猪轮状病毒与传染性胃肠炎病毒核酸免疫研究

将昆明鼠随机分为A,B,C,D4组,各组分别采用含有猪轮状病毒(RV)JL94株VP7基因的重组真核表达质粒pcDNA-VP7,含有猪传染性胃肠炎病毒(TGEV)TH98株N基因的重组真核表达质粒pcDNA-N,pcD-NA-VP7和pcDNA-N、真核表达载体pcDNA3.1(+),肌肉注射3次,每次间隔2周。首次注射前后定期采血,检测血清抗体和外周血淋巴细胞中CD4+,CD8+T细胞数量的变化。A,C组小鼠血清在首免后第14天即可检出针对RVVP7的阳性抗体(P/N≥2.0)。B组小鼠血清在首免后第39天检出针对TGEVN蛋白的阳性抗体(P/N≥2.0)。A,B,C组小鼠在首免后外周血CD4+、CD8+T细胞数量与D组小鼠相比,在不同时间有显著差异(P<0.05),并明显高于D组小鼠。说明猪轮状病毒与传染性胃肠炎病毒核酸免疫后能诱发机体免疫反应。     

猪传染性胃肠炎病毒NSP8蛋白单克隆抗体的制备及抗原表位的初步鉴定

为鉴定猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)NSP8蛋白的抗原表位,本研究对GST-NSP8重组蛋白进行了原核表达,并用纯化后的重组蛋白免疫6周龄BALB/c小鼠,取免疫小鼠脾脏,采用常规杂交瘤细胞融合方法,经3次亚克隆后制备了1株稳定分泌抗NSP8蛋白的单克隆抗体杂交瘤细胞株。分泌的单克隆抗体亚类鉴定其重链为IgG1型,轻链为κ链;杂交瘤细胞培养上清的效价为1∶3 200。Western blotting试验结果表明该单克隆抗体能识别原核及真核表达的NSP8重组蛋白。利用截短表达的方法对NSP8蛋白进行抗原表位的鉴定,初步确定了单克隆抗体针对的抗原表位序列为31SPQILKQLTKAFNIAKSDFEREASV55。本研究制备的单克隆抗体及对抗原表位的鉴定,为TGEV NSP8蛋白相关功能的研究奠定了基础。     

安徽部分地区冬春季仔猪腹泻的流行病学调查与分析

2012年1-3月间,通过定点监测、问卷调查、实地调查和实验室检测等方式,对安徽省16个市、50个县的757个养猪场(户)的仔猪腹泻发病情况进行了流行病学调查,并采集74份发病或病死仔猪样品肠内容物进行实验室检测.结果表明,安徽省仔猪腹泻主要以流行性腹泻、猪传染性胃肠炎为主,其中猪流行性腹泻病毒阳性率为89.2％,猪传染性胃肠炎病毒样阳性率为10.8％,饲养管理条件差和环境气候多变等是冬春季仔猪腹泻多发的重要诱因.     

牛胃肠炎发生原因及防治

胃肠炎是家畜生长中的常见病和高发病,牛具有很高的发病率。造成胃肠炎发生的原因是饲养管理不到位、饲料营养价值较差,饲料不足,牛出现营养不良,身体抵抗能力下降,使消化机能变差,进入消化道中的饲料不能很好的吸收,肠鸣音大,饲料随着肠道的蠕动排出体外。临床上主要表现为腹泻、身体脱水、生长发育不良。由于牛胃肠炎的发生机理还不明确,病因比较复杂,在疾病诊治期间应尝试明确实际的发病愿意,对动物进行对症防治,确保牛健康生长。该文主要论述牛胃肠炎的发生原因和防治措施。     

