Etiology of Ulcerative Dermal Necrosis (UDN) in Brown Trout (Salmo trutta Morpha trutta)-Preliminary Results.

Every year, ulcerative dermal necrosis (UDN) affects salmonids that spend most of their lives in the sea during their migration to the rivers of northern Poland to spawn. The clinical form of the disease manifests itself in ulcerative skin lesions, which lead to significant weakening of the fish and, in most cases, result in their death. This study was carried out on samples taken from sea trout in the Slupia River in northern Poland. In order to identify the pathogen, experiments on the transmission of the disease were carried out, and additional histopathological, microbiological and electron microscopic examinations were performed. As a result of these studies, it was possible to experimentally transfer the disease from sick to healthy fish. The results indicate a complex etiology of the disease (lack of a clearly defined pathogen), in which the change in the environment from salty to freshwater triggers the related changes in skin physiology, which are the main causes of increased susceptibility to the development of the disease.

Distribution of carp edema virus in organs of infected juvenile common carp.

Introduction: The disease caused by carp edema virus (CEV) manifests with lethargy as a primary sign; this observation in koi in Japan gained the disease the name koi sleepy disease (KSD). In the years following the discovery of the virus in Japan, KSD cases have been noted in the UK in koi and common carp. Conducting research in order to expand knowledge of the processes of distribution of CEV in infected fish organs will be helpful for eradication and diagnostic purposes. Material and Methods: Carp edema virus-affected fish with clinical signs of KSD were experimentally cohabited with common carp fry (30 fish). Three fish were euthanised by bath in a 0.5 g L-1 tricaine solution at one week intervals (7, 14, 21 and 28 days post cohabitation). Tissue samples from the brain, gills, spleen, kidney, intestines and skin were collected, and the total DNA was extracted and tested by real-time PCR. Results: By the seventh day post infection, CEV DNA was most often found in the skin, gills and brain and less frequently in the kidney and intestines. In many of the common carp fry, CEV DNA could typically be found in several organs of each individual fish, although it was only found in one sample of spleen tissue. Conclusion: In this experimental study the pathogenesis of the CEV infection process was shown, the high infectivity of CEV was confirmed and the best organs were determined for sampling in CEV-infection experimentation. The real-time PCR method used in our cohabitation experiments was shown to be useful at the clinical and asymptomatic stage of virus infection.

In vitro modelling of the influence of alternative feeds (Hermetia illucens, Arthrospira platensis) on the resistance of different rainbow trout populations (Oncorhynchus mykiss) against the viral haemorrhagic septicaemia virus and Yersinia ruckeri.

Replacing fishmeal, a finite resource with high market demand, in the diet of carnivorous rainbow trout with proteins from alternative sources may be a challenge for these fish. Therefore, this study investigated whether replacing fishmeal with protein derived from Hermetia illucens or Arthrospira platensis could promote disease susceptibility in local trout populations with different growth performance. This was assessed in vitro by measuring susceptibility to infection with the viral haemorrhagic septicaemia virus (VHSV) or the bacterium Yersinia ruckeri. Analysis of fin tissue explants and primary cell cultures from scales from the three trout populations infected in vitro with VHSV and gill explants infected with Y. ruckeri showed no significant differences in virus replication or bacterial counts. Evaluation of the virucidal or bactericidal effect of skin mucus showed a significant reduction in viral load and bacterial count for all samples with mucus addition, but no significant difference was observed between the experimental groups. This study documents no apparent impairment of innate immune mechanisms in the skin and gills of trout after feeding a diet replacing fishmeal with Arthrospira or Hermetia proteins. This underlines the potential of these alternative protein sources for the further development of sustainable trout aquaculture.

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.

Emerging Viral Pathogens in Sturgeon Aquaculture in Poland: Focus on Herpesviruses and Mimivirus Detection.

