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1.
Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non‐pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi‐granulocytes) have the main function in phagocytosis of both pathogenic and non‐pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non‐virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post‐inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non‐virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.  相似文献   

2.
流式细胞术比较研究三种对虾血细胞的分群   总被引：4，自引：1，他引：3  
应用流式细胞术对墨吉对虾(Penaeus merguiensis)、南美白对虾(Penaeus vannamei)、斑节对虾(Penaeus monodon)三种对虾的血细胞类型进行了比较研究。根据其血细胞的前向角散射光(FSC)和侧向角散射光(SSC)特征的不同将血细胞分群,FSC和SSC二维图分析表明三种对虾的血细胞都可分为三个亚群:透明细胞、小颗粒细胞、大颗粒细胞。同时还对各种类型血细胞所占比例进行了比较研究,发现各个亚群血细胞所占组成比例在三种对虾之间具有相似性。其比例均为:小颗粒细胞最多,约为45%~49%;透明细胞次之,约为26%~28%;大颗粒细胞最少,约为10%~12%。  相似文献   

3.
White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.  相似文献   

4.
应用流式细胞术(FCM)测定凡纳滨对虾(Litopenaeus vannamei)不同类型血细胞(透明细胞、小颗粒细胞和大颗粒细胞)的非诱导性和诱导性活性氧(ROS)含量以及非特异性酯酶活性,探讨不同类型血细胞的免疫功能.结果显示,透明细胞的非诱导性ROS含量最低,显著低于小颗粒细胞和大颗粒细胞(P＜0.05),大颗粒细胞的非诱导性ROS含量最高;经脂多糖(LPS)刺激后,透明细胞的ROS含量没有显著变化(P＞0.05),小颗粒细胞和大颗粒细胞的ROS含量显著提高(P＜0.05);大颗粒细胞的酯酶活性显著高于小颗粒细胞和透明细胞(P＜0.05).研究表明,3类血细胞在活性氧含量和酯酶活性的功能上存在差异,两类颗粒细胞的活性氧水平较高,大颗粒细胞的酯酶活性较强.  相似文献   

5.
Argopecten irradians is one of the most important commercial species of Pectinidae family in China. The internal defense system of mollusks consists of circulating haemocytes. In order to characterize the haemocytes of the scallop A. irradians, light and electron microscopical studies were carried out. Four types of haemocytes were recognized: type I small hyalinocytes (2.38 ± 0.08 μm, 30-35%), type II large hyalinocytes (4.41 ± 0.33 μm, 15-20%), type III small granulocytes (4.15 ± 0.26 μm, 20-25%), and type IV large granulocytes (8.26 ± 0.52 μm, 25-30%). Granulocyte types showed smaller N/C ratios than hyalinocytes. The mean haemocyte concentration was about (3.75 ± 0.65) × 107 cells ml− 1 of haemolymph. Among haemocytes, 44.7% are granular and 55.3% are agranular. These gave a relatively systematic classification scheme for haemocytes of A. irradians. Three types of granules were identified: type I, with high electron-density; type II, with low electron-density; and type III, with a middle level of electron-density, based on TEM studies. Different haemocyte types were not separated with DDGC of Percoll in this study. Both granulocytes and hyalinocytes showed a phagocytic response to the two strains of bacteria, Escherichia coli and RLOs. The phagocytic ability of granulocyte was significantly higher (41-48%) than that of hyalinocyte (9.2-11.2%).  相似文献   

6.
采用流式细胞仪对菲律宾蛤仔、中国蛤蜊、文蛤和紫石房蛤4种双壳贝类的血细胞分类进行了比较研究。通过比较4种贝类的前向角散射光(FSC)和侧向角散射光(SSC)双参数点图以及FSC和SSC单参数直方图,发现4种贝类的血细胞均可根据其细胞质中是否有颗粒分为透明细胞和颗粒细胞两种基本类型,中国蛤蜊和紫石房蛤的颗粒细胞又可分为小颗粒细胞和大颗粒细胞两个亚群,菲律宾蛤仔的颗粒细胞又可以区分为小颗粒细胞、中颗粒细胞和大颗粒细胞3个亚群,而文蛤只有颗粒细胞,通过统计分析双参数点图中的各细胞亚群,4种贝类血细胞亚群的含量存在一定差异。  相似文献   

