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1.
Detection of koi herpesvirus DNA in tissues of infected fish   总被引：6，自引：0，他引：6  
A newly recognized herpesvirus, koi herpesvirus or KHV, causes a lethal disease in common carp, Cyprinus carpio , and its colourful strain known as koi or fancy carp. In this study, we report new outbreaks of the disease, present initial characterization of the KHV genome, and describe assays for detection of KHV DNA in infected cells and tissues of infected fish. Restriction endonuclease (RE) profiles of viral DNA derived from two epidemiologically distinct KHV isolates were identical to each other. Cloned KHV BamHI and SphI DNA probes specifically hybridized to KHV DNA, but not to DNAs derived from a variety of other fish herpesviruses. The KHV DNA probes detected KHV DNA in tissues of experimentally infected koi fish by DNA hybridization. The KHV specific polymerase chain assays (PCR) were developed for rapid detection and confirmation of KHV DNA in tissues of infected fish.  相似文献   

2.
Koi herpesvirus (KHV) causes an economically important, highly infectious disease in common carp and koi, Cyprinus carpio L. Since the occurrence of mass mortalities worldwide, highly specific and sensitive molecular diagnostic methods have been developed for KHV detection. The sensitivity and reliability of these assays have essentially focused at the detection of low viral DNA copy numbers during latent or persistent infections. However, the efficacy of these assays has not been investigated with regard to low-level viraemia during acute infection stages. This study was conducted to compare the sensitivity of seven different polymerase chain reaction (PCR) assays to detect KHV during the first hours and days post-infection (hpi; dpi), using lethal and non-lethal sampling methods. The results highlight the limitations of the assays for detecting virus during the first 4 dpi despite rapid mortality in experimentally infected carp. False-negative results were associated with time post-infection and the tissue sampled. Non-lethal sampling appears effective for KHV screening, with efficient detection in mucus samples obtained from external swabs during this early infection period (<5 dpi), while biopsies from gills and kidney were negative using the same PCR assays. Non-lethal sampling may improve the reliability of KHV detection in subclinical, acutely infected carp.  相似文献   

3.
Hybrids of koi, Cyprinus carpio × crucian carp, Carassius carassius and koi × goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV‐3) and developed KHV disease (KHVD). While hybrids of koi × goldfish were partly resistant to mortality following infection by immersion, most koi × crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi × goldfish and koi × crucian carp hybrids as well as in SPF carp.  相似文献   

4.
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5.
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   

6.
Koi herpesvirus (KHV) causes a highly virulent disease affecting carp, Cyprinus carpio L., and poses a serious socio‐economic threat to the UK carp industry. This study aimed to determine the geographic distribution and prevalence of KHV exposed fish in England and Wales through ELISA antibody testing. Only three of the 82 farms sampled produced positive results, suggesting fish farms provide a relatively safe source of fish. Of the 71 ‘high‐risk’ fisheries tested, 26 were positive. All eight geographic areas within England and Wales studied had at least one KHV positive site. Twelve consignments of imported koi carp from seven S.E. Asian countries were tested for KHV antibody. Six consignments from six different countries were positive. Although a high proportion of consignments were positive, the results indicate that lower risk stocks of fish exist that could be sourced by the ornamental carp sector. The study provides evidence that KHV is widespread and prevalent in ‘high‐risk’ fisheries. There are, however, prospects for controlling KHV as English and Welsh farms appear to be relatively free of the virus, and in most cases fish are not moved from fisheries to other waters.  相似文献   

7.
Since its first outbreak in Japan in 2003, koi‐herpesvirus (KHV) remains a challenge to the carp Cyprinus carpio L. breeding industry. In this study, inactivation of KHV in water from carp habitats (carp habitat water) was investigated with the aim of developing a model for rapidly inactivating the pathogen in aquaculture effluent. Experiments with live fish showed that, in carp habitat water, KHV lost its infectivity within 3 days. Indications were that inactivation of KHV was caused by the antagonistic activity of bacteria (anti‐KHV bacteria) in the water from carp habitats. Carp habitat water and the intestinal contents of carp were therefore screened for anti‐KHV bacteria. Of 581 bacterial isolates, 23 showed anti‐KHV activity. An effluent treatment model for the disinfection of KHV in aquaculture effluent water using anti‐KHV bacteria was developed and evaluated. The model showed a decrease in cumulative mortality and in the number of KHV genome copies in kidney tissue of fish injected with treated effluent compared with a positive control. It is thought that anti‐KHV bacteria isolated from the intestinal contents of carp and from carp habitat water can be used to control KHV outbreaks.  相似文献   

