Motile Aeromonas Septicemia And Clinostomum Disease In Farmed Chinese Sucker, Myxocyprinus Asiaticus

Fish diseases have become the main constraint on the development of aquaculture industry as with high-density cultivation.Recent years,the outbreaking of the two diseases,has caused a certain economic loss on the cultivation of Chinese sucker,a unique species of Catostomidae in Asia and one of China's second-class protected animal species,which has important value both in research and economy field in China.In this study,the typical case of bacterial disease of MAS?motile Aeromonas septicemia?and digenetic trematode disease of Clinostomum disease?also named yellow grubs disease?were studied on farmed Chinese sucker?Myxocyprinus asiaticus?.The etiology,pathology and immune response mechanism in fish of these two diseases were explored respectively.These results will shed a light for further understanding of the pathogenesis of MAS,and provide a reference in preventing and controlling MAS and Clinostomum disease in farmed Chinese sucker.1.MAS The pathogen were firstly isolated from viscera and ascites infected with MAS of Chinese sucker juveniles from a nursery in Sichuan province using traditional methods.Then,the pathogens were identified by morphological observation,physiological and biochemical characteristics analysis,phylogenetic tree analysis based on 16S rRNA and cpn60,rpoB,gyrB genes.Then,artificial infection experiments,half lethal dose 50?LD50?test and16 virulence genes detection were carried out to determine the pathogenicity of isolated bacterial strain.Meanwhile,bacterial susceptibility to drugs was carried out with thirty-two drug sensitivity tests.In order to obtain pathological fish of MAS,bacterial suspensions were intraperitoneally injected with 200?L(2.0×10⁸ cfu/mL(LD_{50-24 h}))per healthy juveniles.Samples of trunk kidney,head kidney,spleen and liver were collected for histopathological examination at 4,12 and 24 hpi?hour post-infection?and for ultrastructural examination at 24hpi.In addition,the degree of tissue damage was evaluated by a semi-quantitative description method combining with histopathological analysis.Using melano-macrophage center?MMC?metrics as biological indicators,the response strategies of MMC in different tissues were described by quantitative description method to evaluate the level of tissue immune response.In the end,the transcriptome profiles of the spleen of Chinese sucker were examined at 4 hpi and 19 immune related genes were detected at 4,12 and 24 hpi by qRT-PCR to explore the molecular mechanisms.The main results are as follows:?1?Two dominant colonies were isolated from viscera and ascites of infected Chinese sucker which were then identified as Aeromonas hydrophila?henceforth BBAh1?and A.veronii?BBAv1?based on the results of the physiological biochemistry characteristic test and phylogenetic analysis.?2?The results of artificial infection experiments showed that the two strains both contribute to MAS of Chinese sucker.The strain BBAh1?A.hydrophila?has seven virulent genes including aerA,alt,ascV,hlyA,lip,ompA and aspA.The strain BBAv1?A.veronii?has eight virulent genes including aerA,alt,ascV,hlyA,lip,aexT,ast and flaA.The LD_50-7d value was 1.93×10⁵ cfu/g and 8.77×10⁵ cfu/g for BBAh1 and BBAv1.In addition,BBAh1 was more pathogenicity,while BBAv1 was more enteritis.?3?The results of drug susceptibility tests showed the two strains had the same sensitivity to 28 antibiotics which was corresponded to the fact that they have the same habitats.It is suggested that some drugs including cefotaxime,aztreonam,ceftriaxone and cefuroxime,which are highly sensitive to both strains,should be used to treat MAS of Chinese sucker.?4?Histopathological observations of MAS showed damaged endothelial cell,haemolysis,haemagglutination,MMC response,necrosis,lymphocyte infiltration,enhanced granulocytes cytoplasm acidophilia,vacuolization,concretion and acidophilic degeneration of hepatocytes and erythrocyte atrophy degeneration,etc.Ultrastructural pathological features of MAS showed collapsed blood capillary,mesangial cell swelling,podocyte hyperplasia,structure destruction of the glomerular filtration device.Many damaged cells like eosinophils and lymphocytes were phagocytosed by large numbers of active macrophages.Severely damaged disse space of liver,disappeared glycogen,swelling mitochondria,increased autophagic lysosomes in hepatocytes were also observed in infected juveniles.In conclusion,these results indicate that MAS infection lead to serious pathological changes in tissues'dysfunction of haematopoiesis,excretion and metabolism,which in turn results in death from anoxia,energy shortage and metabolic waste accumulation.?5?MMC response strategy to MAS:The MMC response strategy was almost identical,showing that the increasing number of MMC with relative larger surface area and single MMC area compared with the controls.However,MMC response strategies were different between liver and the above tissues.The number and proportion of MMC only significantly increased at 24 hpi in the liver?P<0.05?.?6?The strategy of MMC response indicate that the spleen plays a dominant role in the immune response,followed by the head kidney and the middle kidney,and while the liver is the weakest.Therefore,we consider that MMC in the spleen may be more sensitive as biomarkers to evaluate physiological status or immune level of fish than those in other tissues.?7?Following A.hydrophila acute infection,1390 DEGs?differentially expressed genes?were obtained from the spleen transcriptome data?q<0.05?,including 907 upregulated and483 downregulated genes.Meanwhile,8 DEGs were randomly selected for qRT-PCR