Establishment Of Pathological Detection Technique For Enterocytozoon Hepatopenaei

Enterocytozoon hepatopenaei(EHP)is an obligate intracellular parasite that can infect a variety of eukaryotes.The pathogen was first discovered in the hepatopancular tubule epithelial cells of the Penaeus monodon which was not known to be a microsporidian in Thailand in 2004.In 2009 Tourtip et al.also discovered this microsporidia from the hepatopancreas of Penaeus monodon,Parasites in different stages of the host cell's cytoplasm can be observed.The alignment of the SSU rRNA sequence homology indicates that the homology of the microsporidia to the B.tylovirus is 84%.Combining its unique ultrastructure,Plasmodium parasite cytoplasmic position and SSU rRNA sequence homology analysis,it was named as S.pneumoniae,and it was identified as a new species of the intestinal epithelial cell microsporidia.As an important pathogen of farmed shrimp,shrimp enterocytopenia has been detected in various places in recent years and is on the rise.The parasite is seriously threatening the development of the entire shrimp aquaculture industry.In this paper,the establishment and comparison of the pathological detection techniques of the enterobacter were reviewed.The spores staining method and fluorescence microscopy of the hepatic worms were established.The special staining method and fluorescent staining method of the intestinal enteroblasts in the tissues were established.The hybrid chromogenic method,which includes two spore detection techniques and three pathological staining techniques,provides technical support and guidance for the rapid clinical diagnosis and future research of EHP and white stool syndrome.(1)EHP spore staining effect comparison and method establishment.The pure spore fluid was isolated and purified.Staining solution of EHP spores was stained with 24 kinds of common stains.24 stains were Giemsa stain,Coomassie brilliant blue R250,Crystal Violet,Gention violet,Methylene blue,Malachite green,Fuchsin basic,Eosin Y alcohol solution,Coomassie brilliant blue G250,Safranine T,Victoria blue B,Methyl green,Carmine B,Eosin Y water soluble,Pyronine G,Brilliant green,Trypan blue,Sudan III,Methyl red,Anilin blue water soluble,Neutral red,Methyl blue,Congo red,Fast green FCF.The experimental results showed that there are 8 kinds of dyeing liquids that can be used for spore staining: Giemsa stain,Coomassie brilliant blue R250,Crystal Violet,Gention violet,Methylene blue,Malachite green,Fuchsin basic anf Eosin Y alcohol solution.Staining solution,malachite green is an aquatic product banned drug,try to avoid the use of other stains can be used for EHP spore staining.The dyeing liquids that can stain spores but have problems such as lighter staining and more background impurities include Coomassie brilliant blue G250,Safranine T,Victoria blue B,Methyl green,Carmine B,Eosin Y water soluble,Pyronine G and Brilliant green 8 kinds of dyeing liquid;stains which are not colored at all and there are problems such as deep background color and excessive impurities,such as Trypan blue,Sudan III,Methyl red,Anilin blue water soluble,Neutral red,Methyl blue,Congo red and Fast green FCF dyeing liquor.(2)Establishment of EHP staining method for hepatopancreas of shrimp.Pathological examination is an important method for diagnosing pathogens and observing pathological changes of the organism.Special staining is the prerequisite for finding pathogens in infected tissues.Good staining helps to distinguish pathogens from tissues,thereby increasing the detection rate of pathogens in tissue sections.In order to screen out a suitable method for the detection of Enterocytozoon hepatopenaei(EHP)in histopathology,we compared the staining effects of eight different staining methods: Hematoxylin-eosin staining,Masson staining,Alcian blue-Periodic acid schiff staining,Toluidine blue staining,Verhoeff-Van Gieson staining,Luxol fast blue staining,Sirius red and Prussian blue staining method,using the hepatopancereas tissues of P.vannamei,and counted the detection rate of EHP.The effects of HE,AB-PAS,TB,EVG,Sirius red and PB staining method was poor,the contrast between the spore and the background was not noticeable,the detection rate of a single visual field was also low.LFB staining makes the spores easy to be identified,but there are losses of spores and tissues staining effect is poor.Masson staining showed that the spores color was clearly visible magenta,the staining effect of the tissue cell structure was better,and the detection rate of the single field was the highest.Comprehensive analysis of the effect of staining and detection rate showed that Masson method was a well suitable method for the staining of EHP spores in host tissues.Therefore,this study provides an effective method for the histopathological study of microsporidiosis.(3)EHP microfluorescence detection technology was established.Microsporidia are closely related to fungi.Some fungal organisms have the characteristics of autofluorescence,and whether microsporidia also has the characteristics of autofluorescence.Whether the shrimp enterocytophages embedded in tissue sections can be caused by autofluorescence if it is detected,if not,whether it can be achieved by means of fluorescent staining,in order to solve these problems,the fourth chapter of the experiment first uses fluorescence microscopy to perform preliminary studies on the characteristics of autofluorescence of shrimp Enterobacter hepatica,and secondly,using fluorescent dyes.The histopathological sections of the parasites of the livers of shrimps were stained to establish a method for histopathological fluorescence staining of the hepatophages of shrimps.The experimental results show that the spores can be autofluorescence,and clear spores and empty spores can be observed under ultraviolet light excitation.The fluorescence observation and photographing can be maintained for about 3 minutes;the sections stained with DAPI fluorescent dye can be clearly observed under the fluorescence microscope.To the spores,and observed at different stages of the body,provides a new research method for the pathological study of EHP.(4)Establishment and comparison of in situ hybridization for EHP.We established and optimized EHP in situ hybridization for the detection of hepatopancreas tissue of shrimp L.vannamei infected with EHP.The same hepatopancreas was used for consecutive 3 sections.Masson staining,DAPI fluorescence staining and in situ hybridization were performed.The staining was performed to compare the advantages and disadvantages of the three pathological detection techniques.The results of in situ hybridization showed that the in situ hybridization can directly show the location of the hepatopancreas tissue lesions,making it black clusters,the more serious the infection,the more dense the black mark,the larger histopathological sections can be diagnosed by the naked eye Whether it is a positive infection or a small amount of spores scattered in the cytoplasm of the host,the spores cannot be marked by the probe as black,but the lumps formed by a large number of scattered spores will also show a black color reaction,and the experimental results are very satisfactory.Sensitivity and specificity.The comparison of the three detection techniques showed that Masson's staining had the best detection effect on spores.Even a small amount of spores scattered in the lumen of the hepatic tubules or a large number of spores in the host cells could be stained in red and detected.The worms in other proliferating stages in the host cells are not easily identified and the detection effect is poor.Fluorescence staining can effectively detect the parasites and spores in different periods,and it is easier to detect spores with different depths of field,and it can also well distinguish the protozoa of the host cytoplasm.However,the disadvantage is that the fluorescence is easily quenched and cannot be long.Observe and save time.The detection of spores by in situ hybridization was poor,but the qualitative detection of the parasites during the proliferation period was more accurate and reliable.It was suitable for studying the path of EHP in hepatopancreas tissues.Researchers can choose the appropriate detection technology based on experimental needs,providing data support for the selection of future research methods.