8.
Hygienic measures such as disinfection are important tools for the maintenance of fish health in aquaculture. While little information is available on the disinfection of water intended for fish containment, Huwa‐San®, a disinfectant used in food and water industries, was used for daily treatment at concentrations of approximately 60 ppm over a total period of 3 months (experiment 1) with a 3‐week treatment‐free interval after 2 months (experiment 2). During this period, koi herpesvirus (KHV) was added to the water of two aquaria, one used as a normal contact control, the other one receiving daily water disinfectant treatments that prevented KHV infection of carp. In the second experiment, Huwa‐San® treatment was interrupted and KHV infection was prevalent. However, when naïve fish were introduced to the same aquarium after re‐application of disinfectant, KHV could not be detected in those naïve fish. Whilst KHV could not be detected in samples where disinfectant had been applied, it was present in samples of naïve fish cohabiting with infection contact control animals which had undergone no disinfectant treatment over experiments 1 and 2. The results presented here show that water treatment with a disinfectant may prevent transmission of infectious KHV to naïve carp cohabited with infected carp.  相似文献   

9.
Koi herpesvirus specifically infects and causes mass mortality on koi and carp, resulting in severe economic losses. In this study, we presented the efficacy of KHV DNA vaccine administration by immersion method on Cyprinus carpio. Two different immersion densities of fish were applied, namely 800 fish L?1 and 1200 fish L?1. Thirty‐day‐old common carp juveniles were immersed for 30 min in the water containing 1.3 × 108 CFU mL?1 of heat‐killed Escherichia coli carrying DNA vaccine encoding glycoprotein‐25, and without vaccination treatment as controls. The challenge test was performed at 30 days post vaccination by injecting 0.1 mL KHV filtrate (10?3 of dilution rate). The result showed that higher relative per cent survival of KHV‐challenged fish was obtained in 800 fish L?1 (P < 0.05). Furthermore, significant specific antibody anti‐KHV response (P < 0.05) was detected on 28 and 36 days post vaccination in 800 and 1200 fish L?1, respectively, compared to the controls there was no specific antibody detected. In conclusion, the KHV DNA vaccine could provide good protection in common carp against KHV infection, which has practical applications in aquaculture practices.  相似文献   

10.
The koi herpesvirus (KHV) has spread worldwide since its discovery in 1998 and causes disease and mortality in koi and common carp populations with a high impact on the carp production industry. Many investigations have been conducted to examine ways of distribution and to identify possible transmission vectors. The answers, however, raise many new questions. In the present study, different wild fish species taken from carp ponds with a history of KHV infection were examined for their susceptibility to the virus. In the tissue of these fish, the virus load was determined and it was tested whether a release of the virus could be induced by stress and the virus then could be transferred to naive carp. Wild fish were gathered from carp ponds during acute outbreaks of virus‐induced mortality in summer and from ponds stocked with carp carrying a latent KHV infection. From these ponds, wild fish were collected during the harvesting process in autumn or spring when the ponds were drained. We found that regardless of season, temperature variation, age and infection status of the carp stock, wild fish from carp ponds and its outlets could be tested positive for the KHV genome using real‐time PCR with a low prevalence and virus load. Furthermore, virus transfer to naive carp was observed after a period of cohabitation. Cyprinid and non‐cyprinid wild fish can therefore be considered as an epidemiological risk for pond carp farms.  相似文献   

11.
Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody‐based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin‐embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post‐infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.  相似文献   

12.
Koi herpesvirus (KHV) poses a significant threat to cultured koi and common carp, both Cyprinus carpio L. Since the first reported case in Israel in 1998, KHV has rapidly spread worldwide. This study investigates the spread of KHV to Taiwan by collecting 49 cases of suspected common carp and koi infections from 2003 to 2005 for analysis. Clinical signs included lethargy, anorexia, increased respiratory movements and uncoordinated swimming. Hyperaemia, haemorrhage on body surface and necrotic gill filaments were recorded. Gill epithelial hyperplasia, necrosis and eosinophilic intranuclear inclusion bodies were observed by histological examination, while virions were detected using transmission electron microscopy. By detecting the presence of the KHV thymidine kinase (TK) gene and the KHV 9/5 gene using polymerase chain reaction (PCR), 37 cases were identified as KHV-positive, and the cumulative mortality of infected fish was 70-100%. Positive cases showed identical sequences for the genes analysed, implying that they were of the same origin. For the KHV 9/5 gene sequence, these cases exhibited 100% identity with the Japanese strain (TUMST1, accession number AP008984) and 99% identity with the Israeli (KHV-I, DQ177346) and US (KHV-U, DQ657948) strains. Additionally, a loop-mediated isothermal amplification (LAMP) assay was performed and found to be more sensitive than PCR tests, suggesting its potential use as a rapid diagnostic method for KHV. This is the first epidemiological study of KHV infection in cultured common carp and koi in Taiwan.  相似文献   

