应用夹心阻断ELISA测定血清中抗狂犬病抗体的研究

本研究建立的检测狂犬病抗体的夹心阻断ELISA,直接利用未经提纯的病毒悬液代替提纯的抗原,并仅用一种酶标抗体,测定了9种动物的血清.本方法敏感度的95%可信限为0.0013～0.0071IU/mL,比小鼠中和试验和微量免疫酶试验均敏感.按阻断50%判定血清ELISA的阴阳性,9种动物的1134份血清中有91份阳性,其中发病点的牛、猪、犬、家鼠血清的阳性率均在10%以上.根据对一定量的抗原阻断率相同时,抗体浓度一致的原理,测定了7种动物的185份血清效价,结果狂犬病抗体浓度在0.01IU/mL以上的为63份.利用夹心阻断ELISA和小鼠中和试验测定了7种动物的23份血清,阳性份数分别为12和11.两种方法均证明家鼠血清中有狂犬病抗体.本研究表明,测定狂犬病抗体的夹心阻断ELISA,简便、敏感、快速,可用于流行病学调查.     

Comparison of antibody responses after vaccination with two inactivated rabies vaccines

Thirty laboratory dogs were randomly assigned to two groups (A and B) of 15 dogs and subcutaneously vaccinated with a single dose of one of two commercially available monovalent inactivated rabies vaccines: RABISIN (Merial, France) (group A) and NOBIVAC Rabies (Intervet International) (group B). Rabies antibodies were measured over a period of 4 months using the fluorescent antibody virus neutralization (FAVN) test. The two vaccines performed differently in terms of magnitude and persistence of rabies antibodies titers in dogs. Two weeks after vaccination, average rabies antibody titers peaked at 2.53 IU/mL (range, 0.17-13.77 IU/mL) and 1.26 IU/mL (range, 0.50-4.56 IU/mL) in groups A and B dogs, respectively. The average FAVN antibody titres against rabies on D28, D56, D84, D112 and D120 were significantly higher in group A than in group B. Although all dogs from group B serologically responded to vaccination, the proportion of dogs with antibody titres >or=0.5 IU/mL dropped significantly after D28 and was statistically significantly lower on D56, D84 and D112 compared to group A dogs. In conclusion, in the context of international trade, the choice of the vaccine and the timing of blood tests are critical factors in achieving successful serological test results after rabies vaccination. RABISIN induces high and sustained antibody titres against rabies, increasing the flexibility for the time of blood sampling after primo-vaccination.     

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.     

A dot enzyme linked immunosorbent assay (dot ELISA): Comparison with standard fluorescent antibody test (FAT) for the diagnosis of rabies in animals

A modified enzyme linked immunosorbent assay (Dot ELISA) is described for visual detection of rabies antigen in animals. The test materials were dotted onto the nitrocellulose paper and allowed to react with rabies antiserum. The bound antigen—anti-body were reacted with a peroxidase conjugated antirabbit immunoglobulin. Positive reactions were easily visualized as brown dots after enzyme degradation of the substrate. A total of 400 specimens from various geographical locations were tested with the dot ELISA technique, and also with the fluorescent antibody test (FAT), which was used as a reference method. The concordance between the two tests was 95.25%. The dot ELISA may have potential applications as a rapid, simple and economical field test in the diagnosis of rabies.     

Rabies diagnosis in western Canada, 1985-1989

The results of laboratory examination of 20460 specimens for the diagnosis of rabies by the standard fluorescent antibody and mouse inoculation tests over a five year period are presented. Specimens were received from British Columbia, Alberta, Saskatchewan, Manitoba, and the Yukon and Northwest Territories. Of those examined, 9.96% were positive. The main reservoirs of rabies were skunks, bats, and foxes. During this observation period a rabies epizootic occurred in skunks in Saskatchewan. When both tests were performed, the fluorescent antibody test was found to agree with the mouse inoculation test in over 99% of cases.     

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.     

Laboratory Diagnosis of Rabies in Western Canada (1968-1977)

The results of laboratory examination of 18,086 specimens for the presence of rabies antigen by the fluorescent antibody and mouse inoculation tests over a ten year period are presented. The submissions were received from British Columbia, Alberta, Saskatchewan, the Yukon and the Northwest Territories. Of those examined, 10.74% were positive: however, the incidence of rabies varied widely in the specimens and species submitted, depending on their origin. The principal wildlife reservoirs of the disease appear to be skunks, foxes and bats. A correlation of almost 99% was obtained between the fluorescent antibody test and the mouse inoculation test, indicating that the diagnostic procedures used were highly reliable in identifying rabies-infected animals.     

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.     

Factors associated with the success of rabies vaccination of dogs in Sweden

Background

United Kingdom, Ireland, Malta and Sweden maintain their national provisions for a transitional period regarding rules concerning rabies vaccination and individual serological test for rabies neutralizing antibodies. The purpose of vaccinating dogs against rabies is to establish pre-exposure immunity and protect individual animals from contracting rabies.The aim of the study was to investigate factors associated with reaching the internationally accepted threshold antibody titre of 0.5 IU/mL after rabies vaccination of dogs.

