我国和欧盟兽药质量监督抽检工作机制对比研究

为更好地完善我国兽药质量监督抽检计划,通过对欧盟药品抽检管理工作机制的研究,本文分析了欧盟和我国兽药监督抽检管理方式的不同,从抽检责任部门、抽检目的、抽检计划内容、抽检计划制定流程、抽检品种的遴选和检测参数的确定等6方面进行了对比分析,探讨我国兽药质量监督抽检工作的管理中可以借鉴的方面。     

A/H1N1研究进展

2009年3以来,包括墨西哥、美国和加拿大在内的许多国家发生了甲型H1N1流感[Swine-origin Influenza A (A/H1N1)]疫情,WHO已于2009年6月20日将此次流感流行的预警级别提升至6级.现已基本明确,引起此次流感疫情的A/H1N1流感病毒是猪流感病毒(Swine Influenza Virus, SIV)的一种新型变异株.此次流感疫情的发生,再次使猪流感成为社会各界关注的焦点之一.本文就甲型H1N1流感的临床表现、病毒特征、相互关系及其对动物卫生监督工作的影响等作一综述.     

H1N1亚型猪流感病毒A/Swine/GD/2/12基因特征分析

2012年从广东省某猪场的疑似流感猪群采集鼻拭子样品,接种鸡胚并收集尿囊液,通过血凝、血凝抑制和RT-PCR,鉴定出1株H1N1亚型猪流感病毒,命名为A/Swine/GD/2/12。RT-PCR扩增得到全基因8个片段,与GenBank收录的参考毒株比对并构建进化树,发现本毒株可能是H1N1亚型重组株,其8个片段与我国猪源和北美地区1985—1992年间的猪源、禽源(A/turkey/IA/1992)和人源(A/Maryland/12/1991)流感毒株在同一个大分支上,其中与我国猪源参考株同源性在95.8%以上,与北美地区的H1N1参考株同源性在94%以上。HA受体位点分析表明,本毒株既具备结合Saα2,6Gal型人类流感病毒SA受体的特点,也有结合Saα2,3Gal型禽类流感病毒SA受体的可能。提示本毒株可能是由北美地区猪源、禽源和人源的H1N1亚型流感病毒重排形成的。HA蛋白裂解位点分析、NS和PB2蛋白位点分析表明,本毒株具备低致病性毒株的特点。小鼠致病性试验进一步证实本毒株能够引起小鼠运动减少、食欲欠佳、体重减轻等表现,但不会引起咳嗽和死亡等严重反应。     

Susceptibility of bacterial pathogens against lincomycin/spectinomycin (1/2), penicillin G/neomycin (1/1), and penicillin G/dihydrostreptomycin (1/1) as determined in the BfT-GermVet monitoring program 2004-2006

A total of 368 bacterial pathogens, including 72 coagulase-positive and coagulase-variable staphylococci, 97 beta-haemolytic streptococci, 51 Escherichia coli, 75 Pasteurella multocida, 25 Mannheimia haemolytica, 25 Pseudomonas aeruginosa, and 23 Arcanobacterium pyogenes, were investigated for their susceptibility to the three combinations of antimicrobial agents lincomycin/spectinomycin (1/2), penicillin G/neomycin (1/1), and penicillin G/dihydrostreptomycin (1/1) in comparison to their susceptibility to the corresponding single substances. When comparing the minimum inhibitory concentrations (MICs) determined for any of the three combinations with those for the single substances, the lowest MIC of one of the two substances usually determined the MIC of the combination.This observation was made for all three combinations and all bacterial pathogens tested.Thus, it is assumed that the combination of lincomycin with spectinomycin as well as that of penicillin with either neomycin or dihydrostreptomycin resulted in an extended spectrum of target bacterial pathogens rather than in an increase in antimicrobial efficacy.     

H5N1亚型猪流感病毒A／Swine／Fujian／1／01感染性克隆构建及其对小鼠致病性研究

2001年从福建某猪场分离到1株H5N1亚型猪流感病毒(SIV)A/Swine/Fujian/1/01(SW/FJ/1/01).SW/FJ/1/01对小鼠具有强致病性,致死率为100%,为进一步研究SW/FJ/1/01对BALB/c小鼠致病性的分子基础,本研究构建了SW/FJ/1/01的8个节段重组质粒构成的反向基因操作系统,成功拯救了病毒(R-SW/FJ/1/01).R-SW/FJ/1/01和野生型SW/FJ/1/01对BALB/c小鼠致病性没有差别.SW/FJ/1/01反向基因操作系统的建立为进一步阐明H5N1亚型SIV对哺乳动物模型BALB/c小鼠的致病机制等方面的研究奠定了基础.     