新发PDCoV和TGEV双重RT-PCR检测方法的建立及初步应用

猪Delta冠状病毒(Porcine deltacoronavirus,PDCoV)是一种新发现的冠状病毒,临床上主要引起感染仔猪出现水样腹泻、呕吐和脱水等症状,和猪传染性胃肠炎病毒(Porcine transmissible gastroenteritis virus,TGEV)感染引起的临床症状极为类似,且临床上PDCoV和TGEV混合感染时有发生,单靠临床诊断很难区分两种疾病,因此建立同时检测且区分PDCoV和TGEV的检测方法具有重要的临床意义.本研究根据GenBank中收录的PDCoVN基因和TGEVN基因序列设计了2对引物,在同一反应体系中同时扩增PDCoV的N基因片段和TGEV的N基因片段,通过对RT-PCR反应条件的优化,建立了同时检测PDCoV和TGEV的双重RT-PCR方法,并对该方法进行了特异性、灵敏性和重复性研究.结果显示,所建立的双重RT-PCR对PDCoV和TGEV的最低检测量分别是3.14×102 copies/μL和3.68× 103 copies/μL,利用该双重RT-PCR方法对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、猪博卡病毒(Porcinebocavirus,PBoV)、猪繁殖与呼吸综合征病毒(Porcine respiratory and reproductive syndrome virus,PRRSV)和猪伪狂犬病毒(P.pseudorabiesvirus,PRV)的扩增结果均为阴性,显示出较好的特异性.为了解所建立的双重RT-PCR方法的临床应用效果,对252份临床样品进行了检测.结果表明,双重RT-PCR检出PDCoV阳性样品31份(阳性率为12.30％),TGEV阳性样品12份(阳性率为4.76％),同时感染PDCoV和TGEV的阳性样品2份(阳性率为0.79％),且检测结果与单一PDCoV和TGEV RT-PCR相符.因此,本研究所建立的双重RT-PCR方法可用于PDCoV和TGEV的临床检测,为进一步研究PDCoV和TGEV的临床鉴别诊断和流行病学调查提供了理论依据和技术支持.     

6种中药成分对猪传染性胃肠炎病毒的体外抑制作用

【目的】为探讨连翘苷等6种中药成分抗猪传染性胃肠炎病毒（TGEV）的作用机制,明确其体外抗病毒作用效果,为抗TGEV的药物筛选提供依据。【方法】利用动物细胞培养技术,采用MTT比色法和细胞病变(CPE)观察法相结合,测定连翘苷、连翘苷元、绿原酸、咖啡酸、丁香酚、丹皮酚6种中药成分不同浓度下对猪睾丸细胞（ST）的毒性作用,通过观察细胞CPE情况,确定药物对细胞作用的最大安全浓度;同时测定病毒的TCID₅₀,用细胞维持液配成100·TCID₅₀病毒悬液备用;将6种药物在最大安全浓度范围内连续2倍倍比稀释后,分别采用先感染病毒后加药、先加药后感染病毒、药与病毒混合后感染细胞3种不同作用方式进行体外增殖抑制试验,各试验组均设置正常细胞对照组和病毒对照组,每组设置3个重复,通过酶标仪测得630 nm处OD值,计算出不同作用方式下中药成分分别对TGEV的抑制率,筛选出抗TGEV活性较好的中药成分,并分别记录抗病毒效果最佳时药物的浓度;6n>种中药成分与病毒作用后,测定TGEVRT-PCR鉴定各个药物单体对病毒RNA合成的抑制情况,进一步明确各中药成分对TGEV的抑制作用。【结果】连翘苷、连翘苷元、绿原酸、咖啡酸、丁香酚、丹皮酚的最大安全浓度分别为320、200、80、125、100、200 μmol·L^-1;抗病毒效果最佳时药物的浓度分别为：160、100、20、62.5、25、100 μmol·L^-1。根据Karber法计算出初始TGEV的TCID₅₀为10^-6.25/0.1 mL;6种中药成分对TGEV在ST细胞上均有一定的增殖抑制作用,其中咖啡酸浓度为62.5 μmol·L^-1时与100·TCID₅₀病毒混合作用后的病毒增殖抑制效果最好,相互作用72 h时,细胞形态依然能够保持圆滑、无固缩、完整,且细胞间轮廓清晰,仅有少量细胞脱落、死亡;此时测上清病毒的TCID₅₀为10^-3.75/0.1 mL,结果显示咖啡酸组病毒含量比病毒对照组10^-6.45/0.1 mL差异极显著（P＜0.01）;通过酶标仪测得630 nm处OD值计算出抑制率能够达到84.4%,咖啡酸直接灭活病毒作用极其显著。其次是连翘苷、连翘苷元、绿原酸、丹皮酚及丁香酚,其病毒滴度分别为：10^-4.75、10^-5.55、10^-5.55、10^-5.65、10^-5.75/0.1 mL,但是这几种中药成分对病毒的抑制作用不够显著,抑制率大多在50%以下。另外,各中药成分对TGEV在ST细胞上的增殖抑制作用均为对TGEV的直接灭活作用最好,其次为对TGEV吸附阻断作用,最后为对TGEV的复制阻断作用。RT-PCR鉴定中药成分对病毒抑制作用,结果显示咖啡酸组条带与病毒对照组相比较暗,病毒效价较低,对病毒抑制作用效果显著;其次是连翘苷、连翘苷元、绿原酸、丹皮酚及丁香酚。【结论】6种中药成分在ST细胞上对TGEV均有一定的增殖抑制作用,其中咖啡酸直接灭活病毒作用最显著,有望被开发成抗病毒药物。     

Protective effect of the AT137RQ and ARQK176 PrP alleles against classical scrapie in Sarda breed sheep

