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1.
C. L. SCHULTZ B. C. LIDGERDING P. E. McALLISTER F. M. HETRICK 《Journal of fish diseases》1986,9(2):117-122
Abstract. Differential incorporation of uridine and uracil was used to assay for mycoplasma contamination in five fish cell lines: bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papillosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The method was not suitable for monitoring BF-2, CHSE-214, FHM, and RTG-2 cell lines because they incorporated uracil. Differential incorporation of uridine and uracil may be applicable for screening EPC cells because only this cell line could distinguish cultures experimentally infected with Mycoplasma orale from cultures known to be free from microbial contaminants. 相似文献
2.
为了明确鳗鲡疱疹病毒(Anguillid herpesvirus, AngHV)的生物学及理化特性,本实验利用一株从欧洲鳗鲡"脱黏败血综合征"病料中分离的AngHV病毒株,研究了其增殖特性及其对主要鱼类细胞系的感染敏感性,进一步分析了其对热、酸碱、氯仿和乙醚等理化因子的耐受性。结果发现,AngHV感染的鳗鲡卵巢细胞系(eel ovary cell line, EO)内可见典型的疱疹病毒样颗粒,细胞出现时序性细胞病变;AngHV可在EO细胞系内稳定传代,较适宜扩繁温度为25～27°C,不能在鲤上皮瘤细胞系(epithelioma papilloma cyprinid cell line, EPC)、草鱼卵巢细胞系(grass carp ovary cell line, CO)、胖头逓肌肉细胞系(fathead minnow cell line, FHM)、大鳞大麻哈鱼胚胎细胞系(chinook salmon embryo cell line,CHSE-214)、虹鳟性腺细胞系(rainbow trout gonad cell line, RTG-2)及蓝鳃太阳鱼细胞系(bluegill fry cell line, BF-2)等鱼类细胞内增殖;理化特性分析表明,37°C处理30 min,AngHV滴度降低不明显,而56°C处理30 min可完全灭活AngHV;AngHV对酸(pH 3.0)敏感,而对碱(pH 10.0)较耐受,同时对氯仿和乙醚敏感。本研究结果可为AngHV的综合防控及疫苗的开发提供参考依据。 相似文献
3.
Jewhurst VA Todd D Rowley HM Walker IW Weston JH McLoughlin MF Graham DA 《Journal of fish diseases》2004,27(3):143-149
A simple method of detecting the presence of the salmonid alphaviruses (SAVs), salmon pancreas disease virus (SPDV) and sleeping disease virus (SDV), from serum samples is described. Using a 96-well tissue-culture plate format, test sera are diluted in medium and added to chinook salmon embryo (CHSE-214) cells. After incubation for 3 days at 15 degrees C, plates are fixed and stained using a monoclonal antibody (mAb)-based immunoperoxidase (IPX) detection system, and virus-infected cells are observed microscopically by white light. Application of this screening test, which is now used routinely in our laboratory in conjunction with an IPX-based virus neutralization (IPX-VN) test for detecting antibodies to SAVs, has resulted in the recovery of 12 additional isolates from salmon sera and four additional isolates from trout sera. A low level of antigenic variation was detected when these SAV isolates were investigated by indirect immunofluorescence using a panel of mAbs raised to reference SPDV and SDV isolates. 相似文献
4.
Abstract. Eight isolates of infectious pancreatic necrosis virus (IPNV) propagated solely in RTG-2 cells (RTG-IPNV) were examined for ability to replicate in FHM cells. Seven isolates replicated in FHM cells; however, the plaque titres were 10- to greater than 100 000-fold less than titres in RTG-2 cells. The ability of IPNV to replicate in FHM cells was shown to result from the presence of a host range variant (FHM-IPNV) that produced plaques in both cell types. Variant-free preparations of two isolates differed in ability to generate FHM-IPNV during a single replication cycle in RTG-2 cells. One isolate did not generate the variant and failed to replicate in FHM cells even upon blind passage.
