Sličnosti i razlike vibrioze kod lubina uzrokovane bakterijama Vibrio anguillarum i Vibrio harveyi

Vibrioses caused by different members of the genus Vibrio are common diseases in aquaculture characterised by systemic infections, high mortalities, and economic losses of fish and shellfish. Farming of European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) has rapidly grown over the last decade in Croatia but its economic efficacy is significantly jeopardized by losses due to Vibrio anguillarum and Vibrio harveyi infection. Therefore, we studied and compared the most prominent similarities and differences in the environmental conditions, clinical signs and conventional and molecular diagnostic methods of vibriosis caused by both bacterial pathogens. Outbreaks in sea bass and sea bream caused by V. anguillarum mostly occur during the spring and autumn, following a fast increase or decrease in the sea temperature, whereas infections with V. harveyi occur during the summer months at a temperature above 20oC. They have a similar clinical appearance in the acute form but in subacute forms they differ. V. anguillarum infection is characterized by massive haemorrhages in the abdominal wall, while during infection with V. harveyi keratitis, corneal opacity and uncoordinated swimming behaviour are noticed. Necropsy disclosed haemorrhages in the liver, intestine and stomach during infection with V. anguillarum, and sero-catarrhal enteritis with distension of the intestine and ascites in the case of V. harveyi. Although molecular tools enable correct identification, in this study we defined the main differences capable of distinguishing V. anguillarum from V. harveyi based on traditional bacteriological identification such as sensitivity to O/129 (10 μg) and novobiocin, and the enzymatic activity of β-galactosidase, arginine dihydrolase and amygdalin fermentation.Vibrioze uzrokovane bakterijama roda Vibrio su česte bolesti u akvakulturi, a karakteriziraju ih sustavne infekcije, visoka smrtnost i ekonomski gubici riba i školjkaša. Uzgoj lubina (Dicentrarchus labrax) i komarče (Sparus aurata) u Hrvatskoj je u posljednjem desetljeću naglo porastao, a ekonomska učinkovitost značajno je ugrožena gubicima zbog infekcije bakterijama Vibrio anguillarum i Vibrio harveyi. Stoga smo proučavali i usporedili okolišne uvjete, kliničke znakove te konvencionalne i molekularne dijagnostičke metode vibrioze uzrokovane s oba bakterijska patogena. Vibrioze uzrokovane bakterijom V. anguillarum kod lubina i komarče uglavnom se javljaju tijekom proljeća i jeseni, nakon brzog porasta ili sniženja temperature mora, dok se infekcije V. harveyi javljaju tijekom ljetnih mjeseci na temperaturama mora iznad 20 °C. Imaju sličnu kliničku sliku u akutnom obliku, ali se u subakutnom obliku razlikuju. Infekciju V. anguillarum karakteriziraju masivna krvarenja po trbušnoj stijenci, dok se kod infekcije V. harveyi uočava keratitis, zamućenje rožnice i nekoordinirano plivanje. Tijekom infekcije bakterijom V. anguillarum, razudbom su vidljiva krvarenja na jetri, crijevima i želucu. U slučaju infekcije s V. harveyi, vidljivi su sero-kataralni enteritis s distenzijom crijeva i ascitesom. Iako molekularna dijagnostika omogućava ispravnu identifikaciju, u ovom istraživanju smo definirali glavne čimbenike koje mogu razlikovati V. anguillarum od V. harveyi na temelju tradicionalne bakteriološke identifikacije kao što su osjetljivost na O/129 (10 μg) i novobiocin, te enzimska aktivnost β -galaktozidaze, arginin dihidrolaze i fermentacija amigdalina

