The complete mitochondrial genome of the pentastomid Linguatula arctica (Pentastomida) from reindeer (Rangifer tarandus) in Northern Norway.

Here, we present the first complete mitochondrial genome of the pentastomid Linguatula arctica collected from the nasal passages of a reindeer (Rangifer tarandus) in Norway. The full length mitochondrial genome of L. arctica, which measures 14,789 bp in length, contains 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. A clear A + T bias is observed in the mitogenome of L. arctica with an overall base composition of 32.6% A, 27.5% T, 32.8% C, and 7,1% G., and a GC content of 39.9%. The gene arrangement is identical to that of previously described pentastomid mitogenomes.

Molecular characterisation of five Sarcocystis species in domestic sheep (Ovis aries) from Spain.

The major aim of the present study was to determine by molecular methods whether the wide and narrow types of macroscopic sarcocysts in Spanish sheep belonged to different species, that is, Sarcocystis gigantea and Sarcocystis medusiformis, respectively. Additionally, we wanted to identify and characterize molecularly the species forming microscopic sarcocysts and determine the phylogenetic placement of all species found. Portions of the oesophagus, diaphragm and hind legs containing macroscopic sarcocysts were collected from slaughtered culled ewes at an abattoir in the Province of Madrid, Central Spain, but both macroscopic and microscopic sarcocysts were isolated for molecular examination. Genomic DNA from 63 sarcocysts (21 macroscopic, 42 microscopic) were examined at the cytochrome c oxidase subunit I gene (cox1), while selected isolates of each species found were further examined at the 18S and 28S ribosomal RNA (rRNA) genes. The 63 sarcocysts comprised five cox1 sequence types, each corresponding to a particular sarcocyst type, and thus represented five Sarcocystis spp. The slender fusiform and thick macrocysts belonged to S. medusiformis and S. gigantea, respectively. The microscopic sarcocysts belonged to Sarcocystis arieticanis, Sarcocystis tenella and a Sarcocystis mihoensis-like species with slanting thorn-like cyst wall protrusions, which was characterised molecularly for the first time. Based on its phylogenetic position, the S. mihoensis-like species probably uses corvids as definitive hosts.

Morphological and molecular characteristics of seven Sarcocystis species from sika deer (Cervus nippon centralis) in Japan, including three new species.

Samples of diaphragm were collected from 53 sika deer from Gifu Prefecture, Japan; 220 sarcocysts were isolated, examined in wet mounts and classified according to their cyst wall protrusions. The sarcocysts were then examined molecularly in order to assign them to different species. All but 11 of the 220 sarcocysts were initially identified by means of a multiplex PCR assay targeting cox1 of five species, whereas the remaining 11 sarcocysts were identified by standard PCR and sequencing. DNA from selected sarcocysts was used for PCR amplification and sequencing of cox1 (59 sequences) and 18S rDNA (23 sequences). The 220 sarcocysts comprised seven major cox1 sequence types or species. Types 4 and 7 were assigned to the known species Sarcocystis pilosa and Sarcocystis ovalis, whereas types 1, 3 and 5 were considered to represent three new species, for which the names Sarcocystis japonica, Sarcocystis matsuoae and Sarcocystis gjerdei have been proposed. Types 2 and 6 were most similar to Sarcocystis tarandi and Sarcocystis taeniata, respectively, but could not be unequivocally assigned to these species. Sarcocysts belonging to S. japonica were macroscopic with fairly thick finger-like protrusions, whereas most sarcocysts of the six other species were microscopic. Sarcocysts of S. cf. tarandi and S. matsuoae were spindle-shaped and possessed thin finger-like cyst-wall protrusions. Sarcocysts of S. pilosa and S. gjerdei had similar hair-like protrusions, whereas those of S. cf. taeniata had a smooth surface. Sarcocysts of S. japonica, S. pilosa, S. cf. tarandi, S. gjerdei, S. matsuoae, S. cf. taeniata and S. ovalis were found in 50 (94.3%), 29 (54.7%), 22 (41.5%), 10 (18.9%), 8 (15.1%), 6 (11.3%) and 1 (1.9%) of the 53 sika deer examined, respectively. An improved multiplex PCR assay targeting cox1 was developed, through which the seven Sarcocystis spp. found in the present study could be identified.

Molecular differentiation of five Sarcocystis species in sika deer (Cervus nippon centralis) in Japan based on mitochondrial cytochrome c oxidase subunit I gene (cox1) sequences.

