Study On Etiology And Prevention Of Bacterial Septicemia In Sturgeons

Sturgeons and paddlefish of the order Acipenseriformes are the only extant large-size Chondrostei species in Osteichthyes,which have extremely high scientific and economic value.Due to the impacts of human activities such as overfishing,construction of hydroelectric dams and habitat destruction,25 out of the 27Acipenseriformes species are in different endangered status all over the word.In order to protect the endangered sturgeons?paddlefish?and meet the market's growing demand for sturgeon caviar,many countries and regions have carried out artificial propagation of sturgeons,and the global sturgeon breeding industry has flourished.China has become the largest sturgeon breeding country in the word since 2003.However,sturgeon diseases have become increasingly prominent following the decline of sturgeon germplasm resources and the improvement of intensive farming,which seriously restricts the protection of endangered sturgeons and sustainable development of sturgeon breeding industry.In this study,pathogenic biology,pathology,pathogenic mechanism investigation were performed on the fatal bacterial septicemia occurred in farmed Chinese sturgeon?Acipenser sinensis?adults and juveniles at three breeding base in Hubei Province.In order to prevent and control the bacterial diseases of sturgeons,we screened the potential probiotics isolated from the gut of healthy Chinese sturgeon and Dabry's sturgeon?Acipenser dabryanus?.The main study methods and results in this study are showed as follows.1.A fatal bacterial septicemia occurred in F₂ generation Chinese sturgeon adults and juveniles at three breeding base located in Hubei Province during 2015 to 2017.Eleven dominant strains were isolated from the liver and spleen of moribund Chinese sturgeon by traditional pathogen isolation and culture methods.Experimental infection tests showed that nine stains were pathogenic to healthy Dabry's sturgeon via intraperitoneal injection.Koch's postulates were fulfilled by reisolation of same pathogenic bacteria from the infected Dabry's sturgeon.Based on the strains morphological characteristics,biochemical characteristics and phylogenetic analysis derived from 16S rRNA,gyrB and rpoD gene sequences,nine isolates were identified as motile Aeromonas spp.,mainly Aeromonas veronii and Aeromonas hydrophila.Meanwhile,histopathological characteristics of the diseased Dabry's sturgeon were observed,and antibiotic susceptibility of two pathogenic bacteria were performed.Histopathology of Dabry's sturgeon infected with A.hydrophila or A.veronii revealed that liver hyperemia,hepatic and splenic necrosis,and enteritis.Antimicrobial susceptibility tests showed that both bacterial strains were equally sensitive to fluoroquinolones antibiotics,and third and fourth generation cephalosporins,but were resistant to oxacillin,ampicillin and penicillin.2.In order to study the pathogenic mechanism of A.veronii and A.hydrophila to sturgeons,the spleen of Dabry's sturgeon infected with A.veronii or A.hydrophila was used for transcriptome profiling analysis.Illumina sequencing generated 410996612clean reads,all of which were assembled de novo into 26939820 Contigs,521713Transcripts and 254211 Unigenes.A total of 5574 differentially expressed genes were identified from transcriptome profiles of Dabry's sturgeon infected with A.hydrophila,of which 2716 were up-regulated and 2854 were down-regulated;a total of 1724differentially expressed genes were identified from transcriptome profiles of Dabry's sturgeon infected with A.veronii,of which 835 were up-regulated and 889 were down-regulated,1087 differentially expressed genes shared in both groups.Further KEGG enrichment analysis of differentially expressed genes from Dabry's sturgeon infected with A.hydrophila showed eight immune-related pathways,including the Toll-like receptor signaling pathway,Phagosome,Cytokine-cytokine receptor interaction,Lysosome,Intestinal immune network for IgA production,Jak-STAT receptor signaling pathway,Herpes simplex infection and Neuroactive ligand-receptor interaction;KEGG enrichment analysis of differentially expressed genes from Dabry's sturgeon infected with A.veronii showed five immune-related pathways,including the Phagosome,Lysosome,Cytokine-cytokine receptor interaction,Herpes simplex infection and Neuroactive ligand-receptor interaction.Six differentially expressed genes?LTBR,MHC2,Integrin,CD40L,V-type ATPase and CDH3?were randomly selected and their expression patterns were largely consistent with qRT-PCR,which clearly validated the reliability of transcriptome analysis.3.In order to investigate the effects of A.hydrophila infection on the intestinal microflora of Dabry's sturgeon,we compared and analysed the intestinal microflora between healthy and diseased?A.hydrophila-infected?Dabry's sturgeon via Illumina Miseq high-throughput sequencing technology.Principal component analysis?PCoA?of unweighted unifrac distance matrices showed that the intestinal microflora of Dabry's sturgeon in healthy and diseased groups distributed in different regions,indicating that A.hydrophila infection caused a significant change in the intestinal microflora of Dabry's sturgeon.Compared with alpha diversity of the intestinal microflora in healthy and diseased groups,we found that invsimpson index was significantly decreased after A.hydrophila infection?P<0.05?.The community composition analysis showed that the relative abundance of Fusobacteria,Tenericutes and Bacteroidetes in diseased group were higher than those in healthy group,but the relative abundance of Firmicutes and Proteobacteria were lower than those in healthy group.In addition,Wilcoxon rank-sum test was used to examine significant differences in abundance of individual OTUs among two groups.Results showed that the relative abundance of Mycoplasma in the intestinal microflora of diseased group was significantly higher than that in healthy group?P<0.05?,while