湖南部分地区犬狂犬病病毒的监测研究

对湖南部分地域的8个县(市、区)的8个城区、乡镇和农村的健康犬抽样采集脑组织标本142份,应用RT-PCR和直接荧光抗体试验(FAT)检测,检测结果142份犬脑样中均不含狂犬病病毒。调查上述监测点及周边近三年狂犬病的免疫情况,免疫率均超过70%。表明湖南部分犬免疫工作较好的地区,通过加大群免疫密度,可以使犬狂犬病病毒携带率降低,这可能与形成免疫屏障有关。     

2010—2011年广西狂犬病监测及免疫效果分析

为了解当前广西家犬狂犬病流行情况,评价防控效果,采集免疫过狂犬病疫苗的犬血清2 972份进行抗体检测。结果:抗体阳性的有2 620份,阳性率为88.16%。另外,采集交易市场或屠宰场表观健康犬的脑组织样品800份,疑似发病犬脑组织样品25份,分别通过RT-PCR方法和小鼠接种试验检测狂犬病病原,结果表观健康犬有11份样品呈阳性,阳性率1.38%,疑似狂犬病犬有5份样品呈阳性,阳性率20%。实施狂犬病疫苗免疫后,广西家犬总体免疫抗体水平良好,表观健康犬携带狂犬病病毒比率显著降低。提示应继续加强农村犬的免疫和管理,有效降低狂犬病的发生。     

2012-2013年广西犬狂犬病监测及免疫效果分析

为了解当前广西家犬狂犬病流行情况,评价防控效果,采集免疫过狂犬病疫苗的犬血清2 325份进行抗体检测,抗体阳性的有1571份,阳性率为67.57%。另外,采集交易市场或屠宰场表观健康犬的脑组织样品1 012份,通过RT-PCR方法检测狂犬病病原,结果表观,健康犬1份样品呈阳性,阳性率为0.099%。结果表明,实施狂犬病疫苗免疫后,广西家犬总体免疫抗体水平良好,表观健康犬携带狂犬病病毒比率下降到极低水平。提示应继续加强农村犬只的免疫和管理,有效降低狂犬病的发生。     

玉林市犬狂犬病流行状况调查

通过调查问卷方式得知2010-2016年玉林市犬只饲养量年均20.13万头,免疫犬年均为13.64万头,免疫密度年均为67.74%,仍存在未经免疫的犬。ELISA方法检测免疫犬抗体阳性率为91.57%(1509/1648),非免疫犬为4.13%(10/242);RT-PCR方法检测520份犬脑组织,病原检出率为5.00%(26/520)。通过数据分析与实际情况结合,表明玉林市犬狂犬病免疫抗体合格率较高,疫苗效果表现稳定,外观健康犬仍携带狂犬病病毒。     

酶联免疫吸附试验夹心法检测梅花鹿狂犬病病毒抗原的研究

以马抗狂犬病固定毒抗体建立的酶联免疫吸附试验夹心法,检测了动物脑组织中的梅花鹿狂犬病病毒抗原。对已知阳性和阴性鼠脑抗原的检测表明,本法具有敏感、特异、快速,简便等特点。实验室检测了50份阳性鼠脑抗原及40份阴性鼠脑抗原,其消光值(OD值)均数(X)分别为0.928和0.209。对5只鹿、3头牛和4只羊脑组织的检测结果皆与其它诊断结果相符;对15只犬的检测发现,其敏感性远远高于补体结合试验的敏感性。     

广西家犬及蝙蝠狂犬病病毒监测与分析

为了解广西狂犬病病毒(RV)感染与分布情况,本研究对采集自广西境内9个市31个县(区)1 007份犬脑组织、1 324份犬唾液拭子及564只野生蝙蝠进行RV检测,结果显示有6个县的犬脑组织检测出RV,阳性率为0.65%(1/153)~5.38%(5/93),县(区)阳性率为27.27%(6/22),广西划分的5个地理区域均有分布。外观健康犬和疑似发病犬脑组织平均阳性率分别为1%(10/1 005)和100%(2/2)。犬唾液拭子和野生蝙蝠均未检测到RV。结果表明广西家犬的RV感染率存在一定的地域性差异,平均感染率较低,但个别县(区)连续几年阳性率均处于较高水平,应加强对犬只的管理和疫苗免疫。     

