Transmission of Cyprinid herpesvirus-3 (CyHV-3) from goldfish to naïve common carp by cohabitation

Cyprinid herpesvirus-3 (CyHV-3) has spread worldwide and has had a major impact on koi and common carp production. Previous studies on the host range of the CyHV-3 found that fish species other than koi and common carp are fully resistant to natural virus exposure. Recently, CyHV-3 was detected in goldfish (Carassius auratus auratus) that were in contact with CyHV-3 infected koi. In the present study, a specific RT-PCR product was amplified from the viral thymidine kinase gene in gills, intestine and brain tissues of CyHV-3 infected goldfish. This implied that CyHV-3 replicated in these goldfish. Also, in the presence of a stress factor such as temperature fluctuation, the CyHV-3 infected goldfish transmitted the virus to cohabitated naïve SPF common carp. CyHV-3 DNA was detected in the cohabitated naïve carp tissues by PCR. The results of this study demonstrate that goldfish is a carrier for CyHV-3, permit virus propagation, and disseminate the virus to susceptible carp causing the disease.     

Nationwide Cyprinid herpesvirus 3 contamination in natural rivers of Japan

Cyprinid herpesvirus 3 (CyHV-3) disease is a significant threat for common and koi carp cultivators and for freshwater ecosystems. To determine the prevalence of CyHV-3 in Japanese rivers, a nationwide survey of all national class-A rivers was undertaken in the Summer of 2008. The virus was concentrated from river water samples using the cation-coated filter method. CyHV-3 DNA was detected in 90 rivers, representing 90% of 103 successfully analysed rivers. More than 100,000 copies of CyHV-3 DNA per litre of sample were detected in four rivers, higher than that reported during the Yura River outbreak in 2007. For CyHV-3-positive rivers, the log CyHV-3 density was negatively correlated with the water temperature on the sampling date and positively correlated with the suspended solids and dissolved oxygen, which are annually averaged for each river. Our results demonstrate that virus detection using molecular biology techniques is a powerful tool for monitoring the presence of CyHV-3 in natural environments.     

Reservoirs of Cyprinid herpesvirus 3 (CyHV-3) DNA in sediments of natural lakes and ponds

Cyprinid herpesvirus 3 (CyHV-3) is a lethal DNA virus that infects common carp and koi. It has caused outbreak of the disease within both aquaculture and natural environmental ecosystems. However, there is not enough understanding of the distribution of CyHV-3 in the natural environments, partly because there is no suitable quantification method. In this study, we tested CyHV-3 extraction methods from sediment and then compared its abundance between sediment and water using real-time PCR. Sediment samples were taken from lake and pond, and total viral DNA was extracted using the viral elution method recommended by the US Environmental Protection Agency (manual method), as well as a commercial DNA extraction kit for soil (commercial kit method) before PCR detection. 7 of 12 (58%) and 5 of 10 (50%) sediment samples showed PCR positive signal for CyHV-3 DNA using the manual method and the commercial kit, respectively, and consistent results were obtained from the samples using the manual method between two independent primer sets. The quantification of CyHV-3 DNA in natural sediment using the manual method and external standard virus revealed that its concentration was 1.2×10(4) to 3.3×10(5) copies DNA/kg. The concentration in sediments was 46-1238 times higher than that in water from the same location, suggesting that sediment could act as a reservoir for CyHV-3 in natural freshwater environments. This is the first report of the existence of CyHV-3 in the sediment of a natural lake or pond.     

Detection of cyprinid herpesvirus-3 DNA in lake plankton

The disease caused by cyprinid herpesvirus-3 (CyHV-3) severely impacts the natural freshwater ecosystem and damages carp and koi farming, however, the pathway of CyHV-3 transmission remains unclear. It is possible that the virus adheres to plankton, which then facilitate viral movement and transmission, and therefore, it is hypothesised that plankton are involved in the disease dynamics. In this study, plankton were collected at eight sites in the Iba-naiko lagoon; we detected and quantified CyHV-3 DNA from plankton samples. The results of the correlation analysis showed a significant positive correlation between CyHV-3 copies and the number of Rotifera, suggesting that CyHV-3 binds to and/or is concentrated by Rotifera. Our results suggest that plankton affect viral ecology in the natural environment.     

Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells

ABSTRACT: Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a mortal and highly contagious disease in common and koi carp. The skin is the major portal of entry of CyHV-3 in carp after immersion in water containing the virus. In the present study, we used in vivo bioluminescence imaging to investigate the effect of skin mucus removal and skin epidermis lesion on CyHV-3 entry. Physical treatments inducing removal of the mucus up to complete erosion of the epidermis were applied on a defined area of carp skin just before inoculation by immersion in infectious water. CyHV-3 entry in carp was drastically enhanced on the area of the skin where the mucus was removed with or without associated epidermal lesion. To investigate whether skin mucus inhibits CyHV-3 binding to epidermal cells, tail fins with an intact mucus layer or without mucus were inoculated ex vivo. While electron microscopy examination revealed numerous viral particles bound on the fins inoculated after mucus removal, no particle could be detected after infection of mucus-covered fins. Finally, anti-CyHV-3 neutralising activity of mucus extract was tested in vitro. Incubation of CyHV-3 with mucus extract reduced its infectivity in a dose dependent manner. The present study demonstrates that skin mucus removal and epidermal lesions enhance CyHV-3 entry in carp. It highlights the role of fish skin mucus as an innate immune protection against viral epidermal entry.     

Cyprinid herpesvirus 3: an interesting virus for applied and fundamental research

Cyprinid herpesvirus 3 (CyHV-3), a member of the family Alloherpesviridae is the causative agent of a lethal, highly contagious and notifiable disease in common and koi carp. The economic importance of common and koi carp industries together with the rapid spread of CyHV-3 worldwide, explain why this virus became soon after its isolation in the 1990s a subject of applied research. In addition to its economic importance, an increasing number of fundamental studies demonstrated that CyHV-3 is an original and interesting subject for fundamental research. In this review, we summarized recent advances in CyHV-3 research with a special interest for studies related to host-virus interactions.     

Feeding Cyprinus carpio with infectious materials mediates cyprinid herpesvirus 3 entry through infection of pharyngeal periodontal mucosa

ABSTRACT: Cyprinid herpesvirus 3 (CyHV-3), also known as Koi herpesvirus, is the etiological agent of a mortal disease in common and koi carp. Recently, we investigated the entry of CyHV-3 in carp using bioluminescence imaging and a CyHV-3 recombinant strain expressing luciferase (LUC). We demonstrated that the skin is the major portal of entry after inoculation of carp by immersion in water containing CyHV-3. While this model of infection mimics some natural conditions in which infection takes place, other epidemiological conditions could favour entry of virus through the digestive tract. Here, we investigated whether ingestion of infectious materials mediates CyHV-3 entry through the digestive tract. Carp were fed with materials contaminated with the CyHV-3 LUC recombinant (oral contamination) or immersed in water containing the virus (contamination by immersion). Bioluminescence imaging analyses performed at different times post-infection led to the following observations: (i) the pharyngeal periodontal mucosa is the major portal of entry after oral contamination, while the skin is the major portal of entry after contamination by immersion. (ii) Both modes of inoculation led to the spreading of the infection to the various organs tested. However, the timing and the sequence in which some of the organs turned positive were different between the two modes of inoculation. Finally, we compared the disease induced by the two inoculation modes. They led to comparable clinical signs and mortality rate. The results of the present study suggest that, based on epidemiological conditions, CyHV-3 can enter carp either by skin or periodontal pharyngeal mucosal infection.     

Surveillance of herpesviruses in koi carp Cyprinus carpio koi and goldfish Carassius auratus cultured in West Bengal,India

Background: Viral diseases create one of the greatest challenges for the rapidly expanding ornamental fish culture sector. In recent years, the host-specific herpesviruses are receiving much attention due to the intensification in aquaculture globally. Cyprinid herpesvirus-2 (CyHV-2, goldfish hematopoietic necrosis virus), and Cyprinid herpesvirus-3 (CyHV-3, koi herpesvirus) are the lethal pathogens of koi carp Cyprinus carpio koi and goldfish Carassius auratus, respectively. In India, West Bengal is the leading state in ornamental fish production and export. Methods: The surveillance of CyHV-2 and CyHV-3 in koi carp and goldfish cultured in West Bengal was carried out between November 2014 and January 2017 as per Office International des Epizooties guidelines. Results: Both fish species were negative for CyHV-3. The CyHV-2 infection was detected in both gill and kidney tissues of apparently healthy and diseased C. auratus from December 2014 to March 2015. Severe necrosis was observed in the gills of infected C. auratus. Coinfection with members of the bacterial genera Aeromonas and Flavobacterium was also observed in the kidney of infected goldfish. The histopathological observations in the kidney of CyHV-2 infected C. auratus demonstrated the pathogenic potential of CyHV-2 and tissue tropism. Conclusions and Clinical relevance: Environmental stressors like low water temperature and the use of wastewater for culture played a vital role in the onset of CyHV-2 infection in the stressed goldfish. The spread of CyHV-2 can likely pose a certain degree of threat to aquaculture especially the unrestricted movement of goldfish. The results of the present study emphasize the necessity of an organized risk assessment to plan for the management strategies on the control and spread of CyHV-2 in Indian aquaculture.     

