A cyst-forming coccidian with large geographical range infecting forest and commensal rodents: Sarcocystis muricoelognathis sp. nov.

BACKGROUND: The geographic distribution and host-parasite interaction networks of Sarcocystis spp. in small mammals in eastern Asia remain incompletely known. METHODS: Experimental infections, morphological and molecular characterizations were used for discrimination of a new Sarcocystis species isolated from colubrid snakes and small mammals collected in Thailand, Borneo and China. RESULTS: We identified a new species, Sarcocystis muricoelognathis sp. nov., that features a relatively wide geographic distribution and infects both commensal and forest-inhabiting intermediate hosts. Sarcocystis sporocysts collected from rat snakes (Coelognathus radiatus, C. flavolineatus) in Thailand induced development of sarcocysts in experimental SD rats showing a type 10a cyst wall ultrastructure that was identical with those found in Rattus norvegicus from China and the forest rat Maxomys whiteheadi in Borneo. Its cystozoites had equal sizes in all intermediate hosts and locations, while sporocysts and cystozoites were distinct from other Sarcocystis species. Partial 28S rRNA sequences of S. muricoelognathis from M. whiteheadi were largely identical to those from R. norvegicus in China but distinct from newly sequenced Sarcocystis zuoi. The phylogeny of the nuclear 18S rRNA gene placed S. muricoelognathis within the so-called S. zuoi complex, including Sarcocystis attenuati, S. kani, S. scandentiborneensis and S. zuoi, while the latter clustered with the new species. However, the phylogeny of the ITS1-region confirmed the distinction between S. muricoelognathis and S. zuoi. Moreover, all three gene trees suggested that an isolate previously addressed as S. zuoi from Thailand (KU341120) is conspecific with S. muricoelognathis. Partial mitochondrial cox1 sequences of S. muricoelognathis were almost identical with those from other members of the group suggesting a shared, recent ancestry. Additionally, we isolated two partial 28S rRNA Sarcocystis sequences from Low's squirrel Sundasciurus lowii that clustered with those of S. scandentiborneensis from treeshews. CONCLUSIONS: Our results provide strong evidence of broad geographic distributions of rodent-associated Sarcocystis and host shifts between commensal and forest small mammal species, even if the known host associations remain likely only snapshots of the true associations.

Morphological and molecular phylogenetic characterization of Sarcocystis kani sp. nov. and other novel, closely related Sarcocystis spp. infecting small mammals and colubrid snakes in Asia.

We investigated the morphology and phylogenetic relationships of novel and previously recognized Sarcocystis spp. infecting small mammals and colubrid snakes in Asia. The nuclear 18S rRNA and mitochondrial cox1 of Sarcocystis sp.1 from mangrove snakes (Boiga dendrophila) in Thailand and Sarcocystis sp.2 from a ricefield rat (Rattus argentiventer) in Sumatra were partially sequenced. Sporocysts of Sarcocystis sp.1 induced development of sarcocysts in experimentally infected rats, which showed a unique ultrastructure that was observed previously by S.P. Kan in rats from Malaysia; therefore, we describe this species as Sarcocystis kani sp. nov. Its integration into the 18S rRNA phylogeny of Sarcocystis spp. cycling between small mammals and colubrid snakes helped clarify relationships among the so-called S. zuoi-complex of molecularly cryptic species: Sarcocystis kani sp. nov., S. sp.2, S. attenuati, S. scandentiborneensis, and S. zuoi were all included in this clade. Tree topology was resolved into dichotomies congruent with the morphological disparities between the taxa. However, cox1 gene sequencing (including newly sequenced S. singaporensis and S. zamani) revealed that Sarcocystis kani, S. attenuati, and S. scandentiborneensis were identical suggesting a recent, common ancestry. To identify other distinctive features, lineage-specific molecular patterns within both genes were examined revealing that all 18S rRNA sequences of the S. zuoi - complex possess a unique, 7-nt long motif in helix 38 of domain V7 that was different in S. clethrionomyelaphis which branched off basally from the complex. Three-dimensional homology modelling of COX1 protein structure identified amino acid substitutions within the barcode area specific for the S. zuoi-complex and substantial divergence in structurally important amino acids between Sarcocystis species of snakes as definitive hosts and other lineages of the Sarcocystidae. We discuss the utility of selected genes for species delimitation of the Sarcocystis spp. under investigation, which probably evolved during recent radiations of their intermediate and definitive hosts.

Examination of Sarcocystis spp. of giant snakes from Australia and Southeast Asia confirms presence of a known pathogen - Sarcocystis nesbitti.

We examined Sarcocystis spp. in giant snakes from the Indo-Australian Archipelago and Australia using a combination of morphological (size of sporocyst) and molecular analyses. We amplified by PCR nuclear 18S rDNA from single sporocysts in order to detect mixed infections and unequivocally assign the retrieved sequences to the corresponding parasite stage. Sarcocystis infection was generally high across the study area, with 78 (68%) of 115 examined pythons being infected by one or more Sarcocystis spp. Among 18 randomly chosen, sporocyst-positive samples (11 from Southeast Asia, 7 from Northern Australia) the only Sarcocystis species detected in Southeast Asian snakes was S. singaporensis (in reticulated pythons), which was absent from all Australian samples. We distinguished three different Sarcocystis spp. in the Australian sample set; two were excreted by scrub pythons and one by the spotted python. The sequence of the latter is an undescribed species phylogenetically related to S. lacertae. Of the two Sarcocystis species found in scrub pythons, one showed an 18S rRNA gene sequence similar to S. zamani, which is described from Australia for the first time. The second sequence was identical/similar to that of S. nesbitti, a known human pathogen that was held responsible for outbreaks of disease among tourists in Malaysia. The potential presence of S. nesbitti in Australia challenges the current hypothesis of a snake-primate life cycle, and would have implications for human health in the region. Further molecular and biological characterizations are required to confirm species identity and determine whether or not the Australian isolate has the same zoonotic potential as its Malaysian counterpart. Finally, the absence of S. nesbitti in samples from reticulated pythons (which were reported to be definitive hosts), coupled with our phylogenetic analyses, suggest that alternative snake hosts may be responsible for transmitting this parasite in Malaysia.