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.
Common carp (Cyprinus carpio) is a very important fish species for warm‐water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi ...herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures.
Noble pen shell (
Pinna nobilis
) is an endemic species and the largest known bivalve in the Mediterranean Sea. By filtering large amounts of water, they maintain a high percentage of organic matter, ...hence playing an important role in the marine ecosystem. The ecological community of pen shells is impressive, and there are numerous microorganisms present in its soft tissues. Since this species is highly endangered due to recently described mass mortalities throughout the Mediterranean, this study was aimed at finding out more about its microbiome. In this study, we identified the predominant bacterial populations of specimens collected at three separate locations along the Eastern Adriatic coast. The predominant bacteria were isolated and 16S rRNA sequencing was performed to identify eight different bacterial genera:
Aestuariibacter
sp.,
Aliivibrio
sp.,
Alteromonas
sp.,
Marinobacter
sp.,
Pseudoalteromonas
sp.,
Rubritalea
sp.,
Thalassospira
sp. and the
Vibrio splendidus
clade. The identified genera are ubiquitous in the marine environment and have previously been described as both beneficial symbionts and potential pathogens in other molluscs. There was a clear difference in the predominant bacterial populations between northern and southern sampling sites, which could be linked to water temperature. These findings indicate the need for expanded sampling over a longer time period, since more exhaustive research would provide information vital to the conservation of this critically endangered species.
The major histocompatibility complex (MHC) includes highly polymorphic genes involved in antigen presentation, which is crucial for adaptive immune response. They represent fitness related genetic ...markers particularly informative for populations exposed to environmental challenges. Here we analyse the diversity and evolutionary traits of MHC class II DQA and DQB genes in the dolphins Stenella coeruleoalba and Grampus griseus from the Mediterranean Sea. We found substantial nucleotide and functional diversity, as well as strong evidence of balancing selection indicated by allele and supertype frequencies, Tajima's D statistics and dN/dS tests. The Risso's dolphin, considered the least abundant in the region, showed the effect of divergent allele advantage at the nucleotide and functional-peptide levels. An outstanding polymorphism was found in the striped dolphin, particularly intriguing in the DQA gene where the Ewens-Watterson test detected a selection sweep that occurred in recent history. We hypothesize that morbillivirus, which has recurrently invaded Mediterranean populations over the last decades, exerted the detected selective pressure.
Noble pen shells (Pinna nobilis) along the Eastern Adriatic coast were affected by mass mortalities similarly to the populations across the Mediterranean basin. Samples of live animals and organs ...originating from sites on Mljet Island on the south and the Istrian peninsula on the north of the Croatian Adriatic coast were analyzed using histology and molecular techniques to detect the presence of the previously described Haplosporidium pinnae and Mycobacterium spp. as possible causes of these mortalities. To obtain more information on the pattern of the spread of the mortalities, a study was undertaken in Mljet National Park, an area with a dense population of noble pen shells. The results of the diagnostic analysis and the velocity of the spread of the mortalities showed a significant correlation between increases in water temperature and the onset of mortality. Moderate to heavy lesions of the digestive glands were observed in specimens infected with H. pinnae. A phylogenetic analysis of the detected Haplosporidium pinnae showed an identity of 99.7 to 99.8% with isolates from other Mediterranean areas, while isolated Mycobacterium spp. showed a higher heterogeneity among isolates across the Mediterranean. The presence of Mycobacterium spp. in clinically healthy animals a few months before the onset of mortality imposes the need for further clarification of its role in mortality events.
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.
The annual production of European flat oysters (Ostrea edulis) in Croatia is about 50 to 65 tons, and it has a long tradition. All Croatian oyster farms are subjected to the national surveillance ...program aiming to detect the presence of Bonamia ostreae and Marteilia refringens according to the Council Directive 2006/88/EC. Within the surveillance program, the first findings of the parasite Bonamia spp. occurred in 2016 in two production areas in the north and south of the Eastern Adriatic coast. The repeated findings of the parasite were noted up to 2020 but also on two additional sites in the north. The parasite was detected by cytological analysis of stained heart smears, histological examination, and PCR. PCR positive samples were sequenced for SSU rDNA gene, and BLAST analysis confirmed infection with Bonamia exitiosa. Attempts to prove the Pacific oyster as a putative vector of the parasite failed. The infection prevalence from 2016 until 2020 ranged from 3.3 to 20% in different sites. No mortalities were reported from the infected sites, and it seemed that infection of flat oysters with B. exitiosa did not affect their health. The study has not shown the source and way of infection spread, which imposes the need for more comprehensive molecular and epidemiological studies.
spp. that cause marine brucellosis are becoming more important, as the disease appears to be more widespread than originally thought. Here, we report a whole and annotated genome sequence of
CRO350, ...a sequence type 27 strain isolated from a bottlenose dolphin carcass found in the Croatian part of the northern Adriatic Sea.
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.
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