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1.
草鱼呼肠孤病毒Ⅱ型是导致草鱼出血病的主要病原体之一,目前在我国广泛流行。本研究根据GenBank中草鱼呼肠孤病毒Ⅱ型基因序列,设计了草鱼呼肠孤病毒Ⅱ型特异性探针和PCR引物,通过优化反应条件,建立了草鱼呼肠孤病毒Ⅱ型一步法TaqMan荧光定量RT-PCR检测方法。试验对反转录和PCR反应体系进行摸索,通过优化的冻干工艺制备出草鱼呼肠孤病毒Ⅱ型全预混RT-PCR试剂,在荧光定量PCR中,冻干试剂的检测灵敏度可达10个模板,是普通PCR的100倍。批次间变异系数小于4%。特异性试验结果表明,该方法可特异性地检出草鱼呼肠孤病毒Ⅱ型,而对鲤春病毒血症病毒、传染性造血器官坏死症病毒无检测信号。该冻干试剂4℃保存12个月,室温25℃保存3个月,37℃保存15d,敏感性仍为10个,与第1d相同;利用手持单通道荧光PCR仪,可在42min内完成核酸诊断。本试验基于荧光定量PCR检测方法,结合核酸扩增体系冻干工艺制备的全预混草鱼呼肠孤病毒Ⅱ型RT-PCR试剂具有耐保存便于运输、准确性高、仪器拓展性好的特点,对草鱼呼肠孤病毒Ⅱ型快速诊断有重要意义。 相似文献
2.
近江牡蛎HSC70基因对溶藻弧菌感染的反应 总被引：1，自引：0，他引：1
人工注射溶藻弧菌(Vibrio alginolyticus)感染近江牡蛎(Crassostrea hongkongensis),通过实时荧光定量RT-PCR方法检测了近江牡蛎6种不同组织器官(外套膜、鳃、消化腺、闭壳肌、心脏和血细胞)中HSC70基因表达量的变化;同时采用平板活菌计数法测定了近江牡蛎不同组织器官中溶藻弧菌菌量的变化。结果显示,人工注射感染溶藻弧菌后,近江牡蛎6种组织器官中HSC70基因表达量均显著性升高(P<0.05),且随时间变化均呈现出先升高后恢复至对照水平的趋势;其中鳃组织中HSC70基因分别在第6小时和第72小时出现2次显著性高表达,且在第72小时的表达量高于第6小时(P<0.05)。在多数组织器官中,HSC70基因出现显著高表达后便急剧下降至对照水平,而在消化腺中高表达持续的时间长达18 h(第6 24小时)。在注射感染溶藻弧菌后第6小时,血细胞中HSC70基因的表达量达到峰值,接近于对照水平的15倍;而在其他组织器官中,该基因表达峰值仅为对照水平的2.5倍左右。人工注射感染溶藻弧菌后3 h,3种组织(消化腺、闭壳肌和血淋巴)中均能检测到溶藻弧菌的积累,其中闭壳肌中含菌量最高,6 h时闭壳肌中含菌量急剧下降,随后又增加并维持在一定水平直至72 h;消化腺中菌量随着时间推移而增加,到达峰值后又逐步下降;血淋巴中,溶藻弧菌含量整体上随时间不断增加,到48 h稳定在较高水平。各组织中HSC70表达量与溶藻弧菌含量之间存在一定的关联性,尤其在溶藻弧菌攻击的靶器官(消化腺)中最为明显。由此可见,病原菌感染可以诱导近江牡蛎HSC70基因高表达,并且HSC70可能参与了机体抗病原菌感染的相关作用,这为进一步研究近江牡蛎HSC70基因在抗病免疫中的作用机制奠定了基础。 相似文献
3.
