Vibriosis caused by Vibrio harveyi: studies on etiopathogenesis and vaccine efficacy in European sea bass (Dicentrarchus labrax)

Vibrio harveyi rappresenta un patogeno batterico emergente per l’acquacoltura marina a livello globale. Nel branzino V. harveyi è stato isolato in episodi di mortalità caratterizzati da atassia natatoria, lesioni cutanee ed enteriche. Un protocollo polifasico di identificazione per V. harveyi è stato standardizzato basandosi su metodi biochimici, molecolari e proteomici ed è stato valutato raffrontando una collezione di 81 isolati di campo di V. harveyi (specie ittiche e bivalvi) con ceppi di referenza di V. harveyi e di altre 22 specie della famiglia Vibrionaceae. La valutazione fenotipica si è basata su prove in macrometodo, micrometodo (API20E) e crescita su terreni TCBS e CHROMagar Vibrio. L’analisi molecolare ha impiegato il sequenziamento del gene housekeeping pyrH, e l’amplificazione specie-specifica del gene toxR. Il profilo proteico è stato determinato mediante MALDI-TOF analizzando il contenuto proteico ribosomiale. E’ stato valutato il profilo di sensibilità/resistenza agli antibiotici su 51 ceppi di V. harveyi, isolati da episodi clinici o da campioni ambientali, mediante metodo Kirby-Bauer e determinazione della minima concentrazione inibente (MIC). È stata indagata mediante infezioni sperimentali (immersione e inoculo intraperitoneale IP) la patogenicità di differenti isolati di V. harveyi in branzino valutando le lesioni anatomo-istopatologiche indotte. È stato inoltre sviluppato un protocollo vaccinale basato su vaccino spento polivalente da somministrare per immersione o per inoculo IP, addizionato con estratto extracellulare ed emulsionato o meno con adjuvante. Sono state condotte prove di sicurezza ed efficacia vaccinale determinando per i vari protocolli la percentuale relativa di sopravvivenza (RPS). Un metodo ELISA indiretto è stato sviluppato per comparare la risposta immunitaria (IgM sieriche) degli esemplari vaccinati con i differenti protocolli, evidenziando una risposta significativamente maggiore nei soggetti inoculati IP con vaccino adjuvato.Vibrio harveyi is an emerging bacterial pathogen for marine aquaculture globally. In the European sea bass V. harveyi has been isolated in episodes of mortality characterized by ataxia, cutaneous and enteric lesions. In the present study, a polyphasic identification protocol for V. harveyi was standardized based on biochemical, molecular and proteomic methods and was evaluated by comparing a collection of 81 V. harveyi field isolates (fish and bivalve species) with V. harveyi reference strains and other 22 species of the family Vibrionaceae. The phenotypic evaluation was based on macromethod and micromethod (API20E) tests and growth on TCBS and CHROMagar Vibrio. Molecular analysis employed the sequencing of the housekeeping pyrH gene, and the species-specific amplification of the toxR gene. The protein profile was determined by MALDI-TOF analyzing the ribosomal protein content. The antibiotic susceptibility profile was evaluated on 51 V. harveyi strains, isolated from clinical episodes or environmental samples, applying the Kirby-Bauer method and determination of the minimum inhibitory concentration (MIC). The pathogenicity of different V. harveyi isolates in sea bass was investigated by experimental infections (immersion and intraperitoneal IP injection) and the induced anatomo-histopathological lesions were evaluated. A vaccine protocol based on a polyvalent bacterin was also developed to be administered by immersion or by IP, enriched with extracellular extract and emulsified or not with adjuvant. Safety tests and vaccine efficacy were carried out by determining the relative survival rate (RPS) for the various protocols. An indirect ELISA method was developed to compare the immune response (serum IgM) of the specimens vaccinated with the different protocols, highlighting a significantly greater response in the specimens inoculated IP with adjuvant vaccine

Viral nervous necrosis in gilthead sea bream (Sparus aurata) caused by reassortant betanodavirus RGNNV/SJNNV : an emerging threat for Mediterranean aquaculture

