Proteomic analysis of carp seminal plasma provides insights into the immune response to bacterial infection of the male reproductive system.

Aeromonas salmonicida is recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. However, the mechanism of immunity to these bacteria in common carp is still not well understood, especially the immune regulation in the gonad to bacterial infection. The aims of our study were to analyze changes in the seminal plasma proteome following A. salmonicida infection in carp males. The observed pathological changes in the tissue (liver, spleen, kidney and testis) morphology and upregulation of immune-related genes (tnfa2, il6a) confirmed the successful infection challenge. Using mass spectrometry-based label-free quantitative proteomics, we identified 1402 seminal plasma proteins, and 44 proteins (20 up- and 24 downregulated) were found to be differentially abundant between infected and control males. Most differentially abundant proteins were involved in the immune response mechanisms, such as acute phase response, complement activation and coagulation, inflammation, lipid metabolism, cell-cell and cell-matrix adhesion, creatine-phosphate biosynthesis and germ cell-Sertoli cell junction signaling. Bacterial infection also caused profound changes in expression of selected genes in the testis and hematopoietic organs, which contributed to changes in seminal proteins. The altered seminal proteins and bacterial proteins in seminal plasma may serve as valuable markers of infection in the testis.

Immunological insights into the resistance of Nile tilapia strains to an infection with tilapia lake virus.

The emergence of viral diseases affecting fish and causing very high mortality can lead to the disruption of aquaculture production. Recently, this occurred in Nile tilapia aquaculture where a disease caused by a systemic infection with a novel virus named tilapia lake virus (TiLV) caused havoc in cultured populations. With mortality surpassing 90% in young tilapia, the disease caused by TiLV has become a serious challenge for global tilapia aquaculture. In order to partly mitigate the losses, we explored the natural resistance to TiLV-induced disease in three genetic strains of tilapia which were kept at the University of Göttingen, Germany. We used two strains originating from Nilotic regions (Lake Mansala (MAN) and Lake Turkana (ELM)) and one from an unknown location (DRE). We were able to show that the virus is capable of overcoming the natural resistance of tilapia when injected, providing inaccurate mortality results that might complicate finding the resistant strains. Using the cohabitation infection model, we found an ELM strain that did not develop any clinical signs of the infection, which resulted in nearly 100% survival rate. The other two strains (DRE and MAN) showed severe clinical signs and much lower survival rates of 29.3% in the DRE strain and 6.7% in the MAN strain. The disease resistance of tilapia from the ELM strain was correlated with lower viral loads both at the mucosa and internal tissues. Our results suggest that the lower viral load could be caused by a higher magnitude of a mx1-based antiviral response in the initial phase of infection. The lower pro-inflammatory responses also found in the resistant strain might additionally contribute to its protection from developing pathological changes related to the disease. In conclusion, our results suggest the possibility of using TiLV-resistant strains as an ad hoc, cost-effective solution to the TiLV challenge. However, as the fish from the disease-resistant strain still retained significant virus loads in liver and brain and thus could become persistent virus carriers, they should be used within an integrative approach also combining biosecurity, diagnostics and vaccination measures.\.

Don't Let It Get Under Your Skin! - Vaccination Protects the Skin Barrier of Common Carp From Disruption Caused by Cyprinid Herpesvirus 3.

