A simple technique for the rapid diagnosis of rabies in formalin-preserved brain

A simple technique is described for the rapid diagnosis of rabies in formalin-preserved brain. Brain tissue was emulsified, washed with phosphate-buffered saline and digested with trypsin. The digested material was stained according to conventional immunofluorescent procedures. Digestion with trypsin markedly enhanced the staining of rabies inclusions and eliminated non-specific staining of formalin-preserved brain. The method seems to be more accurate than histological examination.     

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.     

A case of dumb rabies and trypanosomiasis in an eight-week-old puppy

An eight-week-old male puppy affected with trypanosomiasis and dumb rabies was presented at the University of Nigeria Veterinary Hospital. The dog had a septic wound on the side of the face. The clinical signs suggested trypanosomiasis and this diagnosis was confirmed by laboratory examination. After treatment with Berenil and antibiotics, the puppy apparently recovered, but a week later became anorectous and recumbent. Dumb rabies was suspected and the dog died in a quarantine kennel less than 24 hours later. Negri bodies were detected in brain smears from the dog and a mouse inoculation test was performed to confirm the diagnosis. Dumb rabies poses serious public health hazards to dog owners and their families, as well as to those occupationally exposed to dogs.     

Enzyme immunohistochemical staining of formalin-fixed tissues for diagnosis in veterinary pathology

Disease diagnosis often relies on the detection of specific antigens in tissue specimens. Enzyme-based immunohistochemical stains of formalin-fixed, paraffin-embedded tissues may be used to identify antigens associated with viral, bacterial and protozoal microorganisms, autoimmunity, and neoplasia. The detection of antigens in routinely fixed tissues offers several advantages over other diagnostic techniques. Sample submission is convenient and facilitates safe handling of potential human pathogens. Retrospective studies of stored specimens are possible. The technique is relatively rapid and enables detection of nonviable microorganisms. In addition, the ability to detect antigens in fixed specimens allows simultaneous visualization of the antigen and the histological lesion which may enhance the accuracy of diagnosis.     

狂犬病病毒入胞途径的初步探索

狂犬病病毒(rabies virus,RABV)具有高度嗜神经性,通过识别细胞膜上特异性受体并借助内吞途径侵入细胞。为了探究其入胞途径,本研究在人神经瘤细胞(SH-SY5Y)和非洲绿猴肾上皮细胞(Vero)上,应用氯丙嗪(chlorpromazine)、制霉菌素(nystatin)、巴弗洛霉素a1(bafilomycin a1)、dynasore等抑制剂处理细胞后,进行RABV感染,通过检测病毒N基因拷贝数和RABV效价,对RABV入胞途径进行初步探究。结果显示,RABV通过网格蛋白介导、低pH值和发动蛋白(dynamin)依赖途径侵入SH-SY5Y和Vero细胞,但不通过小窝蛋白依赖途径入胞。这些结果为进一步研究狂犬病病毒感染机制和药物靶点研究提供了新的数据。     

Demonstration of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissues from natural cases of ovine and bovine encephalitis

In the present work, evidence of Listeria monocytogenes antigens based on the avidin-biotin complex (ABC) immunoperoxidase technique was performed on formalin-fixed central nervous system tissues (CNS) from a total of 23 natural cases of encephalitis (four ovine and 19 bovine). Listeria monocytogenes serotype 4 was isolated from 10 of 17 cultured specimens. Meningoencephalitis characterized by focal necrosis, microabscesses, perivascular cuffing, and gliosis with presence of macrophages and/or neutrophils was observed at histological examination. Positive L. monocytogenes antigens were successfully identified by immunohistochemistry (IHC) in the CNS of all 23 cases. Paraffin-embedded tissues assayed were stored up for 17 years. Morbidity of the outbreaks was between 0.3-3% and 0.1-1% for ovine and bovine cases, respectively. In all the ovine cases, flocks involved were under extensive grazing conditions. In nine of the 19 bovine cases (47.3%), supplementation with corn silage was used. The ABC test can help as a practical tool for the diagnosis of natural cases of L. monocytogenes encephalitis on formalin-fixed specimens from ovine and bovine.     

A study of antibody levels in wild ruminants vaccinated against rabies

The authors have vaccinated 22 fallow deer (Dama dama) and 10 mouflons (Ovis ammon musimon) against rabies with an inactivated vaccine: 4 fallow deer with 1 ml, 14 fallow deer and 10 mouflons with 2 ml, 4 animals were kept as controls (fallow deer). The antibody responses were checked by fluorescent foci inhibition carried out on blood samples collected during a two-year period. All the animals developed antibody titres and were still protected after 24 months.     

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。