Viral nervous necrosis (VNN) certainly represents the biggest challenge for the sustainability and the development of aquaculture. A large number of economically relevant fish species have proven to be susceptible to the disease. Conversely, gilthead sea bream has generally been considered resistant to VNN, although it has been possible to isolate the virus from apparently healthy sea bream and sporadically from affected larvae and postlarvae. Unexpectedly, in 2014-2016 an increasing number of hatcheries in Europe have experienced mass mortalities in sea bream larvae. Two clinical outbreaks were monitored over this time span and findings are reported in this paper. Despite showing no specific clinical signs, the affected fish displayed high mortality and histological lesions typical of VNN. Fish tested positive for betanodavirus by different laboratory techniques. The isolates were all genetically characterized as being reassortant strains RGNNV/SJNNV. A genetic characterization of all sea bream betanodaviruses which had been isolated in the past had revealed that the majority of the strains infecting sea bream are actually RGNNV/SJNNV. Taken together, this information strongly suggests that RGNNV/SJNNV betanodavirus possesses a particular tropism to sea bream, which can pose a new and unexpected threat to the Mediterranean aquaculture

Detection and characterization of a rhabdovirus causing mortality in black bullhead catfish, Ameiurus melas

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In the wake of the ongoing mass mortality events: Co-occurrence of Mycobacterium, Haplosporidium and other pathogens in Pinna nobilis collected in Italy and Spain (Mediterranean Sea)

Following the Mass Mortality Events (MMEs) of the pen shell P. nobilis in Campania region and Sicily, a survey of moribund P. nobilis specimens was also conducted in other Italian regions (Campania, Tuscany, Sardinia, and Apulia) and Spain (Catalunya). Histopathological and molecular examination of 27 specimens of P. nobils revealed different types of pathogens associated with tissue lesions, morbidity and mortality. Presence of Mycobacterium, Vibrio species, Haplosporidium pinnae and Perkinsus sp. were detected, differently distributed into the areas. The Mycobacterium sp., previously reported in Campania and Sicily samples, was observed in all the analyzed areas and individuals, associated to systemic inflammatory lesions. In Spain, H. pinnae was observed in 36% of cases, always associated to the Mycobacterium sp. Molecular study using hsp65 genes and Internal Transcriber Spacer ITS support that a new species of Mycobacteria is infecting P. nobilis, close to M. triplex and belonging to the group of M. simiae complex with M. sherrisi. Presence of Perkinsus spp. resembling P. mediterraneus was observed in 2 out of 13 Italian individuals whose presence should be addressed as potential risk for shellfish aquaculture of the area. Vibrio spp. were also detected in some case. The preliminary results of this study suggest that Mycobacterium sp., Vibrio spp., H. pinnae and Perkinsus sp. cooperate to disease pathogenesis, being Mycobacterium and Haplosporidium most of the time involved. Vigilant inspection of those areas where MME is now starting, along with continuous systematic surveys, are crucial to define the spatiotemporal progress of mortality and the role of every single pathogen in the disease outcome.info:eu-repo/semantics/publishedVersio

Corrigendum: In the Wake of the Ongoing Mass Mortality Events: Co-occurrence of Mycobacterium, Haplosporidium and Other Pathogens in Pinna nobilis Collected in Italy and Spain (Mediterranean Sea)

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Cosmopolitan distribution of Endozoicomonas-like organisms and other intracellular microcolonies of bacteria causing infection in marine molluscs

Intracellular microcolonies of bacteria, in some cases developing large extracellular cysts, have been historically reported infecting a wide diversity of economically important mollusc species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterise those organisms, traditionally named as Rickettsia or Chlamydia -like organisms (RLO/CLO), via international collaboration, 98 samples comprising 20 mollusc species were collected over 10 countries and examined using histology and phylogenetic analysis. A 16S rRNA gene amplicon library-based sequencing showed the presence of different species of Endozoicomonas-like organisms (ELO) in all the mollusc species analysed, infecting primarily gill and digestive glands. Co-infections of ELOs with other intracellular bacteria were also observed. Subsequent phylogenetic analysis of Operational Taxonomic Units (OTU) revealed a novel microbial diversity associated with molluscan RLO/CLOs infection distributed along different taxa, including Spirochaetes phyla, Rickettsiales order, Simkaniaceae family, Mycoplasma and Francisella genera, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained. The presence of ELO sequences in the RLO/CLO infection was confirmed by standard PCR, Sanger sequencing, and by in situ hybridisation in a selection of samples. The phylogenetic analysis conducted in this study will allow for further characterization of the microbial community associated with Rickettsia and Chlamydia-like infection in marine molluscs and their correlation with severity of the lesions in order to reveal their role as endosymbionts, commensals or true pathogens.info:eu-repo/semantics/publishedVersio

Invasive parasites and global change: Evidence for the recent and rapid spillover of a potential pathogen of tilapias with a complex, three-host life cycle

