Finding Sarcocystis spp. on the Tioman Island: 28S rRNA gene next-generation sequencing reveals nine new Sarcocystis species

The Tioman Island of Malaysia experienced acute muscular sarcocystosis outbreaks from 2011 to 2014. So far, a previous study based on the 18S rRNA gene sequencing has reported S. singaporensis, S. nesbitti and Sarcocystis sp. YLL-2013 in water samples acquired from the island, thus confirming the waterborne nature of this emerging parasitic disease. This study aimed to improve the detection methods for Sarcocystis, in order to have a clearer picture of the true diversity of Sarcocystis species in Tioman. A new primer set (28S R7F-28S R8 Deg R) was designed to amplify the 28S rRNA gene of Sarcocystis. Subsequently, Sarcocystidae was detected in 65.6% (21/32) of water samples and 28% (7/25) of soil samples acquired between 2014 and 2015 from Tioman. Next-generation sequencing (NGS) on 18 of the positive samples was then performed using amplicons generated from the same primer set. This yielded 53 potentially unique Sarcocystidae sequences (290 bp), of which nine of the most abundant, prevalent and unique sequences were named herein. In contrast, NGS of the 18S rRNA gene V9 hypervariable region of 10 selected samples detected only two Sarcocystis species (160 bp). S. mantioni was the most ubiquitous sequence found in this study.