摘要
血细胞是贝类生物细胞免疫的主要执行者,是抵御外来病原侵袭和环境刺激的主要“屏障”。血细胞数量及胞内酶活的变动可以作为一个用于指示扇贝受病原感染或环境胁迫的免疫指标。本论文研究了栉孔扇贝(Chlamys farreri)经病毒感染和多糖刺激后血细胞数量及胞内酶活的变化规律;分类、分离了栉孔扇贝各种类型血细胞,以此为抗原研制出了抗各种类型血细胞的单克隆抗体;利用单抗制备了检测扇贝血细胞的酶联免疫(ELISA)试剂盒;应用试剂盒监测了扇贝在自然生长及胁迫环境下的血细胞变化;旨在为评估扇贝的健康状况提供手段和理论依据。本论文主要包括以下5部分:
1栉孔扇贝经病毒感染后的血细胞变化用不同浓度(5~0、5~(-1)、5~(-2)、5~(-3)、5~(-4)、5~(-5)、5~(-6)和5~(-7))的栉孔扇贝急性病毒性坏死病毒(AVNV),25℃(病发温度)人工感染扇贝,观察感染后各浓度组的存活率,确定了5~(-3)、5~(-4)和5~(-5)为较适宜的感染浓度。分别以5~(-3)和5~(-5)病毒浓度人工感染扇贝,感染后每天测定血细胞总数(THC)、血细胞内酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、超氧化物歧化酶(SOD)、酚氧化酶(PO)、髓过氧化物酶(MPO)和过氧化物酶(POD)的变化,共测定15天。结果显示:感染后前8、9天,THC显著降低,而ACP、SOD、PO、MPO显著升高,POD无明显规律,ALP未被检测到;感染10天后,各免疫指标逐渐恢复到对照值。此外,5~(-3)浓度对各免疫指标的影响幅度要大于5~(-5)浓度。结果表明:血细胞在应激病毒感染过程中起着关键作用。
2栉孔扇贝经β-葡聚糖刺激后的血细胞变化分别在15℃(最适温度)和25℃对栉孔扇贝注射0.5、1.0、2.0 mg/ml的β-葡聚糖,注射后每12小时测定THC和胞内ACP、ALP、SOD、PO、MPO和POD的变化,共测定7天。结果显示:15℃时,THC在0.5、1.0、2.0 mg/ml组均显著升高;而PO在0.5、1.0、2.0 mg/ml组,ACP,SOD和POD在1.0和2.0 mg/ml组,MPO和ALP分别在1.0和2.0 mg/ml组被显著激活。25℃时,THC只在1.0 mg/ml组显著升高;PO在0.5和1.0 mg/ml组,ACP在1.0和2.0 mg/ml组,SOD在1.0 mg/ml组被显著激活,而POD,MPO和ALP则未被激活或未被检测到。此外,THC、PO、ACP和SOD在25℃时的提升幅度和持续时间不及15℃。结果表明:血细胞在应激高温和β-葡聚糖刺激过程中起着关键作用;而高温能削弱扇贝识别和结合外源刺激物的能力,从而增加了被侵染的机率。
3抗各种类型血细胞的单克隆抗体的研制用Percoll不连续密度梯度(10、20、30、40和50%)离心法分离了栉孔扇贝全血细胞,吉姆萨染色和流式细胞术鉴定了各层血细胞的成分,确定了30-40%界面层为透明细胞,而40-5~0%界面层为颗粒细胞。分别收集透明细胞和颗粒细胞免疫小鼠,细胞融合,间接免疫荧光法(IIFA)筛选,克隆,所得单抗再经流式免疫荧光法(FCIFA)和Western blotting法(WBA)特性分析。结果显示:共筛选克隆得到7株既抗透明细胞和又抗颗粒细胞的单抗1F7,4D5,5C6,4G11,6A6,2H11,4E2,和1株只抗颗粒细胞的单抗6H7,没有得到只抗透明细胞的单抗。单抗1F7的阳性率为82±2.4%,其中阳性透明细胞(PH)占34±2.2%,阳性颗粒细胞(PG)占48±3.1%;它抗原决定簇丰富,能与多个血细胞蛋白结合。单抗4D5的阳性率为76±2.3%,其中PH占37±2.4%, PG占39±1.1%;它能与分子量为16.7,87和96 kDa的血细胞蛋白结合。单抗5C6的阳性率为70±2.6%,其中PH占32±1.9%,PG占38±2.4%;它能与分子量为16.7 kDa的血细胞蛋白结合。单抗6H7的阳性率为62±2.5%,其中PG占54±3.0%,PH仅占8±2.3%,它能与分子量为155 kDa的血细胞蛋白结合。
4扇贝血细胞酶联免疫试剂盒的研制用IIFA,FCIFA和WBA从血细胞单抗库中筛选单抗作为栉孔扇贝血细胞酶联免疫试剂盒的第一抗体。梯度稀释抗原抗体,ELISA棋盘滴定法确定抗原抗体的最佳使用稀释度。高温(25℃)和病毒(5~(-4)AVNV)刺激栉孔扇贝,ELISA检测各刺激条件下的血细胞变化,以常温和高温刺激的比较结果制定扇贝正常状态与受环境胁迫的临界范围,以未感染和病毒感染的比较结果制定扇贝受环境胁迫与病原感染的临界范围,建立评估标准。最后组装制备试剂盒。结果显示:单抗1F7具阳性率高,抗透明细胞和颗粒细胞,及抗原决定簇丰富的特性被确定为试剂盒的第一抗体。抗原和抗体的最佳使用稀释度为抗原稀释2倍,抗体稀释4倍。待检样品OD_(405nm)值>0.57±0.025,属体质正常范围;待检样品OD_(405nm)值<0.47±0.019,属病原感染范围;两者之间为环境胁迫范围。试剂盒包括酶标板,封闭液,单抗1F7,碱性磷酸酶(AP)标记的羊抗鼠抗体,显色液,栉孔扇贝血细胞标准样品及检测结果评估标准等,其灵敏度可达200 ng/ml。
IIFA分析了抗栉孔扇贝颗粒细胞的单抗6H7与虾夷扇贝(Patinopecten yessoensis)、海湾扇贝(Argopecten irradians)颗粒细胞的交叉性,确定了单抗6H7能与这两种扇贝的颗粒细胞特异性结合,但不与透明细胞结合。将单抗6H7选作为扇贝颗粒细胞酶联免疫试剂盒的第一抗体。分别梯度稀释栉孔扇贝、虾夷扇贝、海湾扇贝抗原,梯度稀释单抗6H7,ELISA棋盘滴定法确定抗原抗体的最佳使用稀释度。最后组装制备试剂盒。结果显示:不经稀释的抗原抗体具最佳的结合效果。试剂盒包括酶标板,封闭液,单抗6H7,AP标记的羊抗鼠抗体,显色液,三种扇贝血细胞标准样品等,其灵敏度可达5μg/ml。
5扇贝血细胞酶联免疫试剂盒的应用于2009年3月至2010年1月,每月中旬从山东地区采集栉孔扇贝,测完壳长后,取血,用试剂盒研究栉孔扇贝血细胞变化与生长的关系。用5~(-4)AVNV人工感染栉孔扇贝,感染后用试剂盒每天测定颗粒细胞的变化,共测定9天。将海湾扇贝分成两组:一组每天实施投喂,另一组则不投喂,用试剂盒每周测定两组扇贝颗粒细胞的变化,共测定5周。结果显示:4,5,6月栉孔扇贝生长较快,对应的血细胞数量较多;8,9,10月生长缓慢,对应的血细胞数量则少。病毒感染后第1天,栉孔扇贝颗粒细胞数显著升高;随后急剧下降,于第3天达到最低值;之后开始回升,于第6、7天又显著升高;最后恢复到对照值。饥饿胁迫后,海湾扇贝颗粒细胞数前3周呈依次显著递减的趋势,3周后基本不再下降;投喂组的颗粒细胞数5周内基本维持同一水平。测定结果表明本论文研制的试剂盒具较好的应用效果。