Fish eustrongylidosis: Review Article

Oblići roda Eustrongylides se nalaze širom svijeta u brojnih riba slatkovodnih i morskih ekosustava. Sve vrste ovih u riba opisanih parazita imaju neizravni razvojni ciklus te su ribe drugi međudomaćin ili paratenični nositelj i plijen ribojednih ptica koje su konačni domaćini. Eustrongilidi su oblikom okrugli, glatki, relativno dugački crvi zbog prisustva hemoglobina crvene boje. Veći broj ličinki parazita u ribi može izazvati teške oblike bolesti, utjecati kod nekih vrsta na plodnost i povećati smrtnost. Za dijagnozu eustrongilidoze sigurna metoda je razudba male grupe zaraženih riba te mjesta nalaza i karakteristike parazita u ribi. Potrebno je isključiti druge parazite riba - crve plošnjake, segmentirane trakavice, jednorodne i dvorodne metilje te akantocefale. Za prevenciju eustrongilidoze u riba potrebno je u nekom od razvojnih stadija prekinuti životni ciklus parazita za što je potrebno ukloniti bilo kojeg međudomaćina ili konačne domaćine. Za uzgajane ribe, sanitacija ribnjaka omogućava prevenciju zaraze ribe od ovih parazita. Eustrongilidi riba su identificirani kao uzročnici infekcije ljudi kao slučajnih domaćina te iako je opisan mali broj kliničkih slučajeva, eustongilidoza je priznata zoonoza. Da bi se spriječila infekcija ljudi preporuka je izbjegavati konzumiranje sirove ribe i sirovih ribljih proizvoda. Učinkovita metoda ubijanja parazita je temeljita termička obrada.Nematodes of the genus Eustrongylides can infect many fish species inhabiting freshwater and marine ecosystems worldwide. All species of these parasites described in fish have indirect life cycles and fish are the second intermediate hosts or paratenic hosts, and the prey of fish-eating birds, as the definite hosts. Eustronoglydes are typically coiled, smooth, and relatively long worms that are red in colour due to the presence of haemoglobin. A large number of parasite larvae in fish can cause severe forms of the disease, affecting the fertility of some species and increasing mortality. The best method for diagnosis of fish eustrongylidosis is necropsy of several affected individuals, and identification of the parasite by their morphological characteristics and position in the fish. It is necessary to exclude other fish parasites, including flatworms, segmented tapeworms, monogenean and digenean flukes and acanthocephali. To prevent eustrongylidosis in fish, the parasite life cycle must be interrupted in one of the developing stages, which requires removing any of the intermediate or final hosts. For farmed fish, pond sanitation is important in preventing fish infection. Fish eustrongylides have been identified as causative agents of infection in humans as accidental hosts, and although a small number of clinical cases have been described, eustrongylidosis is a recognized zoonosis. To prevent human infection, it is recommended to avoid consuming of raw fish and raw fish products. An effective method of killing parasites is freezing and heat treatments, under well-defined conditions

Emerging vibriosis of Mediterranean fish caused by the bacterial species Vibrio harveyi: an overview

