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Analysis of seal heartworm (Acanthocheilonema spirocauds) and identification of unknown harbor porpoise (Phocoena phocoena) parasites using molecular techniques
Steven A. Williams
Master of Science
Molecular parasitology, Molecular biology, Marine mammals, Porpoises, Parasite
Abstract. Seal heartworm (Acanthocheilonema spirocauda) is a filarial parasite of phocid seals, mainly the harbor seal (Phoca vitulina). Seal heartworm has been reported in several phocid seal host species, and is distributed throughout the Northern hemisphere (Leidenberger et al., 2007; Measures et al., 1997). Clinical manifestations are very similar to that of classic dog heartworm, including respiratory and cardiac complications (Dailey, 2005). To date, seal heartworm is believed to be restricted to only phocid hosts, and has only been observed in particular species (Felix, 2013; Leidenberger et al., 2007; Measures et al., 1997). Initial molecular results suggested that seal heartworm, or an extremely close relative, is capable of infecting the common harbor porpoise (Phocoenca phocoena), a species that has never been reported to harbor the parasite (Leidenberger, 2007). Preliminary identification of A. spirocauda has relied on small, single bar-coding genes: the nuclear internal transcribed spacer 2 (ITS2), the small ribosomal subunit (18S rRNA, SSU) and the mitochondrial gene cytochrome oxidase subunit 1 (COX1). In addition to these single gene barcodes, the presence of the Porphobilinogen deaminase (PBGD) pseudogene was tested as an potential indicator of genus, as this pseudogene had only been reported in Acanthocheilonema and Dipetalonema species (unpub. data; Keroack et al., 2016). In this study this assumption is proven , and the serendipitous discovery that strongylid porpoise lungworms also contain the Wolbachia derived pseudogene may have major evolutionary implications. PBGD screening is thus not a good diagnostic tool. To create more definitive identifications, recent work has focused on isolating and amplifying full mitochondrial genomes (mtDNA). Thus far, we have amplified and sequenced genomes from representative samples of two strongylid lungworms: Halocercus delphini, Otostrongylis circumlitis, as well as A. spirocauda using long PCR. Additionally the mtDNA of the 9 unknown porpoise parasites was also sequenced using low coverage full genome sequencing. These mitochondrial genomes will enable the definitive identification of seal heartworm in the harbor porpoises, as the gene order between filarial and stongylid parasites differs (Yatawara et al., 2010). Additionally, I have sequenced the full genome of A. spirocauda and developed a simple quantitative real time PCR diagnostic assay that is extremely specific to seal heartworm. These data combined have allowed us to identify these porpoise parasites, and assess whether or not the parasite is spreading outside of its traditional hosts. Although the data show that seal heartworm is not spreading in cetaceans, the data do show that the parasite is spreading to new phocid hosts. This study also highlights the need to improve reference databases to allow for accurate DNA barcoding, which is currently not accurate or effective.
Keroack, Caroline Dana, "Analysis of seal heartworm (Acanthocheilonema spirocauds) and identification of unknown harbor porpoise (Phocoena phocoena) parasites using molecular techniques" (2016). Masters Thesis, Smith College, Northampton, MA.
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161 pages : color illustrations. Includes bibliographical references (pages 120-129)