Bivalves frequently exposed to environmental stress and invasive or pathogenic microbes, and their survival depends to a large extent on the circulating cells collectively known as haemocytes. Haemocyte count and intracellular enzyme activity can be directly or indirectly used to evaluate the physiological state of a host or to estimate its immunity and potential defence capability against disease agents. In this dissertation, variations in haemocytes of scallop Chlamys farreri after viral and polysaccharidal stimulations were investigated. Then, C. farreri haemocytes were classified and separated. Subsequently, monoclonal antibodies (MAbs) against different types of C. farreri haemocytes were produced using the separated haemocytes as antigens. Afterwards, enzyme-linked immunosorbent assay (ELISA) kits for detection scallop haemocytes were prepared by employing these MAbs as primary antibodies. Finally, variations in scallop haemocytes in natural or stressed conditions were detected by ELISA kits. The main aim of this dissertation was to provide promising tool and theoretical basis for the assessment of health conditions of cultured scallops. This dissertation included the following 5 parts:
1 Variation in haemocytes of C. farreri after a viral infection
Different concentrations (5~0, 5~(-1), 5~(-2), 5~(-3), 5~(-4), 5~(-5), 5~(-6) and 5~(-7)) of acute viral necrobiotic virus (AVNV) were used to infect scallops C. farreri at 25°C. After infection, survival rates in each concentrations were counted, after that, among them, three concentrations of 5~(-3), 5~(-4) and 5~(-5) were picked out as the desired infection concentrations. Subsequently, AVNV concentrations of 5~(-3) and 5~(-5) were used to infect scallops C. farreri at 25°C, and total haemocyte count (THC) and activities of acid phosphatase (ACP), alkaline phosphatase (ALP), superoxide dismutase (SOD), myeloperoxidase (MPO), phenoloxidase (PO) and peroxidase (POD) in haemocytes were measured every day till 15 days, respectively. The results showed that THC in viral groups was significantly lower than that in control group, while activities of ACP, SOD, MPO, PO were significantly higher than control group in the first 8 or 9 days, and went back to control group gradually after the 10~(th) day. Moreover, these five immune parameters in group of 5~(-3) varied more than that in group of 5~(-5). Differently, POD activity was reduced sometimes or induced other times, and showed no regular trends, while ALP activity wasn’t detected. The results clearly illustrated that THC and intracellular enzyme activities varied greatly after a viral infection, validating that haemocytes play a crucial role in the process of C. farreri against AVNV invasion.
