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3.
Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 102 CFU mL?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 103 CFU g?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.  相似文献   

4.
A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.  相似文献   

5.
Streptococcus agalactiae is one of the major causative agents of tilapia streptococcosis, which has caused severe economic losses in aquaculture. Rapid and accurate detection of the pathogen is necessary to limit losses because of this disease. In this study, a loop‐mediated isothermal amplification (LAMP) assay was developed for the detection of S. agalactiae. Firstly, a set of four primers was designed to target the cfb gene of S. agalactiae. Then, using Bst DNA polymerase, the LAMP assay was performed at 65 C for 60 min and terminated at 80 C for 10 min in a simple water bath. Positive or negative results could be observed by direct visual inspection. There were no cross reactions with other bacterial species, indicating high specificity of the LAMP assay. The sensitivity of the LAMP assay for detecting S. agalactiae was about 20 cells per reaction. Moreover, the developed closed‐tube step has greatly improved the LAMP system. The LAMP method was also applied to detect S. agalactiae in infected tilapia tissue, demonstrating usefulness in diagnostics. Overall, this study showed that the cfb‐based LAMP assay was an effective method to detect S. agalactiae rapidly.  相似文献   

6.
L Zhou  P Li  S Ni  Y Yu  M Yang  S Wei  Q Qin 《Journal of fish diseases》2017,40(12):1831-1838
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7.
A rickettsia‐like organism, designated NZ‐RLO2, was isolated from Chinook salmon (Oncorhynchus tshawytscha) farmed in the South Island, New Zealand. In vivo growth showed NZ‐RLO2 was able to grow in CHSE‐214, EPC, BHK‐21, C6/36 and Sf21 cell lines, while Piscirickettsia salmonis LF‐89T grew in all but BHK‐21 and Sf21. NZ‐RLO2 grew optimally in EPC at 15°C, CHSE‐214 and EPC at 18°C. The growth of LF‐89 T was optimal at 15°C, 18°C and 22°C in CHSE‐24, but appeared less efficient in EPC cells at all temperatures. Pan‐genome comparison of predicted proteomes shows that available Chilean strains of P. salmonis grouped into two clusters (p‐value = 94%). NZ‐RLO2 was genetically different from previously described NZ‐RLO1, and both strains grouped separately from the Chilean strains in one of the two clusters (p‐value = 88%), but were closely related to each other. TaqMan and Sybr Green real‐time PCR targeting RNA polymerase (rpoB) and DNA primase (dnaG), respectively, were developed to detect NZ‐RLO2. This study indicates that the New Zealand strains showed a closer genetic relationship to one of the Chilean P. salmonis clusters; however, more Piscirickettsia genomes from wider geographical regions and diverse hosts are needed to better understand the classification within this genus.  相似文献   

8.
Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV‐2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV‐2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV‐2. The highly conserved ORF72 of CyHV‐2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA‐LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross‐reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA‐LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV‐2 when resources are limited.  相似文献   

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No gold standard assay exhibiting error‐free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non‐culture assays in three matrices (phosphate‐buffered saline, ovarian fluid and kidney tissue). Non‐culture assays included polyclonal enzyme‐linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane‐filtration FAT, nested polymerase chain reaction (nested PCR) and three real‐time quantitative PCR assays. Injection challenge of specific pathogen‐free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.  相似文献   

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12.
This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 100 amplicon copies per assay (equivalent to 2 × 10?11 ng/µl, Cq value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 100 gene copies in tissues artificially infected and 0.02 × 100 in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.  相似文献   

13.
White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost‐effective strategy for long‐term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 103 fold higher in disease susceptible than that in disease resistant shrimps using real‐time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease‐free shrimp aquaculture industry.  相似文献   