Biological invasions pose a serious threat to local flora and fauna and have negative impacts on ecosystems. Invasive parasites can also cause severe losses in aquaculture. In this article, we provide evidence of the recent spillover of an African parasite with a complex, three-host life cycle that has rapidly and successfully established itself in the Middle East, most likely due to the recent migration of its final hosts (great cormorant) from Africa. This case of parasite introduction into a country with intensive aquaculture is also important from an economic point of view, since large (up to 2 cm long) larvae of this parasite, the cyclophyllidean tapeworm Amirthalingamia macracantha (Cestoda) localised in the liver, can be pathogenic to their fish hosts, including farmed and wild fish, as shown by our histopathological examination of heavily infected fish. Since its first detection in Israel in November 2020, the parasite has spread rapidly and is currently found in both migratory (great cormorant, Phalacrocorax carbo) and non-migratory birds (pygmy cormorant, Microcarbo pygmaeus), as well as in fish intermediate hosts, including farmed tilapia in several farms in Israel and wild cichlids. There are numerous examples of the spillover of introduced parasites, including those that parasitise fish of commercial importance, but have a direct life cycle or use only a single intermediate host. Tilapines are the second most important group of farmed fish in the world after carps and are produced mainly in Southeast Asia, Central and South America. The global spread of great cormorants and the early evidence that pygmy cormorant may also harbour A. macracantha pose the risk of further spread of this invasive parasite to other countries and areas. In addition, global warming and reductions in foraging and resting areas near these waters may allow the parasite to complete its life cycle in new hosts

How much do crayfish plague and microsporidiosis affect the conservation status of the white-clawed crayfish Austropotamobius pallipes complex in Trentino (NE Italy)?

The white-clawed crayfish Austropotamobius pallipes complex is an endangered species, with decreasing trend in distribution and abundance throughout Europe. One of the causes of decline is the widespread invasion of alien crayfish and the associated spread of infectious diseases, primarily of the crayfish plague caused by Aphanomyces astaci. Although this disease usually causes mass mortality in A. pallipes, some wild populations appear to be resistant or/and tolerant towards A. astaci. A further disease, poorly investigated and understood, is microsporidiosis, i.e., the porcelain disease, caused by the microsporidian parasites Astathelohania contejeani and Nosema austropotamobii. We present the results of a 2021-2022 monitoring survey, aimed at mapping the distribution of A. astaci, A. contejeani and N. austropotamobii in the wild populations of A. pallipes in Trentino. We applied non-invasive sampling methods to collect cuticular swabs from 17 of 44 known populations, concurrently collecting eDNA at one of the sampling sites, to investigate the presence of A. astaci and if possible, identify its genotype through molecular analyses. Sixteen 16 specimens from 5 populations showed abdominal muscles with macroscopic signs of porcelain disease, tissue from these specimens were collected and subjected to molecular evaluation to confirm the presence of microsporidia and identify the species. Aphanomyces astaci was detected in 5 populations and the presence of a low pathogenic genotype (genotype A) was confirmed in one of them. The presence of A. contejeani was identified in 5 populations. In 2 of them, N. austropotamobii was detected in co-infection with A. contejeani in the same individual

Hikui disease in nine koi carp (Cyprinus carpio): First description of a cutaneous perivascular wall tumour

Background: Hikui disease is a well known disfiguring disease of koi carp (Cyprinus carpio) primarily affecting fish with red pigmentation. It causes light orange to golden yellow, multifocal to coalescing raised patches, starting from the red cutaneous areas. Some cases respond to surgery or topical treatment, but recurrence is common. Objectives: To describe the clinical and pathological presentation of Hikui disease and its cause. Animals: Nine affected koi carp belonging to private hobbyists. Methods: Eight fish underwent surgery or biopsy; one was euthanized. Tissues were submitted for histology, immunohistochemistry and transmission electron microscopy. Results: Five fish showed typical lesions of Hikui disease, whereas four fish showed an atypical presentation characterized by focal or multifocal, oedematous, dark red cutaneous plaques or nodules. Histology showed unencapsulated, infiltrating and densely cellular neoplasms composed of spindle cells arranged in bundles, rows and whorls frequently centred on capillaries. Immunohistochemistry for smooth muscle actin labelled neoplastic cells in all cases. Ultrastructure showed neoplastic cells with slender cytoplasmic processes encircling the capillaries, a thin basal membrane and occasional plasmalemmal vesicles. Conclusions and clinical importance: All of the data supported a neoplastic process producing perivascular wall tumours. Immunoreactivity to smooth muscle actin and the ultrastructural features were indicative of a pericyte origin (haemangiopericytoma). This is the first report dealing with Hikui disease that has achieved a conclusive diagnosis. The neoplastic nature of this condition suggests the potential usefulness of a surgical approach in the clinical management of less severe cases