Diversity of Sarcocystis parasites in southeastern Baltic Sea catchment ecosystems.

Currently, research on apicomplexan Sarcocystis parasites is mainly carried out by analyzing animal carcasses. However, environmental studies would not only allow faster detection of possible sources of infection but also avoid the use of animals for investigations. Therefore, in the current study, we aimed to identify tested Sarcocystis species in sediment collected from water bodies located in the southeastern Baltic countries. A total of 99 sediment samples were collected during the summer from different types of water bodies in Estonia, Latvia, Lithuania, and Poland. Species-specific nested PCR targeting cox1 gene was used for the detection of selected Sarcocystis species (S. cruzi, S. bovifelis, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. miescheriana, and S. bertrami) infecting livestock. The results showed a statistically lower (p < 0.05) occurrence of Sarcocystis parasites in Estonia (50%) compared to three countries, where the detection rate of Sarcocystis spp. DNA was remarkably higher, ranging from 88 to 100%. Among Sarcocystis species tested, S. cruzi (83.8%) and S. arieticanis (55.6%) using cattle and sheep as their intermediate hosts were most commonly identified. The detection rates of some of the analyzed Sarcocystis species were significantly different in southeastern Baltic countries. It is discussed that the detection rates of certain Sarcocystis species depend not only on the number of animals per 1 km2 but also on various ecological factors and farming practices that differ in the amount of contact domestic animals have with predators and the potential for animals to become infected through natural water or food sources.

Dynamics of Polyamines, Proline, and Ethylene Metabolism under Increasing Cold in Winter Oilseed Rape.

Cold stress is among the most important environmental factors reducing the yield of crops. The present study aimed to investigate the impact of increasing cold stress conditions on winter oilseed rape polyamines, proline, and ethylene metabolism in acclimated and non-acclimated winter oilseed rape. This study was carried out under controlled conditions in the laboratory. The winter oilseed rape hybrid 'Visby' was used in the experiment. Acclimated and non-acclimated plants were subjected to a two-day-long increasing cold (from -1 °C to -3 °C) treatment. HPTLC, RT-qPCR, spectral analysis, and gas chromatography methods were used to analyse the levels of polyamines, gene expression, proline, and ethylene, respectively. This study showed a decrease in putrescine, spermidine, and spermine content during cold acclimation and a decrease in putrescine and spermidine levels at sub-zero temperatures. There were intensive changes in ADC2 gene expression, proline, and ethylene levels in non-acclimated plants: a substantial increase after exposure to -1 °C temperature and a sharp decrease after exposure to -3 °C temperature. The changes in these parameters were lower or absent in acclimated plants. The phenomena observed in this study add new insights to the knowledge about the plant stress response and suggest questions to be answered in the future.

A novel RFLP method for identification of morphologically similar avian Sarcocystis species.

Protozoans of genus Sarcocystis are widespread parasites infecting mammals, birds, and reptiles. Morphology of their sarcocysts is an important criterion for species identification. However, as more and more morphologically similar Sarcocystis species are being found and described, additional methods for their routine diagnostics are needed. We investigated restriction fragment length polymorphism (RFLP) as potential alternative to sequencing data analysis for the identification of Sarcocystis species using birds as an intermediate host. The internal transcribed spacer 1 (ITS1) region sequences of seventeen Sarcocystis species (S. albifronsi, S. anasi, S. calchasi, S. columbae, S. cornixi, S. corvusi, S. cristata, S. halieti, S. falcatula, S. fulicae, S. kutkienae, S. lari, S. lindsayi, S. rileyi, S. turdusi, S.wenzeli, and S. wobeseri) of interest were analysed and five best-fitting endonucleases generating most informative restriction fragments were selected for routine testing. In general, RFLP analyses are always inconclusive as they target very short DNA sequences. However, it can be an irreplaceable technique when fast and cheap identification and discrimination of known species are required, which was our main goal and preliminary results indicate that RFLP could be successfully used when identifying closely related avian Sarcocystis species with just two nucleases.

Investigations on Sarcocystis species in the leg muscles of the bird family Corvidae in Lithuania.

