Seasonal Distribution of Cyprinid Herpesvirus 3 in Lake Biwa,Japan

The seasonal distribution of the cyprinid herpesvirus 3 (CyHV-3) in Lake Biwa, Japan, was investigated. CyHV-3 was distributed all over the lake 5 years after the first outbreak. The mean concentration of CyHV-3 in water showed annual oscillation, with a peak in the summer and a trough in winter. Our results suggested that CyHV-3 is present at high density in reductive environments, such as reed zones and turbid or eutrophic water.A novel fatal disease of fish caused by cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus or carp interstitial nephritis and gill necrosis virus, which infects the common carp (Cyprinus carpio carpio) and ornamental koi (Cyprinus carpio koi) was reported at the end of the 1990s, and it has spread rapidly worldwide (13). In 2003, the first mass mortality in Japan was observed in Lake Kasumigaura, Ibaraki Prefecture (17), and the disease immediately spread all over the country.Lake Biwa is the largest and oldest freshwater lake in Japan. It occupies 670 km², and the total length of the coastline is 241 km. The first and relatively small-scale mortality caused by CyHV-3 was observed in the autumn of 2003 in Lake Biwa, and mass mortality occurred in the following spring, in which more than 100,000 carp died (10). Thus, the disease is a great threat not only to the cultivation industry and koi collectors but also to the natural carp population. Carp is regarded as an ecological engineer that has an impact on freshwater ecosystems, and therefore, mass mortality will affect the entire lake ecosystem (10).The pathogenesis and diagnosis of the disease have been studied intensively, but the dynamics of CyHV-3 has not been clarified. Recently, the major portal of CyHV-3 entry was reported to be fish skin (2), which means that infection via water is possible. Thus, to determine the method by which the virus spreads and to evaluate the infectious risk in the environment, one must clarify the CyHV-3 dynamics in the natural environment. In the present study, we surveyed the seasonal distribution pattern of CyHV-3 in Lake Biwa, Japan, using a quantitative method.     

Anthropogenic and Ecological Drivers of Amphibian Disease (Ranavirosis)

Ranaviruses are causing mass amphibian die-offs in North America, Europe and Asia, and have been implicated in the decline of common frog (Rana temporaria) populations in the UK. Despite this, we have very little understanding of the environmental drivers of disease occurrence and prevalence. Using a long term (1992-2000) dataset of public reports of amphibian mortalities, we assess a set of potential predictors of the occurrence and prevalence of Ranavirus-consistent common frog mortality events in Britain. We reveal the influence of biotic and abiotic drivers of this disease, with many of these abiotic characteristics being anthropogenic. Whilst controlling for the geographic distribution of mortality events, disease prevalence increases with increasing frog population density, presence of fish and wild newts, increasing pond depth and the use of garden chemicals. The presence of an alternative host reduces prevalence, potentially indicating a dilution effect. Ranavirosis occurrence is associated with the presence of toads, an urban setting and the use of fish care products, providing insight into the causes of emergence of disease. Links between occurrence, prevalence, pond characteristics and garden management practices provides useful management implications for reducing the impacts of Ranavirus in the wild.     

Detection of the herpesviral hematopoietic necrosis disease agent (Cyprinid herpesvirus 2) in moribund and healthy goldfish: validation of a quantitative PCR diagnostic method

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen of goldfish Carassius auratus auratus L. that causes herpesviral hematopoietic necrosis (HVHN) disease. The disease is associated with necrosis of hematopoietic tissues and anemia with high mortality. We have developed a real time 5'-nuclease PCR method (Taqman) that quantitatively detects CyHV-2 with a linear response over 8 logs of target concentration. The coefficient of variability on replicate samples tested on different days was 13% and the calculated sensitivity approached 1 target molecule per reaction. The assay does not cross-react with other similar fish herpesviruses, including CyHV-1 (carp pox) and CyHV-3 (koi herpesvirus), but reliably detects known CyHV-2 positive fish. The assay detects CyHV-2 not just in clinical cases of HVHN but also in apparently healthy 1 yr old goldfish fingerlings and even in 3 to 5 yr old broodfish.     

Resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 is influenced by major histocompatibility (MH) class II B gene polymorphism

The role of MH class II B (Cyca-DAB1-like) genes in resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 (CyHV-3), also known as koi herpesvirus (KHV) was analysed. The material consisted of 934 fish from six carp crosses. Fish were challenged with CyHV-3 at an age of 7 and 10 months. During challenge experiments the peak of mortality caused by CyHV-3 was observed at days 8–12 p.i. and the overall cumulative mortality reached 79.9%. Among six Cyca-DAB1-like genotypes, revealed by PCR-RF-SSCP analysis, one genotype (E) was found associated with higher resistance to CyHV-3. Three other genotypes (B, H and J) could be linked to higher susceptibility to CyHV-3. Analysis of the alleles that compose the Cyca-DAB1-like genotypes linked one particular allele (Cyca-DAB1*05) to significantly increased, and two alleles (Cyca-DAB1*02 and Cyca-DAB1*06) to significantly decreased resistance to CyHV-3. Our data indicate that MH class II B genes could be used as potential genetic markers in breeding of common carp for resistance to this virus.     

Widespread and enduring demographic collapse of invasive common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) in the Upper Mississippi River System

Populations of invasive species that undergo rapid expansions after establishment in a new system can also be subject to collapse. Although the dynamics of the establishment and expansion phases and their ecological effects are well documented, substantially fewer studies document collapses despite their importance for understanding invasion dynamics. Two long-term fish monitoring programs sample the fish assemblage of the Upper Mississippi River System. These data provide an opportunity to document the collapse of common carp (Cyprinus carpio), a globally invasive freshwater fish species. Here we describe their population trajectory over several decades and examine several hypotheses to explain the decline, including: boom-bust population dynamics; suppression by native predators; resource exhaustion; improvements in environmental conditions; and disease. The observed trends appear to be most consistent with the hypothesis that disease was the most important factor contributing to the collapse. In particular, cyprinid herpesviruses have been shown to affect common carp in a manner consistent with the observed decreases in catch rates and increases in size distributions. The apparent role of a viral agent in causing the decline of common carp across one of the largest river basins in North America suggests similar collapses may occur elsewhere.     

Experimental study of the impacts of silver carp on plankton communities of eutrophic Villerest reservoir (France)

We examined the impact of five silver carp biomass levels (0, 8, 16, 20, and 32 g m⁻³) on plankton communities and water quality of Villerest eutrophic reservoir (France). We realized the experiments using outdoor mesocosms. The presence of silver carp led to changes in zooplankton and phytoplankton assemblages. High fish biomass strongly reduced cladoceran abundance (through predation). Silver carp inefficiently grazed down particles < 20 μm. More importantly, however, the suppression of herbivorous cladocerans resulted in the increase of small size algae which were relieved from grazing and benefit from high nutrient concentrations. In contrast, in mesocosms without fish, the dominance of cladocerans (mainly Daphnia) controlled small size algae and probably also larger size algae (colonial chlorophytes, cyanobacteria). Thus, the Secchi disc transparency increased markedly. Through cascade effects, the modification of grazers communities led to changes in the utilization patterns of the added nutrients by phytoplankton communities. In high fish biomass treatments, nutrients were more efficiently accumulated into particulate fractions compared with no-fish and low-fish biomass treatments that were characterized by higher dissolved nutrients concentrations. Zooplankton was an essential source of food for silver carp. The productivity of zooplankton sustained a moderate silver carp biomass (up to 16 g m⁻³). In the presence of the highest fish biomass, the productivity of zooplankton was not large enough and silver carps fed on additional phytoplankton. Although mesocosms with high fish biomass were characterized by a slight cyanobacteria development compared with other fish mesocosms, silver carp was not effective in reducing cyanobacteria dominance. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rational Development of an Attenuated Recombinant Cyprinid Herpesvirus 3 Vaccine Using Prokaryotic Mutagenesis and In Vivo Bioluminescent Imaging

Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV-3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV-3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.     