Bakterije roda Vibrio spp. su najčešći uzročnici bolesti akvatičnih životinja. Pripadaju porodici Vibrionaceae u kojoj je jedan od najznačajnih predstavnika klad Vibrio harveyi. V. harveyi se uobičajeno nalazi u morskom okolišu i predstavnik je normalne flore akvatičnih životinja. Brojni sojevi su opisani kao uzročnici bolesti morskih rakova i školjkaša. U posljednje vrijeme V. harveyi nanosi velike štete u uzgoju mediteranskih vrsta riba, posebice u ljetnim mjesecima. V. harveyi je Gram-negativna, halofilna, aerobna ili fakultativno anaerobna, načelno nije zoonotska bakterija, međutim zabilježeni su izdvojeni slučajevi oboljenja ljudi. Neki sojevi su visoko patogeni dok se drugi smatraju uvjetno patogenima. Glavni čimbenici virulencije su bičevi, litički enzimi, kapsula, siderofore, hidrofobni površinski antigeni i sposobnost prianjanja i infekcije epitelnih stanica domaćina. Sposobnost stvaranja biofilma omogućava rezistenciju na antibiotike, a sposobnost bakterija da izvlače željezo iz stanica domaćina je ključna za njihovo preživljavanje. Intracelularni sustav omogućava međustaničnu komunikaciju bakterija. Klinički znaci su slični drugim bakterijskim infekcijama u riba, a obično započinju letargijom i gubitkom apetita. V. harveyi posjeduje urođenu rezistenciju na neke antibiotike. U novije se vrijeme razvija bakteriofagna terapija koja je daje obećavajuće rezultate. Cijepljenje je jedan od najučinkovitijih načina prevencije bolesti i metoda redukcije gubitaka i smanjenja uporabe antibiotika. Dobra proizvođačka praksa koja podrazumijeva optimalne uvjete u uzgojnim jedinicama kao i primjena mjera biosigurnosti su važni u spriječavanju ulaska uzročnika bolesti u farmu i širenja na sve uzgojne jedinice. Sve češće se javljaju infekcije visoko patogenim emergentnim sojevima Vibrio harveyi. Uzevši u obzir činjenicu da je V. harveyi moguće izdvojiti iz okoliša, morskih organizama i probavnog trakta zdravih jedinki, očito je da postoje različiti sojevi iste bakterijske vrste te je potrebno definirati razlike između apatogenih i patogenih sojeva.Bacteria of the genus Vibrio spp. family Vibrionaceae are the most common causative agents of disease in aquatic animals. Economically, the members of the Harvey clade, with eleven closely related bacterial species, are the most important representatives of the genus Vibrio. V. harvey is commonly found in the marine environment and is a normal part of the microflora of aquatic animals. Numerous strains are described as pathogens for crustaceans and molluscs. The prevalence of infection with V. harvey is increasing and affecting more Mediterranean fish species, especially in the summer months. It is a Gram-negative, halophilic, aerobic or facultatively anaerobic bacteria. In general, it does not have zoonotic potential, though cases of human infection caused by this bacterium have been described. Some strains are highly pathogenic while others are considered opportunistic pathogens. The main virulence factors are bacterial flagellum, lytic enzymes, capsule, siderophores, hydrophobic surface antigens and its ability to adhere and infect epithelial host cells. Production of biofilm is a mechanism of antibiotic resistance, and the ability of the bacteria to extract iron from host cells is crucial for their survival. The intracellular system allows intercellular communication between bacteria. Clinical signs are similar to other bacterial infections in fish, usually starting with lethargy and loss of appetite. V. harveyi has an innate resistance to certain antibiotics. Recently, bacteriophage therapy has been developed and is showing promising results. Vaccination is one of the most effective ways of preventing disease, reducing losses and reducing antibiotic use. Good manufacturing practice and application of biosecurity measures are very important to minimizing the risk of introducing an infectious disease and its spread to animals within a facility. More often, infections occur with highly pathogenic emergent strains of V. harvey. Since V. harvey can be extracted from the environment, marine organisms and the digestive tract of healthy individuals, it is evident that there are different strains of the same bacterial species and it is necessary to define the differences between non- pathogenic and pathogenic strains

Prisutnost alohtone kamenice Crassostrea gigas u Jadranskom moru

The Pacific oyster, Crassostrea gigas, was introduced into the Europe in the late 1960s for aquaculture purposes and has since spread extensively in Mediterranean waters. For the first time, screening of the four ecologically most suitable bays along the Eastern Adriatic coast was conducted to detect the possible presence of non-native oysters. Sequencing analysis of a fragment of mitochondrial 16S rRNA was examined to demonstrate the presence of the exotic species C. gigas in the natural environments of the northern and central coastal areas of the eastern Adriatic. The likely routes of invasion are discussed and the importance of monitoring the distribution of feral C. gigas populations emphasized, with a focus on the potential direct and indirect effects on the native European flat oyster, Ostrea edulis.Japanska ili pacifička kamenica, Crassostrea gigas, uvedena je u Europu kasnih 1960-ih za potrebe akvakulture i od tada se intenzivno proširila po Mediteranu. Po prvi put je izvršen pregled četiriju ekološki najprikladnijih zaljeva istočne obale Jadrana kako bi se utvrdila prisutnost alohtone kamenice. Analiza djelomičnog fragmenta mitohondrijske 16S rRNA je korištena kako bi se molekularno identificirala egzotična vrsta C. gigas u prirodnim staništima sjevernih i središnjih obalnih područja istočnog Jadrana. Mogući invazijski putevi su raspravljani kao i važnost praćenja distribucije populacija C. gigas, s naglaskom na potencijalne izravne i neizravne učinke na autohtonu europsku plosnatu kamenicu Ostrea edulis koja obitava u Jadranskom moru