Rapid Detection of Cyprinid Herpesvirus 3 in Latently Infected Koi by Recombinase Polymerase Amplification

Since the emergence of cyprinid herpesvirus 3 (CyHV-3), outbreaks have been devastating to Common Carp Cyprinus carpio and koi (a variant of Common Carp), leading to high economic losses. Current diagnostics for detecting CyHV-3 are limited in sensitivity and are further complicated by latency. Here we describe the detection of CyHV-3 by recombinase polymerase amplification (RPA). The RPA assay can detect as low as 10 copies of the CyHV-3 genome by an isothermal reaction and yields results in approximately 20 min. Using the RPA assay, the CyHV-3 genome can be detected in the total DNA of white blood cells isolated from koi latently infected with CyHV-3, while less than 10% of the latently infected koi can be detected by a real-time PCR assay in the total DNA of white blood cells. In addition, RPA products can be detected in a lateral flow device that is cheap and fast and can be used outside of the diagnostic lab. The RPA assay and lateral flow device provide for the rapid, sensitive, and specific amplification of CyHV-3 that with future modifications for field use and validation could lead to enhanced surveillance and early diagnosis of CyHV-3 in the laboratory and field.

Received September 14, 2015; accepted April 9, 2016 Published online August 2, 2016     

Sensitivity and permissivity of Cyprinus carpio to cyprinid herpesvirus 3 during the early stages of its development: importance of the epidermal mucus as an innate immune barrier

Cyprinid herpesvirus 3 (CyHV-3) causes a lethal disease in common and koi carp (Cyprinus carpio). The present study investigated the ability of CyHV-3 to infect common carp during the early stages of its development (from embryos to fingerlings) after inoculation by immersion in water containing the virus. Fish were inoculated at different times after hatching with a pathogenic recombinant CyHV-3 strain expressing luciferase. The sensitivity and permissivity of carp to CyHV-3 were investigated using in vivo bioluminescence imaging. The susceptibility of carp to CyHV-3 disease was investigated by measuring the survival rate. Carp were sensitive and permissive to CyHV-3 infection and susceptible to CyHV-3 disease at all stages of development, but the sensitivity of the two early developmental stages (embryo and larval stages) was limited compared to later stages. The lower sensitivity observed for the early developmental stages was due to stronger inhibition of viral entry into the host by epidermal mucus. In addition, independent of the developmental stage at which inoculation was performed, the localization of light emission suggested that the skin is the portal of CyHV-3 entry. Taken together, the results of the present study demonstrate that carp are sensitive and permissive to CyHV-3 at all stages of development and confirm that the skin is the major portal of entry after inoculation by immersion in infectious water. The results also stress the role of epidermal mucus as an innate immune barrier against pathogens even and especially at the early stages of development.     

Emergence of fatal European genotype CyHV-3/KHV in mainland China

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a highly infectious causative agent to common carp and koi worldwide. The virus is mainly consisted of European and Asian genotype isolates. To date, no European genotype CyHV-3 has been found emerging in the East and Southeast Asian regions. In late March 2011, an outbreak of CyHV-3 disease occurred in Guangzhou City, Guangdong Province, China, resulting in the deaths of approximately 200 large-sized adult koi within four weeks. One moribund koi was sampled for CyHV-3 isolation. Thus, a CyHV-3 was isolated in KCF-1 cells and designated as KHV-GZ11. Abundant mature or immature virions in infected KCF-1 cells were observed under a transmission electron micrograph. In addition, intra-nuclear inclusion body-like structures with masses of virions were also observed. Based on the TK and ORF136H genes, the sequence analyses revealed that KHV-GZ11 is a distinct European genotype of CyHV-3. Moreover, the infectivity experiment showed that KHV-GZ11 was highly virulent to koi. In summary, we are the first to confirm the emergence of fatal European genotype CyHV-3/KHV in East and Southeast Asia. Our study will provide new insight to explore the virus origin and epidemiology, as well as its pathogenicity.     