猪流感病毒分离株A/swine/Shanghai/3/2014(H1N1)分子特性研究

采用套式PCR检测方法,结合鸡胚分离鉴定,从20份患呼吸道疾病猪的鼻咽拭子样品中分离到1株流感病毒。经亚型鉴定及全基因组序列分析,证实该分离株为H1N1流感病毒,命名为A/swine/Shanghai/3/2014(H1N1)。遗传进化分析表明该分离株与类禽猪流感病毒(Avian-Like H1N1)相似性最高。蛋白序列分析发现,该分离株具有低致病性流感病毒特征,即其HA蛋白的裂解位点为PSIQSR↓GLFGAI。此外,该基因中有7个潜在的糖基化位点,受体结合位点为108(Y)、148~152(GVTAA)、167(W)、197(H)、204~212(DQQ SLYQNA)和238~243(RDQEGR);其中225~228EQAG显示该分离株具有结合SAα2,6Gal受体的能力,证明该分离株具有感染哺乳动物的能力。同时PB2蛋白的627E、701N及NS蛋白的92D位点均证明了该分离株的低致病性和感染哺乳动物的能力。     

H1N2亚型猪流感病毒A／Swine／Guangdong／1／06分离鉴定和对猪致病性研究

2006年5月从广东某大型猪场采集具有流感症状保育猪鼻拭子共98份,无菌常规处理猪鼻拭子后接种MDCK细胞,分离到4株流感病毒.用血凝抑制和PCR方法鉴定均为H1N2亚型.挑选其中一株A/Swine/Guangdong/1/06(H1N2),进行全基因组测序,并与GenBank中相关序列进行比较.核苷酸同源性分析表明:A/Swine/Guangdong/1/06(HIN2)与A/Swine/Guangxi/13/06不同基因之间同源性为96.6%～98.1%.以106EID50的剂量,将H1N2病毒鼻腔感染35日龄仔猪,结果表明H1N2亚型流感病毒可以感染猪上呼吸道.但不表现临床症状.本研究结果对于揭示H1N2亚型猪流感流行规律和病毒的致病机理具有一定的意义.     

瘤胃调节剂在反刍动物生产中的应用研究进展

随着反刍动物规模化养殖的发展,高精料饲喂是促进反刍动物快速生长的常用策略,但这通常会导致动物瘤胃代谢紊乱等问题。瘤胃调节剂作为一类高效、安全、价格低廉的营养代谢调控剂在反刍动物饲养中得到广泛应用,但其使用的效果和作用机制目前还存在着不同的观点。本文综述了瘤胃调节剂在反刍动物中应用的国内外研究进展,以期为今后开展瘤胃代谢调控研究提供参考。     

Morphologic heterogeneity of a strain of swine influenza virus (A/swine/Wisconsin/1/68, Hsw1N1) propagated at different temperatures.

Strain A/swine/Wisconsin/1/68 (WI/68) swine influenza virus (SIV) was propagated in embryonating chicken eggs at 33, 35, or 37 C. The SIV harvested from eggs incubated at 33 C invariably had higher hemagglutination (HA) and egg infectivity titers than did SIV propagated in eggs at the 2 higher temperatures. When SIV inoculum propagated at 33 C was inoculated into separate groups of eggs and incubated at 33, 35, and 37 C, the SIV harvested from inoculum incubated at the 2 higher temperatures had significantly lower infectivity and HA titers than did that propagated at 33 C. By electron microscopy (EM), viral particles of Wi/68 were of various sizes and shapes regardless of the temperature used to propagate the virus. However, in contrast to what was seen in SIV harvested from innoculum incubated at 33 C incubation, pleomorphic shapes and particles with surface abnormalities were much more frequent in SIV harvested from inoculums kept at the 2 higher temperatures. Approximately one-third of the particles from 35 and 37 C incubation either were spikeless or were relatively deficient in surface spikes.     

