Abstract
BackgroundThe recommended screening of rabies in ‘suspect’ animal cases involves testing fresh brain tissue. The preservation of fresh tissue however can be difficult under field conditions and formalin fixation provides a simple alternative that may allow a confirmatory diagnosis. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. The presence of Negri bodies and immunohistochemical detection of rabies virus antigen were evaluated in the cortex, hippocampus, cerebellum and brainstem. The effect of autolysis and artefactual degeneration of the tissue was also assessed.ResultsRabies was confirmed in 53 of 57 (93%) cases by IHC. IHC labelling was statistically more abundant in the brainstem. Negri bodies were observed in 32 of 53 (60.4%) of the positive cases. Although tissue degradation had no effect on IHC diagnosis, it was associated with an inability to detect Negri bodies. In 13 cases, a confirmatory Polymerase chain reaction (PCR) testing for rabies virus RNA was undertaken by extracting RNA from fresh frozen tissue, and also attempted using FFPE samples. PCR detection using fresh frozen samples was in agreement with the IHC results. The PCR method from FFPE tissues was suitable for control material but unsuccessful in our field cases.ConclusionsHistopathological examination of the brain is essential to define the differential diagnoses of behaviour modifying conditions in rabies virus negative cases, but it is unreliable as the sole method for rabies diagnosis, particularly where artefactual change has occurred. Formalin fixation and paraffin embedding does not prevent detection of rabies virus via IHC labelling even where artefactual degeneration has occurred. This could represent a pragmatic secondary assay for rabies diagnosis in the field because formalin fixation can prevent sample degeneration. The brain stem was shown to be the site with most viral immunoreactivity; supporting recommended sampling protocols in favour of improved necropsy safety in the field. PCR testing of formalin fixed tissue may be successful in certain circumstances as an alternative test.
Highlights
The recommended screening of rabies in ‘suspect’ animal cases involves testing fresh brain tissue
At present Polymerase chain reaction (PCR) is not recommended for routine diagnosis by the WHO/ OIE because of the requirement for exacting standards of quality control, but it has a crucial role in epidemiological analysis of virus type [13, 34, 36]
There have been reports of rare Negri body-like inclusions occurring in rabies negative canine brain, in this series inclusions were not observed in IHC negative cases [26, 28]
Summary
The recommended screening of rabies in ‘suspect’ animal cases involves testing fresh brain tissue. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. Confirmation of rabies infection requires laboratory testing of brain tissue. The standard methods for the diagnosis of rabies virus in animals are recommended by the OIE (World Health Organisation for Animal Health) [31, 36]. Histopathologic examination of infected brain tissue can be used to directly identify Negri bodies in neuronal cytoplasm, which is considered pathognomonic for rabies virus infection [23]. The reduced sensitivity in comparison to immunological and molecular tests has resulted in this technique no longer being recommended for primary diagnosis by the WHO or OIE [12, 36]
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