Myxobolus infections of common carp (Cyprinus carpio) in Syrian fish farms

During a survey on Myxobolus infection of pond-cultured common carp in Syria three Myxobolus spp. were found. Myxobolus dispar infected the gill arteries, forming large elongated plasmodia in the gill filaments. The plasmodia of M basilamellaris were located in the gill arches at the base of the filaments. Elongated filiform plasmodia of M. encephalicus were found in the blood vessels of the brain. Despite the common occurrence of the above parasites, no disease symptoms were observed in the infected fish specimens. This is the first report on myxosporean infection of fish from Syrian waters.     

Infectivity of porcine circovirus type 2 DNA in semen from experimentally-infected boars

Porcine circovirus type 2 (PCV2) is an economically important pathogen. It has been demonstrated that PCV2 DNA can be detected in boar semen by PCR; however, the biological relevance of this is unknown. The objectives of this study were to determine if semen positive for PCV2 DNA is infectious (1) in a swine bioassay, or (2) when used for artificial insemination. For the first objective, 4-week-old pigs were inoculated intraperitoneally with PCV2 DNA-negative (bioassay-control; n = 3), PCV2a DNA-positive (bioassay-PCV2a; n = 3), or PCV2b DNA-positive (bioassay-PCV2b; n = 3) raw semen, or PCV2 live virus (bioassay-positive; n = 3), respectively. Pigs inoculated with PCV2 DNA-positive semen and PCV2 live virus became viremic and developed anti-PCV2 antibodies indicating that the PCV2 DNA present in semen was infectious. For the second objective, three Landrace gilts were inseminated with PCV2 DNA-negative semen (gilts-controls) from experimentally-infected boars, and six gilts were artificially inseminated with semen positive for PCV2a DNA (gilts-PCV2a; n = 3) or PCV2b DNA (gilts-PCV2b; n = 3). Serum samples collected from the gilts in all groups remained negative for anti-PCV2 antibodies for the duration of the experiment. In addition, fetal serum samples from all 105-day-gestation fetuses were negative for anti-PCV2 antibodies or PCV2 DNA. Under the conditions of this study, PCV2 DNA-positive semen was not infectious when used to artificially inseminate gilts; however, it was demonstrated to be infectious in a swine bioassay model and therefore is a potential means of PCV2 transmission amongst swine herds.     

Survey on Myxobolus infection of the bleak (Alburnus alburnus L.) in the river Danube and in Lake Balaton

In a three-year survey of myxosporean infections of the bleak (Alburnus alburnus), involving the examination of 205 fish specimens from the River Danube and 50 from Lake Balaton, four Myxobolus species (two gill parasites, one fin parasite and a species parasitising the skeletal muscles) were detected. Two of the species could be identified as M. alburni and M. obesus. Of the other two species, the gill parasite proved to be a hitherto undescribed species which is described here as a new species by the name of M. margitae. One of the two gill-parasitic species, M. obesus, formed plasmodia in the respiratory lamellae of the gill filaments, while the plasmodia of M. margitae n. sp. were formed in the afferent artery of the primary gill filaments. The plasmodia containing spores morphologically identifiable with the species M. alburni were located in the connective tissue between the fin rays. The less frequently found muscle-parasitic Myxobolus species has not been identified precisely. The plasmodia of M. obesus were found in the fish in May and June, while those of M. alburni and M. margitae n. sp. in July and August. The prevalence of infection in fish examined in these periods was 15.5% for M. obesus, 11.5% for M. margitae and 14.0% for M. alburni.     

三聚氰胺对鲤鱼组织SOD、POD和MDA含量的影响

通过将三聚氰胺腹腔注射鲤鱼后测定超氧化物歧化酶(SOD)、过氧化物酶(POD)活性及丙二醛(MDA)含量的变化,探讨其对鲤鱼脑、肝、肾和腮各组织的影响。对50条鲤鱼分别以1、10、100、1 000、2 000mg/L浓度的三聚氰胺腹腔注射,对照组注射等量的生理盐水。以比色法测定肾脏SOD、POD活性和MDA含量的变化。发现鲤鱼各组织中SOD、POD活性先升高后降低;MDA含量呈上升趋势,且始终高于对照组。三聚氰胺对各组织的影响存在剂量-效应关系。表明三聚氰胺影响鲤鱼各组织功能,这可能与各组织中抗氧化酶活性降低和丙二醛含量升高有一定的关系。     

