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1.
Abstract. The sensitivity of the immunoperoxidase (IP) and fluorescent antibody (FA) techniques applied to frozen sections of organs of carp infected with spring viraemia virus (SVCV) was similar, both in respect of the intensity of the reaction and in the detection rate of the antigen. Seven days after a waterborne infection both methods detected the antigen in the kidneys and to a lesser extent in the liver and spleen. Quantification of virus gave a titre of 102.8 to 105.5 TCID50/g of kidney, whereas the agent could not be isolated from liver and spleen tissue. In contrast, at 241/2 days post–infection the viral antigen could be readily detected in liver and spleen but only occasionally in the kidneys using the IP and FA techniques. At this time, liver and spleen showed infectivity titres of 103.8 to 107.2 TCID50/g tissue, whereas the kidneys were found to be free of infective virus. It is concluded that using the IP and FA techniques SVCV can be detected most frequently in the kidneys from 7 to 14 days post–infection and in the liver and spleen from 14 to 24 days post-infection.  相似文献   

2.
Plasma kinetics and tissue sites of degradation for native and chemically modified low density lipoproteins have been investigated in (Oncorhynchus mykiss). Native and modified LDL labelled with125I-tyramine-cellobiose, (a residualizing adduct), were injected intravenously, and plasma and organ samples analyzed. Native LDL were cleared with a half life of about 30 hours, and mainly catabolized in the liver. Acetylation of LDL resulted in accelerated clearance (t1/2=2 h) and catabolism in the kidneys. Methylation of LDL had only minor effects on catabolism. The cellular localization of lipoprotein uptake was visualized in kidney by fluorescence microscopy. Native LDL were endocytozed by spheroid, parenchymal cells, supposedly steroid-producing cells. Acetylated, fluorescent LDL were found in vacuoles of flattened, sinusoidal lining endothelial cells. Our data show that catabolism of native low density lipoproteins in salmonids takes place mainly via hepatic receptors. A scavenger receptor pathway, for modified lipoproteins (mainly localized in the kidney) is also operative in trout.  相似文献   

3.
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4.
Abstract. Radiolabelled (125I, 3H) immunomodulatory laminaran (isolated from Laminaria hyperborea ), a β(1,6)-branched β(1,3)-D-glucan and different radiolabelled sulphated analogues of laminaran were administered peranally to Atlantic salmon, Salmo salar L. Intestinal absorption and tissue distribution were examined by means of radioactive tracer techniques. The intestinal uptake was highest with native laminaran and laminaran with a low degree of sulphatation, while highly sulphated laminarans were poorly absorbed. The tissue distribution analysis revealed high amounts of radiolabelled compound in the liver, and anterior and posterior kidney, whereas the spleen contained low amounts. Peak serum and organ concentrations were reached about 30 min after administration. The results were confirmed by autoradiography of tissue sections from salmon after peranal administration of radiolabelled laminaran. Finally, the peranal administration of fluorescence labelled laminaran revealed epithelial supranuclear fluorescent vacuoles containing laminaran. It is concluded that native laminaran and slightly sulphated laminaran are absorbed from the posterior intestine and that they are distributed to tissues rich in immunocompetent cells. Thus, these compounds may have potential as immunomodulatory feed additives.  相似文献   

5.
Fingerling Atlantic salmon (Salmo solar) fed a production diet deficient in pyridoxine showed several significant effects: increased mortality; behavioral changes; degenerative changes in kidneys, ovaries, and liver; a paucity of thyroid colloid; and hyperplasia of renal hematopoietic tissue. Changes in nerve tissue were equivocal.  相似文献   

6.
Studies on the morphology of the liver of teleosts reflect some controversy in the interpretation of the data, but also provide confirmation of variations in the structure of the organ in several species. Thus, we intend to understand the specific structural organization of the liver of Astyanax altiparanae. Specimens were collected in the city of Andirá, Paraná, Brazil. The livers were processed according to histological routine for inclusion in Paraplast, and the sections were stained with HE and Mallory’s trichrome or followed the protocol for fluorescence immunohistochemistry, anti-cytokeratin. The liver of A. altiparanae was covered by a capsule of connective tissue, without delimiting lobes. The hepatocytes had an arrangement in cords around sinusoids. Melanomacrophage centers were observed. The vascular components and intrahepatic pancreatic acini were distributed between hepatocytes. Presence of cytokeratin was detected in tissues that lined the liver and endothelial cells of sinusoids. The comparison of the liver of A. altiparanae to other characids corroborates with the fact that there is variation in the morphology of the liver even between closely related species. Moreover, it appears that in this species, endothelial cells of sinusoids can synthesize the cytokeratin filaments required for the regulation of blood flow in capillaries in adults.  相似文献   

