Mixed mycobacterial infections in farmed sturgeons

Based on microbiological and histopathological examinations and DNA sequencing, several outbreaks of mycobacteriosis in the reared sturgeons, including Chinese sturgeon (Acipenser sinensis Gray) and Amur sturgeon (Acipenser schrencki), were identified during 2009 to 2010. Forty‐nine isolates of non‐tuberculous mycobacteria(NTM)were isolated from 19 diseased sturgeons. In total, seven species of Mycobacterium were identified, namely, Mycobacterium chelonae, Mycobacterium marinum, Mycobacterium gordonae, Mycobacterium fortuitum, Mycobacterium szulgai, Mycobacterium arupense and Mycobacterium porcinum. Among them, M. marinum was found to be more prevalent (89.5%) compared with the other mycobacterial species. When two molecular biological methods, PCR‐DGGE (denaturing gradient gel electrophoresis) analysis and rpoB gene library sequencing, were used to analyse the mycobacterial DNAs extracted from the diseased fish tissues, mixed infections of two or three mycobacterial species were found being the predominant infection form (94.7%) in sturgeon mycobacteriosis. M. marinum was the only one species that caused sturgeon mycobacteriosis alone. Virulence assay showed that M. marinum possessed stronger pathogenicity to zebrafish killing 100% of fish in 28 days at 10³ cfu/fish than the other species. These results suggested that M. marinum is the major pathogenic bacteria in sturgeon mycobacteriosis. To the best of our knowledge, this study is the first report on mycobacteriosis in farmed Chinese and Amur sturgeons as well as the first isolation of M. porcinum and M. arupense from fish.     

Evidence of fish and human pathogens associated with doctor fish (Garra rufa,Heckel, 1843) used for cosmetic treatment

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G. rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A. hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M. goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well‐known agents of zoonosis. Indeed, M. goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M. goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.     

A new cultivation‐independent tool for fast and reliable detection of Mycobacterium marinum

The Mycobacterium marinum group (MMG) is a class of mycobacteria that includes M. marinum, the cause of chronic systemic infections in fish. This species occasionally causes granulomatous skin lesions in humans. Other members of MMG are mycolactone‐producing mycobacteria (MPM; M. ulcerans, M. shottsii and M. pseudoshottsii). The cultivation‐independent approach presented in this study brings a fast and reliable alternative to classically used cultivation methods. The developed triplex erp/IS2404 qPCR assay is based on a primary species‐specific erp detection, which allows enumeration of MMG in analysed samples, and secondary IS2404 detection is suitable for the differentiation of M. marinum from MPM. The detection of M. marinum in clinical specimens and in artificially contaminated tissue samples has proven its applicability for diagnostic purposes.     

Genital mycobacteriosis caused by Mycobacterium marinum detected in two captive sharks by peptide nucleic acid–fluorescence in situ hybridization

Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)-infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M. marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M. marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60 kDa heat-shock protein gene (hsp65), and peptide nucleic acid–fluorescence in situ hybridization (PNA-FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M. marinum. Because M. marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes.     

Transmission of Mycobacterium chelonae and Mycobacterium marinum in laboratory zebrafish through live feeds

The zebrafish (Danio rerio) is a popular vertebrate model organism used in a wide range of research fields. Mycobacteriosis, caused by Mycobacterium species, is particularly concerning because it is a common disease associated with chronic infections in these fish. Infections are also a source of uncontrolled experimental variance that may influence research results. Live feeds for zebrafish are common and include paramecia (Paramecium caudatum), brine shrimp (Artemia franciscana) and rotifers (Branchionus spp.). Although nutritionally beneficial, live feeds may pose a biosecurity risk. In this study, we investigate transmission of Mycobacterium chelonae and Mycobacterium marinum through these three live feeds. We show that all three live feeds ingest both M. marinum and M. chelonae and can transmit mycobacterial infections to zebrafish. This observation emphasizes the need for live feeds to be included in the consideration of potential biosecurity risks. This study is of importance to other beyond the zebrafish community, including those of additional aquatic models and those using live feeds for other types of aquaculture.     

