THE FIRST OUTBREAK OF VIRAL ENCEPHALOPATHY AND RETINOPATHY IN FARMED SEA BASS (Dicentrarchus labrax) IN SLOVENIA

Viral encephalopathy and retinopathy (VER) is considered a serious disease of several marine fish species, caused by RNA virus belonging to the family Nodaviridae, genus Betanodavirus. The disease is spread almost worldwide and causes significant losses among diseased fish. It is characterised by vacuolation of the central nervous system and the retina. In July 2018, behavioural abnormalities i.e. altered swimming, swirling and vertical floating as well as lethargy and anorexia were observed in farmed sea bass (Dicentrarchus labrax) in the Gulf of Piran (Slovenia), associated with significant mortality. The disease initially occurred in juvenile sea bass, but later market-sized fish also became affected. Diseased fish displayed ocular opacity and multifocal skin ulceration on the head. Emaciation in some fish was also evident. Histopathology revealed characteristic vacuolation in the brain and retina. Performing a RT-PCR and RT-qPCR techniques, we have identified and confirmed the presence of betanodavirus nucleic acid in ocular and brain tissues. In addition, concentrations of the causative agent of VER in spleen and kidney did result in significantly higher viral yield than expected. Phylogenetic analysis showed that Slovenian isolate belongs to RGNNV species of betanodaviruses. Based on the clinical signs, gross and typical microscopic lesions and results of molecular analyses, we can conclude that farmed sea bass from the Gulf of Piran were affected with VER. To the best of our knowledge, this is the first report of VER in Slovenia.Key words: viral encephalopathy and retinopathy; betanodavirus; sea bass; histopathology; RT-qPCR PRVI IZBRUH VIRUSNE ENCEFALOPATIJE IN RETINOPATIJE PRI GOJENIH BRANCINIH (Dicentrarchus labrax) V SLOVENIJI Izvleček: Virusna encefalopatija in retinopatija (VER) je nevarna bolezen številnih vrst morskih rib, ki jo povzroča nevrotropni RNA virus iz družine Nodaviridae, rod Betanodavirus. Bolezen je razširjena skoraj po vsem svetu in povzroča visok pogin okuženih rib. Zanjo so značilne vakuole v centralnem živčnem sistemu in retini. Konec julija 2018 so v ribogojnici v Piranskem zalivu pri brancinih opazili nepravilno plavanje, vrtenje in postavljanje v vertikalno smer ter letargijo in neješčnost, brancini so množično poginjali. Bolezen se je najprej pojavila pri mladicah, nato tudi pri konzumnih kategorijah brancinov. Obolele ribe so imele sivo-motna očesna zrkla ter multifokalne kožne razjede na glavi, posamezne so bile shujšane. S histopatološko preiskavo smo ugotovili značilne vakuole v možganih in retini. Z molekularnima metodama RT-PCR in RT-qPCR smo potrdili prisotnost nukleinske kisline betanodavirusa v očesnem zrklu in možganih. Koncentracije virusa, ki so bile signifikantno višje od pričakovanih, smo ugotovili tudi v vranici in ledvicah. Na podlagi kliničnih znakov, makroskopskih in tipičnih histopatoloških sprememb ter rezultatov molekularnih preiskav lahko zaključimo, da so gojeni brancini v ribogojnici v Piranskem zalivu zboleli za VER. Opisani izbruh je prvi potrjeni primer te bolezni v Sloveniji.Ključne besede: virusna encefalopatija in retinopatija; betanodavirus; brancin; histopatologija; RT-qPCR

Summer Mortality Syndrome Affecting Cultured European Seabass at Kafrelsheikh Province, Egypt

The present work aimed to investigate the causes of summer mortality syndrome affecting cultured European seabass (Dicentrarchus labrax) by examining physiochemical farm water characteristics, isolation, and identification of recovered bacterial pathogens from diseased fish studying the effect of water temperature on stress biomarkers and disease severity. Studied water parameters were normal except ammonia and dissolved oxygen was higher and lower than the standard value. Sixty-two bacterial isolates were recovered from moribund fish and identified as 31 Vibrio fluvialis, 23 Pseudomonas aeruginosa and 8 Staphylococcus aureus isolates. The calculated LD50 of V. fluvialis, P. aeruginosa and S. aureus for D. labrax fingerlings were 4.67 × 107, 2.37 × 106 and 1.38 × 107, respectively. There was a direct correlation between water temperature and mortality rate of fish challenged with V. fluvialis as the mortality rate was 44.44, 50, 66.66, and 83.33% for fish maintained at 27, 30, 33, and 36°C. Plasma cortisol, superoxide dismutase, catalase and malondialdehyde significantly increased when the water temperature exceeded 30°C. The experimentally infected fish showed similar clinical signs and postmortem lesions of naturally diseased fish with no boundary between different pathogens. Antibiogram test indicated that florfenicol was the most effective antibiotic against all the recovered bacterial isolates while all isolates resisted sulfamethoxazole-trimethoprim. Massive degenerative changes observed in the hepatopancreas, posterior kidney and gill tissues of experimentally infected fish.

