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The common gull (Larus canus) and the black-headed gull (Larus ridibundus) are common waterbird species in Lithuania. Until now, the composition of Sarcocystis species in these birds was unknown. The current study aimed to identify Sarcocystis spp. by the morphological examination of sarcocysts found in the muscle tissues and by DNA sequence analysis. Between 2011 and 2019, the leg muscles of 42 common gulls and 63 black-headed gulls were tested for Sarcocystis spp. Based on the methylene blue staining of squashed muscle samples, sarcocysts were detected in six common gulls (14.3%) and in six black-headed gulls (9.5%). Under a light microscope, one type of microcyst was observed. Sarcocysts were thread-like (2860–8250 × 40–180 μm) and had a smooth and thin (0.8–1.4 μm) cyst wall, while bradyzoites were banana-shaped and 5.0–9.2 × 1.3–2.4 μm in size. The sequencing of complete ITS1 showed the presence of S. columbae, S. halieti and S. wobeseri in the common gull and S. columbae and S. halieti in the black-headed gull. The highest intraspecific genetic variability was established for S. halieti, which is characterized by a wide host range. This species is considered to be pathogenic, therefore further histopathological examination of the various organs of gulls is needed.

Abstract Background Members of the genus Sarcocystis are protozoan parasites characterized by a prey–predator two-host life-cycle. Sarcocysts are formed in the muscles or central nervous system of the intermediate host (IH), while sporocysts develop in the small intestine of the definitive host (DH). Various birds of prey have been confirmed to be DH for Sarcocystis spp. Three Sarcocystis species, S. wobeseri, S. halieti and S. falcatula, have been identified in the muscles of birds of prey, of which the latter are known to be pathogenic and can cause encephalitis in various birds. The aim of this study was to identify Sarcocystis spp. in the muscles of birds of prey from Spain. Methods Between 2019 and 2020, muscle tissue samples taken from 59 birds of prey admitted to the Wildlife Recovery Centre in Ilundain (Navarra, Spain) were examined for the presence of Sarcocystis spp. Sarcocysts in fresh squashed samples were morphologically characterized under the light microscope (LM). Sarcocystis spp. were identified by means of 28S ribosomal RNA and internal transcribed spacer 1 sequence analysis. Results Microscopic examination of squashed tissue samples stained with methylene blue revealed the presence of sarcocysts in three of the 59 (5.1%) birds examined. Only one sarcocyst type was observed under the LM. Sarcocysts were thread-like (1050–2160 × 130–158 μm) and had a thin (0.7–1.4 μm) and smooth cyst wall. Septa divided the cysts into compartments filled with banana-shaped (5.9 × 1.7 μm) bradyzoites. On the basis of DNA sequence results, S. halieti was identified in the western marsh harrier (Circus aeruginosus) and the black kite (Milvus migrans) for the first time. Sarcocysts of S. halieti were shorter and wider compared to those observed in the great cormorant (Phalacrocorax carbo) and the herring gull (Larus argentatus). According to current knowledge, S. halieti may infect birds belonging to four different orders: Suliformes, Charadriiformes, Strigiformes and Accipitriformes. Conclusions This is the first report of S. halieti in the western marsh harrier and the black kite as IH. So far, little research has been conducted on birds of prey as IH for Sarcocystis spp. These results indicate that further studies combining morphological, histopathological, and molecular methods are required. Graphical abstract

Abstract Background Birds of the family Laridae have not been intensively examined for infections with Sarcocystis spp. To date, sarcocysts of two species, S. lari and S. wobeseri, have been identified in the muscles of gulls. The aim of the present study was to evaluate the species richness of Sarcocystis in the herring gull, Larus argentatus, from Lithuania. Methods In the period between 2013 and 2019, leg muscles of 35 herring gulls were examined for sarcocysts of Sarcocystis spp. Sarcocystis spp. were characterised morphologically based on a light microscopy study. Four sarcocysts isolated from the muscles of each infected bird were subjected to further molecular examination. Sarcocystis species were identified by means of ITS1 sequence analysis. Results Sarcocysts were detected in 9/35 herring gulls (25.7%). Using light microscopy, one morphological type of sarcocysts was observed. Sarcocysts were microscopic, thread-like, had a smooth and thin (about 1 µm) cyst wall and were filled with banana-shaped bradyzoites. On the basis of ITS1 sequences, four Sarcocystis species, S. columbae, S. halieti, S. lari and S. wobeseri, were identified. Furthermore, it was demonstrated that a single infected herring gull could host two Sarcocystis species indistinguishable under light microscopy. Conclusions Larus argentatus is the first bird species found to act as intermediate host of four Sarcocystis spp. According to current knowledge, five species, S. falcatula, S. calchasi, S. wobeseri, S. columbae and S. halieti can use birds belonging to different orders as intermediate hosts.