Prisutnost alohtone kamenice Crassostrea gigas u Jadranskom moru

The Pacific oyster, Crassostrea gigas, was introduced into the Europe in the late 1960s for aquaculture purposes and has since spread extensively in Mediterranean waters. For the first time, screening of the four ecologically most suitable bays along the Eastern Adriatic coast was conducted to detect the possible presence of non-native oysters. Sequencing analysis of a fragment of mitochondrial 16S rRNA was examined to demonstrate the presence of the exotic species C. gigas in the natural environments of the northern and central coastal areas of the eastern Adriatic. The likely routes of invasion are discussed and the importance of monitoring the distribution of feral C. gigas populations emphasized, with a focus on the potential direct and indirect effects on the native European flat oyster, Ostrea edulis.Japanska ili pacifička kamenica, Crassostrea gigas, uvedena je u Europu kasnih 1960-ih za potrebe akvakulture i od tada se intenzivno proširila po Mediteranu. Po prvi put je izvršen pregled četiriju ekološki najprikladnijih zaljeva istočne obale Jadrana kako bi se utvrdila prisutnost alohtone kamenice. Analiza djelomičnog fragmenta mitohondrijske 16S rRNA je korištena kako bi se molekularno identificirala egzotična vrsta C. gigas u prirodnim staništima sjevernih i središnjih obalnih područja istočnog Jadrana. Mogući invazijski putevi su raspravljani kao i važnost praćenja distribucije populacija C. gigas, s naglaskom na potencijalne izravne i neizravne učinke na autohtonu europsku plosnatu kamenicu Ostrea edulis koja obitava u Jadranskom moru

Confronto tra la composizione chimica e la quantità di metalli pesanti Nella carne di branzino (Dicentrarchus labrax) e di orata (Sparus aurata) dall´allevamento e dalla pesca libera

