A novel quantitative real-time PCR diagnostic assay for seal heartworm (Acanthocheilonema spirocauda) provides evidence for possible infection in the grey seal (Halichoerus grypus)

Abstract

The distinct evolutionary pressures faced by Pinnipeds have likely resulted in strong coevolutionary ties to their parasites (Leidenberger et al., 2007). This study focuses on the phocid seal filarial heartworm species Acanthocheilonema spirocauda. A. spirocauda is known to infect a variety of phocid seals, but does not appear to be restricted to a single host species (Measures et al., 1997; Leidenberger et al., 2007; Lehnert et al., 2015). However, to date, seal heartworm has never been reported in grey seals (Halichoerus grypus) (Measures et al., 1997; Leidenberger et al., 2007; Lehnert et al., 2015). The proposed vector for seal heartworm is Echinophthirius horridus, the seal louse. Seal lice are known to parasitize a wide array of phocid seal species, including the grey seal. With the advent of climate change, disease burden is expected to increase across terrestrial and marine mammals (Harvell et al., 2002). Accordingly, increased prevalence of seal heartworm has recently been reported in harbor seals (Phoca vitulina) (Lehnert et al., 2015). Thus, the need for improved, rapid, and cost-effective diagnostics is urgent. Here we present the first A. spirocauda-specific rapid diagnostic test (a quantitative real-time PCR assay), based on a highly repetitive genomic DNA repeat identified using whole genome sequencing and subsequent bioinformatic analysis. The presence of an insect vector provides the opportunity to develop a multifunctional diagnostic tool that can be used not only to detect the parasite directly from blood or tissue specimens, but also as a molecular xenomonitoring (XM) tool that can be used to assess the epidemiological profile of the parasite by screening the arthropod vector. Using this assay, we provide evidence for the first reported case of seal heartworm in a grey seal.

The Seal Louse (Echinophthirius horridus) in the Dutch Wadden Sea: Investigation of Vector-Borne Pathogens and Morphological Adaptations

“…In this context, E. horridus has previously been described as natural obligate intermediate host of A. spirocauda [19,20,21], and different stages of A. spirocauda larvae were found in dissected E. horridus seal lice [19,21]. So far, the heartworm A. spirocauda has been reported from different phocid species such as harbour seals (P. vitulina), hooded seals (Cystophora cristata), bearded seals (Erignathus barbatus), ribbon seals (P. fasciata), harp seals (P. groenlandica), ringed seals (P. hispida), spotted seals (P. largha), monk seals (Monachus monachus), and recently from grey seals (H. grypus) [20,37,53]. Furthermore, there is a signi cant positive correlation between heartworm infection and infestation of harbour seals with seal lice [21,22].…”

“…The designed nested PCR on basis of the mitochondrial cox1 gene was superior in detection of A. spirocauda-DNA in lice compared to a single PCR and had a con dent sensitivity of ve micro lariae corresponding to 0.5 ng DNA [54]. Recently Keroack et al [37] developed a more sensitive A. spirocauda real-time quantitative PCR based on a highly repetitive genomic DNA repeat identi ed using whole genome sequencing which will improve future monitoring of seal heartworm infections. These authors also identi ed the rst time an A. spirocauda adult worm in a presumed grey seal carcass from the coast of Cape Cod (Massachusetts, USA), supporting our detection of A. spirocauda-DNA in the seal lice collected from the grey seal which implied that grey seals could also get infected.…”

“…Interestingly, one A. spirocauda isolate (PPr11-007; accession nos. cox1 KT899872, ITS2 MG581463) collected from a stranded, deceased harbour seal from the USA west coast (Stinson beach, California) [37] differs from all other A. spirocauda isolates by some indels in the ITS2 region and point mutations in the partial cox1 gene resulting in only 95% and 93% sequence identity respectively (not shown). Thus the California isolate might indicate a subspecies or cryptic species.…”

The seal louse (Echinophthirius horridus) in the Dutch Wadden Sea: investigation of vector-borne pathogens 

“…In this context, E. horridus has previously been proposed to be the natural obligate intermediate host of A. spirocauda [7,8,9], and different stages of A. spirocauda larvae were found in dissected E. horridus seal lice [7,9]. So far, the heartworm A. spirocauda has been reported from different phocid species such as harbour seals, hooded seals (Cystophora cristata), bearded seals, ribbon seals (Phoca fasciata), harp seals (Phoca groenlandica), ringed seals, spotted seals (Phoca largha), monk seals (Monachus monachus), and recently from grey seals [8,33,52]. Furthermore, there is a signi cant positive correlation between heartworm infection and infestation of harbour seals with seal lice [9,10].…”

“…Interestingly, one A. spirocauda isolate (PPr11-007; accession nos. cox1 KT899872, ITS2 MG581463) collected from a stranded, deceased harbour seal from the USA west coast (Stinson beach, California) [33] differs from all other A. spirocauda isolates by some indels in the ITS2 region and point mutations in the partial cox1 gene resulting in only 95% and 93% sequence identity respectively (not shown). The partial 16S sequence of the Mycoplasma species detected in seal lice of three harbour seals in this stuy, was identical with the sequence from an infected patient.…”