Validation of a Field-Portable, Handheld Real-Time PCR System for Detecting Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats.

White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has decimated bat populations across North America. Despite ongoing management programs, WNS continues to expand into new populations, including in US states previously thought to be free from the pathogen and disease. This expansion highlights a growing need for surveillance tools that can be used to enhance existing monitoring programs and support the early detection of P. destructans in new areas. We evaluated the feasibility of using a handheld, field-portable, real-time (quantitative) PCR (qPCR) thermocycler known as the Biomeme two3 and the associated field-based nucleic acid extraction kit and assay reagents for the detection of P. destructans in little brown bats (Myotis lucifugus). Results from the field-based protocol using the Biomeme platform were compared with those from a commonly used laboratory-based qPCR protocol. When using dilutions of known conidia concentrations, the lowest detectable concentration with the laboratory-based approach was 108.8 conidia/mL, compared with 1,087.5 conidia/mL (10 times higher, i.e., one fewer 10× dilution) using the field-based approach. Further comparisons using field samples suggest a high level of concordance between the two protocols, with positive and negative agreements of 98.2% and 100% respectively. The cycle threshold values were marginally higher for most samples using the field-based protocol. These results are an important step in establishing and validating a rapid, field-assessable detection platform for P. destructans, which is urgently needed to improve the surveillance and monitoring capacity for WNS and support on-the-ground management and response efforts.

Two Novel Iflaviruses Discovered in Bat Samples in Washington State.

Arthropods are integral to ecosystem equilibrium, serving as both a food source for insectivores and supporting plant reproduction. Members of the Iflaviridae family in the order Picornavirales are frequently found in RNA sequenced from arthropods, who serve as their hosts. Here we implement a metagenomic deep sequencing approach followed by rapid amplification of cDNA ends (RACE) on viral RNA isolated from wild and captured bat guano in Washington State at two separate time points. From these samples we report the complete genomes of two novel viruses in the family Iflaviridae. The first virus, which we call King virus, is 46% identical by nucleotide to the lethal honeybee virus, deformed wing virus, while the second virus which we call Rolda virus, shares 39% nucleotide identity to deformed wing virus. King and Rolda virus genomes are 10,183 and 8934 nucleotides in length, respectively. Given these iflaviruses were detected in guano from captive bats whose sole food source was the Tenebrio spp. mealworm, we anticipate this invertebrate may be a likely host. Using the NCBI Sequence Read Archive, we found that these two viruses are located in six continents and have been isolated from a variety of arthropod and mammalian specimens.

First Detection of Bat White-Nose Syndrome in Western North America.

White-nose syndrome (WNS) is an emerging fungal disease of bats caused by Pseudogymnoascus destructans. Since it was first detected near Albany, NY, in 2006, the fungus has spread across eastern North America, killing unprecedented numbers of hibernating bats. The devastating impacts of WNS on Nearctic bat species are attributed to the likely introduction of P. destructans from Eurasia to naive host populations in eastern North America. Since 2006, the disease has spread in a gradual wavelike pattern consistent with introduction of the pathogen at a single location. Here, we describe the first detection of P. destructans in western North America in a little brown bat (Myotis lucifugus) from near Seattle, WA, far from the previously recognized geographic distribution of the fungus. Whole-genome sequencing and phylogenetic analyses indicated that the isolate of P. destructans from Washington grouped with other isolates of a presumed clonal lineage from the eastern United States. Thus, the occurrence of P. destructans in Washington does not likely represent a novel introduction of the fungus from Eurasia, and the lack of intensive surveillance in the western United States makes it difficult to interpret whether the occurrence of P. destructans in the Pacific Northwest is disjunct from that in eastern North America. Although there is uncertainty surrounding the impacts of WNS in the Pacific Northwest, the presence of the pathogen in western North America could have major consequences for bat conservation. IMPORTANCE White-nose syndrome (WNS) represents one of the most consequential wildlife diseases of modern times. Since it was first documented in New York in 2006, the disease has killed millions of bats and threatens several formerly abundant species with extirpation or extinction. The spread of WNS in eastern North America has been relatively gradual, inducing optimism that disease mitigation strategies could be established in time to conserve bats susceptible to WNS in western North America. The recent detection of the fungus that causes WNS in the Pacific Northwest, far from its previous known distribution, increases the urgency for understanding the long-term impacts of this disease and for developing strategies to conserve imperiled bat species.

Novel poxvirus in big brown bats, northwestern United States.

A wildlife hospital and rehabilitation center in northwestern United States received several big brown bats with necrosuppurative osteomyelitis in multiple joints. Wing and joint tissues were positive by PCR for poxvirus. Thin-section electron microscopy showed poxvirus particles within A-type inclusions. Phylogenetic comparison supports establishment of a new genus of Poxviridae.

First records of rodent-infesting Cuterebra bot flies parasitizing raccoons (Procyon lotor) in North America.

Larvae of Cuterebra spp. bot flies are specialized parasites of native species of either Rodentia (e.g., mice, rats, and tree squirrels) or Lagomorpha (e.g., rabbits and hares) in the Americas. However, they also infest other native and introduced wildlife, domestic animals, and humans, but which Cuterebra species parasitize these "atypical" hosts has seldom been determined, largely because the larvae are difficult to identify to species without first rearing them to adults. Here, we describe 2 Cuterebra spp. larvae removed from raccoons, Procyon lotor (Carnivora), in Washington (WA larva) and Florida (FL larva). These specimens belong to rodent-infesting species, which possess flattened, multipoint cuticular platelets in contrast to the typically single-pointed, spinelike platelets of lagomorph-infesting species. Based on the number and shape of points on these platelets, and the infestation dates and locations, we tentatively identify the species involved as Cuterebra grisea (WA larva) and C. fontinella (FL larva), which typically parasitize Peromyscus spp. mice. Uncertainty about these species' assignments arises from a lack of information on intraspecific variation in larval characteristics and the apparent lack of larval descriptions for 2 of the 7 Cuterebra species most likely to have parasitized these animals. This report seems to be the first published documentation of raccoons infested by larvae of Cuterebra spp. for the west and Gulf coasts of North America, and of rodent-infesting Cuterebra species parasitizing a species of carnivore.