Isolation and detection of rabies virus in the fecal contents of naturally infected fruit bats

Abstract

The spread of the rabies virus into various tissues, organs and secretions in infected animals occurs with the progression of clinical disease. Fecal samples may have lower viral concentration and the objective of this study was to isolate and detect rabies virus in the fecal contents of fruit bats through virus isolation in cell culture technique and by RT-PCR, hemi-nested RT-PCR (hnRT-PCR) and Real Time RT-PCR. Thirty specimens of the genus Artibeus bats, previously identified as positive for rabies by FAT and inoculation of murine neuroblastoma cell line (N2A) were selected and the intestine was removed from every animal and was scraped off, in order to collect the fecal contents. The fecal contents were weighed, homogenized and diluted 1:10 (w/v) using a diluent, consisting of 0.85% saline solution, supplemented with 2% Bovine Fetal Serum free of rabies virus-specific antibodies and 0.1% of gentamicin sulfate. The suspensions were kept at 4°C for 30 minutes and centrifuged at 800xg for 30 minutes at 4°C, filtered with the aid of 5-ml and 33mm length syringe provided with filter Millex® with porosity of 0.45μm. The suspensions were inoculated into murine neuroblastoma cells (N2A) for viral isolation. For molecular techniques, extraction of total RNA and the reverse transcription were carried out, followed by PCR and hnRT-PCR targeting to gene N. The Real Time RT-PCR technique was performed on the product generated from the reverse transcription. Of the 30 suspensions inoculated, only one (3.33%) was positive for virus isolation. None of the samples was positive by RT-PCR, however, 13 samples (43.33%) were positive for rabies by hnRT-PCR and Real Time RT-PCR techniques. The fact that only one sample was positive by virus isolation can be explained by a variety of interferents found in this substrate, such as the presence of bacteria and also different degraded products of food that can cause inhibition of the reactions. It is also believed that these interferents may influence the results of the RT-PCR, hnRT-PCR and the Real Time RT-PCR techniques, demonstrating that the fecal contents are of the most complex biological samples for amplification techniques used as diagnostic methods. Nevertheless, this study demonstrated that both the hnRT-PCR and Real Time RT-PCR techniques were sensitive for the detection of rabies virus. Thus, we conclude that these techniques can be used as complementary tools in laboratory diagnosis and fecal samples may also be used for diagnosis of rabies.