Molecular characteristics of Indonesian Enterocytozoon hepatopenaei isolates based on sequence analysis of spore wall protein genes

Disease outbreaks caused by Enterocytozoon hepatopenaei (EHP) in Indonesia have been reported in Litopenaeus vannamei farms in North Sumatra, Lampung, West Java, Central Java, East Java, Bali, Lombok and Sulawesi. So far, reports on the molecular characteristics of EHP in Indonesia were limited. Characters of EHP from various sites were very important to describe their epidemiological relationship. The aims of this study were to determine the character of DNA, and to review the phylogeny relationship of EHP originating from various locations in Indonesia based on the Spore Wall Protein coding gene (SWP). 12 EHP isolates were successfully sampled from infected vannamei shrimp from farms, using purposive sampling in Lampung, Karawang, Pangandaran, Sidoarjo, Banyuwangi, Probolinggo, Blitar and Makassar. The EHP isolates were molecularly diagnosed using PCR with pairs of specific SWP_1F and SWP_1R primers, DNA sequencing, nucleotide sequence homology analysis and reconstructed genetic relationship tree. DNA sequence homology analysis demonstrated that the 12 EHP isolates were 100% similar with Enterocytozoon hepatopenaei SWP1 from Thailand and India. The alignment results illustrated that all EHP sequences from the 8 locations were identical. The phylogenetic tree topology provided the information that all samples were from the same clade and spread evenly. Indonesian EHP species from different locations were identical to the nucleotide sequence character among them and with the EHP isolates from Thailand and India. Therefore, Indonesian EHP isolates observed in this study most likely came from the same source, Asia. Spore wall protein coding genes have a good capacity as genetic markers for partial identification and characterization of EHP species, but they are less able to distinguish EHP intraspecies genetic diversity. However, further genetic analysis may be needed, especially for genes that encode effector proteins for virulence or genes that play a role in pathogenicity.