2 Variation in haemocytes of C. farreri after a polysaccharidal stimulation
Scallops C. farreri were treated with 0.5, 1.0 or 2.0 mg/ml ofβ-glucan suspensions at 15°C (the optimum temperature for C. farreri) or 25°C (the temperature at which the disease rate is elevated), then THC, and the activities of PO, ACP, SOD, POD and ALP were measured in haemocytes every 12 h for seven days. The results showed that at 15°C, compared to the control, THC and PO in 0.5, 1.0 and 2.0 mg/ml, ACP, SOD and POD in 1.0 and 2.0 mg/ml, MPO and ALP respectively in 1.0 and 2.0 mg/mlβ-glucan were significantly increased. While at 25°C, THC and SOD in 1.0 mg/ml, PO in 0.5 and 1.0 mg/ml, ACP in 1.0 and 2.0 mg/mlβ-glucan were significantly induced, however, no significant induction of POD or MPO activity was observed, surprisingly, ALP activity was undetectable at 25°C throughout the entire experimental period. Moreover, the extent and duration of inducement as well as the time of peak emergence for these seven immune parameters appeared higher, longer and earlier at 15°C than 25°C in allβ-glucan treatments. These results clearly illustrated that THC and intracellular enzyme activities varied greatly, validating that haemocytes also play a crucial role in the process of C. farreri againstβ-glucan and high water temperature stresses, and high temperature may weaken or delay immune reactions to exogenous stress, thereby increasing the probability of being infected.
3 Production of MAbs against different types of C. farreri haemocytes
Scallop C. farreri haemocytes were separated by Percoll discontinuous density gradient (10, 20, 30, 40 and 50%) centrifugation, and the haemocytes at each resulting interface were collected and identified using flow cytometry and Giemsa staining, finding 30-40% interface of hyalinocytes, and 40-50% of granulocytes. Subsequently, separated hyalinocytes and granulocytes were respectively used as antigens to immunise mice. After cell fusion, screen using an indirect immunofluorescence assay (IIFA) and clone, the resulting MAbs were characterised using a flow cytometric immunofluorescence assay (FCIFA) and western blotting assay (WBA), respectively. The results showed that more than fifty hybridomas secreting positive antibodies were obtained, however, most of the antibodies showed specificities for both hyalinocytes and granulocytes, such as MAb 1F7, 4D5, 5C6, 4G11, 6A6, 2H11 and 4E2, among them one MAb designated as 6H7 presented a specificity only for granulocytes, and no MAbs showed a specificity only for hyalinocytes. MAb 1F7 had a positive rate of 82±2.4%, among them, positive hyalinocytes (PH) of 34±2.2%, positive granulocytes of 48±3.1%, it had abundant epitopes, reacted with many haemocyte proteins. MAb 4D5 was 76±2.3%, PH of 37±2.4%, PG of 39±1.1%, it reacted with three haemocyte proteins of 16.7, 87 and 96 kDa. MAb 5C6 was 70±2.6%, PH of 32±1.9%, PG of 38±2.4%, it reacted with 16.7 kDa haemocyte protein. While MAb 6H7 was 62±2.5%, PG of 54±3.0%, PH of 8±2.3%, it reacted with 155 kDa haemocyte protein.
4 Preparation of ELISA kits for detection scallop haemocytes
Specific MAb suitable for primary antibody of ELISA kit for detection C. farreri haemocytes was firstly screened out using IIFA, FCIFA and WBA. Then the optimal using dilution was found out using ELISA chessboard titration by respective gradient dilution for antigen and screened MAb. Afterward, scallops C. farreri were stimulated by high water temperature (25°C) and 5~(-4)AVNV, respectively, and haemocyte variation in challenged scallops was detected by ELISA. Subsequently, an evaluation criteria was completed by finding a critical range between normal physical and environmental stress from the ELISA data comparison of normal temperature and high temperature group, a critical range between environmental stress and pathogenic infection from the ELISA data comparison of non-infected and infected groups. Finally, ELISA kit was assembled and prepared. The results showed that MAb 1F7 with a high positive rate, abundant epitopes, against both hyalinocytes and granulocytes, was suitable for primary antibody of ELISA kit for detection C. farreri haemocytes. The optimal using dilution of antigen and antibody was that antigen diluted two times, and antibody diluted four times. The critical range between normal physical and environmental stress was 0.57±0.025, between environmental stress and pathogenic infection was 0.47±0.019. This ELISA kit contained ELISA plate, blocking solution, MAb 1F7, alkaline phosphatase labeled goat anti-mouse antibody (AP-GMA), colour developmental solution, standard samples and evaluation criteria, etc., with a sensitivity of 200 ng/ml.
Granulocyte antigenic cross-reactivity of other two scallop species Patinopecten yessoensis and Argoecten irradians was carried out using MAb 6H7 against C. farreri granulocytes, illustrating MAb 6H7 also specifically reacted with the granulocytes of P. yessoensis and A. irradians, not with hyalinocytes, therefore, MAb 6H7 was chosen as primary antibody of ELISA kit for detection scallop granulocytes. After that, the optimal using dilution was found out using ELISA chessboard titration by respective gradient dilution for three antigens and MAb 6H7. Finally, ELISA kit was assembled and prepared. The results showed that not diluted antigen and antibody had a best reaction. This ELISA kit contained ELISA plate, blocking solution, MAb 6H7, AP-GMA, colour developmental solution and standard samples of three scallop species, etc., with a sensitivity of 5μg/ml.