Evaluation of the INNO‐LiPA mycobacteria v2 assay for identification of aquatic mycobacteria

Fifty‐seven isolates of mycobacteria comprising 10 reference strains, 47 field isolates and one non‐Mycobacterium isolate were screened using commercial INNO‐LiPA v2 assay kits. All mycobacteria isolates tested hybridized with the Mycobacterium genus probe on the LiPA strip. All M. marinum, M. fortuitum and M. chelonae reference and field strains and three out of the four M. gordonae isolates hybridized to their corresponding species‐ or complex‐specific probes. Two cultures (a type strain and a field isolate) yielded mixed growth of two mycobacterial species, i.e. M. chelonae and M. fortuitum. A Mycobacterium isolate from one of these cultures was subsequently purified and correctly identified with the kit. However, sequence analysis of the 16S–23S rRNA internal transcribed spacer (ITS) region of various mycobacteria isolates revealed a misidentification of M. shottsii and M. pseudoshottsii with the kit because these isolates reacted with the M. marinum/M. ulcerans probe. Moreover, nine of the 13 field isolates presumed to be M. fortuitum from the results of the kit had closer ITS sequence homology with M. conceptionense, a species which, to our knowledge, has never been reported in fish. These findings highlight the need to redesign the M. fortuitum–M. peregrinum probe included in the INNO‐LiPA assay and to introduce additional complex‐specific probes into the kit. Nevertheless, the kit proved to be a rapid and reliable method for identifying mycobacteria in the aquatic environment and would be particularly useful in laboratories without sequencing facilities.     

Morphology and distribution of granulomatous inflammation in freshwater ornamental fish infected with mycobacteria

Mycobacteriosis in fish is a chronic progressive ubiquitous disease caused by Mycobacterium marinum, M. gordonae and M. fortuitum in most cases. The aim of this study was to describe the morphology and distribution of lesions in 322 freshwater ornamental fish across 36 species. Granulomatous inflammation was diagnosed by gross examination and histopathology testing in 188 fish (58.4%); acid‐fast rods (AFR) were determined in only 96 (51.1%) fish from 19 species after Ziehl–Neelsen staining. The most often affected organs with AFR were the kidney (81.2%), digestive tract (54.1%), liver (48.2%), spleen (45.9%) and skin (21.2%); sporadically, AFR were found in the branchiae (9.4%) and gonads (4.7%). In 14 randomly selected fish originating from four different fish tanks, the distribution of mycobacterial infection was studied by culture examination of the skin, gills, muscle tissue, digestive tract, liver, spleen and kidney. In 12 fish, the species M. marinum, M. gordonae, M. fortuitum, M. triviale, and M. avium subsp. hominissuis (serotypes 6 and 8 and genotype IS901? and IS1245+) were detected; mixed infection caused by different mycobacterial species was documented in five of them.     

The efficacy of MS‐222 as anaesthetic agent in four freshwater aquarium fish species

The efficacy of anaesthetic tricaine methanesulfonate (MS‐222) was evaluated in four freshwater aquarium fish species, Zebrafish (Danio rerio), Guppy (Poecilia reticulata), Discu (Symphysodon discus) and Green swordtail (Xiphophorus helleri). The correct dose of anaesthetic should induce the plane 4 of anaesthesia in less than 180 s, recovery in less than 300 s and must survive when exposed during 30 min to anaesthetic. Fishes were exposed to six concentrations of anaesthetic (75, 100, 125, 150, 200 and 250 mg L^?1) and the time of fish reaching plane 4 of anaesthesia, post exposure recovery, and the percentage of survival when fish were subject to 30 min in the anaesthetic were recorded. The optimal doses varied according to the species: D. rerio – 75, 100 and 125 mg L^?1, P. reticulata – 125, 150 and 200 mg L^?1, S. discus – 75 and 100 mg L^?1 and X. helleri – 125 and 150 mg L^?1. The induction time generally decreased significantly with increasing concentration of MS‐222 for all of the species evaluated. The recovery time had a tendency to increase with the increase of the MS‐222 concentration for D. rerio, P. reticulata and S. discus. On the other hand, X. helleri recovery time decreased with the increase of MS‐222 concentration. MS‐222 proved to be effective in anaesthesia for all the freshwater ornamental species studied. The main results clearly show that the optimal dose to anesthetize is fish species dependent and it is completely wrong to extrapolate optimal anaesthetic concentrations between different species.     

