Morphological and genetic diversity of Sarcocystis species parasitizing gulls (Larus) and corvids (Corvidae) in Lithuania

Members of the genus Sarcocystis are parasitic protozoa widespread in reptiles, birds, and mammals distinguished by a two-host prey-predator life cycle. Asexual multiplication with sarcocyst formation occurs in the intermediate host, while sexual stages develop in the small intestine of the definitive host. Previous studies have confirmed two species of Sarcocystis each in the muscles of gulls and corvids, but a greater diversity of these parasites is assumed in these hosts. The objective of this study was to determine the species richness of Sarcocystis parasites in muscle and intestine samples of Lithuanian gulls and corvids using morphological and molecular analysis. DNA sequence analysis revealed four morphologically indistinguishable Sarcocystis species in the muscle of Lithuanian gulls and three species – in the muscles of the investigated corvids. A new species of Sarcocystis, S. kutkienae, was described and confirmed in common raven, common magpie and hooded crow. The prevalence (p < 0.001) and density (p < 0.05) of Sarcocystis infection were reliably higher in the muscles of corvids than in the muscles of guls. During the study, S. columbae was identified for the first time in the muscles of gulls. For the first time, a pathogenic species S. halieti was identified in muscle samples of gulls and corvids. Also, during this study, for the first time, mixed Sarcocystis species infections were detected in the muscles of gulls and corvids. New intermediate hosts confirmed for five Sarcocystis species. For the first time, 11 Sarcocystis species were identified in the intestine mucosal scrapings of corvids

The Role of Birds of the Family Corvidae in Transmitting Sarcocystis Protozoan Parasites

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

The Richness of <i>Sarcocystis</i> Species in the Common Gull (<i>Larus canus</i>) and Black-Headed Gull (<i>Larus ridibundus</i>) from Lithuania

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

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

Molecular identification of four Sarcocystis species in the herring gull, Larus argentatus, from Lithuania

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

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

The role of mustelids in the transmission of Sarcocystis spp. using cattle as intermediate hosts

Members of the genus Sarcocystis are worldwide distributed protozoan parasites. Sarcocystis infections cause great losses in economically important animals. There is a lack of studies on Sarcocystis in naturally infected wild predators, especially of the family Mustelidae which represent a presumably important group of definitive hosts of these parasites. The objective of the present study was to examine the small intestine samples of various mustelid species from Lithuania serving as a possible source of Sarcocystis spp. using cattle as intermediate hosts. Overall, 84 samples collected from five mustelid species were analyzed. Oocysts/sporocysts of Sarcocystis spp. were detected in 75 animals (89.3%). Using molecular methods four Sarcocystis spp., S. bovifelis, S. cruzi, S. hirsuta and S. hominis were identified, with the first two being the most prevalent. These results indicate that mustelids are involved in the transmission of Sarcocystis spp. using cattle as intermediate hosts. The determined high prevalence of Sarcocystis spp. rates cause concerns about food safety issues. To control the spread of infection, further studies on the way carcasses of cattle or beef waste become accessible to mustelids are needed. There is a lack of research on the role of mustelids in the transmission of various Sarcocystis spp. In the present study we tested the hypothesis that widespread mustelids in Lithuania could be involved in the transmission of Sarcocystis spp. using cattle as intermediate hosts. In 2016-2020, intestinal samples of 84 mustelids were examined. Sarcocystis spp. were identified by species-specific PCR targeting the cox1 gene and subsequent sequencing. Under a light microscope, oocysts/sporocysts of Sarcocystis spp. were observed in 40 samples (47.6%), while using molecular methods, they were detected in 75 animals (89.3%). Four Sarcocystis spp. were identified in the intestinal samples of American mink (Neovison vison), Beech marten (Martes foina), European pine marten (Martes martes), European badger (Meles meles) and European polecat (Mustela putorius). The prevalence of predominant Sarcocystis spp., S. bovifelis (89.3%) and S. cruzi (73.8%) was significantly higher than that of S. hirsuta (3.6%) and S. hominis (1.2%). In an individual sample, most frequently two Sarcocystis spp. were identified (69.0%), then a single species (15.5%) and three species (4.8%). The present study provides strong evidence that mustelids serve as definitive hosts for Sarcocystis spp. using cattle as intermediate hosts

Morphologic and genetic identification of sarcocystis fulicae n. Sp. (apicomplexa: Sarcocystidae) from the eurasian coot (fulica atra)

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 lm wide. Histologically, the cyst wall reached up to 1.2 lm in thickness and seemed smooth. The detected sarcocysts were packed with relatively small banana-shaped bradyzoites that were 6.732.0 lm in size. Ultrastructurally, the cyst wall amounted to 1 lm 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

Molecular Confirmation of <i>Accipiter</i> Birds of Prey as Definitive Hosts of Numerous <i>Sarcocystis</i> Species, including <i>Sarcocystis</i> sp., Closely Related to Pathogenic <i>S</i>. <i>calchasi</i>

The present study aimed to test intestinal scrapings of the Northern Goshawk (Accipiter gentilis) and the Eurasian Sparrowhawk (Accipiter nisus) from Lithuania for S. calchasi and other Sarcocystis species characterised by bird–bird life cycles. The protozoan parasite Sarcocystis calchasi can cause respiratory and neurological diseases in a variety of birds; however, the distribution of this parasite is not well-examined. Sarcocystis species were identified with nested PCR and sequencing of the partial ITS1 region. Sporocysts and/or sporulated oocysts of Sarcocystis spp. were observed in 16 (100%) Northern Goshawks and 9 (56.3%) Eurasian Sparrowhawks. Four species, S. columbae, S. halieti, S. turdusi, and S. wobeseri, were confirmed in the Eurasian Sparrowhawk. Apart from the latter four species, S. calchasi, S. cornixi, S. kutkienae, and S. lari were established in the Northern Goshawk. A higher prevalence of Sarcocystis spp. and species richness in Northern Goshawks is associated with the differences in the diet of two examined Accipiter species. This study is the first report of S. calchasi in Lithuania. Furthermore, the genetically distinct species Sarcocystis spp. 23LTAcc, which is most closely related to S. calchasi, was found in three Northern Goshawks

Molecular identification of Sarcocystis rileyi and Sarcocystis sp. (closely related to Sarcocystis wenzeli) in intestines of mustelids from Lithuania

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)