为了研究致病性粪肠球菌(Enterococcus faecalis)对脊尾白虾(Exopalaemon carinicauda)免疫相关基因表达的影响,分别对健康脊尾白虾注射生理盐水和粪肠球菌,测定了不同时间点血细胞和肝胰腺中酚氧化酶原(proPO)、C型凝集素(CTL)、超氧化物歧化酶(SOD)、组织蛋白酶D(Cat D)和组织蛋白酶L (Cat L)基因的相对表达量变化.结果显示,与对照组相比,感染粪肠球菌后,血细胞和肝胰腺中proPO基因表达量均于6h达到最大值(P＜0.05),随后逐渐降低至对照组水平;血细胞和肝胰腺中CTL基因表达量分别于12h和6h上升至最大值,随后逐渐降低;血细胞中CTL基因表达量于72 h虽有降低,但仍显著高于对照组(P＜0.05);血细胞和肝胰腺中SOD基因表达量分别于6h和3h达到最大值,随后逐渐降低,并于72 h达到正常水平;血细胞和肝胰腺中Cat D基因表达量变化趋势基本一致,呈现先降低后升高、然后显著降低再升高的趋势;血细胞和肝胰腺中Cat L基因表达量分别于6h和3h显著降低,随后逐渐升高至最大值,并于72 h达到对照组水平.研究表明,长时间的粪肠球菌感染对脊尾白虾免疫相关基因表达影响显著,本研究所用免疫相关基因均可作为粪肠球菌感染疾病的监测指标. 相似文献
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为了研究淋巴因子基因(T-cell acute lymphocytic leukemia,tal)在香港牡蛎中的免疫功能,实验采用c DNA末端快速扩增技术(rapid-amplification of cDNA ends,RACE)克隆了香港牡蛎tal的c DNA全长序列,命名为Chtal,该基因全长812 bp,其中5?非编码区17 bp,3?非编码区135 bp,开放阅读框(open reading frame,ORF)660 bp,编码219个氨基酸,相对分子质量25.11 ku,理论等电点5.80。多序列比对和系统进化树结果显示,Chtal与长牡蛎、美洲牡蛎和海蜗牛tal同源性较高,证明了该基因是软体动物tal家族的一个成员。荧光定量PCR结果显示,Chtal在香港牡蛎血淋巴细胞中表达量最高,其次是心脏、鳃和消化腺。在溶藻弧菌和溶血性葡萄球菌刺激后,Chtal表达量显著上调,分别在24和12 h达到最高水平,随着时间的增加该基因的表达量逐渐下降。亚细胞定位结果显示,Chtal在细胞核中表达。活体注射重组TAL蛋白能够显著提高淋巴细胞的数量,通过Edu增殖实验进一步证实了tal具有促进香港牡蛎血淋巴细胞再生的功能。本研究初步揭示了香港牡蛎tal参与了淋巴细胞再生,为深入研究该基因在牡蛎抗病中的功能提供了依据。 相似文献
5.
以牡蛎(Crassostrea gigas)为研究对象,研究了不同超高压处理条件对牡蛎体内微生物的消减作用,分析了冷藏过程中挥发性盐基氮(TVB-N)和 pH 值的变化情况,对比了热加工和超高压处理对牡蛎滋味的影响。结果显示,经300 MPa 处理15 min、400 MPa 处理10、15 min 和500 MPa处理5、10和15 min 后,牡蛎体内的菌落总数均有2个对数以上的减少,表明超高压处理能有效消减牡蛎体内的微生物;牡蛎经300 MPa 处理15 min、400 MPa 处理10 min 和500 MPa 处理5 min,在4℃冷藏条件下保存,货架期延长至15 d;超高压处理能有效抑制牡蛎在4℃贮藏过程中挥发性盐基氮的产生,牡蛎经300 MPa 处理15 min,冷藏20 d 后 TVB-N 值仍小于10 mg/100 g,符合生鲜牡蛎要求;滋味方面,与加热处理相比,超高压处理后牡蛎更接近生鲜牡蛎,尤其300 MPa 处理15 min 的牡蛎同生鲜牡蛎最为接近。研究表明,选取300 MPa 处理15 min 作为牡蛎加工的最佳处理条件,即能有效消减牡蛎体内的微生物,同时能很好地保持牡蛎品质。 相似文献
6.
