Sarcocystis halieti Gjerde & Vikøren & Hamnes 2018

3.3.2. Ribosomal DNA unit of S. halieti

As stated above, one species from the sea eagle, which we have named S. halieti, showed a high identity with GenBank sequences JQ733511–JQ733513 of an unnamed species from the great cormorant in Lithuania (unpublished study by Prakas and co-workers), and is considered to be identical with that species. As regards the complete 18S rRNA gene, which was 1803 bp long, the new sequence (GenBank no. MF 946587) was 100% identical with GenBank sequence JQ733511 from the great cormorant, but it was also completely identical with sequences of Sarcocystis wobeseri (GenBank nos. GQ 922885, GQ922886, HM 159419, EU502869). Moreover, the new sequence of S. halieti differed at only one nucleotide position (A rather than G in position 9 from the 3′ end) from GenBank sequence GQ245670 of S. calchasi, but this difference seems to be due to the inclusion of the terminal reverse primer (Primer B) in that sequence (Olias et al., 2010b). This primer does not match sequences of various Sarcocystis spp. in that position, and it was therefore modified to Primer BSarc (Gjerde, 2014b). Thus, S. calchasi actually has a G in this position as can be ascertained from GenBank sequence KC733718. Hence, at least two species, S. wobeseri and S. calchasi, were identical with S. halieti at the complete 18S rRNA gene. Moreover, sequences of this gene from several other Sarcocystis spp. with birds or carnivores as intermediate hosts (S. columbae, GenBank no. HM 125054; Sarcocystis corvusi, GenBank no. JN256117; S. turdusi, GenBank no. JF975681; S. lutrae, GenBank no. KM 657769; S. lari, GenBank no. JQ733508) differed by only 1–6 nucleotides (99.7–99.9% identity) from that of S. halieti.

The new 1504-bp-long sequence of the partial 28S rRNA gene that was assigned to S. halieti (GenBank no. MF 946610) differed at only one of 1474 overlapping positions (T instead of A at position 445 of new sequence; 99.9% identity) from GenBank sequence JQ733513 of the unnamed species from the great cormorant. As regards other species, the most similar ones were S. columbae (GenBank nos. HM 125053, GU253887; 99.5% identity), S. corvusi (GenBank no. JN256118; 99.5%), S. wobeseri (GenBank nos. GQ 922887, GQ922888, HM 159420, EF 079886; 99.0%), and S. turdusi (GenBank no. JF975682; 99.0%), whereas many other species with birds or carnivores as intermediate hosts showed identities between 98 and 99%.

As regards the ITS1region, a total of eight clones of S. halieti from one isolate were processed and sequenced (GenBank nos. MF 946589–MF946596; Table 1). The full-length sequences, including both primers, were 1083 or 1085 bp long, of which the ITS1 region comprised 828 or 830 bp, and the flanking portions of the 18S and 5.8S rRNA genes, 137 and 118 bp, respectively. The eight clones differed from each other at 1–9 nucleotide positions in the ITS1 region (98.9–99.9% identity within the ITS1 region; 99.2–99.9% identity between full-length sequences). These differences were mainly due to substitutions (single nucleotide polymorphisms, SNPs), but one clone (GenBank no. MF 946596) differed from the seven others in having two deletions. Similarly, at the positions displaying substitutions, only one sequence deviated from the others. Hence, these variations might have been due to polymerase errors during the PCR amplification prior to cloning. The eight clones differed from GenBank sequence JQ733513 of the unnamed species from the great cormorant, which comprised the ITS1 region only, at 11–15 of 830 positions (98.2–98.7% identity). At 11 of these positions, all the eight new sequences differed from that sequence. With respect to other species, the ITS1 region of S. halieti showed the highest identity with this region of an unnamed Sarcocystis sp. from the intestine of A. cooperii (GenBank no. KY348755; 93%), S. columbae (GenBank nos. GU253885, HM 125052; 92%) and S. corvusi (GenBank no. JN256119; 92%), followed by identities of about 84% with several sequences of S. calchasi and S. wobeseri.