Several surveys of Sarcocystis infection in sika deer in Japan have shown a high prevalence, but the identification has been unclear because molecular data have been lacking or have been limited to 18S ribosomal RNA gene sequences. Thus, in our previous study based on such sequences, some Sarcocystis isolates from sika deer were not clearly separated from other species in the phylogenetic analysis. In the present study, we therefore characterized sarcocyst isolates from sika deer (Cervus nippon centralis) at the mitochondrial cytochrome c oxidase subunit I gene (cox1). Moreover, we developed a multiplex PCR based on cox1 sequences of all species found, so that we could rapidly identify sarcocysts of these species. Twenty-one sarcocysts from nine sika deer were examined. Five distinct cox1 sequence types, each with a high sequence identity (> 99%), were found, and the sarcocysts could thus be classified into five species. Based on the sequence comparisons and the phylogeny, Sarcocystis spp. of types 1, 3, and 5 are considered to represent three new species, which were most closely related to Sarcocystis silva/Sarcocystis truncata, Sarcocystis entzerothi, and Sarcocystis iberica/Sarcocystis venatoria, respectively. There was a slight uncertainly whether Sarcocystis sp. with type 2 sequences represented a new species or was identical to Sarcocystis tarandi. Type 4 sequences showed 99% identity with those of Sarcocystis pilosa from sika deer in Lithuania and have therefore been assigned to this species. In the multiplex PCR, type-specific fragments were successfully amplified for all five Sarcocystis spp., indicating that this assay may be useful for a rapid identification of sarcocysts of these species.

Prevalence and molecular characterisation of Sarcocystis miescheriana and Sarcocystis suihominis in wild boars (Sus scrofa) in Italy.

A sample of the diaphragm was collected from each of 100 wild boars legally hunted in the Val Grande National Park in north-western Italy and examined for the presence of Sarcocystis infection by histological and molecular methods. In histological sections, thick-walled sarcocysts consistent with those of Sarcocystis miescheriana were detected in 32 wild boars. Genomic DNA extracted from diaphragm samples was initially subjected to PCR amplification of the internal transcribed spacer 1 (ITS1) region, and 97 wild boars were found to harbour a Sarcocystis infection at this screening. Selected DNA samples were then subjected to PCR amplification and sequencing of the ITS1 region and the 18S and 28S ribosomal RNA (rRNA) genes of the nuclear ribosomal DNA unit, while all positive samples were subjected to PCR amplification of the mitochondrial cytochrome c oxidase subunit I (cox1) gene. S. miescheriana was identified in 97 wild boars (97%), while the zoonotic Sarcocystis suihominis was identified in one wild boar (1%), which also harboured S. miescheriana. Intra-specific sequence variation was found in all four DNA regions of S. miescheriana examined and in the 18S rRNA gene and ITS1 region of S. suihominis. The partial cox1 gene was amplified and sequenced from 72 isolates of S. miescheriana, yielding 43 haplotypes with pairwise sequence identities of 97.6-99.9%. These haplotypes were 79.1-79.8% identical with the cox1 sequence of S. suihominis. Phylogeny based on cox1 sequences placed S. miescheriana and S. suihominis as sister species within a clade comprising mainly Sarcocystis spp. of ruminants with felids as known or presumed definitive hosts. The same was true for the phylogeny based on 18S rRNA gene sequences.

Molecular identification of Sarcocystis halieti n. sp., Sarcocystis lari and Sarcocystis truncata in the intestine of a white-tailed sea eagle (Haliaeetus albicilla) in Norway.

An emaciated white-tailed sea eagle (Haliaeetus albicilla) from Western Norway was found and nursed briefly before it died. The necropsy revealed that the principal cause of death was an inflammation and occlusion of the bile ducts. A secondary finding was the presence in the intestinal mucosa of numerous sporulated Sarcocystis oocysts measuring 21.8-22.8 × 16.0-17.0 µm. The aim of this study was to identify these oocysts to species level using molecular methods. Genomic DNA was extracted from 10 mucosal scrapings containing oocysts and subjected to PCR amplification and sequencing of four DNA regions: the 18S and 28S rRNA genes, the ITS1 region and the cox1 gene. DNA of three previously known Sarcocystis spp. was identified, but only two of these, Sarcocystis halieti n. sp. and Sarcocystis lari, both employing sea birds as intermediate hosts, were considered to have used the sea eagle as a definitive host and to have formed oocysts in its intestine. The third species found, Sarcocystis truncata, employs red deer as intermediate hosts and seems to use felids as definitive hosts based on its phylogenetic position and prevalence. The sea eagle had probably recently ingested portions of one of the latter hosts (red deer or cat/lynx) containing stages (sarcocysts/oocysts) and thus DNA of S. truncata. The species S. halieti and S. lari could only be unambiguously separated from their most closely related congeners on the basis of their ITS1 sequences. This is the first report of Sarcocystis oocysts in sea eagles and the first identification to species level of Sarcocystis oocysts in any type of eagle. The sea eagle also acted as intermediate host of an unidentified Sarcocystis spp. as evidenced by the finding of six thin-walled sarcocysts in a histological section of cardiac muscle.

Morphological and molecular characteristics of six Sarcocystis spp. from red deer (Cervus elaphus) in Spain, including Sarcocystis cervicanis and three new species.

