Setting epidemiological cut-off values for Vibrio harveyi relevant to MIC data generated by a standardised microdilution method.

The lack of internationally harmonised criteria for interpreting the data generated by standardised susceptibility testing methods presents a serious obstacle for the development of prudent use of antimicrobials in aquaculture. The data required to set epidemiological cut-off values for minimum inhibitory concentrations for antibiotic agents against Vibrio harveyi was determined using a standard microdilution method that specified the use of cation-adjusted Mueller Hinton broth and incubation at 28°C for 24 to 28 h. In total, 120 observations were made in 4 independent laboratories from 109 unique isolates. The aggregated data from these laboratories were analysed by the normalised resistance method and by ECOFFinder to calculate epidemiological cut-off values. The data for chloramphenicol, meropenem and sulfamethoxazole were not considered as suitable for analysis. The data for ampicillin indicated that this species is innately resistant to this agent. No acceptable ranges for quality control strains have been set for ceftazidime and, therefore, only provisional cut-off values could be generated for this agent. The epidemiological cut-off values were, however, calculated for the other 6 agents. These values were ≤0.5 µg ml-1 for enrofloxacin, ≤1 µg ml-1 for florfenicol, oxolinic acid and oxytetracycline, ≤4 µg ml-1 for gentamicin and ≤0.5/9.5 µg ml-1 for trimethoprim/sulfamethoxazole. Evidence is presented demonstrating that the data for these 6 antimicrobial agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for V. harveyi.

Epidemiological cut-off values for Vibrio anguillarum MIC and disc diffusion data generated by standardised methods.

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24-28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 µg ml-1), gentamicin (≤4 µg ml-1), oxytetracycline (≤0.25 µg ml-1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 µg ml-1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cut-off values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.

The role of femoral obliquity angle and T1 pelvic angle in predicting quality of life after spinal surgery in adult spinal deformities.

BACKGROUND: Adult spinal deformities (ASD) represent a growing clinical condition related to chronic pain, disability and reduction in quality of life (QoL). A strong correlation among spinal alignment, spinopelvic parameters and QoL after spinal fusion surgery in ASD patients was thoroughly investigated over the last decade, However, only few studies focused on the relationship between lumbo-pelvic-femoral parameters - such as Femoral Obliquity Angle (FOA), T1 Pelvic Angle (TPA) and QoL. METHODS: Radiological and clinical data from 43 patients surgically treated with thoracolumbar posterior spinal fusion for ASD between 2015 and 2018 were retrospectively analyzed. The primary outcomes were the correlation between preoperative spino-pelvic-femoral parameters and postoperative clinical, functional outcomes and QoL. Secondary outcomes were: changes in sagittal radiographic parameters spino-pelvic-femoral, clinical and functional outcomes and the rate of complications after surgery. RESULTS: Using Spearman's rank correlation coefficients, spinopelvic femoral parameters (FOA, TPA, pre and post-operative) are directly statistically correlated to the quality of life (ODI, SRS-22, pre and post-operative; > 0,6 strong correlation, p <  0.05). Stratifying the patients according pre preoperative FOA value (High FOA ≥ 10 and Normal/Low FOA <  10), those belonging to the first group showed worse clinical (VAS: 5.2 +/- 1.4 vs 2.9 +/- 0.8) and functional outcomes (ODI: 35.6+/- 6.8 vs 23.2 +/- 6.5) after 2 years of follow-up and a greater number of mechanical complications (57.9% vs 8.3% p <  0.0021). CONCLUSION: Based on our results, preoperative FOA and TPA could be important prognostic parameters for predicting disability and quality of life after spinal surgery in ASD patients and early indicators of possible spinal sagittal malalignment. FOA and TPA, like other and better known spinopelvic parameters, should always be considered when planning corrective surgery in ASD patients.

Fish photobacteriosis-The importance of rapid and accurate identification of Photobacterium damselae subsp. piscicida.

MALDI-TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on-target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24-hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48-hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI-TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate-dependent.

Helicobacter apri sp. nov., isolated from wild boars.

