| The Apostichopus japonicus is the profitable maricultural animal in the northern coastal areas of China with the largest artificial farming scale,which is of high medicinal and commercial values.In recent years,as artificial farming of Apostichopus japonicus has rapidly developed,the skin ulcer syndrome caused by Vibriosis as the main pathogenic bacteria has led to serious economic loss for Apostichopus japonicus farming.According to statistics,this disease resulted in an economic loss up to 2 billion yuan only from 2003 to 2005.In addition,mortality of the larva Apostichopus japonicus which suffered from this disease exceeded 90%.and thus,it has become a difficult problem that restricts farming development of Apostichopus japonicus.Several studies have demonstrated Vibrio alginolyticus as one of the major pathogens causing the disease.Phage therapy is to utilize the lysing effect of the phage on the target host bacteria and take the initiative to reduce the density of pathogens,thereby reducing or avoiding the pathogen infection or pathogenesis and achieving the purpose of prevention and control of pathogens.The present study indicates that aquaculture environment is ideal for phage application.As the high fluidity of water media can improve the dispersal ability of phage,and the phage is featured by safety,no poisoning,high efficiency,low cost and short development cycle,the phage is very suitable to be applied as a kind of antibiotic substitutes to prevent and control the pathogen in aquaculture activities.This study takes the pathogenic Vibrio alginolyticus of Apostichopus japonicus as the host to screen the lytic phage and examine the bactericidal effect of phage on Vibrio alginolyticus in aquaculture environment.Main work in this study is as follows:(1)A strain of non-lysogenic Vibrio alginolyticus is isolated from the lesion of Apostichopus japonicus suffering from skin ulceration disease,and is verified to have the single-strain pathogenicity effect on healthy Apostichopus japonicus(the median lethal dose is 9.44×103 for intraperitoneal injection,and 1.82×106 CFU/mL for immersion).The Vibrio alginolyticus is resistant to Ampicillin and Streptomycin.Two lytic phage of Vibrio alginolyticus,namely PVA1 and PVA2,are morphologically obtained and observed as the phages with tails.Among them.PVA1 is belonged to Podoviridae,while PVA2 belonged to Myoviridae;PVA1 has the latent period of 20 min,with the burst size of 108 PFU/cell,while the latent period of PVA2 is about 30 min and the burst size is 98 PFU/cell.Two phages can inhibit growth of Vibrio alginolyticus within 12 h.The most suitable pH value within 6.0?8.0;(2)According to the whole genome sequencing results,the phage PVA1 is double-stranded linear DNA genome with a total length of 41529 bp,a GC content of 43.7%and without tRNA coding gene.75 open reading frames are predicted,accounting for 93.32%of the whole genome;21 functional genes are predicted,which can divide the phage genome into four structural regions:DNA packaging,DNA replication and regulation and other function.The result of BLAST indicates no other phages which are homeotic to PVA1 genome;(3)The bioinformatics analysis indicates an endolysin gene existed in the phage PVA1 genome.The results show that the predicted protein size is 171 aa and the molecular weight is 19.41 kDa.In addition,the activity domain of glycosyl hydrolase is present to residues 6?94,and a peptidoglycan binding domain is present to residues 5 8?15 9,which is the typical functional domain layout of Gram-positive bacteria phage endolysin.After the gp60-coded endolysin gene is cloned and expressed in E.coli BL21 cell,the recombinant endolysin is found to have partially soluble expression and the molecular weight is less than 20 kDa;the turbidimetric method is used to detect the cracking activity of recombinant endolysin,which is estimated to be 1184±163 U/mg that is significantly lower than that of commercial hen egg white lysozyme;(4)PVA1,PVA2 and the mixture of both are applied to conduct preventive test against the Apostichopus japonicus which is treated by bathing of pathogenic Vibrio alginolyticus.The results show that the mixture of PVA1 and PVA2 with 1.0 x 107 PFU/mL has the best protective effect on the Apostichopus japonicus and manifested no significant difference with the Doxycycline test group with 5 mg/L.According to the results of therapeutic test where different phages and antibiotics are injected into Apostichopus japonicus that is treated by intraperitoneal injection of Vibrio alginolyticus,the therapeutic effect is better as long as the phage is earlier applied to the treated Apostichopus japonicus.However,the survival rate of the phage group is significantly lower than that of the Doxycycline test group.Compared with injection of Vibrio alginolyticus,injection of the mixture which is composed of Vibrio alginolyticus and phage into coelomic fluid of healthy Apostichopus japonicus can significantly reduce the activity of lysozyme,acid phosphatase and peroxidase and enhance the activity of nitric oxide synthase;(5)After the phage lyophilized powder is added to basic diet of the Apostichopus japonicus and the Apostichopus japonicus is fed for 60 days,the results reveal that addition of PVA1,PVA2 and the mixture of both can not significantly improve the growth performance of Apostichopus japonicus.However,it can improve the activity of non-specific immune related enzymes in the Apostichopus japonicus coelomic fluid.The feeding of diet added with phage do not cause significant effect on the number of vibrio in the intestinal tract,but can significantly improve the ability of Apostichopus japonicus to resist from infection of Vibrio alginolyticus. |
PDF Full Text Request