Molecular characterisation of Sarcocystis lutrae n. sp. and Toxoplasma gondii from the musculature of two Eurasian otters (Lutra lutra) in Norway

Abstract

Sarcocysts were detected in routinely processed histological sections of skeletal muscle, but not cardiac muscle, of two adult male otters (Lutra lutra; Mustelidae) from northern Norway following their post-mortem examination in 1999 and 2000. The sarcocysts were slender, spindle-shaped, up to 970 μm long and 35-70 μm in greatest diameter. The sarcocyst wall was thin (∼ 0.5 μm) and smooth with no visible protrusions. Portions of unfixed diaphragm of both animals were collected at the autopsies and kept frozen for about 14 years pending further examination. When the study was resumed in 2013, the thawed muscle samples were examined for sarcocysts under a stereo microscope, but none could be found. Genomic DNA was therefore extracted from a total of 36 small pieces of the diaphragm from both otters, and samples found to contain Sarcocystidae DNA were used selectively for PCR amplification and sequencing of the nuclear 18S and 28S ribosomal (r) RNA genes and internal transcribed spacer 1 (ITS1) region, as well as the mitochondrial cytochrome b (cytb) and cytochrome c oxidase subunit 1 (cox1) genes. Sequence comparisons revealed that both otters were infected by the same Sarcocystis sp. and that there was no genetic variation (100 % identity) among sequenced isolates at the 18S and 28S rRNA genes (six identical isolates at both loci) or at cox1 (13 identical isolates). PCR products comprising the ITS1 region, on the other hand, had to be cloned before sequencing due to intraspecific sequence variation. A total of 33 clones were sequenced, and the identities between them were 97.9-99.9 %. These sequences were most similar (93.7-96.0 % identity) to a sequence of Sarcocystis kalvikus from the wolverine in Canada, but the phylogenetic analyses placed all of them as a monophyletic sister group to S. kalvikus. Hence, they were considered to represent a novel species, which was named Sarcocystis lutrae. Sequence comparisons and phylogenetic analyses based on sequences of the 18S and 28S rRNA genes and cox1, for which little or no sequence data were available for S. kalvikus, revealed that S. lutrae otherwise was most closely related to various Sarcocystis spp. using birds or carnivores as intermediate hosts. The cox1 sequences of S. lutrae from the otters were identical to two sequences from an arctic fox, which in a previous study had been assigned to Sarcocystis arctica due to a high identity (99.4 %) with the latter species at this gene and a complete identity with S. arctica at three other loci when using the same DNA samples as templates for PCR reactions. Additional PCR amplifications and sequencing of cox1 (ten sequences) and the ITS1 region (four sequences) using four DNA samples from this fox as templates again generated cox1 sequences exclusively of S. lutrae, but ITS1 sequences of S. arctica, and thus confirmed that this arctic fox had acted as intermediate host for both S. arctica and S. lutrae. Based on the phylogenetic placement of S. lutrae, the geographical location of infected animals (otters, arctic fox) a...

Molecular characterisation of three regions of the nuclear ribosomal DNA unit and the mitochondrial cox1 gene of Sarcocystis fusiformis from water buffaloes (Bubalus bubalis) in Egypt

“…Four DNA regions were amplified in the following manner as described previously (Gjerde and Josefsen 2015), except for the replacement of primer SU1F with a new primer: The (i) complete (∼1,875 bp) 18S rRNA gene was amplified in two overlapping fragments using primer pairs ERIB1/S2r and S3f/ Primer Bsarc; the (ii) complete ITS1 region and short segments of the flanking 18S and 5.8S rRNA genes (∼850 bp) were amplified with the new forward primer SFF (5′-TGTC ACGGTTGTTGTACAACAG-3′) and reverse primer 5.8SR2; (iii) an ∼1,650-bp-long portion of the 28S rRNA gene was amplified with primer pair KL1/KL3; and (iv) an ∼1,100-bp-long portion of cox1 was amplified with forward primer SF1 and reverse primer SR9 (most samples) or COIRm. Sequences of previously used primers are given in Gjerde and Josefsen (2015). The new forward primer SFF targeted a variable region located 96-117 nucleotides from the 3′-end of the 18S rRNA gene of S. fusiformis (KR186116-KR186123), in which this species differs considerably from other sequenced Sarcocystis spp.…”

Sarcocystis oreamni, n. sp. (Apicomplexa: Sarcocystidae) from the mountain goat (Oreamnos americanus)

“…Sequences analysis at single marker alone may not be enough because some species have more intra-species sequences variation in a given region as demonstrated recently in S. lutrae (Gjerde and Josefsen 2015). Therefore, two nuclear DNA regions (18S rRNA, 28S rRNA) of new species, S. oreamni were amplified and sequenced to identify and show relationship with other Sarcocystis species.…”

Abundance Detection and Molecular Characterization of Toxoplasma gondii by SAG1 Gene in Rodents and Cattle of Golestan Province, Northeast of Iran

“…In 2013, Cong detected house sparrows toxoplasmosis in China, Khademvatan detected birds toxoplasmosis in southwest of Iran and Ortega found pigs toxoplasmosis in Mexico [22][23][24][25][26]. Most of the [26][27][28][29][30]. In recent years have not perform any studies to detect rodents and cattle toxoplasmosis at the same time in Golestan area.…”

“…This study showed important role of rodents and cattle in dissemination of toxoplasmosis in humid area. In this study we showed that the SAG1 gene was very important marker to detection of abundance of zoonotic toxoplasmosis and brain or heart tissue was main tissues to follow SAG1 gene from Tachyzoites of Toxoplasma parasite, also the rodents and cattle were very important reservoirs in dissemination of toxoplasmosis to human in Golestan area, northeast of Iran [26][27][28][29][30].…”