Although three species of Sarcocystis, S. cornixi, S. corvusi and S. kutkienae, have been described in corvids, molecular studies of sarcocysts isolated from these birds are incomplete. Leg muscles of 83 corvids, 35 hooded crows (Corvus cornix), 21 western jackdaws (Coloeus monedula), 11 rooks (Corvus frugilegus), 9 common ravens (Corvus corax), 4 common magpies (Pica pica) and 3 Eurasian jays (Garrulus glandarius), from Lithuania were examined for the presence of Sarcocystis spp. in the present study. In methylene blue-stained squashed samples, sarcocysts were detected in 26 birds (31.0%). Under a light microscope, two morphological types of sarcocysts were distinguished (type A and type B). Sarcocysts of type A had a smooth and thin (about 1 µm) cyst wall, while cysts of type B were characterised by a thicker (1.4-2.5 µm) cyst wall. Based on ITS1 sequence comparison, sarcocysts of type A were identified as S. halieti and Sarcocystis sp. ex Corvus corax, whereas cysts of type B belonged to S. kutkienae and S. cornixi. Furthermore, it was demonstrated that a single bird could host two different Sarcocystis spp. Sarcocystis halieti was detected in corvids for the first time in the common raven and the hooded crow. Also, this study presents the first evidence of S. kutkienae in the hooded crow and the common magpie, and S. cornixi in the western jackdaw. Sarcocystis sp. ex Corvus corax was genetically characterised using almost complete 18S rDNA, partial 28S rDNA and complete ITS1 sequences. Sarcocystis sp. ex Corvus corax clustered together with S. columbae, S. corvusi and S. halieti in phylogenetic trees reconstructed using 28S rDNA and ITS1 sequences.

Invasive American mink (Neovison vison) as potential definitive host of Sarcocystis elongata, S. entzerothi, S. japonica, S. truncata and S. silva using different cervid species as intermediate hosts.

Canids and scavenger birds were shown to act as definitive hosts of numerous Sarcocystis species using members of the Cervidae family as an intermediate host, whereas definitive hosts spreading closely related S. elongata, S. entzerothi, S. japonica, S. matsuoae, S. rangiferi, S. truncata, S. silva and S. tarandi remain unknown. In the current study, the intestine samples of 40 American minks (Neovison vison) were molecularly tested for the presence of the above-mentioned Sarcocystis spp. Species-specific PCR of cytochrome c oxidase subunit I (cox1) fragments and subsequent sequencing revealed the presence of sporocysts/oocysts of five species, S. elongata (n=2), S. entzerothi (n=10), S. japonica (n=4), S. silva (n=13) and S. truncata (n=21) in the analysed samples. Sarcocystis infection was confirmed in 32/40 (80%) examined samples. In addition, half of the infected animals (50%) were infected with multiple Sarcocystis species suggesting that American minks had access to meat of different deer species, such as roe deer, red deer and sika deer. This causes concern about compliance of hunters and game processing companies with game waste management rules. Further research on the involvement of mustelids in the transmission of various Sarcocystis spp. from different geographical locations is needed.

Molecular identification of Sarcocystis species in diaphragm muscle tissue of European mouflon (Ovis gmelini musimon) from Austria.

Previous morphological studies suggested that mouflon may have sarcocysts similar to those of sheep. However, to date, no molecular-based studies of the species of Sarcocystis infecting mouflon have been done. The present study identified Sarcocystis species in diaphragm muscle samples from 20 European mouflon (Ovis gmelini musimon). Molecular identification using the cox1 sequence analysis was performed on sarcocysts excised from muscle tissue and on DNA from digested muscle samples. Both frequency and intensity of infection in mouflon were high with 19 of 20 animals testing Sarcocystis positive and > 50 cysts per gram of tissue recovered from 10 of the 19 Sarcocystis positive animals. Molecular analysis revealed dominant Sarcocystis tenella (18/19 animals) and Sarcocystis arieticanis (1/19 animals), whose known intermediate hosts are sheep. In addition, Sarcocystis capracanis, which is known to form sarcocysts in goats, was detected in two animals. The results of this study demonstrated the digestion method to be superior over the direct isolation of sarcocysts for the molecular identification of Sarcocystis species in a certain host. Future research of Sarcocystis diversity in wild ovine and caprine species is needed.