The genus Sarcocystis is a group of numerous protozoan parasites having a two-host life cycle. Based on laboratory experiments and/or phylogenetic analysis results it was shown that seven Sarcocystis spp. producing sarcocsyts in bird tissues are transmitted via predatory placental mammals. To date the role of small mammals of the family Mustelidae in the distribution of avian Sarcocystis spp. have not been studied. During the current investigation, intestinal mucosa scrapings of 115 mustelids belonging to five species were tested for S. albifronsi, S. anasi, S. rileyi, and S. wenzeli infecting anseriforms and chickens. Microscopically, free sporocysts, sporulating oocysts, and loose oocysts were found in 61 samples (53.0%). Using nested PCR targeting the ITS1 region and sequencing, S. rileyi was confirmed in eight American minks, two European polecats and single European badger. Sarcocystis sp. was identified in one American mink and one European pine marten. Based on the partial ITS1 region this parasite showed that 100% identity to pathogenic Sarcocystis sp. caused a fatal infection in backyard chickens from Brazil. Phylogenetically, the Sarcocystis sp. identified in our study was most closely related to S. wenzeli parasitising domestic fowl (Gallus domesticus).

Members of the family Corvidae are ecologically flexible omnivorous birds, particularly adaptive to urban habitats, and living in proximity to humans; these birds may serve as definitive hosts (DH) for Sarcocystis spp., but research about this is lacking. In the present study, intestinal samples from 91 corvids collected in Lithuania were molecularly tested by species-specific PCR targeting the ITS1 and cox1 genes and subsequently sequenced for the presence of Sarcocystis spp. Under a light microscope, oocysts of Sarcocystis spp. were observed in 43 samples (47.3%), while molecular methods, detected Sarcocystis spp. in 77 birds (84.6%). Eleven Sarcocystis spp. (S. columbae, S. cornixi, potentially pathogenic S. halieti, S. kutkienae, S. lari, S. turdusi, S. wobeseri, S. arctica, S. lutrae, S. ovalis, and S. oviformis) were identified in the intestinal samples from six corvid species from Lithuania. Infections with multiple Sarcocystis spp. were detected in 79.2% of the infected corvid birds. Three of the identified Sarcocystis spp. use corvids as intermediate hosts (IH); therefore, corvids may serve as IH and DH of the same Sarcocystis species. Based on molecular results and on corvid diet, omnivorous corvids may play an important role in transmitting Sarcocystis spp.

One morphologic type of sarcocyst was found in 26% (7/27) of Eurasian Coots (Fulica atra) and was described as Sarcocystis fulicae n. sp. using morphologic, 18S ribosomal (r)DNA, 28S rDNA, and internal transcribed spacer 1 (ITS1) analysis. By light microscope, cysts were ribbon-shaped and measured 7.3 mm in length by 116 μm wide. Histologically, the cyst wall reached up to 1.2 μm in thickness and seemed smooth. The detected sarcocysts were packed with relatively small banana-shaped bradyzoites that were 6.7×2.0 μm in size. Ultrastructurally, the cyst wall amounted to 1 μm and had many conical protrusions but seemed almost smooth in some places. The parasitophorous vacuolar membrane appeared undulating, with knob-like blebs and invaginations. The cyst wall belonged to type 1d. Morphologically, cysts of S. fulicae differed considerably from cysts of Sarcocystis atraii previously described in the same host but were indistinguishable from those of Sarcocystis corvusi, Sarcocystis lari, and Sarcocystis wobeseri, which use birds as intermediate hosts. According to the phylogenetic and ecologic data, birds of prey, mostly the Western Marsh Harrier (Circus aeruginosus) and the White-tailed Eagle (Haliaeetus albicilla), are presumed to be definitive hosts of S. fulicae. [ABSTRACT FROM AUTHOR]