Samples of muscle tissue from the diaphragm, oesophagus and/or heart of eight adult red deer (Cervus elaphus hispanicus) from the Quintos de Mora Park in Toledo, Central Spain, were screened for sarcocysts by means of the compression method. From positive samples, individual sarcocysts were excised and examined in wet mounts under a light microscope (LM) in order to study their basic morphology before being preserved for molecular studies. In all red deer examined, only microscopic sarcocysts were found. Those in the diaphragm and oesophagus were spindle-shaped and about 1 × 0.1 mm in size, while those in cardiac muscle were sac-like and 500-800 × 80-180 µm. By LM, the sarcocysts either had densely packed, about 8-µm-long, hair-like protrusions (type 1), sparsely distributed indistinct projections (fuzzy outline; type 2) or no visible protrusions (smooth surface; type 3). In cardiac muscle, only sarcocysts without visible protrusions were found. One of the latter sarcocysts was examined by scanning electron microscopy (SEM) and found to possess thin ribbon-like protrusions. Forty-eight sarcocysts isolated from the diaphragm, oesophagus and heart of one red deer, as well as 55 sarcocysts from the heart of three other red deer, 103 sarcocysts in total, were characterized molecularly through PCR amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of the mitochondrial genome, revealing the presence of six major cox1 sequence types. Each type comprised either a single sequence (three types) or a collection of several identical or nearly identical sequences. From selected isolates possessing each of these cox1 sequence types, the complete 18S ribosomal RNA (rRNA) gene was amplified and sequenced directly and/or after cloning of the 5' end half. Supported by the sequence data from the latter gene, as well as the morphology of the sarcocysts from which the sequences originated, the six cox1 sequence types were considered to represent six separate Sarcocystis spp. Two cox1 sequence types were identified as belonging to the previously characterized species Sarcocystis hjorti (one sequence/sarcocyst) and Sarcocystis linearis (38 sequences/sarcocysts), respectively, whereas four sequence types were new. One of the latter types was assigned to the previously named species Sarcocystis cervicanis from red deer, since this sequence type was obtained from 52 sarcocysts from cardiac muscle, which matched the original morphological description (smooth surface) and habitat of this species. The remaining three sequence types were assigned to the three new species Sarcocystis iberica (one sequence/sarcocyst) Sarcocystis venatoria (10 sequences/sarcocysts) and Sarcocystis morae (one sequence/sarcocyst), respectively. The two species S. iberica and S. venatoria shared the same sarcocyst morphology (type 1) and habitat (diaphragm) and had virtually identical 18S rRNA gene sequences, but differed by 4% at cox1, which was considered sufficient to regard them as separate species. The single sarcocyst of S. morae (from the oesophagus) examined by LM had a smooth wall and this species was therefore believed to have the same type of ribbon-like protrusions (ultrastructurally) as sarcocysts of S. cervicanis and S. linearis, which were also the species most closely related to S. morae at cox1. Thus, there seems to be three species with similar ribbon-like cyst wall protrusions in red deer (S. cervicanis, S. linearis, S. morae), as well as three species with similar hair-like protrusions (S. hjorti, S. iberica, S. venatoria). Sarcocysts of S. cervicanis were only identified in cardiac muscle, whereas sarcocysts of S. linearis were found mainly in the diaphragm and oesophagus and rarely in the heart. The relative number of cox1 haplotypes was greater among sequences/isolates of S. linearis (17/38) than among isolates of S. cervicanis (7/52). Four of the species examined (S. cervicanis, S. linearis, S. iberica, S. venatoria) possessed considerable intra-isolate (intra-genomic) sequence variation (insertions/deletions, substitutions) in the 18S rRNA gene.

Morphological and molecular characterization of four Sarcocystis spp., including Sarcocystis linearis n. sp., from roe deer (Capreolus capreolus) in Italy.

