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1.
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   

2.
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   

3.
采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)、横向流动试纸条技术(Lateral flow dipstick,LFD),建立一种鳗利斯顿氏菌快速检测方法。针对鳗利斯顿氏菌金属蛋白酶基因设计一套特异性引物及异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记的探针,结合生物素标记的环介导等温扩增技术扩增反应和横向流动试纸条技术对鳗利斯顿氏菌进行检测;比较LAMP-AGE、LAMP-LFD和PCR-AGE的灵敏度;选取4株鳗利斯顿氏菌和6株非鳗利斯顿氏菌验证LAMP-LFD特异性。试验结果表明,应用LAMP-LFD,能在30 min内完成鳗利斯顿氏菌的检测;LAMP-LFD检测的灵敏度为7.7 cfu/ml,LAMP-AGE和PCR-AGE检测的灵敏度均为77 cfu/ml;4株鳗利斯顿氏菌均呈阳性反应,其他6株非鳗利斯顿氏菌均为阴性。应用LAMP-LFD检测鳗利斯顿氏菌特异性强、灵敏度高、并且操作安全、简便、快捷。  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.
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6.
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   

7.
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8.
Cyprinid herpesvirus 3 (CyHV‐3) is the aetiological agent of a serious and notifiable disease afflicting common and koi carp, Cyprinus carpio L., termed koi herpesvirus disease (KHVD). Significant progress has been achieved in the last 15 years, since the initial reports surfaced from Germany, USA and Israel of the CyHV‐3 virus, in terms of pathology and detection. However, relatively few studies have been carried out in understanding viral replication and propagation. Antibody‐based affinity has been used for detection of CyHV‐3 in enzyme‐linked immunosorbent assay and PCR‐based techniques, and immunohistological assays have been used to describe a CyHV‐3 membrane protein, termed ORF81. In this study, monoclonal antibodies linked to N‐hydroxysuccinimide (NHS)‐activated spin columns were used to purify CyHV‐3 and host proteins from tissue samples originating in either CyHV‐3 symptomatic or asymptomatic fish. The samples were next analysed either by polyacrylamide gel electrophoresis (PAGE) and subsequently by electrospray ionization coupled to mass spectrometry (ESI‐MS) or by ESI‐MS analysis directly after purification. A total of 78 host proteins and five CyHV‐3 proteins were identified in the two analyses. These data can be used to develop novel control methods for CyHV‐3, based on pathways or proteins identified in this study.  相似文献   

9.
Vibrio nigripulchritudo is considered one of the major pathogens threatening shrimp aquaculture. In this study, we developed a novel and highly specific quantitative loop-mediated isothermal amplification (Q-LAMP) assay. A set of four specific primers were designed targeting the V. nigripulchritudo intergenic spacer region. The reaction time and temperature were optimized for 60 min at 63°C. Quantitative analysis was then performed by measuring the turbidity of the reaction solution using a real-time turbidimeter, allowing for quantification of the initial DNA concentration with a sensitivity of 102 copy numbers equivalent to 2.3 colony forming units/ml or 0.3 fg/μl. The LAMP assay was able to specifically detect two representative strains of V. nigripulchritudo, whereas other Vibrio and non-Vibrio species were not amplified. A standard curve was generated for V. nigripulchritudo by plotting the threshold time (T t) versus the log of bacterial number. A high correlation coefficient (R 2 = 0.9749) was observed for the Q-LAMP reaction. In conclusion, Q-LAMP assay is a sensitive, rapid, and simple tool that can be used for the detection and quantification of V. nigripulchritudo in shrimp, thereby facilitating surveillance of vibriosis infection.  相似文献   

10.
Cyprinid herpesvirus 2 (CyHV‐2) is the causative agent of herpesviral haematopoietic necrosis (HVHN) in goldfish, Carassius auratus, and Prussian carp, C. auratus gibelio. In this study, we investigated virus persistence in goldfish experimentally infected with CyHV‐2. Virus DNA presence in organs was monitored in survivors reared at a virus permissive temperature and also in survivors treated with a non‐permissive temperature for 4 days, initiated at three different time points post‐infection in order to obtain fish with different virus loads. We detected virus DNA in all organs tested at 51 days post‐infection (dpi) and in the spleen, trunk kidney and gills of survivors at 81 dpi, although the virus load in fish influenced the subsequent number of organs that tested positive for virus DNA. In addition, some organs dissected from four out of five asymptomatic survivors tested positive by PCR following incubation in vitro in a medium for 5 days. Following inoculation with the homogenate of PCR‐positive kidney incubated in vitro, one of the three inoculated fish died, showing that the detected virus by PCR produced infectious particles. This study suggests that CyHV‐2 can establish a persistent infection in some organs, especially the spleen and trunk kidney, and that asymptomatic surviving fish can be a source of infection.  相似文献   