Sažetak Svrha ovog istraživanja bila je usporediti kemijski sastav i količinu teških metala u mesu lubina i komarče iz uzgoja i slobodnog ulova. Ukupno je analizirano 30 uzoraka komarče i 30 uzoraka lubina, konzumne veličine, od čega je polovica uzoraka bila iz uzgoja, a polovica iz slobodnog ulova. Analize kemijskog sastava pokazale su da kod obje vrste ribe postoji statističku značajna razlika u količini vode, bjelančevina, masti i kolagena između jedinki iz ulova i uzgoja. Utvrđena je 4 odnosno 8 puta veća količina masti u mesu uzgojenih jedinki lubina i komarče u odnosu na jedinke iz ulova. Količine teških metala kadmija (Cd) i olova (Pb), kod obje vrste ribe te neovisno o podrijetlu ribe, bile su manje od najvećih dopuštenih količina propisanih hrvatskim i EU pravilnicima. Za razliku od količine Cd i Pb, količina žive (Hg) određena u pojedinim uzorcima obje vrste ribe iz ulova bila je veća od NDK, ukazujući na zdravstvenu neispravnost određenih jedinki. Budući da sadržaj teških metala u mišićnom tkivu riba može varirati u ovisnosti o vrsti, spolu, starosti, godišnjem dobu, načinu prehrane i dostupnosti hrane, u cilju cjelovite procjene potencijalnih opasnosti uslijed konzumacije navedenih vrsta riba potrebno je provesti opsežnije istraživanje, uzimajući u obzir navedene čimbenike.The aim of this research was to compare chemical composition and content of heavy metals in the meat of sea bass and gilt-head sea bream, both in farmed fish and from free fishing. A total of 30 samples of gilt-head sea bream and 30 samples of sea bass of commercial size, out of which half the samples were farmed fish and the other half were from free fishing. Chemical composition analyses have shown that in both kinds of fish there is a statistically significant difference in content of water, protein, fat and collagen between the samples from free fishing and farmed fish. There was determined four times, i.e. eight times higher content of fat in the meat of farmed samples of sea bass and gilt-head sea bream in comparison to samples of farmed fish. Contents of heavy metals, cadmium (Cd) and lead (Pb) with both fish species and regardless of the origin of fish, were lower than the maximum quantities allowed proscribed by Croatian and EU regulations. As opposed to the quantities of Cd and Pb, the quantity of mercury (Hg) determined in certain samples of both fish species from free fishing was higher than maximum quantity allowed, indicating to the fact that some samples were not safe for consumption. Considering the fact that the content of heavy metals in muscle tissue of fish can vary depending on species, sex, age, season, diet and food availability, in the goal of the total assessment of potential dangers due to consummation of the abovementioned fish species, it is necessary to conduct a more detailed research, taking into account the listed factors.Zusammenfassung Der Zweck dieser Untersuchung war, den Vergleich der chemischen Zusammensetzung und der Inhaltsmenge der schweren Metalle im Fleisch von Seebarsch und Goldbrasse aus der Zucht und aus dem freien Fang zu machen. Insgesamt wurden 30 Muster Seebarsch und 30 Muster Goldbrasse von Konsumgröße analysiert, die Hälfte davon war aus der Zucht und die andere Hälfte aus dem freien Fang. Die Analyse der chemischen Zusammensetzung hat gezeigt, dass bei beiden Fischsorten ein statistisch bedeutender Unterschied hinsichtlich Wassermenge, Eiweißstoffe, Fette und Collagen bei dem Fisch aus der Zucht und aus dem freien Fang besteht. Es wurde für beide Sorten eine 4 bis 8 Mal größere Fettmenge im Fleisch von Fischen aus der Zucht als im Fleisch von Fischen aus dem freien Fang festgestellt. Die Menge von Schwermetallen Kadmium (Cd) und Blei (Pb) war bei beiden Sorten niedriger als die kroatischen und die EU Vorschriften als Größtmenge vorschreiben, u.zw. ohne Rücksicht darauf, ob die Fische aus der Zucht oder aus dem freien Fang stammten. Zum Unterschied von Cd und Pb Mengen, war die festgestellte Quecksilbermenge (Hg) in manchen Mustern beider Fischsorten aus dem Fang größer als NDK, was auf die gesundheitliche Unrichtigkeit einiger Einzelfische hinweist. Da der Inhalt von Schwermetallen im Muskelgewebe der Fische variieren kann, u.zw. in Bezug auf Sorte, Geschlecht, Alter, Jahreszeit, Nahrung und Nahrungszugänglichkeit, ist es nötig bei angeführten Fischssorten umfangreiche Untersuchungen durchzuführen. Dies erweist sich als Schlußfolgerung wegen der potentiellen Gefahren hinsichtlich des Konsums der angeführten Fischsorten.Sommario Lo scopo di questa ricerca era paragonare la composizione chimica e la quantità di metalli pesanti nella carne di orata e quella di branzino dall\u27allevamento e dalla pesca libera. Sono stati analizzati 30 campioni di branzino e 30 campioni di orata, di misura consumo, la metà delle quali proveniva dall\u27allevamento e l\u27altra dalla pesca libera. Le analisi del contenuto chimico hanno mostrato che da tutti i due i tipi di pesce c\u27era una notevole differenza in statistica nella quantità dell\u27acqua, delle proteine, dei grassi e del collagene tra le entità dalla pesca e quelle dall\u27allevamento. È stata rivelata da 4 a 8 volte maggiore quantità dei grassi nella carne delle entità d\u27orata e branzino dall\u27allevamento rispetto alle entità dalla pesca. Le quantità di metalli pesanti, il cadmio (Cd) e il piombo (Pb), da ambedue tipi di pesce, e indipendentemente dalla loro provenienza, erano minori dalle quantità massime consentite prescritte nei regolamenti croati e dell\u27Unione europea. A differenza della quantità del cadmio e del piombo, la quantità del mercurio (Hg) determinata in certi campioni di tutte e due i tipi di pesce dall\u27allevamento era maggiore della quantità massima consentita, e questo vuol dire che certe entità non erano conformi alla sicurezza sanitaria. Siccome il contenuto di metalli pesanti nel tessuto muscolare può variare rispetto al tipo, sesso, età, stagione, regime alimentare e del cibo accessibile, bisogna fare degli studi più dettagliati che, allo scopo di valutazione del pericolo potenziale del consumo di branzino e orata, prenderanno in considerazione i suddetti indicatori

El porcentaje de grasas y composición de ácidos grasos en el lubina de mercado (dicentrarchus labrax) criado en la zona del mar Mediterráneo

