Investigation of the brain, brainstem, eye, and lacrimal gland in animals naturally infected with the rabies virus using pathological, immunohistochemical, and molecular methods

This study aimed to investigate pathological changes in the eye, lacrimal gland, optic nerve, brain, and cerebellum tissues of animals naturally infected with rabies virus using histopathological, immunohistochemical, and molecular methods. The study material consisted of head samples from 92 animals (89 domestic and 3 wild mammals) brought to the Rabies Diagnostic Laboratory with suspicion of rabies from 13 provinces in the Eastern and Southeastern Anatolia regions (Batman, Bingöl, Bitlis, Diyarbakır, Elazığ, Hakkari, Malatya, Mardin, Muş, Siirt, Şırnak, Tunceli, and Van). Rabies diagnosis in the samples was confirmed by Direct Fluorescent Antibody Test (DFA) and qRT-PCR. Following routine histopathological procedures, eye, lacrimal gland, brain, and cerebellum tissues were evaluated; the eye and lacrimal gland were also examined immunohistochemically using TLR3, TLR4, IL-6, IL-1β, TNFα, HSP70, CD3, and CD20 markers. Molecular analyses were performed using qRT-PCR and LN34 testing. Macroscopically, hemorrhage, edema, and congestion were observed in the brain, while no significant changes were detected in the eye or lacrimal gland. Histopathological examination revealed limited lesions in the retinal layers and, in 10 cases, Negri bodies in the ganglion cells. In the lacrimal gland, lesions were generally characterized by inflammatory infiltrates in the interstitial areas. In the cerebrum and cerebellum, nonsuppurative encephalitis was noted, characterized by perivascular mononuclear cell infiltration, gliosis, neuronal necrosis, and Negri bodies. Statistically, according to immunohistochemical findings, IL-6 immunoreactivity did not show a significant difference between the eye and lacrimal gland (p>0.05), while significant differences were observed in CD20 and HSP70 levels (p=0.0029 and p<0.0001, respectively). In molecular analyses, the presence of viral nucleic acid was detected in the vitreous using qRT-PCR (p=0.0412). Furthermore, RABV nucleic acid was successfully amplified in FFPE brain tissues using the LN34 test (p=0.0011). In conclusion, the findings suggest that rabies can affect the eye and lacrimal glands. The detection of viral material in the vitreous suggests that viral spread occurs in these areas, and that the vitreous can be used as an alternative diagnostic material. Furthermore, the usability of FFPE brain tissues for diagnostic purposes has been confirmed. These findings demonstrate the potential use of tears for antemortem diagnosis and highlight the need for more detailed studies on this topic.