11.
Neoparamoeba perurans is the causative agent of amoebic gill disease (AGD). Two loop-mediated isothermal amplification (LAMP) assays targeting the parasite 18S rRNA and the Atlantic salmon EF1α, used as internal control, were designed. The N. perurans LAMP assay did not amplify close relatives N. pemaquidensis and N. branchiphila, or the host DNA. This assay detected 106 copies of the parasite 18S rRNA gene under 13 min and 103 copies under 35 min. Five “fast-and-dirty” DNA extraction methods were compared with a reference method and further validated by TaqMan™ qPCR. Of those, the QuickExtract buffer was selected for field tests. Seventy-one non-lethal gill swabs were analysed from AGD-clinically infected Atlantic salmon. The pathogen was detected under 23 min in fish of gill score >2 and under 39 min for lower gill scores. About 1.6% of the tests were invalid (no amplification of the internal control). 100% of positives were obtained from swabs taken from fish showing gill score ˃3, but only ~50% of positives for lower gill scores. The present LAMP assay could be implemented as a point-of-care test for the on-site identification of N. perurans; however, further work is required to improve its performance for lower scores.  相似文献   

12.
Cyprinid herpesvirus 3 (CyHV‐3) or koi herpesvirus (KHV) is a virulent viral infection in common carp and koi. The disease has caused global epizootic and economic loss in fish aquaculture and in the wild. Clinacanthus nutans (Burm. f.) Lindau is a well‐known medicinal plant used in Thai traditional medicine. Virucidal effects of the plant extract against human herpes simplex virus have been reported. In this study, C. nutans crude extract was tested for antiviral activities against CyHV‐3 in koi carp. Results showed effective antiviral activity against CyHV‐3 pre‐ and post‐infection. The 50% lethal concentration (LC50) of extract was higher than 5 mg/ml. The 50% effective dose (ED50) was 0.99 mg/ml, 0.78 mg/ml, 0.75 mg/ml and 0.71 mg/ml at 1, 2, 3 and 4 hr pre‐infection, respectively. The ED50 from post‐infection tests was 2.05 mg/ml and 2.34 mg/ml at 0 and 24 hr, respectively. These results demonstrated that crude extract expressed antiviral activity against CyHV‐3 and can be applied as a therapeutic agent in common carp and koi aquaculture.  相似文献   

13.
Cyprinid herpesvirus 2 (CyHV‐2) is the main pathogen responsible for causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid‐based diagnostic methods have been applied, no stable and sensitive immunological diagnostic approaches have been reported. In this study, to detect CyHV‐2 in clinical samples using immunological methods, recombinant ORF72 protein (pORF72), encoded by the CyHV‐2 ORF72 gene, was used as a capture antigen to identify blood and tissues infected with CyHV‐2. First, ORF72 gene was amplified from the CyHV‐2 genome and cloned into a PGEX‐4t‐3 expression vector to produce pORF72 in Escherichia coli. The purified pORF72 was used as an immunogen to prepare monoclonal antibodies. The Western blotting assays revealed that the monoclonal antibody could specifically identify the pORF72. Furthermore, an immunohistochemical protocol and a blood smear method were established to detect CyHV‐2 in carps. The results indicate that the monoclonal antibody against pORF72 could be utilized as an effective detection tool for haematopoietic necrosis disease in Carassius auratus gibelio.  相似文献   

14.
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15.
Massive mortalities of Carassius auratus (L.) occurred in a farm in France during summer 2014. Fish presented anorexia, loss of scales and large amounts of mucus on the gills. Necrosis of the distal tip of the filament and the lamellae, combined with fusion of the lamellae, was observed, as well as necrosis in the hematopoietic organs and in the digestive tract. The histological examination led to hypothesize the implication of a virus in the mortality. The presence of cyprinid herpesvirus 2 (CyHV‐2) in dead fish was demonstrated by amplification and sequencing of portions of the DNA polymerase and helicase genes, both sequences exhibiting 100% identity with CyHV‐2 from Japan. In an attempt to find genetic markers of variation, two regions containing tandem repeats in the Japanese genome were amplified from a virus‐positive sample from the present outbreak. A first region (mB) was fully identical to the Japanese isolate. However, the second region (mA) exhibited a range of deletions and substitutions compared to CyHV‐2 from Japan. This is the first report of CyHV‐2 in France in association with mortality of goldfish and the first identification of a molecular marker for its tracing.  相似文献   