Investigation and management of an outbreak of multispecies mycobacteriosis in Australian lungfish (Neoceratodus fosteri) including the use of triple antibiotic treatment

Disease due to non‐tuberculous mycobacteria (NTM) is common in fish. Current recommendations focus on outbreak management by depopulating entire fish stocks and disinfecting tanks. Treatment is not advocated. Treatment may be appropriate, however, where individual, valuable fish are concerned. ZSL London Zoo managed an outbreak of mycobacteriosis in a valuable group of imported F1 captive‐bred Australian lungfish (Neoceratodus fosteri) by depopulation, isolation, extensive testing and daily oral antibiotic treatment. Four species of Mycobacterium (M. marinum, M. fortuitum, M. chelonae and M. peregrinum) were involved in this outbreak, each with unique antibiotic sensitivities. Triple therapy with rifampicin, doxycycline and enrofloxacin for 8 months was the most effective antibiotic combination, resulting in full disease resolution. No side effects were noted and, more than 18 months post‐treatment, no recurrence had occurred. This is the first report of mycobacterial disease in lungfish and the first report of a polymycobacterial outbreak in fish involving these four species of Mycobacterium. This report demonstrates the value of extensive isolation and identification. Also, as therapies currently advised in standard texts did not reflect the antibiotic sensitivity of the NTM found in the fish reported here, we recommend that antibiotic treatment should always be based on sensitivity testing.     

Characterization of spaC‐type Erysipelothrix sp. isolates causing systemic disease in ornamental fish

Since 2012, low‐to‐moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram‐positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep‐PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species.     

Immunohistochemical and Taqman real-time PCR detection of mycobacterial infections in fish

Real‐time PCR and immunohistochemistry (IHC) assays were developed to detect fish mycobacterial infections at the genus level, based on the RNA polymerase β subunit (rpoB) gene and polyclonal anti‐Mycobacterium rabbit serum, respectively. The PCR assay positively identified a number of pathogenic mycobacteria including Mycobacterium abscessus, M. avium ssp. avium, M. bohemicum, M. chelonae ssp. chelonae, M. farcinogenes, M. flavescens, M. fortuitum ssp. fortuitum, M. gastri, M. gordonae, M. immunogenicum, M. malmoense, M. marinum, M. montefiorense, M. phlei, M. phocaicum, M. pseudoshottsii, M. salmoniphilum, M. senegalense, M. shottsii, M. smegmatis, M. szulgi and M. wolinskyi. A detection limit equivalent to 10² cfu g^?1 was registered for M. salmoniphilum‐infected fish tissue. The IHC precisely localized both free and intracellular mycobacteria in tissues and detected mycobacterial infections down to 10² cfu g^?1 tissue. Both assays were found to be more sensitive than Ziehl–Neelsen (ZN) staining, where the detection limit was below 8 × 10³ cfu g^?1 tissue. Although specificity testing of the real‐time PCR against a panel of non‐Mycobacterium spp. revealed a degree of cross‐reaction against pure DNA extracted from Nocardia seriolae and Rhodococcus erythropolis, no cross‐reactions were identified (by either real‐time PCR or IHC) on testing of formalin‐fixed paraffin‐embedded (FFPE) tissues confirmed to be infected with these bacteria. The broad applicability of both assays was confirmed by analysis of FFPE tissues from a range of fish species infected with diverse Mycobacterium spp. The results indicate that both assays, alone or in combination, constitute sensitive tools for initial, rapid diagnosis of mycobacteriosis in fish. This should in turn allow rapid application of more specific studies, i.e. culture based, to identify the specific Mycobacterium sp. involved.     

Relative prevalence of Mycobacterium marinum in fish collected from aquaria and natural freshwaters in central Europe

A survey was carried out on occurrence of Mycobacterium marinum in fish kept in aquaria and those living in their natural environment. Species‐specific qPCR targeting the erp and IS2404 genes together with the conventional culture method were used. The analysis of 72 ornamental fish (n = 216 samples: gills, muscle and intestine) collected from aquaria revealed the presence of M. marinum in 30 individuals (41.7%) of whom 17 (23.6%) were later culture positive. Culture‐independent detection revealed the presence of M. marinum in 16 of 83 environmental samples (19.3%) collected in aquaria. The presence of viable M. marinum cells was later confirmed in 5 samples (6.0%). No qPCR or culture positivity was observed when 123 groundwater fish and their corresponding environmental samples (n = 142) were analysed.     

