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1.
Abstract

The etiological agent of bacterial coldwater disease (BCWD), Flavobacterium psychrophilum, can be transmitted both vertically and horizontally. Outbreaks of BCWD can result in significant losses in salmonid aquaculture. Reduction of outbreaks in fry may be possible through implementation of a management strategy in which progeny of heavily infected broodstock are culled from the general population. Diagnostic assays to quantify F. psychrophilum concentrations in tissue samples and confirm presence of the bacterium in ovarian fluid have been previously validated. In the current study, these assays were used to screen 60 female Rainbow Trout Oncorhynchus mykiss and 60 female Coho Salmon O. kisutch broodstock at two aquaculture facilities. Eyed eggs from 10 female broodstock (five fish from each facility) exhibiting graded levels of infection were transferred to the University of Idaho and monitored through early life stages for the presence of F. psychrophilum. Female Rainbow Trout broodstock were not positive for F. psychrophilum by enzyme-linked immunosorbent assay (ELISA) and prevalence was low in these progeny. However, ELISA optical density values for kidney correlated to F. psychrophilum prevalence in progeny (r = 0.938, P < 0.05) of Coho Salmon. Nested PCR on ovarian fluid was not a reliable indicator of vertical transmission in either species as broodstock ovarian fluid results did not correlate to F. psychrophilum prevalence in eyed eggs. Further research with these assays is necessary; however, results from this study indicate that broodstock screening may be a potential tool for evaluating F. psychrophilum infection levels, which could become an important component for disease management.

Received December 19, 2012; accepted December 8, 2013  相似文献   

2.
为了开发基于嗜冷黄杆菌CRISPR/Cas系统的基因组编辑技术,本研究对嗜冷黄杆菌的CRISPR/Cas系统结构及其作用机制进行生物信息学分析。从GenBank数据库中获得8株嗜冷黄杆菌的全基因组序列,利用CRISPRCasFinder软件查找成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)结构和CRISPR相关(Cas)蛋白在嗜冷黄杆菌基因组上的数量和分布;利用CRISPRFinder软件分析CRISPR结构的重复序列和间隔序列,并使用Mega X软件对cas基因核苷酸序列做相似性对比;通过与重复序列配对,获得反式激活crRNA (trans-activating CRISPR RNA,tracrRNA)与重复序列配对序列,使用ARNold软件预测tracrRNA基因的终止子;使用BPROM软件预测tracrRNA基因和crRNA前体(pre-CRISPR RNA,pre-crRNA)的启动子;使用Clustal X软件对所有的间隔序列做相似性对比,并使用CRISPRTarget软件对独特的间隔序列配对从而获得原间隔序列(protospacers)及原间隔序列邻近基序(protospacer adjacent motif,PAM)序列;使用WebLogo软件使PAM序列可视化。结果显示,8株嗜冷黄杆菌均含有1个完整的CRISPR/Cas9系统,由1个CRISPR结构和3个Cas蛋白组成。CRISPR结构由短而重复的序列即重复序列和短而可变的序列即间隔序列相间排列组成;重复序列大小为46 bp,核苷酸序列高度保守;间隔序列大小在29~31 bp之间,数量在20~41个之间。Cas蛋白含有Cas9、Cas1和Cas2,并且cas基因核苷酸序列高度保守。8株嗜冷黄杆菌的tracrRNA基因均位于cas9基因上游并且核苷酸序列相似性为100%。tracrRNA上有一段大小为24 bp的核苷酸序列,其中23个核苷酸与重复序列完全配对。每个重复序列均含有一个较短的启动子,可单独启动pre-crRNA的转录。不同菌株的间隔序列比对结果表明,新获得的间隔序列可以插入到嗜冷黄杆菌CRISPR结构的5'端或内部。在65个独特的间隔序列中,13个间隔序列能够配对上原间隔序列,这些原间隔序列均来源于噬菌体或质粒。原间隔序列上游侧翼序列分析结果表明,嗜冷黄杆菌Cas9识别的PAM序列是5‘-GANTTTT-3’。以上结果表明嗜冷黄杆菌的CRISPR/Cas9系统理论上可以开发适用于嗜冷黄杆菌的基因组编辑技术。  相似文献   

