Isolation of Pathogenic Bacteria Aeromonas hydrophila from. Acipenser baerii and Immune Effect of Its whole Cell Vaccine

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1 Chinese Journal of Zoology 2008,43 (6) :19 3 ( ) : ( Acipenser baerii) X1, (LC 50 ) cfuπml, ; ATB 16S rdna,x1 ( Aeromonas hydrophila) ;,X1 ATCC35654 ( :X ),99 %0130 %, X1,, X1, X1,, X1 X1,50 %, X1, X1 X1, 70 %,X1 : ; ; ; :S ; S94215 :A : (2008) Isolation of Pathogenic Bacteria Aeromonas hydrophila from Acipenser baerii and Immune Effect of Its whole Cell Vaccine LI Yuan2Yuan CAO Hai2Peng DENGLu YANG Xian2Le 3 ( Aquatic Pathogen Collection Center of Ministry of Agriculture, Shanghai Fisheries University, Shanghai , China) Abstract :A pathogenic strain X1 was isolated from Siberian Sturgeon ( Acipenser baerii ) suffering with bacterial septicaemia. The median lethal concentration (LC 50 ) of strain X1 was cfuπml to healthy Siberian Sturgeon, which showed that strain X1 had rather strong virulence. Strain X1 was identified as Aeromonas hydrophila by physiological and biological studies and 16S rdna sequence analysis,which indicates that it had close relativity with A. hydrophila strain ATCC35654 (locus number :X ),with a high homology of 99 %. Strain X1 was inactivated by 0130 % formalin and its whole cell vaccine was made to immune Siberian Sturgeon with injection. The experiment results showed that whole cell vaccine of A. hydrophila strain X1 could obviously enhance the level of serum anti2a. ( No. 2006BAD03B0506),(No. B ), (No ),( KFT200825) ; 3,E2mail edu. cn ;,,; : ; E2mail com : ,:

2 2 Chinese Journal of Zoology 43 hydrophila antibodies - and the total counts of immunoglobulin,total proteins,as well as lysozyme. In addition,whole cell vaccine effectively prevented Siberian Sturgeon from being infected by A. hydrophila. The relative survival percent of Siberian Sturgeon was 50 %. Further,whole cell vaccine with FIA improved relative survival percent to 70 %, showing more effective immunity effect. Therefore,there is a potential to apply whole cell bacteria A. hydrophila strain X1 to prevention and cure of Siberian Sturgeon suffering with bacterial septicaemia. Key words : Acipenser baerii ; Aeromonas hydrophila ; Whole cell vaccine ; Immune effect ( Acipenser baerii) (Acipenserdae), 26, [1 ] 1996, ( A. schrenckii), 2 [2 ],, ( Aeromonas hydrophila) [3 ],, [46 ],28 24 h,,tsa,,, ,,, cfuπml, X1,, :,, X1,,, 1 111,5070 g,1316 cm, (Freund s adjuvant incomplete,fia), ; (), ,,, 75 %,, (tryptose soya agar,tsa),012 ml 8, d,, cfuπml 212

3 6 : cfuπml cfuπml cfuπml,,012 ml 8, d, [7 ] (LC 50 ) 11214, 16S rdna, NCBI BLASTN GenBank + EMBL + DDBJ + PDB 16S rdna, BioEdit 710 Mega 410 ; ATB 113,30 24 h cfuπ ml, 0130 %(),30, 24 h, ,3, + FIA,3, cfuπml 012 ml, + FIA 1 1 FIA 012 ml 7 d + FIA,,, [8 ] d + FIA 5,, 2 h rπmin 15 min, [9] d + FIA 5,,2 h 4, rπmin 15 min,- 20, [10 ],- 20, () d + FIA 5,,2 h 4, rπmin 15 min, d + FIA 10, cfuπml, 012 ml, 7 d,, ( relative percent survival,rps) :RPS = (1 - Π ) 100 % 115 SPSS 1115 ANOVA,Duncan s , X1 X2 X3 X4,, X1, 7 d, 100 %(1),,,,

4 4 Chinese Journal of Zoology 43 X1 ( %) (log (cfuπml) ) : y = x ( 1), X1 (LC 50 ) cfuπml,devesa [11 ],X1 1 Table 1 Results of artificial infection test of isolated strain Bacteria strain X1 X2 X3 X4 Injection concentration (cfuπml) Number of fish () Number of fish died () 1 d 2 d 3 d 4 d 5 d 6 d 7 d Mortality ( %) 1 Fig. 1 The relation curve of sturgeon mortality with logarithm of bacterial concentration 212 X1 16S rdna NCBI, ( Aeromonas sp. ) 16S rdna,98 %99 %, X1 ATCC35654 ( :X ) (2),ATB X1,X1, 99 %(2) 16S rdna, X1 ( Aeromonas hydrophila) Test item 2 ATB X1 Table 2 The identification of strain X1 by ATB bacteria identification instrument Result Test item Result Test item Oxidase + Citrate - Saccharose + D2 D2glucose + Histidine + Maltitol + Salicin Ketogenic substance - Itaconic acid - D2 D2melibiose Oxhydryl2butyrate - Suberate - L2 L2fucose Keto2gluconate - Malonate - D2 D2sorbierite Oxhydryl2benzoas - Acetas - L2L2arabinose + Rhamnose + DL2 DL2lactate + Propionate - N2 N2acetyl glucosamine + L2 L2alanine + Caprate - L2 L2proline + Glycogen + Valerate + Inositol - Mannitol + L2 L2serine + + ;- + Denotes positivity ;- Denotes negativity. Result