Aeromonas salmonicida infection levels in pre‐ and post‐stocked cleaner fish assessed by culture and an amended qPCR assay

Due to increasing resistance to chemical therapeutants, the use of ‘cleaner fish’ (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de‐licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A‐layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real‐time PCR (qPCR), targeting the A. salmonicida A‐layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A‐layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild‐caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre‐ and post‐stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded.     

Virulence‐associated factors in Vibrio cholerae non‐O1/non‐O139 and V. mimicus strains isolated in ornamental fish species

During recent decades, ornamental fish have proven to be one of the fastest growing categories of pets in Europe. In this framework, we evaluated both the potential pathogenic and zoonotic risks caused by 53 Vibrio cholerae non‐O1/non‐O139 and a Vibrio mimicus strain isolated from ornamental fish species mostly originating from South‐East Asia countries between 2000 and 2015 in Italy. All the strains were firstly identified at species level by biochemical, phylogenetic and mass spectrometry (matrix‐assisted laser desorption ionization time of flight) methods, and then studied to reveal the presence of the main virulence and colonization‐associated factors, as ctxA, ace, zot, stn/sto, toxR, rtxA, hlyA and tcpA by multiplex and single endpoint PCR assays. Findings showed that 21 of 54 strains harboured at least one virulence factor with a predominance for the toxR⁺, rtxA⁺ and hlyAET⁺ genotype. Interestingly, the V. mimicus strain harboured the colonization factor and the CTX prophage receptor, tcpA, indicating the ability to capture and integrate it in its genome increasing its pathogenicity. Although these enterotoxins can sporadically cause gastroenteritis, the results highlight their probable involvement in causing severe implications for public health, suggesting the need for an European microbiological monitoring.     

Distribution of mycobacteria in clinically healthy ornamental fish and their aquarium environment

Some mycobacterial species (particularly Mycobacterium marinum) found in aquarium environments may cause chronic diseases in fish and cutaneous infections in humans, the so-called 'fish tank granuloma'. The presence and distribution of mycobacterial species in clinically healthy aquarium fish and their environment has not been adequately explored. The present study analysed the occurrence of mycobacteria in a decorative aquarium (Brno, South Moravia) and in five aquaria of a professional fish breeder (Bohumin, North Moravia). After Ziehl-Neelsen staining, acid-fast rods (AFR) were observed in six (14.3%) and mycobacteria were detected by culture in 18 (42.9%) of 42 tissue samples from 19 fish. Sixty-five samples of the aqueous environment from all six aquaria were examined; AFR were found in 16 (24.6%) and mycobacteria were detected by culture in 49 (75.4%) samples. Forty-one (70.7%) of 58 selected mycobacterial isolates were identified biochemically as follows: M. fortuitum, M. flavescens, M. chelonae, M. gordonae, M. terrae, M. triviale, M. diernhoferi, M. celatum, M. kansasii and M. intracellulare. The clinically important species for humans and fish, M. marinum, was not detected. Mycobacterium kansasii was isolated from one sample of the aquarium environment from North Moravia, which is a region of the Czech Republic with endemic incidence of M. kansasii in water. The incidence of other conditionally pathogenic mycobacterial species in healthy fish and in all investigated constituents of the aquarium environment including snails and crustaceans used for fish feeding, was quite high. Accordingly, mycobacterial species from aquarium environments may serve as a possible source of infection for both aquarium fish and immunodeficient fish handlers.     

Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)₃] and Group IV [with Al(OH)₃ and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.     

Chemical,bioactive properties and in vitro digestibility of spray‐dried fish silages: Comparison of two discard fish (Equulites klunzingeri and Carassius gibelio) silages

The fermented fish silages produced with Streptococcus spp., Lactobacillus brevis, Lactobacillus plantarum, Pediococcus acidilactici and Enterococcus gallinarum, and formic acid silages were compared for production of two discard fish silages (Equulites klunzingeri and Carassius gibelio). The E/NE ratio of spray‐dried fish silages was determined in range of 0.80–1.10 for E. klunzingeri and 0.80–0.90 for C. gibelio silages. Pediococcus acidilactici and En. gallinarum groups had greater antioxidant activity than other silage groups. The DPPH radical scavenging ability was found as 6.14%–14.71% and 6.99%–13.36% for E. klunzingeri and C. gibelio silages, respectively. The OMD, ME and NE_L values were determined in range of 69.74%–80.08%, 6.38–8.65 MJ/kg DM and 6.45–7.49 MJ/kg DM, respectively for spray‐dried E. klunzingeri silages and 81.18%–86.62%, 8.97–9.61 MJ/kg DM and 7.61–8.08 MJ/kg DM, respectively, for spray‐dried C. gibelio silages. According to the nutritional and chemical evaluation, spray‐dried fish silages have great potential as a feed components because of high rate of digestibility and nutritious components.     

