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.     

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.     

Mycobacteriosis caused by Mycobacterium marinum in reared mullets: first evidence from Sardinia (Italy)

Mycobacterium marinum is a slow‐growing non‐tuberculous mycobacterium, and it is considered the most common aetiologic agent of mycobacteriosis in wild and cultured fish. The diagnosis is principally made by histology when positive Ziehl–Neelsen stain granulomas are detected. The aim of this study was to investigate the occurrence of mycobacteriosis in extensively cultured Mugilidae of two lagoons (Cabras and San Teodoro) from Sardinia by the use of histology, microbiology, PCR and DNA sequencing. Nine of 106 mullets examined were affected by mycobacteriosis, and the spleen was the most affected organ. The histology detected higher rate (100%) of infection in spleen than the culture and PCR (75% and 62.5%, respectively). The sequencing of hsp65 gene identified M. marinum as the primary cause of mycobacteriosis in the mullets examined. Mullets affected by mycobacteriosis were mainly fished in the San Teodoro lagoon characterized by critical environmental conditions. Histology remains the most common method in detecting fish affected by mycobacteriosis, and PCR‐based methods are essential for species identification. Our finding are worthy of attention because mycobacteriosis caused by M. marinum in reared mullets was evidenced for the first time in Sardinia, suggesting that this disease may be underestimated also in other cultured fish species.     

A sensitive FRET probe assay for the selective detection of Mycobacterium marinum in fish

Mycobacterium marinum is the causative agent of mycobacteriosis in wild and cultured fish and of atypical infection in humans. For the diagnosis of M. marinum , cultural and traditional polymerase chain reaction (PCR) methods are currently used. However, these protocols, although able to discriminate within Mycobacterium spp., have proved to be time-consuming or difficult to carry out. For this reason, the aim of this study was to obtain a rapid and specific diagnostic tool to quantify fish Mycobacterium spp. or to discriminate M. marinum from other mycobacteria. A primary PCR amplification with SYBR Green had a detection limit (dl) of 10² Mycobacterium DNA copies with a log-linear quantification range up to 10⁴ ( R ² = 0.99). The second PCR using FRET probes, flanking a region containing species specific nucleotide variations, was designed and validated with synthetic erp gene fragments corresponding to different mycobacterial species, different whole mycobacteria suspensions, experimentally infected fish tissues, tissues from experimentally infected fish, and samples of cultured fish. The results show that the FRET probes demonstrate a high specificity as the melting curve analysis allowed efficient discrimination of M. marinum from Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium pseudoshottsii , Mycobacterium shottsii and Mycobacterium ulcerans . The kidney is the organ with the strongest detection signal and using fish tissues the method has a mean sensitivity of 50 DNA copies/PCR.     

Mycobacteria in aquarium fish: results of a 3‐year survey indicate caution required in handling pet‐shop fish

Fish are commonly infected with non‐tuberculous mycobacteria (NTM), which should be regarded as potential pathogens when handling aquarium fish and equipment. This study examined 107 aquarium fish from pet shops. Cultivation of the fish samples using different selective media was conducted for identification of NTM. Isolates were identified using the GenoType Mycobacterium common mycobacteria and additional species assays, sequencing of the 16S rRNA and rpoB genes, and real‐time PCR assay for identification of Mycobacterium (M.) marinum. Among the investigated fish, 79.4% (85/107) were positive for mycobacteria, with 8.2% (7 of 85) having two mycobacterial species present. Among the positive fish, the common pathogens M. marinum, Mycobacterium fortuitum (M. fortuitum group) and Mycobacterium chelonae were identified in approx. 90% of fish and other NTM species in 10%, including Mycobacterium peregrinum/septicum, Mycobacterium gordonae, Mycobacterium arupense, Mycobacterium kansasii, Mycobacterium ulcerans and Mycobacterium setense. The well‐known human pathogen M. marinum was present in 10.6% of the positive fish (9 of 85). The species of mycobacteria identified in the study are not only recognized as aquarium fish pathogens, but can also cause pathology in humans. Microbiological and clinical communities should therefore be sensitized to the role of NTM in infections associated with exposure to aquarium fish.     