7.
At present the most common and most devastating disease of shrimp is caused by the white spot syndrome virus (WSSV), which has spread throughout the world mainly by different species of crustaceans carrying the virus. After experimental injection of Penaeus monodon with a known copy number of WSSV in the abdominal muscle, the rate of viral progression in different tissues at 12, 24, 36 and 48 hpi (hours post infection) was assessed using quantitative real‐time PCR. At 12 hpi the viral load was highest in haemocytes followed by pleopod, muscle and gills whereas at 48 hpi, the gills, the main target of WSSV, showed the highest viral load followed by pleopod, muscle and haemocytes. Viral copy number in the haemocytes was the lowest beyond 12 hpi indicating a remarkable reduction in the rate of viral replication in haemocytes compared with other tissues. The viral load in haemocytes, though increased again beyond 36 hpi, never surpassed the load in the other tissues. The real‐time PCR assay with its high sensitivity and wide dynamic range make it ideal for detecting low‐level WSSV infections that can occur in apparently healthy P. monodon.  相似文献   

8.
The effect of plants and probiotics on the survival and immune response of Litopenaeus vannamei challenged with the white spot syndrome virus (WSSV) was evaluated. A probiotic mixture (PM), plant extract (PE) or powdered plants (PP) were added to feed with the attractant Dry Oil®. An experiment was conducted with five treatments in triplicate. Shrimp (weighing 11.70±2.5 g) were cultured in 120 L plastic tanks and fed twice a day with commercial feed plus additives or with commercial feed plus WSSV. Animals were monitored for the occurrence of WSSV using single‐step and nested PCR. The PM and PP added to the commercial feed showed high survival, a decrease in WSSV prevalence in shrimp and an increase in the activity of lysosomal enzymes, N‐acetyl‐β‐glucosaminidase and acid phosphatase. The total haemocyte count in shrimp treated with PM was significantly higher than that in the control group (treatment I) and in shrimp fed with PE. The results of the present work indicate that PP and PM are good candidates for use as feed additives against WSSV in shrimp cultures.  相似文献   

9.
Abstract. The circulating haemocytes of the tanner crab, Chionocetes bairdi , were studied and classified as hyalinocytes, intermediate granulocytes and eosinophilic granulocytes. Differential haemocyte counts of normal crabs and crabs infected with an apparently pathogenic unnamed ascomycete fungus revealed a shift in the hyalinocyte-granulocyte ratio in infected crabs. There was a highly significant increase in the percentage of granulocytes and a corresponding decrease in hyalinocytes in infected crabs. This shift was thought to be caused by a marked increase in the relative number of eosinophilic gianulocytes. There was also a highly significant statistical relationship between the severity of the internal fungus infection, established by histopathological analysis of the major organ systems, and the increased number of circulating eosinophilic granulocytes. The results of the study indicate that differential haemocyte counts may be a valuable tool in monitoring the health of C. bairdi populations.  相似文献   

10.
In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA‐VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune‐related genes were examined to estimate the effect of dsRNA‐VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA‐VP28 treatment, whereas MDA content did not change significantly. Among the ten immune‐related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA‐VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA‐VP28 stimulation indicate that these immune‐related genes were involved in dsRNA‐VP28‐induced innate immunity in shrimp.  相似文献   

11.
Immune cells were identified and their interaction towards Vibrio alginolyticus, V. parahaemolyticus and V. anguillarum was studied in vitro in the penaeid shrimp, Penaeus indicus. Haemocytes were divided into agranulocytes, semi-dense granulocytes and dense granulocytes according to their morphology. Agranulocytes (100%) and 0.3–0.7% of granulocytes were actively involved in coagulation. Granulocytes were involved in in vitro phagocytosis and encapsulation of foreign materials. Phagocytosis was enhanced by prior opsonization of bacteria with cell-free shrimp haemolymph. Semi-dense granulocytes were phagocytic towards V. alginolyticus with and without opsonization at the rate of 91.1% and 83.1%, respectively ( P < 0.05 ). Granulocyte death observed after 2 h with opsonized haemolymph was 26.1%. About 64.5% of dense granulocytes and 23.2% of semi-dense granulocytes were actively involved in encapsulation, forming capsules. A spectrophotometric nitroblue tetrazolium (NBT) reduction assay was used to demonstrate the production of superoxide anions (O2−) by shrimp haemocytes. All the Vibrio spp. were able to induce superoxide anions (O2−) during phagocytosis. Live Vibrio sp. induced O2− production in haemocytes in a dose-dependent manner. Significant activity was detected with a 40:1 bacteria to haemocyte ratio ( P < 0.05 ). NBT reduction assay for measuring the post-phagocytic killing mechanism in shrimp haemocytes might be a valuable tool for monitoring shrimp health and immunological studies.  相似文献   

