Tissue Distribution And Transmission Of Myxobolus Honghuensis In Infected Gibel Carp Carassius Auratus Gibelio

Myxobolus honghuensis belongs to Cnidaria,Myxozoa and Myxobolidae,which is the important pathogen of the pharyngeal myxosporidiosis,and can often cause a large number of deaths in cultured gibel carp Carassius auratus gibelio(Bloch).Due to the lack of research on the route of infection transmission and the law of pathogen occurrence,there has been no scientific and effective prevention and control measures for this kind of disease.In this study,a sensitive and specific single-tube semi-nested PCR method was established for the detection of Myxobolus honghuensis,and microscopic examination,histopathology,PCR detection and fluorescence in situ hybridization(FISH)were used to analyze the tissue distribution and transmission pathways of gibel carp infected by Myxobolus honghuensis.The experimental results of this study are as follows:(1)A single tube semi-nested PCR assay to detect and monitor the infection of M.honghuensis was developed with two new designed primer pairs,which were designed based on 18 S r DNA.The specificity,sensitivity,repeatability and applicability of the assay were seriously evaluated.The results indicated that the amplification was positive only in the M.honghuensis sample,and the other myxosporeas and muscle of Carassius auratus gibelio(Bloch)were all negative;the lowest detection was 4.2 copy/μL of the target gene.The clinic samples detection showed that M.honghuensis present in the ovaries,kidneys and spleens of seem-healthy gibel carp from epizootic zone,with postive ratio 40 %,32 % and 8 %respectively.The detection rate of this method was significantly higher than that of common PCR,and this method can be applied to the early rapid detection of M.honghuensi in the clinical samples of gibel carp.(2)This study has collected and examined the muscle,pseudobranch,gills,head kidney and other 11 organs by microscopic,singal-tube semi-nested PCR and histopathological section.Microscopical exam showed that the sporocyst and mature spores were most detected in the pharynx,pseudobranch and gills of diseased fish,while the mature spores were enclosed with yellow nodal and most detected from pseudobranch and first efferent branchial artery in the asymptomatic gibel carp.PCR results showed M.honghuensis could be detected in the most tested organs in diseased fish except muscle,and pseudobranch had the highest infection rate(100 %).In asymptomatic gibel carp,positive results were detected in pharynx,pseudobranch,head kidney,kidney,spleen and ovary,and the highest infection rate(26.7 %)also was from the pseudobranch.Tissue sections of the diseased gibel carp of microscope showed M.honghuensis forming numbers of sporocysts between the pharynx wall and skull,and some near the pharyngobranchials.The pseudobranch was seriously destructed by the sporscysts,and the sporogonic cells of M.honghuensis could be observed near the left pseudobranch filament.(3)In this study,34 allogynogenetic gibel carp were artificially propagated and hatched under the recirculating aquaculture conditions in the laboratory and one-month culture experiment was carried out.The ovary,pseudobranch,oocyte and progeny fry 3,15 and 30 days post hatching were detected by single-tube semi-nested PCR,fluorescence quantitative PCR,and oligodeoxynucleotide fluorescence in situ hybridization(FISH)was used to accurately locate Myxobolus honghuensis in infected tissues and offspring fry of gibel carp.The PCR detection results showed that there were Myxobolus honghuensis infection in the ovaries,pseudobranch,oocytes of the allogynogenetic gibel carp,and in the progeny fish fry15 and 30 days post hatching.The results of FISH showed that,the trophozoites of Myxobolus honghuensis were distributed in the ovarian connective tissue near the pseudobranch,the lymphoid tissue of the kidney and spleen of the allogynogenetic gibel carp.In the yolk membrane of the progeny fish fry of 3 days post hatching in the head kidney and kidney of the progeny fish fry of 15 days post hactching,there were distributed the trophozoites of Myxobolus honghuensis.In summary,in this study,a new method for early detection of Myxobolus honghuensis was successfully established,firstly found that the pseudobranch may be the main parasitic and sporogonic site of M.honghuensis to parasite and mature development,and preliminary analysis shows that the Myxobolus honghuensis has a vertical transmission route in allogynogenetic crucian carp.