Fresh (frozen/thawed) muscle samples from four 2-12-year-old roe deer (Capreolus capreolus) from the Sondrio province in north-eastern Italy were examined under a dissecting microscope, and about 180 sarcocysts were isolated and identified to morphological type in wet mounts by light microscopy (LM). Seventy-seven of these sarcocysts were subsequently examined by molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of all isolates, as well as PCR amplification, cloning and sequencing of the complete18S ribosomal RNA (rRNA) gene of two isolates of each species found. By LM, three major sarcocyst types were recognised: spindle-shaped sarcocysts, 0.5-3 mm long, either with no clearly recognisable protrusions (thin-walled) or with finger-like protrusions (thick-walled); and slender, thread-like sarcocysts, 2-3 mm long, with hair-like protrusions. Sequencing of cox1 revealed that the sarcocysts belonged to four different species. Those with no visible protrusions either belonged to Sarcocystis gracilis (n = 24) or to a Sarcocystis taeniata-like species (n = 19), whereas those with finger- and hair-like protrusions belonged to Sarcocystis silva (n = 27) and Sarcocystis capreolicanis (n = 7), respectively. The 19 cox1 sequences of the S. taeniata-like species, comprising five haplotypes, differed from each other at 0-16 of 1038 nucleotide positions (98.5-100% identity). They differed from 25 previous cox1 sequences of S. taeniata from moose and sika deer (with 98.0-100% intraspecific identity), at 33-43 nucleotide positions (95.9-96.8% interspecific identity), and there were 20 fixed nucleotide differences between the two populations. In the phylogenetic analysis based on cox1 sequences, the two populations formed two separate monophyletic clusters. The S. taeniata-like species in roe deer was therefore considered to represent a separate species, which was named Sarcocystis linearis n. sp. At the 18S rRNA gene, however, the two species could not be clearly separated from each other. Thus, there was considerable intraspecific sequence variation in the 18S rRNA gene of S. linearis (98.1-99.9% identity between 24 sequences), which was similar both in magnitude and nature to the variation previously found in this gene of S. taeniata. The new 18S rRNA gene sequences of S. linearis shared an identity of 97.9-99.6% with those of S. taeniata (overlap between intra- and interspecific identity), and in the phylogenetic tree, sequences of the two species were interspersed. By scanning electron microscopy (SEM), the sarcocysts of S. linearis were found to possess regularly spaced, thin and narrow ribbon-like cyst wall protrusions (about 2.8-3.2 µm long, 0.3-0.4 µm wide and about 0.02-0.03 µm thick), terminating in a plate-like structure of the same thickness but with an elliptic outline (about 0.3-0.4 µm wide and 0.7-0.9 µm long). The terminal plates were connected in the middle with the band-like portion of the protrusions like the board of a seesaw (tilting board). The terminal plates of adjacent protrusions were neatly arranged in a hexagonal pattern resembling tiles on a roof. Together, they formed an outer roof-like layer facing the surrounding cytoplasm of the host cell and completely covering the band-like proximal portion of the protrusions, which overlapped and were stacked in three to four layers close to the cyst surface. The sarcocyst morphology of S. linearis was consistent with that of an unnamed Sarcocystis sp. in roe deer previously found by transmission electron microscopy in several countries, including Italy. A few sarcocysts of S. gracilis and S. silva were also examined by SEM, confirming the presence of regularly distributed, short knob-like protrusions in S. gracilis (as seen in previous SEM studies) and revealing tightly packed, erect 6-7-µm-long villus-like protrusions having regularly distributed round depressions on their surface in S. silva. The sequencing of cox1 of 7, 24 and 27 new isolates of S. capreolicanis, S. gracilis and S. silva, respectively, recovered 7, 11 and 10 new haplotypes from each of the three species and expanded our knowledge on the intraspecific sequence variation at this marker. Similarly, the study revealed a more extensive intragenomic sequence variation at the 18S rRNA gene of S. capreolicanis and S. silva than known from previous studies and confirmed a near absence of such variation in the 18S rRNA gene of S. gracilis.

Domestic cats (Felis catus) are definitive hosts for Sarcocystis sinensis from water buffaloes (Bubalus bubalis).

The definitive hosts of Sarcocystis sinensis in water buffaloes have hitherto been unknown, but the close similarity of this species to the cat-transmitted Sarcocystis bovifelis in cattle suggested they were felids. In a previous study, two domestic cats were fed macroscopic sarcocysts of Sarcocystis fusiformis contained within or dissected from the esophageal muscles of water buffaloes, while no microscopic sarcocysts of S. sinensis were noticed. Both cats started shedding small numbers of sporocysts 8-10 days post infection (dpi) and were euthanized 15 dpi. Using a PCR-based molecular assay targeting the mitochondrial cox1 gene of S. fusiformis, both cats were shown to act as definitive hosts for this species. In the present study, DNA samples derived from oocysts/sporocysts in the intestinal mucosa of both cats were further examined by PCR for the presence of S. sinensis using 2 newly designed primers selectively targeting the cox1 gene of this species. All 6 DNA samples examined from each cat tested positive for S. sinensis. A 1,038-bp-long portion of cox1 was amplified and sequenced as 2 overlapping fragments from 5 of these DNA samples. The 5 sequences shared 99.3-100% identity with 7 previous cox1 sequences of S. sinensis obtained from sarcocysts in water buffaloes. Additionally, amplification of the ITS1 region with primers targeting various Sarcocystis spp., yielded amplicons of 2 different lengths, corresponding to those obtained from sarcocyst isolates of S. sinensis and S. fusiformis, respectively. This is the first study to show that cats act as definitive hosts for S. sinensis.

Molecular differentiation of Sarcocystis buffalonis and Sarcocystis levinei in water buffaloes (Bubalus bubalis) from Sarcocystis hirsuta and Sarcocystis cruzi in cattle (Bos taurus).

