６μｇ／Ｌ、４．２６μｇ／Ｌ,９５％置信区间分别为１．９３￣２．４２μｇ／Ｌ、３．８２￣４．７５μｇ／Ｌ,离体条件下对草鱼种脑Ｍｇ＾２＋－ＡＴＰ酶活性抑制显著,呈一定的浓度     

猪圆环病毒2型和3型双重荧光定量PCR检测方法的建立

为建立一种同时检测猪圆环病毒2型(PCV2)和猪圆环病毒3型(PCV3)的方法,参照GenBank上已登录的PCV2 Cap基因和PCV3 Cap基因的保守序列,设计特异性引物和TaqMan探针,通过优化反应条件,建立了同时检测PCV2和PCV3的双重荧光定量PCR检测方法,并对其进行特异性、灵敏性、可重复性检验.特异...     

猪圆环病毒2型重组病毒ZJ-R感染性克隆的构建

利用无缝克隆方法构建ZJ-R全长基因组的单拷贝分子克隆以及双拷贝串联分子克隆;通过生物信息学技术预测ZJ-R病毒结构蛋白的B细胞抗原表位,人工合成选定的表位肽,然后偶联KLH免疫新西兰大白兔制备多克隆抗体;免疫组化试验证实该多抗与转染PK15细胞的ZJ-R双拷贝串联分子克隆具有反应性。结果表明构建的ZJ-R双拷贝串联分子克隆具有感染性。     

The Estimation of Soil Trace Elements Distribution and Soil-Plant-Animal Continuum in Relation to Trace Elements Status of Sheep in Huangcheng Area of Qilian Mountain Grassland,China

The purpose of the present study was to survey contents of trace elements of Cu, Mn, Fe, and Zn in the surface layer （0-20 cm） in the soil, pasture and serum of sheep in Huangcheng area of Qilian mountain grassland, China. Also the soil-plantanimal continuum was analyzed. Soil （n=300）, pasture （n=60）, and blood serum samples from sheep （n=480） were collected from Huangcheng area of Qilian mountain grassland, China. The contents of trace element in the samples were analyzed by atomic absorption spectrophotometer after digestion. The soil trace elements density distribution shows a ladder-like pattern distribution. Equations developed in the present study for prediction of Fe （R2=0.943） and Zn （R2=0.882） had significant R2 values.     

猪圆环病毒2型的致病机理和疫苗研究进展

为了深入了解猪圆环病毒2型的致病机理和疫苗研究进展,介绍了猪圆环病毒2型感染引起猪免疫系统的损伤过程,其他病原对该过程的促进和诱导作用,刺激免疫系统影响病毒复制和临床病例发生,综述了有关疫苗研究情况。国内外在猪圆环病毒2型的致病机理和疫苗研究进展上有很多相关研究报道,可供借鉴。研究为探讨运用疫苗途径来预防控制该病的发生提供了依据。     

云南地区部分猪场猪呼吸道疾病综合症（PRDC）患病猪群中PRRSV，PCV-2，CSFV，PRV混合感染调查

 针对云南省不同地区不同季节猪呼吸道疾病综合征进行流行病学调查,根据其临床特征,在不同规模养殖场共采集1164份血清样品,利用ELISA和RT-PCR方法检测其猪瘟抗原CSFV,猪繁殖与呼吸综合征抗原PRRSV,猪伪狂犬病PRV gE抗体及猪圆环病毒2型PCV-2特异抗体。结果表明：各季节和不同生长阶段猪群存在一种及两种以上的病毒混合感染,四重感染相对较少。从全年看单独感染占39.10%,PCV-2感染率最高,其次是PRV,PRRSV和CSFV;二重感染占26.13%,以PCV-2+PRV最常见,其次是PCV-2+ PRRSV、PRV+PRRSV;三重感染占8.05%,PCV-2+ PRRSV+PRV最常见;有四重感染出现,仅占1.57%。从季节来看,春季和夏季混合感染最高,分别为76.53%和60.74%,冬季和秋季的混合感染率分别为59.50%和53.55%。从年龄和性别看,育肥猪的混合感染率高于仔猪,分别为68.66%和61.11%,种公猪的感染率高于母猪,分别为70.00%和55.51%。说明4种病毒有单独感染或混合感染,推测其中PCV-2在混合感染中可能充当免疫抑制的角色,混合感染使PRDC症状更明显,死亡率增高。     