First description of Sarcocystis species infecting Barbary sheep (Ammotragus lervia).

Barbary sheep (Ammotragus lervia) is a North African native wild Caprinae, introduced in the 1970s in new territories such as Spain, the USA, and Mexico. Here, we describe Sarcocystis species in Barbary sheep. Sarcocysts were found in 19 out of 56 adult A. lervia in Southern Spain and characterized morphologically and molecularly. By light microscopy, sarcocysts had thin (< 1 µm) or thick (> 2 µm) walls. By transmission electron microscopy, sarcocysts with thick walls had Type 14 villar protrusions corresponding to S. tenella/S. capracanis of domestic sheep (Ovis aries) or goats (Capra hircus). Sarcocysts with thin walls had Type 7b villar protrusions that corresponded to S. arieticanis/S. hircicanis of domestic sheep or goats. Molecular analyses allowed the identification of only thick-walled Sarcocystis species. Six sarcocysts were assigned to S. tenella (99.2-100% and 95.6-100% sequence similarity within 18S rRNA and COI, respectively) and 19 sarcocysts were assigned to S. capracanis (98.5-99.8% and 97.9-99.0% sequence similarity within 18S rRNA and COI, respectively). Further studies are needed for taxonomic identification of sarcocysts in Barbary sheep because Sarcocystis species in sheep and goats are not cross transmissible despite morphological similarities.

Population Structure of Fusarium graminearum Isolated from Different Sources in One Area over the Course of Three Years.

Fusarium head blight (FHB) is an important crop disease worldwide and is mainly caused by members of the Fusarium graminearum species complex. F. graminearum sensu stricto is the most common cosmopolitan and predominant FHB causal agent in Europe. Thus far, the majority of studies have focused on the primary hosts (wheat and barley) of this pathogen, while the relationships between other sources of infection remain unclear. We monitored and sampled test fields over the course of 3 years and acquired 804 F. graminearum isolates from different sources: primary hosts and other plant species included in the crop rotations, weeds from the test fields, decaying plant residue, soil samples, and crop seed. Of these isolates, 73.3% had the 15-acetyldeoxynivalenol genotype and 26.7% had the 3-acetyldeoxynivalenol genotype. F. graminearum isolation rates from weeds (>50%) were much higher than from soil (< 10%) or decaying plant matter (4%). Variable number of tandem repeat markers were used for population analysis. Noticeable genetic differentiation was detected between isolates from living plants and soil biome. In contrast, absence of any noticeable division between primary and alternative plant host communities indicates the importance of weeds and other segetal plants for FHB control and prevention.

Molecular and morphological description of Sarcocystis kutkienae sp. nov. from the common raven (Corvus corax).

Until now, two Sarcocystis species, S. cornixi and S. corvusi, were known to employ members of the family Corvidae as intermediate hosts. Between 2013 and 2019, having examined leg muscles of 23 common ravens in Lithuania, sarcocysts were detected in 18 birds (78.3%). Using light microscopy, transmission electron microscopy (TEM), and molecular analysis (three genetic loci, 18S rDNA, 28S rDNA, and ITS1), sarcocysts found in the common raven were described as a new species S. kutkienae. Under a light microscope, the observed sarcocysts were ribbon-shaped (1500-8147 × 53-79 µm) and had a wavy striated cyst wall that reached up to 1.5 µm. Lancet-shaped bradyzoites were 7.7 × 2.2 µm (6.1-9.0 × 1.2-3.0 µm) in size. Ultrastructurally, the sarcocyst wall was 1.5-1.8 µm in thickness and had conical-like protrusions with minute invaginations of a parasitophorous vacuolar membrane. The cyst wall was type 1e-like. Limited genetic variability was observed between the 18S rDNA and 28S rDNA sequences of S. kutkienae and other Sarcocystis spp. using birds as intermediate hosts. In contrast, S. kutkienae could be clearly identified by comparing sequences. At this locus, sequences of S. kutkienae shared the highest similarity (89.5-89.7%) with those of S. cornixi. Phylogenetic analysis showed that S. kutkienae was most closely related to Sarcocystis spp. that employs birds as intermediate and definitive hosts. The issue relating to which species might serve as definitive hosts of S. kutkienae in Lithuania is addressed.