3.
Experimental infections and lethal dose 50% (LD 50) evaluation were conducted in rainbow trout fingerlings, using a virulent strain of Flavobacterium psychrophilum processed and stored or maintained in different ways; lyophilisation, freezing at -80 degrees C, maintenance in enriched Anacker and Ordal (EAO) medium at 4 degrees C, revival and subsequent in vivo passages in fish. Experiments were performed 1, 8 and 23 months after storing the bacteria. Out of a total of 12 cultures revived for experimentation, one failed to grow and another was found to express modified properties including decreased virulence in spite of in vivo passages. In all other cases, whatever the conditions of preservation, virulence was fairly well maintained after 1 and 8 months of storage. In the last test, after 23 months, the bacteria maintained in the EAO medium at 4 degrees C were found significantly attenuated. Conversely, lyophilised and frozen bacteria only expressed a slight increase in LD0. It was concluded that virulent strains of F. psychrophilum were likely to retain their properties without special provisions within limited periods of time, and that both lyophilisation and freezing at -80 degrees C were reliable methods for long-term preservation of virulence.  相似文献   

4.
Abstract

Juvenile rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta acclimated to freshwater or salinities of 9.0‰ or less were exposed to Yersinia ruckeri, the bacterial pathogen that causes enteric redmouth disease (ERM). Both species of fish were kept in the same recirculating systems after bacterial exposure. Rainbow trout mortality was significantly (P < 0.05) different in each salinity: 96.5% in freshwater, 89.5% in water of 1.1‰ salinity, 81.3% in 3.0‰ salinity, and 75.0% in 9.0‰ salinity (model SE = 1.0). All deaths occurred between 3 and 12 d after exposure to Y. ruckeri. Only 2.3% of brown trout in all salinities died, and differences among treatments were not significant. For both fish species, Y. ruckeri was isolated from liver, spleen, and trunk kidney of fish dying during this experiment, and lesions of rainbow trout were consistent with ERM. Yersinia ruckeri was not isolated from brown trout surviving for 21 d after bacterial exposure but was isolated from 3 of 24 surviving rainbow trout; a polymerase chain reaction assay detected the DNA of Y. ruckeri in 3 additional rainbow trout survivors. Neither the lesions of fish with ERM nor the percentage of surviving fish subclinically infected with Y. ruckeri was affected by salinity. Bacterial growth in vitro was not affected by low (≤9.0‰) salinity; however, bacterial adhesion to polystyrene was significantly reduced as salinity increased. Although mortality caused by Y. ruckeri was significantly lower for rainbow trout in water with slightly increased salinity, none of the salinities tested was effective in preventing serious losses caused by this pathogen in recirculating systems.  相似文献   

5.
四种药物对虹鳟鱼苗的毒性研究   总被引:12,自引:1,他引:11  
虹鳟鱼是一种具有极高营养价值和食用价值的冷水性鱼类 ,随着养殖的发展 ,虹鳟鱼的病害也日益严重 ,生产上因使用药物种类和剂量不当而造成虹鳟鱼死亡的现象时有发生。由于缺乏虹鳟鱼对某些药物的敏感性资料 ,文章对虹鳟鱼苗进行了 4种常用药物的急性毒性试验。结果表明 ,虹鳟鱼苗对四种药物的敏感性依次为孔雀石绿 (现已禁用 ) >高锰酸钾 >硫酸铜与硫酸亚铁合剂 >溴氯海因 ,4种药物对虹鳟鱼苗的安全浓度分别为 0 .0 2 5mg/L,0 .2 1 7mg/L,0 .574mg/L,0 .82 7mg/L。溴氯海因对虹鳟的安全浓度远高于常用治疗浓度 ( 0 .3 mg/L ) ,是虹鳟较理想的治疗药物 ;硫酸铜与硫酸亚铁合剂对虹鳟的安全浓度为略低于常用治疗浓度 ( 0 .7mg/L) ,是治疗浓度的 82 % ,故使用时要准确掌握用量 ;高锰酸钾和孔雀石绿对虹鳟的安全浓度远低于常用治疗浓度 (分别为 2 mg/L和 0 .1 mg/L ) ,所以不宜作为虹鳟鱼病的防治药物  相似文献   

