王軼南,戰(zhàn)文斌,唐小千,邢 婧

(中國(guó)海洋大學(xué)海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室,山東青島266003)

白斑癥病毒(WSSV)對(duì)克氏原螯蝦血細(xì)胞的感染規(guī)律*

王軼南,戰(zhàn)文斌**,唐小千,邢 婧

(中國(guó)海洋大學(xué)海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室,山東青島266003)

用WSSV粗提液注射感染克氏原螯蝦,感染后每24 h采集螯蝦血細(xì)胞。血細(xì)胞經(jīng)與抗WSSV單克隆抗體及FITC標(biāo)記的羊抗鼠抗體結(jié)合反應(yīng)后,應(yīng)用流式細(xì)胞儀檢測(cè)WSSV對(duì)血細(xì)胞的感染,同時(shí)記錄螯蝦累積死亡率及血細(xì)胞密度。結(jié)果表明,感染W(wǎng)SSV后螯蝦1～7 d的累積死亡率分別為3.3%,13.3%,16.7%,36.7%,70.0%,90%和100%;1～5 d血細(xì)胞被感染比例分別為6.47%,6.93%,10.65%,26.08%和4.94%;1～5 d被感染細(xì)胞的平均熒光強(qiáng)度分別為10.7,10.89,11.71,13.77和15.47;1～6 d血細(xì)胞密度分別為(7.56±0.30),(5.60±0.24),(4.21±0.30),(1.45±0.26),(1.21±0.21)和(1.14±0.18)×106個(gè)/mL,陰性對(duì)照組螯蝦血細(xì)胞密度為(5.34±0.22)×106個(gè)/mL?？梢?感染W(wǎng)SSV后螯蝦血細(xì)胞密度呈現(xiàn)先升后降的趨勢(shì),至感染后第6天血細(xì)胞密度僅為對(duì)照組的21.3%;被感染血細(xì)胞內(nèi)的病毒量始終呈上升趨勢(shì)、達(dá)到最高感染率時(shí)螯蝦處于瀕死狀態(tài),表明血細(xì)胞的WSSV感染與螯蝦死亡密切相關(guān)。

克氏原螯蝦;血細(xì)胞;對(duì)蝦白斑癥病毒

對(duì)蝦白斑癥病毒(WSSV)病流行廣泛、危害嚴(yán)重,自暴發(fā)以來的近20 a一直倍受關(guān)注[1-2]。目前,探明WSSV致病機(jī)理、尋找預(yù)防和控制該病已逐漸成為研究的熱點(diǎn)[3-4]。已有研究表明血細(xì)胞是WSSV侵染的主要靶細(xì)胞,并在病毒對(duì)各組織器官的感染中起重要作用[5-6],因此研究WSSV對(duì)宿主血細(xì)胞的感染特性及其與宿主死亡的關(guān)系,對(duì)了解WSSV致病機(jī)理具有重要意義。研究發(fā)現(xiàn)斑節(jié)對(duì)蝦(Penaeus monodon)、凡納濱對(duì)蝦(Litopenaeus vannamei)、墨吉對(duì)蝦(Penaeus merguiensis)、寬大太平螯蝦(Pacif astacus leniusculus)以及印度對(duì)蝦(Penaeus indicus)等感染W(wǎng)SSV后血細(xì)胞數(shù)量均發(fā)生變化[5-9],并且不同類型血細(xì)胞對(duì)WSSV的易感性存在差異[7,10],此外,感染W(wǎng)SSV后的斑節(jié)對(duì)蝦和印度對(duì)蝦血細(xì)胞具有明顯的細(xì)胞程序性壞死現(xiàn)象[9]。本研究的前期成果是應(yīng)用抗WSSV的單克隆抗體和FITC標(biāo)記的羊抗鼠抗體的免疫熒光技術(shù)及流式細(xì)胞儀檢測(cè)了24 h內(nèi)WSSV對(duì)克氏原螯蝦(Procambarus clarkia)血細(xì)胞的感染及感染強(qiáng)度,建立了螯蝦血細(xì)胞內(nèi)病毒感染的檢測(cè)方法[11]。在此基礎(chǔ)上,本文研究了克氏原螯蝦血細(xì)胞被WSSV感染到瀕死之間的感染規(guī)律及與螯蝦死亡的關(guān)系,以期為進(jìn)一步探明WSSV的致病機(jī)理提供參考。