河南省南阳地区黄牛狂犬病防制研究

由河南省南阳地区患“怪叫病”黄牛脑组织分离鉴定了5株狂犬病病毒,建立了检测狂犬病病毒抗原的夹心间接斑点酶联免疫吸附试验、检测狂犬病病毒抗体和中和抗体的夹心阻断酶联免疫吸附试验和微量免疫酶试验.应用所建立的方法,结合小鼠中和试验,对疫区牛、马、猪、羊、犬、猫、鸡、鼠和蝙蝠9种动物的1138份血清标本进行了检测,结果发现阳性率达12.65%,其中疫点内牛、猪、犬、猫和鼠的阳性率更高,为20%左右.根据动物群中较高阳性率,亦即隐性感染动物的存在,提出了南阳地区黄牛狂大病除因疯犬或带毒犬咬伤所致者外,可能还有因其他动物如鼠等咬伤甚至非咬伤感染途径的存在.在确定病性以及上述流行病学调查的基础上,实施了以管(制)、免(疫)、灭(扑杀)为中心的综合防制措施,经3年的工作,收到了明显的防制效果.     

成都市区犬猫狂犬病病毒和弓形虫感染的调查

为了解四川省成都市区家养犬、猫狂犬病病毒及弓形虫的感染情况,采用商品化狂犬病病毒和弓形虫抗原快速检测试纸卡,对2010年8～10月间来自成都市区的103份家养犬以及75份家猫的唾液和血液样品进行检测;同时采用文献报道的PCR方法对家养犬血液样品中的弓形虫核酸进行检测。结果显示,103份家养犬唾液样品狂犬病病毒抗原均为阴性,而75份家猫唾液样品中,检出阳性样品5份(阳性率6.7%),可疑样品7份;103份家养犬血液样品弓形虫抗原阳性样品33份(32.0%),可疑样品22份,75份家猫血液样品中,检出阳性样品2份(阳性率2.7%),可疑样品3份。弓形虫核酸PCR检测结果显示,96份家养犬血液样品弓形虫核酸阳性样品57份(阳性率59.4%),与弓形虫抗原阳性和可疑样品总和所占比例基本一致(53.4%)。提示应重视源于家猫的狂犬病病毒和家养犬弓形虫对人的威胁。     

重庆市2009-2010年动物狂犬病检测及流行病学调查

用RT-PCR方法检测重庆地区2009—2010年发生的36份临床疑似狂犬病脑组织样品和969份临床健康犬唾液拭子样品,并利用流行病学调查方法对阳性样品进行疫情溯源。结果从20份临床疑似狂犬病样品中检测出狂犬病毒,流行病学调查显示未免疫的流浪犬是主要的传染源,而农民(55.95%)和儿童(30.95%)是最容易受到狂犬攻击的人群。从969份临床健康犬唾液拭子和犬脑组织样品中未检出阳性样品。因此重庆地区狂犬病防控应加强对犬的免疫及流浪犬的管理,实行强制性疫苗免疫,加强对儿童和农民的狂犬病知识宣传和安全防护工作。     

山东省某规模猪场猪伪狂犬病毒变异毒株的分离与鉴定

2015年山东省烟台市某免疫伪狂犬病疫苗Bartha-K61猪场暴发疑似伪狂犬病疫情,临床表现为妊娠母猪繁殖障碍,仔猪神经症状。脑组织病料样品接种BHK21细胞,分离获得1株伪狂犬病病毒(PRV),命名为PRV-YT株。该病毒株gE基因与JS-2012、HN1201和ZJ01株序列同源性为99.4%～99.8%,且位于同一遗传进化分支。选择70日龄PRV阴性健康猪接种PRV-YT株,发病率和死亡率均达100%(5/5),均表现严重脑组织、肝脏与肺脏损伤,表明PRV-YT株为猪伪狂犬病病毒变异强毒株。本研究为制定猪伪狂犬病防控方案提供了重要基础。     