First molecular characterization of Sarcocystis miescheriana in wild boars (Sus scrofa) from Latvia.

Various muscle samples of wild boar (Sus scrofa) from Latvia were studied for the presence of Sarcocystis infection by means of morphological and molecular methods. Sarcocysts were detected in 122 out of 140 (87.1%) wild boar examined. According to the morphological appearance of sarcocysts, the observed cysts belonged to one morphological type and resembled Sarcocystis miescheriana. Twenty-three sarcocysts isolated from the muscles of Latvian wild boars were molecularly characterized at 18S rRNA, ITS1 and cox1. Additionally, eight sarcocysts obtained from Lithuanian wild boars were subjected to molecular analysis in order to compare intraspecific genetic variability. The amplified 18S rRNA region using newly designed primers is sufficiently variable to separate S. miecheriana from S. suihominis. All Latvian and Lithuanian isolates were confirmed belonging to S. miescheriana. No genetic variation was detected within 18S rRNA and ITS1. By contrast, the high intraspecific genetic variability of S. miescheriana was observed within cox1 since each newly obtained sequence represented a unique haplotype. The comparison made using S. miescheriana isolates from Italian and Japanese wild boar and Chinese domestic pig revealed the genetic similarity of the samples depending on their geographical distances. The current study provides the first detection of Sarcocystis infection in wild boars from Latvia and molecular characterization of S. miescheriana.

Morphological and molecular description of Sarcocystis ratti n. sp. from the black rat (Rattus rattus) in Latvia.

Rodents have been widely studied as intermediate hosts of Sarcocystis; however, only a few reports on these parasites in the black rat (Rattus rattus) are known. Having examined 13 black rats captured in Latvia, sarcocysts were found in skeletal muscles of two mammals and were described as Sarcocystis ratti n. sp. Under a light microscope, sarcocysts were ribbon-shaped, 0.9-1.3 × 0.09-0.14 mm in size and had a thin (0.8-1.3 µm) and smooth cyst wall. The lancet-shaped bradyzoites were 8.3 × 4.3 (7.5-9.3 × 3.9-4.8) µm. Under a transmission electron microscope, the cyst wall was up to 1.3 µm thick, wavy, the ground substance appeared smooth, type 1a-like. Morphologically, sarcocysts of S. ratti were somewhat similar to those of S. cymruensis, S. rodentifelis, and S. dispersa-like previously identified in the brown rat (Rattus norvegicus). On the basis of 18S rDNA, 28S rDNA, and cox1, significant genetic differences (at least 2.3, 4.5, and 5.8%, respectively) were observed when comparing S. ratti with other Sarcocystis species using rodents as intermediate hosts. While ITS1 sequences of S. ratti were highly distinct from other Sarcocystis species available in GenBank. Phylogenetic and ecological data suggest that predatory mammals living near households are definitive hosts of S. ratti.

Sarcocystis species identification in the moose (Alces alces) from the Baltic States.

Various muscle tissue samples from 60 moose (Alces alces) in the Baltic region were examined for Sarcocystis species. Sarcocysts were detected in 49 out of 60 (81.7%) moose investigated. Six species, Sarcocystis alces, Sarcocystis hjorti, Sarcocystis linearis, Sarcocystis silva, Sarcocystis ovalis, and Sarcocystis sp., were identified using light microscopy (LM), and DNA sequence analysis (cox1 and 18S rDNA). Sarcocysts of S. alces, S. ovalis, and S. hjorti were studied using transmission electron microscopy (TEM); sarcocyst walls of S. alces, S. ovalis, and S. hjorti were type 25, type 24, and type 7a, respectively. Sarcocystis linearis previously found in roe deer and red deer was also shown to use moose as an intermediate host for the first time. The unknown Sarcocystis sp. was rare and might employ another main intermediate host. Phylogenetic results demonstrated that Sarcocystis sp. was most closely related to Sarcocystis tarandivulpes, using canids as definitive hosts.