Modeling the potential for managing invasive common carp in temperate lakes by targeting their winter aggregations

Common carp (Cyprinus carpio) is one of world’s most invasive fish and managers have long searched for practical control strategies for this species. In temperate systems, common carp forms large winter aggregations that can be located with telemetry and removed with seine nets. This has been viewed as an excellent management possibility, but its success has been mixed. Using a modeling approach, we demonstrate that the usefulness of winter seining in controlling common carp in temperate North American lakes depends on whether carp populations are driven by one of two distinct recruitment dynamics. In lakes where carp can easily recruit within systems from which they are being removed, such as within productive lakes with poor communities of micropredators, winter seining is unlikely to be effective. Even very high removal rates (90 % adults annually) were not sufficient to reach management goal (biomass <100 kg/ha) in such systems. However, in regions with strong predatory communities where carp can recruit only in outlying, seasonally unstable marshes, removal rates as low as 30 % annually or 50 % every other year were able to reduce carp biomass below the management threshold. Such removal rates are achievable as they fall within the range of empirically measured values. Because many carp populations are driven by external recruitment dynamics, strategically conducted winter removal could be used to control this species in a large number of systems across temperate North America and elsewhere.     

Cyprinid herpes virus-3 (CyHV-3) bears genes of genetically distant large DNA viruses

A large DNA virus, designated koi herpes virus (KHV), carp interstitial nephritis gill necrosis virus (CNGV) and Cyprinid herpes virus-3 (CyHV-3), causes massive mortality of carp. Morphologically, the virus resembles herpes viruses, but it contains a genome of ca 295 kbp, larger than that of any Herpesviridae member. Interestingly, three CyHV-3 genes, thymidylate monophosphate kinase (TmpK), ribonucleotide reductase and thymidine kinase, which are involved in deoxynucleotide tri-phosphate synthesis, resemble those of pox viruses. In addition to the TmpK gene, which is nonexistent in the genome of herpes viruses, CyHV-3 contains a B22R-like gene, exclusively expressed by pox viruses. These results raise questions on the phylogenic origin of CyHV-3.     

The outbreak of carp disease caused by CyHV-3 as a model for new emerging viral diseases in aquaculture: a review

Aquacultured koi and common carp (Cyprinus carpio) are intensively bred for ornamental purposes and for human consumption worldwide. The carp and koi farming industries have suffered enormous economic losses over the past decade due to an epizootic disease caused by Cyprinus herpesvirus-3 (CyHV-3) also known as koi herpesvirus and carp interstitial nephritis gill necrosis virus. CyHV-3 is a large dsDNA virus, morphologically similar to herpesviruses, yet contains genetic elements similar to those of pox, irido- and herpesviruses. Considering the phylogenic distance between CyHV-3 and higher vertebrate herpesviruses, CyHV-3 represents the prototype of viruses assigned to the novel family Alloherpesviridae. Although emergence of a new virus rarely initiates a pandemic so severe that it reduces the life expectancy of a population, CyHV-3 is exceptional because of its enormous impact on the world carp population. High population density is the major contributing factor to the epizootic disease caused by CyHV-3.     

The cestode Atractolytocestus huronensis (Caryophyllidea) continues to spread in Europe: new data on the helminth parasite of the common carp

The caryophyllidean tapeworm Atractolytocestus huronensis Anthony, 1958, originally described from the common carp Cyprinus carpio carpio L. from North America, has recently been introduced into Europe (UK, Hungary). In the present study, the dispersion of the tapeworm in other countries of Central Europe (Slovakia and the Czech Republic) is described, including its first observation in the common carp of the Tisa River, southeastern Slovakia. Because of the transboundary location of the river, there are possible consequences of the rapid distribution of the parasite to other regions throughout the Danube River basin. The parasite has also been found in cultured carp from fishponds in South Bohemia, from where the carp are imported to many European countries. Rapid dissemination of this cestode in Europe indicates its ability to colonise new regions, and represents another example of man-made introduction of potential pathogens of carp and other farmed fish. The morphology of the A. huronensis specimens found is compared with that of specimens from Hungary and North America as well as that of Atractolytocestus sagittatus (Kulakovskaya and Akhmerov, 1965) from the same fish host in eastern Asia.     

