养殖锦鲤鲤鱼浮肿病的检测与鉴定

北京房山某锦鲤养殖场锦鲤发生大量死亡,患病锦鲤呈烂鳃、肾脏糜烂、身体浮肿;症状类似锦鲤疱疹病毒（koi herpes virus,KHV）感染,但经PCR检测排除了KHV感染。为进一步确定病原,对发病鱼进行了临床症状观察、病毒分离、细菌分离培养、病鱼组织超薄切片电镜观察和PCR扩增等检测。通过病鱼组织超薄切片电镜观察,在肾脏内可见200 nm×400 nm的痘病毒样颗粒。抽提病毒核酸后,用已知的鲤鱼浮肿病毒（carp edema virus,CEV）的保守序列设计2对引物进行PCR扩增,扩增出548和180 bp片段,测序结果和GenBank公布的CEV H504株序列完全一致。根据发病鱼临床症状和实验室检测结果,最终确定该病为CEV引起的鲤鱼浮肿病。这是在中国首次发现的鲤鱼浮肿病。     

Testing and Identification of Carp Edema Virus Disease in Cultured Koi

An acute infectious diseases occurred in a koi farm in Fangshan district, Beijing, and it resulted in mortalities of more than 50%.The main symptoms of sick koi were gills necrosis, kidney erosion and edema,which were similar to the clinical signs of koi herpes virus disease (KHVD).But PCR tests showed negative results for KHV. For further diagnosis, bacterial cultures, transmission electron microscopy studies, virus isolation and PCR tests were used. Electron microscopic observation revealed pox virus particles having a size of about 200 nm×400 nm in the kidney. 548 and 180 bp fragments were amplified from organs of sick koi by PCR method using specific primers of carp edema virus (CEV). The fragments were sequenced and analysed. The results showed that they were shared 100% nucleotide identity with CEV-H504. All the results indicated that this disease was carp edema virus disease, caused by a kind of pox virus, CEV. This was the first report on the CEV of cultured koi in China.     

基于牡蛎疱疹病毒DNA聚合酶基因的巢式PCR检测方法的建立及应用

为了建立适用于Os HV-1不同变异株的检测方法,在牡蛎疱疹病毒(Os HV-1)3个变异株全基因组序列比对的基础上,筛选到牡蛎疱疹病毒基因组中高度保守的DNA聚合酶(DNA polymerase)基因,据此设计巢式PCR引物,优化PCR反应体系和条件,建立了基于Os HV-1 DNA聚合酶基因的巢氏PCR检测方法(P-n PCR检测方法),利用P-n PCR与Cn PCR检测方法对不同年份和宿主来源的Os HV-1疑似感染样本进行检测。结果显示,Pn PCR检测方法能稳定地检出100拷贝/μL的病毒DNA;P-n PCR较C-n PCR检测方法具有更强的特异性和更高的检出率。研究表明,本研究建立的P-n PCR检测方法适用于Os HV-1不同变异株的检测,可为该病毒的检测和流行病学调查提供可靠的技术支持。     

Efficacy of statistical process control procedures to identify deviations in continuously measured physiological and behavioral variables in beef heifers resulting from an experimentally combined viral–bacterial challenge

The objective of this experiment was to determine if statistical process control (SPC) procedures coupled with remote continuous data collection could accurately differentiate between animals experimentally inoculated with a viral–bacterial (VB) challenge or phosphate buffer solution (PBS). Crossbred heifers (N = 38; BW = 230 ± 16.4 kg) were randomly assigned to treatments by initial weight, average daily gain (ADG), bovine herpes virus 1, and Mannheimia haemolytica serum titers. Feeding behavior, dry matter intake (DMI), animal activity, and rumen temperature were continuously monitored remotely prior to and following VB challenge. VB-challenged heifers exhibited decreased (P < 0.01) ADG and DMI, as well as increased (P < 0.01) neutrophils and rumen temperature consistent with a bovine respiratory disease (BRD) infection. However, none of the heifers displayed overt clinical signs of disease. Shewhart and cumulative summation (CUSUM) charts were evaluated, with sensitivity and specificity computed on the VB-challenged heifers (n = 19) and PBS-challenged heifers (n = 19), respectively, and the accuracy was determined as the average of sensitivity and specificity. To address the diurnal nature of rumen temperature responses, summary statistics (mean, minimum, and maximum) were computed for daily quartiles (6-h intervals), and these quartile temperature models were evaluated separately. In the Shewhart analysis, DMI was the most accurate (95%) at deciphering between PBS- and VB-challenged heifers, followed by rumen temperature (94%) collected in the 2nd and 3rd quartiles. Rest was most the accurate accelerometer-based traits (89%), and meal duration (87%) and bunk visit (BV) frequency (82%) were the most accurate feeding behavior traits. Rumen temperature collected in the 3rd quartile signaled the earliest (2.5 d) of all the variables monitored with the Shewhart, followed by BV frequency (2.8 d), meal duration (2.8 d), DMI (3.0 d), and rest (4.0 d). Rumen temperature and DMI remained the most accurate variables in the CUSUM at 80% and 79%, respectively. Meal duration (58%), BV frequency (71%), and rest (74%) were less accurate when monitored with the CUSUM analysis. Furthermore, signal day was greater for DMI, rumen temperature, and meal duration (4.4, 5.0, and 3.7 d, respectively) in the CUSUM compared to Shewhart analysis. These results indicate that Shewhart and CUSUM charts can effectively identify deviations in feeding behavior, activity, and rumen temperature patterns for the purpose of detecting sub-clinical BRD in beef cattle.     