the relative abundance of Bacillus was significantly lower than that in healthy group?P<0.05?.4.The aim of this study was to isolate and screen the potential probiotic Bacillus spp.from the intestine of healthy Chinese sturgeon and Dabry's sturgeon for enhancing immunity and disease resistance of sturgeons.Results showed that three strains?BSth-1,BSth-4 and BSth-5?were isolated from healthy Dabry's sturgeon;one strain?BSth-19?was isolated from healthy Chinese sturgeon.Based on the strains morphological characteristics,biochemical characteristics and phylogenetic analysis derived from 16S rRNA and gyrB gene sequences,four isolates were identified as Bacillus subtilis.Four isolates were further investigated for activity against pathogenic bacteria,enzyme-producing activity in vitro,resistance to stress conditions of intestinal tract?low pH and high concentration bile?and animal safety test,which are important for probiotics to be used as feed additives.Among the isolates,B.subtilis BSth-5 and BSth-19 exhibited antimicrobial effects against four Chinese sturgeon pathogens,including A.hydrophila,A.veronii,Aeromonas media and Streptococcus iniae.The cell number of B.subtilis BSth-5 and BSth-19 changed little after 2 h of exposure to pH=3.0or fresh Dabry's sturgeon bile at 5.0%.Meanwhile,B.subtilis BSth-5 and BSth-19produced extracellular protease,cellulose and lipase.And it was proved that B.subtilis BSth-5 and BSth-19 were harmless after injection to Dabry's sturgeon.To evaluate the effects of B.subtilis on growth performance,non-specific immunity,intestinal digestive enzyme activity and disease resistance of Dabry's sturgeon.A total of 360 healthy Dabry's sturgeon juveniles were randomly divided into 3 groups with 3 replicates in each group and 40 fish in each replicate.One group?C?of Dabry's sturgeon was fed a control diet and two groups were fed experimental diets containing 2.0×10⁸ CFU/g BSth-5?T1?or BSth-19?T2?for 8 weeks.No significant differences in final weight,weight gain rate and special growth rate were observed in the T1 and T2 groups compared to the control group?P>0.05?,but a significant improvement in survival rate was detected after 4 and 8 weeks of feeding in the T1 and T2 groups?P<0.05?.Serum total antioxidant capacity?T-AOC?,total superoxide dismutase?T-SOD?activity and IgM levels were significantly higher in the T1 and T2 groups compared to the control group after 8 weeks?P<0.05?.Moreover,serum lysozyme?LZM?activity was significantly higher in the T1 group relative to the control group during the whole experiment period?P<0.05?,while the differences were not significant between the T2and control groups?P>0.05?.Serum malondialdehyde?MDA?levels in the T1 and T2groups were significantly lower than those in the control group after 4 weeks?P<0.05?.Intestinal protease,amylase and lipase activities of Dabry's sturgeon fed diet T1 and diet T2 were higher than those of fish fed the control diet after 8 weeks,but the differences were not significant?P>0.05?.The challenge tests showed that survival rate was highest in the T1 group?66.67%?,followed by the T2 group?53.33%?and then the control group?33.33%?.5.To evaluate the effects of B.subtilis BSth-5 and BSth-19 on the intestinal microflora of Dabry's sturgeon,we compared and analysed the intestinal microflora between the T1 and T2 groups and the control group via Illumina Miseq high-throughput sequencing technology after 8 weeks of feeding.Principal component analysis?PCoA?of unweighted unifrac distance matrices showed that the intestinal microflora of Dabry's sturgeon in the T1,T2 and control groups distributed in different regions,indicating that B.subtilis BSth-5 and BSth-19 have significantly changed the intestinal microflora of Dabry's sturgeon.Further analysis showed alpha diversity of the intestinal microflora of Dabry's sturgeon in the T1 and T2 groups were higher than those in the control group,and the differences were significant between the T1 and control groups?P<0.05?.The community composition analysis showed that the relative abundance of Fusobacteria,Tenericutes and Bacteroidetes in the T1 and T2 groups were higher than those in the control group,but the relative abundance of Proteobacteria was lower than that in the control group.In addition,Wilcoxon rank-sum test was used to examine significant differences in abundance of individual OTUs between the T1 and T2 groups and the control group.Results showed that the relative abundance of potential pathogenic bacteria?Aeromonas?in the intestinal microflora of the T1 and T2groups were significantly lower than that in the control group?P<0.05?,while the relative abundance of potential probiotics?Bacillus,Lactobacillus and Bifidobacterium?were significantly higher than those in the control group.To the best of our knowledge,this study is the first report of A.veronii and A.hydrophila causing bacterial septicemia in cultured Chinese sturgeon.Potential drugs for prevention and treatment of bacterial septicemia were found based on antimicrobial susceptibility tests.Illumina sequencing showed A.hydrophila could inhibit the expression of antigen-presenting-related factors and cause Dabry's sturgeon cell apoptosis.By comparing the intestinal microflora between healthy and A.hydrophila-infected Dabry's sturgeon,it was found that Bacillus spp.may be potential probiotics in the intestinal tract of Dabry's sturgeon.Two potential probiotic B.subtilis from the intestine of healthy Chinese sturgeon and Dabry's sturgeon were added to the basal diet and fed with Dabry's sturgeon juveniles for 8 weeks.Dietary supplementation with BSth-5 and BSth-19 could enhance the survival rate,antioxidant activity,serum immunity and disease resistance in Dabry's sturgeon juveniles.Meanwhile,dietary supplementation with BSth-5 and BSth-19 could increase the relative abundance of the potential probiotics in the intestinal microflora of Dabry's sturgeon juveniles and reduce the relative abundance of the potential pathogens.