Recently, Poland has become a leading producer of sturgeon meat and caviar in Europe and is one of the largest in the world. The growing importance of this branch of aquaculture means that diseases of these fish, especially viral ones, are becoming the object of interest for ichthyopathologists. In recent years, there have been increasing reports of health problems in the dynamically developing sturgeon farming. The greatest risk appears to be emerging infectious diseases that are caused by viruses and that can become a serious threat to the development of the aquaculture industry and the success of sturgeon restitution programs undertaken in many European countries, including Poland. In this paper, an attempt was made to determine the spread of the two most important groups of viruses in Polish sturgeon farming: These include the herpesviruses and sturgeon nucleocytoplasmic large DNA viruses (sNCLDV), in particular, mimiviruses. In the years 2016-2020, 136 samples from nine farms were collected and tested by using the WSSK-1 cell line, PCR and Real Time PCR methods. All results were negative for herpesviruses. Out of the samples, 26% of the samples have been tested positive for mimiviruses. Sanger sequencing of mimiviruses demonstrated their affiliation with AciV-E. The sequence characterization confirmed the presence of both V1 and V2 lineages in Polish fish facilities, but variant V2 seems to be more widespread, as is observed in other European countries.

Genome Sequence of a Ranavirus Isolated from a Red-Eared Slider (Trachemys scripta elegans) in Poland.

The red-eared slider (RES) ranavirus (RESRV) was isolated from a free-ranging RES turtle that died with evidence of respiratory disease. The RESRV genome sequence (106,878 bp) was determined, and phylogenetic analysis revealed that it is a common midwife toad virus (CMTV) strain. This study is the first report of CMTV in RES.

Phylogenetic characterization of Polish isolates of infectious pancreatic necrosis virus in salmonid fish.

INTRODUCTION: Infectious pancreatic necrosis virus belongs to the genus Aquabirnavirus and family Birnaviridae. By VP2 gene similarity, aquatic birnavirus is clustered into seven genogroups. The aim of this study was to genetically analyse IPN viruses occurring on Polish fish farms. MATERIALS AND METHODS: Samples from freshwater fish mostly from 2012 to 2013 and from northern Poland were examined for the presence of IPN virus using isolation on cell cultures, real-time RT-PCR and RT-PCR. Fragments of 1,377 and 1,079 bp of the VP2 and VP5 genes, respectively, were sequenced, and the results were assembled into one consensus and analysed by Geneious software. The same VP2 gene region was compared and a phylogenetic tree generated by the neighbour-joining method and MEGA6 software. RESULTS: All tested Polish isolates belonged to genogroup 5, like other European Spajurup isolates. CONCLUSION: Our findings prove that there is only one IPN virus genogroup in Poland. Polish isolates show close relationships with each other. There is a close relationship between Polish isolates and isolates from Turkey, Spain and Iran. Isolate 57 is a separate branch related to isolates from the United States and Taiwan. This points to the likelihood of past virus introduction via import of stock from those countries.

Potential vector species of carp edema virus (CEV).

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

Type I interferon responses of common carp strains with different levels of resistance to koi herpesvirus disease during infection with CyHV-3 or SVCV.

Carp from breeding strains with different genetic background present diverse levels of resistance to viral pathogens. Carp strains of Asian origin, currently being treated as Cyprinus rubrofuscus L., especially Amur wild carp (AS), were proven to be more resistant to koi herpesvirus disease (KHVD; caused by cyprinid herpesvirus 3, CyHV-3) than strains originating from Europe and belonging to Cyprinus carpio L., like the Prerov scale carp (PS) or koi carp from a breed in the Czech Republic. We hypothesised that it can be associated with a higher magnitude of type I interferon (IFN) response as a first line of innate defence mechanisms against viral infections. To evaluate this hypothesis, four strains of common carp (AS, Rop, PS and koi) were challenged using two viral infection models: Rhabdovirus SVCV (spring viremia of carp virus) and alloherpesvirus CyHV-3. The infection with SVCV induced a low mortality rates and the most resistant was the Rop strain (no mortalities), whereas the PS strain was the most susceptible (survival rate of 78%). During CyHV-3 infection, Rop and AS strains performed better (survival rates of 78% and 53%, respectively) than PS and koi strains (survival rates of 35% and 10%, respectively). The evaluation of virus loads and virus replication showed significant differences between the carp strains, which correlated with the mortality rate. The evaluation of type I IFN responses showed that there were fundamental differences between the virus infection models. While responses to the SVCV were high, the CyHV-3 generally induced low responses. Furthermore, the results demonstrated that the magnitude of type I IFN responses did not correlate with a higher resistance in infected carp. In the case of a CyHV-3 infection, reduced type I IFN responses could be related to the potential ability of the virus to interfere with cellular sensing of foreign nucleic acids. Taken together, the results broaden our understanding of how common carp from different genetic strains interact with various viral pathogens.

