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.


Background
Protozoan parasites of the genus Sarcocystis are cystforming coccidians having an obligatory two-host preypredator life-cycle [1,2]. Asexual multiplication occurs in the intermediate host (IH), whereas sexual multiplication takes place in the small intestine of the definitive host (DH) [2,3]. Thus far over 200 Sarcocystis species have been described; however, a much higher number or species diversity of these parasites is presumed [1,4].
Birds may serve as intermediate or definitive hosts for many Sarcocystis species [1,[5][6][7][8]. More than 25 Sarcocystis species have been identified using birds as intermediate hosts [1,9]. Two species, S. falcatula and S. calchasi are highly pathogenic for their intermediate hosts. Some species, such as S. falcatula, S. calchasi and S. wobeseri are not strictly specific to the intermediate host and could form sarcocysts in birds of several different orders [10][11][12][13]. By contrast, other species like S. fulicae, S. lari and S. ramphastosi are strictly specific to a single bird species [14][15][16].
Herring gulls are opportunistic predators of marine invertebrates, fishes, insects and birds, as well as opportunistic scavengers of dead animals and garbage [17,18]. To date, only two Sarcocystis species, S. lari and S. wobeseri, have been described in birds of the family Laridae [15,19]. The present study provides molecular identification of four Sarcocystis species from L. argentatus that are morphologically indistinguishable under light microscopy examination.

Collection of samples
A total of 35 herring gulls, road-killed and received from taxidermists between 2013 and 2019 were studied. Leg muscles were examined for the presence of sarcocysts.

Morphological analysis
The prevalence and intensity of infection with Sarcocystis spp. was evaluated in methylene blue-stained preparations. For this purpose, 28 oat-size fragments (about 1 g) of muscles were cut-off, stained with 0.2% methylene blue solution, clarified with 1.5% acetic acid solution and pressed in a glass compressor. After squeezing of fresh muscle tissues, sarcocysts were excised with the help of preparation needles and then morphologically characterized under a light microscope (LM).

DNA extraction and PCR
Four sarcocysts were extracted from the leg muscles of each infected bird and subjected to light microscopy and molecular investigation. For the molecular analysis, sarcocysts were placed in individual 1.5 ml tubes containing 20 μl of 96% ethanol and kept at − 20 °C. Genomic DNA was extracted from individual sarcocysts using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the manufacturer's recommendations.
The complete ITS1 region was amplified using the SU1F/5.8SR2 primer pair [20]. Each PCR mixture consisted of 25 μl, containing 12.5 μl of Dream-Taq PCR Master Mix (Thermo Fisher Scientific, Waltham, US), 0.5 μM of each primer, 0.02 μg template DNA and nuclease-free water. The cycling conditions began with one cycle at 95 °C for 5 min followed by 35 cycles of 94 °C for 45 s, 60 °C for 60 s and 72 °C for 80 s, and a final extension step at 72 °C for 7 min. PCR products were evaluated using a 1.5% agarose gel and visualized via UV light after staining with 0.05 µg/ml ethidium bromide. Amplified DNA fragments were purified with exonuclease ExoI and alkaline phosphatase FastAP (Thermo Fisher Scientific).

DNA sequencing, sequence alignment and phylogenetic analysis
Sequencing reactions were performed using the Big-Dye Terminator v3.1 Cycle Sequencing Kit and the 3500 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) according to the manufacturer's recommendations. PCR products were sequenced directly using the PCR forward and reverse primers. The ITS1 sequences obtained in this study were compared with those of various Sarcocystis spp. using the Nucleotide BLAST program (megablast option). Sequences were aligned using the MUSCLE algorithm implemented in MEGA7 [21] software. The TOPALi v2.5 software [22] was used to select a nucleotide substitution model with the best fit to the aligned sequence dataset and to construct the phylogenetic tree under the Bayesian inference. Sequences for Sarcocystis spp. from L. argentatus generated in the present study are deposited in the GenBank database under the accession numbers MN450338-MN450373.
Surprisingly, the comparison of ITS1 sequences showed that the morphologically similar sarcocysts belonged to four different species of Sarcocystis, S. columbae, S. halieti, S. lari and S. wobeseri (Fig. 1). In the phylogenetic tree, the examined Sarcocystis spp. were placed into single-species clusters with a maximum support value. Based on ITS1, 833-bp long sequences of S. columbae obtained from L. argentatus (GenBank: MN450338-MN450339) demonstrated 99.9-100% identity with those of S. columbae (Gen-Bank: GU253885, HM125052) from the wood pigeon (Columba palumbus). The BLAST analysis revealed that 860-bp long sequences of S. lari from L. argentatus (MN450357-MN450364) shared 99.1-100% identity with those of S. lari from the black-backed gull Based on ITS1 sequences, S. columbae was identified in one out of nine infected birds. Two other species, S. lari and S. wobeseri, were confirmed in two and three herring gulls, respectively; whereas the most common  Table 1).
The morphological analysis of sarcocysts isolated from herring gulls indicated that S. columbae, S. halieti, S. lari and S. wobeseri are indistinguishable based on the size of sarcocysts and bradyzoites, as well as the thickness of the sarcocyst wall (Table 2). For instance, S. wobeseri had the thickest sarcocysts wall and S. columbae was distinguished by the thinnest cyst wall. However, morphological parameters of the four Sarcocystis species overlapped and it was impossible to discriminate these parasites under LM.