12.
Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.  相似文献   

13.
通过对流式细胞术(FCM)检测虾类血细胞总数(THC)的条件和方法进行优化,为虾类血细胞学研究提供快捷、准确的测定方法。2012年7-9月,应用SYBR Green I作为荧光染料标记完整血细胞,设置3个不同染料浓度(1×、10×和100×),测定不同孵育时间下染色细胞比例的变化。结果显示,染料终浓度为10×时染色效果最佳,其最佳孵育时间为60 min;应用建立的FCM方法和显微计数方法测定10尾凡纳滨对虾(Litopenaeus vannamei)的THC,平均值分别为(16.68±1.57)×106个/m L和(15.09±1.76)×106个/m L,2种方法测定结果的相关性极显著(R2=0.8064,P<0.01)。凡纳滨对虾经不同浓度(0.5 mg/L和5.0 mg/L)的Cd2+胁迫,利用建立的FCM方法测定Cd2+胁迫下对虾THC的变化。结果显示,0.5 mg/L和5.0 mg/L Cd2+胁迫下,对虾THC随着胁迫时间的延长不断下降,胁迫48 h时分别下降至对照组的78.7%和64.7%,可见Cd2+胁迫对虾类血细胞产生毒性,抑制了血细胞活性,表明该方法适用于虾类的血细胞学研究。  相似文献   

14.
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15.
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16.
Redox potential represents the intensity of anaerobic condition in the pond sediment, which may affect the dominant microbial transformations of substances, the toxins production, mineral solubility, as well as the water quality in the sediment–water interface inhabited by the shrimp. This study evaluates the effect of sediment redox potential in conjunction with stocking density on shrimp production performance, immune response and resistance against white spot syndrome virus (WSSV) infection. A completely randomized two factors experimental design was applied with three different sediment redox potential, i.e. ?65, ?108 and ?06 mV, and two shrimp densities, i.e. low (60 shrimp m?2) and high (120 shrimp m?2). Shrimp juveniles with an initial mean body weight of 5.32 ± 0.22 g were maintained in semi‐outdoor fibre tanks (270 L in capacity) for 35 days of experimental periods. At the bottom of each tank, 5‐cm deep soil substrate with different redox potential was added according to the treatments. The survival and biomass production were significantly reduced at ?206 mV sediment redox potential, regardless of stocking density. Highly negative sediment redox potential (?206 mV) and higher stocking density significantly reduced total haemocyte counts and phenoloxydase activity, and shrimp resistance to WSSV infection. We recommend to maintain the redox potential of pond sediment at a level of more than ?206 mV.  相似文献   

17.
The gene coding for translationally controlled tumour protein (TCTP) was polymerase chain reaction amplified from haemocyte cDNA of Indian shrimp, Penaeus indicus, and sequenced. The N‐terminal region, a conserved one among all the TCTPs, was shown to have one substitution at position 37, in the Indian isolate. Besides this, there were two substitutions in the C‐terminal region (135, 149), exclusive to the Indian isolate. Phylogenetic analysis suggested a close relatedness of TCTP from P. indicus to Fenneropenaeus chinensis compared with other isolates. Translationally controlled tumour protein gene expression was found to be elevated in the haemocytes of WSSV‐infected shrimps compared with the uninfected ones. However, tissues from the infected shrimps did not exhibit any detectable levels of TCTP expression.  相似文献   

18.
White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow‐out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV‐positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m‐PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m‐PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively.  相似文献   

19.
Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 104 to 9.0 × 1010 WSSV copies μg–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 109 WSSV copies μg–1 of DNA were detected which is equivalent to 5.7 × 1010 WSSV copies g–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).  相似文献   

20.
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   

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