The purpose of the present study was to obtain sarcocysts of Sarcocystis buffalonis and Sarcocystis levinei from water buffaloes and characterize the isolates by molecular methods in order to determine whether the two species were genetically different from Sarcocystis hirsuta and Sarcocystis cruzi, respectively, from cattle, which had been characterized before. About 35 macroscopically visible (3-4 × 1-2 mm) and 20 barely visible (1-3 × 0.2 mm) sarcocysts were excised from the esophagus of 18 naturally infected and freshly slaughtered adult water buffaloes at three slaughterhouses in Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were first characterized at the mitochondrial cytochrome c oxidase subunit I gene (cox1) gene through PCR amplification and direct sequencing. Selected isolates were subsequently further characterized at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit by direct sequencing or cloning. Only six of the isolated macroscopic sarcocysts belonged to S. buffalonis, whereas the others belonged to Sarcocystis fusiformis. Twelve of the smaller cysts belonged to S. levinei and seven to Sarcocystis sinensis. The characterization of the sarcocysts of S. sinensis and some of the sarcocysts of S. fusiformis have been reported before. Fifteen additional sarcocyst isolates of S. fusiformis were characterized at cox1 in the present study and found to be identical or closely similar to previous isolates. At cox1, the sequence identity between the six isolates of S. buffalonis was 99.8-100 % (two haplotypes), whereas the identity between the 12 isolates of S. levinei was 99.0-100 % (10 haplotypes). The identity between cox1 sequences of S. buffalonis and S. hirsuta (n = 56) was 92.9-93.6 % (on average 93.4 %), and the identity between cox1 sequences of S. levinei and S. cruzi (n = 22) was 92.9-94.0 % (on average 93.5 %). The phylogenetic analyses placed with high support the cox1 sequences of S. buffalonis and S. hirsuta into two monophyletic sister groups, and the same was true for the cox1 sequences of S. levinei and S. cruzi. Hence, the study established that S. buffalonis and S. levinei are distinct species different from S. hirsuta and S. cruzi, respectively. Nucleotide sequences of S. buffalonis could be distinguished from those of S. hirsuta also at the 28S rRNA gene (clearly different) and the ITS1 region (small and uncertain difference) but not at the 18S rRNA gene. Sequences of S. levinei could be distinguished from those of S. cruzi both at the 18S and 28S rRNA genes (ITS1 region not examined). However, the cox1 gene was superior to the 18S and 28S rRNA genes as regards the ability to unambiguously delimit the species within each species pair, since at the latter markers, the number of consistent nucleotide differences between the species was low and there was a slight overlap between the intraspecific and interspecific sequence divergence. Comparison of the newly generated 18S rRNA gene sequences of S. levinei from water buffaloes with similar sequences deposited in GenBank suggested that S. levinei and S. cruzi are not strictly intermediate host specific but might occasionally infect cattle and water buffaloes, respectively.

The resurrection of a species: Sarcocystis bovifelis Heydorn et al., 1975 is distinct from the current Sarcocystis hirsuta in cattle and morphologically indistinguishable from Sarcocystis sinensis in water buffaloes.

In the mid-1970s, it was established through transmission experiments and ultrastructural studies of sarcocysts by transmission electron microscopy (TEM) that cattle was the intermediate host of three Sarcocystis spp. using dogs, cats and humans, respectively, as definitive hosts. The cat-transmitted species with microscopic sarcocysts was initially named Sarcocystis bovifelis, but it was soon renamed Sarcocystis hirsuta, since it was considered to be identical with a previously named species. In recent years, an apparently new species has been detected in cattle in several countries by molecular methods and TEM and found by both methods to be indistinguishable from Sarcocystis sinensis in water buffaloes. This species was recently named Sarcocystis rommeli. Beginning in August 2014, a thorough review of papers comprising TEM micrographs of thick-walled sarcocysts in cattle was made in order to determine whether S. sinensis-like sarcocysts had been reported previously under other designations. Surprisingly, the review showed that the species S. bovifelis Heydorn et al., 1975 as described from cattle in Germany was S. sinensis-like and that indistinguishable sarcocysts had also been found in cattle in New Zealand and Canada in the 1980s. However, in the New Zealand study, these small sarcocysts were erroneously thought to represent developmental stages of a species with ultrastructurally similar but macroscopic sarcocysts, since the macroscopic cysts were found to be infective for cats. Thus, in the late 1980s, the cat-transmitted S. bovifelis, after having been renamed S. hirsuta, was erroneously synonymised with a second cat-transmitted species in cattle and then slid into obscurity until recently being rediscovered as a S. sinensis-like species in cattle and then named S. rommeli. Following the erroneous synonymisation, the name S. hirsuta has consistently been used for a taxon with macroscopic sarcocysts, and this usage should be continued. The name S. bovifelis should again be used not only for the species originally described from cattle in Germany but also for morphologically indistinguishable taxa recently reported from cattle under the names S. sinensis and S. rommeli. Because of the morphological similarity between S. bovifelis and S. sinensis, it is likely that cats also act as definitive hosts for S. sinensis. The present paper also gives a thorough review of all research in the 1970s pertaining to S. bovifelis, including its development in cats and cattle; a review of reports of S. bovifelis-like sarcocysts in cattle, water buffaloes and other hosts; and a review of reports of the taxon currently named S. hirsuta in cattle. The usage of the name S. sinensis versus Sarcocystis dubeyi for the S. bovifelis-like taxon in water buffaloes is discussed, and the latter name is found to represent a nomen dubium since the original description concerned a mixture of a S. sinensis- and a Sarcocystis hominis-like species. Based on available transmission (TEM) and scanning electron microscopy (SEM) images, the three-dimensional configuration of the cyst wall protrusions of S. bovifelis/S. sinensis and the current S. hirsuta has been inferred and is described. The protrusions of S. bovifelis/S. sinensis are shaped like soft plastic tubes, having a cylindrical basal portion and a flattened distal portion, making them prone to fold over. The protrusions of the current S. hirsuta are thin, flattened and flexible rectangular structures (like a soft cover note book), which are attached to the cyst surface with a narrow stalk. The appearance of both types of protrusions in ultrathin sections viewed by TEM is highly dependent on how the sarcocysts and the protrusions themselves have been sectioned.

