Development of a copro-diagnostic molecular tool for the detection and identification of amphibian infecting helminths

UoM administered thesis: Master of Philosophy

  • Authors:
  • Lucas Huggins

Abstract

Worldwide at least a third of amphibians are currently categorised as being threatened with extinction, with many species experiencing population declines at an unprecedented rate. The causes of such synchronised amphibian losses are varied but recent research has begun to highlight a growing role that macroparasites are playing in this decline. However, diagnosing parasite infection in the field can be problematic and has principally relied on collection and euthanasia of hosts, followed by necropsy and morphological identification of parasites in situ. To improve upon this, the current study developed a non-invasive PCR-based methodology for sensitive detection and identification of nematode infecting amphibians by detecting environmental DNA released in faeces or shed eggs. 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. Used in conjunction this DNA extraction protocol and primer pair was tested on a wide range of faecal samples from both captive and wild amphibians, showing great promise at detecting parasitic nematode infections. New infections, such as a Railletnema nematode infestation in poison dart frogs from London Zoo were uncovered, as well as a similar infection from Mantella cowani frogs in the wild. These results demonstrate the utility of our developed protocol for revealing previously undetected parasitic infections from amphibians in both captive colonies and in situ. Furthermore, the method was proven to function equally well on reptile samples. When our new primers were compared to previously reported nematode specific primers in the literature they were demonstrated to have greater sensitivity and specificity for nematode DNA. Our developed eDNA approach mitigates problems associated with microscopic identification, such as the necessity for researcher expertise and could also provide insight into the dynamics of parasite infection in wild amphibians, underscoring links to their declines. In addition, the work within this thesis raises awareness of some of the issues relating to the diagnostic use of eDNA from faeces, for example the contamination of samples by free-living organisms.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date31 Dec 2016