Methods

The study was a prospective single cohort study including 6,789 samples from Swedish dogs vaccinated with commercially available vaccines in Sweden, and the dog''s antibody responses were determined by the OIE approved FAVN test. Information on potential risk factors; breed, age, gender, date of vaccination, vaccine label and the number of vaccinations, was collected for each dog. Associations between the dependent variable, serological response ≥ 0.5 IU/mL or < 0.5 IU/mL and each of the potential risk factors were investigated using logistic regression analysis.

Results

Of 6,789 vaccinated dogs, 6,241 (91.9%) had an approved test result of ≥ 0.5 IU/mL. The results of the multivariable logistic regression analysis showed that vaccinating with vaccine B reduced the risk of having antibody titres of < 0.5 IU/mL by 0.2 times compared with vaccination using vaccine A. Breed size was found significant as an interaction with number of vaccinations and age at vaccination as an interaction with day of antibody testing after last vaccination. In summary, larger breeds were at higher risk of having antibody titres of < 0.5 IU/mL but if vaccinated twice this risk was reduced. Moreover, there were a increased risk for dogs < 6 months of age and > 5 years of age to have antibody titres of < 0.5 IU/mL, but this was affected by number of days from vaccination till testing.

Conclusions

The probability of success of rabies vaccinations of dogs depends on type of vaccine used, number of rabies vaccinations, the breed size of the dog, age at vaccination, and number of days after vaccination when the antibody titres are tested. The need for a booster vaccination regimen is recommended for larger breeds of dog.     

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.     

Absence of rabies encephalitis in a raccoon with concurrent rabies and canine distemper infections

Concurrent infection of a raccoon by rabies and canine distemper viruses is described. Fluorescent antibody (FA) test demonstrated rabies antigen in the brain of this animal, however, histologically only lesions characteristic of canine distemper infection were seen. We recommend testing tissues for rabies of animals that histologically are positive for canine distemper.     

Results of vaccination of Asian elephants (Elephas maximus) with monovalent inactivated rabies vaccine

OBJECTIVE: To evaluate the humoral immune response of Asian elephants to a primary IM vaccination with either 1 or 2 doses of a commercially available inactivated rabies virus vaccine and evaluate the anamnestic response to a 1-dose booster vaccination. ANIMALS: 16 captive Asian elephants. PROCEDURES: Elephants with no known prior rabies vaccinations were assigned into 2 treatment groups of 8 elephants; 1 group received 1 dose of vaccine, and the other group received 2 doses of vaccine 9 days apart. All elephants received one or two 4-mL IM injections of a monovalent inactivated rabies virus vaccine. Blood was collected prior to vaccination (day 0) and on days 9, 35, 112, and 344. All elephants received 1 booster dose of vaccine on day 344, and a final blood sample was taken 40 days later (day 384). Serum was tested for rabies virus-neutralizing antibodies by use of the rapid fluorescent focus inhibition test. RESULTS: All elephants were seronegative prior to vaccination. There were significant differences in the rabies geometric mean titers between the 2 elephant groups at days 35, 112, and 202. Both groups had a strong anamnestic response 40 days after the booster given at day 344. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed the ability of Asian elephants to develop a humoral immune response after vaccination with a commercially available monovalent inactivated rabies virus vaccine and the feasibility of instituting a rabies virus vaccination program for elephants that are in frequent contact with humans. A 2-dose series of rabies virus vaccine should provide an adequate antibody response in elephants, and annual boosters should maintain the antibody response in this species.     

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。     

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.     

Immune response to Japanese rabies vaccine in domestic dogs

To evaluate the immune response induced by Japanese rabies vaccine for veterinary use as international units (IU), we measured levels of rabies antibody in serum samples from dogs by the rapid fluorescent focus inhibition test (RFFIT). In dogs immunized with a reference vaccine (potency level of 3.1 IU/ml), prepared by the same method as that used to produce commercial vaccine, and its dilutions (1 : 2 or 1 : 4), neutralizing-antibody levels increased to 1.0-2.0 IU/ml over a period of 1 month and then decreased to 0.2-1.5 IU/ml over a period of 1 year after the first vaccination and showed a remarkable increase to 12-47 IU/ml after the second vaccination. Sixty-five (74.7%) of the 87 serum samples from domestic dogs that were tested were seropositive (> or =0.1 IU/ml). However, the seropositive rate in dogs less than 1-year old at the time of vaccination was low (57.1%), and the antibody levels in these dogs were not sufficiently high for the rabies antibody titre in serum to be maintained for 1-year. Levels of rabies antibody in all serum samples were also measured by the virus neutralizing test (VNT), and a strong correlation (r > 0.95) was found between the results of the RFFIT and those of the VNT.     

