Epidemiological Investigation,In Vitro Culture And Morphological Observation Of Perkinsus Beihaiensis In Shellfish

Perkinsus spp.is a kind of highly pathogenic protozoan pathogens in shellfish.Infection with high-intensity can cause large-scale shellfish death and cause great harm to the shellfish breeding industry.As the open-cultivated characteristics of the majority of shellfish make it very difficult to control and prevent the disease,it is particularly important to investigate the epidemics of the shellfish pathogens for in-depth understanding of its’occurrence.Through the seasonal monitoring of mollusks along the coastal areas of Guangdong province,we explored the temporal and spatial changes of Perkinsus;On the basis of our early preliminary Perkinsus cultured experiments using by liquid thioglycolate culture medium（RFTM）,this study explored the effects of different antibiotic concentrations in the culture medium on variation of the number of Perkinsus hypnospore stage;The life history of the worm during in vitro culture was observed using an optical microscope and a scanning electron microscope.The main research results are as follows:1.Common PCR（Polymerase Chain Reaction）technology was used to investigate the seasonal dynamics and analyse the temperal and spatial distribution of Perkinsus spp.among five mainly cultivated shellfish in six aquaculture locations along the coast of Guangdong Province.Two Perkinsus spp.were detected in the investigation,Perkinsus beihaiensis and Perkinsus olseni,respectively.The prevalence of P.beihaiensis was significantly higher than P.olseni.Among five shellfish species sampled,except Crassostrea angulate,all sampling shellfish species were detected the infection of Perkinsus spp.Crassostrea hongkongensis and Haliotis diversicolor were infected by two Perkinsus spp.,while Pinctada martensii and Chlamys nobilis were infected just by P.olseni.The prevalence of Perkinsus spp.in the areas of western Guangdong,including Zhanjiang,Yangjiang,and Jiangmen,was 17.17%,significantly higher than the areas of eastern Guangdong,including Shanwei,Jieyang,and Shantou.The prevalence of Perkinsus spp.in summer and autumn was 15.67%,significantly higher than that in winter and spring.2.In this study,four groups of liquid mercaptoethanol medium ARFTM medium with different antibiotic content were set up.Perkinsus beihaiensis were cultured in a vacuum environment.The number of Perkinsus beihaiensis in the culture medium was observed on the second day and the seventh day to verify the effect of pachychenia on the growth of the spores in ARFTM medium.The results showed that when ARFTM was used for cultivating Perkinsus in a vacuum environment,the medium without antibiotics can effectively prevent the emergence of bacteria and other parasites within a certain period of time.The medium without antibiotics was beneficial to the growth of Perkinsus beihaiensis;The concentration of chloramphenicol at 25μg·mL^-1,nystain at 25 U·mL^-1,penicillin and streptomycin at 250U·mL^-1were the most suitable concentrations for the growth of Perkinsus beihaiensis hyphospores in this study;High concentrations of antibiotics would inhibit the growth of hyphospores of Perkinsus beihaiensis.3.ARFTM medium and DME/F12 medium were used to culture Perkinsus cells in vitro,and optical microscope and electron microscope were used to observe the morphological variation of Perkinsus beihaiensis at different growth stages.the result showed that in addition to trophozoites,hyphospores and zoospores,there was also a Perkinsus vermiform schizonts in the life history of the schizophrenia.This stage was a new form of the dormant spores developing into zoosporangium,which drags a tail-shaped cell with a length of about 150μm for the head globular cell.This stage was a new form of Perkinsus reproduction.