verification to confirm the consistency with that of transcriptome data.?8?1390 differentially expressed genes were significantly enriched in 16 secondary GO categories and 15 KEGG pathways which were mostly related to immune response,these GO terms including interleukin-1 receptor binding?GO:0005149?,interleukin-1 receptor complex?GO:0045323?,cytokine receptor binding?GO:0005126?,inflammatory response?GO:0006954?,growth factor receptor binding?GO:0070851?and immune response?GO:0006955?,etc.15 KEGG pathways,mainly including NF-kappa B signaling pathway?ko04064?,TNF signaling pathway?ko04668?,NOD-like receptor signaling pathway?ko04621?,Toll-like receptor signaling pathway?ko04620?and Inflammatory bowel disease?ko05321?,etc.?9?The qRT-PCR results showed that many immune-related components are involved in the antibacterial process,such as pathogen recognition receptors?TLR2,TLR4,TLR6 and PGRP5?,interleukins?IL1B,IL6,IL8,IL10,IL11 and IL12?,chemokines?CXCL1,CXCL2and CXCL8?,activity regulation molecules of immune cell?CD68,CD200,CD247,CSF3 and CSF3R?and complements?C6 and C7?.?10?On the basis of transcriptom and qRT-PCR data,we can speculate the immune response mechanism in fish faced to A.hydrophila infection as follows.Pathogen-associated molecular patterns?PAMP?are recognized by pathogen recognition receptors like TLRs and PGRP5 to promote cytokines secretion of receptor cell,such as IL1,TNF which then activated the NF-kB signaling pathway through activating its upstream MAPK signal pathway.Next,activated NF-kB entered the nucleus and binds to specific sequence upstream of target gene to initiate transcription.Herein,intense immune response was activated to resist further infection of pathogens.Many pathological features appeared,including immune cells proliferation,active phagocytosis,strong MMC response and infiltration of inflammatory cells.However,MAPK and NF-kB signaling pathway may initiate a negative regulatory mechanism to prevent excessive inflammation.2.Clinostomum disease In this part,148 infected Chinese sucker juveniles were randomly sampled from the natural aquafarm for parasites detection at the laboratory,where the growth index?total length,body length and weight?were also measured.The infected juveniles were dissected,and the number and sites of the parasitic cysts were recorded for each juvenile.The prevalence,average intensity,infection frequency and infection intensity were calculated subsequently.The metacercariae from the cysts were preserved in 70%ethanol for morphological and molecular analysis?CO1 and ITS?.We focused on the first intermediate host?snails?and the final host?piscivorous birds?for parasite life-cycle investigations at the infected aquafarm.The main results are listed as follows:?1?To our knowledge,this is the first report of C.complanatum as intermediate hosts in M.asiaticus and even in Catostomidae.Using morphological description method combined with molecular markers,the genital complex feature was proved to be more reliable in than other morphological characteristics in identifying Clinostomum.CO1?6.87%?is more efficient than ITS?1.68%?due to its higher ratio of the variation sites.?2?A total of ninety-six juveniles?64.9%?were infected from the selected samples with the number of parasites varying between 1 and 7?mean:2.33±1.38?per fish.Overall,the parasites appeared higher infection frequency?0.557?and infection intensity?0.545?in the head than in the trunk and tail.?3?There was no significant difference in total length and body length between infected groups and control groups?P>0.05?.While the body weight and condition factor in the infected groups?weight=6.44±0.19 g;condition factor=1.70±0.03 g/cm3?were significantly lower than in the control groups?weight=10.36±0.51 g;condition factor=2.51±0.03 g/cm3??P<0.01?.?4?In conclusion,the life-cycle of C.complanatum in the aquafarm was complete for Radix swinhoei and Galba pervia snails are the first intermediate host,M.asiaticus as the second intermediate host and Egretta garzetta,Ardeola bacchus and Alcedo bengalensis birds as the final hosts.?5?Suggestions for Clinostomum disease control and prevention:1)splash the quicklime to kill the snails at the end of the year and clear snails in the pond and feeding canals completely.2)set the bird screens on the fry and fingerling ponds.In conclusion,for the first time,virulent A.hydrophila?strain BBAh1?and A.veronii?strain BBAv1?with strong pathogenicity were isolated from infected Chinese sucker with MAS in our current study,and also find some possible drugs to treat it according to drug sensitivity test results.Through observations of histological structure and ultrastructure changes of kidney,spleen and liver before and after A.hydrophila acute infection,we found a series of new pathological features of MAS,and MMC response to MAS.Meanwhile,the transcriptomic profiles of the spleens from infected and control groups in Chinese sucker and the qRT-PCR results of 19 immune related genes showed that pattern recognition receptors,such as TLRs and PGRP5,play important role in pathogen recognition at the early stage of A.hydrophila infection which was critical for activating its downstream signaling pathway and triggering immune response.In addition,our research found that the life-cycle of C.complanatum in the aquafarm was complete,and M.asiaticus,even Catostomidae,is as the intermediate host.Last but not least,we provide some suggestions for Clinostomum disease prevention,such as cleaning ponds,killing snails and setting the bird screens.Our results have expanded the research field in fish diseases and may provide some references for the disease control and prevention of M.asiaticus.