A novel copro-diagnostic molecular method for qualitative detection and identification of parasitic nematodes in amphibians and reptiles

• Published in: PloS one 2017-09, Vol.12 (9), p.e0185151
• Main Authors: Huggins, Lucas G, Michaels, Christopher J, Cruickshank, Sheena M, Preziosi, Richard F, Else, Kathryn J
• Format: Article
• Language: English
• Subjects: Amphibia; Amphibians; Amphibians - parasitology; Animals; Anthropogenic factors; Biodiversity; Biodiversity loss; Bioindicators; Biology; Biology and Life Sciences; Climate change; Deoxyribonucleic acid; Diagnostic systems; DNA; DNA - analysis; DNA - genetics; DNA Primers - genetics; Ecosystems; Eggs; Endangered & extinct species; Environmental DNA; Euthanasia; Extinct species; Extinction; Feces - parasitology; Fragments; Frogs; Genetic aspects; Habitat fragmentation; Health aspects; Identification; Identification and classification; Indicator species; Infections; Manures; Medical diagnosis; Medicine and Health Sciences; Methods; Mice; Molecular Diagnostic Techniques - methods; Morphology; Nematoda - genetics; Nematoda - physiology; Nematode Infections - diagnosis; Nematodes; Parasites; Polymerase chain reaction; Primers; Protocol; Reptiles; Reptiles - parasitology; Research and analysis methods; Roundworms; Species extinction; Trends
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0185151

Anthropogenic disturbance via resource acquisition, habitat fragmentation and climate change, amongst other factors, has led to catastrophic global biodiversity losses and species extinctions at an accelerating rate. Amphibians are currently one of the worst affected classes with at least a third of species categorised as being threatened with extinction. At the same time, they are also critically important for many habitats and provide man with a powerful proxy for ecosystem health by acting as a bioindicator group. Whilst the causes of synchronised amphibian losses are varied recent research has begun to highlight a growing role that macroparasites are playing in amphibian declines. However, diagnosing parasite infection in the field can be problematic, principally relying on collection and euthanasia of hosts, followed by necropsy and morphological identification of parasites in situ. The current study developed a non-invasive PCR-based methodology for sensitive detection and identification of parasitic nematode DNA released in the faeces of infected amphibians as egg or tissue fragments (environmental DNA). A DNA extraction protocol optimised for liberation of DNA from resilient parasite eggs was developed alongside the design of a novel, nematode universal, degenerate primer pair, thus avoiding the difficulties of using species specific primers in situations where common parasite species are unknown. Used in conjunction this protocol and primer pair was tested on a wide range of faecal samples from captive and wild amphibians. The primers and protocol were validated and detected infections, including a Railletnema nematode infection in poison dart frogs from ZSL London Zoo and Mantella cowani frogs in the wild. Furthermore, we demonstrate the efficacy of our PCR-based protocol for detecting nematode infection in other hosts, such as the presence of pinworm (Aspiculuris) in two tortoise species and whipworm (Trichuris muris) in mice. Our environmental DNA approach mitigates problems associated with microscopic identification and can be applied to detect nematode parasitoses in wild and captive hosts for infection surveillance and maintenance of healthy populations.