1 材料與方法

1.1 材料及試劑

600尾健康克氏原螯蝦購(gòu)于青島南山水產(chǎn)品市場(chǎng),體長(zhǎng)8～10 cm,體質(zhì)量20～25 g,經(jīng)PCR檢測(cè)為WSSV陰性,實(shí)驗(yàn)前經(jīng)5 d暫養(yǎng)。自然感染W(wǎng)SSV的對(duì)蝦取自乳山對(duì)蝦養(yǎng)殖場(chǎng),于-80℃保存?？筗SSV單克隆抗體為本實(shí)驗(yàn)室研制[12]。異硫氰酸熒光素(FITC)標(biāo)記的羊抗小鼠Ig購(gòu)自Sigma公司。

1.2 螯蝦的人工感染

將螯蝦分為感染觀察組及感染流式檢測(cè)取樣組,并分別設(shè)WSSV感染組與對(duì)照組。將自然感染W(wǎng)SSV對(duì)蝦的鰓與0.01 mol/L磷酸鹽緩沖液(PBS,p H=7.4)體積比按1∶10勻漿,勻漿液經(jīng)差速離心(400×g,15 min,4℃;800×g,15 min,4℃)后,取上清用無菌PBS稀釋1 000倍,經(jīng)0.45μm微孔濾膜過濾除菌,經(jīng)螯蝦的腹部皮下注射感染,每尾50μL,陰性對(duì)照組注射同體積無菌PBS,空白對(duì)照不注射(見表1)。

表1 人工感染及實(shí)驗(yàn)分組Table 1 Artificial infection and experiment groups

1.3 螯蝦感染程度觀測(cè)

人工感染后,每24 h記錄觀察組螯蝦的死亡情況、繪制累積死亡率曲線。取死亡螯蝦的鰓絲冰凍包埋后,制備冰凍切片,以正常螯蝦鰓為陰性對(duì)照。在螯蝦鰓的冰凍切片上滴加15μL抗WSSV混合單克隆抗體,置37℃濕盒中孵育45 min;PBS洗滌3次,每次5 min。洗滌后滴加15μL FITC標(biāo)記的羊抗鼠Ig抗體(1∶256),37℃濕盒中孵育45 min;同上洗滌。甘油封片后熒光顯微鏡下觀察拍照。

1.4 血細(xì)胞感染程度檢測(cè)

人工感染后,每24 h從感染組中隨機(jī)取15只螯蝦,用裝有預(yù)冷提取液(抗凝劑:8%多聚甲醛=3∶1)的10 mL注射器抽取血淋巴[11],混合血淋巴后使用血球計(jì)數(shù)板顯微計(jì)數(shù),計(jì)算血細(xì)胞密度,每個(gè)樣本重復(fù)3次計(jì)數(shù),取平均值。同樣從陰性對(duì)照組中每24 h隨機(jī)取10尾螯蝦,抽取血淋巴作為對(duì)照。

上述血細(xì)胞樣品于4℃放置20 min后離心(600 r/min,15 min,4℃),用提取液重懸沉淀,調(diào)整細(xì)胞密度至5×106個(gè)/mL。取2 mL血細(xì)胞懸液(約1×107個(gè)血細(xì)胞)再次離心,沉淀用0.1%Triton-X100重懸,4℃通透10 min;PBS離心洗滌1次后用2%牛血清白蛋白(BSA)4℃封閉30 min;PBS離心洗滌3次后加入0.5 mL抗WSSV的混合單抗,4℃孵育45 min;PBS離心洗滌3次后加入0.5 mL FITC標(biāo)記的羊抗鼠Ig抗體(1∶256),4℃孵育45 min,同上洗滌,最后用PBS重懸血細(xì)胞,調(diào)整密度至1×106個(gè)/mL,使用Coulter EPICS XL流式細(xì)胞儀及軟件FCS Express3進(jìn)行檢測(cè)分析,以全部血細(xì)胞為對(duì)象(對(duì)主細(xì)胞群設(shè)門,圈入95%以上細(xì)胞)做流式FL1直方圖,以陰性對(duì)照螯蝦血細(xì)胞為對(duì)照,分析感染后各取樣時(shí)間點(diǎn)的病毒感染情況,包括被感染細(xì)胞百分率(Percent of gated cells)及平均熒光強(qiáng)度(Mean fluorescence intensity,MFI)[11]。

2 結(jié)果

2.1 螯蝦感染死亡情況及WSSV的感染檢測(cè)

人工感染1 d后螯蝦開始出現(xiàn)死亡,感染7 d后全部死亡;同時(shí),感染初期,螯蝦死亡較為緩慢,感染4 d后,螯蝦死亡率明顯加快。隨著感染時(shí)間延長(zhǎng),螯蝦累積死亡率分別為3.3%(1 d),13.3%(2 d),16.7%(3 d),36.7%(4 d),70%(5 d),90%(6 d),100%(7 d)(見圖1)。感染組的死亡螯蝦鰓經(jīng)免疫熒光檢測(cè),呈現(xiàn)點(diǎn)狀特異性綠色熒光,為WSSV感染陽性(見圖2A)。陰性對(duì)照及空白對(duì)照均有1尾死亡,經(jīng)間接免疫熒光檢測(cè)為WSSV感染陰性(見圖2B)。

