Study On The Protozoon Of Oyster And Its Rapid Detection Method

Perkinsus, Bonamia, Marteilia refringens, Mikrocytos, Haplosporidium nelsoni andHaplosporidiu.costale are the important parasitic pathogens of farmed shellfish. They werefound in many countries and regions and included in the Aquatic Animal Diseases List byWorld Organisation for Animal Health (OIE) and the Asia-Pacific aquaculture network center(NACA). These protozoa were investigated of farming oysters on southeast coastal of China.It is compared and analysis ITS gene sequences of Perkinsus in different regions. Andestablish a multiplex PCR assay for detecting Perkinsus, Haplosporidium.nelsoni andBonamia. These are provided important references and technical support for variation,diseaseand the distribution of shellfish protozoa in Chinese coastal.A series of examination and detection methods were established in this study.(1) Oysterswere collected from Xiamen coastal and used the ray's fluid thioglycollate medium (RFTM)culture method to culturing perkinsus hypnospore. The hypnospore were cultured successfullyand stained by giemsa, Lugol and no staining to observe the morphology. At the same time,we observed perkinsus hypnospore breeding with two split ways.(2) Six common shellfishprotozoosis PCR detection methods and species identification methods were established usingthe recommended primer of OIE. And use these methods to test491samples of oysters whichwere collected from eight regions of the southeast coast of China in Fujian Province,Guangdong Province and Hainan Province. The results show that these oysters are detectedcommonly Perkinsus,Bonamia, Haplosporidium nelsoni and Haplosporidium costale withoutMarteilia refringens and Mikrocytos. And different parts have big difference infection rate ofchese protozoa. perkinsus infection rates are high in Fujian regions, Xiamen up to47.83%.Bonamia infection rates are more than66.7%in Sanya and Haikou regions. Haplosporidiumnelsoni and Haplosporidium costale infection rates of Guangdong and Hainan regions aremore than the average infection rate of28.05%,38.29%. The results of species identificationshow that Bonamia were all the Bonamia ostrea, the majority of perkinsus were perkinsusolseni, only two sample of oysters were perkinsus beihaiensis n.sp in the survey.(3) PCRmethod was established to amplified complete sequences of internal transcribed spacer (ITS) of perkinsus. And the ITS of perkinsus in the eight different regions were cloned, sequencedand made multiple alignment and homology analysis. The results showed that perkinsusolseni and perkinsus beihaiensis n.sp ITS sequence was713bp and738bp.5.8S sequence washighly conserved. Intraspecific and interspecific of perkinsus nucleotide differences is mainlymanifested in ITS-1and ITS-2region. The homology analysis showed that ITS sequences ofperkinsus olseni was between100%and99.3%. Two sample of perkinsus beihaiensis n.spwas97.9%.(4) Two pairs of specific primers were designed to the conserved sequence ofPerkinsus olseni and Haplosporidium.nelsoni. Bonamia primers used the OIE recommended.It was establish a multiplex PCR assay for rapid detection these three protozoa. As little as10pg of the recombinant plasmid templates can be detected and without interference by otherprotozoa, bacteria and shellfish. The results of clinical test showed that the results wereconsistent with the test results of OIE PCR detection method. But the method was moreefficiently and rapidly.At present the information about the popular of shellfish protozoa in our country is verylittle. Bonamia, Marteilia refringens, Haplosporidium.nelsoni and Haplosporidium.costaleMikrocytos are more less especially. But the protozoa surveys and species identification ofsoutheast coast can make up the gaps. A multiplex PCR was establish for simultaneousdetection of Perkinsus olseni, Bonamia, and Haplosporidium.nelsoni. Perkinsus beihaiensisn.sp ITS complete sequence was obtained for the first time by the PCR amplification methodwhich we establish. And it made up for the blank in corresponding field. These were theinnovation and characteristic in the study. It laid the foundation for further study of theshellfish protozoa.