5 6 : S rdna Fig. 2 The phylogenetic tree based on 16S rdna sequence ; ; ; Aeromonas hydrophila : ; Aeromonas punctata : ; Aeromonas media : ; Aeromonas veronii : ; Aeromonas molluscorum Aeromonas hydrophila sub sp. decolorations : ; X1 : [ EU ] The figure is constructed by maximum likelihood method ; The value of bootstrap confidence level of nodes are indicated above the branch ; The numbers behind the species denote locus number. X11 Aeromonas hydrophila[ EU ]. 213 (3), X1 35 d,, , 4215 % ( P < 0105), X1, 42 d, + FIA, , %( P > 0105) 214 (4), X1 35 d,,( ) gπl, 20 % ( P < 3 Fig. 3 Changes of antibody titer with time in serum of Siberian Sturgeon 0105),35 d, + FIA, ( ) gπl, 2512 % ( P < 0105)

6 6 Chinese Journal of Zoology 43 X1, 4 Fig. 4 Changes of total protein with time in blood serum of Siberian Sturgeon 215 ( 5), X1 28 d,, ( ) gπl, 36 % ( P < 0105), X1, 28 d, + FIA,( ) gπl, %( P < 0105) 216 (6), X1 7 d,,( ) UΠml, 4414 % ( P < 0105),X1, 5 Fig. 5 Changes of immunoglobulin with time in blood serum of Siberian Sturgeon 7 d, + FIA,( ) UΠml, %( P < 0105) 6 Fig. 6 Changes of lysozyme content with time in serum of Siberian Sturgeon 217 (3), X1 X1, 50 %, X1, X1 X1

7 6 : 7 3 Table 3 The relative survival percent of Siberian Sturgeon infected with whole cell vaccine Group Control + FIA Whole cell + FIA Whole cell Concentration (cfuπml) Number of fish died 1 d 2 d 3 d 4 d 5 d 6 d 7 d Mortality ( %) Relative survival percent ( %), 70 % ,,,,,,,,,,,,, ( A. gueldenstaeti) ( Huso huso A. ruthenus ) ( H. dauricus), ( A. caviae ) ( Plesiomonas shigelloides) [1214 ], X1,, LC cfuπml, 16S rdna,, ATB,,,,,, [15 ], X1, 16S rdna, X1 TS622 [16,17 ], [18 ], ( Ctenopharyngodon idellus ) ( Carassius auratus) ( Siniperca chuatsi) : [4 ],, 3 d,7 d [8 ], 3 d,, X1 7 d,6,, [4 ] [8 ],

8 8 Chinese Journal of Zoology 43,X1, [4 ], ( A. salmonicida), Kolman [19,20 ],, 6, [21 ] Kolman [19,20 ], ( ) ( ) ( ) gπl ( ) gπl, X1 [22 ],,, [23 ],,, Kolman Lukyanenko [19,20,23 ],,X1,, [4 ],, X1, ( ),X1 [8 ] [24 ],,,, [25 ] [ 1 ] Ruban G I. Species structure, contemporary distribution and status of the Siberian sturgeon Acipenser baerii. Environ Biol Fishes,1997,48 : [ 2 ],..,2005,12(4) : [ 3 ],..,2007,3(33) : [ 4 ],,..,2007, 14 (5) : [ 5 ],,..,2007,31 (1) : [ 6 ],,..,2004,5 (11) : [ 7 ],,.. :, 2002, [ 8 ],,.. ( ),1999,14 (2) : [ 9 ],,. ( ). :,1999, [10 ],,..,2006,30 (4) : [11 ] Devesa S. First Report of Vibriosis in Turbot ( Scophthalmus maximus) Cultured in Northwestern Spain : Fish and Shellfish Pathology. New York :Academic Press,1985, [12 ]..,2001, 31 (5) :40411 [13 ],..,2003,33(2) :16171 [14 ],,..,2007,37 (2) :53561 [15 ],,..,2004,32(3) : [16 ],,..,2007,14 (5) :

9 6 : 9 [17 ],,.., 2006,22 (4) : [18 ],..,2003,30(5) :56611 [ 19 ] Kolman H, Siwicki A K, Kolman R. Influence of O2antigen Aeromonas salmonicida on non2specific and specific immune responses in Siberian sturgeon, Acipenser baeri Brandt. Arch Polish Fish,1999,7(1) : [20 ] Kolman H. Primary humoral response in Siberian sturgeon after exposure to anti2furunculosis bacterin. Czech J Anim Sci, 2002,47 (5) : [21 ],..,2006,30 (6) : [22 ],,..,2001,23(3,4) :1171 [23 ] Lukyanenko V I. Immunologiya Ryb :Vrozhdjonnyj Immunitet. Moscow :Agropromizdat,1989, [24 ],..,2006,28 (1) : [25 ],..,2005, 33 (3) : (Society for Conservation Biology, SCB) ,,,,,,, 30,,,,,,,,,,,,,,,,,,, ; http :ΠΠscb2009. ioz. ac. cnπ Cnindex. asp,,, ,,, :SCB2009 (:100101),: , ,: ,E2mail : ac. cn!

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