IPNV propagated in AS cells (AS-IPNV) was similar to RTG-IPNV, producing equal numbers of plaques in RTG-2 and AS cells but failing to form plaques in FHM cells. However, IPNV propagated in BF-2 cells (BF-IPNV) was similar to FHM-IPNV in producing equal numbers of plaques in all three cell lines.
RTG-IPNV and FHM-IPNV were identical in size, morphology and density in CsCl but differed in plaque size distribution and neutralization by specific antiserum.
Restriction of the replication of RTG-IPNV in FHM cells was partially explained by a reduction of 75% in adsorption to FHM cells. However, the synthesis of RTG-IPNV antigens was reduced 90 to >99% in FHM cells. 相似文献
IPNV propagated in AS cells (AS-IPNV) was similar to RTG-IPNV, producing equal numbers of plaques in RTG-2 and AS cells but failing to form plaques in FHM cells. However, IPNV propagated in BF-2 cells (BF-IPNV) was similar to FHM-IPNV in producing equal numbers of plaques in all three cell lines.
RTG-IPNV and FHM-IPNV were identical in size, morphology and density in CsCl but differed in plaque size distribution and neutralization by specific antiserum.
Restriction of the replication of RTG-IPNV in FHM cells was partially explained by a reduction of 75% in adsorption to FHM cells. However, the synthesis of RTG-IPNV antigens was reduced 90 to >99% in FHM cells. 相似文献
5.
《Aquaculture (Amsterdam, Netherlands)》1986,56(1):1-21
Isolation and in vitro characteristics of a significant obligate intracellular eukaryotic pathogen (rosette agent) of chinook salmon (Oncorhynchus tshawytscha) are described. The pathogen was isolated by primary culture of infected spleen tissue followed by inoculation onto the chinook salmon embryo CHSE-214 cell line. Chicken and rabbit antibody was produced to a soluble antigen derived from the rosette agent. Mortalities and the disease were experimentally reproduced by inoculating chinook salmon with the isolate. The pathogen was reisolated 27 days post-inoculation from moribund fish. Identity of the isolate and the disease-causing organism was further supported by documenting antigenic and morphologic identity. Tested in CHSE-214 cells, the organisms are sensitive to the antibiotics oxytetracycline, chloramphenicol, amphotericin-B and nystatin. A ratio of about one organism per tissue culture cell at inoculation was required to develop an easily detectable infection. Adsorption of organisms to tissue culture cells at inoculation did not enhance the pathogen's infectivity. The pathogen replicated at an increasing rate from 5°C to 20°C. It could not be stored by freezing but storage in tissue culture at 5°C for at least 5 months proved possible. Early passages of the organism showed a high degree of host specificity in tissue culture which decreased in later passages. The taxonomic affinities of the organism are discussed. 相似文献
6.
Laboratory studies were carried out to investigate the cultural characteristics of salmonid alphaviruses (SAV) from Atlantic salmon (AS, Salmo salar) and rainbow trout (RT, Oncorhynchus mykiss), particularly in relation to cell line and temperature. In an initial study, SAV was isolated from 12 viraemic sera and passaged in Chinook salmon embryo (CHSE‐214) cells at 15 °C. Geometric mean titres (GMT) after initial isolation were found to be significantly higher (P < 0.05) relative to those after two or four passages. Primary isolation of SAV was conducted from 12 viraemic sera (six AS and six RT) in seven different cell lines at 15 °C: CHSE‐214, rainbow trout gonad (RTG‐2), TO (derived from Atlantic salmon head kidney leucocytes), salmon head kidney (SHK‐1), blue fin‐2 (BF‐2), fat head minnow (FHM) and Epithelioma papulosum cyprini (EPC). Overall, significant differences were found between cell lines in both the numbers of strains where growth was detected and in the GMT obtained. For both AS and RT strains, GMT values were significantly (P < 0.01) higher in both TO and BF‐2 cells relative to the others, including CHSE‐214 and RTG‐2, the cell lines conventionally used for SAV. The effects of temperature of incubation (4, 10, 15 and 20 °C) on growth in TO, CHSE‐214 and RTG‐2 were investigated. In TO and RTG‐2 growth was optimal at 15 °C, whereas in CHSE‐214 results at 10 and 15 °C were more similar. Little or no growth was detected at 4 or 20 °C. 相似文献
7.
