Lyssa excreta: Defining parameters for fecal samples as a rabies virus surveillance method.

It is not possible to systematically screen the environment for rabies virus (RABV) using current approaches. We sought to determine under what conditions RABV is detectable from feces and other accessible samples from infected wildlife to broaden the number of biological samples that could be used to test for RABV. We employed a recently-developed quantitative RT-PCR assay called the "LN34 panlyssavirus real-time RT-PCR assay", which is highly sensitive and specific for all variants of RABV. We harvested and tested brain tissue, fecal, and/or mouth swab samples from 25 confirmed RABV positive bats of six species. To determine if rabies RNA lasts in feces sufficiently long post-defecation to use it as a surveillance tool, we tested fecal samples from 10 bats at the time of sample collection and after 24 hours of exposure to ambient conditions, with an additional test on six bats out to 72 hours. To assess whether we could pool fecal pellets and still detect a positive, we generated dilutions of known positives at 1:1, 1:10, 1:50, and 1:200. For six individuals for which matched brain, mouth swab, and fecal samples were tested, results were positive for 100%, 67%, and 67%, respectively. For the first time test to 24 hours, 63% of feces that were positive at time 0 were still positive after 24 hours, and 50% of samples at 72 hours were positive across all three replicates. Pooling tests revealed that fecal positives were detected at 1:10 dilution, but not at 1:50 or 1:200. Our preliminary results suggest that fecal samples hold promise for a rapid and non-invasive environmental screening system.

Endangered Nectar-Feeding Bat Detected by Environmental DNA on Flowers.

Leptonycteris nivalis (the Mexican long-nosed bat) is an endangered nectar-feeding bat species that follows "nectar corridors" as it migrates from Mexico to the southwestern United States. Locating these nectar corridors is key to their conservation and may be possible using environmental DNA (eDNA) from these bats. Hence, we developed and tested DNA metabarcoding and qPCR eDNA assays to determine whether L. nivalis could be detected by sampling the agave flowers on which it feeds. We sampled plants with known bat visitations in the Sierra Madre Oriental in Laguna de Sanchez (LS), Nuevo León, Mexico, and in the Chisos Mountains in Big Bend National Park, TX, USA (CB). A total of 13 samples included both swabs of agave umbels and cuttings of individual flowers. DNA metabarcoding was performed as a PCR multiplex that targeted bats (SFF-COI), arthropods (ANML-COI), and plants (ITS2 and rbcL). We targeted arthropods and plants in parallel with bats because future metabarcoding studies may wish to examine all the pollinators and plants within the nectar corridor. We developed and tested the sensitivity and specificity of two qPCR assays. We found that both DNA metabarcoding and qPCR were highly successful at detecting L. nivalis (11 of 13 for DNA metabarcoding and 12 of 13 for qPCR). Swabs and flower cuttings and both qPCR assays detected the species over four replicates. We suggest that L. nivalis leaves substantial DNA behind as it forages for nectar. We also suggest that future studies examine the time since sampling to determine its effect on detection success. The DNA metabarcoding multiplex will be useful for parallel questions regarding pollination ecology, while, with further testing, the qPCR assays will be effective for large-scale sampling for the detection of migration corridors and foraging areas. This work may be relevant to other nectar-feeding bat species, which can likely be detected with similar methodologies.