16.
Early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) is currently the most significant disease of shrimp in farms of Vietnam, Thailand, Malaysia, China and Mexico, and there is a great risk that it may spread to other shrimp farming countries. Although, an array of sophisticated detection tools for AHPND available, there is a need for a sensitive, simple and rapid detection method. In this study, a simple, sensitive, rapid and polyclonal antibody‐based farmer‐friendly flow‐through assay (FTA) test has been developed for the detection of AHPND pathogen. The recombinant Photorhabdus insect‐related (Pir) A toxin‐like protein of AHPND pathogen was used to immunize rabbits at 21‐day interval observed for highest antibody titre after third booster by ELISA. The raised rabbit antiserum was purified by affinity chromatography and characterized by Western blot. The antiserum showed no cross‐reactivity with AHPND‐free Vibrio parahaemolyticus, V. anguillarum, White Spot Virus (WSV), Aeromonashydrophila and Aphanomycesinvadans. This polyclonal rabbit antiserum was used to develop a farmer‐friendly FTA test for the detection of AHPND pathogen. This simple FTA testis is more sensitive and could detect PirAVP toxin up to 0.121 µg/ml, compared with 0.242 µg/ml by immunodot assay. Furthermore, FTA test requires only 8–10 min for completion, compared with 3 hr by immunodot thus found to be more sensitive, specific and cost‐effective. Collectively, sensitive FTA test would help shrimp farmers to take real‐time management decisions, especially emergency harvest and finally be a better hope for the prevention of AHPND.  相似文献   

17.
Cyprinid herpesvirus 3 (CyHV‐3) is an alloherpesvirus, and it is the aetiological agent of koi herpesvirus disease. Although the complex morphogenic stages of the replication cycle of CyHV‐3 were shown to resemble that of other members of the Herpesvirales, detailed analysis of the sequence and timing of these events was not definitively determined. This study describes these features through a time course using cyprinid cell cultures (KF‐1 and CCB) infected with CyHV‐3 (KHV isolate, H361) and analysed by transmission electron microscopy. Rapid viral entry was noted, with high levels of intracellular virus within 1–4 h post‐infection (hpi). Intranuclear capsid assembly, paracrystalline array formation and primary envelopment of capsids occurred within 4 hpi. Between 1 and 3 days post‐infection (dpi), intracytoplasmic secondary envelopment occurred, as well as budding of infectious virions at the plasma membrane. At 5–7 dpi, the cytoplasm contained cytopathic vacuoles, enveloped virions within vesicles, and abundant non‐enveloped capsids; also there was frequent nuclear deformation. Several morphological features are suggestive of inefficient viral assembly, with production of non‐infectious particles, particularly in KF‐1 cells. The timing of this alloherpesvirus morphogenesis is similar to other members of the Herpesvirales, but there may be possible implications of using different cell lines for CyHV‐3 propagation.  相似文献   

18.
  1. Species identification of sharks under catch or trade regulations is important for law enforcement and species conservation. Rapid detection of Convention on the International Trade in Endangered Species (CITES)-listed species is needed for on-site screening.
  2. Species-specific primers were designed to target three mitochondrial genes (ND2, COI, and CytB) in both the simplex and multiplex loop-mediated isothermal amplification (LAMP) assay for the pelagic thresher shark (Alopias pelagicus), the bigeye thresher shark (Alopias superciliosus), and the scalloped hammerhead shark (Sphyrna lewini), respectively. Another primer set designed to target S. lewini was used for detection-limit testing of the LAMP assay.
  3. The refined direct multiplex LAMP was used to detect the three CITES-listed shark species and omitted the lengthy DNA extraction process. A homogenizer was used to release the DNA from the shark tissues, and a simplex or multiplex LAMP reaction was conducted for 30 min in an incubator at 65°C using species-specific primer sets.
  4. Positive LAMP reactions showed a colour change from pink to yellow, whereas negative reactions showed no colour change. Multiplex LAMP assays were performed using 84 samples, which successfully identified the target and non-target species and provided a fast (<1 h), simple, and reliable method to distinguish three CITES-listed shark species from the other non-target species, for either fresh or dry fin products.
  5. Results of this study and the method developed will play a critical role in assisting fishery agencies and customs officials in identifying the illegal catch and trade of CITES-listed shark species.
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19.
The pathogenesis of cyprinid herpesvirus‐3 (CyHV‐3) was studied using different lineages of carp/koi. After exposure to the virus, infected cells were first found in the skin by histopathology and by in situ hybridization. The epidermis of the skin was most severely damaged and often sloughed off in the fish sampled on days 5 through 8, and the fish that were highly sensitive to the virus died within 8 or 10 days after infection. Serum osmolality of the infected fish, particularly just before death, was significantly lower, suggesting that the osmotic shock consequent on the damage to the skin was the direct cause of the acute deaths. On the other hand, clinical and histopathological observations indicate that the carp of a less sensitive lineage most probably died of viral encephalitis around 3 weeks after infection. For these fish, the largest number of infected cells was found in the central nervous system (CNS) sampled on day 12. A substantial amount of viral genome was found in the CNS of carp surviving more than 1 year after the infection. Thus, the CNS is probably a major target for CyHV‐3, and the virus can persistently infect the CNS, presumably establishing latency.  相似文献   

20.
As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)‐based enzyme‐linked apta‐sorbent assay (VA2‐ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2‐ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2‐ELASA could specifically identify V. alginolyticus, but not other non‐target bacterial strains. VA2‐ELASA could detect V. alginolyticus at the concentration of 5 × 104/ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2‐ELASA in this study. It took less than one hour to accomplish the detection process by VA2‐ELASA. The characteristics of specificity, sensitivity and easy operation make VA2‐ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.  相似文献   

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