E Tobback A Decostere K Hermans W Van den Broeck F Haesebrouck K Chiers 《Journal of fish diseases》2010,33(3):197-209
In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non‐virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE‐214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non‐virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin‐D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non‐virulent strains were generally serum sensitive. 相似文献
8.
辽宁某种鱼场从日本民间引进虹鳟鱼发眼卵,鱼苗上浮时暴发流行病,发病急,死亡快,死亡率达90％以上,连续几年春季鱼苗上浮时,都有这种急性传染病发生,死亡非常严重,有时上浮的稚鱼全部死亡,致使该场几年鱼苗生产中断,造成严重的经济损失,89年我们为此进行了流行病学和病原学的调查,用CHSE—214细胞从发病濒死的稚鱼中分离到一株传染性胰脏坏死病毒(Infectious pancreatic Necnosis Virus简称IPNV),其行态学特性,理f{二性和生物学特性与国外IPNV的相关报道基本一致,新分离的IPNV(代号IPNV—5)在CHSE,PG,RTG—2等细胞上增殖良好,且可引起良好的病变,病毒滴度达10~(5(?)5)TCID50/0.05ml,通过血清中和试验和ELISA检测,新分离的病毒可鉴定为IPNV—SP株,病毒对乙醚不敏感、耐酸,耐碱,对热比较稳定,0.5％的胰蛋白酶不能完全灭活,病毒的复制不受FUDR的抑制,电镜观察,病毒粒子存在细胞质中,病毒颗粒呈二十面体,无囊膜,92个壳粒,直径为67nm,属RNA病毒。将分离到的病毒回归缝康的虹鳟稚鱼苗,有明显的感染力并能复制出与天然发病时有相同的症状和死亡率,将死亡鱼病料接种CHSE细胞回收到了IPNV。 相似文献
9.
S‐I Kitamura S‐I Ohtake J‐Y Song S‐J Jung M‐J Oh B‐D Choi K Azumi E Hirose 《Journal of fish diseases》2010,33(2):153-160
‘Soft tunic syndrome’ causes mass mortality in the edible ascidian Halocynthia roretzi in Korean and Japanese aquaculture. In histopathological comparison, there were no specific differences between diseased specimens from Korea and Japan, indicating that soft tunic syndrome occurring in Korea and Japan is the same disease. No bacterial or protozoan cells were microscopically detected in either healthy or diseased tunics suggesting they are not the direct causes of soft tunic syndrome. Attempts were made to isolate virus from affected ascidians taking into account temperature conditions in which soft tunic syndrome is most prevalent in the field. However, no viruses were isolated from diseased or non‐diseased specimens using chinook salmon embryo (CHSE‐214), flounder fin (FFN) or epithelioma papillosum cyprini (EPC) cell lines. 相似文献
10.
Abstract. An unidentified mycoplasma was isolated from cultures of Atlantic salmon (AS) cells and implicated as the cause of cytopathic effects (CPE). The agent did not visibly affect RTG-2 cells. Experimental infection of RTG-2 cells with Acholeplasma laidlawii resulted in increased cellular granularity and destruction. Scanning electron microscopic (SEM) examination of infected RTG-2 and AS cultures revealed typical mycoplasmas distributed on cell surfaces and a marked effect on the cell surface topography, characterized by a loss of microvilli and cellular processes. SEM also revealed mycoplasmal contamination that was not detected by culturing. Comparisons of contaminated and uncontaminated RTG-2 cells showed enhanced (2–100 fold) replication of infectious pancreatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV) in the presence of A, laidlawii . 相似文献
11.