Spill-over of European bat lyssavirus type 1 into a stone marten (Martes foina) in Germany

European bat lyssavirus type 1 (EBLV-1, genotype 5) is known to endemically circulate in insectivorous bat populations in Germany. In August 2001, a rabies suspect stone marten (Martes foina) was found in the city of Burg (Saxony-Anhalt, Germany) and was sent to the regional veterinary laboratory for routine rabies diagnosis. Whereas brain samples repeatedly tested negative in the fluorescent antibody test for classical rabies virus (genotype 1), the mouse inoculation test and the rabies tissue culture inoculation test yielded positive results. Rabies viral RNA was also detected in the stone marten brain sample both by nested and heminested RT-PCR specific for the nucleoprotein gene and for the nucleoprotein phosphoprotein junction of rabies virus. The amplification products were sequenced to genotype the isolate. Sequence data obtained from the first-round RT-PCR products were analysed and the suspect stone marten isolate was confirmed as a rabies related virus (EBLV-1a). Phylogenetic comparison with sequences from recent genotype five isolates from Germany and Denmark showed that it was closely related to a previous isolate of EBLV-1 from a serotine bat in Saxony-Anhalt obtained in the same year in an area adjacent to the place where the EBLV-1 infected stone marten was found. Both EBLV-1 isolates share a 99.5% identity. This is the first report of an EBLV-1a spill-over from an insectivorous bat into wildlife in Europe.     

Spill‐over of European Bat Lyssavirus Type 1 into a Stone Marten (Martes foina) in Germany

European bat lyssavirus type 1 (EBLV‐1, genotype 5) is known to endemically circulate in insectivorous bat populations in Germany. In August 2001, a rabies suspect stone marten (Martes foina) was found in the city of Burg (Saxony‐Anhalt, Germany) and was sent to the regional veterinary laboratory for routine rabies diagnosis. Whereas brain samples repeatedly tested negative in the fluorescent antibody test for classical rabies virus (genotype 1), the mouse inoculation test and the rabies tissue culture inoculation test yielded positive results. Rabies viral RNA was also detected in the stone marten brain sample both by nested and heminested RT‐PCR specific for the nucleoprotein gene and for the nucleoprotein phosphoprotein junction of rabies virus. The amplification products were sequenced to genotype the isolate. Sequence data obtained from the first‐round RT‐PCR products were analysed and the suspect stone marten isolate was confirmed as a rabies related virus (EBLV‐1a). Phylogenetic comparison with sequences from recent genotype five isolates from Germany and Denmark showed that it was closely related to a previous isolate of EBLV‐1 from a serotine bat in Saxony‐Anhalt obtained in the same year in an area adjacent to the place where the EBLV‐1 infected stone marten was found. Both EBLV‐1 isolates share a 99.5% identity. This is the first report of an EBLV‐1a spill‐over from an insectivorous bat into wildlife in Europe.     

检测狂犬病病毒抗原的夹心间接Dot—ELISA的建立和应用研究

本研究建立了检测狂犬病病毒抗原的夹心间接斑点酶联免疫吸附试验(SI-Dot-ELISA).本法检出狂犬病病毒抗原的最低浓度为:0.01IU/mL的标准抗原,1:100000 (相当于20 LD_(50))的狂犬病病毒CVS株和鹿8202株的鼠脑悬液,1:400(相当于7906TCID_(50)/mL)的狂犬病病毒SAG株的细胞培养物.对多种健康动物的组织、健康细胞培养物以及犬瘟热病毒、犬腺病毒等检测均为阴性.用SI—Dot—ELISA、夹心间接ELISA(SI—ELISA)和小鼠脑内接种3种方法,检测了388份材料,检出的阳性份数分别为173、171和179,经统计学处理,3种方法在狂犬病病毒抗原的检测上,可以相互代替.甲醛灭活病毒不影响本方法的检测,而加入氢氧化铝胶后的病毒悬液则不能用本法检测.本法适用于狂犬病的诊断、流行病学调查、疫苗效价的测定和实验研究中病毒抗原的测定.     