5 Application of ELISA kits for detection scallop haemocytes
Scallops C. farreri were collected monthly from Shandong area during the period from March 2009 to January 2010. After measurement of shell height, a relationship between haemocyte count and growth was investigated using ELISA kit. Scallops C. farreri were infected with 5~(-4) AVNV, and granulocyte count was measured using ELISA kit every day for 9 days. Scallops A. irradians were divided into two groups: one group fed every day, the other one not fed, afterward, granulocyte count in two group was measured using ELISA kit every week for 5 weeks. The results showed that in April, May and June, C. farreri grew fast, correspondingly, in this period, C. farreri had a large number of haemocytes, while in August, September and October, C. farreri grew slowly, with a small number of haemocytes. After AVNV infection, C. farreri granulocytes count significantly increased on day 1, then decreased on days 2, 3 and 4, thereafter, rebounded and approached to a second peak on day 6, finally went down gradually to the control level on day 8. After starvation stress, A. irradians granulocyte count significantly decreased in first 3 weeks, thereafter, decreased slightly, basically kept at a stable level, while in fed group, granulocyte count was maintained at the same level within 5 weeks. Conclusively, the results of these applications showed that ELISA kits developed in this dissertation had a promising prospect.
引文
[1] Ai QH, Mai KS, Zhang L, et al. Effects of dietaryβ-1, 3-glucan on innate immune response of large yellow croaker, Pseudosciaena crocea. Fish Shellfish Immunol, 2007, 22:394–402.
[2] Aladaileh S, Nair, SV, Birch D, et al. Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function. J Invertebr Pathol, 2007, 96:48–63.
[3] Allam B, Ford SE. Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: I. Haemocyte changes following in vitro challenge. Fish Shellfish Immunol, 2006, 20:374–383.
[4] Andrews PC, Krinsky N. Quantitative determination of myeloperoxidase using tetramethyl -benzidine as substrate. Anal Biochem, 1982, 127:346–350.
[5] Armstrong PB, Rossner MT, Quigley JP. Anα2-macroglobulinlike activity in the blood of chelicerate and mandibulate arthropods. J Exp Zool, 1985, 236:1–9.
[6] Ashida M, S(o|¨)derh(a|¨)ll K. The prophenoloxidase activating system in crayfish. Comp Biochem Physiol, 1984, 77:21–26.
[7] Auffret M. Bivalve hemocyte morphology. Am Fish Soc Spec Publ, 1988, 18:169–177.
[8] Bachère, E, Mialhe E, Noёl D, et al. Knowledge and research prospects in marine mollusc and crustacean immunology. Aquaculture, 1995, 132:17–32.
[9] Bayne CJ. Phagocytosis and non-self recognition in invertebrates. Phagocytosis appears to be an ancient line of defense. Bioscience, 1990, 40:723–731.
[10] Bayne CJ, Moore MN, Carefoot TH, et al. Hemolymph functions in Mytilus californianus: the cytochemistry of hemocytes and their responses to foreign implants and hemolymph factors in phagocytosis. J Invertebr Pathol, 1979, 34:1–22.
[11] Boyd JN, Burnett LE. Reactive oxygen intermediate production by oyster hemocytes exposed to hypoxia. J Exp Biol, 1999, 202:3135–3143.
[12] Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye binding. Anal Biochem, 1976, 72:248–254.
[13] Bubel A, Moore MN, Lowe D. Cellular responses to shell damage in Mytilus edulis L. J Exp Mar Biol Ecol, 1977, 30:1–27.
[14] Carballal MJ, Villalba A, López C. Seasonal variation and effects of age, food availability, size, gonadal development, and parasitism on the hemogram of Mytilus galloprovincialis. J Invertebr Pathol, 1998, 72:304–312.
[15] Chang CF, Su MS, Chen HY, et al. Dietaryβ-1,3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus. Fish Shellfish Immunol, 2003, 15:297–310.
[16] Chen MY, Yang HS, Delaporte M, et al. Immune condition of Chlamys farreri in response to acute temperature challenge. Aquaculture, 2007a, 271:479–487.
[17] Chen MY, Yang HS, Delaporte M, et al. Immune responses of the scallop Chlamys farreri after air exposure to different temperatures. J Exp Mar Biol Ecol, 2007b, 345:52–60.
[18] Cheney DPA. A summary of invertebrate leucocyte morphology with emphasis on blood elements of the Manila clam, Tapes semidecussata. Biol Bull, 1971, 140:353–368.
[19] Cheng TC. A classification of molluscan hemocytes based on functional evidences. Comp Pathobiol, 1984, 6:111–146.
[20] Cheng TC. Bivalves. In: N. A. Ratcliffe and A. F. Rowley, Editors, Invertebrate Blood Cells, Academic Press, London, 1981, pp. 233–299.
[21] Cheng TC. Hemocytes: forms and functions. In: Kennedy VS, Newell RIE, Eble AF, editors. The Eastern Oyster Crassostrea virginica. Maryland Sea Grant: College Park, MD, USA; 1996, pp. 299–333.
[22] Cheng TC. The role of lysosmal hydrolases in molluscan cellular response to immunologic challenge. Comp Pathbio1, 1978, 4:59–71.
[23] Chu F-LE. Defence mechanisms in marine bivalves. In: Fingerman M, Nagabhushanam R, editors. Recent Advances in Marine Biotechnology. Immunobiology and Pathology vol. 5, Science Publishers, Enfield, NH, Plymouth, UK, 2000, pp. 1–42.
[24] Dalmo RA, B(?)gwald J.β-glucans as conductors of immune symphonies. Fish Shellfish Immunol, 2008, 25:384–396.
[25] Das BK, Debnath C, Patnaik P, et al. Effect ofβ-glucan on immunity and survival of early stage of Anabas testudineus (Bloch). Fish Shellfish Immunol, 2009, 27:678–683.