7.
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   

8.
During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).  相似文献   

9.
A fluorescent in situ hybridization (FISH) method was developed for detection of infectious pancreatic necrosis virus (IPNV) in paraffin-embedded tissues of Atlantic salmon, Salmo salar L. Several methods of probe labelling and detection were evaluated and found unsuitable for FISH because of tissue autofluorescence. Likewise, the use of avidin to detect biotin-labelled probe was obviated by the presence of endogenous biotin. An existing approach, using digoxigenin (DIG)-labelled probes and detection by anti-DIG antibody-labelled with alkaline phosphatase, was modified to use a fluorescent substrate, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate/4-chloro-2-methylbenzene diazonium hemi-zinc chloride salt (HNPP/Fast Red TR). This improved method allowed sensitive detection of IPNV target, without interference from autofluorescence or endogenous alkaline phosphatase. Furthermore, the reporter produces a discrete, non-fading signal, which is particularly suitable for analysis by confocal microscopy.  相似文献   

10.
The field use of a staphylococcal coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples from Atlantic salmon, Salmo salar L., was evaluated. The COA test was compared with an immunohistochemical (IHC) method for the detection of clinical outbreaks of infectious pancreatic necrosis (IPN). The present paper describes the evaluation of 320 COA test results performed at local fish health laboratories in Norway from 1994 to 1996, and COA test results from two infection trials with IPNV. The agreement between the COA test and the IHC was very good. The agreement beyond chance, measured as kappa values, was 0.74 in individuals and 0.90 in pooled samples. Thus, the COA test was suited for the detection of outbreaks of IPN. Covert infections with IPNV remained undetected by the COA test. The minimum IPNV titre needed to obtain a positive COA test was ≈ 105 TCID50 mL–1.  相似文献   

11.
Amyloid associated with pancreatic adenocarcinoma was discovered in two captive adult tricolour sharkminnows Balantiocheilus melanopterus Bleeker found dead in a freshwater display. Enlarged abdomens expanded by bloody ascitic fluid and grossly visible masses of abnormal tissue were present surrounding sections of the stomach and intestine. Histologically, the masses were composed of areas of well‐organized exocrine pancreatic acini interspersed with cords of poorly differentiated, spindle‐shaped cells that compressed and effaced normal parenchyma. These cells possessed small numbers of cytoplasmic zymogen granules; the exocrine nature of these cells was confirmed using transmission electron microscopy (TEM). Fibrovascular connective tissue of the hepatopancreas and mesenteries was expanded by lightly eosinophilic, hyaline, homogeneous acellular material. Similar material greatly expanded the tunica media of large blood vessels in the hepatopancreas. After staining with Congo red or thioflavin T, this material exhibited red–green dichroism under polarized light or bright green fluorescence under ultraviolet light (255 nm), respectively. The non‐branching fibrils, of indeterminate length, had an approximate diameter of 10–20 nm using TEM. Although exocrine pancreatic neoplasia is relatively common in fish, the presence of amyloid is not. To our current knowledge, the latter has not yet been described in association with a neoplastic lesion in fish.  相似文献   

12.
Abstract. A rainbow trout population of infectious pancreatic necrosis virus carriers was studied over a one-year period using both homogenization and co-cultivation for virus isolation. The percentage of virus-yielding fish was high between March and June, but declined during the latter part of the year. This was diametrically opposite to the trend in the serum antibody levels indicating that the marked humoral immune response resulted in a very significant reduction in the virus titres. The highest isolation rate was obtained from the kidneys after co-cultivation underlining the very high sensitivity of this method for virus detection. Virus was occasionally isolated from the faeces indicating that this may well be a possible avenue for horizontal transmission of the virus. No virus was ever detected in gonadal tissue; the mechanism of vertical transmission of the virus is still very poorly understood.  相似文献   

