| Nile Tilapia is an important farmed fish in China,which accounts for more than half of the world’s fish harvest.Streptococcus agalactiae(Group B Streptococcus,GBS),a bacterial pathogen,leads to severe economic losses of Nile Tilapia in China,where controlling and purification is urgent needed for the Ⅲ classic of aquatic animal disease.Research on GBS disease has drew enough attention in clinical applications,such as vaccine and medicine,whilst the fundamental research remains poor.Fish spleen,a main site of invasive GBS infections,is an essential immune organ with important hematopoietic function in fish.The aim of the thesis was systematically pathological studied the spleen that was infected by GBS in Nile Tilapia,to facilitate both fundamental research and clinical applications in the near future.The first objective was histological observation of healthy Nile Tilapia spleens and its blood-spleen barrier Positioning,as positive control.Secondly,established a damageable scoring system through dynamic pathology observation of the GBS infected spleen,to stage pathological phases and provide biopsy guidance for followed study on spleen pathology.Thirdly,observed interaction between splenic cells and GBS by ultra-structural pathological technique,for exploring pathogenesis of GBS and possible molecular mechanisms of spleen injury.Fourthly,RNA-seq of four different pathological stages of spleen was carried out,and analyzed relative signaling pathways and key genes to support the study of molecular pathology of GBS-infected spleen.Lastly,investigated immune functions of all stages of injury spleen.The specific experimental contents are explained in this abstract.1.Normal structure of the Nile tilapia spleenSplenic structure of Nile tilapia was observed by gross anatomy,corrosion,histological and ultra-structural techniques.The results demonstrated that the spleen of Nile tilapia is a separate organ,which lies between the former intestine and stomach.Spleen capsule is thin,and its trabeculae was poorly developed.In addition,spleen is comprised of white pulp and red pulp,and the boundary between red and white pulp is not clear.The white pulp,within clear ellipsoids,contains lymphocytes vesicles and a small amount of melano-macrophage centers(MMCs).The ellipsoid is composed of cuboidal endothelial cells,reticular fibers and phagocytes.Whereas,red pulp consisted of splenic cords and red blood cells(RBCs).The splenic cords that surrounded by lymphocytes and mononuclear-macrophages were made up of reticular cells.Only deformed RBCs could pass through the holes of splenic cords.Reticular fibers were mainly distributed in the splenic cords and around the blood vessels,while collagen fibers mainly appeared in the splenic cords and around the splenic arteries.Therefore,based on the distribution of vessels of spleen and its microstructure,the spleen can be generally divided into three zones,inner zone,middle zone and outer zone.To locate the blood-spleen barrier,vital staining was performed with trypan blue.Results showed that trypan blue congregated in the endothelial cells of ellipsoids after intraperitoneal injection.In contrast,it precipitated on the reticular fiber around the ellipsoid after intravenous injection.These hinted that the blood-spleen barrier of the Nile tilapia is located at ellipsoid,and it is made up of endothelial cells and reticular fibers of ellipsoids.2.Dynamic pathology of the Nile tilapia spleen post GBS infectionInfected Nile tilapia with 1×107 CFU GBS.Spleen was sampled at an interval of 4 hours during the infected time(72 hours).Analyzed general pathology and histopathology of spleen,and the dynamic distribution of GBS.The general pathological changes were showed through obvious change of splenic index and capsule.Spleen index reduced obviously in 4-8 hours post injection(hpi),followed by a remarkable increase in 12-20 hpi,then returned to normal during 24-36 hpi,and significantly increased in 40-72 hpi.Besides,capsule was seen to be covered by a white cellulose membrane in 40-72 hpi.The histopathological changes were summarized as follow.Ellipsoid aggregated during 4-8 hours after invasion,with emptiness splenic sinus,and increased lymphocytes and MMCs.In12-20 hpi,endotheliocytes of ellipsoid presented vacuoles denaturalization,and juvenile Erythrocytes increased,unlike lymphocytes and MMCs decreased.During 24-32 hpi,reticular cells around ellipsoid proliferated,but lymphocytes and MMCs decreased greatly.Hyperplastic nodules formed in ellipsoids were observed at 36-40 hpi.During 44-72 hpi,the hyperplastic nodules developed into exudative nodules or necrotic nodules.Capsule and parenchyma below the capsule presented ribbon-like necrosis,with a clear boundary formed beside normal parenchyma within 44-72 hpi.On the