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

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

Biochemical and molecular characterization of three serologically different Vibrio harveyi strains isolated from farmed Dicentrarchus labrax from the Adriatic Sea

Vibrio harveyi is recognized as one of the major causes of vibriosis, a disease that threatens the long-term sustainability of aquaculture. Current research shows that the Mediterranean strains of V. harveyi are serologically heterogeneous, though research comparing the traits of different strains is scarce. This study aims to describe the biochemical, physiological and genetic characteristics of three serologically different strains of V. harveyi isolated from farmed European Sea bass (Dicentrarchus labrax) from the Adriatic Sea. A total of 32 morphological and biochemical markers were examined and, the susceptibility to 13 antimicrobials tested, and then compared the results of high-throughput sequencing and in silico analyses. This study also presents the first whole genome sequences of V. harveyi isolated from European sea bass. A large number of nonsynonymous variations were detected among sequences of the three strains. The prediction analysis of resistance genes did not correspond with the in vitro antimicrobial susceptibility tests. Six virulence genes previously unrelated to virulence of vibrios were detected in all three studied strains. The results show that differences were detected at every level of comparison among the three studied strains isolated from the same fish species originating from a small geographic area

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

Spinal Deformities in Grass Goby (Zosterisessor Ophiocephalus) from the Karin Sea

Zbog učestalog pojavljivanja deformacija kralježnice u različitih uzrasta slobodno živuće populacije glavoča travaša iz Karinskog mora, rengenografski i patomorfološki opisani su utvrđeni deformiteti. Ustanovljena je učestalost njihovog pojavljivanja i utvrđene su histološke promjene u aksijalnom mišićju uzorkovanom u području deformiteta. U istraživanom uzorku glavoča travaša deformacije kralježnice utvrđene su u 11,2% primjeraka, te učestalije u manjih riba. Rengenografski na standardnim projekcijama i makroskopski na preparatima utvrđena su različita povijanja kralježnice (lordoza, skolioza i kifoza) koja se pojavljuju samostalno ili u međusobnim kombinacijama, te brojne promjene na tijelima kralježaka. Histološki nalaz u aksijalnom mišićju uključuje fibrozu intersticija i atrofiju mišićnih vlakana te nametnika roda Kudoa. To je ujedno i prvi nalaz ovog nametnika u glavoča travaša. Za pretpostaviti je da opisane deformacije kralježnice u glavoča travaša nastaju u ranom stadiju razvoja kao prilagodba organizma patološkim promjenama u aksijalnom mišićju, odnosno kao posljedica dugotrajne pojačane mišićne aktivnosti zbog nepravilnog savijanja tijela tijekom plivanja.Because of an increased incidence of spinal deformities in different age classes in natural populations of the grass goby in the Karin Sea, these skeletal changes were examined using radiology and pathomorphology analysis. In addition, histological analysis of the axial muscle was carried to determine the association of the spinal deformity with changes in the axial muscle. Spinal deformities were determined in 11.2% of total grass gobies examined, and were more frequent in smaller fish. Various spinal deformities (lordosis, scoliosis and kyphosis) and numerous changes in the vertebral bodies and associated spine were observed on standard X-ray projections and macroscopically on preparations. Histological examination revealed: interstitial fibrosis, muscle atrophy and parasites of the genus Kudoa. This is also the first finding of this parasite in the grass goby. It is reasonable to assume that the observed changes in the grass goby occur at an early age as a result of adaptation to pathological changes in axial muscle i.e. improper body bending during swimming or prolonged increased workload

Spinal Deformities in Grass Goby (Zosterisessor Ophiocephalus) from the Karin Sea

Zbog učestalog pojavljivanja deformacija kralježnice u različitih uzrasta slobodno živuće populacije glavoča travaša iz Karinskog mora, rengenografski i patomorfološki opisani su utvrđeni deformiteti. Ustanovljena je učestalost njihovog pojavljivanja i utvrđene su histološke promjene u aksijalnom mišićju uzorkovanom u području deformiteta. U istraživanom uzorku glavoča travaša deformacije kralježnice utvrđene su u 11,2% primjeraka, te učestalije u manjih riba. Rengenografski na standardnim projekcijama i makroskopski na preparatima utvrđena su različita povijanja kralježnice (lordoza, skolioza i kifoza) koja se pojavljuju samostalno ili u međusobnim kombinacijama, te brojne promjene na tijelima kralježaka. Histološki nalaz u aksijalnom mišićju uključuje fibrozu intersticija i atrofiju mišićnih vlakana te nametnika roda Kudoa. To je ujedno i prvi nalaz ovog nametnika u glavoča travaša. Za pretpostaviti je da opisane deformacije kralježnice u glavoča travaša nastaju u ranom stadiju razvoja kao prilagodba organizma patološkim promjenama u aksijalnom mišićju, odnosno kao posljedica dugotrajne pojačane mišićne aktivnosti zbog nepravilnog savijanja tijela tijekom plivanja.Because of an increased incidence of spinal deformities in different age classes in natural populations of the grass goby in the Karin Sea, these skeletal changes were examined using radiology and pathomorphology analysis. In addition, histological analysis of the axial muscle was carried to determine the association of the spinal deformity with changes in the axial muscle. Spinal deformities were determined in 11.2% of total grass gobies examined, and were more frequent in smaller fish. Various spinal deformities (lordosis, scoliosis and kyphosis) and numerous changes in the vertebral bodies and associated spine were observed on standard X-ray projections and macroscopically on preparations. Histological examination revealed: interstitial fibrosis, muscle atrophy and parasites of the genus Kudoa. This is also the first finding of this parasite in the grass goby. It is reasonable to assume that the observed changes in the grass goby occur at an early age as a result of adaptation to pathological changes in axial muscle i.e. improper body bending during swimming or prolonged increased workload

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’ 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

Is the Illegal Trade of Glass Eels (Anguilla anguilla) Increasing the Spread of Disease? A Case of EVEX

The European eel (Anguilla anguilla) is a catadromous species that inhabits the rivers of the Adriatic watershed in Croatia. It is a critically endangered fish species, according to the IUCN, due to its declining abundance in European rivers caused by overfishing and trafficking and by diseases caused by nematodes, pathogenic bacteria and viruses. An illegal parcel of glass eels was confiscated at the Zagreb Airport and was intended to re-populate Croatian rivers. Barcoding was employed to determine species affiliation, and a thorough health check was carried out. This study reports the evaluation of gross lesions, histological findings, and EVEX virus isolation and identification. Since the confiscated glass eels were of unknown origin and given the serological and genetic similarities of EVA and EVEX, we designed primers and probes for almost whole genome sequencing to elucidate the origin of glass eels based on viral phylogeny. Bayesian phylogeny showed that the isolated strain had the most common ancestor with a Danish isolate and likely evolved from the French isolate of EVEX. These findings are discussed in light of the divergence of recently isolated strains and their possible contribution to the decrease of the abundance of the European eel in European waters