13.
In recent years, infectious disease caused by Streptococcus agalactiae has increased in aquaculture, threatening the healthy development of tilapia and resulting in substantial economic losses. Cultivating disease‐resistant tilapia by transgenic technology is one strategy that has been developed to address this issue. Here, we used a transposon system to investigate gene expression and tissue lytic activity in transgenic zebrafish expressing the tilapia lysozyme. The transpose vector contained the Tgf2 transposon skeleton, tilapia heat shock protein 70 promoter (Hsp70) and two target genes (tilapia C‐type lysozyme 3 [lysozyme‐C3] and green fluorescent protein [GFP]). The transgenic zebrafish F0 generation was obtained by microinjecting the donor vector and transposase mRNA into fertilized zebrafish eggs. There was 45% green fluorescence in the zebrafish F0 generation, which was spread over the entire body based on microscope observation. The F0 generation was reared to sexual maturity, at which point individuals were mated with wild‐type zebrafish to produce the F1 generation. Tilapia lysozyme‐C3 expression was detected in the liver of F1 generation by RT‐PCR and western blotting, but was not detected in the skin, intestine, or muscle. Significantly higher liver bacteriolysis activity was detected in transgenic zebrafish compared with wild‐type zebrafish. Therefore, transgenic zebrafish may have an increased potential for bacterial resistance.  相似文献   

14.
A method for the rapid serological identification of infectious pancreatic necrosis (IPN) virus using the complement fixation test, is described. The test uses a simple, easily prepared tissue culture antigen. Methods used for preparation of suitable antisera for the test and their inclusion in a polyvalent antiserum are described.  相似文献   

15.
The objective of this study was to investigate the toxic effects of buprofezin insecticide on Nile tilapia (Oreochromis niloticus). The fish were exposed to buprofezin at 100 mg/L for 28 days. Compared to control, activity of serum transferases and levels of urea and creatinine showed significant increases. Oxidative stress was recorded manifested by elevated levels of malondialdehyde (MDA), reduced concentrations of reduced glutathione (GSH) and inhibition of activities of superoxide dismutase (SOD) and catalase (CAT) in liver and kidney. Examination of peripheral RBCs revealed elevated frequency of micronucleated cell. Interleukin 1 beta (IL‐1β) gene was upregulated in liver, muscle and brain, while that of cyclooxygenase 2 (COX‐2) gene increased in liver and muscle, but not in brain. Histopathological alterations were recorded in liver, kidneys, brain, gills, pancreas, spleen, intestine, muscle and ovaries. The immunohistochemical detection of caspase‐3 in the liver revealed no differences between treated and control groups; however, the expression of inducible nitric oxide synthase (iNOS) was demonstrated in hepatocytes and hepatopancreas in buprofezin‐treated group compared to control. It has been concluded that the tissue damage induced by buprofezin in Nile tilapia is mediated by oxidative stress and inflammatory response but not by apoptosis.  相似文献   

16.
ABSTRACT:   Green fluorescent protein ( GFP ) and red fluorescent protein ( RFP ) genes regulated by the medaka skeletal muscle actin promoter were microinjected into fertilized d-rR medaka eggs to establish transgenic medaka lines. Intense fluorescence was detected in skeletal muscle. During development, GFP and RFP became detectable in anterior somites at the 12- and 30-somete stages, respectively. After hatching, intense fluorescence in skeletal muscle enabled individual fish to be identified under normal lighting without fluorescent microscopy. Fluorescence was also observed in the gills and esophagus of the adult fish. These data indicated that medaka lines are convenient not only for the study of skeletal muscle but also for the identification of cells or individuals in various studies.  相似文献   

17.
利用RT-PCR方法扩增出IPNV-ZYX分离株主要结构蛋白VP2的抗原表位区基因(616 bp), 命名为IPNV VP2 COE, 将其克隆到pCold TF表达载体中构建重组质粒pCold TF-VP2 COE, 在大肠杆菌BL21(DH5α)感受态表达, 经SDS-PAGE电泳分析, 表达蛋白约78 ku, 用镍离子亲和层析柱纯化该蛋白, 制备抗血清, 间接ELISA结果显示, IPNV (ATCC VR-1318)细胞培养物与鼠抗VP2 COE蛋白血清发生特异性反应, 效价为1∶12 800; 间接免疫荧光结果显示, 鼠抗VP2 COE血清可与黑龙江某渔场已知感染IPNV虹鳟肝组织产生特异性的荧光, 以上两项结果表明, 表达IPNV VP2 COE蛋白具有良好的免疫原性和免疫反应性, 为IPNV检测方法的建立及疫苗的制备提供理论依据  相似文献   