[26] Donaghy L, Kim BK, Hong HK, et al. Flow cytometry studies on the populations and immune parameters of the hemocytes of the Suminoe oyster, Crassostrea ariakensis. Fish Shellfish Immunol, 2009, 27:296–301.
[27] Duchemin MB, Fournier M, Auffret M. Seasonal variations of immune parameters in diploid and triploid Pacific oysters, Crassostrea gigas (Thunberg). Aquaculture, 2007, 264:73–81.
[28] Dyrynda EA, Pipe RK, Ratcliffe NA. Sub-populations of haemocytes in the adult and developing mussel, Mytilus edulis, identified by monoclonal antibodies. Cell Tissue Res, 1997, 289:527–536.
[29] Feng SY. Cellular defense mechanisms of oysters and mussels. Am Fish Soc Spec Public, 1988, 18:153–168.
[30] Feng SY. Pinocytosis of proteins by oyster leucocytes. Biol Bull, 1965, 129:95–105.
[31] Feng SY, Feng JS. The effect of temperature on cellular reactions of Crassostrea virginica to the injection of avian erythrocytes. J Invertebr Pathol, 1974, 23:22–37.
[32] Foley DA, Cheng TC. Degranulation and other changes of molluscan granulocytes associated with phagocytosis. J Invertebr Pathol, 1977, 29: 321–325.
[33] Ford SE, Ashton-Alcox AK, Kanaley SA. Comparative cytometric and microscopic analyses of oyster hemocytes. J Invertebr Pathol, 1994, 64:114–122.
[34] Franchini A, Ottaviani E. Repair of molluscan tissue injury: role of PDGF and TGF-b. Tissue Cell, 2000, 32:312–321.
[35] Fu CL, Song WB, Li Y. Monoclonal antibodies developed for detection of an epizootic virus associated with mass mortalities of cultured scallop Chlamys farreri. Dis Aquat Org, 2005, 65:17–22.
[36] González F, Esther Fárez-Vidal M, Arias JM, et al. Partial purification and biochemical properties of acid and alkaline phosphatases from Myxococcus coralloides D. J Appl Microbiol, 1994, 77:567–573.
[37] Hine PM. The inter-relationships of bivalve haemocytes. Fish Shellfish Immunol, 1999, 9:367–385.
[38] Holmblad T, S(o|¨)derh(a|¨)ll K. Cell adhesion molecules and antioxidative enzymes in a crustacean, possible role in immunity. Aquaculture, 1999, 172:111–123.
[39] Janeway CA. The role of microbial pattern recognition in self:nonself discrimination in innate and adaptive immunity. In: Hoffmann, J.A., Janeway, C.A., Natori, S. (Eds.), Phylogenetic Perspectives in Immunity, the Insect Host Defence. RG Landes, Austin, TX, 1994, pp. 115–122.
[40] Jayaraj SS, Thiagarajan R, Arumugam M, et al. Isolation, purification and characterization ofβ-1,3-glucan binding protein from the plasma of marine mussel Perna viridis. Fish Shellfish Immunol, 2008, 24:715–725.
[41] Jiravanichpaisal P, Lee BL, S(o|¨)derh(a|¨)ll K. Cell-mediated immunity in arthropods: Hematopoiesis, coagulation, melanization and opsonization. Immunobiology, 2006, 211:213–236.
[42] Jiravanichpaisal P, S(o|¨)derh(a|¨)ll K, S(o|¨)derh(a|¨)ll I. Effect of water temperature on the immune response and infectivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish. Fish Shellfish Immunol, 2004, 7:265–275.
[43] J(?)rgensen JB, Robertson B. Yeastβ-glucan stimulates respiratory burst activity of Atlantic Salmon (Salmo salar L.) macrophages. Dev Comp Immunol, 1995, 19:43–57.
[44] Lemaire N, Pellerin J, Fournier M, et al. Seasonal variations of physiological parameters inthe blue mussel mytilus spp. from farm sites of eastern Quebec. Aquaculture, 2006, 261:729–751.
[45] Ling E, Yu XQ. Prophenoloxidase binds to the surface of haemocytes and is involved in haemocyte melanization in Manduca sexta. Insect Biochem Mol Biol, 2005, 35:1356–1366.
[46] Lopez C, Carballal MJ, Azevedo C, et al. Differential phagocytic ability of the circulating haemocyte types of the carpet shell clam Ruditapes decussatus (Mollusca: Bivalvia). Dis Aquat Org, 1997, 30:209–215.
[47] Mar Costa M, Novoa B, Figueras A. Influence ofβ-glucans on the immune responses of carpet shell clam (Ruditapes decussatus) and Mediterranean mussel (Mytilus galloprovincialis). Fish Shellfish Immunol, 2008, 24:498–505.
[48] Martello LB, Friedman CS, Tjeerdema RS. Combined effects of pentachlorophenol and salinity stress on phagocytic and chemotactic function in two species of abalone. Aquat Toxicol, 2000, 49:213–225.
[49] Matozzo V, Marin MG, Cima F, et al. First evidence of cell division in circulating haemocytes from the Manila clam Tapes philippinarum. Cell Biol Int, 2008, 32:865–868.
[50] Matozzo V, Monari M, Foschi J, et al. Exposure to anoxia of the clam Chamelea gallina. I: Effects on immune responses. J Exp Mar Biol Ecol, 2005, 325:163–174.
[51] Matozzo V, Rova G, Marin MG. Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses. Fish Shellfish Immunol, 2007, 23:732–746.