Gill oxidative stress and histopathological biomarkers of pollution impacts in Nile tilapia from Lake Mariut and Lake Edku, Egypt

Various oxidative stress and histopathological biomarkers in gill tissues of Nile tilapia Oreochromis niloticus were investigated. Fish were collected from four sites that differ in their extent of pollution load, including heavy metals: the southeast basin (SEB), main basin (MB), and northwest basin (NWB) of Lake Mariut; and Boughaz El-Maadiya, a channel in Lake Edku. The oxidative stress biomarkers that were analyzed included lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), and glutathione redox cycle enzymes (glutathione peroxidase [GPx] and glutathione reductase [GR]). Levels of reduced glutathione (GSH) were also evaluated. Gill morphology was analyzed by light microscopy and scanning electron microscopy (SEM). Gill LPO was significantly higher in gill tissues of fish collected from the more heavily contaminated MB (40.0%) and NWB (51.4%) sites than in gill samples from the less-contaminated (reference) site, the SEB. Gill LPO in fish from Lake Edku was intermediate but was not significantly higher (17.1%) than the reference. The activities of antioxidant enzymes and the redox-sensitive thiol compound GSH were significantly lower in gill samples from the disturbed sites than in samples from the reference site. Specifically, SOD in MB, NWB, and Lake Edku samples; CAT and GPx in NWB samples; and GR activity and GSH content in MB and NWB samples were lower than those in SEB samples. In most cases, gill tissues from Lake Edku fish had intermediate levels of antioxidants. The main histopathological alterations observed in gills were epithelial lifting, hyperplasia and hypertrophy of the respiratory epithelium, lamellar fusion, and aneurysms. In addition, SEM results demonstrated transformation of the surface structure of epithelial pavement cells. Pathological reactions in the gills of Nile tilapia were most severe at the MB and NWB sites. Our findings suggest that Nile tilapia responded differently according to the environmental stress index in each sampling area. This study is the first to report gill oxidative stress and histopathologies in Nile tilapia from Egyptian aquatic environments.     

全鱼基因在鲫鱼体内的整合与转录

以鲤鱼金属硫蛋白启动子与大麻哈鱼生长激素基因重组的融合全鱼基因（ｃｏｍｍｏｎｃａｒｐｍｅｔａｌｏｔｈ－ｉｏｎｅｉｎｐｒｏｍｏｔｅｒａｎｄｔｒｏｕｔｓａｌｍｏｎｇｒｏｗｔｈｈｏｒｍｏｎｅｇｅｎｅ，ｃＭＴｓＧＨ）为外源基因，通过显微注射法将其线性片段导入鲫鱼受精卵内，研究了其整合与转录效率。结果表明，全鱼基因在鲫鱼基因组中的整合率为３６．４％（１６／４４），对转基因鱼阳性的ＲＮＡ样本进行Ｎｏｒｔｈｅｒｎ印迹杂交检测，转录率为２５％（１／４）。由此认为，该全鱼基因可以作为转基因鱼研究和应用的外源基因。     

Phenotypic plasticity is maintained despite geographical isolation in an African cichlid fish,Pseudocrenilabrus multicolor

Gene flow among populations in different selective environments should favor the evolution of phenotypic plasticity over local adaptation. Plasticity in development is a common response to long‐term hypoxia in some widespread African fishes, including Pseudocrenilabrus multicolor, a cichlid that exploits both normoxic (high oxygen) rivers/lakes and hypoxic (low oxygen) swamps. Previous studies have shown that fish from normoxic and hypoxic sites differ in many traits, including gill size, brain size and body shape, and that much of this variation reflects developmental plasticity. However, these earlier studies focused on areas in Uganda where gene flow between swamp and river or lake populations is high. In this study we tested the hypothesis that P. multicolor from a relatively isolated lake population (Lake Saka, Uganda) exhibit low levels of plasticity in traits related to oxygen uptake. Multiple broods of P. multicolor from Lake Saka were reared under low and high dissolved oxygen, and traits related to gill size, brain mass and body shape were quantified. Surprisingly, both gill size and brain mass showed high levels of developmental plasticity. We suggest that high levels of plasticity, particularly in the gill size of P. multicolor, reflects low costs of maintaining the plastic response, even in relatively isolated populations.