6.
Abstract

During 1996 and 1997, 112 rainbow trout Oncorhynchus mykiss and 204 brown trout Salmo trutta, all young of the year, were sampled from a 40-km study area of the upper Colorado River and were examined for gill parasites. Ambiphrya, Chilodonella, Ichthyobodo, Apiosoma, Trichodina, Trichodinella, Tripartiella, Epistylis, and an unidentified cochliopodid amoeba were the representative protozoan genera observed on fish examined. Significant month–year–species interactions (P = 0.0295) were revealed, reflecting the changes in infestation prevalence among months, years, and species of salmonid. Greater ectoparasite richness was observed in downstream sections of the study area, most notably near Hot Sulphur Springs, Colorado. Peaks of infestation intensity and ectoparasite richness occurred in August and September of both years, presumably because of high mean water temperatures and low flows during that time.  相似文献   

7.
In order to investigate methods for controlling systemic bacterial coldwater disease (CWD), bacteriophages that infect Flavobacterium psychrophilum were isolated by the enrichment method from pond water collected from Japanese ayu farms. The five phages isolated were classified as members of Myoviridae (PFpW-3, PFpC-Y), Podoviridae (PFpW-6, PFpW-7), and Siphoviridae (PFpW-8) and had highly variable patterns of infectivity for different F. psychrophilum isolates (n = 128). The stability tests of the phages in different waters, pHs and temperatures were assessed, and the results indicated that none of the phages were affected by ayu farm conditions. Among the phages, PFpW-3 had high infectivity for F. psychrophilum isolated from ayu and other fish and demonstrated sufficient survivability in the stability tests. Thus, PFpW-3 and its indicator strain N2-3 were inoculated into cytophaga broth at different doses of multiplicity of infection (MOI) and proved to be efficient for the reduction of bacterial growth. This study may be the basis for a further evaluation of phage therapy in the treatment of CWD in Japanese ayu farms.  相似文献   

8.
Abstract

A method was developed to experimentally induce saprolegniasis in rainbow trout Oncorhynchus mykiss. The development of a reliable method to produce infected fish is essential to efforts to determine the efficacy of various antifungal treatments. Three methods for inducing saprolegniasis were evaluated in waters containing known concentrations of Saprolegnia parasitica zoospores. These methods included application of the following stressors to fish: (1) abrasion and dewatering, (2) water temperature increase, and (3) a combination of abrasion, dewatering, and temperature increase. Neither physical abrasion nor temperature increase stress alone was effective for inducing saprolegniasis. Only 25.9% of fish stressed by abrasion and dewatering alone became infected. Application of both abrasion and temperature stress, however, induced saprolegniasis in 77.8% of fish tested. Most of these fish became infected after 5 d of stress treatments. No fish became infected or died in the positive control group (not stressed but exposed to S. parasitica zoospores) or the negative control group (not stressed or challenged). This method should enable researchers to induce saprolegniasis in rainbow trout to study its pathogenesis or to test the efficacy of antifungal treatments. In conducting efficacy studies, it is important that therapeutic treatments begin promptly after the first signs of saprolegniasis are observed because the disease can progress very quickly and often results in mortality.  相似文献   

9.
Abstract

A lectin that agglutinates rabbit red blood cells (RBCs) and human type B RBCs was isolated from ova of rainbow trout Oncorhynchus mykiss. Hemagglutination of rabbit RBCs was inhibited completely by 10 mM L-rhamnose but not by certain other sugars, 100 mM EDTA, or 100 mM 2-mercaptoethanol. Partial purification of this hemagglutinating material was achieved by affinity chromatography of an H2O-dialyzed yolk homogenate on rhamnose-linked Sepharose. A polyacrylamide gel electrophoresis (SDS-PAGE) performed on this sample revealed two polypeptides with approximate molecular masses of 19 kilodaltons (kDa) and 30 kDa. By fast-phase liquid chromatography, proteins with a molecular mass of less than 20 kDa were separated from other elements of the affinity-purified hemagglutinating material. These proteins were found to lack hemagglutinating activity. When a western blot with rabbit antilectin antiserum was performed against yolk extract, rainbow trout serum, or yolk from larvae, a 30-kDa polypeptide was detected within all three samples. If the rainbow trout serum and egg lectins are the same molecule, then the biological function of the rainbow trout egg lectin may include host defense or perhaps a basic, homeostatic mechanism such as glycoprotein transport  相似文献   

