Wildlife pathogen detection: evaluation of alternative DNA extraction protocols

Accurate detection of wildlife pathogens is critical in wildlife disease research. False negatives or positives can have catastrophic consequences for conservation and disease-mitigation decisions. Quantitative polymerase chain reaction is commonly used for molecular detection of wildlife pathogens. The reliability of this method depends on the effective extraction of the pathogen’s DNA from host samples. A wildlife disease that has been in the centre of conservationist’s attention is the amphibian disease Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Here, we compare the efficiency of a spin column extraction kit (QIAGEN), commonly used in Bd DNA extraction, to an alternative spin column kit (BIOKÈ) used in extractions from other types of samples, which is considerably cheaper but not typically used for Bd DNA extraction. Additionally, we explore the effect of an enzymatic pre-treatment on detection efficiency. Both methods showed similar efficiency when extracting Bd DNA from zoospores from laboratory-created cell-cultures, as well as higher efficiency when combined with the enzymatic pre-treatment. Our results indicate that selecting the optimal method for DNA extraction is essential to ensure minimal false negatives and reduce project costs.

The Invasive Bank Vole (Myodes glareolus): A Model System for Studying Parasites and Ecoimmunology during a Biological Invasion

The primary driver of the observed increase in emerging infectious diseases (EIDs) has been identified as human interaction with wildlife and this increase has emphasized knowledge gaps in wildlife pathogens dynamics. Wild rodent models have proven excellent for studying changes in parasite communities and have been a particular focus of eco-immunological research. Helminth species have been shown to be one of the factors regulating rodent abundance and indirectly affect disease burden through trade-offs between immune pathways. The Myodes glareolus invasion in Ireland is a unique model system to explore the invasion dynamics of helminth species. Studies of the invasive population of M. glareolus in Ireland have revealed a verifiable introduction point and its steady spread. Helminths studies of this invasion have identified enemy release, spillover, spillback and dilution taking place. Longitudinal studies have the potential to demonstrate the interplay between helminth parasite dynamics and both immune adaptation and coinfecting microparasites as M. glareolus become established across Ireland. Using the M. glareolus invasion as a model system and other similar wildlife systems, we can begin to fill the large gap in our knowledge surrounding the area of wildlife pathogen dynamics.

Soil Reservoir Dynamics of Ophidiomyces ophidiicola, the Causative Agent of Snake Fungal Disease

Wildlife diseases pose an ever-growing threat to global biodiversity. Understanding how wildlife pathogens are distributed in the environment and the ability of pathogens to form environmental reservoirs is critical to understanding and predicting disease dynamics within host populations. Snake fungal disease (SFD) is an emerging conservation threat to North American snake populations. The causative agent, Ophidiomyces ophidiicola (Oo), is detectable in environmentally derived soils. However, little is known about the distribution of Oo in the environment and the persistence and growth of Oo in soils. Here, we use quantitative PCR to detect Oo in soil samples collected from five snake dens. We compare the detection rates between soils collected from within underground snake hibernacula and associated, adjacent topsoil samples. Additionally, we used microcosm growth assays to assess the growth of Oo in soils and investigate whether the detection and growth of Oo are related to abiotic parameters and microbial communities of soil samples. We found that Oo is significantly more likely to be detected in hibernaculum soils compared to topsoils. We also found that Oo was capable of growth in sterile soil, but no growth occurred in soils with an active microbial community. A number of fungal genera were more abundant in soils that did not permit growth of Oo, versus those that did. Our results suggest that soils may display a high degree of both general and specific suppression of Oo in the environment. Harnessing environmental suppression presents opportunities to mitigate the impacts of SFD in wild snake populations.

