鸭肝组织中DHBcAg表达水平定量免疫组织化学方法的建立

目的：建立鸭肝组织中鸭乙肝病毒核心抗原（DHBcAg）表达水平定量的免疫组织化学方法。方法比较热修复、微波修复、胃蛋白酶修复和不修复四种不同抗原修复方法的免疫组化检测技术,建立 DHBcAg抗原修复的最佳方案。采用Image-Pro Plus 6.0软件,设置优化图像采集参数,通过扣除空白区域的面积计算二氨基联苯胺（DAB）黄染的平均光密度（MD）,客观评价不同切片中阳性表达的程度。采用该方法对 DHBV感染的鸭肝组织中 DHBcAg表达水平进行定量,评价DHBcMAb-TAT PTD穿膜抗体的抗病毒作用。结果抗原不修复的方式进行 DHBcAg免疫组化染色优于其他三种抗原修复的方法。建立优化的图像采集参数,进行DHBcAg抗原表达的 MD值计算。比较受试麻鸭治疗前后肝组织中DHBcAg的 MD差值,结果显示随着DHBcMAb-TAT PTD穿膜抗体给药剂量的增加,鸭肝细胞内 DHBcAg的表达逐渐降低,存在明显剂量依赖关系。表明该方法可有效评价药物对DHBcAg的影响。结论建立DHBcAg抗原不修复的定量免疫组织化学方法可以更加客观、准确的评价DHBcAg表达水平。     

万乃洛韦抗鸭乙型肝炎病毒的实验研究

目的 观察万乃洛韦在体内抗鸭乙型肝为病毒（DHBV）的作用,为临床治疗乙型肝炎病人提供实验依据。方法 采用重庆麻鸭乙型肝炎动物模型,用万乃洛韦治疗1个月,检测用药前后血清中的DHBV DNA及转氨酶、肝转氨酶、肝细胞病理、肝细胞中的DHBV DNA等。结果 万乃洛韦用药1月能使血清DHBV DNA总体水平显著降低（P＜0．05或P＜0．01）,各剂量组血清DHBV DNA有阴转现象。肝脏病理检查及血清转氨酶 检查发现,万乃洛韦对肝组织无明显影响,大剂量用药组ALT虽有轻度升高,但停药后即恢复正常。Southem Blot检测肝组织内DHBV DNA,提示该药能抑制病毒在肝内的复制,但不能彻底清除超螺旋DNA。结论 万乃洛韦在体内有抗鸭乙型肝炎病毒的作用。     

Comparative study of DHBV DNA levels and endogenous DNA polymerase activity in naturally infected ducklings in France

Duck hepatitis B virus (DHBV) was found in the serum of 1-6% of Pekin ducklings originated from French commercial flocks. The viremia was followed in the serum of 5 ducklings over a span of 3 mth by monitoring the levels of DHBV DNA and the endogenous DNA polymerase (DNAp) activity. The DHBV DNA levels in serum were quantified either by the DNA dot hybridization technique including counting of retained radioactivity, or by successive dilutions of each serum sample followed by DNA hybridization. The counting of the retained radioactivity was plotted on a curve and its evolution compared with that of viral DNAp activity. DHBV DNA levels in serum, estimated by both methods paralleled those of the DNAp activity, which peaked at the 4th or 5th week posthatch to decrease and fluctuate thereafter. Occasional discordance between DHBV DNA levels and the endogenous DNAp activity was observed, which could be correlated with the degree of repair of the single stranded gap of serum DHBV DNA. Parallel follow up studies comparing quantitative estimations of serum viral DNA and of DNAp activity, as presented here, may provide some clues for the understanding of the mechanisms involved in the establishment of the HEPA DNA virus carrier state. Such comparative studies may also be crucial for optimal monitoring of antiviral drugs in both human clinical trials and animal experimental studies.     

基于物种特异性PCR方法的鸡内金真伪鉴别

目的:建立鉴别鸡内金及其常见伪品的物种特异性聚合酶链式反应(PCR)方法,对全国中药材及饮片专项抽验任务中所抽取的鸡内金饮片进行真伪鉴别,评价市场上鸡内金的真伪情况。方法:根据鸡、鸭、鹅的12S序列差异,设计或优化物种特异性PCR鉴别引物,对影响PCR结果的主要因素退火温度,PCR循环次数,模板DNA浓度,Taq酶种类等进行方法学考察和优化。使用优化的特异性PCR方法对鸡、鸭、鹅内金样品进行DNA分子鉴别。结果:在进行方法学考察确定最优鉴别条件的基础上,当PCR退火温度为55℃,循环次数为30次的条件下,当使用文中筛选得到的鸡物种特异性鉴别引物时,所测鸡内金饮片均检出约为273 bp的特异性扩增条带,混伪品鸭内金和鹅内金均无相应扩增条带;当使用鸭和鹅鉴别引物时,均未检出相应的扩增条带。结论:位点特异性PCR方法能够快速准确的鉴别出鸡内金真伪品,可用于全国中药材及饮片专项抽验任务中鸡内金的真伪鉴定,目前市场上用鸭内金和鹅内金充鸡内金现象较少,质量评价较好。     