Tag retention and survival of juvenile bighead carp implanted with a dummy acoustic tag at three temperatures

Bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix (together, the bigheaded carps) are invasive fishes in North America that have resulted in substantial negative effects on native fish communities and aquatic ecosystems. Movement and behavior of adult bigheaded carps has been studied previously using telemetry, while similar studies with juvenile bigheaded carps have yet to be attempted. Recent technological advances in telemetry transmitters has increased the availability of tags sufficiently small enough to implant in juvenile carps. However, the effects of surgical implantation of telemetry tags on juvenile bigheaded carps have not been evaluated. We determined tag retention and survival associated with surgical implantation of acoustic telemetry tags into juvenile bighead carp (range 128–152 mm total length) at three temperatures (13, 18, and 23°C). In addition, we assessed the effect of surgically implanted transmitters on the fitness, defined as changes in weight or critical swimming speed, of carp implanted with transmitters. Survival was high among tagged fish (85%) with 47% of tags retained at the conclusion of the 45‐day study. No substantial decline in fitness of the fish was observed in tagged fish compared to untagged fish.     

Transmission dynamics of an emerging infectious disease in wildlife through host reproductive cycles

Emerging infectious diseases are major threats to wildlife populations. To enhance our understanding of the dynamics of these diseases, we investigated how host reproductive behavior and seasonal temperature variation drive transmission of infections among wild hosts, using the model system of cyprinid herpesvirus 3 (CyHV-3) disease in common carp. Our main findings were as follows: (1) a seroprevalence survey showed that CyHV-3 infection occurred mostly in adult hosts, (2) a quantitative assay for CyHV-3 in a host population demonstrated that CyHV-3 was most abundant in the spring when host reproduction occurred and water temperature increased simultaneously and (3) an analysis of the dynamics of CyHV-3 in water revealed that CyHV-3 concentration increased markedly in breeding habitats during host group mating. These results indicate that breeding habitats can become hot spots for transmission of infectious diseases if hosts aggregate for mating and the activation of pathogens occurs during the host breeding season.     

Quantification of Cyprinid Herpesvirus 3 in Environmental Water by Using an External Standard Virus

Cyprinid herpesvirus 3 (CyHV-3), a lethal DNA virus that spreads in natural lakes and rivers, infects common carp and koi. We established a quantification method for CyHV-3 that includes a viral concentration method and quantitative PCR combined with an external standard virus. Viral concentration methods were compared using the cation-coated filter and ultrafiltration methods. The recovery of virus-like particles was similar for the two methods (cation-coated filter method, 44% ± 19%, n = 3; ultrafiltration method, 50% ± 3%, n = 3); however, the former method was faster and more suitable for routine determinations. The recovery of seeded CyHV-3 based on the cation-coated filter method varied by more than 3 orders of magnitude among the water samples. The recovery yield of CyHV-3 was significantly correlated with that of the seeded λ phage, and the average ratio of λ to the CyHV-3 recovery yield was 1.4, indicating that λ is useful as an external standard virus for determining the recovery yield of CyHV-3. Therefore, to quantify CyHV-3 in environmental water, a known amount of λ was added as an external standard virus to each water sample. Using this method, CyHV-3 DNA was detected in 6 of the 10 (60%) types of environmental water tested; the highest concentration of CyHV-3 DNA was 2 × 10⁵ copies liter⁻¹. The lowest recovery limit of CyHV-3 DNA was 60 copies liter⁻¹. This method is practical for monitoring CyHV-3 abundance in environmental water.Cyprinid herpesvirus 3 (CyHV-3) is a lethal DNA virus that infects the common carp (Cyprinus carpio L.) and koi carp (C. carpio koi). The occurrence of the disease in the United Kingdom has been dated to 1996, following outbreaks in the United States, Israel, Europe, and South Asia (10), and has afflicted cultured ornamental and common carps, causing severe losses to fish breeders, retailers, and hobbyists (28). Therefore, the characterization and diagnosis of the disease have been the subject of intensive research (15). In recent years, the mortality of wild carp has been reported in natural freshwater environments (11, 18, 23). In Lake Biwa in Japan, 60 to 80% of the wild carp population (>100,000) died in 2004, presumably due to CyHV-3 infection (Shiga Prefectural Government, http://www.pref.shiga.jp/g/suisan-s/seika/files/seikah1711.pdf [in Japanese]) (18). The mass mortality of wild carp can directly and indirectly affect community composition and environmental ecosystems (18). Nevertheless, the occurrence of the disease and the means of transmission of CyHV-3 in the natural environment are still not well understood.CyHV-3 is present in several organs of infected fish, such as the intestines, kidneys (7), and gills (29). CyHV-3 is also detected in droppings (3); therefore, infected fish are suspected of releasing CyHV-3 into natural waters. Seasonal variation and the spatial distribution of CyHV-3 may be important for understanding the transmission routes and mechanisms by which CyHV-3 spreads. However, the lack of a reliable method for quantifying CyHV-3 in environmental water precludes our elucidation of how this disease spreads.In general, the concentration of a pathogen in environmental water is considerably lower than that found in host bodies. Therefore, a CyHV-3 concentration method is required to detect and quantify the virus in environmental water. Several methods have been developed for determining concentrations of viruses in water samples. Ultrafiltration can concentrate a pathogen from a large volume of water in <100 liters (27, 35). An alternative method involving the use of electronegative or electropositive microporous adsorbent filters has also been used to concentrate viruses from environmental water (1, 8). The mechanism of concentration in this method is based on electrostatic interactions. Haramoto et al. established a cation-coated filter method in which viruses that had been trapped were eluted with NaOH solution (pH 10.8) instead of the conventional solution, beef extract, which inhibits the PCR (12, 13). The concentrated viruses can then be used for PCR-mediated identification. Using this method, they succeeded in the qualitative detection of CyHV-3 DNA from river water samples (13).Viral recovery during concentration is influenced by soluble organic compounds (33, 34) and salts (31) in the water, which may vary in each sample. Therefore, quantification of the viral DNA from concentrated environmental water samples has been difficult. Because sediments contain many substances that influence DNA recovery, Mumy and Findlay developed a method for the routine determination of DNA extraction efficiency using an external DNA recovery standard, as follows: λ DNA was added to sediments, the total DNA was extracted, and the amount of target DNA recovered was determined by quantitative PCR (22).In this study, we established a method for quantifying CyHV-3 in environmental water using a viral concentration method and TaqMan PCR combined with an external standard virus. To choose a suitable viral concentration method, we compared the viral recovery yields between the ultrafiltration and cation-coated filter methods, and the procedure was modified to increase sensitivity. We then confirmed that the recovery yields of CyHV-3 and the external standard virus λ from different environmental waters throughout the procedure were positively correlated. Finally, we applied this method to environmental water samples taken from Lake Biwa and Takaragaike Pond in Japan at 3 years and 1 month, respectively, after an outbreak of the disease for the detection and quantification of CyHV-3.     

