Immunohistochemical investigation of canine lymph nodes collected during a rabies outbreak in South Africa

Rabies is a fatal zoonosis that impairs host immune function, yet effects on peripheral lymphoid architecture are poorly defined. During a 2021-2022 rabies virus (RABV) outbreak in South Africa, we collected cervical lymph nodes from 36 rabies-suspect dogs; RABV RNA was detected in 27. Canine distemper virus RNA was detected in a subset across both RABV-positive (RABV+) and RABV-negative (RABV-) groups and was not associated with clinical-sign count. We set up a computer-assisted histological analysis tool to quantify germinal-centre (GC) nucleus density and immunohistochemistry for CD20, PNA and IBA1 to profile B cells, GC activity and macrophages. Within the outbreak cohort, GC density and marker-based metrics did not differ between RABV+ and RABV- dogs. Two healthy dogs were included as reference tissues; values in outbreak dogs were generally lower, but these contrasts are contextual given the limited, non-matched controls. This study provides a reproducible framework for quantifying immune cell organization in field-collected tissues during natural RABV exposure and highlights the need for larger, geographically matched control groups and complementary functional immune measurements.