Vaccination is the best form of protecting fish against viral diseases when the pathogen cannot be contained by biosecurity measures. Vaccines based on live attenuated viruses seem to be most effective for vaccination against challenging pathogens like Cyprinid herpesvirus 3. However, there are still knowledge gaps how these vaccines effectively protect fish from the deadly disease caused by the epitheliotropic CyHV-3, and which aspects of non-direct protection of skin or gill integrity and function are important in the aquatic environment. To elucidate some elements of protection, common carp were vaccinated against CyHV-3 using a double deletion vaccine virus KHV-T ΔDUT/TK in the absence or presence of a mix of common carp beta-defensins 1, 2 and 3 as adjuvants. Vaccination induced marginal clinical signs, low virus load and a minor upregulation of cd4, cd8 and igm gene expression in vaccinated fish, while neutralisation activity of blood serum rose from 14 days post vaccination (dpv). A challenge infection with CyHV-3 induced a severe disease with 80-100% mortality in non-vaccinated carp, while in vaccinated carp, no mortality was recorded and the virus load was >1,000-fold lower in the skin, gill and kidney. Histological analysis showed strongest pathological changes in the skin, with a complete destruction of the epidermis in non-vaccinated carp. In the skin of non-vaccinated fish, T and B cell responses were severely downregulated, inflammation and stress responses were increased upon challenge, whereas vaccinated fish had boosted neutrophil, T and B cell responses. A disruption of skin barrier elements (tight and adherence junction, desmosomes, mucins) led to an uncontrolled increase in skin bacteria load which most likely exacerbated the inflammation and the pathology. Using a live attenuated virus vaccine, we were able to show that increased neutrophil, T and B cell responses provide protection from CyHV-3 infection and lead to preservation of skin integrity, which supports successful protection against additional pathogens in the aquatic environment which foster disease development in non-vaccinated carp.

It is everywhere-A survey on the presence of carp edema virus in carp populations in Germany.

Carp edema virus (CEV) is the causative agent of koi sleepy disease (KSD), a serious gill disease affecting common carp, Cyprinus carpio, and its ornamental variety, koi. After recent detections of the virus in various countries around the world, KSD has emerged as a new global disease in carp. However, the prevalence of the infection in carp populations in a given geographical region has not been studied thoroughly. The present communication reports an investigation into the presence of CEV in carp and koi populations in Germany. For this purpose, gill samples collected from carp and koi populations suffering from gill diseases or collected for a routine examination of their health status were tested for the presence of CEV by PCR. In total, 651 fish samples from 401 carp or koi cases were examined in 2015 and 2016, additional 118 samples from previous studies were included in the examination. CEV was detected in archive samples from carp dating back to 2007, and in koi samples dating back to 2009. From 2015 to 2016, CEV was detected in 69% of cases from carp populations examined from the main carp-producing areas in Germany, and in 41% of the examined cases from koi populations from all over Germany. Clinical KSD occurred mainly from April to June in carp populations at water temperatures ranging from 8 to 12°C and in koi populations at water temperatures ranging from 18 to 22°C. Most fish from clinically affected carp or koi populations harboured high virus loads of above 10,000 copies of CEV-specific DNA per 250 ng DNA, while gills from fish of other fish species from the ponds, including goldfish, grass carp and European perch were found CEV negative or harboured a low virus load. A phylogenetic analysis revealed the presence of multiple CEV variants from genogroup I in carp and genogroup II in koi populations in Germany. Genetically identical genogroup I isolates were detected in carp from different geographical locations in Germany and in other European carp populations. Some German genogroup II variants were identical to variants previously recorded from koi in Asian and other European countries. The data presented here show that CEV is highly prevalent in German common carp and koi populations and implies the spreading of this virus by intense trading of common carp and koi without necessary risk mitigating measures. As infections with this virus may induce serious disease, CEV diagnostic should be included in health surveillance and disease monitoring programmes.

Association of the alga Cladogonium sp. with a multifactorial disease outbreak in dwarf shrimp (Neocaridina davidi).

This study outlines a multifactorial disease outbreak in a population of the freshwater shrimp Neocaridina davidi, with the focus on a rarely described parasitic alga. Within this multifactorial disease outbreak, low but consistent mortality was observed. During microscopic examination, an infection of the shrimp with bacterial and fungal-like agents was diagnosed. Furthermore, the green alga Cladogonium sp. was found in pleopodal regions. The alga compromised the body surface of the shrimp, and its rhizoids penetrated the chitin shell and reached into the subcutaneous tissue. This might be a first indication of a parasitic lifestyle. In addition to a morphological description, sequencing data are presented which allow the taxonomic classification of the organism within the order Trentepohliales.

Koi sleepy disease as a pathophysiological and immunological consequence of a branchial infection of common carp with carp edema virus.