14.
Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus‐2 (CyHV‐2), has affected the commercial production of the goldfish Carassius auratus and gibelio carp Carassius auratus gibelio. High water temperature treatments are reported to reduce the mortality rate of infected goldfish and elicit immunity in the survivors. To define the mechanism by which this intervention induces resistance, clonal ginbuna Carassius auratus langsdorfii, which is closely related to both species and has been used in fish immunology, may represent a promising model species. In this study, we investigated the susceptibility of clonal ginbuna strains to CyHV‐2 and the effect of high water temperature treatment on infected ginbuna and goldfish. Experimental intraperitoneal infection with CyHV‐2 at 25 °C caused 100% mortality in ginbuna strains, which was accompanied by histopathological changes typical of HVHN. Both infected ginbuna S3n strain and goldfish, exposed to high temperature for 6 days [shifting from 25 °C (permissive) to 34 °C (non‐permissive)], showed reduced mortalities after the 1st inoculation, and subsequent 2nd virus challenge to 0%, indicating induction of immunity. It was concluded that ginbuna showed a similar susceptibility and disease development in CyHV‐2 infection compared to goldfish, suggesting that ginbuna can be a useful fish model for the study of CyHV‐2 infection and immunity.  相似文献   

15.
Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real‐time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real‐time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra‐ and interassay coefficient of variation (CV) values ranged from 0.42–1.95%, demonstrating that the assay has good reproducibility. There was not any cross‐reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real‐time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real‐time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples.  相似文献   

16.
Carp oedema virus (CEV) and koi herpes virus (KHV) are of major concern to common carp breeders and koi enthusiasts worldwide. The viruses cause diseases that exhibit similar external signs; thus, it is difficult to distinguish between them clinically. In this study, we developed and optimized rapid and accurate single‐ and multiplex isothermal diagnostic tools, based on recombinase polymerase amplification (RPA), for detection and differentiation of CEV and KHV. The assays were combined with a lateral flow dipstick to enable visual detection of amplification products and simplify post‐amplification analysis. Both CEV‐ and KHV‐RPA assays were specific for their target virus. The lower detection limits of the assays were similar to those of established diagnostic PCR tests for the viruses. A sample preparation method was optimized to eliminate the need for total DNA extraction from fish tissues. The estimated time to perform these RPA assays, from receiving the sample to having a result, is 50 min, compared to 10 and 7 hr for CEV‐ and KHV‐PCR tests, respectively. The assays can be performed in field situations to improve screening of fish and reduce spread of these viruses and thereby enhance the common carp and koi industries.  相似文献   

17.
A better understanding of the relationships between oceanic environments and fishing conditions could make the utilization of fish more efficient, profitable, and sustainable. The current lack of high‐precision subsurface seawater information has long been a constraint on fishery research. Using near‐real‐time Argo observations, this paper presents a new approach called gradient‐dependent optimal interpolation. This approach provides daily subsurface oceanic environmental information according to fishery dates and locations. An experiment was conducted in the western and central Pacific Ocean using yellowfin tuna (YFT) catch data in August 2017. The results of seawater temperature and salinity represented differences of less than ±0.5°C and ±0.05, respectively, according to verification of error analysis and truth‐finding comparisons. After applying the constructed temperature and salinity profiles, we described the relationship between subsurface information and yellowfin tuna catch distribution. Statistical analysis revealed that yellowfin tuna were more adapted to warmer and saltier seawater. At the near‐surface (<5 m), the most suitable temperature was 28–29°C, although yellowfin tuna can endure a temperature range from 11 to 12°C at a depth of 300 m. The corresponding upper boundary of the thermocline was approximately 75 m, with a mean strength of 0.074°C/m, and the most suitable salinity for yellowfin tuna was 34.5–36.0 at depths shallower than 300 m. These results indicated that the constructed subsurface information was very close to the true values and they had high spatial and temporal accuracy.  相似文献   