狂犬病病毒磷蛋白单抗的制备与应用

以灭活的狂犬病病毒CVS株细胞毒免疫BALB/c小鼠,通过间接ELISA法和Western-blot筛选获得针对磷蛋白的单克隆抗体4株：1C9、4B10、2G12、4G5,其中1C9针对氨基端保守表位。以亲和层析法纯化1C9单抗腹水,异硫氰酸荧光黄标记制备荧光抗体。以1C9磷蛋白荧光抗体与本实验室研制的狂犬病核蛋白免疫荧光抗原检测试剂盒,对本实验室收集的501份疑似狂犬病鼬獾、蝙蝠、犬和黄鼬的脑组织样品进行直接免疫荧光平行检测。结果显示,两种检测手段对基因1型狂犬病毒的检出结果完全一致,而蝙蝠源Irkut病毒仅能以磷蛋白单抗1C9检出。本研究成功获得了与我国现有不同基因型狂犬病毒良好反应的抗狂犬病磷蛋白单抗,并应用于狂犬病的直接免疫荧光检测,为狂犬病诊断提供了敏感性和可靠性良好的诊断试剂。     

抗狂犬病毒单克隆抗体的纯化及标记

对一株稳定、特异、高效的单克隆抗体（mAb）进行了纯化和荧光素（FITC）的标记,利用标记产物与已有的抗狂犬病毒核蛋白单克隆抗体标记物相比较,效果良好,二者可以结合（或单独）使用;应用标记好的荧光抗体与FITC-抗狂犬病毒免疫球蛋白比较进行血清样品效价测定实验,来确定纯化、标记后的单克隆抗体的灵敏度,结果表明本实验制备的单克隆抗体与FITC-抗狂犬病毒免疫球蛋白具有相同的灵敏度,该单克隆抗体测得的血清样品效价结果与FITC-抗狂犬病毒免疫球蛋白测得的结果相同。因此,此荧光抗体可应用于狂犬病病毒检测（FAT）、病毒毒力测定（TCID50）、中和抗体实验（FAVN）等方面的检测实验,此荧光抗体可为狂犬病的相关研究提供有力的保障。     

A case study of rabies diagnosis from formalin-fixed brain material

Rabies is caused by several Lyssavirus species, a group of negative sense RNA viruses. Although rabies is preventable, it is often neglected particularly in developing countries in the face of many competing public and veterinary health priorities. Epidemiological information based on laboratory-based surveillance data is critical to adequately strategise control and prevention plans. In this regard the fluorescent antibody test for rabies virus antigen in brain tissues is still considered the basic requirement for laboratory confirmation of animal cases. Occasionally brain tissues from suspected rabid animals are still submitted in formalin, although this has been discouraged for a number of years. Immunohistochemical testing or a modified fluorescent antibody technique can be performed on such samples. However, this method is cumbersome and cannot distinguish between different Lyssavirus species. Owing to RNA degradation in formalin-fixed tissues, conventional RT-PCR methodologies have also been proven to be unreliable. This report is concerned with a rabies case in a domestic dog from an area in South Africa where rabies is not common. Typing of the virus involved was therefore important, but the only available sample was submitted as a formalin-fixed specimen. A real-time RT-PCR method was therefore applied and it was possible to confirm rabies and obtain phylogenetic information that indicated a close relationship between this virus and the canid rabies virus variants from another province (KwaZulu-Natal) in South Africa.     

A Comparative Study of the RAPINA and the Virus‐Neutralizing Test (RFFIT) for the Estimation of Antirabies‐Neutralizing Antibody Levels in Dog Samples

The mass vaccination of dogs against rabies is a highly rational strategy for interrupting the natural transmission of urban rabies. According to the World Organization for Animal Health (OIE) and the World Health Organization (WHO), the immunization of at least 70% of the total dog population minimizes the risk of endemic rabies. Knowledge of the virus‐neutralizing antibody (VNA) level against the rabies virus (RABV) is required to evaluate protective immunity and vaccine coverage of dogs in the field. The rapid focus fluorescent inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test are recommended by OIE and WHO to determine the VNA levels in serum. However, these tests are cell culture based and require the use of live viruses and specialized equipment. The rapid neutralizing antibody test (RAPINA) is a novel, immunochromatographic test that uses inactivated virus to estimate the VNA level qualitatively. It is a simple, rapid and inexpensive, although indirect, assay for the detection of VNA levels. The RAPINA has shown good positive and negative predictive values and a high concordance with the RFFIT results. In this study, we compared the performance of the two tests for evaluating the vaccination status of dogs in the Philippines, Thailand and Japan. A total of 1135 dog sera were analysed by the RAPINA and compared to the VNA levels determined by the RFFIT. The overall positive and negative predictive values of the RAPINA were 96.2–99.3% and 84.5–94.8%, respectively, with a concordance (kappa) of 0.946–0.97 among the three countries. The RAPINA results were highly homologous and reproducible among different laboratories. These results suggest that this test is appropriate to survey vaccination coverage in countries with limited resources.