other hand,in 4-8 hpi,the interior surface of blood vessels injured,and the reticular fibers shrunk and gathered.Next,the reticular fibers then disrupted and dissolved during 12-44 hpi.It finally disappeared in 48-72 hpi,as well as the collagen fibers dissolved gradually.GBS was initially observed in the endotheliocytes of ellipsoids at 20 hpi.The number of GBS increased in 24-48 hpi,and positioned within ellipsoidal endothelial cells.At the following 52-72 hpi,GBS decreased in most sampled spleens.According to the histopathological changes and GBS quantities,the pathological procedures of spleen were classified into four stages,and they were the early phase,characterized by the aggregation of ellipsoids;the reaction phase,characterized by the increase of juvenile RBCs;the Lag phase,characterized by the hyperplastic nodules;and the peril phase,characterized by the necrotic nodules.3.Effects of GBS dose on pathological stages of Nile tilapia spleenInjected three Nile Tilapia groups(Namely group A,B and C)with three different doses(1×106 CFU,1×107 CFU or 1×108 CFU))of GBS,separately.Spleen was sampled at the time of 4,8,24,72 hpi for each group.Besides,spleens of group A were also collected on the 7th and 14th days post GBS injection.Next,analyzed their general pathology,histopathology and the quantity of GBS for pathological staging.Results revealed that the death of group A,B,C occurred on 10th day,40th hour and 16th hour post injection,respectively.Spleens of group A experienced the early phase in 4-8 hpi,the reaction phase in 24-72 hpi,and the peril phase on 7th day post injection.However,spleens of group C showed both early phase and reaction phase in 4-8 hpi,and both the Lag phase and the peril phase in 24-72 hpi.Importantly,acute necrosis was observed in 8-24 hpi in group C as a new phase.Moreover,the phases classification of group B was as the same as in the point 2.Quantification of GBS with sip-qPCR,and the data indicated the infection time for each group as followed.No GBS was detected during the whole infection time in group A.For group B,GBS was initially detected at 8 hpi,and achieved the highest amount at 24 hpi,then reduced in 72 hpi.The changes of GBS amount in group C remained the same trend as it in group B likewise,but with higher bacterial content all the time than group B.As a consequence,the dose of GBS affected pathological stages and bacterial content of injury spleen,respectively.Notably,the 5th pathological stage,acute necrosis phase,was found in the spleen of Nile tilapia injected with 1 x 108 cfu GBS.4.Ultrastructural pathology of the Nile tilapia spleen post GBS infectionTransmission electron microscope was used to observe the interactions between splenic cells and GBS in all five pathological stages.The images demonstrated some ultrastructural pathological changes.In the early phase,vasoconstriction of splenic ellipsoid capillary(EC)was found,with a migration of monocytes moving from the EC to splenic parenchyma;Less bacteria were observed,and the injury of splenic cells were not obvious.During the reaction phase,the primary and secondary lysosomes increased in phagocytic cells;A large number of bacteria and bacterial debris wrapped in single,double or even multi-layer phospholipid membrane were observable;Mitochondrial swollen and nuclear membrane expanded in some phagocytes and lymphocytes;A small number of GBS survived and proliferated in some cells.In the Lag phase,the number of GBS was obvious lower in cells,and reticulocytes around the ellipsoids proliferated.In the peril phase,the number of bacteria continued to decrease.Endotheliocytes of EC were necrosis,and shed into the vascular lumens.In the acute necrosis phase,GBS filled the endotheliocytes of ellipsoid;and proliferated in the splenic cells and mesenchyme.The Erythrocytes were involved in GBS killing.Furthermore,within the whole infection processes,abundant capsules were only observed around live GBS,while no capsules were surrounded dead bacteria.Observation of the morphological changes of splenic cells in acute necrosis phase with scanning electron microscope indicated that erythrocytes seemingly bulged to be deformationand blocked the splenic cords;GBS adhered on reticular fibers.As has been noted,the endotheliocytes of ellipsoid,RBCs and phagocytes played important roles in phagocytosis of GBS.However,GBS could damage these cells and protect itself from phagocytosis with generating surface capsule.5.Analysis of the Nile tilapia spleen post GBS infection by RNA-seqTranscriptome analysis of GBS-invasive spleen in reaction phase(Lym),acute necrosis phase(Nec),Lag phase(Hem)and peril phase(Hyp)was performed through Illumina HiSeqTM 2500 high throughput sequencing platform,separately.In short,the transcriptome profile revealed that a