Three isolates (A19T, C21 and F12) with spiral-shaped cells and one bipolar sheathed flagellum were obtained from gastric mucosa and caecal contents of three different wild boars (Sus scrofa) and subjected to a polyphasic taxonomic study. A genus-specific PCR showed that these isolates belonged to the genus Helicobacter. Phylogenetic analysis based on 16S rRNA, 60-kDa heat-shock protein (HSP60) and atpA genes demonstrated they formed a novel lineage within this genus. Pairwise 16S rRNA, HSP60 and atpA gene sequence comparisons of the three isolates revealed 99.7, 99.4 and 99.9 % similarity, respectively, among the three isolates; the 16S rRNA gene of isolate A19T shared 98.5 % sequence similarity with its nearest validly named neighbouring species, Helicobacter mastomyrinus (to the type strain MIT 97-5577T). The taxonomic uniqueness of the wild boar isolates was confirmed by protein analysis performed by matrix-assisted laser desorption/ionization time-of-flight MS and by a distinctive biochemical profile. These data indicated that isolates A19T, C21 and F12 represent a novel taxon, for which the name Helicobacter apri sp. nov. is proposed, with isolate A19T (=DSM 28990T=LMG 28471T) as the type strain.

FIRST ISOLATION OF TENACIBACULUM MARITIMUM IN A CAPTIVE SAND TIGER SHARK (CARCHARIAS TAURUS).

This report describes a case of the first isolation of Tenacibaculum maritimum from a captive-bred adult female sand tiger shark (Carcharias taurus) housed at the Cattolica Aquarium (Italy). The animal showed, between the second dorsal fin and the precaudal pit, skin lesions characterized by the presence of abundant whitish necrotic tissue. Through routine bacteriological examination, a bacterium was isolated from a skin lesion and subsequently identified as T. maritimum by phenotypic characters and species-specific polymerase chain reaction. The antimicrobial sensitivity of the isolated strain was evaluated for 11 antimicrobial agents by disk diffusion method. Antibiotic therapy was conducted with enrofloxacin at 10 mg kg(-1) i.m. on alternate days for 10 days. One month after the end of treatment skin lesions showed complete resolution and the shark recovered completely. The case presented here represents the first report of infection by T. maritimum in a sand tiger shark and highlights the potential pathogenic role of this microorganism in elasmobranchs kept in an aquarium.

Disseminated mycobacteriosis in a stranded loggerhead sea turtle (Caretta caretta).

A loggerhead sea turtle (Caretta caretta) was found stranded alive along the Adriatic coast close to Ancona, Italy, displaying obtundation, tachypnea, and increased respiratory effort. It died a few hours after admission, and a postmortem examination was immediately performed. Miliary yellowish nodules were evident in the liver, and a lower number in the heart, stomach, and gut wall. Hundreds of whitish nodules were scattered in the lungs, with the majority of the pulmonary parenchyma being replaced by the lesions. Histologically, all nodular lesions consisted of a small central area of necrosis with acid-fast bacilli surrounded by epithelioid cells, macrophages, and lymphocytes. Giant cells were found in the spleen and the liver. Kidneys, lungs, liver, spleen, brain, and skin lesions were inoculated aseptically onto general isolation media and selective isolation media for mycobacteria. The isolate showed a restriction pattern identical to Mycobacterium chelonae by polymerase chain reaction-restriction fragment length polymorphism. To the best of the authors' knowledge, this is the first description of a disseminated infection caused by a potentially pathogenic mycobacteria in a stranded, free-ranging loggerhead sea turtle. Veterinary staff and biologists who handle sea turtles with suspected mycobacterial disease should protect themselves appropriately.

Occurrence and genetic diversity of Arcobacter butzleri in an artisanal dairy plant in Italy.

The present study aimed to investigate the presence, distribution, and persistence of Arcobacter spp. in an artisanal dairy plant and to test the isolates to determine their different genotypes in the processing plant and in foods. Samples were collected in an artisanal cheese factory on four occasions between October and December 2012. Food samples (raw milk, ricotta cheese, mozzarella cheese, and conditioning liquid), water samples, and environmental samples were analyzed by the culture method; isolates were identified by multiplex PCR and genotyped by pulsed-field gel electrophoresis (PFGE) analysis. Arcobacter butzleri was isolated from 29 out of 59 samples (46.6%), 22 of which were from environmental samples and 7 of which were from food samples. Cluster analysis divided the strains into 47 PFGE patterns: 14 PFGE clusters and 33 unique types. Our findings indicate that the plant harbored numerous A. butzleri pulsotypes and that the manual cleaning and sanitation in the studied dairy plant do not effectively remove Arcobacter. The recurrent isolation of A. butzleri suggests that the environmental conditions in the dairy plant constitute a good ecological niche for the colonization of this microorganism. In some cases, the presence of indistinguishable strains isolated from the same facilities on different sampling days showed that these strains were persistent in the processing environment.

Isolation of Arcobacter species in water buffaloes (Bubalus bubalis).