Evidence for Vertical Transmission of Novel Duck‐Origin Goose Parvovirus‐Related Parvovirus

In 2015, novel duck‐origin goose parvovirus‐related parvovirus (N‐GPV) infection progressively appeared in commercial Cherry Valley duck flocks in North China. Diseased ducks were observed to have beak atrophy and dwarfism syndrome (BADS). A previous study showed that a high seropositive rate for N‐GPV indicated a latent infection in most breeder duck flocks. To investigate this possibility in hatching eggs collected from N‐GPV‐infected breeder ducks, 120 eggs were collected at various stages of embryonic development for viral DNA detection and an N‐GPV‐specific antibody test. N‐GPV DNA was present in nine hatching eggs, eleven duck embryo and eight newly hatched ducklings. Of the newly hatched ducklings, 58.33% (21/36) were seropositive. Further, two isolates were obtained from a 12‐day‐old duck embryo and a newly hatched duckling. N‐GPV infection did not reduce the fertilization rate and hatchability. These results indicate possible vertical transmission of N‐GPV and suggest that it may be transmitted from breeder ducks to ducklings in ovo.     

Identification of a common conserved neutralizing linear B-cell epitope in the VP3 protein of waterfowl parvoviruses

ABSTRACT

Waterfowl parvoviruses (WPVs) including goose parvovirus (GPV), novel GPV-related virus (NGPV) and Muscovy duck parvovirus (MDPV) cause significant economic losses and epizootic threat to the waterfowl industries, and little is known about the B-cell epitopes of WPVs. In this study, a monoclonal antibody (mAb) 5B5 against the VP3 protein of NGPV was used to identify the possible epitope in the three kinds of WPVs. The mAb 5B5 had neutralizing activities to the three viruses, and reacted with the conserved linear B-cell epitopes of ⁴³⁸LHNPPP⁴⁴³ in VP3 protein of GPV, NGPV and MDPV. To the authors’ best knowledge, this is the first report on identification of the common conserved neutralizing linear B-cell epitope on VP3 protein of three different WPVs, which would facilitate the development of a novel immunodiagnostic assay for rapid detection of WPV infection.     

Extensive chondroid bone in juvenile duck limbs hints at accelerated growth mechanism in avian skeletogenesis

Modern altricial birds are the fastest growing vertebrates, whereas various degrees of precocity (functional maturity) result in slower growth. Diaphyseal osteohistology, the best proxy for inferring relative growth rates in fossils, suggests that in the earliest birds, posthatching growth rates were more variable than in modern representatives, with some showing considerably slow growth that was attributed to their assumed precocial flight abilities. For finding clues how precocial or altricial skeletogenesis and related growth acceleration could be traced in avian evolution, as a case study we investigated the growing limb diaphyseal histology in an ontogenetic series of ducks which, among several other avian taxa, show a combination of altricial wing and precocial leg development. Here we report the unexpected discovery that chondroid bone, a skeletal tissue family intermediate between cartilage and bone, extensively contributes to the development of limb bone shaft in ducks up to at least 30 days posthatching age. To our knowledge, chondroid bone has never been reported in such quantities and with an ontogenetically extended deposition period in post-embryonic, non-pathological periosteal bone formation of any tetrapod limb. It shows transitional cellular/lacunar morphologies and matrix staining properties between cartilage and woven bone and takes a significant part in the diametric growth of the limb bone shaft. Its amount and distribution through duckling ontogeny seems to be associated with the disparate functional and growth trajectories of the altricial wings vs. precocial legs characteristic of duck limb development. The presence of isogenous cell groups in the periosteal chondroid bone implies that cartilage-like interstitial growth took place before matrix mineralization complementing appositional bone growth. Based on these characteristics and on its fast formation rate in all previously reported normal as well as pathological cases, we suggest that chondroid bone in ducks significantly accelerates diametric limb bone growth. Related to this growth acceleration, we hypothesize that chondroid bone may be generally present in the growing limb bones of modern birds and hence may have key skeletogenic importance in achieving extreme avian growth rates and placing birds among the fastest growing vertebrates. Thus, we encourage future studies to test this hypothesis by investigating the occurrence of chondroid bone in a variety of precocial and altricial bird species, and to explore the presence of similar tissues in the growing limbs of other extant and extinct tetrapods in order to understand the evolutionary significance of chondroid bone in accelerated appendicular skeletogenesis.     

