狂犬病快速诊断方法的建立和应用

根据狂犬病病毒核蛋白基因保守区序列设计1对引物，建立了用于狂犬病病毒特异性核酸检测的RT-PCR技术。该技术可从狂犬病病毒CVS株、8202株和SRV9株的含毒细胞培养物及鼠脑组织中。扩增出443bp的核酸片段，检出核酸的敏感性约为3Pg。对30份不同种动物脑组织的检测结果与小鼠脑内接种试验（MIT）的测定结果完全吻合，但前者可在3h内直接对组织匀浆进行诊断，具有快速、简便和敏感的优点。     

狂犬病快速诊断方法的建立和应用

根据狂犬病病毒核蛋白基因保守区序列设计1对引物，建立了用于狂犬病病毒特异性核酸检测的RT-PCR技术。该技术可从狂犬病病毒CVS株、8202株和SRV9株的含毒细胞培养物及鼠脑组织中，扩增出443bp的核酸片段，检出核酸的敏感性约为3pg。对30份不同种动物脑组织的检测结果与小鼠脑内接种试验(MIT)的测定结果完全吻合，但前者可在3h内直接对组织匀浆进行诊断，具有快速、简便和敏感的优点。     

The Use of Fluorescent Antibody Staining in the Diagnosis of Rabies

Brain material from 750 domestic and wild animals submitted to this laboratory for rabies diagnosis was studied by the following three methods: a) microscopic examination of Williams'' stained impressions, b) mouse inoculation test, and c) microscopic examination of impressions stained with fluorescein-tagged antibodies. The purpose of this investigation was to compare the sensitivity of the fluorescent antibody technique with that of two classical methods, when applied to the routine diagnosis of rabies. From the results obtained by one or the other method of study, 175 specimens were diagnosed as positive. Of these, only 58 (33 per cent) were detected by the examination of Williams'' stained impressions. On the other hand, two rabid cases were missed by the mouse inoculation test, and four by the fluorescent antibody technique. Without being completely reliable, the last two methods proved to be almost equally sensitive and much more so than the examination of Williams'' stained impressions.     

A tissue culture infection test in routine rabies diagnosis.

A cell culture infection test was developed for the isolation of rabies virus from field cases submitted for rabies diagnosis. The procedure involved the addition of a suspension of suspect brain tissue to a suspension of murine neuroblastoma cells in 96-well microtiter plates. The cultures were then incubated at 35-36 degrees C for four days at which time they were fixed, stained with a fluorescein-labelled hamster antirabies antibody conjugate and examined with a fluorescence microscope. Rabies antigen in cells was readily visible as brilliant, apple-green fluorescent particles. This technique was compared with the standard mouse inoculation test and was at least as sensitive to infection with small amounts of virus, required a much shorter test period and was substantially more economical than the mouse inoculation test. The new cell culture test is now in use at this laboratory, replacing the mouse inoculation test.     

Demonstration of Rabies Antigen in Salivary Glands of Rabies Suspected Animals

Submaxillary salivary glands from 129 rabies suspected animals were studied by the following methods: a) microscopic examination of frozen sections stained by the Fluorescent antibody technique (FAT), and b) mouse infectivity test (MIT). Flourescent antibody staining of frozen sections from the salivary glands of rabid animals proved to be a satisfactory method for demonstrating rabies antigen, when compared with the mouse infectivity test.     

湖南湘西地区外观健康犬携带狂犬病病毒的调查研究

为了解湖南湘西地区外观健康犬携带狂犬病病毒的情况,我们于2005年9月从湘西狂犬病疫点采集38份扑杀的外观健康的犬脑组织,并于2006年1月在湘西农贸市场采集了168份外观健康的犬脑组织。RT-PCR和直接荧光抗体试验(FAT)检测表明共有5份犬脑标本为阳性,并用乳鼠脑内接种的方法分离得到了5株狂犬病病毒毒株,分子流行病学分析表明这5株病毒均为野毒。在这5份阳性标本,疫点有4份,检出率为10.5%,市场1份,检出率为0.59%。我们的调查结果表明疫点的外观健康犬携带狂犬病病毒的检出率与国内的其它报道接近,而从市场采集的标本的检出率却要低很多。     

Rabies – epidemiology,pathogenesis, public health concerns and advances in diagnosis and control: a comprehensive review

Rabies is a zoonotic, fatal and progressive neurological infection caused by rabies virus of the genus Lyssavirus and family Rhabdoviridae. It affects all warm-blooded animals and the disease is prevalent throughout the world and endemic in many countries except in Islands like Australia and Antarctica. Over 60,000 peoples die every year due to rabies, while approximately 15 million people receive rabies post-exposure prophylaxis (PEP) annually. Bite of rabid animals and saliva of infected host are mainly responsible for transmission and wildlife like raccoons, skunks, bats and foxes are main reservoirs for rabies. The incubation period is highly variable from 2 weeks to 6 years (avg. 2–3 months). Though severe neurologic signs and fatal outcome, neuropathological lesions are relatively mild. Rabies virus exploits various mechanisms to evade the host immune responses. Being a major zoonosis, precise and rapid diagnosis is important for early treatment and effective prevention and control measures. Traditional rapid Seller's staining and histopathological methods are still in use for diagnosis of rabies. Direct immunofluoroscent test (dFAT) is gold standard test and most commonly recommended for diagnosis of rabies in fresh brain tissues of dogs by both OIE and WHO. Mouse inoculation test (MIT) and polymerase chain reaction (PCR) are superior and used for routine diagnosis. Vaccination with live attenuated or inactivated viruses, DNA and recombinant vaccines can be done in endemic areas. This review describes in detail about epidemiology, transmission, pathogenesis, advances in diagnosis, vaccination and therapeutic approaches along with appropriate prevention and control strategies.     

