Abstract. Although a fast and reliable fluorescent antibody (FA) test for rabies diagnosis is available, ideally the diagnostic procedure requires fresh or frozen brain tissues. In some instances (and particularly for retrospective studies), fresh or frozen tissues may not be available. In such cases, immunohistochemical tests may be utilized. Because such tests have been used only in a limited number of cases, their diagnostic reliability has not been thoroughly evaluated. This study documents the results of a streptavidin-biotin complex (ABC) immunoperoxidase test on formalin-fixed, paraffin-embedded brain tissues of domestic and wild animals that were positive on FA test for rabies prior to fixation. Thirty-nine of 40 rabies cases were positive with the ABC technique. Based on these preliminary results, the ABC technique appears reliable. However, more corroborative test results are needed before the ABC method can be used as a routine diagnostic procedure, especially with field samples and negative controls.The eastern USA is enzootic for raccoon (Procyon lotor) rabies. 1 Within this area, the disease is compartmentalized in wildlife and on many occasions has spilled over into the domestic animal population. 7 The fluorescent antibody (FA) test for rabies diagnosis is a fast and reliable laboratory test. 6 However, one disadvantage is that ideally the diagnostic procedure requires unfixed (fresh or frozen) brain tissue.the ABC technique for rabies antigen detection has not been used routinely, its reliability as a diagnostic test has not been documented. Therefore, the following study was conducted to correct this deficiency. The validity of the ABC technique for diagnosis of rabies was tested on a variety of domestic and wild animals that were previously found to be positive for rabies by the FA test. Recently, the Laboratory of Large Animal Pathology (University of Pennsylvania) was confronted with 2 instances where rabies was suspected in animals, but unfixed tissue samples were unavailable. In the first case, a puppy had bitten a child; because the dog was unvaccinated and was from a rabies-enzootic area, it was euthanized. The referring veterinarian removed the brain from the carcass and placed it in formalin. In the second case, a neurologically impaired cat from a nonenzootic area was euthanized and necropsied, and tissues were fixed in formalin for histopathology. Microscopic examination revealed an extensive nonsuppurative meningoencephalitis, and rabies was considered as a differential diagnosis. In both cases, only formalin-fixed tissues were available. Materials and methodsSeveral methods have been previously described for the detection of rabies antigen in formalin-fixed, paraffin-embedded tissue sections. 2-5,9,12 An immunoperoxidase method utilizing the streptavidin-biotin complex (ABC) has been used for a retrospective survey of rabies in equine necropsy cases 7 and for documenting the distribution of lesions and rabies antigen in tissues of raccoons that had contracted the disease. 8 Because...

Abstract. Histopathologic (hematoxylin and eosin [HE]) and immunoperoxidase (streptavidin-biotin complex) methods were used for examination of formalin-fixed tissues of rabid raccoons from an enzootic area of Pennsylvania. Extensive morphologic lesions of rabies encephalitis were present in the cerebrum and the brain stem regions. Negri bodies were detected by both methods and were present in the brain (cerebral cortex, hippocampus, brain stem, cerebellum, and cervical spinal cord) and in the ganglia of the trigeminal nerves. The viral inclusions were also seen in ganglion cells in the tongue, parotid salivary glands, pancreas, intestines, and adrenal glands. These sites were not associated with any inflammatory cellular infiltrate. The immunoperoxidase method was superior to HE for the detection of Negri bodies. Because lesions of rabies encephalitis were consistently observed in the cerebrum, brain stem, and cervical spinal cord regions, these areas of the brain should be included when raccoons are examined by the fluorescent antibody test for rabies. Rabies in raccoons was first reported in the United Materials and methods States in 1936;3 only sporadic cases were diagnosed until the 1950s when the first cases from what is now Two raccoons (nos. 1 and 2) that were showing neurologic an enzootic area in central coastal Florida were recsigns and were from a rabies enzootic area of York County, Pennsylvania, were utilized for this investigation. Raccoon ognized. 8 In recent years, a new focus of a major epi-1 was seen exhibiting neurologic signs during daylight hours zootic among raccoons in the mid-Atlantic region of and was shot by the local wildlife authorities. The second the United States has been established.' raccoon was also seen during daylight and was trapped alive.In 1990, for the first time raccoons outnumbered It appeared lethargic, had mucoid diarrhea, and died on the skunks as the primary rabies reservoir in the United second day of captivity. Both carcasses were submitted to States. Despite the intensity of this recent outbreak, the University of Pennsylvania New Bolton Center for necpathologic distribution of lesions in natural cases of 4 The fixed brain was cut at approximately 2-mm transverse sections. All brain sections and tissue sections of the tion should enable diagnosticians, when presented with other organs were embedded in paraffin, sectioned at 5 µm, a suspect rabies case, to select appropriate tissue sam-and stained with hematoxylin and eosin (HE). Selected parples of brain for diagnosis; especially when conducting affin sections of cerebral cortex, hippocampus, cerebellum, routine surveillance for rabies activity in an area. The brain stem, trigeminal (Gasserian) ganglion, tongue, parotid second aim of the study was to document the presence salivary gland, pancreas, adrenal glands, and intestines (duor absence of neuronal inclusions (Negri bodies) in odenum) were also stained by the streptavidin-biotin comvarious organs of rabid raccoons, to explore the pos- Negri bodies in the bra...