Gills of fish are involved in respiration, excretion and osmoregulation. Due to numerous interactions between these processes, branchial diseases have serious implications on fish health. Here, "koi sleepy disease" (KSD), caused by carp edema virus (CEV) infection was used to study physiological, immunological and metabolic consequences of a gill disease in fish. A metabolome analysis shows that the moderately hypoxic-tolerant carp can compensate the respiratory compromise related to this infection by various adaptations in their metabolism. Instead, the disease is accompanied by a massive disturbance of the osmotic balance with hyponatremia as low as 71.65 mmol L-1, and an accumulation of ammonia in circulatory blood causing a hyperammonemia as high as 1123.24 µmol L-1. At water conditions with increased ambient salt, the hydro-mineral balance and the ammonia excretion were restored. Importantly, both hyponatremia and hyperammonemia in KSD-affected carp can be linked to an immunosuppression leading to a four-fold drop in the number of white blood cells, and significant downregulation of cd4, tcr a2 and igm expression in gills, which can be evaded by increasing the ion concentration in water. This shows that the complex host-pathogen interactions within the gills can have immunosuppressive consequences, which have not previously been addressed in fish. Furthermore, it makes the CEV infection of carp a powerful model for studying interdependent pathological and immunological effects of a branchial disease in fish.

Impact of a reduced water salinity on the composition of Vibrio spp. in recirculating aquaculture systems for Pacific white shrimp (Litopenaeus vannamei) and its possible risks for shrimp health and food safety.

Tropical shrimp, like Litopenaeus vannamei, in land-based recirculating aquaculture systems (RAS) are often kept at low water salinities to reduce costs for artificial sea salt and the amount of salty wastewater. Although these shrimp are tolerant against low salinities, innate immunity suppression and changes in the microbial composition in the water can occur. As especially Vibrio spp. are relevant for shrimp health, alterations in the species composition of the Vibrio community were analysed in water from six RAS, run at 15‰ or 30‰. Additionally, pathogenicity factors including pirA/B, VPI, toxR, toxS, vhh, vfh, tdh, trh, flagellin genes and T6SS1/2 of V. parahaemolyticus were analysed. The Vibrio composition differed significantly depending on water salinity. In RAS at 15‰, higher numbers of the potentially pathogenic species V. parahaemolyticus, V. owensii and V. campbellii were detected, and especially in V. parahaemolyticus, various pathogenicity factors were present. A reduced salinity may therefore pose a higher risk of disease outbreaks in shrimp RAS. Because some of the detected pathogenicity factors are relevant for human health, this might also affect food safety. In order to produce healthy shrimp as a safe food for human consumption, maintaining high water salinities seems to be recommendable.

Characterization of carp seminal plasma Wap65-2 and its participation in the testicular immune response and temperature acclimation.

Two functionally distinct isoforms of warm-temperature acclimation related 65-kDa protein (Wap65-1 and Wap65-2) with a role in the immune response are present in fish. To our knowledge, contrary to Wap65-1, Wap65-2 has neither been isolated nor functionally characterized in carp especially in reproductive system. The aim of this study was to characterize Wap65-2 and ascertain its functions in immune response and temperature acclimation within reproductive system. Wap65-2 corresponded to one of the most abundant proteins in carp seminal plasma, with a high immunologic similarity to their counterparts in seminal plasma of other fish species and a wide tissue distribution, with predominant expression in the liver. The immunohistochemical localization of Wap65-2 to spermatogonia, Leydig cells, and the epithelium of blood vessels within the testis suggests its role in iron metabolism during spermatogenesis and maintenance of blood-testis barrier integrity. Wap65-2 secretion by the epithelial cells of the spermatic duct and its presence around spermatozoa suggests its involvement in the protection of spermatozoa against damage caused by heme released from erythrocytes following hemorrhage and inflammation. Our results revealed an isoform-specific response of Wap65 to temperature acclimation and Aeromonas salmonicida infection which alters blood-testis barrier integrity. Wap65-2 seems to be related to the immune response against bacteria, while Wap65-1 seems to be involved in temperature acclimation. This study expands the understanding of the mechanism of carp testicular immunity against bacterial challenge and temperature changes, in which Wap65-2 seems to be involved and highlights their potential usefulness as biomarkers of inflammation and temperature acclimation.