This is the first report of Arcobacter spp. in rectal fecal samples from healthy water buffaloes (Bubalus bubalis) reared on a dairy farm. Arcobacter species were isolated after enrichment, and isolates were identified at species level by multiplex-polymerase chain reaction assay. Thirty samples were examined and Arcobacter spp. were isolated from 96.7% of water buffaloes tested: 38 Arcobacter spp. isolates were obtained, with A. cryaerophilus as the dominant species followed by A. butzleri and A. skirrowii. Nine animals (31%) were colonized by more than one Arcobacter species. The present study indicates that water buffaloes can harbor a variety of Arcobacter spp. and that healthy buffaloes may act as hosts. Water buffalo fecal shedding of Arcobacter spp. may be of significance to human health, considering the potential fecal contamination during harvesting of raw milk and slaughtering.

Sale of raw milk in northern Italy: food safety implications and comparison of different analytical methodologies for detection of foodborne pathogens.

The safety of raw milk sold in Northern Italy was investigated in relation to hygiene quality parameters and presence of Salmonella spp., Listeria monocytogenes, thermotolerant Campylobacter, and Verocytotoxin producing Escherichia coli O157:H7. The performance of different analytical methods used-official culture method (ISO), modified Bacteriological Analytical Manual cultural method (mBAM), and polymerase chain reaction (PCR)-was evaluated. The presence of Mycobacterium avium subsp. paratuberculosis (Map) was investigated only by PCR. All samples met regulations for alkaline phosphatase and inhibitory substance, while 18% and 44.8% of samples collected from vending machines had, respectively, somatic cell count (SCC) >300,000/mL and total bacterial count (TBC) >50,000 CFU/mL. The correlation between hygienic quality parameters in samples collected from bulk tank and vending machines showed a significant increase of TBC in vending machines meaning that raw milk was mishandled during distribution and sale. All pathogens investigated were detected in raw milk sold at vending machines; a total of five samples (5%) had at least one pathogen, of which two were detected by PCR and three by mBAM. None of the samples was positive by cultural ISO methods. Even if the comparison of analytical methods showed that none performs significantly better than the others, testing a higher volume of milk (25 versus 210 mL) affects significantly the detection rate of pathogens. Three samples (3%) were positive for Map, suggesting that raw milk is a significant source of Map exposure for consumers. The observed TBC increase and the detection of several pathogenic bacteria pose questions on the safety of raw milk; the use of ISO seems inefficient in detecting a low contamination level of pathogens in milk and consequently not appropriate as official method for testing. In order to ensure consumer's safety, a new approach for the raw milk chain is required.

The Animal-Visitor Interaction Protocol (AVIP) for the assessment of Lemur catta walk-in enclosure in zoos.

Animal-Visitor Interactions (AVI) are activities offered by zoos and other tourism facilities, in which visitors come into close contact with animals. These activities can promote conservational and educational content, raise conservation mindedness and responsibility for the environment and animal welfare, but if not properly managed can jeopardize visitors' and animals' well-being and conservation efforts. The Animal-Visitor Interaction assessment Protocol (AVIP) has been designed to perform an integrated and multidisciplinary assessment of these activities, encompassing the "One Health, One Welfare" approach. AVIP throughout six different steps allows to assess the effects of AVIs both on animals, visitors, and the staff involved. Results can assist zoos to improve management decisions, ensure a transparent evaluation of their activities and promote conservation education goals. Lemurs walk-in enclosures have become increasingly popular among zoos, nevertheless studies focused on their assessment are still scarce. To validate AVIP to this particular AVI, we applied it to assess a walk-in enclosure hosting five Lemur catta in an Italian zoo. Results of behavioural and physiological analyses suggested no changes in animal welfare level and the Animal Welfare Risk Assessment showed low animal welfare risks. Two Visitor Experience Surveys were used to interview 291 visitors, showing that the assessed AVI could help promote the zoo's conservation objectives and visitor education. Risk Assessment found low and medium risks to the health and safety of visitors. Results were then combined to perform a final ethical assessment. Some potential ethical concerns were detected, but the outcomes indicated that these conflicts were well managed. In the context of recent findings AVIP demonstrated its potential for application also in assessing AVIs involving primates. Our findings confirmed the usefulness of AVIP in assessing and monitoring AVIs, allowing to gain key information in a single process on multiple welfare-related parameters, educational impact, safety of the main stakeholders involved, and ethical concerns.

Campylobacter vulpis sp. nov. isolated from wild red foxes.