[52] Misra CK, Das BK, Mukherjee SC, et al. Effect of long term administration of dietaryβ-glucan on immunity, growth and survival of Labeo rohita fingerlings. Aquaculture, 2006, 255:82–94.
[53] Misra CK, Das BK, Pradhan J, et al. Changes in lysosomal enzyme activity and protection against Vibrio infection in Macrobrachium rosenbergii (De Man) post larvae after bath immunostimulation withβ-glucan. Fish Shellfish Immunol, 2004, 17:389–395.
[54] Mix MC. A general model for leukocyte cell renewal in bivalve mollusks. Mar Fish Bull, 1976, 38:37–41.
[55] Moore MN, Lowe DM. The cytology and cytochemistry of the hemocytes of Mytilus edulis and their response to experimentally injected carbon particles. J Invertebr Pathol, 1977, 29:18–30.
[56] Mount AS, Wheeler AP, Paradkar RP, et al. Hemocyte-mediated shell mineralization in the eastern oyster. Science, 2004, 304:297–300.
[57] Mu(n|~)oz P, Meseguer J,ángeles Esteban M. Phenoloxidase activity in three commercialbivalve species. Changes due to natural infestation with Perkinsus atlanticus. Fish Shellfish Immunol, 2006, 20:12–19.
[58] Noёl D, Pipe R, Elston R, et al. Antigenic characterization of hemocyte subpopulations in the mussel Mytilus edulis by means of monoclonal antibodies. Mar Biol, 1994, 119:549–556.
[59] Ottaviani E, Franchini A, Cassanelli S, et al. Cytokines and invertebrate immune responses. Biol Cell, 1995, 85:87–91.
[60] Pipe RK. Generation of reactive oxygen metabolites by the haemocytes of the mussel Mytilus edulis. Dev Comp Immunol, 1992, 16:111–122.
[61] Pipe RK, Coles JA. Environmental contaminants influencing immune function in marine bivalve molluscs. Fish Shellfish Immunol, 1995, 5:581–595.
[62] Robertsen B, Engstad RE, J(?)gensen JB.β-Glucans as immunostimulants in fish. In: Stolen, J.S., Fletcher, T.C. (Eds.), Modulators of Fish Immune Responses, Models for Environmental Toxicology, Biomarkers, and Immunostimulators. SOS Publications, Fair Haven, 1994, pp. 83–99.
[63] Ruddell CL. Elucidation of the nature and function of the granular oyster amebocytes through histochemical studies of normal and traumatized oyster tissues. Histochemie, 1971, 26:98–112.
[64] Russell-Pinto F, Reim(a|~)o R, De Sousa M. Haemocytes in Cerastoderma edule (Mollusca, Bivalvia): distinct cell types engage in different responses to sheep erythrocytes. Fish Shellfish Immunol, 1994, 4:383-397.
[65] Schlenk D, Martinez PG, Livingstone DR. Studies on myeloperoxidase activity in the common mussel, Mytilus edulis L. Comp Biochem Physiol C, 1991, 99:63–68.
[66] Seki N, Muta T, Oda T, et al. Horseshoe crab (1,3)-beta-D-glucan-sensititive coagulation factor G. A serine protease zymogen heterodimer with similarities toβ-glucan-binding proteins. J Biol Chem, 1994, 269:1370–1374.
[67] Smolowitz RM, Miosky D, Reinisch CL. Ontogeny of leukemic cells of the soft shell clam. J Invert Pathol, 1989, 53:41–51.
[68] S(o|¨)derh(a|¨)ll K, Cerenius L. Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol, 1998, 10:23–28.
[69] S(o|¨)derh(a|¨)ll K. Fungal cell wall beta-1,3-glucans induce clotting and phenoloxidase attachment to foreign surfaces of crayfish hemocyte lysate. Dev Comp Immunol, 1981, 5:565–573.
[70] Song SJ. The Clinician's Handbook. Shanghai Scientific and Technical Publishers, 1991,(185 pp. in Chinese)
[71] Sparks AK. Inflammation and wound repair in bivalve mollusks. Am Fish Soc Spec Public, 1988, 18:139–152.
[72] Stefano GB, Digenis A, Spector S, et al. Opiate-like substances in an invertebrate, an opiate receptor on invertebrate and human immunocytes, and a role in immunosuppression. Proc Natl Acad Sci US A, 1993, 90:11099–11103.
[73] Stefano GB, Smith EM, Hughes TK. Opioid induction of immunoreactive intedeukin-1 in Mytilus edulis and human immunocytes: an interleukin-1-like substance in invertebrate neural tissue. J Neuroimmunol, 1991, 32:29–34.
[74] Su JG, Song, LS, Xu W, et al. cDNA cloning and mRNA expression of the lipo -polysaccharide and beta-1,3-glucan-binding protein gene from scallop Chlamys farreri. Aquaculture, 2004, 239:69–80.
[75] Sung HH, Chang HJ, Her CH, et al. Phenoloxidase activity of hemocytes derived from Penaeus monodon and Macrobrachium rosenbergii. J Invertebr Pathol, 1998, 71:26–33.
[76] Sung HH, Kou GH, Song YL. Vibriosis resistance induced by glucan treatment in tiger shrimp (Panaeus monodon). Fish Pathol, 1994, 29:11–17.
[77] Suzuki T. Functions of hemocytes during the wound healing process in the pearl oyster. NOAA Technical Report NMFS, 1991, 111:109–112.