蛋白酶抑制因子是极其多样的蛋白质或多肽,能抑制蛋白酶的水解活性。研究发现蛋白酶抑制因子能通过抑制病原蛋白酶活性,从而抑制病原的入侵。I84蛋白酶抑制因子家族是MEROPS数据库中新增的一个家族,其部分成员在免疫防御过程中的作用得到了一定的揭示。为探究I84蛋白酶抑制因子家族在长牡蛎中的分布和功能状况,实验鉴定了长牡蛎中23个潜在的I84家族基因,根据系统进化树分析,挑选了5个同源基因进行时空表达和功能分析。首先,利用克隆技术验证了长牡蛎中5个I84家族同源基因CgSi3、CgSi5、CgSi6、CgSi16和CgSi19可表达性。结果显示,5个基因在外套膜、闭壳肌、性腺、血淋巴细胞、肝胰腺和鳃等6个组织中均表达,但肝胰腺中表达量显著高于其他组织。同时,5个基因在长牡蛎幼体不同发育阶段表达模式不同,其中各基因在受精卵时期均不表达,CgSi3表达量则在眼点幼虫期显著上升后下降,而CgSi6在壳顶幼虫期开始表达后,表达量随长牡蛎发育而持续增加。另外,对牡蛎进行病原相关模式分子(PAMPs)注射后,5个基因也表现出不同的表达模式,其中LPS和PGN注射后CgSi6表达量变化明显,而poly (... 相似文献
7.
The Pacific oyster Crassostrea gigas is a sessile bivalve that inhabits the intertidal zone and therefore frequently exposed to air during the tidal cycle. It
is highly adaptive to hypoxic conditions. We have studied the physiological state of oysters during long-term exposure to
air. The oysters became hypoxic when exposed to air or hypoxic seawater. The 50% lethal time of oysters exposed to air at
4, 15 and 20°C was 47.8, 15.9 and 12.2 days, respectively. The hemolymph pH decreased by day 3; however, it showed a slight
increase by day 5 at both 4 and 20°C. The adenylate energy charge (AEC) values decreased rapidly on the first day of air exposure
in the adductor muscle, mantle, gill and body trunk, and these decreases were accompanied by decreases in ATP concentrations
and increases in AMP concentrations. The AEC values in all of the tissues had fallen to below 30% by day 50 of air exposure
at 4°C. These data suggest that the energy state of oysters deteriorates rapidly with air exposure. Consequently, AEC values
may be useful indices of the physiological state of the oyster during long-term exposure to air. 相似文献
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A synthetic antibacterial peptide from Mytilus galloprovincialis reduces mortality due to white spot syndrome virus in palaemonid shrimp 总被引：3，自引：0，他引：3
White spot syndrome virus (WSSV) isolated from Penaeus monodon was found to be highly infective for the western Mediterranean shrimp, Palaemon sp. Using polymerase chain reaction (PCR), it was demonstrated that such shrimp are not naturally carriers of WSSV. Following challenge with virus, mortality reached 100% 3.5-4 days after injection at 22 degrees C. Incubation of infected shrimp at 10 degrees C totally suppressed the mortality which rapidly developed when shrimp were returned to 18 or 22 degrees C. Preincubation of WSSV with mature synthetic mytilin significantly reduced shrimp mortality with a 50% efficient dose of about 5 microM. Survival of shrimp was not due to the development of an active mechanism of defence as re-injection of WSSV produced the same mortality pattern. Mortality was probably due to WSSV replication as dot blot failed to detect viral DNA in the injection sample but was positive 1 day post-injection. Protection by mytilin was by interaction at the virus level, preventing replication as no WSSV nucleic acid was detected by PCR even after 7 days in shrimp injected with WSSV preincubated with 10 or 50 microM mytilin. 相似文献
11.