Morphological and genetic characterisation of Sarcocystis halieti from the great cormorant (Phalacrocorax carbo).

Having examined 19 great cormorants (Phalacrocorax carbo) hunted in Lithuania, sarcocysts were found in the muscles of two birds. Sarcocysts detected were examined using light microscopy (LM), transmission electron microscopy (TEM), and 18S rDNA, 28S rDNA, ITS1, cox1, and rpoB sequence comparison. Based on the molecular analysis, mainly of the ITS1 region, sarcocysts were identified as Sarcocystis halieti. This is the first Sarcocystis species characterised in the great cormorant. Under the LM sarcocysts were ribbon-shaped, very long and thin (the largest fragment found amounted to 6.5 × 0.1 mm) with a smooth and thin (up to 1.2 µm) cyst wall. Banana-shaped bradyzoites were 7.2 × 1.9 (6.3-8.2 × 1.4-2.4) µm. Under TEM, the cyst wall was wavy, 0.8- to 1.2-µm thick. The comparison of 12 species demonstrated cox1 and rpoB to be unsuitable for the identification of Sarcocystis spp. using birds as intermediate hosts.

Molecular identification of Sarcocystis lutrae (Apicomplexa: Sarcocystidae) in muscles of five species of the family Mustelidae.

Carnivores usually act as definitive hosts of Sarcocystis species. However, the number of reports on sarcocyst formation in musculature of predators is on the increase. In the present study, muscle samples of 68 mustelids collected in Lithuania were examined for sarcocysts of Sarcocystis species. Sarcocysts were detected in diaphragm, tongue and limb muscles of ten animals (14.7%) but were not discovered in the heart. Based on 18S rDNA, 28S rDNA, cox1 and ITS1 sequence analysis, Sarcocystis lutrae was identified in three American minks (Neovison vison), two beech martens (Martes foina), three Eurasian badgers (Meles meles), one Eurasian otter (Lutra lutra) and one European polecat (Mustela putorius). The intraspecific variability of this Sarcocystis species was determined only in ITS1 region. Based on the phylogenetic analysis, no clear separation of S. lutrae by intermediate hosts or geographical locations was established. This paper represents the first identification of S. lutrae in the American mink, the beech marten and the European polecat. Current results indicate that S. lutrae is a common species in the muscles of various European mustelids.

Morphological and molecular identification of Sarcocystis spp. from the sika deer (Cervus nippon), including two new species Sarcocystis frondea and Sarcocystis nipponi.

Diaphragm muscles of 25 sika deer (Cervus nippon) farmed in Lithuania were examined for sarcocysts of Sarcocystis species. Two new Sarcocystis species, Sarcocystis frondea and Sarcocystis nipponi, were observed using light microscopy (LM) and transmission electron microscopy (TEM) and characterized by 18S ribosomal DNA (rDNA) and subunit I of cytochrome c oxidase (cox1) sequence analyses. By LM, sarcocysts of S. frondea and S. nipponi were ribbon-shaped and had finger-like sarcocyst wall protrusions, respectively. Under TEM, protrusions of S. frondea were about 9 × 1-1.5 µm, filled with clearly visible electron-dense substance and microtubules, type 39-like. Whereas, protrusions (about 9 × 0.2 µm) of S. nipponi arose from dome-shaped bases were filled with microtubules extending to the ground substance layer, type 9o-like. Moreover, three known Sarcocystis spp., Sarcocystis entzerothi, Sarcocystis ovalis, and Sarcocystis truncata previously described in other cervids as intermediate hosts, were characterized in sika deer. The cox1 was more suitable than 18S rDNA delimitating closely related Sarcocystis species from cervids. The phylogenetic results suggest that scavenger birds could be definitive hosts of S. frondea. According to the summarized morphological data on Sarcocystis found in the sika deer, such host should harbor at least nine different Sarcocystis species.