During a sampling of wild red foxes (Vulpes vulpes) for the detection of Epsilonproteobacteria, 14 strains were isolated from the caecal contents of 14 epidemiologically-unrelated animals. A genus-specific PCR indicated that the isolates belonged to the genus Campylobacter. Based on the results of a species-specific PCR, the isolates were initially identified as C. upsaliensis. However, multi-locus sequence typing (MLST) revealed that the isolates were significantly different from the C. upsaliensis present in the MLST database. A polyphasic study, including conventional biochemical and tolerance characteristics, morphology by transmission electron microscopy (TEM), MALDI-TOF analysis, and genetic comparisons based on partial 16S rDNA and atpA gene sequences, was undertaken. Finally, the complete genome sequence of the type strain 251/13T and the draft genome sequences of the other isolates were determined. Average nucleotide identity, average amino acid identity and in silico DNA-DNA hybridization analyses confirmed that the isolates represent a novel taxon for which the name Campylobacter vulpis sp. nov. is proposed, with isolate 251/13T (=CCUG 70587T = LMG 30110T) as the type strain. In order to allow a rapid discrimination of C. vulpis from the closely-related C. upsaliensis, a specific PCR test was designed, based on atpA gene sequences.

An Ethical Assessment Tool (ETHAS) to Evaluate the Application of Assisted Reproductive Technologies in Mammals' Conservation: The Case of the Northern White Rhinoceros (Ceratotherium simum cottoni).

Assisted reproductive technologies (ARTs) can make a difference in biodiversity conservation. Their application, however, can create risks and raise ethical issues that need addressing. Unfortunately, there is a lack of attention to the topic in the scientific literature and, to our knowledge, there is no tool for the ethical assessment of ARTs in the context of conservation that has been described. This paper reports the first applications of the Ethical Assessment Tool (ETHAS) to trans-rectal ovum pick-up (OPU) and in vitro fertilization (IVF) procedures used in a northern white rhinoceros (Ceratotherium simum cottoni) conservation project. The ETHAS consists of two checklists, the Ethical Evaluation Sheet and the Ethical Risk Assessment, and is specifically customized for each ART procedure. It provides an integrated, multilevel and standardized self-assessment of the procedure under scrutiny, generating an ethical acceptability ranking (totally, partially, not acceptable) and a risk rank (low, medium, high), and, hence, allows for implementing measures to address or manage issues beforehand. The application of the ETHAS to the procedures performed on the northern white rhinoceros was effective in ensuring a high standard of procedures, contributing to the acceptability and improved communication among the project's partners. In turn, the tool itself was also refined through an iterative consultation process between experts and stakeholders.

First description of the adult stage of Clinostomum cutaneum Paperna, 1964 (Digenea: Clinostomidae) from grey herons Ardea cinerea L. and a redescription of the metacercaria from the Nile tilapia Oreochromis niloticus niloticus (L.) in Kenya.

The combined use of morphological and molecular studies allowed for the first time the recognition and description of the adult stage of Clinostomum cutaneum Paperna, 1964 from the grey heron Ardea cinerea L. in Kenya. A redescription of the metacercaria that infect Nile tilapia Oreochromis niloticus niloticus (L.) from the same aquatic environment is also presented. C. cutaneum differs from all other species of Clinostomum Leidy, 1856 in the shape of its uterus. Sequencing the rRNA confirmed the morphological similarity between adults from the grey heron and the metacercarial stage from tilapia, and a level of genetic similarity with the other previously sequenced Clinostomum spp. was observed. The need for a reorganisation of Clinostomum using both morphological and molecular methods is highlighted.

Mycobacterium pseudoshottsii in Mediterranean Fish Farms: New Trouble for European Aquaculture?

Mycobacterium pseudoshottsii, a slow-growing mycobacterium closely related to M. marinum, has been isolated only in wild fish in the United States and in Japanese fish farms to date. Here, we report cases of mortality in three farmed fish species (Dicentrarchus labrax, Sparus aurata, and Sciaenops ocellatus) caused by M. pseudoshottsii in Italy. Samples underwent necropsy, histology, and culture with pathogen identification based on PCR and sequencing of housekeeping genes (16S rRNA, hsp65, rpoB). Multifocal to coalescing granulomatous and necrotizing inflammation with acid-fast bacilli were observed in the parenchymatous organs, from which M. pseudoshottsii was isolated and identified. Phylogenetic analysis confirmed the results of gene sequencing and allowed subdivision of the isolates into three distinct groups. M. pseudoshottsii poses a potential threat for Mediterranean aquaculture. Its origin in the area under study needs to be clarified, as well as the threat to the farmed fish species.

The life cycle of Myxobolus lentisuturalis (Myxozoa: Myxobolidae), from goldfish (Carassius auratus auratus), involves a Raabeia-type actinospore.