圖1 螯蝦感染W(wǎng)SSV后累積死亡率的變化Fig.1 Variety of accumulative mortality of crayfish after WSSV infected

圖2 螯蝦鰓的WSSV間接免疫熒光檢測(cè)Fig.2 Immunofluorescence assay of WSSV infection of crayfish gill

2.2 血細(xì)胞的數(shù)量及感染強(qiáng)度變化

顯微計(jì)數(shù)觀察結(jié)果顯示,陰性對(duì)照組螯蝦的血細(xì)胞密度約為(5.34±0.22)×106個(gè)/mL,在感染W(wǎng)SSV后第1、2天,血細(xì)胞密度出現(xiàn)初始的升高,分別為(7.56±0.30)和(5.60±0.24)×106個(gè)/mL,然后從第3天起血細(xì)胞密度急劇下降,分別為(4.21±0.30),(1.45±0.26),(1.21±0.21)和(1.14±0.18)×106個(gè)/mL,感染后第6天血細(xì)胞密度僅為陰性對(duì)照組的21.3%(見圖3)。

圖3 螯蝦感染W(wǎng)SSV后血細(xì)胞密度的變化Fig.3 Variety of haemocyte indensity of crayfish infected with WSSV

流式檢測(cè)得出的直方圖顯示,螯蝦在感染W(wǎng)SSV后,被感染血細(xì)胞的比例在1～3 d內(nèi)增加緩慢,分別為:6.47%,6.93%和10.65%,3 d后急劇增多,在第4天達(dá)到高峰,感染率為26.08%,之后急劇降低,感染后第5天血細(xì)胞感染率僅為4.94%(見圖4,圖5)。

圖4 感染W(wǎng)SSV后螯蝦血細(xì)胞的流式檢測(cè)Fig.4 FCM analysis of haemocytes of WSSV infected crayfish

圖5 螯蝦感染W(wǎng)SSV后被感染血細(xì)胞比例的變化Fig.5 Variety of infected rate of haemocytes after WSSV infected

血細(xì)胞熒光信號(hào)的流式檢測(cè)數(shù)據(jù)顯示,被感染血細(xì)胞的平均熒光強(qiáng)度逐漸升高,感染后1～5 d分別為10.7,10.89,11.71,13.77,15.47,表明被感染細(xì)胞的病毒數(shù)量持續(xù)升高,且上升速度在3 d后加快(見圖6)。

圖6 被感染血細(xì)胞平均熒光強(qiáng)度的變化Fig.6 Variety of mean fluorescence intensity of the infected haemocytes

3 討論

人工感染W(wǎng)SSV后,隨時(shí)間延長(zhǎng)螯蝦的累積死亡率逐漸上升。為探明血細(xì)胞的病毒感染規(guī)律,本研究采取了以下措施來確保實(shí)驗(yàn)數(shù)據(jù)的準(zhǔn)確性:第一,取活螯蝦的血細(xì)胞進(jìn)行檢測(cè)分析;第二,保證足量的取樣基數(shù);第三,設(shè)立螯蝦感染觀察組,以與血細(xì)胞的病毒感染情況進(jìn)行比較。另外,感染6 d后螯蝦累積死亡率已達(dá)到90%,因而停止流式采樣與檢測(cè)。

已有多項(xiàng)研究表明,宿主感染W(wǎng)SSV后血細(xì)胞數(shù)量較感染前明顯降低[5-9];也有研究發(fā)現(xiàn)凡納濱對(duì)蝦及寬大太平螯蝦隨WSSV感染時(shí)間延長(zhǎng),血細(xì)胞數(shù)量呈現(xiàn)先升高后降低的趨勢(shì)[6,8]。本研究顯示,感染W(wǎng)SSV后第1天,克氏原螯蝦的血細(xì)胞數(shù)量較感染前出現(xiàn)明顯升高,之后開始降低。從第3天開始,下降速度明顯加快。其原因分析為:在WSSV感染克氏原螯蝦的初期,WSSV刺激造血組織加速產(chǎn)生血細(xì)胞,此時(shí)血細(xì)胞因病毒感染而產(chǎn)生的崩解滯后于血細(xì)胞的產(chǎn)生速度,使得血細(xì)胞密度高于感染前[6];隨著感染程度的加深,血細(xì)胞被感染率增加,WSSV在血細(xì)胞中高強(qiáng)度的增殖直接導(dǎo)致血細(xì)胞的崩解死亡,同時(shí),宿主為防御病毒而引發(fā)的細(xì)胞凋亡也是導(dǎo)致宿主血細(xì)胞數(shù)量急劇減少的主要原因[9]。另外,數(shù)據(jù)顯示,感染后期克氏原螯蝦的血細(xì)胞密度僅為陰性對(duì)照組的21.3%,而Sahul Hameed等的研究結(jié)果表明斑節(jié)對(duì)蝦和印度對(duì)蝦感染W(wǎng)SSV后,瀕死時(shí)血細(xì)胞的總數(shù)仍不低于未感染時(shí)總數(shù)的1/2[9],兩者數(shù)量變化的差異,推測(cè)是宿主和感染方式不同造成的。