[78] Tang BJ, Liu BZ, Wang XM, et al. Physiological and immune responses of zhikong scallop Chlamys farreri to the acute viral necrobiotic virus infection. Fish Shellfish Immunol, 2010, 29:42–48.
[79] TirapéA, Bacque C, Brizard R, et al. Expression of immune-related genes in the oyster Crassostrea gigas during ontogenesis. Dev Comp Immunol, 2007, 31:859–873.
[80] Vargas-Albores F, Yepiz-Plascencia G. Beta glucan binding protein and its role in shrimp immune response. Aquaculture, 2000, 191:13–21.
[81] Vetvicka V, Yvin JC. Effects of marineβ-1,3 glucan on immune reactions. Int Immuno -pharm, 2004, 4:721–730.
[82] Xiao J, Ford SE, Yang HS, et al. Studies on mass summer mortality of cultured zhikong scallops (Chlamys farreri Jones et Preston) in China. Aquaculture, 2005, 250:602–615.
[83] Xing J, Zhan WB. Hemocyte type of Chinese scallop Chlamys farreri. Am Fish Soc Symp, 2003, 38:301–307.
[84] Xing J, Zhan WB, Zhou L. Endoenzyme associated with haemocyte types in the scallop (Chlamys farreri). Fish Shellfish Immunol, 2002, 13:271–278.
[85] Xu B, Chen M Y, Yang H S, et al. Starvation-induced changes of hemocyte parameters inthe zhikong scallop Chlamys farreri. J Shellfish Res, 2008, 27(5):1195–1200.
[86] Xue Q, Renault T. Monoclonal antibodies to European flat oyster Ostrea edulis hemocytes: Characterization and tissue distribution of granulocytes in adult and developing animals. Dev Comp Immunol, 2001, 25:187–194.
[87] Yu JH, Song JH, Choi MC, et al. Effects of water temperature change on immune function in surf clams, Mactra veneriformis (Bivalvia: Mactridae). J Invertebr Pathol, 2009, 102:30–35.
[88] Zhang WZ, Wu XZ, Wang M. Morphological, structural, and functional characterization of the haemocytes of the scallop, Argopecten irradians. Aquaculture, 2006, 251:19–32.
[89]艾海新,王崇明,王秀华,等.栉孔扇贝急性病毒性坏死症病原人工感染研究.中国水产科学, 2003,10(5):386–391.
[90]陈全震,杨俊毅,王小谷,等.皱纹盘鲍血细胞的亚显微结构及分类研究.水产学报,2001,25(6):492–495.
[91]陈茹,林志雄,刘琳琳,等.用地高辛标记的PCR-ELISA技术快速检测转基因鱼.中国水产科学,2001,8(4):13–16.
[92]陈蜀娜,石安静.河蚌外套膜的组织化学定位及运输途径的研究.四川大学学报(自然科学版),1994,31:175–178.
[93]陈松林,邓文涛.用酶联免疫吸附受体法检测鱼类生长激素的生物活性.水产学报,1995,19( 3) :217–224.
[94]董玉华,刘仁沿,许道艳,等.酶联免疫吸附方法分析海水和贝类中的滴滴涕及代谢物.水产科学,2007,26(4):229–233.
[95]樊景凤,梁玉波,宋立超,等.贝类帕金虫间接酶联免疫吸附测定法的建立.海洋环境科学,2006,25(2):62–64.
[96]樊甄姣,刘志鸿,杨爱国.氨氮对栉孔扇贝血淋巴活性氧含量和抗氧化酶活性的影响.海洋水产研究,2005,26(1):23-27.
[97]樊甄姣,杨爱国,刘志鸿. Cu~(2+)对栉孔扇贝体内几种免疫因子的影响.中国水产科学,2004,11(6):576-579.
[98]方平,李英文.酶联免疫吸附法在水产养殖中的应用.水利渔业,2004,24(1):12–14.
[99]郭皓.免疫方法在藻毒素及贝毒素检测中的应用.卫生研究,1999,28( 2):52–55.
[100]韩贻仁.分子细胞生物学.科学出版社,2001,pp 224–234.
[101]贺桂珍,李赟,宋微波,等.栉孔扇贝病原感染与病害发生关系探讨.水产学报,2003,27(3):273–277.
[102]胡曦璇,石安静.椭圆背角无齿蚌珍珠囊形成过程中钙代谢的初步研究.四川大学学报(自然科学版),1994,31:145–148.
[103]江育林,于平.用酶联免疫吸附试验快速检测虹鳟的传染性胰脏坏死病病毒.水生生物学报,1990,14(3):276–279.
[104]李子牛,邢婧,林听听,等.栉孔扇贝D形幼虫血细胞发生特点的研究.中国海洋大学学报,2010,40(9):61–64.
[105]刘莉.溶酶体的形成与作用及其研究近况.黑龙江畜牧兽医,2003,(3):19–19.
[106]刘世良,麦康森.贝类免疫系统和机理的研究进展.海洋学报,2003,25(2):95–105.
[107]刘英杰,王崇明,朱明壮,等.栉孔扇贝类立克次体自然感染调查及人工感染试验.中国水产科学, 2002,9(4):346–352.
[108]马洪明,刘晓伟,麦康森,等.盐度突降对栉孔扇贝( Chlamys farreri)抗病力指标的影响.高技术通讯,2006,16(7):746–751.
[109]牟海津,江晓路,刘树青,等.免疫多糖对栉孔扇贝酸性磷酸酶、碱性磷酸酶和超氧化物歧化酶活性的影响.青岛海洋大学学报,1999,29 (3):463–468.