H5N1亚型禽流感病毒A/Guangxi/1/2005株抗药机制的研究

为研究H5N1亚型禽流感病毒(AIV)的抗药机制,本研究选取Clade2.3.4亚群中一株对金刚烷胺敏感的人源AIV A/Guangxi/1/2005(H5N1)(S-GX05),用抗流感病毒药物金刚烷胺对其进行定向诱导,筛选出一株抗药性病毒株,命名为R-A/Guangxi/1/2005(R-GX05)。通过全基因测序并与S-GX05全基因序列进行对比,结果显示S-GX05只在其M2蛋白中有一个氨基酸位点发生突变,即A30P;抗药性鉴定这两株病毒的半数药物抑制浓度(IC50)分别为0.9μM和48.9μM,表明R-GX05对金刚烷胺表现出一定程度的抗性。动物实验证实,这两株病毒对BALB/c小鼠的致病性基本一致,均表现出高致病性,其MLD50分别为4.7 log10 EID50和5.0 log10 EID50,两株病毒在小鼠体内各组织脏器中的分布及增殖能力也基本相同。这些结果表明,S-GX05在药物压力下产生抗药性后,并未引起其它生物学特性的改变。A30P的发现为进一步从分子水平上研究H5N1亚型AIV的抗药机制及新型抗流感新药的研发奠定了基础。     

新城疫病毒分离株APMV1/chicken/China/JL-11/02的致病性研究

将在吉林省分离到的一株新城疫病毒APMV1／chicken／china／JL-11／02(基因Ⅶ型)蚀斑纯化后，与我国标准毒株F48E9分别接种42日龄无抗体SPF雏鸡和已获得LaSota疫苗、Clone30疫苗(基因Ⅱ型)抗体的高免雏鸡(抗体效价为1：64～1：128)。结果表明无抗体雏鸡感染JL—11和F48E9后，144h内全部死亡，病死鸡的大部分组织中都检测到病毒；但JL—11与F48E9对高免抗体鸡致死效果不同，JL—11的致死率为28．5％，F48E9的致死率为7．14％。由此可见，生产中常用的ND疫苗并不能完全保护目前流行株的攻击，这可能是养禽生产中高免抗体鸡群发生免疫失败的主要原因之一。为此建议应在传统疫苗免疫的基础上进行新型流行株灭活苗的加强免疫，以提高免疫效果，降低ND的发生率。     

First recovery of A/equine/Fontainebleau/1/79 influenza viruses in Italy

In Italy epizootics of equine influenza often occur, but no virus isolation has been reported since 1971. This paper describes the antigenic and biochemical characterization of two equine influenza viruses isolated in Italy from 1985 to 1989. The virus isolates were shown to differ antigenically from earlier strains of the same subtype, A/equine/Miami/1/63 (H3N8). Monoclonal antibody analysis showed that the haemagglutinins of these strains were serologically indistinguishable from A/equine/Fontainebleau/1/79, a variant of A/equine/Miami, never isolated in Italy before. One of the two virus isolates was obtained from a horse immunized with a bivalent inactivated influenza vaccine, not containing A/equine/Fontainebleau/79 antigens.

The vaccine failure underlines the importance of antigenic relatedness between currently circulating viruses and vaccine strains. Therefore, to improve the protection afforded by equine immunization, the vaccine composition should be decided according to the results of a virological surveillance activity, systematically conducted among horses.     

H5N1亚型禽流感病毒A/goose/Guangdong/1/96的NS基因对鸡致病能力的影响

为了解H5N1亚型禽流感病毒经自然途径感染SPF鸡后，病毒的致病能力与NS基因的关系，本文主要从病理学角度比较了两株利用反向基因操作技术拯救的病毒RGSGD／1／96和RGSGD／1／2NS的致病能力。虽然只有NS基因不同，但是这两株病毒经鼻腔感染4周龄SPF鸡后表现出完全不同的致病能力，RGSGD／1／96对鸡的致死率为100％，感染鸡只的各组织脏器均可发现严重的病理损伤，该病毒在鸡体内复制能力很强，感染后3d、6d，各组织脏器均可发现大量的病毒抗原；GSGD／1／2NS对鸡的致死率为0，病毒在感染鸡体内只引起肺间质少量淋巴细胞浸润，免疫组织化学检查未发现病毒抗原信号。由于两株病毒只有NS基因不同，说明NS基因决定了H5N1亚型禽流感病毒A／goose／Guangdong／1／96对SPF鸡的致病能力。     

Pandemic A/H1N1/2009 influenza virus in swine, Cameroon, 2010

Although swine origin A/H1N1/2009 influenza virus (hereafter "pH1N1″) has been detected in swine in 20 countries, there has been no published surveillance of the virus in African livestock. The objective of this study was to assess the circulation of influenza A viruses, including pH1N1 in swine in Cameroon, Central Africa. We collected 108 nasal swabs and 98 sera samples from domestic pigs randomly sampled at 11 herds in villages and farms in Cameroon. pH1N1 was isolated from two swine sampled in northern Cameroon in January 2010. Sera from 28% of these herds were positive for influenza A by competitive ELISA and 92.6% of these swine showed cross reactivity with pandemic A/H1N1/2009 influenza virus isolated from humans. These results provide the first evidence of this virus in the animal population in Africa. In light of the significant role of swine in the ecology of influenza viruses, our results call for greater monitoring and study in Central Africa.     