18.
Although breeding of rare shell colour variants has drawn widespread attention from shellfish breeders, the potential disadvantages of their adaptive capacity have been ignored in practice. To explore the difference in adaptive capacity between orange shell variant (OSO) and commercially cultured population (CPO) of the Pacific oyster Crassostrea gigas at early life stage, the development to D‐larvae and larval survival and growth (just 23 and 30°C for larval experiment) of them were compared under different temperature (16, 23 and 30°C) and salinity (17, 25 and 33 psu) combinations. In this study, at 23°C and 25 psu, for both OSO and CPO there was no difference in fertilization rates and survival (p > .05) (mean percentages of D‐larvae after fertilized 40 hr ≥ 95.00%; mean larval survival rates on day 10 > 80.00%). However, the percentage of D‐larvae of CPO at 40 hr was significantly (p < .05) higher than OSO at temperatures of 16 and 30°C and 25–33 psu and 17 psu at 23°C. Similarly, CPO has a better larval survival on day 10 and growth than OSO at salinities of 17 and 33 psu at 23°C. Overall, our results indicate that OSO can have an equally good performance like CPO at early life stage under optimal condition (23°C; 25 psu), but the potential disadvantages in adaptive capacity will be shown at suboptimal conditions. These findings can guide future hatchery breeding of OSO, and suggest the potential disadvantages in adaptive capacity in rare colour variants need more attention in further breeding.  相似文献   

19.
A need to improve larval rearing techniques led to the development of protocols for catecholamine‐induced settlement of flat oyster, Ostrea angasi, larvae. To further refine these techniques and optimize settlement percentages, the influence of salinity or temperature on development of O. angasi larvae was assessed using epinephrine‐induced metamorphosis. Larvae were reared between salinities of 15–35 and temperatures between 14.5 and 31°C. The greatest percentage survival, growth, development occurred when larvae were reared between 26 and 29°C and between salinities of 30 and 35. Larvae reared outside this salinity and temperature range exhibited reduced growth, survival and/or delayed development. Short‐term (1 h) reduction in larval rearing temperature from 26°C to 23.5°C significantly increased larval metamorphosis without affecting larval survival. Short‐term (1 h) increase in larval rearing temperature from 26°C to 29 and 31°C decreased larval survival and metamorphosis. To ensure repeatability in outcomes, tests showed that larvae sourced from different estuaries did not vary significantly in their metamorphic response to short‐term temperature manipulation and epinephrine‐induced metamorphosis.  相似文献   

20.
Nile tilapia (Oreochromis niloticus) juveniles were fed diets containing 13 g/kg total polyunsaturated fatty acids (PUFAs) at different n‐3/n‐6 dietary ratios (0.2, 0.5, 0.8, 1.3 and 2.9) for 56 days, at 28°C. Subsequently, fish were submitted to a winter‐onset simulation (22°C) for 33 days. PUFA n‐3/n‐6 dietary ratios did not affect fish growth at either temperature. At 28°C, tilapia body fat composition increased with decreasing dietary PUFA n‐3/n‐6. Winter‐onset simulation significantly changed feed intake. The lowest dietary n‐3/n‐6 ratio resulted in the highest feed intake. At both temperatures, body concentrations of α‐linolenic acid, docosahexaenoic acid, eicosatrienoic acid and docosapentaenoic acid decreased as dietary n‐3/n‐6 decreased. Body concentrations of eicosapentaenoic acid (EPA, 20:5 n‐3) increased with decreasing concentrations of dietary EPA. The n‐6 fatty acids with the highest concentrations in tilapia bodies were linoleic acid and arachidonic acid (ARA, 20:4 n‐6). At 28°C, SREBP1 gene expression was upregulated in tilapia fed the lowest n‐3/n‐6 diet compared to tilapia fed the highest n‐3/n‐6 ratio diet. Our results demonstrate that a dietary PUFA of 13 g/kg, regardless of the n‐3/n‐6 ratio, can promote weight gains of 2.65 g/fish per day at 28°C and 2.35 g/fish per day at 22°C.  相似文献   

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