Co‐infection of rainbow trout (Oncorhynchus mykiss) with infectious hematopoietic necrosis virus and Flavobacterium psychrophilum

Co‐infection of rainbow trout with infections haematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum is known to occur, and it has been speculated that a combined infection can result in dramatic losses. Both pathogens can persist in fish in an asymptomatic carrier state, but the impact of co‐infection has not been well characterized or documented. In this study, it was hypothesized that fish co‐infected with F. psychrophilum and IHNV would exhibit greater mortality than fish infected with either pathogen alone. To test this, juvenile rainbow trout were co‐infected with low doses of either IHNV or F. psychrophilum, and at 2 days post‐initial challenge, they were given a low dose of the reciprocal pathogen. This combined infection caused high mortality (76.2%–100%), while mortality from a single pathogen infection with the same respective dose was low (5%–20%). The onset of mortality was earlier in the co‐infected group (3–4 days) when compared with fish infected with F. psychrophilum alone (6 days) or IHNV (5 days), confirming the synergistic interaction between both pathogens. Co‐infection led to a significant increase in the number of F. psychrophilum colony‐forming units and IHNV plaque‐forming units within tissues. This finding confirms that when present together in co‐infected fish, both pathogens are more efficiently recovered from tissues. Furthermore, pathogen genes were significantly increased in co‐infected groups, which parallel the findings of increased systemic pathogen load. Extensive tissue necrosis and abundant pathogen present intracellularly and extracellularly in haematopoietic tissue. This was pronounced in co‐infected fish and likely contributed to the exacerbated clinical signs and higher mortality. This study provides novel insight into host–pathogen interactions related to co‐infection by aquatic bacterial and viral pathogens and supports our hypothesis. Such findings confirm that mortality in fish exposed to both pathogens is greatly elevated compared to a single pathogen infection.     

Occurrence of Mycobacterium spp. in ornamental fish in Italy

The occurrence of Mycobacterium spp. in freshwater and marine ornamental fish was studied in Italy from June 2002 to May 2005. Two surveys were carried out, one of aquarium fish sent to the Laboratory for diagnosis, and the other of prevalence of infection by mycobacteria in ornamental fish imported into Italy. Bacterial isolation was carried out from the spleen, kidney and liver, and the isolates were subsequently identified by biochemical tests. In the first survey, 387 fish were examined and Mycobacterium spp. were isolated from 181 (46.8%) fish. In the second survey 127 batches of ornamental fish from different countries were examined. Mycobacterium spp. were isolated from 38 (29.9%) batches. The following species were found: M. fortuitum, M. peregrinum, M. chelonae, M. abscessus, M. marinum, M. gordonae, M. nonchromogenicum and M. interjectum. There was a high prevalence of infection independent of the presence of macroscopic lesions. Mycobacterium fortuitum and M. chelonae were more prevalent than M. marinum in the samples examined.     

Limited performance of MALDI‐TOF for identification of fish Aeromonas isolates at species level

The aim of this study was to evaluate the usefulness of the MALDI‐TOF MS to identify 151 isolates of Aeromonas obtained mostly from diseased fish. MALDI‐TOF MS correctly identified all isolates to the genus level but important differences in the percentage of isolates correctly identified depending on the species were observed. Considering exclusively the first identification option, Aeromonas bestiarum, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii and Aeromonas sobria were the best identified with results >95%. However, considering the first and second identification options, the only species that showed values >90% was A. hydrophila. Overall, when the database was supplemented with 14 new spectra, the number of accurate identifications increased (41% vs. 55%) and the number of inconclusive identifications decreased (45% vs. 29%), but great differences in the success of species‐level identifications were found. Species‐distinctive mass peaks were identified only for A. hydrophila and A. bestiarum (5003 and 7360 m/z in 95.5% and 94.1% of their isolates, respectively). This work demonstrates the utility of MALDI‐TOF MS for Aeromonas identification to the genus level, but there is no consistency for the accurate identification of some of the most prevalent species implicated in fish disease.