Experimental exposure of zebrafish, Danio rerio (Hamilton), to Mycobacterium marinum and Mycobacterium peregrinum reveals the gastrointestinal tract as the primary route of infection: a potential model for environmental mycobacterial infection

The natural route by which fish become infected with mycobacteria is unknown. Danio rerio (Hamilton) were exposed by bath immersion and intubation to Mycobacterium marinum and Mycobacterium peregrinum isolates obtained from diseased zebrafish. Exposed fish were collected over the course of 8 weeks and examined for the presence of mycobacteriosis. Mycobacteria were consistently cultured from the intestines, and often from the livers and spleens of fish exposed by both methods. Mycobacteria were not observed in the gills. Histological analysis revealed that fish infected with M. marinum often developed granulomas accompanied by clinical signs of mycobacteriosis, while infection with M. peregrinum infrequently led to clinical signs of disease. Passage of the bacteria through environmental amoebae (Acanthamoeba castellani) was associated with increased growth of M. peregrinum over the course of 8 weeks, when compared to infection with the bacteria not passed through amoebae. The results provide evidence that zebrafish acquire mycobacteria primarily through the intestinal tract, resulting in mycobacterial dissemination.     

Development of a quantitative real-time PCR for the detection of Tenacibaculum maritimum and its application to field samples

The development and the application of a quantitative real‐time PCR for the detection of Tenacibaculum maritimum are described. A set of primers and probe was designed to amplify a 155‐bp fragment specific to the T. maritimum 16S rRNA gene. The test was shown to be very sensitive, able to detect as little as 4.8 DNA copies number μL^?1. In addition, the assay was found to have a high degree of repeatability and reproducibility, with a linear dynamic range (R² = 0.999) extending over 6 log₁₀ dilutions and a high efficiency (100%). The assay was applied to DNA samples extracted from 48 formalin‐fixed paraffin‐embedded (FFPE) Atlantic salmon, Salmo salar, gill tissues showing varying degrees of gill pathology (scored 0–3) and from 26 jellyfish samples belonging to the species Phialella quadrata and Muggiaea atlantica. For each sample, the bacterial load was normalised against the level of the salmonid elongation factor alpha 1 (ELF) detected by a second real‐time PCR using previously published primers and probe. Tenacibaculum maritimum DNA was detected in 89% of the blocks with no signs of gill disease as well as in 95% of the blocks with mild‐to‐severe gill pathology. Association between bacterial load and gill pathology severity was investigated. T. maritimum DNA was detected at low level in four of the 26 jellyfish tested.     

Ocular localization of mycobacterial lesions in tank‐reared juvenile cobia,Rachycentron canadum

Severe clinical mycobacteriosis with consistent ocular lesion localization was diagnosed in a population of 800 juvenile tank‐reared Cobia (Rachycentron canadum) which experienced a sudden increase in mortality approximately 5 months after arriving into Trinidad and Tobago from Florida, USA. Moderate daily mortality (15–20 animals per day) persisted for just over 1 month. Moribund fish displayed circling behaviour and had an open‐mouth gape upon death. Fish consistently presented with bilateral exophthalmia, corneal cloudiness and hyphema. Non‐branching acid‐fast rods were detected in aqueous humour touch preparations. Histological analysis revealed severe bilateral intra‐ocular granulomatous responses in all specimens. Mycobacterium sp. was identified using a real‐time PCR assay detecting the RNA polymerase β‐subunit (rpoB) gene in different tissue samples. Specimens did not present with characteristic granulomatous responses usually seen in viscera. To the best of our knowledge, this represents only the third documentation of piscine mycobacterial infection presenting with only localized ocular lesions, and the second documented case of mycobacteriosis in cobia. It is, however, the first documentation of an ocular presentation of mycobacteriosis in a marine species and is the first documentation of such a presentation in cobia.     

Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae , two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities.     

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.     

