Sarcocystis lari

3.3.3. Ribosomal DNA unit of S. lari

The new sequence of the complete 18S rRNA gene from isolate Ha1.8 (GenBank no. MF 946588), which was 1804 bp long, was 100% identical with GenBank sequence JQ733508 of S. lari from sarcocysts in the great black-backed gull in Lithuania (Prakas et al., 2014). These sequences shared the highest identities with sequences of S. turdusi (99.8%), S. halieti, S. calchasi and S. wobeseri (99.7%); followed by those of S. corvusi and S. lutrae (99.6%).

The new 1506-bp-long sequence of the partial 28S rRNA gene from isolate Ha1.8 (GenBank no. MF 946611) was 100% identical with GenBank sequence JQ733508 of S. lari from sarcocysts in the great black-backed gull, followed by an identity of 98.8% with sequences of S. calchasi (GenBank no. FJ 232949) and S. wobeseri (GenBank nos. GQ 922887, GQ922888, HM 159420, EF079886), and 98.6% identity with a sequence of S. turdusi (GenBank no. JF975682). The new sequence of S. lari was 98.3% identical with the new sequence of S. halieti.

Concerning the ITS1region, a total of 14 clones from isolate Ha1.8 were processed and sequenced, but two clones were identical and therefore 13 clones were submitted to GenBank (accession nos. MF946597–MF946609; Table 1). These sequences were 1115–1117 bp long, of which the ITS1 region comprised 859–861 bp, and the portions of the flanking 18S and 5.8S rRNA genes, comprised 138 and 118 bp, respectively. The new sequences differed from each other at 1–11 nucleotide positions in the ITS1 region (98.7–99.9% identity). These differences were due to several substitutions (SNPs) and three 1-bp-long indels. In the variable positions caused by the substitutions and one of the indels, only one sequence deviated from the remaining 12 sequences, and thus they could have been due to polymerase errors during PCR amplification. As regards the two other indels, several clones possessed a deletion compared to the other sequences, but one of these indels occurred in a stretch of 9–10 consecutive Gs and the other in a stretch of 10–11 consecutive Ts, and thus could have been the result of polymerase slippage during amplification or sequencing. The 13 new sequences differed at 3–8 positions (99.1–99.8% identity) in the ITS1 region from GenBank sequence JQ733509 of S. lari, but there was only one position at which all the new sequences differed from the latter sequence. Hence, the new sequences from the sea eagle were considered to belong to S. lari. The identity with the ITS1 region of other Sarcocystis spp. was 80% or less, including an identity of about 74% with the new sequences of S. halieti and about 70% identity with sequences of S. lutrae (GenBank nos. KM 657773– KM 657805).