[110]钱冬,陈月英,沈锦玉,等.应用酶联免疫吸附法检测暴发病病原——嗜水气单胞菌的研究.水产养殖,(4):14–17.
[111]任伟成,王崇明,孙世春,等.栉孔扇贝急性病毒性坏死病毒荧光定量PCR检测方法的建立和应用.中国水产科学,2009,16(4):564–571.
[112]石安静,陈继群,文秀英.淡水育珠蚌外套膜表皮细胞分泌方式的研究.水生生物学报,1994,18(4):369–375.
[113]史成银,黄倢,宋晓玲.对虾皮下及造血组织坏死杆状病毒单克隆抗体的ELISA快速检测.中国水产科学,1999,6(3):116–118.
[114]孙虎山,李光友.脂多糖对栉孔扇贝血清和血细胞中的7种酶活力的影响.海洋科学,1999,(4):54–58.
[115]孙虎山,李光友.大肠杆菌感染后栉孔扇贝血淋巴中7种酶活力的变化.海洋科学,1999,(5):40–44.
[116]孙虎山,李光友.大肠杆菌感染后栉孔扇贝血淋巴中7种酶活力的变化.海洋科学,1999,(5):40–44.
[117]孙虎山,李光友.免疫多糖对栉孔扇贝血淋巴中氧化酶活力的影响.高技术通讯,2001,(5) :10–12.
[118]孙虎山,李光友.硒化卡拉胶和酵母葡聚糖对栉孔扇贝血淋巴中两种水解酶活力的影响.海洋与湖沼,2002,33(3):245–249.
[119]汪岷,戴继员,张士璀,等.用ELISA检测中国对虾的白斑综合症病毒( WSSV).海洋学报,2000,22(2):132–136.
[120]王崇明,王秀华,艾海新,等.栉孔扇贝大规模死亡致病病原的研究.水产学报,2004,28(5):548–553.
[121]王崇明,王秀华,宋晓玲,等.栉孔扇贝一种球形病毒的分离纯化及其超微结构观察.水产学报,2002,26(2):180–184.
[122]王崇明,杨冰.应用双抗夹心ELISA法检测皱纺盘鲍致病病原—创伤弧菌的研究.海洋水产研究,1999,20(1):30–34.
[123]王军,鄢庆枇,苏永全,等.养殖大黄鱼副溶血弧菌的酶联免疫吸附法研究.台湾海峡,2001,20( 3):346–351.
[124]王如才,王昭萍,张建中.海水贝类养殖学.青岛海洋大学出版社,1998,pp 155.
[125]王秀华,王崇明,黄捷.间接免疫荧光法检测栉孔扇贝急性病毒性坏死症病毒.中国水产科学,2005,12(l):38–42.
[126]王宜艳,孙虎山,刘玉坤.硒化壳聚糖对栉孔扇贝血细胞中部分免疫因子的影响.海洋通报,2007,26(2):117–120.
[127]王运涛,相建海.栉孔扇贝大规模死亡的原因探讨.海洋与湖沼,1999,30 (6):770–774.
[128]吴礼龙,康惠.草鱼出血病的快速ELISA诊断试验.内陆水产,1997,(4):2–3.
[129]吴谦,何雅军.酶免疫技术在牡蛎制剂质量研究中的应用.中药新药与临床药理,1997,8( 3):159–161.
[130]邢婧.栉孔扇贝(Chlamys farreri)血细胞及其免疫特性研究:[博士学位论文].青岛:中国海洋大学,2002.
[131]邢婧,战文斌.栉孔扇贝稚贝造血组织的研究.水产学报,2005,29:145–149.
[132]邢婧,战文斌,周丽.栉孔扇贝(Chlamys farreri)血细胞类型及抗菌力的研究.青岛海洋大学学报,2003,33(1):41–46.
[133]叶玫,吴成业,刘海新,等.酶联免疫吸附法在水产品安全检测中的应用.上海水产大学学报,2002,11(2):171–175.
[134]杨文鸽,薛长湖,徐大伦,等.β-1,3-葡聚糖及其羧甲基衍生物对海湾扇贝免疫功能的影响.水产学报,2006,30(2):236–240.
[135]喻冰冰.虾夷扇贝(Patinopecten yessoensis)血细胞的形态及分布:[硕士学位论文].青岛:中国海洋大学,2009.
[136]于瑞海,王如才,田传远,等.栉孔扇贝大面积死亡的原因分析及预防探讨.海洋湖沼通报,1998,3:69–72.
[137]袁勤生,谢卫华,姚菊芳,等.连苯三酚自氧化法测定超氧化物歧化酶活性的改进.医药工业,1988,19(5):217.
[138]翟玉梅,丁秀云,李光友.软体动物血细胞及体液免疫研究进展.海洋与湖沼, 1998,29(5):558–562.
[139]张峰,李光友.贝类血细胞活性氧体内防御作用的研究进展.海洋科学,1999,2:16–19.
[140]张福绥,杨红生.山东沿岸夏季栉孔扇贝大规模死亡原因分析.海洋科学,1999a,1:44–47.
[141]张福绥,杨红生.栉孔扇贝大规模死亡问题的对策与应急措施.海洋科学,1999b, (2):38–42.
[142]张玺,齐钟彦.贝类学纲要.科学出版社, 1961,pp 240.
[143]赵晓联,赵春城,蔡建荣,等.酶联免疫吸附测定法在饲料毒素检测中的应用.中国饲料,2000,(6):21–22.