Antibiotic treatment of zebrafish mycobacteriosis: tolerance and efficacy of treatments with tigecycline and clarithromycin

Zebrafish (Danio rerio) are a popular model organism used in a growing number of research fields. Maintaining healthy, disease‐free laboratory fish is important for the integrity of many of these studies. Mycobacteriosis is a chronic bacterial infection caused by several Mycobacterium spp. and is the second most common disease found in laboratory zebrafish. Current mycobacteriosis control measures recommend the removal of infected fish and in severe outbreaks, depopulation. These measures can be effective, but less disruptive measures should be assessed for controlling mycobacteriosis, particularly when valuable and rare lines of fish are affected. Here, the in vivo efficacy of two drug candidates, tigecycline (1 μg g^?1) and clarithromycin (4 μg g^?1), was tested in adult zebrafish experimentally infected with Mycobacterium chelonae. We assessed both short (14 day)‐ and long‐term (30 day) treatments and evaluated fecundity and pathological endpoints. Fecundity and histology results show that zebrafish tolerated antibiotics. Antibiotic treatments did not significantly impact the prevalence of acid‐fast granulomas; however, the severity of infections (acid‐fast granuloma intensity) was significantly decreased following treatments.     

Identification of Mycobacterium spp. isolated from snakehead, Channa striata (Fowler), and Siamese fighting fish, Betta splendens (Regan), using polymerase chain reaction–reverse cross blot hybridization (PCR–RCBH)

A variety of methods have been used to identify Mycobacterium spp. isolated from snakehead and Siamese fighting fish, including biochemistry, mycolic acid profiles and antibody-based methods. However, these methods are unable to differentiate between different species of Mycobacterium . Polymerase chain reaction (PCR) followed by reverse cross blot hybridization (RCBH) was adapted in this study to speciate aquatic mycobacteria. The method was highly specific for Mycobacterium spp. and identified the bacteria to species level with a detection limit of 100 fg DNA, equivalent to 20 mycobacteria. Twenty-nine isolates previously collected and cultured from Siamese fighting fish (10 isolates) and snakehead (19 isolates) during outbreaks of mycobacteriosis were analysed using PCR–RCBH. Six of the Siamese fighting fish isolates and nine of the snakehead isolates were identified as Mycobacterium fortuitum , while the remainder were classified as M. marinum . Notably, two isolates recovered from snakehead and Siamese fighting fish, previously identified as M. poriferae and M. piscicida , respectively, were confirmed to be M. fortuitum .     

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.     

High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10⁰ amplicon copies per assay (equivalent to 2 × 10^?11 ng/µl, C_q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 10⁰ gene copies in tissues artificially infected and 0.02 × 10⁰ in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.     

A comparative molecular study of the presence of “Candidatus arthromitus” in the digestive system of rainbow trout,Oncorhynchus mykiss (Walbaum), healthy and affected with rainbow trout gastroenteritis

Observations were made using histopathological techniques in conjunction with a nested polymerase chain reaction (PCR) protocol for the specific detection of “Candidatus arthromitus” on DNA extracted from wax‐embedded tissues and fresh digestive contents of rainbow trout. Samples positive for “Candidatus arthromitus” DNA included fish with rainbow trout gastroenteritis (RTGE), clinically normal cohabiting fish, and apparently healthy controls from RTGE positive and RTGE negative sites. The results obtained from the PCR were confirmed by nucleotide sequencing. “Candidatus arthromitus” DNA was found in distal intestine as well as in sections of pyloric caeca, suggesting that both these locations are appropriate for molecular detection of “Candidatus arthromitus” DNA in trout. Furthermore, rainbow trout fry distal intestinal samples from two different hatcheries where RTGE had not been reported were also positive. Differences in “Candidatus arthromitus” DNA detection between paraffin wax‐embedded and fresh digestive content samples from the same fish suggested that it may be predominantly epithelium‐associated in healthy trout. Parallel histopathological observations indicated that pyloric caeca are the preferred site for visualizing segmented filamentous bacteria (SFB) in trout with RTGE. The results of this study showed that the presence of SFB was not invariably associated with clinical disease and that more information is required to understand the role of these organisms.     