Increased detection of rabies virus in bats in Ceará State (Northeast Brazil) after implementation of a passive surveillance programme

The intensification of dog, cat and livestock vaccination campaigns significantly reduced rabies cases in humans and domestic animals in Ceará State, Brazil. However, sylvatic animals—bats (order Chiroptera), wild canids, raccoons and non‐human primates— remain as reservoirs for the virus. Our hypothesis is that surveillance and monitoring of rabies virus in bats, especially passive surveillance, is of fundamental importance, besides the implementation of health education and strengthening of surveillance actions in humans exposed to aggressions. Thus, we assessed the occurrence of rabies virus in animals focusing on bats, before and after launching of the Sylvatic Rabies Surveillance Program in 2010. Surveillance data from the 184 municipalities of Ceará State were analysed, collected during the periods 2003–2010 (active surveillance) and 2011–2016 (passive surveillance), respectively. A total of 13,543 mammalian samples were received for rabies diagnosis from 2003 to 2016. Of these, 10,960 were from dogs or cats (80.9%), 1,180 from bats (8.7%), 806 from other sylvatic animals (foxes, marmosets, raccoons; 6.0%) and 597 from herbivores (cattle, goats, sheep, equines, pigs; 4.4%). A total of 588 (4.3%) samples were positive for rabies. About 8.4% (99/1,180) of the bat samples were infected with rabies virus, 92 (92.9%) of these were from non‐haematophagous bat species and 7 (7.1%) from haematophagous species. The number of bat samples received and infection rates increased considerably, after a shift from active surveillance (9/355 [2.5%] samples positive), to passive surveillance (90/825 [10.9%] samples positive). Surveillance of rabies virus in bats is fundamental for human and domestic animal health in Ceará State. Bats have to be considered as targets in surveillance and control programmes. Virus lineages should be characterized to increase knowledge on transmission dynamics of sylvatic rabies virus to domestic animals and the human population, and to provide additional evidence for planning and implementation of improved control measures.     

Demonstration of rabies viral antigen in paraffin tissue sections: comparison of the immunofluorescence technique with the unlabeled antibody enzyme method

The immunofluorescence technique and the peroxidase-antiperoxidase method were used to demonstrate rabies antigen in a retrospective study on formalin-fixed, paraffin-embedded brain tissues from 34 naturally infected wild and domestic animals. Rabies was confirmed with immunofluorescent staining on fresh brain tissue at the time of necropsy of the animals. There was a perfect correlation (serial sections from a given brain area were always positive by both methods), but the peroxidase-antiperoxidase technique was preferred, since no trypsin digestion was required. Twenty six of the 34 animals were immunohistochemically positive and had encephalitis, and in 21 of these 26, the hematoxylin and eosin-stained sections contained detectable intracytoplasmic inclusion bodies in at least 1 brain area. Of the remaining 8 animals (with no inflammatory lesions), 7 were positive for rabies antigen and 2 had no inclusion bodies. Rabies antigen was apparent in 62% of the brain areas in which inclusion bodies were not found in the corresponding hematoxylin and eosin stained sections. Thus, together with the inclusion body positive areas, which were all immunohistochemically positive, it was possible to diagnose rabies in a total 84% of the areas examined. Both techniques greatly facilitate the diagnosis of rabies and may be a reliable help to the diagnostic pathologist when only formalin-fixed tissues are available. However, the methods should not be considered substitutes for the immunofluorescence technique and the mouse inoculation test with fresh brain tissue.