Molecular characterisation of Sarcocystis bovifelis, Sarcocystis bovini n. sp., Sarcocystis hirsuta and Sarcocystis cruzi from cattle (Bos taurus) and Sarcocystis sinensis from water buffaloes (Bubalus bubalis).

About 200 individual sarcocysts were excised from 12 samples of cattle beef from five countries (Argentina, Brazil, Germany, New Zealand, Uruguay) and tentatively identified to species or cyst type on the basis of their size and shape and cyst wall morphology. Genomic DNA was extracted from 147 of these sarcocysts and used initially for PCR amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit I gene (cox1) in order to identify the sarcocysts to species and/or sequence type. In addition, seven Sarcocystis sinensis-like sarcocysts collected from the oesophagus of water buffaloes in Egypt were examined at cox1 for comparative purposes. Based on the results from the cox1 marker, selected sarcocyst isolates from both hosts were further characterised at one to three regions of the nuclear ribosomal (r) DNA unit, i.e. the complete 18S rRNA gene, the complete internal transcribed spacer 1 (ITS1) region and the partial 28S rRNA gene. This was done in order to compare the results with previous molecular identifications based on 18S rRNA gene sequences and to evaluate the utility of these regions for species delimitations and phylogenetic inferences. On the basis of sarcocyst morphology and molecular data, primarily the cox1 sequences, four Sarcocystis spp. were identified in the samples of cattle beef. Twenty-two microscopic sarcocysts (1 × 0.1 mm) with hair-like protrusions were assigned to Sarcocystis cruzi, 56 macroscopic sarcocysts (3-8 × 0.5 mm) with finger-like protrusions were assigned to Sarcocystis hirsuta and 45 and 24 microscopic sarcocysts (1-3 × 0.1-0.2 mm) with finger-like protrusions were assigned to Sarcocystis bovifelis and Sarcocystis bovini n. sp., respectively. Sarcocysts of S. cruzi were identified in samples of beef from Argentina and Uruguay; sarcocysts of S. hirsuta in samples from Argentina, Brazil, Germany and New Zealand; sarcocysts of S. bovifelis in samples from Argentina and Germany; and sarcocysts of S. bovini in samples from Argentina and New Zealand. The microscopic sarcocysts from water buffaloes were confirmed to belong to S. sinensis. The cox1 sequences of S. bovifelis and S. bovini, respectively, shared an identity of 93-94 % with each other, and these sequences shared an identity of 89-90 % with cox1 of S. sinensis. In contrast, the intraspecific sequence identity was 98.4-100 % (n = 45), 99.3-100 % (n = 24) and 99.5-100 % (n = 7) for sequences of S. bovifelis, S. bovini and S. sinensis, respectively. In each of the latter three species, an aberrant type of cox1 sequences was also identified, which was only 91-92 % identical with the predominant cox1 type of the same species and about 98 % identical with the aberrant types of the two other species. These aberrant cox1 sequences are believed to represent non-functional nuclear copies of the mitochondrial genes (numts or pseudogenes). They might be used as additional markers to separate the three species from each other. Sequencing of a considerable number of clones of S. bovifelis, S. bovini and S. sinensis from each of the three regions of the rDNA unit revealed intraspecific sequence variation in all loci in all species and particularly in the ITS1 locus (78-100 % identity). As regards the 18S rRNA gene, it was possible to separate the three species from each other on the basis of a few consistent nucleotide differences in the less variable 3' end half of the gene. A comparison of the new sequences with GenBank sequences obtained from S. sinensis-like sarcocysts in cattle in other studies indicated that previous sequences derived from cattle in Germany and Austria belonged to S. bovifelis, whereas those derived from cattle in China belonged to S. bovini. On the basis of the new 28S rRNA sequences, it was possible to separate S. sinensis from S. bovifelis and S. bovini, whereas the latter two species could not be separated from each other. Based on ITS1 sequences, the three species were indistinguishable. Phylogenetic analysis using maximum parsimony placed with fairly high support cox1 sequences of S. bovifelis, S. bovini and S. sinensis, respectively, into three monophyletic clusters, with S. bovifelis and S. bovini being a sister group to S. sinensis. In contrast, phylogenies based on each of the three regions of the rDNA unit did not separate sequences of the three species completely from each other. Characterisation of cox1 of 56 isolates of S. hirsuta from four countries revealed only 13 haplotypes and an intraspecific sequence identity of 99.3-100 %. In the three regions of the rDNA unit, there was more extensive sequence variation, particularly in the ITS1 region. The 22 cox1 sequences of S. cruzi displayed a moderate intraspecific variation (98.6-100 %), whereas there was no variation at the 18S rRNA gene among 10 sequenced isolates. Sequencing of 16 clones of the partial 28S rRNA gene of S. cruzi yielded two markedly different sequence types, having an overall sequence identity of 95-100 %.

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.

A total of 33 macroscopically visible (3-11 × 1-5 mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3-99.9 %. In comparison, the sequence identity was 98.8-99.0 % among eight complete 18S rRNA gene sequences (1,873-1,879-bp long), 98.1-100 % among 28 complete ITS1 sequences (853-864-bp long) and 97.4-99.6 % among five partial 28S rRNA gene sequences (1,607-1,622 bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93 % sequence identity at cox1 versus interleaved sequences and 98.7-99.1 % sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8-10 days post-infection (dpi) and were euthanized 15 dpi. Sporocysts isolated from the intestinal mucosa of both cats were identified molecularly as belonging to S. fusiformis through PCR amplification and sequencing of the partial cox1. The two sporocyst-derived cox1 sequences were identical with the most common sarcocyst-derived cox1 haplotype.