A New Outbreak in Sea Bass Farming in Turkey: Aeromonas veronii

Turkey produces most of the European sea bassin Europe and bacterial diseases are the main cause of economic loss during the production cycle. This research presents the first extended study of the Aeromonas veronii infection in sea bass on the Turkish coast of the Aegean Sea. An outbreak was observed in three different sea bass farms and diseased fish with clinical symptoms were sampled. Abdominal distention, hemorrhages on the body and anus, enlarged spleen and liver, and hemorrhages in the internal organs were detected from clinical and pathological examinations. Biochemical and molecular identification confirmed the pathogen to be A.veronii.The histopathological observations demonstrated that the pathogen caused bacterial colonies in the fibrous connective tissue, granuloma, and vacuolar degeneration.The primary causes of the disease were proved through an infection experiment. 80% and 90% mortality were calculated in 0.85 x 108 CFU ml-1 and 1.28 x 108 CFU ml-1 experimentally infected groups with clinical and pathological signs of the disease respectively. Recently, the pathological symptoms of the disease had been confused with pasteurellosis infection in cage farms but the presence of A.veronii has been confirmed in the current study. A detailed study is needed to investigate the overall status of the disease in the Aegean Sea in order to design an appropriate preventive strategy.

CARP EDEMA VIRUS DISEASE IN SERBIA - DISEASE OUT OF CONTROL

A poxvirus named carp edema virus (CEV), is the causative agent of carp edema virus disease (CEVD), which is an emerging disease of global concern that may cause high rates of morbidity and mortality in ornamental koi and common carp and. Common carp (Cyprinus carpio) is the most important fish species for warm‐water aquaculture in Serbia. CEVD was first time detected in Serbia in 2017. During 2017-2020 period, increasing number of CEVD outbreaks in carp farms is reported. Carp were collected from farms in different regions of Serbia from 2017 to 2020. The fish were sampled for disease diagnosis because they exhibited lethargy and anorexia, which eventually led to mortality. Mortality started with clinical signs of hypoxia while fish swam slowly without escaping reactions. The gills were pale and covered with a thick mucus layer. In advanced cases, the lesions in the gills turned into a necrotizing form. A moderate to high amount of opportunistic freshwater bacteria were isolated from gills of diseased fish. By performing real-time PCR, CEV was detected in 38 samples of diseased carps from 21 carp farms. These outbreaks further confirm the spread of CEVD and the need for practitioners to be vigilant for outbreaks of this disease. To prevent further spreading of the disease, it is very important to introduce CEV testing before fish movement. To avoid further transmission of the virus to common carp populations in Serbia, testing of CEV should become part of fish disease surveillance programs. Fish health service should be aware of the presence of CEV in Serbia which may result in high losses in carp aquaculture. Action should be taken also to prevent transmission of CEV to carp populations in open waters.

Genotypic and Phenotypic Characterization of Highly Alkaline-Resistant Carnobacterium maltaromaticum V-Type ATPase from the Dairy Product Based on Comparative Genomics

Although Carnobacterium maltaromaticum derived from dairy products has been used as a lactic acid bacterium industrially, several studies have reported potential pathogenicity and disease outbreaks. Because strains derived from diseased fish and dairy products are considered potentially virulent and beneficial, respectively, their genotypic and phenotypic characteristics have attracted considerable attention. A genome-wide comparison of 30 genome sequences (13, 3, and 14 strains from diseased aquatic animals, dairy products, and processed food, respectively) was carried out. Additionally, one dairy and two nondairy strains were incubated in nutrient-rich (diluted liquid media) and nutrient-deficient environments (PBS) at pH 10 to compare their alkaline resistance in accordance with different nutritional environments by measuring their optical density and viable bacterial cell counts. Interestingly, only dairy strains carried 11 shared accessory genes, and 8 genes were strongly involved in the V-type ATPase gene cluster. Given that V-type ATPase contributes to resistance to alkaline pH and salts using proton motive force generated via sodium translocation across the membrane, C. maltaromaticum with a V-type ATPase might use nutrients in food under high pH. Indeed, the dairy strain carrying the V-type ATPase exhibited the highest alkaline resistance only in the nutrient-rich environment with significant upregulation of V-type ATPase expression. These results suggest that the gene cluster of V-type ATPase and increased alkaline resistance of dairy strains facilitate adaptation in the long-term ripening of alkaline dairy products.

Management Practices, Farmers’ Knowledge of Diseased Fish, and Their Occurrence in Fish Farms in Nyeri County, Kenya

