| Firstly,we established cadmium(Cd)and Aeromonas hydrophila stress model in zebrafish(Danio rerio).Zebrafish were exposed to different concentration of CdCl2(0,2,4,6,8 and 10 mg/L)and observed for 96 h.LC50 of Cd was calculated based on the accumulating death ratio.After confirmation of virulence gene existence in A.hydrophila,each zebrafish was injected with 50μL different concentration of bacteria(0,9.458×105,9.458×106,1.892×107,9.458×107 and 9.458×108 cfu/ml)and observed for 7 d.LD50 of A.hydrophila was determined.Secondly,we evaluated the stability of eight candidate reference genes in five tissues of zebrafish exposed to different concentrations of Cd(0,0.23 and 1.15 mg/L)and the best four stable reference genes were further assessed at 12 h and 9 d post infection respectively.At last,in order to explore the profiles of early pro-inflammatory cytokine production in zebrafish infected with A.hydrophila after exposure to Cd,the mRNA levels of il-1β,il-6,il-8 and tnf-αin kidney,spleen and liver were investigated at 12 h post injection with PBS or bacteria after 96-h exposure to different concentrations of Cd(0,0.23,1.15 and 5.75 mg/L)by real-time PCR.Later,according to the results above,the mRNA levels of il-1β,il-6,il-8 and tnf-αin the three organs were detected at 0,6,12,24 and 48 h post injection with bacteria after 96-h Cd(0 and 5.75mg/L)exposure.Results showed that:First,the stress model of cadmium and A.hydrophila in zebrafishBased on the accumulating death ratio,the LC50 of Cd for 96 h and LD50 of A.hydrophila for 7 d were 5.8 mg/L and 2.7×106 cfu respectively.Second,stability evaluation of reference genes in zebrafish exposed to cadmium and subsequently infected by A.hydrophilaThe best four suitable reference genes in the five selected organs after exposure to Cd were:kidney:rplp2>rpl7>β-actin>ef1αspleen:β-actin>gapdh>ef1α>rpl13α;liver:rpl7>rpl13α>β-actin>ef1α;gills:rplp2>gapdh>rnf7>ef1α;intestine:ef1α>rnf7>rplp2>rpl13α.The later expression profiles showed that efl a in spleen and kidney,rpl13αin liver and rplp2 in intestine were the most suitable reference genes at 12 h and 9 d post injection with A.hydrophila following Cd exposure.In gills,the expression of gapdh was more stable than ef1αafter 9 days of bacteria challenge while ef1αshowed a higher stability than gapdh at 12 h post injection.Third,the immune response to A.hydrophila in zebrafish exposed to cadmiumIn different concentration groups,the mRNA levels of the four genes(il-1β,il-6,il-8 and tnf-α)in subgroups injected with bacteria were higher than the PBS subgroups and most of them presented significant difference.In different PBS subgroups,it did not show any statistical differences,whereas in high concentration subgroup(5.75 mg/L)with bacterial injection the mRNA levels of il-1β(p<0.01),il-6(p<0.01)and il-8(p<0.01)in kidney,il-1β(p<0.05)and il-8(p<0.01)in spleen and liver were significant higher than in control group injected with bacteria.During infection time,the mRNA levels of the investigated pro-inflammatory cytokines in three immuno-related organs(kidney,spleen and liver)increased at first and demonstrated reverse"U" shape.In kidney,the mRNA levels of il-1β,il-6,il-8 and tnf-a at high Cd group were higher than corresponding control group at different infection time:il-6 at 6 hpi(hours post injection)and 48 hpi showed significant(p<0.05)or highly significant(p<0.01)difference;il-8 and tnfa at 48 hpi presented highly significant(p<0.01)difference.In spleen,il-1βat 0 hpi presented significant(p<0.05)difference.In liver,il-1βat 0 hpi and 48 hpi showed highly significant(p<0.01)difference;il-6,il-8 and tnf-αat 48 hpi showed significant(p<0.05)or very significant(p<0.01)difference.Conclusions:Firstly,the higher concentration of Cd and bacteria,the earlier of zebrafish death and the higher accomulating mortality.It indicated that the lethal effect was concentration dependent.In high concentration of Cd the zebrafish showed signs of hyperemia or bleeding at abdomen or tail.The infected fish were expiratory dyspnea and abdominal swelling or even bleeding.Secondly,different tissues have different suitable reference genes when zebrafish was exposed to Cd and the subsequently pathogenic insults for qPCR method.It emphasized the importance of reference gene evaluation for studies using qPCR,in particular when investigations involve factors not explored previously.Thirdly,the immuno-related organs kidney,spleen and liver could response to bacteria no matter if the Cd in or off and the response trend was unchanged;High concentration of Cd could trigger response of early pro-inflammatory cytokines in kidney and liver to A.hydrophila,while not in spleen. |
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