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

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) and the distribution of carnivores/raptors which may have interacted with them, the white-tailed eagle (Haliaeetus albicilla) seems to be a possible definitive host of S. lutrae. Some of the muscle samples from both otters were shown to harbour stages of Toxoplasma gondii through PCR amplification and sequencing of the entire ITS1 region (five isolates) and/or the partial cytb (eight isolates) and cox1 (one isolate). These sequences were identical to several previous sequences of T. gondii in GenBank. Thus, both otters had a dual infection with S. lutrae and T. gondii.

Molecular characterisation of Sarcocystis rileyi from a common eider (Somateria mollissima) in Norway.

The breast and leg muscles of a common eider (Somateria mollissima; Anatidae: Anseriformes) from northern Norway contained numerous grossly visible cigar-shaped sarcocysts measuring about 5 × 1 mm. Light microscopic examination of isolated sarcocysts revealed that they were encapsulated by a thin fibrous layer, underneath which there was a thin and fairly smooth cyst wall with no visible protrusions. The cystozoites were straight, spindle-shaped and about 13 µm long. Genomic DNA was extracted from 12 excised sarcocysts and each DNA isolate was subjected to PCR amplification of one to four loci: the 18S and 28S ribosomal RNA genes (four isolates), the internal transcribed spacer 1 (ITS1) region (six isolates) and the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (12 isolates). At the three nuclear loci, the new sequences showed 99.9-100% sequence identity with previous sequences of Sarcocystis rileyi from the mallard duck in Lithuania and USA, and they could therefore be assigned to this species. At cox1, the new sequences of S. rileyi were most similar to Sarcocystis arctica and Sarcocystis neurona, but the most closely related Sarcocystis spp. in birds have not been sequenced at this locus. There was no sequence variation at any locus between the 4-12 examined isolates of S. rileyi. This is the first genetically verified record of S. rileyi in the common eider, as well as in any bird species in Norway. The phylogenetic placement of S. rileyi was inferred separately from 28S rRNA gene and cox1 sequences, and similar results were obtained in both analyses.

Morphological and molecular characteristics of four Sarcocystis spp. in Canadian moose (Alces alces), including Sarcocystis taeniata n. sp.

Individual sarcocysts were isolated from fresh or alcohol-fixed muscle samples of two moose from Alberta, Canada, and examined by light (LM) and scanning electron microscopy (SEM) and molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the complete18S rRNA gene and the partial cytochrome c oxidase subunit I gene (cox1). By LM, four sarcocyst types were recognized, and the sequencing results showed that each type represented a distinct species, i.e. Sarcocystis alces, Sarcocystis alceslatrans, Sarcocystis ovalis and Sarcocystis taeniata n. sp. The finding of S. alceslatrans and S. ovalis has been reported briefly previously, but further details are provided here, including the ultrastructure of sarcoysts of S. alceslatrans as seen by SEM. The species S. alces was found for the first time in Canadian moose, whereas the finding of S. taeniata is the first record of this species in any host. The sarcocysts of S. taeniata were sac-like and about 1,000-1,100 × 60-80 µm in size. By LM, the cysts had a thin and smooth wall with no visible protrusions, whereas SEM revealed that the cyst surface had sparsely but regularly distributed, thin ribbon-like protrusions, about 2 µm long and 0.2 µm wide, lying flat against the surface and leaving most of the cyst surface naked. Similar protrusions have previously been reported from Sarcocystis grueneri in reindeer, which was found by sequence comparisons and phylogenetic analyses to be the species most closely related to S. taeniata. The phylogenetic analyses further suggested that S. taeniata, like S. alces and S. alceslatrans, use canids as definitive hosts, whereas corvid birds are known definitive hosts for S. ovalis. In contrast to the three other species found, S. taeniata displayed considerable intra-specific and intra-isolate sequence variation (substitutions, insertions/deletions) in certain regions of the 18S rRNA gene.

Sarcocystis species in red deer revisited: with a re-description of two known species as Sarcocystis elongata n. sp. and Sarcocystis truncata n. sp. based on mitochondrial cox1 sequences.

In a previous investigation, five Sarcocystis species were described from Norwegian red deer and believed to be conspecific with species occurring in either reindeer or moose based on sarcocyst morphology and nucleotide sequences of the nuclear ribosomal DNA unit. The aim of the present study was to characterize numerous isolates of these sarcocyst types at the mitochondrial cytochrome c oxidase subunit I gene (cox1) in order to corroborate or refute previous species designations of Sarcocystis in red deer. The Sarcocystis tarandi- and Sarcocystis rangiferi-like taxa in red deer and reindeer, respectively, were thoroughly compared by sequencing 14-27 isolates of each type. Sequence comparisons revealed four distinct sequence types, which by phylogenetic analyses were placed in four monophyletic groups according to host origin, and they were therefore considered to represent four separate species. The two taxa of this type in red deer were named Sarcocystis elongata and Sarcocystis truncata, respectively. Sequencing of many isolates of Sarcocystis hjorti and Sarcocystis ovalis from red deer and moose confirmed that these species occur in both hosts. A revised description of the two new species is given and the current knowledge concerning all six Sarcocystis species in red deer is reviewed.