Validation of an eDNA-based method for the detection of wildlife pathogens in water

Monitoring the occurrence and density of parasites and pathogens can identify high infection-risk areas and facilitates disease control and eradication measures. Environmental DNA (eDNA) techniques are increasingly used for pathogen detection due to their relative ease of application. Since many factors affect the reliability and efficacy of eDNA-based detection, rigorous validation and assessment of method limitations is a crucial first step. We evaluated an eDNA detection method using in situ filtration of large-volume water samples, developed to detect and quantify aquatic wildlife parasites by quantitative PCR (qPCR). We assessed method reliability using Batrachochytrium dendrobatidis, a pathogenic fungus of amphibians and the myxozoan Tetracapsuloides bryosalmonae, causative agent of salmonid proliferative kidney disease, in a controlled experimental setup. Different amounts of parasite spores were added to tanks containing either clean tap water or water from a semi-natural mesocosm community. Overall detection rates were higher than 80%, but detection was not consistent among replicate samples. Within-tank variation in detection emphasises the need for increased site-level replication when dealing with parasites and pathogens. Estimated parasite DNA concentrations in water samples were highly variable, and a significant increase with higher spore concentrations was observed only for B. dendrobatidis. Despite evidence for PCR inhibition in DNA extractions from mesocosm water samples, the type of water did not affect detection rates significantly. Direct spiking controls revealed that the filtration step reduced detection sensitivity. Our study identifies sensitive quantification and sufficient replication as major remaining challenges for the eDNA-based methods for detection of parasites in water.

A non-invasive method to assess environmental contamination with avian pathogens: beak and feather disease virus (BFDV) detection in nest boxes

Indirect transmission of pathogens can pose major risks to wildlife, yet the presence and persistence of wildlife pathogens in the environment has been little studied. Beak and feather disease virus (BFDV) is of global conservation concern: it can infect all members of the Psittaciformes, one of the most threatened bird orders, with infection often being lethal. Indirect transmission of BFDV through contaminated nest hollows has been proposed as a major infection source. However, data on whether and for how long nest sites in the wild remain contaminated have been absent. We determined the BFDV status of birds (parents and nestlings) for 82 nests of Crimson Rosellas, Platycercus elegans and Eastern Rosellas, Platycercus eximius. In 11 of these nests (13.4%, 95% confidence interval 6.9–22.7), we found an infected parent or nestling. Using nest swabs, we then compared BFDV presence at three points in time (before, during and after breeding) in three groups of nest boxes. These were nest boxes occupied by infected birds, and two control groups (nest boxes occupied by uninfected birds, and unoccupied nest boxes). Detection of BFDV on nest swabs was strongly associated with the infection status of parents in each nest box and with the timing of breeding. During breeding, boxes occupied by BFDV-positive birds were significantly more likely to have BFDV-positive nest swabs than boxes occupied by BFDV-negative birds; nest swabs tested BFDV-positive in 80% (28.4–99.5) of nests with parental antigen excretion, 66.7% (9.4–99.2) of nests occupied by parents with BFDV-positive cloacal swabs and 66.7% (22.3–95.7) of nests occupied by parents with BFDV–positive blood. 0% (0–52.2) of nests with BFDV–positive nestlings had BFDV–positive nest swabs. Across all boxes occupied by BFDV-positive birds (parents or nestlings), no nest swabs were BFDV–positive before breeding, 36.4% (95% CI 10.9–69.2) were positive during breeding and 9.1% (0.2–41.3) remained positive after breeding. BFDV was present on nest swabs for up to 3.7 months. Our study provides novel insights into the potential role of nest cavities and other fomites in indirect transmission of BFDV, and possibly other pathogens, and offers a non-invasive method for surveillance of pathogens in wild bird populations.

Non-invasive surveys of mammalian viruses using environmental DNA

ABSTRACTEnvironmental DNA (eDNA) and invertebrate-derived DNA (iDNA) have been used to survey biodiversity non-invasively to mitigate difficulties of obtaining wildlife samples, particularly in remote areas or for rare species. Recently, eDNA/iDNA have been applied to monitor known wildlife pathogens, however, most wildlife pathogens are unknown and often evolutionarily divergent.To detect and identify known and novel mammalian viruses from eDNA/iDNA sources, we used a curated set of RNA oligonucleotides as viral baits in a hybridization capture system coupled with high throughput sequencing.We detected multiple known and novel mammalian RNA and DNA viruses from multiple viral families from both waterhole eDNA and leech derived iDNA. Congruence was found between detected hosts and viruses identified in leeches and waterholes.Our results demonstrate that eDNA/iDNA samples represent an effective non-invasive resource for studying wildlife viral diversity and for detecting novel potentially zoonotic viruses prior to their emergence.