培养基成分对试管米麻增殖的影响

在不同天然营养附加成分组合及不同生长调节剂组合的培养基上,培养试管米麻,探讨培养基成分对试管米麻增殖的影响.结果表明:促进试管米麻生长的天然营养附加成分组合为20g*L-1菌材液+100g*L-1香蕉汁;促进试管米麻生长的生长调节剂组合为1.0mg*L-16-BA+0.2mg*L-1NAA;促进试管米麻快速增殖的最适培养基是:1/2MS+1.0mg*L-16-BA +0.2mg*L-1NAA+20g*L-1菌材液+100g*L-1香蕉汁.     

针对非洲猪瘟病毒MGF360-13L基因的TaqMan荧光定量PCR的建立

[目的]建立一种以MGF360-13L基因为靶标的实时荧光定量PCR检测非洲猪瘟病毒(African swine fever virus,ASFV)的方法,为非洲猪瘟(African swine fever,ASF)的诊断、MGF360-13L基因缺失毒株鉴别、病毒分离鉴定、基因功能研究提供技术支持.[方法]首先,以A...     

猪伪狂犬病、猪瘟、猪繁殖与呼吸综合症、圆环病毒2型的相关性研究

2003-2004年对广东省猪伪狂犬病(PR)、猪瘟(CSF)、猪繁殖与呼吸综合症(PRRS)、圆环病毒2型(PCV2)病原或野毒抗体调查发现,当前猪场这4种病的混合感染严重.对PR、PCV2、PRRS、CSF的动态流行情况进行相关性分析,结果表明:PR与PCV2之间存在较强的正相关性(P=0.012),相关系数为0.648,互谱图显示,PR与PCV2之间存在同时发生的关系.PCV2与CSF存在较弱的相关性,它们的互谱图表现为:PCV2的流行会微弱影响着未来7个月(负相关)、4个月(正相关)时CSF的流行情况,但这种影响是不显著的(P>0.05).     

猪圆环病毒感染相关疾病及防控措施

猪2型圆环病毒是引起仔猪断奶多系统衰竭综合征、猪皮炎肾病综合征、猪呼吸道疾病综合征、先天性震颤等多种疾病的主要病原体,且能诱发多种病毒和(或)细菌混合及继发感染,给养猪业带来了巨大的经济损失。就这些与猪2型圆环病毒感染相关的疾病分别作简要概述,并从疫苗免疫和饲养管理方面强调防控措施,以期为养殖场防控该病提供参考。     

Molecular Characterization and SYBR Green I-Based Quantitative PCR for Duck Hepatitis Virus Type 1

To determine the genomic sequence of a duck hepatitis virus type 1 （DHV-1） strain, real-time quantitative polymerase chain reaction （RTQ-PCR） assay based on SYBR Green I technology was developed to target 3D gene of DHV-1, Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization, and strain DHV-1 R genetic organaiztion is 5＇ untranslated region （UTR）-VP0-VP3-VPI-2A1-2A2-2B-2C-3A-3B-3C-3D-3＇ UTR, DHV-1 R has close relationship with Parechovirus, and has 95.1-99.1% nucleotide sequence identity with other DHV-1 strains. Based on the DHV-1 sequences in GenBank, three pairs of specific primers were designed to amplify DHV-1 using real-time PCR. The results showed that real-time PCR Tm value is 85.6℃ and the real-time PCR provides a broad dynamic range, detecting from 102 to 109 copies of DHV-1 cDNA per reaction. No cross-reactions were found in specimens containing DPV, AIV and NDV. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus. All results showed that the real-time PCR has high sensitivity and specificity to detect DHV-1 using SYBR Green I dissociation curve analysis, isolates can be distinguished by their melting temperature. These methods are rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.