Search

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., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

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.\., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Adamek, Matras, Rebl, Stachnik, Falco, Bauer, Miebach, Teitge, Jung-Schroers, Abdullah, Krebs, Schröder, Fuchs, Reichert and Steinhagen.)

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.

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., (© 2018 John Wiley & Sons Ltd.)

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., (© 2018 John Wiley & Sons Ltd.)

The effect of dietary β-glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard ® , a β-1,3/1,6-glucan, or a β-glucan-free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10 9 colony-forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT-PCR-DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β-glucan compared to carp from the control group. In β-glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β-glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β-glucan may provide protection against infections of the gut in carp., (© 2018 John Wiley & Sons Ltd.)

Shewanella spp. are Gram-negative, rod-shaped, motile bacteria that are widely distributed in marine and freshwater environments. The bacteria are present in the physiological microflora of fish from temperate waters and are known as fish spoilage species. From clinically healthy fish and from fish with skin ulcerations, Shewanella spp. is regularly isolated, indicating a possible role as fish pathogen. In this study, 74 isolates of Shewanella spp. were analysed. For species identification, biochemical techniques, 16S rRNA sequencing, MALDI-TOF MS and the Sherlock Microbial Identification System (MIS) based on the composition of fatty acid ethyl esters were compared. The phylogenetic relationship, cytotoxicity in vitro and resistance against antibiotics were tested. The most reliable method for species identification was 16S rRNA sequencing. From diseased fish, clinically healthy fish and the aquatic environment, different Shewanella species were isolated. This indicates that Shewanella spp. is widespread in the aquatic milieu and acts as a secondary pathogen. The virulence of Shewanella spp. is probably not depending on the species but on the isolate itself. Many isolates of Shewanella spp. were showing multiresistances against antibiotic substances, especially in samples derived from retailers and in routine diagnostics, all Shewanella spp. should therefore be tested for resistances against antibiotic agents., (© 2017 John Wiley & Sons Ltd.)

Carp edema virus disease (CEVD), also known as koi sleepy disease, is caused by a poxvirus associated with outbreaks of clinical disease in koi and common carp Cyprinus carpio. Originally characterised in Japan in the 1970s, international trade in koi has led to the spread of CEV, although the first recognised outbreak of the disease outside of Japan was not reported until 1996 in the USA. In Europe, the disease was first recognised in 2009 and, as detection and diagnosis have improved, more EU member states have reported CEV associated with disease outbreaks. Although the structure of the CEV genome is not yet elucidated, molecular epidemiology studies have suggested distinct geographical populations of CEV infecting both koi and common carp. Detection and identification of cases of CEVD in common carp were unreliable using the original PCR primers. New primers for conventional and quantitative PCR (qPCR) have been designed that improve detection, and their sequences are provided in this paper. The qPCR primers have successfully detected CEV DNA in archive material from investigations of unexplained carp mortalities conducted >15 yr ago. Improvement in disease management and control is possible, and the principles of biosecurity, good health management and disease surveillance, applied to koi herpesvirus disease, can be equally applied to CEVD. However, further research studies are needed to fill the knowledge gaps in the disease pathogenesis and epidemiology that, currently, prevent an accurate assessment of the likely impact of CEVD on European koi and common carp aquaculture and on wild carp stocks.

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.

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.

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.

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.

Background: Infections with carp edema virus, a pox virus, are known from Japanese koi populations since 1974. A characteristic clinical sign associated with this infection is lethargy and therefore the disease is called "koi sleepy disease". Diseased koi also show swollen gills, enophthalmus, and skin lesions. Mortality rates up to 80 % are described. For a long period of time, disease outbreaks seemed to be restricted to Japan. However, during the last years clinical outbreaks of koi sleepy disease also occurred in the UK and in the Netherlands., Case Presentation: In spring 2014 koi from different ponds showing lethargic behavior, skin ulcers, inflammation of the anus, enophthalmus, and gill necrosis were presented to the laboratory for diagnosis. In all cases, new koi had been purchased earlier that spring from the same retailer and introduced into existing populations. Eleven koi from six ponds were examined for ectoparasites and for bacterial and viral infections (cyprinid herpesviruses in general and especially koi herpesvirus (KHV) known formally as Cyprinid herpesvirus 3 (CyHV-3); and Carp Edema Virus). In most of the cases parasites were not detected from skin and gills. Only opportunistic freshwater bacteria were isolated from skin ulcers. In cell cultures no cytopathic effect was observed, and none of the samples gave positive results in PCR tests for cyprinid herpesviruses. By analyzing gill tissues for CEV in seven out of eleven samples by a nested PCR, PCR products of 547 bp and 180 bp (by using nested primers) could be amplified. An outbreak of Koi Sleepy Disease was confirmed by sequencing of the PCR products. These results confirm the presence of CEV in German koi populations., Conclusion: A clinical outbreak of "koi sleepy disease" due to an infection with Carp Edema Virus was confirmed for the first time in Germany. To avoid transmission of CEV to common carp testing of CEV should become part of fish disease surveillance programs.