Water disinfection by ozonation has advantages over UV irradiation in a brackish water recirculation aquaculture system for Pacific white shrimp (Litopenaeus vannamei).

By keeping tropical shrimp, like Litopenaeus vannamei, in recirculating aquaculture systems (RAS), valuable food for human consumption can be produced sustainable. L. vannamei tolerates low salinities, and therefore, the systems can operate under brackish water conditions. The stabilization of the microbial community in RAS might be difficult under high organic loads, and therefore, water treatment measures like UV irradiation or ozone application are commonly used for bacterial reduction. To investigate the impact of these measures, the effects of UV irradiation and ozone application were studied in small-scale brackish water RAS with a salinity of 15‰ stocked with L. vannamei. UV reactors with 7 and 9 W were used, and by ozonizers with a power of 5-50 mg/hr, the redox potential in the water was adjusted to 350 mV. Ozone had a stabilizing effect on the microbial composition in the water and on biofilms of tank surfaces and shrimp carapaces, prevented an increase of nitrite and accelerated the degradation of nitrate in the water. UV irradiation led to changes in the microbial composition and was less effective in optimizing the chemical water quality. Thus, the use of ozone could be recommended for water treatment in brackish water RAS for shrimp.

Effect of disinfection with peracetic acid on the microbial community of a seawater aquaculture recirculation system for Pacific white shrimp (Litopenaeus vannamei).

When tropical shrimps are kept in recirculating aquaculture systems (RAS), one of the limiting factors is the maintenance of a sufficient water quality, and therefore, often disinfectants like peracetic acid (PAA) are added to the water either as prophylactic or treatment measure. In this study, PAA in concentrations of 0.1 mg/L, 1 mg/L and 10 mg/L was applied continuously for 56 days to small-scale seawater RAS stocked with Litopenaeus vannamei. Treatment with 0.1 mg/L did not result in a reduction in the total bacterial amount and therefore was not effective. A concentration of 10 mg/L led to significant changes in the chemical water parameters already after 2 days and was therefore not recommendable. A concentration of 1 mg/L led to increased levels of ammonia and nitrite within 2 days and to a significant increase in the bacterial amount in the water, most probably due to an enhanced growth of heterotrophic bacteria. The microflora showed significant fluctuations, and there were indications that the welfare of the shrimps was affected. Using 1 mg PAA/L for prophylactic use is therefore also not recommendable but might be an alternative option for short-term treatment in cases of disease outbreaks.

Is humane slaughtering of rainbow trout achieved in conventional production chains in Germany? Results of a pilot field and laboratory study.

BACKGROUND: Rainbow trout, Oncorhynchus mykiss, is an important fish in European freshwater aquaculture. This industry sector is dominated by small family-owned enterprises located in rural areas. A large percentage of rainbow trout produced by these small enterprises is marketed directly and killed on demand and not processed in commercial processing plants. EU and national regulations stipulate that fish shall be stunned prior to killing and slaughter. The overall objective of this study was to monitor how stunning interventions were integrated into the production chains of German conventional trout aquaculture in order to safeguard animal welfare during stunning and killing. For this, the stunning and slaughtering processes were monitored on 18 rainbow trout farms in various German federal states. During the on-farm research, (i) the stunning success, (ii) injuries related to the stunning procedure, (iii) duration between stunning and killing, and (iv) visible responses at the time of slaughtering were assessed as welfare indicators. In addition, haematological and biochemical blood parameters were measured as indicators for physiological stress. Due to the fact that stunning interventions should induce a loss of consciousness in fish, in a laboratory study, it was examined whether the absence of the brainstem/ behavioural responses, opercular movements (OM) or eye-rolling reflex (vestibulo-ocular reflex, VOR) was correlated with the stage of insensibility. RESULTS: The majority of rainbow trout farms applied manual percussion (38%) or electrical stunning (48%), while on 14% of the farms, the fish were stunned by electrical stunning which was immediately followed by manual percussion. After percussive stunning, about 92.3% of the rainbow trout displayed no OM or VOR as brainstem/ behavioural indicators of consciousness. This percentage varied on farms which applied electrical stunning. While on the majority of farms, 95 to 100% of the fish were unconscious according to the observation of brainstem/ behavioural indicators, the stunning intervention was less effective on farms where rainbow trout were stunned at current densities below 0.1 A dm2 or for a few seconds only. The laboratory study confirmed that the absence of brainstem/ behavioural indicators correlated with the absence of visually evoked responses (VER) of the brain to light stimuli as a neuronal indicator of insensibility. Therefore, the brainstem/ behavioural signs can be used to interpret the stage of insensibility in rainbow trout. A stage of insensibility could safely be induced by exposing portion-sized rainbow trout to an electric current density above 0.1 A dm2. This was not influenced by the orientation of the electric field. CONCLUSIONS: In conventional aquaculture, rainbow trout can effectively be stunned by manual percussion or electrical stunning. Consciousness can be monitored by the absence of opercular movements or the eye-rolling reflex, which are lost approximately at the same time as neurological responses like VER. For safeguarding animal welfare during stunning and killing of rainbow trout in conventional production processes, the stunning process requires careful attention and the operating personnel need to be trained in using the stunning devices and recognising indicators of consciousness.

