Establishment of CARD-FISH and Dot-ELISA Assays for Diagnosis of the Parasitic Dinoflagellate Hematodinium perezi.

The parasitic dinoflagellate in Hematodinium genus is a serious pathogen infecting many economically important crustacean species and resulting in epidemic 'milk diseases' in Portunus trituberculatus along Chinese coast. The outbreaks of Hematodinium epidemics resulted in large economic losses in mariculture crustaceans for local farmers. To prevent and control the Hematodinium epidemics in marine crustacean fisheries, it is essential to develop new methods for diagnosis of Hematodinium parasites. In the present study, a catalysed reporter deposition fluorescent in situ hybridisation (CARD-FISH) and a dot enzyme-linked immunosorbent assay (Dot-ELISA) methods were successfully developed to specifically detect in vitro microspores and in vivo trophonts of Hematodinium. perezi, respectively. The newly developed CARD-FISH was proved to be a specific, reliable and accurate method for detection of waterborne transmission stage (microspores) of H. perezi. The developed Dot-ELISA was capable of specifically and sensitively detecting in vivo trophonts of H. perezi in haemolymph of P. trituberculatus. The positive rate of haemolymph samples from 30 cultured P. trituberculatus detected by Dot-ELISA (53.3%) was comparable to PCR analysis (56.7%), with the accuracy evaluated as 94% as normalised by PCR assays (default accuracy 100%). The major findings in the present study will contribute to preventing and controlling Hematodinium epidemics in cultured P. trituberculatus.