18.
Abstract. Cyprinid herpesvirus 1 (CHV) or Herpesvirus cyprini was virulent for carp, Cyprinus carpio L., fry following 1 h immersion in water at 20 °C. Cumulative mortality for carp fry was 86–97% in 2-week-old common carp, 20% in 4-week-old fancy carp, and 0% in both 8-week-old common and fancy carp. The virus did not produce mortality in fry of crucian carp, grass carp or other cyprinids. It was also oncogenic in carp, inducing papillomas to the extent of 55% among both common and fancy carp fry. The neoplasms appeared 5–6 months after carp had been exposed to the virus by immersion and recurred at an incidence of 83% in carp 7·5 months post-desquamation of the tumour. The CHV was reisolated from all moribund fish and from all survivors. It also induced papillomas at an incidence of 13% in adult mirror carp and at 10% in adult fancy carp 5 months after intraperitoneal inoculation of 105 TCID50 ml-1 fish. The virus was rcisolated only from the ncoplastic tissue and not from internal organs. The neoplasms were normally located on fin, skin or mandible, at the intraperitoneal inoculation site. Specific fluorescence for CHV antigen was frequently detected in the gills, liver, kidneys and intestine of 2-week-old fry from 3 to 21 days following challenge with CHV. It was found in greater concentrations in experimentally induced papillomata on 2-week-old carp fry survivors examined 24 weeks after challenge than in naturally occurring neoplasms.  相似文献   

19.
Infectious pancreatic necrosis (IPN) is a very serious viral disease in terms of its impact on production of Atlantic salmon, Salmo salar L., fry and post‐smolts. Post‐smolts of Atlantic salmon were injected with infectious pancreatic necrosis virus (IPNV) and cohabited with naive fish to produce natural infection. Cohabitant fish were sampled every 2 days, up to day 36 post‐infection (p.i.). From 90 cohabitant fish, 11 (12.2%) were positive by immunohistochemistry (IHC). The first detection of IPNV by IHC occurred on day 16 p.i. which coincided with the onset of mortality in this group. Besides the pancreas, the liver was found to be a key target organ for IPNV. For the first time, the virus was observed in the islets of Langerhans and in the kidney corpuscles of Stannius which suggests that the virus could affect the fish’s metabolism. The liver of two fish, which showed the most widespread presence of IPNV by IHC, had a pathology including focal necrosis and widespread presence of apoptotic hepatocytes, many of which did not stain for virus by IHC. Up‐regulation of cytokine gene expression was found only in the IHC‐positive (IHC+ve) fish and reflected the level of infection as determined by IHC positivity of the liver. In most fish, interferon (IFN), Mx, γIFN and γIP were up‐regulated in liver and kidney, while only IFN and Mx were up‐regulated in gill. IL1β and TNFα were not induced in any tissue. The gill showed variable levels of constitutive expression of IL1β and γIFN. The two fish with liver pathology had the highest level of IFN expression, especially relative to the level of Mx expression, in the liver compared with the other IHC+ve fish which did not have a liver pathology. The results suggest that following widespread infection of hepatocytes, the cells may over‐produce IFN, resulting in apoptosis of neighbouring cells with subsequent death from liver failure.  相似文献   

20.
为探究带鱼酶解蛋白亚铁螯合肽对泥鳅免疫特性的影响及其在泥鳅体内的代谢分布情况,通过在泥鳅饲料中添加不同比例带鱼酶解蛋白亚铁螯合肽,喂养40 d,检测泥鳅消化酶活性、血清和肝脏生理生化指标,运用活体成像技术检测FITC标记的带鱼酶解亚铁螯合肽在泥鳅体内的分布。结果显示,亚铁螯合肽添加量为2 g/kg时,脂肪酶活性极显著高于对照组;添加量为1 g/kg时,淀粉酶和胃蛋白酶活性显著高于正常对照组;各实验组胰蛋白酶活性相比于对照组无显著性差异。血清指标中超氧化物歧化酶和溶菌酶在添加量为2 g/kg时,活性显著高于正常对照组;相对与正常对照组,过氧化氢酶在添加量为1 g/kg表现为显著性差异,2 g/kg的添加量表现为极显著性差异;丙二醛在添加量为2 g/kg显著低于正常对照组。活体成像技术发现FITC标记的带鱼酶解蛋白亚铁螯合肽会较长时间滞留在泥鳅体内,荧光强度主要集中在泥鳅身体上部,推测带鱼酶解蛋白亚铁螯合肽被泥鳅吸收后主要分布在肝脏、胆囊等器官内。研究表明,饲料中添加1~2g/kg的带鱼酶解蛋白亚铁螯合肽能提高养殖泥鳅的消化道酶活性,增强泥鳅的非特异性免疫。  相似文献   

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