本文結(jié)果顯示,感染W(wǎng)SSV后螯蝦被感染血細(xì)胞的比例在1～3 d內(nèi)緩慢增加,其后急劇增多、并在第4天時(shí)達(dá)到高峰后驟降。作者認(rèn)為,病毒對(duì)血細(xì)胞的感染率可直接反映宿主體內(nèi)病毒的增殖程度。本試驗(yàn)中,血細(xì)胞的最高感染比例約為26%,此時(shí)螯蝦的累積死亡率不到40%;之后螯蝦死亡率升高,但血細(xì)胞的病毒感染比例下降,當(dāng)螯蝦累積死亡率達(dá)到70%時(shí),血細(xì)胞感染率僅為4.94%。綜合螯蝦死亡率與血細(xì)胞感染率的變化可以推測(cè),血細(xì)胞感染比例達(dá)到26%后,WSSV在螯蝦體內(nèi)的增殖已接近其致死的臨界點(diǎn),因而此后螯蝦開始死亡,并且,由于被感染細(xì)胞的大量崩解死亡,血細(xì)胞感染率隨之急劇降低。本文采用流式細(xì)胞儀檢測(cè)的樣品均為活螯蝦的血細(xì)胞、并以完整細(xì)胞為對(duì)象,實(shí)驗(yàn)結(jié)果表明血細(xì)胞被WSSV感染的程度與螯蝦死亡密切相關(guān)。

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Infection Process of WSSV in Haemocytes of Procambarus clarkii

WANG Yi-Nan,ZHAN Wen-Bin,TANG Xiao-Qian,XINGJing
(The Key Laboratory of Mariculture,Ministry of Education,Ocean University of China,Qingdao 266003,China)

Procambarus clarkiiwere injected with WSSV and then haemocytes were collected every 24 h.After reacted with WSSV specific monoclonal antibodies and secondary antibody conjugated with FITC,the haemocytes were detected for WSSV infection using a flow cytometer.Meanwhile,the accumulative mortalities and the haemocyte densities were also recorded.Results showed that after WSSV infection,the accumulative mortalities ofProcambarus clarkiiwere 3.3%,13.3%,16.7%,36.7%and 70.0%,respectively,from day 1 to day 7;the infected rates of haemocyte were 6.47%,6.93%,10.65%,26.08%and 4.94%,respectively,from day 1 to day 5;the mean fluorescence intensities of infected haemocytes were 10.7,10.89,11.71,13.77 and 15.47,respectively,from day 1 to day 5;the haemocyte densities were(7.56±0.30),(5.60±0.24),(4.21±0.30),(1.45±0.26),(1.21±0.21)and(1.14±0.18)×106cells/mL,respectively,from day 1 to day 6,while the haemocyte density of negative control was(5.34±0.22)×106cells/mL.The results indicated that afterProcambarus clarkiiinfected with WSSV,the density of haemocytes in haemolymph rose at first and then declined,and till day 6 the value was only 21.3%of that in control;and the WSSV amount in the infected haemocytes kept rising;the infected rate of haemocyte increased to the highest value whenProcambarus clarkiiwent into moribund stage,which revealed that the WSSV infection degree of haemocytes inProcambarus clarkiihas a close relationship with its mortality.

Procambarus clarkii;haemocytes;WSSV

S917

A

1672-5174(2010)09-056-05

國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃項(xiàng)目(2006CB101806);國(guó)家高技術(shù)研究發(fā)展計(jì)劃項(xiàng)目(2006AA100312)資助

2009-11-27;

2010-04-26

王軼南(1980-),女,助理研究員,主要從事水產(chǎn)動(dòng)物病害與免疫學(xué)研究,現(xiàn)于大連海洋大學(xué)工作。E-mail:wisteriayn@gmail.com

**通訊作者:E-mail:wbzhan@ouc.edu.cn

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