In this study, fish farmers’ management practices, occurrence, and knowledge of fish diseases in Nyeri County, Kenya, were evaluated. Fish farming management practices for small-scale farmers in Kenya have numerous challenges which have led to disease occurrence and reduced production. Moreover, the impact and association of these challenges to farmers’ knowledge of fish diseases and their burden has not been fully studied. A semistructured questionnaire was used to capture farmers’ biodata, fish species farmed, and farmers’ management practices such as handling of nets, pond fertilization, and disposal of fish waste. Farmers’ knowledge of fish diseases was based on their ability to identify independent and dependent variable indicators. Independent variables included clinical signs, decreased feeding, bulging eyes, floating on water, abdominal swelling, bulging eyes, abnormal skin color, reduced growth, and abnormal swimming with fish death as were the dependent variable. A total of 208 farmers were interviewed and included those of tilapia (134), mixed tilapia and catfish (40), catfish (22), rainbow trout, and five dams under cooperative management. Tilapia was the most kept fish species (66.8%) followed by polyculture of tilapia and catfish (20%) and rainbow trout (2%). Most respondents were male (78.5%) over 51 years of age (50%). Fifty percent of the respondents had secondary school education. There was a significant association between deaths and sharing of nets in Kieni East subcounty ( p = 0.0049 , chi-square), while on-farm fish waste disposing appeared to cause higher deaths compared to burning of the waste although not statistically significant ( p = 0.13 ). Few respondents observed decreased feed uptake (<20%) and poor growth. Fifty-seven percent of farmers reported mortalities. Fish poor growth, floating in water, and management practices in subcounties had significant effect on fish deaths. The farmers had knowledge of signs of diseased fish, but there was paucity of knowing the specific causes of disease. Farmers need to be empowered on best aquaculture husbandry to avoid disease transmission and specific fish disease signs to enhance proper reporting of disease for subsequent mitigation measures.

A First Report of Aeromonas veronii Infection of the Sea Bass, Lateolabrax maculatus in China

The sea bass, Lateolabrax maculatus is commercially farmed in Zhuhai, located in the Guangdong Province of China. L. maculatus in aquaculture have suffered acute death, characterized by ulcerations on the body surface, congestion, and hemorrhage in internal organs such as liver, kidney, and spleen. The dominant infecting strain of bacteria isolated from the kidneys of diseased fish was identified as Aeromonas veronii (strain 18BJ181). This identification was based on analysis of morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. Drug sensitivity testing showed that the strain 18BJ181 isolate was resistant to four antibacterial drugs, including amoxicillin, madinomycin, penicillin and sulfamethoxazole, while moderately sensitive to erythromycin and rifampicin. The detection of growth characteristics showed that the strain 18BJ181 exhibited adaptability to the environment. In addition, some virulence genes, such as aer, act, gcaT, tapA and fla, were detected in the strain 18BJ181. The median lethal dosage of the strain 18BJ181 isolate in L. maculatus was 8.5 × 105 and 4.2 × 105 cfu/g under the conditions of intraperitoneal injection and intramuscular injection, respectively. The experimentally induced infection showed that the 18BJ181 isolate caused considerable histological lesions in L. maculatus, including tissue degeneration, necrosis, and different degrees of hemorrhage. These results provided evidence for a more comprehensive understanding of A. veronii strain 18BJ181 infection in L. maculatus.

Dissecting the localization of Tilapia tilapinevirus in the brain of the experimentally infected Nile tilapia (Oreochromis niloticus)

Tilapia tilapinevirus or tilapia lake virus (TiLV) is an emerging virus that inflicts significant mortality on farmed tilapia globally. Previous studies reported detection of the virus in multiple organs of the infected fish; however, little is known about the in-depth localization of the virus in the central nervous system. Herein, we determined the distribution of TiLV in the entire brain of experimentally infected Nile tilapia. In situ hybridization (ISH) using TiLV-specific probes revealed that the virus was broadly distributed throughout the brain. The strongest positive signals were dominantly detected in the forebrain (responsible for learning, appetitive behavior, and attention) and the hindbrain (involved in controlling locomotion and basal physiology). The permissive cell zones for viral infection were observed mostly to be along the blood vessels and the ventricles. This indicates that the virus may productively enter into the brain through the circulatory system and widen broad regions, possibly through the cerebrospinal fluid along the ventricles, and subsequently induce the brain dysfunction. Understanding the pattern of viral localization in the brain may help elucidate the neurological disorders of the diseased fish. This study revealed the distribution of TiLV in the whole infected brain, providing new insights into fish-virus interactions and neuropathogenesis.

Diagnosis of Aeromonas sobria and Saprolegnia sp. co-infection in rainbow trout fry (Oncorhynchus mykiss)

The aim of the study was to determine a considerable level of mortalities occurred in rainbow trout fry (Oncorhynchus mykiss) cultured in a research hatchery located in Marmara Region/Turkey. Totally 18 individuals (1-8 g) were investigated by using bacteriological and mycological methods. The affected fish showed skin darkening, prolapses and fungal mats at the base of the fins. According to morphological and biochemical characteristics of the isolates, all of them were identified as Aeromonas sobria. Saprolegnia sp. was also observed in squash preparations of the skin. All isolates were determined to be sensitive against florfenicol, enrofloxacin and ciprofloxacin. Histopathologically, vacuolar degeneration, haemorrhage and hyperaemia in the liver, tubular necrosis, periglomerular edema, and multifocal melanomacrophage deposits in the kidney and depletion of white pulp in the spleen were determined. Distal type epithelial cells hyperplasia was also observed in the gills. In diseased fish, motile Aeromonad septicaemia was described and Saprolegnia sp. was also detected in fins and skin lesions but it was considered to be a secondary infection.