| Odontobutis potamophila is a unique economic fish that widely distributed in the freshwater of China,such as the Yangtze River,the Qiantang River and the Minjiang River systems,which has a delicious taste and rich in nutrition.Unfortunately,with expansion of the cultivation and water environment deterioration,epidemic infectious diseases of O.potamophila has emerged in recent years,which have caused serious economic losses of O.potamophila industry.At present,most of the researches are focused on the reproductive biology and culture technology of O.potamophila in China,but the researches on its disease are limited.The results of this study on epidemiological investigation,etiology and histopathology showed that Aeromonas veronii was a major pathogen which caused infection in O.potamophila.In addition,the immune response of O.potamophila against A.veronii was explored by transcriptomic analysis and single-cell transcriptome analysis,which provided theoretical support for prevention and control of the diseases caused by A.veronii in O.potamophila.The main results of this study are as followed:1.In May 2021,outbreaks of mass mortalities occurred in O.potamophila farms in Changshu of Jiangsu Province,the clinical signs of the diseased fish showed hemorrhage on fins,ulceration on the dorsal and abdomen.To investigate the reasons,the bacterial isolates from the diseased O.potamophila exhibited the same phenotypic traits and biochemical characteristics,and the pathogenicity of the dominant bacteria was determined by an artificial infection test,histopathological observation,virulence gene and virulence factors detection.The drug resistance of the isolated bacteria was determined as well.The results showed that the pathogen causing the mass death of O.potamophila was A.veronii,and the median lethal dosage(LD50)of the isolate Stl3-1 for O.potamophila determined as 4.5×105 CFU/mL.Histopathological analysis revealed that the isolate Stl3-1 caused considerable histological lesions in the fish,including tissue cell degeneration,necrosis,and inflammatory cell infiltrating.Detection of virulence-related genes showed that A.veronii Stl3-1 was positive for exu,ompA,lip,flaH,flgA,flgM,flgN,gcaT,hlyA,aer,act and tapA.Detection virulence factors of the A.veronii Stl3-1 showed that A.veronii Stl3-1 produced haemolysin,caseinase,amylase,gelatinase,esterase and lecithase activities.The drug resistance analysis showed that the isolate A.veronii Stl3-1 was resistant to 11 drugs including penicillin G,norfloxacin and compound sulfamethoxazole,etc.;intermediary to 8 drugs including ciprofloxacin,enrofloxacin and levofloxacin,etc.;sensitive to 16 drugs such as florfenicol,doxycycline hyclate and cefoperazone,etc.2.In order to explore the immune response of O.potamophila after A.veronii infection,changes of immune-related genes of O.potamophila were analyzed in response to pathogenic bacterial infection.We performed transcriptome analysis of the O.potamophila head kidney tissue at 12 h and 24 h post-infection(hpi).After assembly and annotation,11,3282 high quality unigenes were obtained from RNA-seq data,and differential expression analysis revealed that there were 164 significantly differential expressed genes(DEGs)at 12 h post infection,with 121 up-regulated and 43 down-regulated genes,while there were 364 significantly differential expressed genes(DEGs)at 24 h post infection,with 211 up-regulated and 153 down-regulated genes.Among these DEGs,some well-known immune-related genes were up-regulated significantly,including MHCHB,Myd88,TLR,SOD,IL1RAPL2、IL4I1、Jak2、IL8 and IL10,etc,GO analysis showed some DEGs were clustered into immune related subcategories such as response to stimulus,immune system process and antioxidant activity,etc.These DEGs were enriched into 99 KEGG pathways at 12hpi,while enriched into 264 KEGG pathways at 24hpi,including a core set of immune related pathways notably in IL-17 signaling pathway,C-type lectin receptor signaling pathway,etc.To further study the immune response of O.potamophila after A.veronii infection,the expression level of four selected immune-related genes(MHCIIB,Myd88,TLR and SOD)based on the transcriptomic data were monitored at different point of time in liver,gill,spleen and head kidney by quantitive real-time PCR(qRT-PCR).The results showed that the expression levels of MHCIIB,Myd88,TLR and SOD were significantly upregulated from 6 to 72 h in liver,gill,spleen and head kidney after A.veronii infection.These results showed that these immune-related genes participated in the immune response and can be considered as potential indexes for monitoring health status of O.potamophila.3.To further investigate the immune response characteristics of O.potamophila in response to A.veronii infection,in this study,10×Genomics high-throughput single-cell transcriptome sequencing was performed on head kidney tissue samples from O.potamophila infected with A.veronii for 24 h and uninfected groups to analyze the role of immune cells in resistance to A.veronii infection in O.potamophila head kidney tissues.The results showed that the total number of cells measured was 13,382,of which 7,174 cells were detected in the head kidney tissue of the A.veronii infected group(TAV)and 6,208 cells were detected in the control group(CK),including B cells,granulocytes,endothelial cells and macrophages.The dimensionality reduction and cluster analysis of granulocytes revealed that a significant increase in neutrophils and a decrease in eosinophils in the head kidney tissue of TAV group,and further differential enrichment analysis of the gene expression of granulocyte subpopulations revealed that O.potamophila could enhance ribosome biosynthesis by activating neutrophils to resist the invasion of A.veronii.The enrichment analysis of B-cell subpopulation showed that RNA biosynthesis process,Thelper cell differentiation,etc.were significantly enriched,suggesting that the head kidney B-cells were actived under the stimulation of A.veronii and showed the characteristics of adaptive immune response against pathogens to resist A.veronii infection.4.The increasing level of ammonia nitrogen could affect organism immune,which will further lead to the increase of susceptibility to pathogens and disease outbreak of O.potamophila.In this study,the effects of ammonia-N stress(0,1,4 and 8 mg/L)on mortalities,activities of immunity-related enzymes and the expression of immune-related genes of O.potamophila infected with A.veronii(4.5×105 CFU/mL)were determined to reveal the relationship between ammonia stress and disease outbreaks.The results showed that the mortality of O.potamophila after A.veronii infection were 27.5%,37.5%,47.5%and 60.0%in the 0,1,4 and 8 mg/L groups,respectively.The immunity-related enzymes activity experiment showed that the activity of SOD,ACP,AKP,LZM and GSH-PX declined gradually with the elevation of ammonia,and the activity of SOD,ACP,AKP,LZM and GSH-PX in the 8 mg/L group compared to control declined by 29.99%,54.26%,49.00%,62.08%and 55.53%,respectively.In addition,the immune-related genes(MHCIIB,Myd88,SOD and TLR)of A.veronii-infected O.potamophila under ammonia stress were monitored at different points of time in spleen,head kidney and gill.The results showed that the expression level of these genes were significantly down-regulated(P<0.05).The results indicated that ammonia stress can cause depression in the immunity of O.potamophila,and subsequently increase their susceptibility to A.veronii infection,which will provide theoretical support for prevention and control the diseases caused by A.veronii in O.potamophila.In this study,the characterization of virulence properties of A.veronii isolated from diseased O.potamophila was clarified;the important immune indicators were screened by transcriptome sequencing and single cell sequencing techniques,which can be used as potential indexes for monitoring fish resistance to bacterial infection.These results will provide theoretical support for prevention and control the diseases caused by A.veronii in O.potamophila. |
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