Localization of the rabies virus antigen in Merkel cells in the follicle-sinus complexes of muzzle skins of rabid dogs

Highlights

• Dog muzzle skin follicle-sinus complex is useful for postmortem rabies diagnosis.

• Rabies virus antigen was detected in the outer root sheath at the ring sinus.

• Staining of Merkel cell markers overlapped with the rabies virus antigen.

• Merkel cells are targets of the rabies virus in rabid dogs.

Abstract

The direct fluorescent antibody test (dFAT) on fresh brain tissues is the gold standard for rabies virus antigen detection in dogs. However, this method is laborious and holds a high risk of virus exposure for the experimenter. Skin biopsies are useful for the diagnosis of humans and animals. In mammals, the tactile hair, known as the follicle-sinus complex (FSC), is a specialized touch organ that is abundant in the muzzle skin. Each tactile hair is equipped with more than 2,000 sensory nerve endings. Therefore, this organ is expected to serve as an alternative postmortem diagnostic material. However, the target cells and localization of rabies virus antigen in the FSCs remain to be defined. In the present study, muzzle skins were obtained from 60 rabid dogs diagnosed with rabies by dFAT at the Research Institute of Tropical Medicine in the Philippines. In all dogs, virus antigen was clearly detected in a part of the outer root sheath at the level of the ring sinus of the FSCs, and the majority of cells were positive for the Merkel cell (MC) markers cytokeratin 20 and CAM5.2. Our results suggest that MCs in the FSCs of the muzzle skin are a target for virus replication and could serve as a useful alternative specimen source for diagnosis of rabies.

Introduction

Rabies is a zoonotic disease caused by the rabies virus. The virus is transmitted to humans via rabid animals and causes acute encephalomyelitis after a variable incubation period. More than 150 countries and territories currently report rabies infections in humans, with over 55,000 deaths annually, of which approximately 34,500 occur in Asian countries (WHO, 2013). In the Philippines, domestic dogs are the primary reservoir of rabies and more than 98% of human rabies deaths are due to dog bites (Dimaano et al., 2011).

The most widely used test for rabies diagnosis is the direct fluorescent antibody test (dFAT), which is recommended by both the OIE and WHO (OIE, 2009, WHO, 2013). The sensitivity of the dFAT is 100% when the brain samples are in a fresh state, and definitive diagnosis can be completed within 2 h. However, the sensitivity lowers and can be unsuitable when the brain samples have started to decompose under warm temperature (David, 2012, Kamolvarin et al., 1993, McElhinney et al., 2014). In addition, sampling of the dog brain is laborious, carries a high risk of virus exposure, and requires expensive equipment. Therefore, alternative specimens for postmortem diagnosis of rabid dogs by low-cost, simple tests with low virus-exposure risk are needed in rabies-endemic Asian countries.

Skin biopsies are useful for ante- and postmortem diagnosis of humans (Blenden et al., 1986) and animals (Blenden et al., 1983, Smith et al., 1972). The virus antigen can be detected in the peripheral nerves surrounding the hair follicles, and the positive rate increases as infection progresses (Blenden et al., 1986). In animals, the tactile hair, known as the follicle-sinus complex (FSC), is a specialized touch organ that is abundant in the muzzle skin. The tactile hair is embedded in the blood sinus, which plays an important role in tactile sensitivity and assists the animal in exploring its environment (Esteves et al., 2009, Marshall et al., 2014). Each tactile hair is equipped with more than 2,000 sensory nerve endings (Halata, 1993). After infection of the central nervous system (CNS), the rabies virus centrifugally spreads to the peripheral tissues via the sensory nerve. Therefore, tactile hairs are expected to serve as an alternative postmortem diagnostic material. In addition, obtaining tactile hair follicles from the muzzle skin of dead dogs is easy and practical, and does not require any specialized and expensive equipment. However, the target cells and localization of rabies virus antigen in the FSCs remain to be defined. The present study were undertaken to provide information on the target cells and localization of rabies virus antigen in the FSCs in the muzzle skins of rabid dogs.