The French oyster production of Crassostrea gigas is based on two sources of spat: wild-caught (WC) and hatchery-produced (HP). Massive mortality related to the ostreid herpesvirus type 1 (OsHV-1) has affected both sources in France since 2008. We investigated the mortality in juvenile C. gigas due to the horizontal transmission of OsHV-1 within (separated condition) and between (mixed condition) the two spat sources in three environments from April to June 2010. In the separated condition, no mortality was observed in the HP batches, while the WC batches experienced moderate to high mortality (40–80 %). In contrast, the WC and HP batches experienced high mortality in all tested environments for the mixed condition. At the beginning of the trial, the HP batches were all negative for OsHV-1 DNA detection by real-time PCR, while the WC batches were all positive for OsHV-1 DNA detection by real-time PCR, even though the percentage of virus DNA-positive oysters and viral load were low. During the experiment, all batches that exhibited mortality were positive for OsHV-1 with a high viral load, while OsHV-1 was never detected for the HP batches of the separated condition. Together, our results demonstrated that OsHV-1 was horizontally transmitted from the WC oysters to the HP oysters. Our study is the first to indicate that the mortality related to OsHV-1 in HP oysters can be avoided using ponds or tanks. However, these oysters were always protected from OsHV-1, and HP oysters could also experience mortality and spread the disease similar to the WC oysters if such care is not used. Finally, the persistence of OsHV-1 at a sub-clinical level in certain oysters supports the hypothesis that the virus can be reactivated and cause viral replication. The use of the two spat sources is discussed to better understand the spread of the disease among oyster stocks. 相似文献
12.
This study investigated potential application of lactic acid bacteria (LAB) in depuration for reducing Vibrio parahaemolyticus in oysters. Lactobacillus plantarum ATCC 8014, which exhibited strong bactericidal effects against V. parahaemolyticus in vitro, was added to artificial seawater for depuration of Pacific oysters (Crassostrea gigas) inoculated with V. parahaemolyticus BE 98-2029 (O3:K6) to levels of about 104 MPN/g at 15 ± 1 and 10 ± 1°C. Application of L. plantarum ATCC 8014 treatment (107 CFU/mL) in oyster depuration did not enhance reductions of V. parahaemolyticus in oysters depurated at 15 ± 1°C but significantly decreased (p < 0.05) levels of V. parahaemolyticus in oysters depurated at 10 ± 1°C after 5 days (3.40 log reductions) when compared with controls (2.75 log reductions). It is not clear if a competitive exclusion by LABs to compete with V. parahaemolyticus binding sites in oyster tissues plays a role in the reduction of V. parahaemolyticus in the oysters. Further studies utilizing different types of LABs in oyster depuration might provide additional knowledge for application of LAB in depuration for decontaminating V. parahaemolyticus in oysters. 相似文献
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Production of inbred larvae through self‐fertilization using oocytes and cryopreserved sperm from the same individuals after sex reversal in eastern oyster Crassostrea virginica 
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下载免费PDF全文 The eastern oyster Crassostrea virginica can change sex which makes self‐fertilization possible if sperm can be cryopreserved. In this study, small (~1 year old) and large (~2–3 years old) oysters were biopsied for sperm collection. Survival of the biopsied oysters after 1 year was 50% for small oysters and 17% for large oysters. Oocytes were collected from sex‐reversed females, and self‐fertilized with cryopreserved sperm. Of the 24 cryopreserved samples, 14 individuals had ≤1% fertility when crossed with oocytes from unrelated females, indicating that the cryopreserved sperm had reduced fertility. The other 10 individuals had a fertility of 39 ± 25% when crossed with oocytes from unrelated females (non‐selfing), but showed a significantly lower success of self‐fertilization (12 ± 16%) (P = 0.008), while aliquots of the same oocytes had a fertilization of 83 ± 11% when crossing with fresh sperm. Larvae were produced at day 3 in the self‐fertilized families (12–94% of the fertilized oocytes), and survived to eyed‐larvae stage at days 11–14. Genotyping with 9 microsatellite markers confirmed that the larvae resulted from self‐fertilization in four families. This study demonstrated the feasibility of creating self‐fertilized inbred lines of oysters by use of non‐lethal sperm collection and cryopreservation. 相似文献
15.