Diagnostic methods for identifying different Aeromonas species and examining their pathogenicity factors, their correlation to cytotoxicity and adherence to fish mucus.

Aeromonas spp. are ubiquitous in the aquatic environment, acting as facultative or obligate pathogens for fish. Identifying Aeromonas spp. is important for pathogenesis and prognosis in diagnostic cases but can be difficult because of their close relationship. Forty-four already characterized isolates of Aeromonas spp. were analysed by 16S rRNA gene sequencing, by gyrase B sequencing, by analysing their fatty acid profiles, by biochemical reactions and by MALDI-TOF MS. To determine their pathogenicity, cytotoxicity, adhesion to mucus and the expression of 12 virulence factors were tested. The susceptibility of the isolates towards 13 different antibiotics was determined. MALDI-TOF MS was found to be an acceptable identification method for Aeromonas spp. Although the method does not detect all species correctly, it is time-effective and entails relatively low costs and no other methods achieved better results. A high prevalence of virulence-related gene fragments was detected in almost all examined Aeromonas spp., especially in A. hydrophila and A. salmonicida, and most isolates exhibited a cytotoxic effect. Single isolates of A. hydrophila and A. salmonicida showed multiple resistance to antibiotics. These results might indicate the potentially pathogenic capacity of Aeromonas spp., suggesting a risk for aquatic animals and even humans, given their ubiquitous nature.

Recommendations for identifying pathogenic Vibrio spp. as part of disease surveillance programmes in recirculating aquaculture systems for Pacific white shrimps (Litopenaeus vannamei).

Due to their pathogenic potential, identifying Vibrio species from recirculating aquaculture systems (RAS) for Pacific white shrimp (Litopenaeus vannamei) is of great importance to determine the risk for animal's as well as for the consumer's health. The present study compared identification results for a total of 93 Vibrio isolates, including type strains and isolates from shrimp aquaculture. Results from biochemical identifications, 16S rRNA sequencing, sequencing of the uridylate kinase encoding gene pyrH and analysis of the protein spectra assessed by MALDI-TOF MS were compared. The results achieved by these different methods were highly divergent for many of the analysed isolates and for several Vibrio spp difficulties in reliably identifying occurred. These difficulties mainly resulted from missing entries in digital databases, a low number of comparable isolates analysed so far, and high interspecific similarities of biochemical traits and nucleotide sequences between the closely related Vibrio species. Due to the presented data, it can be concluded that for identifying Vibrio spp. from samples in routine diagnostics, it is recommended to use MALDI-TOF MS analysis for a quick and reliable identification of pathogenic Vibrio sp. Nevertheless, editing the database, containing the main spectra of Vibrio is recommended to achieve reliable identification results.

Flavobacteria as secondary pathogens in carp suffering from koi sleepy disease.