Investigation of the prevalence of neurologic equine herpes virus type 1 (EHV-1) in a 23-year retrospective analysis (1984–2007)

A single nucleotide polymorphism in the equine herpesvirus 1 (EHV-1) DNA polymerase gene (ORF30 A₂₂₅₄ to G) has been associated with clinical signs of equine herpes myeloencephalopathy (EHM). The purpose of our study was to determine the odds ratio for this genetic marker and EHM using a panel of field isolates from North America collected over the past twenty-three years. EHV-1 isolates cultured at the Cornell University Animal Health Diagnostic Laboratory from 1984 to 2007 were retrieved along with their clinical histories. DNA was extracted from these EHV-1 cultures and allelic discrimination was performed using real-time PCR. The results were confirmed by sequencing of the target region in ORF30. PCR and sequencing were in 100% agreement and showed that 19 out of the 176 isolates had the ORF30 G₂₂₅₄ allele (11%), of which16 were EHM cases and 3 respiratory or abortion cases. The odds of having neurologic disease with the ORF30 G₂₂₅₄ genotype were computed as 162 times greater than those with the opposite allele ORF30 A₂₂₅₄ (95% confidence interval: 35–742). Despite this strong statistical significance, 24% (5/21) of horses with neurologic disease in our study population harbored the “non-neurologic” form of the allele (ORF30 A₂₂₅₄), suggesting that other factors may also contribute to the onset of EHM.     

金鱼疱疹病毒敏感细胞的体外培养

采用组织块法和胰蛋白酶消化法对金鱼细胞进行体外培养和传代，研究了高倍稀释，培养液中的小牛血清浓度以及冷冻保存等因子对显示较高的增殖能力的来源于鳍的细胞GFF的影响进行了试验；并在其进行金鱼疱疹病毒接种、病毒传代培养等试验和电子显微镜观察。结果不论是高倍稀释，还是在低浓度小牛血清培养液H－MEM－5中培养，细胞均具有足够的增殖速率，而且冷冻保存细胞在解冻后的培养仍然具有较好的增殖能力，细胞的存活率在85％以上，使此细胞的冷冻保存成为可能；同时接种细胞出现了CPE且病毒传代培养的细胞内和培养液中均观察到病毒颗粒，表明GFF细胞可以支持病毒的增殖，为研究金鱼病奠定了基础。     

更昔洛韦眼用凝胶和阿昔洛韦滴眼液治疗单疱病毒性角膜炎疗效观察

目的探讨更昔洛韦眼用凝胶和阿昔洛韦滴眼液治疗单疱病毒性角膜炎的临床效果.方法单疱病毒性角膜炎患者104例均为单眼发病.根据治疗方案分为两组,采用阿昔洛韦滴眼液治疗患者52例为对照组,采用更昔洛韦眼用凝胶治疗患者52例为观察组,疗程3周,随访1a,比较两组患者的治疗时间、治疗效果、不良反应及复发情况.结果观察组患者浅层型、深层型、混合型单疱病毒性角膜炎治疗时间均明显少于对照组,观察组患者总有效率明显高于对照组,观察组患者不良反应发生率、复发率均明显低于对照组,差异具有统计学意义（P〈0.05）.结论更昔洛韦眼用凝胶和阿昔洛韦滴眼液均是治疗单疱病毒性角膜炎的有效药物.更昔洛韦眼用凝胶的治疗效果更好,可明显缩短患者的治疗时间,不良反应少,且复发率低,值得临床推广使用.     

禽疱疹病毒活载体疫苗研究进展

禽疱疹病毒主要包括α疱疹病毒亚科的鸡马立克氏病毒( Mareks disease virus, MDV)、传染性喉气管炎病毒( infectious laryngotracheitis virus, IBDV )和鸭肠炎病毒( duck enteritis virus, DEV )。作为疱疹病毒家族的成员，禽疱疹病毒基因组庞大，存在多个复制非必需区，可容纳多个外源基因的插入，是表达其他禽类病原抗原基因的理想载体。总结了禽疱疹病毒的构建方法、外源基因表达水平，并对MDV、IBDV和DEV 三种禽疱疹病毒的不同重组病毒疫苗免疫效果评价进行归纳，旨在为畜禽传染病防治提供参考依据。