We describe the pathologic and immunohistochemical findings in five cases of rabies in the insectivorous Pallas's mastiff bat (Molossus molossus) in the city of Patos, state of Paraíba, northeastern Brazil. All cases were found during the day, in different neighborhoods, prostrate and unable to fly. No significant lesions were observed at necropsy or on histologic examination. The brains of the five bats showed strong immunohistochemical labeling for rabies virus in the form of large corpuscles or multiple clusters of granules within the perikarya of neurons in different areas of the brain, mainly in the cerebral cortex and cerebellar Purkinje cells. Three bats tested by direct immunofluorescence and mouse inoculation were also positive for rabies. These data demonstrate that M. molossus may be infected with the rabies virus and develop rabies, even without histologic lesions in the central nervous system, and can be a source of infection for humans and domestic animals.
Rabies is a viral disease affecting mammals, causing an acute and fatal encephalomyelitis following a bite or scratch from another rabies virus–infected mammal. In Brazil, infective hosts are mainly domestic carnivores or bats (Kotait et al. 2007). Bats have a relevant role in the chain of rabies transmission (FAO 2011), and insectivorous, frugivorous, omnivorous, pollinivorous, and piscivorous bats, including the insectivorous Pallas's mastiff bat (Molossus molossus), are infected with rabies virus (Páez et al. 2003; Jackson and Wunner 2007). In the US, between 1986 and 2011, 44 of 67 cases of rabies were associated with nonhematophagous bats (CDC 2013). In instances in which transmission is associated with nonhematophagous bats, the bite is sometimes not noticed by the patient at an early stage, and postexposure prophylaxis is not effective (Kotait et al. 2007).
In Brazil, rabies has been diagnosed by a fluorescent antibody test (FAT) and mouse inoculation test (MIT) in 42 species of hematophagous and nonhematophagous bats, including Pallas's mastiff bat (Sodré et al. 2010); however, there are no detailed studies of rabies in these species that characterize their histologic and immunohistochemical features. We describe the pathologic and immunohistochemical findings of rabies in five cases of rabies in Pallas's mastiff bat diagnosed at the Laboratory of Animal Pathology, Federal University of Campina Grande in the semiarid region of Paraíba, Brazil.
Bats were identified according to a taxonomic key (Gregorin and Taddei 2002), euthanized, and necropsied. Fragments from organs of the thoracic and abdominal cavities and central nervous system (CNS) were collected and fixed in 10% buffered formalin. The fragments were embedded in paraffin, cut into 5-µm sections, and stained with hematoxylin and eosin for histopathologic analysis. Fragments of the cerebrum, cerebellum, brainstem, and spinal cord of three cases were examined by a FAT (Dean et al. 1996) and MIT (Koprowski 1996).
Paraffin blocks with CNS fragments were selected and sent for immunohistochemical analysis. After paraffin removal and tissue rehydration, antigen retrieval was performed with citrate solution (pH 6.0) in a microwave oven at full power for 10 min. A polyclonal antibody for rabies virus produced in goats (fluorescein isothiocyanate-conjugated antibody [FICA, #5199, Chemicon, Billerica, Massachusetts, USA]) served as the primary antibody and was diluted 1∶1,000 in phosphate-buffered saline with Tween 20 (PBST) prior to incubation for 60 min at 37 C. The biotinylated secondary antibody and streptavidin-biotin-peroxidase complex (LSAB+System HRP, Dako, Troy, Michigan, USA) were incubated at room temperature for 30 min and labeled by addition of the liquid diaminobenzidine+substrate-chromogen system (Dako) and counterstained with Harris hematoxylin. Histologic sections of confirmed cases of rabies in cattle (Bos taurus) were used as a positive control. As a negative control, the same sections were used, and the primary antibody was replaced by PBST.
The five bats were identified as M. molossus. All were found in the morning or late afternoon, prostrate and unable to fly, in different neighborhoods of the city of Patos. At necropsy, no gross lesions were observed to justify the clinical signs. Microscopically, there were no changes observed in the organs of the thoracic or abdominal cavities or the CNS. The brains of the five bats showed strong positive immunohistochemical labeling for rabies virus in the form of large corpuscles or multiple clusters of granules within the perikarya of neurons in several areas of the brain, mainly the cerebral cortex and cerebellar Purkinje cells (Fig. 1A). The three cases submitted to FAT and MIT were positive for rabies.
Our results demonstrate that insectivorous bats identified as M. molossus were infected by rabies virus (i.e., all five cases were positive by immunohistochemistry). Additionally, three of those cases submitted to FAT and MIT were also positive by these techniques. Although histopathology is an important tool in the diagnosis of rabies, it is rarely used in bats, and diagnoses are usually made by FAT and MIT (FAO 2011; CDC 2013). In other species, immunohistochemistry is an important method for rabies diagnosis, especially for cases in which nonsuppurative encephalitis and inclusion bodies are not observed (Maxie and Youssef 2007) and when it is not possible to perform FAT and MIT. According to Tao et al. (2008), the sensitivity and specificity of immunohistochemistry are equivalent to the FAT and real-time PCR.
An important observation in these bats was the absence of nonsuppurative meningoencephalitis and inclusion bodies in the five cases, suggesting that the absence of histologic lesions, a rare event in other species (Maxie and Youssef 2007), is common in Pallas's mastiff bats. In contrast, in Australian frugivorous bats (Pteropus spp.), rabies causes meningoencephalitis and nonsuppurative ganglioneuritis with rare or absent inclusion bodies (Hooper et al. 1999). In some species, such as cattle, a lack of histologic lesions in cases of rabies has been reported, although such cases are positive by immunohistochemistry.
In most cases, the clinical signs of rabies in bats are associated with paresis or paralysis, although some rabid bats may also present aggressiveness (FAO 2011). The prostration and inability to fly observed in the Pallas's mastiff bats are most likely related to muscle paralysis involving the CNS. According to Uieda et al. (1995), muscle paralysis is a common finding in bats infected with rabies, which explains the inability to fly. Clinical signs in bats generally reflect changes in behavior and motor function progressing rapidly to death within hours or days. Both suborders, Microchiroptera and Megachiroptera, have clinical signs including unusual aggressiveness, tolerance to humans, fighting with other bats, increased activity and avoiding shelter during the day, biting, and vocalization; they are often found on the ground unable to fly (Kotait et al. 2007).
All the bats examined in this study were collected from homes, which reflects the risk of rabies transmission to humans. Over the past few years, the importance of nonhematophagous bats in the maintenance of rabies in the urban environment has been demonstrated, as they are responsible for accidental infection of dogs (Canis lupus familiaris) and cats (Felis catus) (Kotait et al. 2007). Rabies transmission to humans by frugivorous and insectivorous bats has not been confirmed yet, although these bats have become a major source of human rabies in areas where rabies has been controlled in domestic carnivores (FAO 2011) and in areas where there are no hematophagous bats. In southern Brazil, rabies was reported in a domestic dog infected by a virus variant associated with the insectivorous Brazilian free-tailed bat (Tadarida brasiliensis) (Batista et al. 2009). In Chile, a human case of rabies was caused by the same variant (Favi et al. 2002).
Rabies is enzootic in Pallas's mastiff bats in the city of Patos, serving as a possible source of infection for humans and domestic animals. These bats may develop the disease even without microscopic lesions in the CNS. Immunohistochemistry is an effective tool for the diagnosis of rabies in insectivorous bats.