Analiziran je udjel masti i sastav masnih kiselina lubina (Dicentrarchus labrax) uzgajanog na više uzgajališta na području Jadrana te uzorkovanog u jesensko-zimskom razdoblju na tržištu Republike Hrvatske od studenog 2013. do ožujka 2014. Analiza je provedena na ukupno 64 uzoraka lubina konzumne veličine. Rezultati su pokazali da postoje statistički značajne razlike (p<0,05) po grupama uzoraka odnosno mjesecima uzorkovanja i to u udjelu ukupne masti i udjelu skupina masnih kiselina. Utvrđene razlike mogu se pripisati različitom sustavu uzgoja i načinu ishrane, ali i razdoblju odnosno mjesecima uzorkovanja. Među višestruko nezasićenim masnim kiselinama, dominantne su bile omega-6 masne kiseline, a vrijednosti omega-3 masnih kiselina pokazale su se manjim u usporedbi sa drugim istraživanjima, vjerojatno zbog načina ishrane. Dobiveni omjer omega-6 i omega-3 masnih kiselina iznosio je 3,04±0,61, u skladu sa zdravstvenim preporukama (3-5:1).The fat content and fatty acid composition of sea bass (Dicentrarchus labrax) cultivated on different fish farms in the Adriatic sea and sampled during the autumn-winter period, on the Croatian market since November 2013 until March 2014, were evaluated. A total of 64 samples of sea bass of commercial size were analyzed. The results showed that there were statistically significant differences (p<0.05) among sampled groups in the proportion of total fat content and composition of fatty acid groups. These differences can be attributed to the different farming systems and diets the fish were fed on, but also the sampling period. Among the polyunsaturated fatty acids omega-6 fatty acids were dominant, and the value of omega-3 fatty acids has proven to be lower than those reported in other studies, possibly because of diet. The resulting omega-6/omega-3 ratio was 3.04±0.61, according to the health recommendations (3-5:1).Es wurden Fettanteil und Fettsäurenzusammensetzung im Seebarsch (Dicentrarchus labrax) gezüchtet in mehreren Zuchtstätten in der Adria analysiert, u.zw. die im Herbst/Winter, vom November 2013 bis März 2014, auf dem Markt der Republik Kroatien zu Musterprobe genommen wurden. Die Analyse wurde auf Mustern von insgesamt 64 Seebarschstücken von üblicher Konsumgröße durchgeführt. Die Resultate haben gezeigt, dass statistisch bedeutende Unterschiede (p<0,05) nach Mustergruppen bzw. Monaten der Musterproben bestehen, u.zw. dies sowohl im gesamten Fettanteil als auch im Anteil der Fettsäurengruppen. Die vorgefundenen Unterschiede können sowohl dem verschiedenen Zuchtsystem und verschiedener Fütterungsart, als auch der Zeitperiode bzw. den Monaten, in denen die Musterproben genommen wurden, zugeschrieben werden. Unter mehrfach ungesättigten Fettsäuren dominierten Omega-6 Fettsäuren. Die Werte von Omega-3 Fettsäuren waren niedriger im Vergleich mit anderen Untersuchungen, möglichst wegen der Fütterungsart. Das Verhältnis zwischen Omega-6 und Omega-3 Fettsäuren war 3,04±0,61, dies im Einklang mit Gesundheitsempfehlungen (3-5:1).Fueron analizados el porcentaje de grasas y la composición de ácidos grasos en el lubina (Dicentrarchus labrax) criado en varios viveros en la zona del Mar Mediterráneo y fueron tomadas las muestras en el mercado de Croacia durante el período del otoño e invierno, desde el noviembre de 2013 hasta el marzo de 2014. El análisis fue hecho en 64 muestras en total en lubina del tamaño optimal para el consumo. Los resultados mostraron la existencia de diferencias stadísticamente significantes (p<0,05) en cuanto a los grupos de las muestras, es decir en cuanto a los meses de muestreo en porcentaje de grasa en total y en porcentaje de grupos de ácidos grasos. Diferencias detectadas pueden ser atribuidas al sistema de crianza tanto como al método de alimentación diferentes y período o sea los meses en los que fue hecho el muestreo. Entre los ácidos grasos poliinsaturados dominan omega-6 ácidos grasos. Los valores de los omega-3 ácidos grasos fueron más bajos que en otras investigaciones, probablemente debido al sistema de alimentación. La proporción obtenida entre omega-6 y omega-3 ácidos grasos fue 3,04±0,61, de acuerdo con las recomendaciones sanitarias (3-5:1)