10.
Abstract

A primary intraperitoneal (IP) vaccination of extracellular products (ECP) from Mycobacterium spp. (strains TB40, TB267, or Mycobacterium marinum) mixed with Freund's incomplete adjuvant and followed by a secondary IP injection at 8 weeks resulted in the elevation of both the nonspecific and the specific immune responses of rainbow trout Oncorhynchus mykiss. Increased nitroblue tetrazolium and phagocytosis activity were observed in these fish; peaks in activity occurred at weeks 2 and 6 after primary immunization with a third peak at week 10. Lysozyme activity, on the other hand, peaked at weeks 2 and 8 after primary immunization except in the TB40-immunized group. A third peak of lysozyme activity was observed at week 10 after primary immunization. The activity of the specific immune response was monitored by an enzyme-linked immunosorbent assay and Western blot. The results indicate that antibodies to the ECP of Mycobacterium spp. were present in rainbow trout serum and that they reacted with major ECP antigens at 65 and 16 kDa (kilodaltons) as well as with some minor antigens at 48, 46, and 40 kDa.  相似文献   

11.
Abstract

Laboratory studies were conducted on the susceptibility of different strains of Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss to infection with the monogenean Gyrodactylus salaris. This parasite, probably originating from the Baltic Sea region, is known to minimally affect Neva River (Baltic Sea) Atlantic salmon. However, following its introduction into Norway, G. salaris has caused severe mortality and morbidity among Norwegian Atlantic salmon, which are considered a highly susceptible strain. The cohabitation experiment included one stock of rainbow trout and four different strains of Atlantic salmon from the Baltic Sea region (Mörrum River, Sweden), Europe (Skjern River, Denmark; Conon River, Scotland), and North America (Bristol Cove River, Canada). Fish were exposed to a Norwegian strain of G. salaris, and parasite population development and distribution were monitored for 7 weeks. Rainbow trout exhibited low susceptibility to G. salaris infection, whereas Conon River and Skjern River Atlantic salmon were highly susceptible and exhibited high mortality rates. Mörrum River Atlantic salmon exhibited intermediate susceptibility and low mortality. Bristol Cove River Atlantic salmon harbored relatively low parasite numbers, but fish mortality was high. Our experiment showed that the Danish Skjern River strain of Atlantic salmon is highly susceptible to G. salaris infection, further supporting the hypothesis that Atlantic Ocean strains are more susceptible to G. salaris infection than are Baltic strains.  相似文献   

12.
Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, threatens wild and propagated salmonids worldwide and leads to substantial economic losses. In addition to being horizontally transmitted, F. psychrophilum can be passed from infected parents to their progeny, furthering the negative impacts of this pathogen. In Michigan, both feral and captive salmonid broodstocks are the gamete sources used in fishery propagation efforts. A 5-year study was initiated to follow the prevalence of systemic F. psychrophilum infections in feral broodstocks of four species (steelhead Oncorhynchus mykiss [potadromous Rainbow Trout]; Coho Salmon O. kisutch; Chinook Salmon O. tshawytscha; and Atlantic Salmon Salmo salar) residing in three Great Lakes watersheds. Additionally, captive broodstocks of four species (Rainbow Trout, Brown Trout Salmo trutta, Lake Trout Salvelinus namaycush, and Brook Trout Salvelinus fontinalis) maintained at two facilities were assessed for the presence of F. psychrophilum. The resultant offspring from each broodstock population were sampled for F. psychrophilum infections multiple times throughout hatchery residency. Using selective flavobacterial culture and PCR confirmation, F. psychrophilum was detected in all broodstocks except the captive Lake Trout and Brook Trout. Logistic regression analysis demonstrated that among the infected feral broodstocks, Chinook Salmon from the Lake Michigan watershed had the highest prevalence of systemic F. psychrophilum infection (mean = 63.2%). Among the captive broodstocks, the Gilchrist Creek strain of Brown Trout had the highest infection prevalence (mean = 5%). Collectively, the captive broodstocks were found to have significantly lower infection prevalence than the feral broodstocks. Despite the high prevalence of systemic F. psychrophilum infections in many broodstock populations, the bacterium was rarely detected in their progeny during hatchery rearing. However, heavy losses associated with clinical BCWD outbreaks did occur. Collectively, our results reinforce that BCWD continues to threaten Great Lakes basin salmonids.

