Ocular and Lacrimal Gland Involvement in Rabies: Histopathological and Molecular Evidence from Naturally Infected Animals

Rabies is still a fatal zoonosis, and post-mortem confirmation is mainly based on examination of brain tissue.However, collection of brain material may be difficult under field conditions and may pose a biosafety concern.We investigated ocular involvement in naturally infected animals by histopathology, immunohistochemistry, and molecular testing.Histopathology was conducted on 92 animals that tested positive for rabies via DFA.Ocular and lacrimal gland specimens (30 each) were independently selected for immunohistochemistry based on tissue availability, preservation, and adequacy to evaluate IL-6, IL-1β, TNFα, TLR3, TLR4, HSP70, CD3, and CD20.Molecular testing was performed on separate subsets of 50 fresh brain, 50 FFPE brain, and 50 vitreous humor samples selected based on specimen availability and analytical suitability.Typical lesions of rabies were observed, such as nonsuppurative meningoencephalitis, focal gliosis, and neuronal necrosis.There was no statistically significant difference in IL-6 positivity between ocular and lacrimal gland samples (P>0.05).HSP70 positivity was more frequent in lacrimal glands compared to ocular tissues (86.7% vs. 13.3%;P<0.0001), as was CD20 positivity (46.7% vs. 13.3%;P=0.0029).Rabies virus RNA was detected in 35/50 vitreous samples (70%) by LN34 qRT-PCR.The Ct values were generally higher in FFPE brain specimens than in fresh brain specimens.Detection of viral RNA in vitreous humor supports ocular involvement in centrifugal spread from the central nervous system.However, 15 samples of vitreous humor from DFA-positive animals were negative, suggesting that vitreous humor cannot be considered as an independent diagnostic specimen, but its potential role is supplementary and needs further evaluation.