The capacity of oysters to regulate energy metabolism‐related processes may be key to their resilience against ocean acidification 下载免费PDF全文
下载免费PDF全文 Priscila Goncalves Kelli Anderson David Andrew Raftos Emma Louise Thompson 《Aquaculture Research》2018,49(5):2059-2071
Bivalve molluscs, such as oysters, are threatened by shifts in seawater chemistry resulting from climate change. However, a few species and populations within a species stand out for their capacity to cope with the impacts of climate change‐associated stressors. Understanding the intracellular basis of such differential responses can contribute to the development of strategies to minimise the pervasive effects of a changing ocean on marine organisms. In this study, we explored the intracellular responses to ocean acidification in two genetically distinct populations of Sydney rock oysters (Saccostrea glomerata). Selectively bred and wild type oysters exhibited markedly different mitochondrial integrities (mitochondrial membrane potential) and levels of reactive oxygen species (ROS) in their hemocytes under CO2 stress. Analysis of these cellular parameters after 4 and 15 days of exposure to elevated CO2 indicated that the onset of intracellular responses occurred earlier in the selectively bred oysters when compared to the wild type population. This may be due to an inherent capacity for increased intracellular energy production or adaptive energy reallocation in the selectively bred population. The differences observed in mitochondrial integrity and in ROS formation between oyster breeding lines reveal candidate biological processes that may underlie resilience or susceptibility to ocean acidification. Such processes can be targeted in breeding programs aiming to mitigate the impacts of climate change on threatened species. 相似文献
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The distribution and expression of lymphocystis disease virus (LCDV) vaccine, on the basis of DNA vaccine (pEGFP-N2-LCDV0.6 kb) construction, were analyzed in tissues of the Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR studies indicated that the vaccine-containing plasmids were distributed in injected muscle, muscle located opposite the injection site, hind intestine, gill, spleen, head kidney, liver and gonad 7 days after vaccination. However, these vaccine-containing plasmids disappeared by 90 days following vaccination. Fluorescent microscopy observations revealed that green fluorescence appeared in muscle, muscle located at the opposite side of the injection site, hind intestine, gill, spleen, head kidney and liver of fish 36 h after vaccination, and that green fluorescence did not appear in control tissue. The green fluorescence became weaker at 60 days post-vaccination, however, it remained detectable in the spleen 90 days post-vaccination. Results from RT-PCR studies indicated that the Mcp gene is expressed in all tissues of vaccinated fish 7-20 days after vaccination. These results demonstrate that the DNA vaccine is distributed and expressed in different tissues of vaccinated fish, and therefore, may have provided an antigen producing specific immune response. 相似文献
17.
Christophe Lambert Philippe Soudant Lionel Dgremont Maryse Delaporte Jeanne Moal Pierre Boudry Frdric Jean Arnaud Huvet Jean-Franois Samain 《Aquaculture (Amsterdam, Netherlands)》2007,270(1-4):276-288
High variability among individuals is often encountered when hemocyte characteristics are measured in bivalves. Such variability is suspected to result partly from genetic factors. In this study, hemocyte characteristics of six families of Crassostrea gigas were compared by flow cytometry at one sampling date in October 2001. These families were obtained from a nested, half-sibling cross design, and reared from July to October 2001 at three sites distributed along the French Atlantic coast from north to south: Baie des Veys (Normandy), Rivière d'Auray (Brittany) and Ronce (Marennes-oléron Basin, Poitou Charentes).
Among the 15 measured hemocyte characteristics, production of reactive oxygen species (ROS) of untreated hemocytes (maintained in filtered sterile seawater) and treated hemocytes (zymosan at 20 particles per hemocyte, and with Vibrio sp. S322 at 50 bacteria per hemocyte) was the most notable differences between families. This supports the existence of a genetic basis, at least partly, for the hemocyte characteristics of oysters, and especially for ROS production.