Abstract. A 5-year (1985-1989) retrospective immunohistochemical study was conducted using an avidinbiotin complex (ABC) immunoperoxidase method to demonstrate Sarcocystis neurona in histologically suspect cases of equine protozoa1 myeloencephalitis (EPM). Primary antibodies against S. neurona and S. cruzi were utilized for the ABC technique. The findings were compared with those from cases in which the organisms were detected by examination of hematoxylin and eosin (HE)-stained neuronal sections. HE-stained sections detected the presence of the organisms in 20% of the suspect cases; whereas the ABC technique confirmed the presence of S. neurona in 51% and 67% of the cases by S. neurona and S. cruzi antibodies, respectively. A review of clinical case histories showed that 21/47 (45%) of the EPM horses with parasites in the tissue sections had prior treatment with antiprotozoal drugs and/or steroids. Using the test results of S. neurona and S. cruzi as a standard reference, HE test sensitivity based on examination of up to 30 neuronal sections per case was only 25%, and test specificity was 91%. Equine protozoal myeloencephalitis (EPM) is a progressive disease of horses affecting the central nervous system (CNS).1,2,4,6 The causative agent is a protozoan, Sarcocystis neurona.5 An antemortem diagnostic test for the disease is not yet available. Postmortem diagnosis is based on the presence of characteristic histopathologic lesions and on the identification of the etiological agent.2,9 However, the detection of the protozoan by histopathology is laborious and often unrewarding 1,9 In a recent retrospective investigation of horses necropsied at the Laboratory of Large Animal Pathology, New Bolton Center (NBC), Kennett Square, Pennsylvania, over 20% of the cases had neurologic disease.7 Of these neurologic cases, 34% were considered to have an infectious cause (protozoal, viral, fungal, or bacterial). Within this group, EPM was the most frequent etiologic diagnosis based on the presence of characteristic microscopic lesions.2,4,9 However, the etiologic agent, S. neurona, 5 was identified in only 14 of the 70 positive cases of EPM. 7In the present study, we report of the immunohistochemical findings on these 70 histologically suspect cases of EPM. Materials and methodsClinical case histories and necropsy reports of 70 cases with lesions histopathologically compatible with EPM that were diagnosed between 1985 and 1989 at the NBC were examined. These records showed that an average of approximately 30 neuronal hematoxylin and eosin (HE)-stained sections were examined per case. Two histologic sections with the presence of S. neurona (determined from the necropsy report) or, in cases where the organisms were not observed, 2 sections with the most severe and extensive morphologic lesions from each of the suspect EPM cases were selected for immunohistochemistry. Consecutive tissue sections from the selected paraffin blocks (2 per horse) were cut at 5 µm and stained by an avidin-biotin complex (ABC) immunoperoxidase method ...

Abstract. Ten raccoons were divided into two random groups (groups 1 and 2) of five animals each. Group 1 raccoons were inoculated intramuscularly in the masseter muscle with a raccoon rabies virus isolate obtained from a natural case of raccoon rabies from the northeastern USA. Group 2 raccoons were infected by a similar route with a Latin American canine isolate of rabies virus. Raccoons either died suddenly or developed neurologic signs compatible with rabies. Clinical signs of rabies in group 1 raccoons were more severe than in group 2. Raccoons in group 1 either died acutely or were euthanized within 25 days ( ± SD = 20.6 ± 2.7 days) postinfection, whereas all group 2 raccoons showed neurologic signs and were euthanized within 17 days (14.2 ± 2.2 days) postinfection. Light microscopic findings revealed extensive nonsuppurative encephalitis predominantly located in the cerebrum and brain stem of raccoons in group 1, whereas in group 2 raccoons the lesions were confined to the brain stem regions. In group 1 raccoons, Negri bodies were commonly seen on hematoxylin and eosin (HE)-stained sections of brain and in ganglion cells of 5 other tissues (trigeminal nerve, salivary glands, duodenum, pancreas, adrenal gland). Negri bodies, however, were either absent or were only occasionally observed in corresponding tissues of raccoons infected with the canine strain (group 2). Paraffin-embedded tissue sections were also examined for Negri bodies by an immunoperoxidase test, which revealed results similar to the HE findings. Results of this study are compared with histopathologic and immunohistochemical findings in raccoons naturally infected with rabies.A major rabies epizootic is currently in progress neuronal and in nonneuronal organs. 9 These different among raccoons in the eastern USA. 18 In response to morphologic presentations by 2 unrelated isolates of this outbreak, an oral vaccinia-rabies glycoprotein (V-rabies virus prompted us to further investigate this RG) recombinant virus vaccine has been developed. phenomenon. In vaccinated raccoons, the V-RG vaccine produces protective immunity for at least 6 months. 22 To date the vaccine has been field tested for raccoons in 5 northeastern states 21 and Florida and for coyotes in Texas.During experimental evaluation of the V-RG vaccine, nonvaccinated control raccoons that succumbed to rabies infection had histopathologic changes confined to the brain stem, 22 whereas naturally infected rabid raccoons from the eastern rabies enzootic area had microscopic lesions that were distributed predominantly in the cerebrum. 9 Similarly, in the experimental raccoons, there was a paucity of Negri bodies within neurons, whereas in the naturally exposed raccoons there were numerous Negri bodies present within both Received for publication March 16, 1995. This investigation documents clinicopathologic findings in raccoons that were experimentally infected with a raccoon or a canine rabies isolate. The study also enabled a comparison of neuropathologic changes (including imm...

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