We studied a natural infection of the oligochaete Branchiura sowerbyi Beddard, 1892 with the Raabeia-type actinosporean stage of Myxobolus lentisuturalis Dyková, Fiala et Nie, 2002 which infected goldfish Carassius auratus auratus (L.) in Italy, using molecular analysis of the SSU rRNA gene. The existence of intraoligochaete development shows that this parasite follows the life-cycle pattern described by Wolf and Markiw (1984) for Myxobolus cerebralis. Histological examinations of the goldfish infected by M. lentisuturalis showed at low magnification the presence of two bilateral crescent-shaped masses in the dorsal epaxial muscle. These lesions were not circumscribed, presented irregular edges and infiltrated the underlying bundles of skeletal muscle and interstitial tissue. At higher magnification, disappearance of muscle fibres and substitution of the muscle tissue with Myxobolus spores and plasmodia were observed.

A Protocol for the Ethical Assessment of Wild Animal-Visitor Interactions (AVIP) Evaluating Animal Welfare, Education, and Conservation Outcomes.

Due to the popularity of wild animal-visitor interactions (AVIs), there is a need for an ethical assessment of their impact on animal welfare, education, and conservation. The protocol presented in this study is designed to evaluate such interactions on an integrated level, using a transparent analysis of all the aspects involved, including all the stakeholders and the potential conflicts of values. The protocol consists of a six-step process encompassing dedicated data acquisition and a specific ethical assessment. When the protocol was applied to assess a "giraffe feeding" interaction, steps devoted to data acquisition found that animal welfare risks were low, and that visitors described giraffes with emotionally linked descriptors more often after the interaction. The net promoter score, which refers to how likely visitors would recommend to a friend to join the animal-visitor interaction, was 74%. The subsequent ethical assessment, which consisted of a comparison of the results of the previous steps with an ethical matrix highlighting the ideal situation for all stakeholders' interests, allowed the overall identification of the ethical concerns entailed by the interaction. A final ethical checklist of the examined AVI had a "yes" in entries regarding animal welfare, emotional, and conservation mindedness outcomes and ethical assessment.

Humboldt penguins' feathers as bioindicators of metal exposure.

Avian feathers have the potential to accumulate trace elements originating from contaminated food and polluted environments. In fact, in feathers, metals bind to keratin, a sulphur-containing protein for which several metals have a strong affinity. Here, the concentrations of 18 essential and non-essential elements were investigated in a Humboldt penguin (Spheniscus humboldti) colony housed at the Acquario di Cattolica (Italy). This species is listed as vulnerable in the Red List of the International Union for Conservation of Nature. According to the literature, there is usually a link between metal levels in the diet of birds and levels detected in their feathers. Thus, metals were also determined in the penguins' food (capelin, Mallotus villosus). We hypothesize that the controlled conditions in which birds are kept in captivity, and the homogeneous diet that they follow could allow a better understanding of metal bioaccumulation (such as mercury) or bio-dilution (such as arsenic) in the marine food chain, indicated by penguins' feathers. Moreover, comparisons with our previous investigations performed on an ex-situ African penguin (Spheniscus demersus) colony suggest that penguins living indoors have lower body burden of metals than those living outdoors. Indeed, environmental contaminants usually found in areas subjected to anthropogenic impact, where zoos and aquaria are often located, are not accumulated to levels of concern.

Feathers of Humboldt penguin are suitable bioindicators of Rare Earth Elements.

Rare earth elements (REEs), also called lanthanides, are emerging contaminants worldwide, due to their unique physical and chemical characteristics that make them essential in a variety of industrial applications. However, there is still a gap in the knowledge of occurrence and accumulation of REEs in biota, and no investigations have yet been performed in penguin feathers, which have already been widely utilized as a non-invasive tool for the biomonitoring of trace elements. The concentrations of 16 REEs were investigated in a colony of Humboldt penguins (Spheniscus humboldti) housed at the Acquario di Cattolica (Italy). Multielement determination of REEs was performed by an Inductively Coupled Plasma-Mass Spectrometer after a microwave digestion of feathers. As this colony lives indoors in a controlled environment, it was the ideal choice for studying lanthanide occurrence in penguin feathers. Since there is a strict link between metal levels in feathers and the diet of penguins, their food (capelin) was also tested for REEs. Chondrite normalized values revealed the same pattern for REEs in feathers and fish, but REE concentrations were an order of magnitude higher in penguin feathers, demonstrating the suitability of this tissue as a bioindicator of REEs.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream.

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48-72 hours.