Detection of Neoparamoeba perurans by duplex quantitative Taqman real-time PCR in formalin-fixed, paraffin-embedded Atlantic salmonid gill tissues

The development and the application of a quantitative duplex real‐time PCR for the detection of Neoparamoeba perurans and the elongation factor α 1 gene (ELF) of Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), are described. A set of primers and probe was designed to amplify a 139‐bp fragment specific to the N. perurans 18S rRNA gene. The test was shown to be very sensitive, being able to detect as little as 13.4 DNA copies per μL corresponding to 0.15 fg of template DNA. In addition, the reaction that detected N. perurans was found to have a high degree of repeatability and reproducibility, to have a linear dynamic range (R² = 0.999) extending over 5 log₁₀ dilutions and to have a high efficiency (104%). The assay was applied to DNA samples extracted from 48 formalin‐fixed, paraffin‐embedded (FFPE) salmon gill tissues showing varying degrees of gill histopathology and amoebic gill disease (AGD)‐type histopathology ranging from absent to severe (each scored 0–3). Neoparamoeba perurans DNA was detected in all the blocks where AGD‐type histopathology was diagnosed microscopically and in 43.6% of the blocks showing signs of gill pathology. The association between parasitic load and gill histopathology and AGD‐type histopathology severity was also investigated. This study also describes the development and the application of a second real‐time PCR for the generic detection of Neoparamoeba spp., Page, 1987. A set of primers and probe conserved among the Neoparamoeba spp. was designed to amplify a 150‐bp fragment within the 18S rRNA gene. Applied to N. perurans‐negative gill tissues, the method was used to exclude the presence of other Neoparamoeba spp. in those blocks where gill pathology was observed microscopically.     

Granulomatous lesions in a wild mullet population from the eastern Ligurian Sea (Italy): mycobacteriosis vs. pseudotuberculosis

Mycobacterium spp. and Photobacterium damselae subsp. piscicida are recognized as the most frequent causative agents of granulomatous lesions in fish. Although frequent episodes of mycobacterial infections have been reported in wild fish worldwide, only sporadic cases have been documented to date in Italy. To investigate for the presence of lesions referable to mycobacteriosis and to identify the mycobacterial species involved, a total of 159 wild mullets were fished from the eastern coast of the Ligurian Sea, killed and necropsied. Liver and spleen samples were collected from all fish for histopathological and microbiological analyses. Molecular investigations for identification of Photobacterium damselae subsp. piscicida were performed. Gross examination revealed granulomatous lesions in one animal; microscopically, 42.14% of fish displayed granulomas with various histological features, 19.50% resulted positive at Ziehl–Neelsen staining, and were confirmed as mycobacterial lesions by culture. The identified colonies were characterized as M. fortuitum, M. abscessus, M. flavescens, M. chelonae, M. septicum and M. nonchromogenicum. In all, 35% of animals resulted positive for Photobacterium damselae subsp. piscicida. These data suggest widespread mycobacterial infection also by Photobacterium damselae subsp. piscicida infections in wild fish. Moreover, the pathogenicity of some mycobacterial species, previously considered as saprophytic, was demonstrated.     

Immunohistochemical diagnosis of tenacibaculosis in paraffin‐embedded tissues of Senegalese sole Solea senegalensis Kaup, 1858

A sensitive and specific immunohistochemical technique was developed to improve the diagnosis of tenacibaculosis and to better understand its pathogenesis. Senegalese sole Solea senegalensis Kaup, 1858 were inoculated subcutaneously with a bacterial suspension of Tenacibaculum maritimum, and samples were taken at different hours post‐inoculation. Sections from different organs were used as positive controls. In addition, a total of 128 field samples from different organs collected from tenacibaculosis outbreaks were used. Tenacibaculum maritimum antigens were detected in several organs of experimentally infected Senegalese sole and in at least one of the tissues from fish suffering from natural tenacibaculosis previously confirmed by culture and PCR‐based methods. In fish collected during outbreaks, a strong positive reaction was detected in ulcerative skin areas. Moreover, bacterial antigen was identified inside scale pockets and in sites of the skin with mild lesion. In kidney and spleen, evident immunostaining of bacterial antigen was detected in both naturally and experimentally infected fish. Besides, the presence of T. maritimum in the intestinal tract without associated histological changes suggests that this organ may act as a reservoir for T. maritimum. The results of this study confirm the usefulness of IHC for the diagnosis of tenacibaculosis in paraffin‐embedded tissues.