Nalaz DNK nametnika Minchinia mytili u dagnjama (Mytilus galloprovincialis) iz Jadranskog mora nakon masovnog uginuća

We investigated a mass mortality event of Mediterranean mussels (Mytilus galloprovincialis) in the Novigrad Sea, Croatia during winter 2022/2023. The mortality rate was up to 70% and was initially linked to an influx of freshwater from the Zrmanja River. Despite mitigation measures that included placing mussels deeper to avoid the influence of freshwater, mortalities continued. Subsequent analysis of 64 samples using cytology, histolology and real-time PCR to exclude listed diseases, yielded negative results. PCR testing for Haplosporidium pinnae revealed the presence of a haplosporidian-like DNA resembling Minchinia mytili. In the study of predominant bacteria, Psychrobacter sp., Colwellia sp., and Vibrio splendidus were detected. Histological examination showed no haplosporidium structures, and in situ hybridisation with probes for detection of Haplosporiidae did not confirmed the presence of M. mytili in mussel tissue. However, our study reports the first detection of M. mytili DNA in the Adriatic Sea, emphasising the need for extensive research and further analysis to determine the exact cause of these mass mortality events and the origin of the haplosporidium DNA.Ovo istraživanje opisuje masovno uginuće dagnji u Novigradskom moru tijekom zimskog razdoblja. Kod izbijanja bolesti zabilježeno je ugibanje do 70 % populacije i isprva je bilo povezano s dotokom slatke vode iz rijeke Zrmanje. Međutim, čak i uz mjere ublažavanja, ugibanje dagnji se nas- tavilo. Za potrebe istraživanja je pretraženo je ukupno 64 uzoraka pojedinačnih dagnji, korištena je metoda PCR u stvarnom vremenu da bi se isključile poznate bolesti dagnji, a to je dalo negativne rezul- tate. Daljnjim istraživanjem, otkrivena je prisutnost DNK nametnika Minchinia mytili iz porodice Haplosporidiidae. Analiziranjem dominantnih bakterija otkriveno je da prevladavaju Psychrobacter sp., Col- wellia sp. i Vibrio splendidus. Histološkim pregledom nije utvrđena prisutnost struktura haplosporidija, a in situ hibridizacija je isto tako dala negativne rezu- ltate. Ovo istraživanje označava prvi opis DNK M. mytili u Jadranskom moru na mediteranskoj dagnji (Mytilus galloprovincialis), naglašavajući potrebu za opsežnijim istraživanjem i daljnjom analizom da bi se utvrdio točan uzrok masovnog ugibanja dagnji u Novigradskom moru tijekom zimskog razdoblja

Transmission Pathways of the VNN Introduced in Croatian Marine Aquaculture

Due to the insufficient capacity of Croatian hatcheries, marine aquaculture depends on the importation of fry from different countries in the Mediterranean basin. Importation enables a risk of spreading pathogenic agents. Viral nervous necrosis (VNN), caused by betanodavirus is devastating for the farming of European sea bass. We described a VNN outbreak that occurred in Croatia in 2014. After the diagnosis of VNN in sea bass fry introduced from the same hatchery to five unconnected marine farms at the Adriatic Coast, we performed surveillance within one of the affected farms. It resulted in proven horizontal spreading of the virus within the farm and to feral fish around farm cages. Real-time RT-PCR tested samples showed the dependence of the virus&rsquo; proliferation to the water temperature and the fish age. The highest mortality rates were noted during higher sea temperatures. Phylogenetic analysis of partial sequences of RNA1 and RNA2 supported the hypothesis that the virus was introduced to all studied farms from the same hatchery. Moreover, phylogenetic analysis of the whole genome sequences of infected farmed sea bass and thicklip mullet showed high similarity and it is unlikely that infection in Croatian sea bass farms has originated from wild reservoirs, as the first positive record in wild mullet was recorded after the disease outbreak