Detection of rabies viral antigens in non-autolysed and autolysed tissues by using an immunoperoxidase technique

The objectives of this study were to determine the potential of an immunoperoxidase technique involving the avidin-biotin complex (ABC) stain for the diagnosis of rabies in fresh tissues and compare it with other standard methods, including the fluorescent antibody test (FAT), haematoxylin and eosin and Seller's stain, and to investigate its capacity to detect rabies antigen in autolysed tissues. Samples of non-autolysed brain from 81 domestic and wild animals suspected of having rabies were examined. Rabies antigen was detected by FAT in 41 of these samples and Negri bodies were detected in 40 (97.6 per cent) of them by the immunoperoxidase technique, in 25 by haematoxylin and eosin and in 22 by Seller's stain. The sensitivity of the immunoperoxidase technique decreased as the tissues were left to autolyse; after two days it was 91.2 per cent, after four days 70.6 per cent, and after seven days 11.8 per cent.     

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.     

狂犬病病毒荧光定量RT-PCR快速检测试剂盒的评价及应用

基于目前在我国人群和动物中流行的狂犬病病毒（RV）均属于基因Ⅰ型,在本室所建立的基因Ⅰ型RV荧光定量RT-PCR方法的基础上,组装了可以便捷使用的RV快速检测试剂盒。对4 577份临床样品的检测结果表明,该试剂盒的检测灵敏度达4.68个TCID50,与《狂犬病防治技术规范》规定的套式RT-PCR灵敏度相当,而且稳定性良好,可以冷冻保存8个月以上。所研制的荧光定量RT-PCR快速检测试剂盒具有操作简便、快速、特异性强、灵敏度高、重复性好、稳定性强等优点,既适合单个临床样品的确诊,又适合RV流行病学监测的大规模筛查。     

Evaluation of five different methods for routine diagnosis of rabies

Brain tissue from 187 animals of different species was investigated by means of fluorescent antibody test, peroxidase anti-peroxidase technique, mouse inoculation test and cell culture technique for a diagnosis of rabies. With peroxidase anti-peroxidase technique the rabies specific reaction comprised inclusion bodies and a granular staining of the cytoplasm of affected cells. A specific positive reaction was found only in neurons, in which perikaryon as well as cell processes were affected. Fluorescent antibody test and peroxidase anti-peroxidase technique detected 98% each, mouse inoculation test 95% and cell culture technique 81% of the rabies positive animals. In conclusion, peroxidase anti-peroxidase technique allows a highly reliable diagnosis of rabies when only formalin-fixed and paraffin-embedded material is available. Histopathological alterations comprising Negri bodies, inflammatory and degenerative lesions were encountered in 53% of the rabies positive brains.     

Polymerase chain reaction and other laboratory techniques in the diagnosis of long incubation rabies in Australia

SUMMARY Blood and post-mortem tissues from a 10-years-old girl were submitted to the Australian Animal Health Laboratory. Clinical signs and histopathological lesions had suggested a diagnosis of rabies, but, an unusually long incubation period of at least 5 years did not encourage such a diagnosis. Serological examinations by the rapid fluorescent focus inhibition test revealed a dramatic increase in rabies virus-neutralising antibody during the 10-day period of hospitalisation. The results of a fluorescent antibody test on brain smears, and an immunoperoxidase test on formalin-fixed sections of brain were also consistent with a diagnosis of rabies. Attempts to isolate virus were unsuccessful. Polymerase chain reactions (PCRs) were conducted on a 10% suspension of a post-mortem sample from the patient's brain, using primers based on the published sequence of the Pasteur virus strain of rabies virus. 413 and 513 bp fragments from the nucleoprotein gene and a 403 bp fragment from the glycoprotein gene were amplified. Subsequent sequencing of these fragments, and comparison with equivalent regions of known rabies viruses, confirmed that the fragments originated from a virus belonging to the rabies virus serotype. This case demonstrated the advantage of using a range of laboratory techniques to obtain a definitive diagnosis. In particular, a PCR-based test may allow a diagnosis, even in the face of conditions that preclude virus isolation such as apparently occurred in this case. Finally, this case demonstrated that an unusually long incubation period should not discourage a tentative clinical diagnosis of rabies.     

Rabies in Western Canada 1978-1984

The results of laboratory examination of 22,503 specimens for the diagnosis of rabies by the standard fluorescent antibody and mouse inoculation tests over a seven year period are presented. Specimens were received from British Columbia, Alberta, Saskatchewan, the Yukon and Northwest Territories. Of the 1,445 positive cases, 10.50% involved human contact. The main reservoirs of rabies were skunks, bats and foxes. The reliability of the fluorescent antibody test for the diagnosis of rabies was reaffirmed by agreement with the mouse inoculation test in over 99% of cases.     

Comparison of three diagnostic detection methods for tuberculosis in French cattle

Three additional techniques (Ziehl-Neelsen, auramine O/rhodamine and immunostaining using polyclonal anti-Mycobacterium bovis) to hematoxylin-eosin histopathology were evaluated for bovine tuberculosis diagnosis on 39 samples from several slaughterhouses. The immunohistochemichal technique was more sensitive and could detect a greater number of positive cattle. It has about the same sensibility as the bacteriology but it was faster.