为查明养殖鲤（Cyprinus carpio）突然大批发病死亡的原因，对鲤病样品进行了细胞攻毒、空斑测定、电镜观察，以及鱼体感染等实验。先以患病鲤的组织匀浆液，经过滤后，分别接种到草鱼鳍条细胞（GCF）、鲤上皮瘤细胞（EPC）等14种培养细胞中。利用倒置显微镜观察显示，在1-2d内，该病鱼组织匀浆液可使其中9种细胞出现典型的细胞病变。收集出现病变的细胞液（即病毒悬液），进一步进行病毒滴度检测、空斑测定和鱼体感染实验。结果显示，在GCF细胞上的病毒滴度为10^7.3TCID50／mL；在FHM，TSB和GCO等细胞中可产生直径1～4mm的圆形空斑，空斑的大小与宿主细胞的种类和接种的病毒浓度有关。通过对感染细胞制备的超薄切片和病毒负染样品进行电镜观察，显示这是一类呈典型子弹头样的弹状病毒颗粒。感染了病毒悬液的鲫和鲤先后在第2天和第3天开始出现病症，间隔1～2d后发病的鱼开始死亡，至第14天，两种感染鱼的死亡率均达到83．3％。收集人工感染后濒死的鲫和鲤，分别制备组织匀浆液，回接感染鱼类培养细胞，24h内能使其出现与原发病鲤组织匀浆液所引起的类似的细胞病变。因此证实患病鲤是由病毒病原感染所致。[中国水产科学，2006，13（4）：617—623] 相似文献
12.
G Kumar M Saleh A‐A S Abdel‐Baki S Al‐Quraishy M El‐Matbouli 《Journal of fish diseases》2014,37(5):443-449
Heterosporis saurida is a microsporidian that infects lizardfish, Saurida undosquamis (Richardson, 1848), in the Arabian Sea. Spores were isolated from infected lizardfish and used to infect derived fish cell lines: common carp brain (CCB), epithelioma papulosum cyprinid (EPC), fathead minnow epithelial (FHM), rainbow trout gonad (RTG), bluegill fry (BF‐2) and chinook salmon embryo (CHSE). Non‐fish cell lines were also tested that include: insect (SF‐9), rabbit (RK‐13) and African green monkey (Vero E6). No growth of H. saurida was observed in any fish cell line, SF‐9 or Vero E6 cell lines. H. saurida spores grew only in RK‐13 cell line and were detected by immunofluorescence. Developmental stages of H. saurida were seen in RK‐13 cells by light and transmission electron microscopy, and species identification was confirmed by sequencing. This study demonstrated that H. saurida was able to proliferate in the mammalian RK‐13 cell line, which thus represents an in vitro model for conducting molecular genetics and cell–pathogen interaction studies of Heterosporis. 相似文献
13.
为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。 相似文献
14.
15.
A rapid immunoperoxidase-based virus neutralization assay for salmonid alphavirus used for a serological survey in Northern Ireland 总被引：1，自引：0，他引：1
A modified virus neutralization (VN) assay was developed to replace an existing assay read on the presence or absence of virus-induced cytopathic effect (CPE). The modified assay used a monoclonal antibody to salmon pancreas disease virus as the first layer of an immunoperoxidase (IPX)-based immunostaining technique to detect viral growth. The IPX-based VN assay required only 3 days to perform, and the adoption of a 96-well microtitre format facilitated a high throughput of samples requiring small volumes of serum, cells and virus. When 352 sera from farmed salmon and 302 sera from farmed trout were tested by both the modified and the original CPE-based assays, overall correlations of 97.72 and 96.03% were, respectively, obtained (96.94% combined). When the modified assay was used to test 188 sera collected from wild salmonids in freshwater river systems in Northern Ireland, no positive results were recorded. 相似文献
16.