13.
Although koi herpesvirus (KHV) has a history of causing severe economic losses in common carp and koi farms, there are still no treatments available on the market. Thus, the aim of this study was to test exopolysaccharides (EPS) for its antiviral activity against KHV, by monitoring inhibition and cytotoxic effects in common carp brain cells. These substances can be easily extracted from extracellular algae supernatant and were identified as groups of sulphated polysaccharides. In order to reach this aim, Arthrospira platensis, which is well known for its antiviral activity of intra‐ and extracellular compounds towards mammalian herpesviruses, was investigated as standard organism and compared to commercial antiviral drug, ganciclovir, which inhibits the viral DNA polymerization. The antiviral activity of polysaccharides of A. platensis against KHV was confirmed in vitro using qualitative assessment of KHV life cycle genes, and it was found by RT‐PCR that EPS, applied at a concentration of >18 μg mL?1 and a multiplicity of infection (MOI) of 0.45 of KHV, suppressed the viral replication in common carp brain (CCB) cells even after 22 days post‐infection, entirely. Further, this study presents first data indicating an enormous potential using polysaccharides as an additive for aquacultures to lower or hinder the spread of the KHV and koi herpesvirus disease (KHVD) in future.  相似文献   

14.
2013年4月初低温下,吉林省白山地区某网箱养殖场鲤（Cyprinus carpioL．）大量死亡,病死鲤无明显临床病变。采用PCR扩增技术检测了这些病鱼,结果表明,该低温爆发的疾病为锦鲤疱疹病毒病（KHVD）。这种病常爆发在水温为18-28℃的春秋季,冰下爆发还未有报道。本次锦鲤疱疹病毒病的鉴定,对于锦鲤疱疹病毒感染鲤温度条件的变化和疾病的预防提供了参考。  相似文献   

15.
Common carp, Cyprinus carpio L., exposed to koi herpesvirus (KHV) may become persistently infected and populations containing such virus-infected individuals may transmit the virus to other fish when co-habited. Detection of virus-infected fish in a population is thus critical to surveillance and control programmes for KHV. A study was therefore designed to detect anti-KHV serum antibodies, with an enzyme-linked immunosorbent assay, in common carp following experimental exposures to KHV under varying environmental conditions. The study determined that a proportion of fish within a population experimentally exposed to KHV (at least 10–25%) develop high antibody titres (1/1600 or greater) to the virus, and this immunological response was detectable for several months (observed at the termination of the experiments at 65, 46 and 27 weeks post-exposure). Furthermore, this response was detected in one population of fish that did not succumb to a high level of mortality when maintained at water temperatures that were non-permissive for KHV. Elevating the water temperatures to permissive conditions for KHV resulted in recurrence of disease despite the presence of anti-virus antibodies, suggesting that serum antibodies alone are not protective under the conditions of our trials.  相似文献   

16.
During a PCR‐based CEV survey in Poland in 2015–2017, the virus was detected in many farms both in clinical and asymptomatic cases and in common as well as in koi carp (Cyprinus carpio). In order to evaluate the potential carrier role of fish species that share the same habitats with carp, an experimental trial was performed. Investigations carried out on specimens of bleak (Alburnus alburnus), crucian carp (Carassius carassius), European perch (Perca fluviatilis), Prussian carp (Carassius gibelio), roach (Rutilus rutilus) and tench (Tinca tinca) cohabited with CEV‐infected carp yielded positive results. These species of fish were experimentally cohabited with CEV‐infected common carp at a temperature of 16°C ± 1. Material from the brain, gills, spleen, kidneys, intestine and skin was investigated for the presence of CEV DNA. Similar investigations were performed with uninfected fish designated controls. Samples were tested for CEV by qPCR.  相似文献   

17.
Koi herpesvirus (KHV) is the aetiological agent of an emerging disease (KHVD) associated with mass mortalities in koi and common carp and reported from at least 30 countries. We report the first detection of KHV from koi in Iran using clinical, histopathological and molecular studies. KHV‐infected fish showed reduced swimming activity, sunken eyes and increased mucus production on skin and fins. On post‐mortem examination, gill necrosis was observed in the majority of fish. Histopathologically, the gill showed diffuse necrosis of the branchial epithelial cells. Margination of chromatin was detected in gills, kidney, heart, spleen, intestine and brain. In addition, sequence analyses of the TK gene, ORF 136 and marker I and II, demonstrates that Iranian KHV isolates were identical and classified as variant A1 of TUSMT1 (J strain) and displayed the I++II+ allele of this Asian genotype.  相似文献   

18.
Common carp (Cyprinus carpio) is a very important fish species for warm-water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures.  相似文献   

19.
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   

20.
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   

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