Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.

Severe Perkinsea infection (SPI) is an emerging disease that has been linked to mass die-offs of tapdoles across a broad geographic range in the USA. The exact relationship between Perkinsea infection and SPI disease remains undefined and untested. It is not known if certain co-factors (e.g. co-infection with other pathogens, exposure to pollution, host microbiome composition) contribute to disease progression, or if differences in host susceptibility affect the distribution of Perkinsea infection and SPI disease. We have yet to determine if the two known lineages of amphibian Perkinsea (i.e. a putatively pathogenic lineage and a putatively non-pathogenic lineage) are distinct strains, or if they are genetic variants of the same strain. Finally, we must continue to explore the extent of the parasite’s geographic range, particularly in regions with high amphibian diversity and/or a large number of threatened species. This thesis aims to elucidate some of the unknown aetiological and epidemiological aspects of Perkinsea infection and SPI disease in amphibians. These aims were addressed with a variety of experimental approaches. We investigated the aetiology and transmission of SPI disease by experimentally infecting healthy tadpoles with Perkinsea parasites, and by feeding tissue from putatively infected tadpoles to healthy tadpoles. We developed a field-compatible duplex qPCR assay for the rapid detection of the two lineages of amphibian Perkinsea, to be used as an epidemiological tool for monitoring amphibian health. We used phylogenetic analysis to investigate Perkinsea infection in captive tadpoles from the UK and wild tadpoles from Panama, a global centre of amphibian diversity and a biosphere greatly affected by amphibian decline. We used amplicon-based metagenomics to compare the intestinal eukaryotic microbiomes of healthy and Perkinsea-infected tadpoles. Our experimental infections did not result in a pathogenic interaction in our sample, and we did not detect ribosomal activity in the parasites. The results for the cannibalism feeding experiment were obscured by a pre-existing cryptic infection in our sample. Our results suggest that Perkisnea pathogenicity is limited, or is only manifest under specific ecological circumstances. Our optimization efforts confirmed that our novel duplex qPCR assay is specific, efficient, and sensitive in most conditions, thereby supporting its application as a tool for amphibian health surveys, and means to improve understanding of the dynamics of Perkinsea infection and SPI disease. We found molecular evidence of Perkinsea infection in a wide host range in Panama, and in the UK aquaculture samples, thereby expanding amphibian Perkinsea’s known biogeographical range and providing a broader base for risk assessment with a view to conservation. The results of our microbiome comparison did not reveal any major compositional differences in the intestinal eukaryotic microbiomes between healthy and Perkinsea-infected tadpoles; however, more specific taxonomic ranks need to be investigated before conclusions can be made. The outcomes of these experiments offer novel insights into under-studied aetiological and epidemiological aspects of this emerging protistan disease of amphibians. These insights can potentially improve amphibian management strageies to help mitigate the impact of Perkinsea infection and SPI disease, and possibly prevent disease outbreaks.