Received April 6, 2015; accepted August 25, 2015  相似文献   


13.
Flavobacterium columnare and F. psychrophilum are major fish pathogens that cause diseases that may require antimicrobial therapy. Choice of appropriate treatment is dependent upon determining the antimicrobial susceptibility of isolates. Therefore we optimized methods for broth microdilution testing of F. columnare and F. psychrophilum to facilitate standardizing an antimicrobial susceptibility test. We developed adaptations to make reproducible broth inoculums and confirmed the proper incubation time and media composition. We tested the stability of potential quality-control bacteria and compared test results between different operators. Log phase occurred at 48 h for F. columnare and 72–96 h for F. psychrophilum, confirming the test should be incubated at 28°C for approximately 48 h and at 18°C for approximately 96 h, respectively. The most consistent susceptibility results were achieved with plain, 4-g/L, dilute Mueller–Hinton broth supplemented with dilute calcium and magnesium. Supplementing the broth with horse serum did not improve growth. The quality-control strains, Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658, yielded stable minimal inhibitory concentrations (MIC) against all seven antimicrobials tested after 30 passes at 28°C and 15 passes at 18°C. In comparison tests, most MICs of the isolates agreed 100% within one drug dilution for ampicillin, florfenicol, and oxytetracycline. The agreement was lower with the ormetoprim–sulfdimethoxine combination, but there was at least 75% agreement for all but one isolate. These experiments have provided methods to help standardize antimicrobial susceptibility testing of these nutritionally fastidious aquatic bacteria.

Received June 24, 2015; accepted October 2, 2015.  相似文献   


14.
Abstract

Dissolved oxygen accounted for a significant proportion of the variation in mortality rate of rainbow trout Oncorhynchus mykiss acclimated for 10 weeks to a range of dissolved oxygen levels and challenged with Yersinia ruckeri. Moderate levels of oxygen supersaturation (150%) resulted in greater cumulative mortality (17.9%) among fish exposed to a 0.5-h static bath challenge with Y. ruckeri (1 × 107 colony-forming units/mL). In contrast, hypoxic (70%) and normoxic (100%) levels of dissolved oxygen resulted in 12.8% and 10% cumulative mortality. Higher oxygen levels provided no additional “protection” against infection by Y. ruckeri in this study.  相似文献   

15.
Florfenicol (FF) is employed in fish farms to contest or prevent bacterial infections. However, this pharmaceutical may produce reactive oxygen species that may cause biochemical changes in antibiotic-treated fish. The aim of this study was to evaluate the effect of FF on Rainbow Trout Oncorhynchus mykiss treated for 10 d with 7.5 and 15 mg/kg FF followed by a withdrawal period of 5 d. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glyoxalase I and glyoxalase II, total glutathione, lactic dehydrogenase, and alkaline phosphatase were investigated in the livers of treated and untreated fish. A general impairment of antioxidant enzymes and metabolic indicators was measured in FF-treated Rainbow Trout. Onset of oxidative damage may have occurred during the antibiotic treatment as a consequence of the effect of FF toxicity at mainly the highest dose. Nevertheless, the rise in levels of total glutathione and glutathione S-transferase even after the withdrawal period may shield the antibiotic-mediated oxidative processes.