Sarcocystis entzerothi n. sp. from the European roe deer (Capreolus capreolus).

In the present study, we describe Sarcocystis entzerothi n. sp. from the European roe deer (Capreolus capreolus) based on the microscopical and DNA analysis. By light microscopy (LM), cysts of S. entzerothi were spindle-shaped with pointed tips, 950-1900 × 70-150 µm in size and had 5-6 µm long finger-like cyst wall protrusions. Cyst wall of S. entzerothi by transmission electron microscopy (TEM) was type 10a-like; villar protrusions were up to 1.2 µm wide, densely packed, lying about 0.1 µm between each other, had profuse microgranules and microfilaments, parasitophorous vacuolar membrane had many minute invaginations, and the ground substance layer measured up to 0.4 µm. This species is morphologically similar to Sarcocystis silva, previously found in the roe deer and the moose (Alces alces). By LM, cysts of S. silva were cigar-shaped with blunted tips, measured 1000-1500 × 130-184 µm, and had 7-8 µm long finger-like cyst wall protrusions. Under TEM, S. silva had no clear differences from S. entzerothi in their cyst wall ultrastructure. Having examined six roe deer hunted in Lithuania, cysts of S. entzerothi and S. silva were identified in four and two animals, respectively. These two Sarcocystis species could be morphologically differentiated according to the shape of the cysts and the length of protrusions. The species examined showed 95.6-96.1 % and 85.6-86.9 % sequence identity within 18S ribosomal DNA (rDNA) and cox1, respectively, and therefore they could be clearly distinguished by means of molecular methods. It should be noted that in the 18S rDNA phylogenetic tree, S. entzerothi from the roe deer was placed together with one sequence of Sarcocystis sp. from the Lithuanian red deer (Cervus elaphus) demonstrating the same species. Based on 18S rDNA and cox1 sequences, S. entzerothi was more closely related to Sarcocystis species transmitted via felids than canids.

Morphological and molecular characterization of Sarcocystis taeniata and Sarcocystis pilosa n. sp. from the sika deer (Cervus nippon) in Lithuania.

The diaphragm muscles of eight sika deer (Cervus nippon) bred in Lithuania were examined for Sarcocystis cysts. Two Sarcocystis species, Sarcocystis taeniata, which were previously reported in Canadian moose (Alces alces) and Argentinean red deer (Cervus elaphus), and Sarcocystis pilosa n. sp. were described using light microscopy (LM), transmission electron microscopy (TEM), 18S ribosomal DNA (rDNA), and subunit I of cytochrome c oxidase (cox1) sequences analysis. By LM, cysts of S. taeniata were 424.8 × 57.9 (200-837 × 30-100) µm in size and had a thin (up to 1 µm) and smooth cyst wall, while short ribbon-like protrusions arising from broadened cone-shaped bases were seen under TEM. Cysts of S. pilosa (by LM) were ribbon-shaped, measured 848.5 × 63.8 (350-1700 × 30-125) µm and had thin 7-8-µm long hair-like protrusions. By TEM, cyst wall was type 7a-like; protrusions arose from 0.3 µm wide dome-shaped base with minute indentations of the parasitophorous vacuolar membrane near it, the surface of protrusions seemed to be smooth, and the ground substance layer was thin (0.18-0.22 µm). The 18S rDNA, in contrast to the cox1, lacked variability to discriminate S. pilosa from closely related Sarcocystis hjorti from the red deer and moose. S. taeniata, but not S. pilosa, showed a considerable intraspecific variation in both genes analyzed. The phylogenetic analyses based on 18S rDNA and cox1 sequences suggest that canids are definitive hosts of both S. taeniata and S. pilosa. This paper represents the first identification of Sarcocystis species in the sika deer by morphological and molecular methods.

Molecular identification of Sarcocystis rileyi sporocysts in red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in Lithuania.