Host microRNA analysis in cyprinid Herpesvirus-3 (CyHV-3) infected common carp.

BACKGROUND: The mechanism of latency and the ability of the cyprinid herpesvirus 3 (CyHV-3) to establish life-long infections in carp remains poorly understood. To explain the role of miRNAs in this process we applied a range of molecular tools including high-throughput sequencing of RNA libraries constructed from the blood samples of infected fish followed by bioinformatic and functional analyses which show that CyHV-3 profoundly influences the expression of host miRNAs in vivo. RESULTS: We demonstrated the changed expression of 27 miRNAs in the clinical phase and 5 in the latent phase of infection. We also identified 23 novel, not previously reported sequences, from which 8 showed altered expressions in control phase, 10 in clinical phase and 5 in latent phase of infection. CONCLUSIONS: The results of our analysis expand the knowledge of common carp microRNAs engaged during CyHV-3 infection and provide a useful basis for the further study of the mechanism of CyHV-3 induced pathology.

Potential Role of Different Fish Species as Vectors of Koi Herpesvirus (CyHV-3) Infection.

INTRODUCTION: Koi herpesvirus (KHV) has infected farmed common carp in Poland clinically and asymptomatically since 2004. The role of non-carp species as vectors of virus transmission is well known except for in the case of KHV. The aim was to better understand this virus' infection and transmission pathways in common carp, looking at the potential vector role of fishes kept with them. MATERIAL AND METHODS: Eight species were experimentally infected with KHV by immersion in a suspension at 20°C ±1 and transferred to a tank after 45 minutes. Specimens were euthanised at intervals up to 56 days post infection (dpi) and tissue was examined for KHV DNA. Surviving infected fishes were introduced at intervals, each time into a separate tank, to naïve common carp for experimental infection. These were observed daily for symptoms, sacrificed along with controls after three months, and dissected to provide tissue samples. Also fish from 14 species collected from a farm with a history of KHV were sampled from 3 to 22 months after disease was confirmed. Organ sections from single fish were collected in a single tube. RESULTS: Viral DNA was detected in tench and roach samples up to 49 dpi, but in three-spined stickleback and stone maroko samples only up to 14 dpi. Transmission of KHV to naïve carp occurred after cohabitation. KHV DNA was detected in three fish species three months after the farm outbreak. CONCLUSION: We confirmed that grass and Prussian carp, tench, roach, and brown bullhead can transfer the virus to naïve common carp.

First record of the occurrence of "gill disease" in Mytilus edulis trossulus from the Gulf of Gdansk (Baltic Sea, Poland).

We are presenting the first report of the occurrence of "gill disease" in Mytilus edulis trossulus from the southern Baltic Sea, Gulf of Gdansk (Poland). The disease preliminary diagnosis was based on the presence of white and yellow "spots" causing deep indentations in the gills with degeneration, destruction and necrosis of gill filaments. Average prevalence of gill erosion in the blue mussels population was 15.8%. According to other authors, gill erosion may affect over 80% of the bivalve population resulting in mortality rates of up to 40%. The origin of the gill erosion remains unknown, but viruses are most likely involved in the etiology of these pathological conditions. The disease as such may indicate a decrease in the immunological resistance of organisms to infections and inflammations directly or indirectly caused by harmful factors in the ambient environment. Furthermore, the occurrence of numerous pathologies in bivalves is a particular problem in the Gulf of Gdansk being a low biodiversity ecosystem. Pathologies reduce bilvalves' reproduction ability, worsen their physiological condition and increase their mortality rate. Those factors may also pose a significant ecological danger and lead to negative alterations of the ecosystem.