Received December 22, 2015; accepted May 26, 2016 Published online October 28, 2016  相似文献   


16.
The etiological agent of bacterial cold-water disease, Flavobacterium psychrophilum, can cause significant losses of salmonid fishes in aquaculture facilities. Few studies describing the value of media components on the growth of F. psychrophilum are available in the literature. We therefore conducted a study that began with the standard enriched Anacker-Ordal broth (EAO) and over the course of multiple iterations evaluated the effects of various media supplements by adding or subtracting them from the base EAO medium. Different media formulations were made, and samples were removed from each broth formulation every 24 h for 72 h. From those samples we determined bacterial density by measuring absorbance values with a spectrophotometer. The medium with the highest absorbance value from one iteration was used as the base medium in the next iteration. Using this iterative approach, we determined that sodium acetate, calcium chloride, and magnesium sulfate inhibit growth and that maltose has no effect on the proliferation of the bacterium. The addition of skimmed milk (0.2%) and horse serum (1%) appears to provide a slight improvement in bacterial proliferation. Variations in agar concentration had no effect on the growth of the bacterium. Even though the addition and removal of some ingredients increased the mean absorbance values, the benefit of these substitutions was not significant. Even so, we found that the growth of F. psychrophilum in EAO was better than that in two other widely used media: tryptone-yeast extract salts and maltose infused tryptone-yeast extract salts.  相似文献   

17.
Abstract

Fry of rainbow trout Oncorhynchus mykiss were exposed to serotype VR-299 of infectious pancreatic necrosis virus (IPNV) by using a standardized immersion challenge. In concurrent experiments, fish were monitored for 11 d for excretion of IPNV or monitored for 9 d for excretion and transmission of IPNV to susceptible rainbow trout fry. Immersion-challenged fish began excreting virus within 2 d after challenge. The rate of IPNV excretion per fish increased steadily from about day 4 to day 8 and then decreased. Virus concentrations in tissues of immersion-challenged fish increased exponentially. Susceptible fish became infected with IPNV within 4 d after being introduced to immersion-challenged fish (e.g., 2 d after the challenged fish began excreting virus). By 9 d, 84% of the susceptible fish were infected with IPNV.  相似文献   

18.
CASE DESCRIPTION: A cohort of 35,200, 13-week-old, female rainbow trout at a fish farm was evaluated because of a 2-week history of anorexia and lethargy and a mortality rate of approximately 100 fish/d. CLINICAL FINDINGS: Affected fish were lethargic and thin and had disequilibrium, bilateral exophthalmia, pale red gills and kidneys, red-tinged coelomic fluid, and pale brown livers. Some fish were differentially pigmented bilaterally. The presumptive diagnosis was bacterial or viral septicemia. The definitive diagnosis was rainbow trout fry syndrome caused by infection with Flavobacterium psychrophilum. TREATMENT AND OUTCOME: A strategy for controlling the outbreak based on reducing pathogen numbers in affected tanks and reducing pathogen spread among tanks was developed. The option of treating with antimicrobial-medicated feed was discussed with the farmer, but was declined. After changes were made, mortality rate declined quickly, with no more deaths within 10 days after the initial farm visit. CLINICAL RELEVANCE: Bacterial coldwater disease is the most common manifestation of infection with F psychrophilum in fingerling and adult rainbow trout. However, the organism can also cause rainbow trout fry syndrome. This condition should be included on a list of differential diagnoses for septicemia in hatchery-reared rainbow trout fry.  相似文献   

19.
20.
Abstract

Blood parameters, disease resistance, and the immune response were sequentially evaluated in rainbow trout Oncorhynchus mykiss with proliferative kidney disease (PKD). The fish were maintained under laboratory conditions, and the study group went through a full cycle of the disease. Hematological and serological changes occurred primarily in those fish with severe kidney lesions. Fish infected with the parasite that causes PKD demonstrated a greater resistance to bacterial challenge, and their immune responses were heightened when compared with those of uninfected fish. These data suggest that PKD alone is not a predisposing factor for secondary infections if the fish does not incur severe renal lesions.  相似文献   

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