Muscular sarcocystosis in two arctic foxes (Vulpes lagopus) due to Sarcocystis arctica n. sp.: sarcocyst morphology, molecular characteristics and phylogeny.

The arctic fox (Vulpes lagopus) is a critically endangered species in Norway, and therefore, the small population is closely monitored, and most foxes found dead are subjected to necropsy. In two deceased foxes, thin-walled muscular sarcocysts were first detected in histological sections, and numerous sarcocysts were later found in frozen and thawed muscle samples from Fox 1. These sarcocysts measured 1-12 × 0.1-0.25 mm and had closely spaced, short, knob-like protrusions, giving the cysts a serrated outline. Genomic DNA was extracted from eight isolated sarcocysts (Fox 1) and two muscle samples (Fox 2) and subjected to polymerase chain reaction amplification at four loci: the nuclear 18S and 28S ribosomal RNA genes and internal transcribed spacer 1 region and the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Both foxes were infected by the same Sarcocystis sp., which displayed little or no genetic variation at the three nuclear loci (99.9-100% identity) and slightly more variation at cox1 (99.4-100% identity). Sequence comparisons and phylogenetic analyses revealed that this species was distinct from other named Sarcocystis spp. but was closely related to various species using avian intermediate hosts and possibly identical to an unnamed species reported from two American dogs. The species described from the two arctic foxes was named Sarcocystis arctica n. sp.

Phylogenetic position of Linguatula arctica and Linguatula serrata (Pentastomida) as inferred from the nuclear 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit I gene.

Genomic DNA was isolated from a Linguatula serrata female expelled from a dog imported to Norway from Romania and from four Linguatula arctica females collected from semi-domesticated reindeer from northern Norway and subjected to PCR amplification of the complete nuclear 18S rRNA gene and a 1,045-bp portion of the mitochondrial cytochrome c oxidase subunit I gene (cox1). The two species differed at two of 1,830 nucleotide positions (99.9% identity) of the complete 18S rRNA gene sequences and at 102 of 1,045 nucleotide positions (90.2% identity) of the partial cox1 sequences. The four isolates of L. arctica showed no genetic variation in either gene. The new cox1 primers may facilitate the diagnosis of various developmental stages of L. arctica and L. serrata in their hosts. In separate phylogenetic analyses using the maximum likelihood method on sequence data from either gene, L. arctica and L. serrata clustered with members of the order Cephalobaenida rather than with members of the order Porocephalida, in which the genus Linguatula is currently placed based on morphological characters. The phylogenetic relationship of L. arctica, L. serrata and other pentastomids to other metazoan groups could not be clearly resolved, but the pentastomids did not seem to have a sister relationship to crustaceans of the subclass Branchiura as found in other studies. A more extensive taxon sampling, including molecular characterisation of more pentastomid taxa across different genera, seems to be necessary in order to estimate the true relationship of the Pentastomida to other metazoan groups.

Phylogenetic relationships among Sarcocystis species in cervids, cattle and sheep inferred from the mitochondrial cytochrome c oxidase subunit I gene.

Coccidian parasites in the genus Sarcocystis have a two-host life cycle, and have traditionally been identified on the basis of morphological features of the sarcocyst stage in their intermediate hosts. Additional molecular species identification, delimitation and phylogeny of Sarcocystis spp. have been based mainly on the nuclear ssrRNA gene. This gene is well suited for discrimination between more distant species but less so for closely related species. The objective of this study was therefore to establish the mitochondrial cytochrome c oxidase subunit I gene (cox1) as a novel genetic marker for Sarcocystis spp. and assess its utility for species identification and delimitation. New primers were developed and 1,020-1,095 bp long cox1 sequences were obtained from 155 isolates of 22 Sarcocystis spp. from cattle, sheep, red deer, reindeer, roe deer and moose, and used for phylogenetic reconstructions. For 18 species, the intraspecific and interspecific sequence identities were 98.5-100% and 58-92%, respectively. The four other species had previously been regarded as two species (Sarcocystis rangiferi, Sarcocystis tarandi), each infecting both reindeer and red deer. From cox1 data, each of those appeared to be two separate species, with S. rangiferi and S. tarandi being restricted to reindeer. Thus, cox1 sequences seem to perform better than ssrRNA gene sequences for delimitation of closely related species. The 22 species were distributed in three major clades according to their definitive hosts as in phylogenetic trees obtained from the ssrRNA gene. There were only minor differences in the branching order of different taxa between the trees obtained from either gene. This study has successfully established cox1 as a novel genetic marker for future research on Sarcocystis spp. It has also provided the first published molecular identification of Sarcocystis gigantea and Sarcocystis tenella in Norwegian sheep, and of Sarcocystis hirsuta and Sarcocystis sinensis in Argentinean cattle.