The thread-like shape of the present specimens, as well as its asexual development in the striated muscles of the intermediate host, a heteroxenous life cycle and the presence of metrocytes and bradyzoites in the sarcocyst indicate that they belong to the genus Sarcocystis [18]. This parasite, despite the small sample size (n = 4), showed a relatively high prevalence in the examined birds thus suggesting that its presence is very common in turacos and that the latter act as its natural intermediate host. This finding represents the first worlwide report of a Sarcocystis sp. in a member of the family Musophagidae.
As above mentioned, there are 25 valid species of Sarcocystis using birds as intermediate hosts, namely: S. accipitris Černá et Kvašňovská, 1986, S. albifronsi Kutkiené, Prakas, Sruoga et Butkauskas, 2012, S. alectoributeonis Pak, Sklyarova et Pak, 1989b, S. alectorivulpes Pak, Sklyarova et Pak, 1989b, S. anasi Kutkiené, Prakas, Sruoga et Butkauskas, 2012, S. atraii El-Morsey, El-Seify, Desouky, Abdel-Aziz, El-Dakhly, Kasem, Abdo, Haridy, Skai et Yanai, 2015, S. calchasi Olias, Gruber, Hafez, Heydorn, Mehlhorn et Lierz, 2010, S. chloropusae El-Morsey, El-Seify, Desouky, Abdel-Aziz, Sakai et Yanai, 2015, S. columbae Olias, Olias, Lierz, Mehlhorn et Gruber, 2010, S. cornixi Kutkiené, Prakas, Sruoga et Butkauskas, 2009, S. corvusi Prakas, Kutkiené, Butkauskas, Sruoga et Žalakevičius, 2013, S. falcatula Stiles, 1893, S. fulicae Prakas, Butkauskas, Švažas, Juozaityté-Ngugu et Stanevičius, 2018, S. halieti Gjerde, Vikøren et Hamnes, 2018, S. horvathi Rátz, 1908, S. kirmsei Garnham, Duggan et Sinden, 1979, S. lari Prakas, Kutkiené, Butkaukas, Sruoga et Žalakevičius, 2014, S. lindsayi Dubey, Rosenthal et Speer, 2001, S. phoeniconaii Göbel, Erber et Grimm, 1996, S. ramphastosi Dubey, Lane et van Wilpe, 2004, S. rileyi (Stiles, 1893) Minchin, 1903, S. sulfuratusi Dubey, Lane et van Wilpe, 2004, S. turdusi Kutkiené, Prakas, Butkauskas et Sruoga, 2012, S. wenzeli (Wenzel, Erber, Boch et Schellner, 1982) Odening, 1997, and S. wobeseri Kutkiené, Prakas, Sruoga et Butkauskas, 2010. Most of these species were morphologically described and differ from the new species in the size of sarcocyst and wall ultrastructure. Specifically, S. cristata sp. nov. has a cyst wall with villar protrusions under LM like in S. albifronsi, S. anasi, S. atraii, S. chloropusae and S. rileyi, but differ in the size of sarcocysts (1.348 mm vs. 4.00, 5.00, 0.165−0.850, 0.650 and 5.00−7.00 mm, respectively).
Molecular analysis of the four loci supported the identification of the present sequences as belonging to a new species. Groups of related species in the phylogenetic trees were formed, in one, mostly by Sarcocystis spp. with a bird of prey (e.g., hawks, eagles) as definitive host [see e.g, 9, 19−23]; while second grouped the new species and others allegedly using mammals as definitive hosts, such as S. albifronsi in Arctic fox (Alopex lagopus) from Lithuania [24], S. anasi and S. rileyi in V. vulpes and racoon dog (Nyctereutes procyonoides) from Lithuania [25] and Germany [26], S. atraii probably in American mink (Neovison vison) and red fox (Vulpes vulpes) from Egypt [27], and S. chloropusae maybe in V. vulpes [28]. The clade formed by S. cristata sp. nov. and these latter 5 congeners is a well-supported and recurrent group of species reported in several previous works [e.g., 22, 27], despite being of different cyst wall type (S. albifronsi and S. anasi type 9, S. atraii type 24, S. chloropusae type 10, S. rileyi type 23) and bird orders (e.g., Anseriformes, Gruiformes, Musophagiformes). Differences in the number and type of Sarcocystis species grouped with S. cristata sp. nov. are related to the availability of sequences in GenBank.
As above mentioned, the great blue turaco is an endemic bird species to West and Central Africa, where it apparently has few natural enemies. Therefore, the life cycle of S. cristata sp. nov. is unknown, but great blue turacos get probably infected after ingestion of sporocyst in food (leaves, buds, insects, flowers) or water, thus acting as intermediate hosts. Ants, cockroaches and crickets are considered food sources by many birds, and can serve as paratenic hosts for internal parasites such as Sarcocystis [see 29]. The fact that the new species clustered with other avian Sarcocystis spp., whose definitive hosts are mammals could indicate that, some terrestrial carnivorous mammals (e.g., mongoose, linsang or side-striped jackal) might act as definitive hosts for it. These predatory mammals commonly occur in the same region and feed on arboreal birds, although nothing is known about its role as predators of turacos.
Shape, cyst wall type, and other morphological and morphometric data have been reported in almost all avian species, but they are insufficient to complete their specific identification [22]. In fact, Levine [18] wrote that “...the fine structure of the sarcocyst wall may change with age and is not considered necessarily satisfactory for separating species”. Recently, Prakas et al. [34] identified one morphological type of Sarcocystis in the herring gull Larus argentatus from Lithuania, but indeed it represented four different species. On the other hand, genetic markers have proved to be more reliable, although 18S rRNA, 28S rRNA and cox1 were sometimes unable to separate species [see 9, 31]. In this case, ITS1 region was the most conclusive locus to clearly separate the new species from its congeners, while other loci were scarcely variable to distinguish species, although all genetic markers supported the designation of the new species. Apparently, ITS1 region is more sensitive to the genetic differences among species, like mentioned by Prakas et al. [22], who also found a similar utility of molecular analysis while studying S. fulicae in Fulica atra from Lithuania.
We do not know whether birds were infected before importation to Czech Republic or they got parasites while captive. However, the presence of great turacos in zoos and private breeding facilities and their potential escaping or releasing to wild should be monitored, since these birds could spread sarcocysts to native carnivorous mammals in the region. The death cause of turacos was neither determined, but the occurrence of Sarcocystis in birds should be diagnosed since a related species (S. rileyi) has been reported as causing sarcocystosis in the skeletal muscle (rice breast) of ducks [32]. Future studies may be focused on the proper parasitic diagnosis before importation and thus preventing the economic losses related to the trade of turacos around the world.