Cortez-San Martin M González-Contreras A Avendaño-Herrera R 《Journal of fish diseases》2012,35(6):431-436
Streptococcus phocae is a beta-haemolytic bacterium that causes systemic infections in Atlantic salmon, Salmo salar L., cultured in southern Chile and also in seals. In this study, the host-pathogen interaction between S. phocae and seven types of cell lines (fish and mammalian) was examined using an indirect fluorescent antibody and confocal microscopy (CM). Chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), salmon head kidney (SHK-1) and Atlantic salmon kidney were used as the fish cell lines, while human cervix epithelial adenocarcinoma (HeLa), African green monkey kidney fibroblast (Cos-7) and mouse leukaemic monocyte macrophage (Raw 264.7) were included as mammalian cell lines. Streptococcus phocae type strain ATCC 51973(T) and isolates LM-08-Sp and P23 were selected as representatives from the salmon and seal host, respectively. For the CM examination, monolayers seeded on round coverslips were studied at 2- and 20-h post-inoculation (pi). The results showed that there is no common infectivity pattern between the three S. phocae strains at 2-h pi and the cell lines tested, regardless of the source of isolation (seal or salmon). All S. phocae strains could internalize and were found inside the fish and mammalian cell cytoplasm after 20-h pi. Regardless of the cells studied (fish or mammal) and incubation (2 and 20 h), S. phocae was never observed inside the nuclei. Seal and salmon isolates showed the highest number of bacteria entering into the primate cell lines (HeLa and Cos-7) from 2-h pi, while ATCC 51973(T) was not found outside or inside the HeLa and Cos-7 cells. 相似文献
17.
Abstract. Five isolates of a rhabdovirus associated with a severe ulcerative disease of striped snakehead, Ophicephalus striatus, and other freshwater fish in S.E. Asia were examined for growth in a range of cold and warm water fish cell lines and for serological relationships to other pathogenic fish rhabdoviruses. All five isolates grew in AS, BF-2, snakehead, O. striatus and O. micropeltis, and climbing perch, Anabas testudineus, cell lines. CHSE-214, RTG-2, EPC, Nile tilapia, Oreochromis niloticus, and grass carp Ctenopharyngodon idella, cell lines were all refractory to infection. Cross neutralization tests showed the ulcerative disease rhabdovirus (UDRV) to be serologically distinct from VHSV, IHNV, EVA, EVX, SVCV, PFRV and perch rhabdovirus. UDRV appears to be a new distinct pathogen of freshwater fish in S.E. Asia. 相似文献
18.
Abstract. Steelhead trout, Oncorhynchus mykiss (Walbaum), fry were experimentally infected with infectious haematopoietic necrosis virus (IHNV) Round Butte 1983 (Type 1). Fry were sampled daily, before and during the epizootic. Fish tissues were tested for infectious virus by tissue culture assay and for IHNV nucleocapsid protein by alkaline phosphatase immunohistochemistry (APIH). The progression of virus through the tissues was followed by APIH until the fourteenth day. Viral infection progressed from two major sites: from the gills into the circulatory system; and from the oral region into the gastrointestinal tract and then into the circulatory system. Once in the blood, virus was disseminated to virtually every organ. Progression of IHNV within and between organs is discussed. 相似文献
19.
Valentin Eckart Takuya Yamaguchi Kati Franzke Sven M. Bergmann Pierre Boudinot Edwige Quillet Motokazu Kawanobe Neila Alvarez de Haro Uwe Fischer 《Journal of fish diseases》2019,42(2):181-187
The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV‐3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin‐4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis. 相似文献
20.
为了查明鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,Cy HV-2)的理化与生物学特性以及病毒在细胞内的超微形态发生过程,利用新建立的对Cy HV-2敏感的异育银鲫脑组织细胞系(Gi CB),对Cy HV-2的理化及生物学特性进行了详细研究,比较了不同来源鱼类细胞系对Cy HV-2感染的敏感性,并对体外培养细胞中Cy HV-2病毒粒子及其超微形态发生过程进行了电镜观察。结果显示,Cy HV-2对热、酸、碱、有机溶剂和冻融敏感;常用鱼类细胞系EPC、RTG-2、Koi-Fin、CIK、CCK、PF-Fin对Cy HV-2的感染不敏感,特异性巢式PCR检测盲传至第7代Cy HV-2细胞培养物,结果均为阴性;Cy HV-2在Gi CB细胞中的增殖动态研究结果表明:病毒感染细胞经过12 h的隐晦期,24 h开始进入对数生长期,96 h病毒滴度达到最高值(107.52±0.26 TCID50/m L),然后进入平台期;透射电子显微镜观察结果显示,Cy HV-2感染细胞可分为吸附与侵入、复制与装配、成熟与释放3个主要过程,病毒进入对数生长期后,被感染细胞内可见形态典型的疱疹病毒颗粒。 相似文献