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

© 2017 Huggins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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

Assessment of the nematicidal potential of vermicompost, vermicompost tea, and urea application on the potato-cyst nematodes Globodera rostochiensis and Globodera pallida

The addition of organic material to the soil can be an effective alternative to the environmentally unsafe chemical treatments that are used to control plant parasitic nematodes. We evaluated the effects of vermicompost alone, and aqueous solutions of vermicompost (vermicompost tea) either alone or mixed with urea, on the development and survival of two potato-cyst nematodes: Globodera rostochiensis (pathotype Ro1) and G. pallida (pathotype Pa2) and on the growth parameters of the host potato plants. Soil amendments with these materials significantly decreased the number of cysts · 400 g–1 of both species in the soil, the number of eggs and juveniles · cyst–1 of both species, and the number of eggs and juveniles · g–1 of both species in the soil, relative to the untreated controls. The suppressive effect was significantly higher at the highest dose than the lowest treatment dose, for all tested materials. Globodera rostochiensis was more sensitive to all the tested materials than G. pallida. The aqueous solutions of vermicompost alone or in combination with urea were more effective than the solid vermicompost used alone, for controlling both species. Vermicompost and the vermicompost teas had positive effects on plant fresh stem weight and stem height. The application of vermicompost tea instead of the solid vermicompost, substantially decreased the amount of material needed. These amendments are thus promising for the control of potato-cyst nematodes in sustainable agricultural systems