Discussion
In the present study four Sarcocystis species, S. columbae, S. halieti, S. lari and S. wobeseri, were identified in L. argentatus from Lithuania. These species had thread-like sarcocysts with a smooth cyst wall and were indistinguishable from one another under LM. Previously two Sarcocystis species were recorded in gulls: S. wobeseri was detected in L. argentatus [19] and S. lari was described based on material from L. marinus [15]. To our knowledge, S. columbae and S. halieti are detected in gulls for the first time in our study. Sarcocysts of Sarcocystis sp. detected in the muscles of the California gull (Larus californicus) from Canada had a thin (0.8 μm) and smooth cyst wall [5]. In Kazakhstan, Pak & Eshtokina [23] discovered sarcocysts with a thin and smooth cyst wall and banana-shaped bradyzoites in the black-headed gull (L. ridibundus) and the common gull (L. canus). Thus, the morphology of sarcocysts observed in the gulls from Canada and Kazakhstan is quite similar to those recorded in the present study.
The results of the present study indicate that not only S. falcatula, S. calchasi and S. wobeseri [10][11][12][13] but also S. columbae and S. halieti could form sarcocysts in birds belonging to different orders. Sarcocysts of S. columbae have previously been detected in the woodpigeon C. palumbus (Columbiformes) and S. halieti has been detected in P. carbo (Suliformes) [11,24]. Haliaeetus albicilla and the Eurasian sparrow hawk (Accipiter nisus) have been confirmed as definitive hosts for S. halieti [6,25]. Accipiter nisus does not prey on adult great cormorants and mainly feeds on small passerines [26]. Consequently, the range of the intermediate hosts of S. halieti might be much wider, whereas S. lari has been identified only in gulls, in L. marinus and in L. argentatus so far. Hence, further studies are needed to reveal the intermediate host specificity of avian Sarcocystis species.
Sarcocystis species richness detected in L. argentatus in the present study is greater than that found in other bird species. Anas platyrhynchos serves as an intermediate host for three Sarcocystis species, S. anasi, S. rileyi and S. wobeseri [10,15,[27][28][29][30]. According to current knowledge, other birds can be involved as intermediate hosts for one or two Sarcocystis species [14-16, 31, 32]. The richness of Sarcocystis species observed in L. argentatus can be related to the wide geographical distribution  and great variety of feeding habitats of this bird species, where herring gulls might ingest sporocysts shed by the definitive hosts [33,34]. It should be noted, that breeding colonies of L. argentatus are often located in the areas that are also used by other gull species, cormorants and ducks, acting as intermediate hosts of S. lari, S. halieti and S. wobeseri, respectively [35]. The morphology of the sarcocysts wall is the main diagnostic feature for morphological separation of Sarcocystis species in intermediate hosts [1]. Under LM, a thin and smooth sarcocyst wall was described for several avian Sarcocystis species, S. calchasi, S. columbae, S. corvusi, S. halieti, S. fulicae, S. lari and S. wobeseri. These species also share similar sarcocyst wall structure under the transmission electron microscope [10,11,15,16,36,37]. Thus, Sarcocystis species discussed are apparently morphologically indistinguishable. To the best of our knowledge, our study provides first evidence for several Sarcocystis spp. with a very similar morphological appearance under LM using a single bird species as an intermediate host. We have also demonstrated that one bird might host two Sarcocystis species, which could not be distinguished under LM. It should be emphasized, that the conclusions about Sarcocystis spp. richness in certain bird species might be misleading if only one sarcocyst is isolated for molecular identification. In 2011, our research group detected sarcocysts in the neck and leg muscles of four out of 11 herring gulls examined [19]. Under LM, one morphological type of sarcocyst was observed and only one excised cyst was subjected to molecular examination. At that time, it was assumed that sarcocysts detected in four herring gulls belonged to S. wobeseri. In contrast, the present study showed that L. argentatus can act as an intermediate host for four Sarcocystis species. Hence, when seeking to determine Sarcocystis species richness in birds, several sarcocysts should be isolated from each infected individual.

Conclusions
In the present study four Sarcocystis species, S. columbae, S. halieti, S. lari and S. wobeseri were identified in L. argentatus from Lithuania by means of ITS1 sequence analysis. Detected Sarcocystis species were morphologically indistinguishable under LM. In comparison with other bird species, L. argentatus has the highest Sarcocystis species richness. The results of the present study showed that S. columbae and S. halieti could use birds of different orders as intermediate hosts. It was revealed that muscles of a single herring gull could be infected with two Sarcocystis species indistinguishable under LM; therefore, in order to determine Sarcocystis species richness in bird intermediate hosts, or at least within genus Larus, we recommend molecular characterization of several sarcocysts isolated from each infected individual.