The intestinal myxosporean parasite Enteromyxum leei causes severe desquamative enteritis in gilthead sea bream (Sparus aurata) (Teleostei) that impairs nutrient absorption causing anorexia and cachexia. In fish, as in terrestrial vertebrates, intestinal goblet cells are responsible for the adherent mucus secretion overlying epithelial cells, which constitutes a first line of innate immune defense against offending microorganisms but serves also as substrate and nutrient source for the commensal microflora. The secreted intestinal mucus of parasitized (n = 6) and unexposed (n = 8) gilthead sea bream was isolated, concentrated, and subjected to downward gel chromatography. Carbohydrate and protein contents (via PAS and Bradford stainings), terminal glycosylation (via lectin ELISA), and Aeromonas hydrophila and Vibrio alginolyticus adhesion were analyzed for the isolated intestinal mucins. Parasitized fish, compared with unexposed fish, presented intestinal mucus mucins with a lower glycoprotein content and glycosylation degree at the anterior and middle intestine, whereas both glycoprotein content and glycosylation degree increased at the posterior intestine section, though only significantly for the total carbohydrate content. Additionally, a slight molecular size increase was detected in the mucin glycoproteins of parasitized fish. Terminal glycosylation of the mucus glycoproteins in parasitized fish pointed to an immature mucin secretion (N-acetyl-α-D-galactosamine increase, α-L-fucose, and neuraminic-acid-α-2-6-galactose reduction). Bacterial adhesion to large-sized mucus glycoproteins (>2,000 kDa) of parasitized fish was significantly lower than in unexposed fish.

A novel host innate immune defence mechanism against invading pathogens, namely the formation of neutrophil extracellular traps (NETs), has recently been discovered. These NETs are described as DNA fibres released by dying neutrophils, which are able to entrap and kill various microbes. Here we studied the effect of the feed additive β-glucan, namely MacroGard(®), on the degradation of NETs by the important fish pathogen Aeromonas hydrophila. Therefore, common carp (Cyprinus carpio) head kidney cells consisting of approximately 45% neutrophils were isolated and treated with or without β-glucan. The degradation of NETs after co-incubation with A. hydrophila was analysed by immunofluorescence microscopy. The data show that A. hydrophila is able to degrade NETs and that treatment of cells with β-glucan significantly protects the NETs against bacterial degradation. Control experiments revealed that β-glucan augments nuclease activity of the bacteria at the same time while protecting the NETs against its degradation. In conclusion the data indicate that β-glucan might affect the composition and stabilisation of NETs and thereby protecting them against degradation by A. hydrophila nuclease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aim: To identify and characterize six chitinolytic bacterial strains isolated from ornamental fish., Methods and Results: Six different isolates of Deefgea chitinilytica were detected in healthy as well as diseased ornamental fish in Germany over a period of 2 years. Bacterial strains were identified using 16S rRNA partial gene sequencing and further characterized using different biochemical microtest systems and additional standard biochemical tests., Conclusion: We show that commercially available biochemical microtest systems are useful for identification of D. chitinilytica, supplemented by 16S rRNA partial gene sequencing. Furthermore, this study provides new information about the occurrence of D. chitinilytica, as this is the first isolation of D. chitinilytica from animals and first described isolation in Europe., Significance and Impact of the Study: Deefgea chitinilytica may be isolated regularly in fish diagnostic laboratories. Therefore, accurate identification of this bacterial species is important. Involvement of D. chitinilytica in opportunistic infections of aquatic organisms cannot be excluded and has to be further investigated., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)