长江中游典型草型湖泊与藻型湖泊鲫种群结构和生长比较(英文)

浅水湖泊牛山湖和月湖均位于长江中游武汉市境内,前者水质清澈、沉水植物茂盛,属典型的草型湖泊;后者长期受生活污水和工业废水排放的影响,水质恶劣、沉水植物稀少,属典型的藻型湖泊。文章选择这两个湖泊的共同优势鱼类——鲫(Carassius auratus)作为研究对象,通过比较两类不同湖泊环境条件下鲫种群结构和生长上的差异,评估人类活动引起的水环境改变对鱼类种群生存状况的影响。2006年春季(3—5月),于牛山湖和月湖各采集鲫种群样本713尾和641尾,研究结果表明:(1)牛山湖和月湖鲫种群的全长频数分布经kolmogorov-Smirnov检验均符合正态分布,但前者的分布范围(63—271 mm)大于后者(72—191 mm),前者的全长峰值(190 mm)亦大于后者(110 mm);(2)牛山湖鲫种群的年龄组成(6个年龄组)较月湖(仅3个年龄组)完整,但月湖各龄鲫的全长均显著大于牛山湖(P<0.05);(3)两个湖泊性成熟鲫的雌、雄鱼比均随全长的增长而增加,即小个体性成熟鲫以雄鱼为主,大个体性成熟鲫以雌鱼为主,该现象在月湖更为明显;(4)牛山湖和月湖鲫全长–体重回归关系(W=a Lb)分别为W=0.00001 L3.060(R2=0.989)和W=0.000006 L3.246(R2=0.971),协方差分析表明两个湖泊鲫种群的b值差异极显著(P<0.01);另一方面,月湖鲫种群各全长组雌、雄鱼的肥满度均显著大于牛山湖(P<0.05)。作者认为影响牛山湖和月湖鲫生长和种群结构的主要因素是两个湖泊饵料组成和捕捞压力等方面的差异。