“发物”对H22肝癌小鼠瘤体质量、免疫功能及病理影响的实验研究

目的比较母鸡、草鸭、黄鱼、羊肉4种“发物”对H22肝癌小鼠瘤体质量、免疫功能及肿瘤病理的影响。方法选择BALB／c小鼠30只,5只作为正常组,其余接种H22细胞株复制肝癌模型。模型小鼠随机分为生理盐水组、1号组（母鸡）、2号组（草鸭）、3号组（黄鱼）和4号组（羊肉）,共5组,每组5只;生理盐水组给予0．9％NaCl溶液,其余各组分别给予相应食物的水煎液。观察各组瘤体质量、免疫功能及肿瘤病理变化情况。结果①与生理盐水组比较,1号组（母鸡）和4号组（羊肉）小鼠瘤体质量差异有统计学意义（P〈0．05）,抑瘤率分别达39．9％和35．2％。②与生理盐水组比较,各“发物”干预组荷瘤小鼠CD3＋、CD4＋、CD8＋差异均无统计学意义（P〉0．05）。③与生理盐水组比较,1号组（母鸡）和4号组（羊肉）IFN-γ水平显著升高,差异有统计学意义（P〈0．05）,4号组（羊肉）IL-4水平显著升高（P〈0．01）。④4号组（羊肉）荷瘤小鼠肿瘤细胞多出现较明显坏死,组织中血管少;1号组（母鸡）荷瘤小鼠肿瘤细胞浸润较多,可见病理性核分裂相,细胞排列紧密;2号组（草鸭）、3号组（黄鱼）荷瘤小鼠肿瘤细胞浸润明显,组织中间见较多淋巴细胞浸润,后者较前者肿瘤血管更丰富。结论羊肉适合于肿瘤患者食用,母鸡次之,草鸭再次之,黄鱼则不宜食用。     

苯丙氨酸二肽类化合物Y101对DHBV-DNA的影响

目的探讨苯丙氨酸二肽类化合物Y101对感染鸭乙肝病毒的北京雏鸭体内外DHBV-DNA表达的影响。方法90只感染DHBV的北京雏鸭随机分为对照组、Y101剂量组和拉米夫定组,各组雏鸭于给药前(d 0)、给药5 d(d 5)、10d(d 10)及停药后3 d(P 3)经腿胫静脉取血,分离血清,斑点杂交法测定鸭血清中DHBV-DNA的含量;利用灌流法分取感染鸭乙肝病毒的北京雏鸭的原代肝细胞,采用QuickEx-tract DNA Extraction Soln 1.0提取鸭原代肝细胞Y101剂量组、拉米夫定对照组和正常对照组细胞内和细胞上清中的DHBV-DNA,用TaqMan探针实时荧光定量法检测DHBV-DNA含量,观察Y101对其抑制作用。结果 Y101对体内外DHBV-DNA都具有良好的抑制作用,体内0.05、0.025 g.kg-1剂量组抑制作用明显,体外80、60 mg.L-1剂量组抑制作用最强,DNA含量明显下降(P<0.05)。结论Y101可以有效的抑制DHBV-DNA的表达,抗病毒作用明显。     

拉米夫定联合原儿茶酸对鸭乙肝病毒的体外抑制

目的:研究原儿茶酸(PA)、拉米夫定(3TC)单用以及联合对鸭HBV(DHBV)感染、复制不同时期的影响。方法:利用感染DHBV的鸭原代肝细胞模型,从病毒感染的不同时期研究PA、3TC单用及合用的抗病毒效果。结果:PA、3TC单用及其合用,分别于DHBV感染前,感染的同时及感染后给药,均可有效抑制DHBV对鸭原代肝细胞的感染。虽然PA与3TC合用对DHBV的预防与阻断作用与单用3TC相比没有显著差异,但对DHBV的吸附与进入,感染活性与复制能力,以及肝细胞中AST,ALT酶活的抑制能力均显著强于单用3TC组。结论:PA与3TC药物组合不仅有比二者单用更强的抗DHBV作用,而且对DHBV所致鸭原代肝细胞损伤也有更强的保护作用。