Despite the fact that Sarcocystis rileyi is one of the earliest described species of the genus Sarcocystis forming macrocysts in ducks, the life cycle of this species is still unknown in Europe. Sarcocystis spp. oocysts/sporocysts were observed in faeces of four of 23 (17.4 %) and in small intestine mucosal scrapings of four of 20 (20.0 %) red foxes (Vulpes vulpes) and in small intestine mucosal scrapings of seven of 13 (53.8 %) raccoon dogs (Nyctereutes procyonoides) hunted in Lithuania. A very small number of Sarcocystis sporocysts measuring 11.9 × 8.3 µm (n = 5) was found in faecal samples, whereas considerably more sporulated Sarcocystis oocysts and free sporocysts were detected in the small intestines of red foxes and raccoon dogs. These sporocysts measured 12.9 × 8.1 µm (n = 16) and 12.1 × 8.1 µm (n = 54) in red foxes and raccoon dogs, respectively. Using species-specific PCR and subsequent sequencing, internal transcribed spacer 1 (ITS-1) region partial sequences of oocysts/sporocysts from small intestine mucosal scrapings of six raccoon dogs and three red foxes were identified as belonging to S. rileyi. The present study provides strong evidence showing that the red fox and the raccoon dog can serve as final hosts of S. rileyi in Europe; however, transmission experiments are needed for the ultimate approval.

Description of Sarcocystis lari sp. n. (Apicomplexa: Sarcocystidae) from the great black-backed gull, Larus marinus (Charadriiformes: Laridae), on the basis of cyst morphology and molecular data.

A morphological type of Sarcocystis cysts found in one of two examined great black-backed gull, Larus marinus (Linnaeus) (Laridae), is considered to represent a new species for which the name Sarcocystis lari sp. n. is proposed and its description is provided. The cysts are ribbon-shaped, very long (the largest fragment found was 6 mm long) and relatively narrow (up to 75 microm). Under a light microscope the cyst wall reaches up to 1 microm and seems to be smooth. Using a computerized image analysis system, knolls, which resemble protrusions on the wall surface, are visible. Lancet-shaped cystozoites measure in average 6.9 x 1.4 microm (range 6.3-7.9 microm x 1.2-1.5 microm) in length. Observed using Transmission electron microscopy (TEM), the cyst wall is wavy and measures up to 1.2 microm in thickness. The parasitophorous vacuolar membrane has regularly arranged small invaginations. Cyst content is divided into large chambers by septa. Sarcocystis lari sp. n. has type-1 tissue cyst wall and is morphologically indistinguishable from other bird Sarcocystis species characterized by the same type of the wall. On the basis of 18S rRNA gene, 28S rRNA gene and ITS-1 region sequences, S. lari is a genetically distinct species, being most closely related to avian Sarcocystis species whose definitive hosts are predatory birds.

The Distribution of Sarcocsytis Species Described by Ungulates-Canids Life Cycle in Intestines of Small Predators of the Family Mustelidae.

PURPOSE: Using molecular techniques, we have previously shown that carnivorous mammals of the family Mustelidae might be common definitive hosts for various protozoan Sarcocystis species. In the present study we aimed to unravel whether Sarcocystis species using ungulates as intermediate hosts and canids or felids as definitive hosts can be found in intestine of mustelids. METHODS: Small intestine samples of 93 individual mustelids of five different species from Lithuania were examined. Sarcocystis species were identified based on species-specific PCR and subsequent cox1 sequencing. RESULTS: Six Sarcocystis species (S. arieticanis, S. bertrami, S. capracanis, S. capreolicanis, S. linearis and S. morae) defined by ungulate-canid life cycle were detected for the first time in small intestines of mustelids. By contrast, the prevalence of Sarcocystis characterised by ungulate-felid life cycle was low (3.2%). Overall, 76% of the examined animals were positive for at least one of the studied Sarcocystis species. Four species, S. arieticanis, S. bertrami, S. capracanis and S. morae were most commonly found, with the detection rate of about 40%. CONCLUSIONS: The current finding, in addition to our previous studies, suggests that mustelids play an important role in the spread of various Sarcocystis species.