猪流感病毒A/swine/Guangdong/2/2012(H1N1)毒株攻毒BALB/c小鼠细胞因子动态变化的研究

为了测定H1N1亚型猪流感病毒(swine influenza virus,SIV)对小鼠的致病性,本试验对A/swine/Guangdong/2/2012(H1N1)株SIV HA基因进行克隆及遗传分析,并将SIV尿囊液经鼻腔感染6周龄BALB/c小鼠,观察感染后小鼠的一般状况、器官系数和组织病理学变化,在病毒感染后第1、3和7天使用荧光定量PCR测定小鼠肺脏、脾脏、脑组织中7种细胞因子mRNA的表达量,研究其对小鼠的致病特性。结果显示,该病毒属于经典SIV,病毒经鼻腔感染后可引起小鼠活动减少、采食量降低,但无咳嗽和死亡;病理组织学变化为肺间隔较正常组织明显增厚,毛细血管明显扩张充血,周围肺泡腔呈代偿性肺气肿;小鼠肺脏、脾脏组织样本中IFN-α、IFN-β、IP-10、IL-1β、TNF-α、IRF-3和IL-10 mRNA含量在感染后第3天均显著升高(P<0.05),而脑组织样本中IL-1β和IL-10在小鼠攻毒后第3和7天均显著上调(P<0.05)。     

禽流感病毒分离株A/Goose/Guangdong/1/96(H5N1)全基因克隆及序列分析

本研究用无特定病原体（ＳＰＦ）鸡胚增殖禽流感病毒鹅体分离株Ａ／Ｇｏｏｓｅ／Ｇｕａｎｇｄｏｎｇ／１／９６（Ｈ５Ｎ１）（ＧＤ１／９６）,提取病毒基因组总ＲＮＡ,运用反转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术分别扩增该病毒分离株的８个基因片段的ｃＤＮＡ,分别克隆后进行序列测定。序列分析结果表明,所扩增到的８个片段均包含相应的病毒基因的完整开放阅读框架。ＧＤ１／９６株与１９９７年香港禽流感事件中的４株香港流感病毒分离株（分别来自人、鸡、鸭和鹅）的ＨＡ基因的高度同源性说明它们可能起源于同一种系,但ＮＳ基因的差异说明它们分属于不同的基因群系。ＧＤ１／９６株与香港流感病毒分离株的核苷酸和推导的氨基酸序列的同源性较低（６３．９％￣９８．１％）,而香港流感病毒分离株之间的核苷酸和氨基酸序列的同源性很高（９６．５％￣１００％）,且香     

禽流感分离株GD/1/96(H5N1)非结构蛋白结构建模

为预测H5N1亚型禽流感病毒(AIV)NS1蛋白全长结构,本研究以A/Goose/Guangdong/1/96(H5N1)株NS1蛋白氨基酸序列为目标序列,通过同源建模以及蛋白质结构叠合预测出NS1蛋白全长单体以及二聚体初始结构,采用约束分子动力学方法对初始结构进行优化,并利用立体化学、折叠可靠性、残基包装质量等评判方法对优化后的模型进行评估。结果表明所建立的NS1蛋白全长单体及二聚体结构模型具有很高的可信度,可用于基于结构的功能研究及虚拟筛选等工作。     

禽流感病毒株A/duck/Fujian/31/2007(H5N1)株M1基因的克隆及序列分析

参照GenBank公布的禽流感病毒基质蛋白M1基因序列,设计合成了1对特异性引物,采用RT-PCR法成功扩增并克隆了禽流感病毒M1基因。序列测定结果为M1基因cDNA全长759 bp,编码252个氨基酸。将其序列与数株甲型流感病毒M1基因序列进行比较,M1基因核苷酸同源性为99.1%~99.6%,推导氨基酸同源性为98.8%~99.6%,生物信息学分析表明,M1基因编码的蛋白质具有亲水性,有很强的抗原性,M1蛋白共有6个潜在的N-糖基化位点,可能存在3个蛋白激酶C磷酸化位点,存在2个酪蛋白激酶Ⅱ磷酸化位点。