cluster of 6295 differential genes presented in the infected spleen,in which 364 differential genes were sharing by the four-infected phases.For the pathological phases Lym,Nec,Hem and Hyp,834,1578,232 and 568 specific differential genes were read in each pathological phase,respectively.Analyzed these differential genes with GO enrichment assay.It was found that the infected differential genes and shared differential genes were significantly enriched in the process of immune response and antigen presentation,respectively.Also,specific differential genes in Lym,Nec,and Hem,were significantly enriched in the immune and anti-inflammatory reactions,catabolism and cell proliferation,respectively.Besides,the specific differential genes in Hyp were not notably enriched in both molecular functions and biological processes.On the other hand,enrichment analysis of KEGG signaling pathway of those differential genes.Results showed that the infected differential genes and the shared differential genes were greatly enriched in immunity pathways and the signal transduction pathways.In addition,the specific differential genes in Lym,Nec,and Hem were significantly enriched in the pathways of cytokine-cytokine,TNF signaling,the fatty acid degradation signaling pathway,and the pathways related to cell cycle progress.In contrast,the specific different genes in Hyp were not significantly enriched in any signaling pathway.Screened differential genes(with FPKM>100)involved in immunity,inflammatory and hematopoiesis in the shared differential genes and specific differential genes of all pathological stages.Consequently,cathepsin L was significant different among the shared differential genes.Also,the main significant differential genes in Lym were CXCL10 and type IV collagenase;IL-8 and complement C1q subunit B were greatly different in Nec;MHC I and DDIT were notably different in Hem;and MHC Ⅱγ,mannose-specific lectin,cathepsin D,CD 209,and hemoglobin a were significantly different in Hyp.The results indicated that the differential genes in Lym mainly enriched in immune system,and it may relate to the proliferate of lymphocytes;the pathway related to cell cycle progress in hem changed significantly,it may in the relation of cell proliferation;and the fatty acid degradation signaling pathway may associate to the necrosis of splenic cells.6.Effect of GBS infection on immunological function of Nile tilapia spleenThe data read from transcriptome profiling of PRRS(TLRs,NLRs,RLR,CLRs)of spleen byRNA-Seq implied the effects of GBS infection.The expression of TLRs was notably more than the other PRRS genes.Among the TLRs,TLR5 was significantly up-regulated in Nec,Lym and Hem;Otherwise,the expression of TLR13 was greatly down-regulated in Nec and Lym;TLR1 was lower expressed in Nec,Lym and Hyp.The TLRs sequences were compared with the TLRs in other fish and human in database ofNCBI and UniPort,with construction of a phylogenetic tree by Neighbor-joined(NJ)using Mega 4.1 software.The results showed that TLR5 of tilapia,human,zebrafish etc.were clustered into a group,which suggested their TLR5 sharing similar functions.As the mRNA expression of the downstream molecules IL-1β and IL-8 mediated by TLR5 in Nec were much more than that in other groups.It is speculated that the increasing expression of IL-1βand IL-8 led to excessive inflammation and acute necrosis of spleen.Analysis of immune-related factors,including C3,MHCⅡα,CSF1R,TCRα,TCRβ,MHCI,RAG1,IgM,and qRT-PCR was performed on the genes with significant changes.Accordingly,the mRNA level of C3,TCRα,TCRβ,CSF1R,RAG 1 and MHCI hardly changed before and after infection;The mRNA level of MHC Ⅱ was significantly down-regulated in Hyp andHem;However,the mRNA of IgM was expressed more in all the phases after GBS infection.The results suggested that the humoral immunity in spleen was the main immune response after GBS infection,and IgM was an important immune molecule.Furthermore,prokaryotic cloned and expressed slgM of Nile Tilapia,and prepared rabbit anti-tilapia IgG antibody,to examine serum IgM titer and IgM distribution in the spleen through ELISA and immunofluorescence.Accordingly,prokaryotic sIgM was successfully expressed in BL21-PET32-sIgM.High affinity and strong specificity sIgM-IgG was obtained by sIgM-immunized rabbits.As the results of ELISA,the expression of IgM protein did not rise in sera nor spleen in all the phases,and the signal of IgM immunofluorescence did not change visually.These implied that GBS inhibited the translation of IgM.Above all,the splenic structure and function were damaged seriously after GBS infection,among which the hyperplasia might be an important cause for chronic death,and excessive inflammation might facilitate acute death. |
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