The susceptibility of sheep to scrapie is under the control of the host’s prion protein (PrP) gene and is also influenced by the strain of the agent. PrP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H) are the main determinants of susceptibility/resistance of sheep to classical scrapie. They are combined in four main variants of the wild-type ARQ allele: VRQ, AHQ, ARH and ARR. Breeding programmes have been undertaken on this basis in the European Union and the USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report the results of a multi-flock study showing the protective effect of polymorphisms other than those at codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 378 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the PrP gene. The distribution of variations other than those at the standard three codons, between scrapie cases and negative controls, was statistically different in all flocks. In particular, the AT₁₃₇RQ and ARQK₁₇₆ alleles showed a clear protective effect. This is the first study demonstrating a protective influence of alleles other than ARR under field conditions. If further investigations in other sheep breeds and with other scrapie sources confirm these findings, the availability of various protective alleles in breeding programmes of sheep for scrapie resistance could be useful in breeds with a low frequency of the ARR allele and would allow maintaining a wider variability of the PrP gene.     

State-of-the-art review of goat TSE in the European Union, with special emphasis on PRNP genetics and epidemiology

Scrapie is a fatal, neurodegenerative disease of sheep and goats. It is also the earliest known member in the family of diseases classified as transmissible spongiform encephalopathies (TSE) or prion diseases, which includes Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE), and chronic wasting disease in cervids. The recent revelation of naturally occurring BSE in a goat has brought the issue of TSE in goats to the attention of the public. In contrast to scrapie, BSE presents a proven risk to humans. The risk of goat BSE, however, is difficult to evaluate, as our knowledge of TSE in goats is limited. Natural caprine scrapie has been discovered throughout Europe, with reported cases generally being greatest in countries with the highest goat populations. As with sheep scrapie, susceptibility and incubation period duration of goat scrapie are most likely controlled by the prion protein (PrP) gene (PRNP). Like the PRNP of sheep, the caprine PRNP shows significantly greater variability than that of cattle and humans. Although PRNP variability in goats differs from that observed in sheep, the two species share several identical alleles. Moreover, while the ARR allele associated with enhancing resistance in sheep is not present in the goat PRNP, there is evidence for the existence of other PrP variants related to resistance. This review presents the current knowledge of the epidemiology of caprine scrapie within the major European goat populations, and compiles the current data on genetic variability of PRNP.     

以乳酸杆菌为载体的猪传染性胃肠炎病毒S基因A、D抗原位点DNA疫苗的构建

应用pRc/CMV2真核质粒骨架,先后插入猪传染性胃肠炎病毒(TGEV)S基因AD抗原位点片段和乳酸杆菌复制子基因Rep.8014,构建了pRc/CMV2-SAD-Rep.8014真核表达穿梭质粒。将其转染PK-15细胞,RT-PCR检测到S基因A D抗原位点的mRNA表达。随后,将pRc/CMV2-SAD-Rep.8014质粒电转化猪源的Lactobacillus acidophilusSW1株,成功构建了以乳酸杆菌为载体的带有TGEV S基因AD抗原位点的DNA疫苗株L.acidophilusTGES-1。本研究结合了乳酸杆菌胃肠道常在共生菌的优势和TGE呈现肠道局部发病的特点,达到益生性与免疫预防的双重功效,对乳酸杆菌口服DNA疫苗进行了有益的探索。     

Cutaneous transmissible venereal tumor without genital involvement in a prepubertal female dog

An 11-month-old prepubertal crossbreed female dog was presented with multiple nodular lesions disseminated over the cervical, back, flank, and abdominal regions. The lesions were ulcerated and cauliflowerlike, or nodular and subcutaneous, measuring up to 13 cm in diameter. Cytologic preparations of one of the lesions revealed a uniform population of round to oval cells, with lightly basophilic cytoplasm that contained multiple distinct vacuoles. Frequent mitotic figures and occasional lymphocytes were also observed. The cytologic diagnosis was cutaneous transmissible venereal tumor (TVT) in a progressing growth phase. This was confirmed by histologic and immunohistochemical findings. Vaginal TVT was diagnosed later in the dog's mother. TVT is a contagious neoplasm of sexually mature dogs that usually is transmitted by coitus and affects the genital mucosa. To our knowledge, this is the first report of naturally occurring multicentric TVT in a prepubertal female dog and also is unique in its exclusively cutaneous (no mucosal) involvement. We speculate that transmission of neoplastic cells occurred during cohabitation and social/mothering behavior between the dogs. Despite the atypical clinical presentation, response to chemotherapy with vincristine was excellent, leading to complete regression of the neoplasm without relapse after 6 months.