Koi sleepy disease (KSD) is a disease with increasing importance in global common carp aquaculture. Despite the fact that carp edema virus (CEV) is most likely the causative agent of KSD, the disease often presents itself as multifactorial with several parasites and bacteria species present on gills, skin or in internal organs. Therefore, in this study, we analysed and presented initial results on an interaction of flavobacteria and CEV in the development of clinical KSD in carp suffering from proliferative gill disease. We examined selected field samples from Germany and Hungary and confirmed the presence of CEV and flavobacteria co-infections in subset of the samples. In several infection experiments, we studied the transfer and dynamics of both infections. Furthermore, we analysed which Flavobacterium species could be isolated from KSD-affected fish and concluded that Flavobacterium branchiophilum is a possible copathogen. Antibiotic treatment experiments showed that CEV seems to be the primary pathogen causing an insult to the gills of carp and by these enabling other pathogens, including F. branchiophilum, to establish co-infections. Despite the fact that F. branchiophilum co-infection is not required for the development of clinical KSD, it could contribute to the pathological changes recorded during the outbreaks.

Stunning of common carp: Results from a field and a laboratory study.

BACKGROUND: Common carp Cyprinus carpio is an important food fish in Central Europe, which in some regions is consumed as part of local tradition. The majority of carp are sold by small retailers and not processed in commercial processing plants. The overall objective of this study was to monitor how animal welfare is safeguarded during the stunning and slaughtering of carp for retail sale. For this, the stunning and slaughtering process was monitored on 12 carp farms. Four welfare-related parameters were assessed: (i) stunning success, (ii) injuries related to the applied stunning method, (iii) time between stunning and slaughter, and (iv) visible responses of carp during slaughtering. In addition, indicators of physiological stress were measured. In order to analyse whether the absence of behavioural indicators of consciousness after electrical stunning was correlated with unconsciousness a complementary laboratory study was performed. Here, carp were exposed to electrical current densities between 0.09 and 0.41 A/dm2. The presence of behavioural responses and visually-evoked responses (VER) in the electro-encephalogram in response to light flashes as indicators for an absence of consciousness was recorded. RESULTS: The carp farms applied manual percussive (18%) or electrical (23%) stunning methods, while the majority of farms used a combination of electrical stunning immediately followed by manual percussive stunning (59%). In the latter condition, 92.6% of stunned carp displayed no behavioural indicators of consciousness and significantly fewer injuries related to mishits compared to sole percussive stunning. In the laboratory study, behavioural indicators of consciousness recovered in carp between 1 and 9 min following removal of the electrical current. However, VER could be recorded already at 30 ± 8 s post stunning. This indicates a fast recovery of carp from electrical stunning when exposed to current densities in the range of those generated by commercially available stunning instruments for fish. CONCLUSIONS: Under field conditions, percussion (applied manually) and electrical stunning might be poor inducers of unconsciousness before slaughter, while a combination was most effective. In order to undertake improvements in electrical stunning, further investigations into the current density, required for inducing prolonged insensibility in carp during electrical stunning, are needed.

Comparison of PCR methods for the detection of genetic variants of carp edema virus.

The infection of common carp and its ornamental variety, koi, with the carp edema virus (CEV) is often associated with the occurrence of a clinical disease called 'koi sleepy disease'. The disease may lead to high mortality in both koi and common carp populations. To prevent further spread of the infection and the disease, a reliable detection method for this virus is required. However, the high genetic variability of the CEV p4a gene used for PCR-based diagnostics could be a serious obstacle for successful and reliable detection of virus infection in field samples. By analysing 39 field samples from different geographical origins obtained from koi and farmed carp and from all 3 genogroups of CEV, using several recently available PCR protocols, we investigated which of the protocols would allow the detection of CEV from all known genogroups present in samples from Central European carp or koi populations. The comparison of 5 different PCR protocols showed that the PCR assays (both end-point and quantitative) developed in the Centre for Environment, Fisheries and Aquaculture Science exhibited the highest analytical inclusivity and diagnostic sensitivity. Currently, this makes them the most suitable protocols for detecting viruses from all known CEV genogroups.

