DETECTION OF A MICROSPORIDIAN PARASITE (Perezia sp.) IN PENAEID SHRIMP FROM MADAGASCAR, MOZAMBIQUE AND THE KINGDOM OF SAUDI ARABIA THROUGH HISTOLOGICAL, IN SITU HYBRIDIZATION AND PCR ANALYSES

Samples of microsporidia-infected shrimp exhibiting clinical signs of cotton shrimp disease were collected from Madagascar, Mozambique, and the Kingdom of Saudi Arabia (KSA) during 2005 to 2014. The tails of the infected shrimp appeared opaque and whitish; and subsequent histological examination revealed the presence of inclusions and mature spores in tissues of skeletal muscle, hepatopancreas, gills, heart, lymphoid organ. By a PCR targeting the 18S rRNA gene from infected samples, a 1.1 kb DNA fragment was amplified. Nucleotide sequence of the 18S rRNA gene is 94% identical to Perezia nelsoni that infects Penaeus setiferus from USA. A phylogenetic analysis showed this microsporidium of cotton shrimp disease clustered with Perezia nelsoni. We thus tentatively named this parasite as Perezia penaei. Its 18S rRNA gene sequence was 100% identical among isolates from Madagascar and KSA, indicating that these parasites were likely originated from a common source within the region of Red Sea and Indian Ocean. A 443-bp fragment of the 18S rRNA gene was cloned and labeled with digoxigenin and in situ hybridized to tissue sections of infected P. monodon from Madagascar and Mozambique, and P. indicus from KSA. The results showed that the probe only reacted to the inclusions and mature spores in the infected shrimp samples. This assay was specific, did not react to another microsporidium, Enterocytozoon hepatopenaei (EHP). We also developed a PCR assay from the 18S rRNA gene region, this PCR was shown to be specific to Perezia penaei, did not react to 2 other parasitic pathogens, an amoeba and EHP, nor to genomic DNA of various crustaceans including polychaetes, squids, crabs and krill.