Among the six families analyzed, three have shown high survival during summer (named as “resistant”, mean mortality 5.2%) and three experienced high mortality during summer (named as “susceptible”, 30.6% mean mortality). Families showing high or low survival to summer mortality had similar hemocyte characteristics, regardless of the environmental conditions or reproductive state. Resistant families were observed to have higher total hemocyte counts and lower production of ROS than susceptible families. Moreover, ROS production of hemocytes from susceptible families was diminished significantly more by pathogenic Vibrio than that of resistant families. However, this study demonstrates also that rearing site strongly affected the hemocyte characteristics of all families of oysters, most notably hemocyte concentration and morphology (size and granularity), production of reactive oxygen species (ROS), and susceptibility to the cytotoxic activity of the pathogenic Vibrio sp. S322 (50 bacteria/hemocyte). Food availability and reproductive state are the most probable explanations for the site differences observed. Finally, it appeared difficult to link oyster survival during summer mortality to hemocyte profiles evaluated at one sampling date; other relevant indicators would probably help explaining oyster survival during summer mortality events. 相似文献
18.
Liang Song Xiaoxu Li Steven Clarke Ting Wang Kriston Bott 《Aquaculture International》2007,15(5):351-362
To further improve the technology used in Pacific oyster farming, information is required on the response of different sized
and aged oysters to various environmental changes. In this study a neutral red retention (NRR) assay was used to investigate
the effects of size and age on the response of Pacific oysters to changes in water temperature and their recovery after exposure
to different air temperatures. Results from moving oysters directly between water temperatures of 5°C and 15°C, 10°C and 20°C
and 15°C and 25°C demonstrated that different water temperature change affect the lysosomal membrane integrity differently.
The NRR times of large and small oysters transferred directly between 10°C and 20°C initially decreased significantly, and
then increased to levels corresponding to the new temperature. In addition, NRR times in large oysters responded at a significantly
slower rate than small oysters when they were transferred from 5°C and 25°C to 15°C water and between 10°C and 20°C water.
Results from the air exposure experiments showed that, after exposure to air temperatures of 5°C, 15°C or 25°C, the lysosomal
membrane integrity of large oysters recovered at a slower rate in 15°C water compared to small oysters. It therefore appears
necessary to develop different management strategies for large (old) and small (young) oysters. Results from this and previous
research also indicate that the NRR assay could potentially be used to develop a model to monitor and predict the performance
of oysters on farms. 相似文献
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Imad G. Saoud David B. Rouse Richard K. Wallace Jeffrey Howe Blan Page 《Journal of the World Aquaculture Society》2000,31(4):640-650
Abstract.— Oyster spat settlement at four oyster reefs in Mobile Bay, Alabama, USA, was studied from August through October 1998 and May through mid November 1999. Spat collectors at the reefs were replaced every 2 wk and spat-set estimated as number of oysters per meter square per day. Water quality data at Fish River Reef was monitored using remote sensors. Spat-set data revealed significant variation between the four sites and between the 2 yr. Spat settlement was 5 to 10 times greater at the other three reefs than at Fish River Reef. Dates and intensity of oyster settlement at Fish River Reef were different from dates and intensity of oyster settlement at Shell Bank Reef, both on the eastern side of the bay. However, settlement was similar between Cedar Point Reef and White House Reef, both on the western side of the bay. Spat set appears to occur 3 wk after a rapid decline in water temperature, provided adequate oxygen concentrations are present at the time of settlement. Data collected suggest that intensity of settlement at Fish River Reef is considerably less than at other reefs in this study but could be adequate to reestablish the reef, if cultch and environmental conditions are suitable. The data also suggest that the source of larval oysters at Fish River Reef is different from the source of larval oysters at the other sites tested in the present study. 相似文献