Experimental infections of different carp strains with the carp edema virus (CEV) give insights into the infection biology of the virus and indicate possible solutions to problems caused by koi sleepy disease (KSD) in carp aquaculture.

Outbreaks of koi sleepy disease (KSD) caused by carp edema virus (CEV) may seriously affect populations of farmed common carp, one of the most important fish species for global food production. The present study shows further evidence for the involvement of CEV in outbreaks of KSD among carp and koi populations: in a series of infection experiments, CEV from two different genogroups could be transmitted to several strains of naïve common carp via cohabitation with fish infected with CEV. In recipient fish, clinical signs of KSD were induced. The virus load and viral gene expression results confirm gills as the target organ for CEV replication. Gill explants also allowed for a limited virus replication in vitro. The in vivo infection experiments revealed differences in the virulence of the two CEV genogroups which were associated with infections in koi or in common carp, with higher virulence towards the same fish variety as the donor fish. When the susceptibility of different carp strains to a CEV infection and the development of KSD were experimentally investigated, Amur wild carp showed to be relatively more resistant to the infection and did not develop clinical signs for KSD. However, the resistance could not be related to a higher magnitude of type I IFN responses of affected tissues. Despite not having a mechanistic explanation for the resistance of Amur wild carp to KSD, we recommend using this carp strain in breeding programs to limit potential losses caused by CEV in aquaculture.

First outbreak of an infection with infectious spleen and kidney necrosis virus (ISKNV) in ornamental fish in Germany.

In 2014, infectious spleen and kidney necrosis virus (ISKNV), a member of the genus Megalocytivirus, was detected for the first time in ornamental fish in Germany. Since 2013, angelfish Pterophyllum spp. originating from Colombia have experienced significant epizootics in a number of German retailers' facilities. The diseased fish showed symptoms such as increased ventilation, swollen gills, and ulcerations of the skin. In 2014, diseased angelfish P. altum and platys Xiphophorus maculatus maintained in the same recirculating system were examined. Histopathological lesions included hypertrophic cells, single-cell necrosis, and an inflammatory infiltration of granulocytes, lymphocytes, and macrophages in liver, spleen, and kidney. Transmission electron microscopy revealed the presence of numerous polygonal viral particles (150 nm in diameter) within the cytoplasm of enlarged cells. A PCR assay for the detection of megalocytiviruses amplified 777 bp of major capsid protein gene that was 100% identical to ISKNV. This is the first report of an ISKNV outbreak in Germany that most probably was introduced by infected angelfish from Colombia. To our knowledge, this is the first report of ISKNV detected in fish imported from South America. Given the lethal nature of megalocytiviruses, proper biosecurity would seem prudent in countries like Germany where these emerging pathogens are not established.

Concentration of carp edema virus (CEV) DNA in koi tissues affected by koi sleepy disease (KSD).

Carp edema virus (CEV), the causative agent of 'koi sleepy disease' (KSD), appears to be spreading worldwide and to be responsible for losses in koi, ornamental varieties of the common carp Cyprinus carpio. Clinical signs of KSD include lethargic behaviour, swollen gills, sunken eyes and skin alterations and can easily be mistaken for other diseases, such as infection with cyprinid herpesvirus 3 (CyHV-3). To improve the future diagnosis of CEV infection and to provide a tool to better explore the relationship between viral load and clinical disease, we developed a specific quantitative PCR (qPCR) for strains of the virus known to infect koi carp. In samples from several clinically affected koi, CEV-specific DNA was present in a range from 1 to 2,046,000 copies, with a mean of 129,982 copies and a median of 45 copies per 250 ng of isolated DNA, but virus DNA could not be detected in all clinically affected koi. A comparison of the newly developed qPCR, which is based on a dual-labelled probe, to an existing end-point PCR procedure revealed higher specificity and sensitivity of the qPCR and demonstrated that the new protocol could improve CEV detection in koi. In addition to improved diagnosis, the newly developed qPCR test would be a useful research tool. For example, studies on the pathobiology of CEV could employ controlled infection experiments in which the development of clinical signs could be examined in parallel with a quantitative determination of virus load.