Extension of the known distribution of a novel clade C betacoronavirus in a wildlife host

Disease surveillance in wildlife populations presents a logistical challenge, yet is critical in gaining a deeper understanding of the presence and impact of wildlife pathogens. Erinaceus coronavirus (EriCoV), a clade C Betacoronavirus, was first described in Western European hedgehogs (Erinaceus europaeus) in Germany. Here, our objective was to determine whether EriCoV is present, and if it is associated with disease, in Great Britain (GB). An EriCoV-specific BRYT-Green® real-time reverse transcription PCR assay was used to test 351 samples of faeces or distal large intestinal tract contents collected from casualty or dead hedgehogs from a wide area across GB. Viral RNA was detected in 10.8% (38) samples; however, the virus was not detected in any of the 61 samples tested from Scotland. The full genome sequence of the British EriCoV strain was determined using next generation sequencing; it shared 94% identity with a German EriCoV sequence. Multivariate statistical models using hedgehog case history data, faecal specimen descriptions and post-mortem examination findings found no significant associations indicative of disease associated with EriCoV in hedgehogs. These findings indicate that the Western European hedgehog is a reservoir host of EriCoV in the absence of apparent disease.

Macroecology of birds potentially susceptible to West Nile virus

Zoonotic diseases transmitted by wildlife affect biological conservation, public and animal health, and the economy. Current research efforts are aimed at finding wildlife pathogens at a given location. However, a meta-analytical approach may reveal emerging macroecological patterns in the host–pathogen relationship at different temporal and spatial scales. West Nile virus (WNV) is a pathogen with worldwide detrimental impacts on bird populations. To understand macroecological patterns driving WNV infection, we aimed to recognize unknown competent reservoirs using three disease metrics—serological prevalence (SP), molecular prevalence (MP) and mortality (M)—and test if these metrics are correlated with the evolutionary history, geographical origin of bird species, viral strain, time–space and methodology. We performed a quantitative review of field studies on birds sampled for WNV. We obtained 4945 observations of 949 species from 39 countries. Our analysis supported the idea that MP and M are good predictors of reservoir competence, and allowed us to identify potential competent reservoirs. Furthermore, results indicated that the variability of these metrics was attributable to phylogeny, time–space and sample size. A macroecological approach is needed to recognize susceptible species and competent reservoirs, and to identify other factors driving zoonotic diseases originating from wildlife.

Assessing the contributions of intraspecific and environmental sources of infection in urban wildlife: Salmonella enterica and white ibis as a case study

Conversion of natural habitats into urban landscapes can expose wildlife to novel pathogens and alter pathogen transmission pathways. Because transmission is difficult to quantify for many wildlife pathogens, mathematical models paired with field observations can help select among competing transmission pathways that might operate in urban landscapes. Here we develop a mathematical model for the enteric bacteria Salmonella enterica in urban-foraging white ibis ( Eudocimus albus ) in south Florida as a case study to determine (i) the relative importance of contact-based versus environmental transmission among ibis and (ii) whether transmission can be supported by ibis alone or requires external sources of infection. We use biannual field prevalence data to restrict model outputs generated from a Latin hypercube sample of parameter space and select among competing transmission scenarios. We find the most support for transmission from environmental uptake rather than between-host contact and that ibis–ibis transmission alone could maintain low infection prevalence. Our analysis provides the first parameter estimates for Salmonella shedding and uptake in a wild bird and provides a key starting point for predicting how ibis response to urbanization alters their exposure to a multi-host zoonotic enteric pathogen. More broadly, our study provides an analytical roadmap to assess transmission pathways of multi-host wildlife pathogens in the face of scarce infection data.