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1.
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.  相似文献   

2.
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3.
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.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout syndrome in freshwater salmonid fish worldwide, generating injuries and high mortality rates. Despite several studies on this bacterium, the infection mechanism remains unknown due to limitations in the employed animal models. In this work, we propose using zebrafish (Danio rerio) as a model for studying bacterial pathogenicity. To substantiate this proposal, zebrafish infection by F. psychrophilum strain JIP 02/86 was characterized. Zebrafish larvae were infected using the bath method, and morphological changes and innate immune system activation were monitored using transgenic fish. Salmonid-like infection phenotypes were observed in 4.74% of treated larvae, as manifested by fin, muscle and caudal peduncle damage. Symptomatic and dead larvae accounted for 1.35% of all challenged larvae. Interestingly, infected larvae with no infection phenotypes showed stronger innate immune system activation than specimens with phenotypes. A failure of function assay for myeloid factor pu.1 resulted in more infected larvae (up to 43.5%), suggesting that low infection rates by F. psychrophilum would be due to the protective actions of the innate immune system against this bacterium in zebrafish larvae. Our results support the use of zebrafish as an infection model for studying F. psychrophilum. Furthermore, the percentage of infected fish can be modulated by disturbing, to varying extents, the differentiation of myeloid cells. Using this evidence as a starting point, different aspects of the infection mechanism of F. psychrophilum could be studied in vivo.  相似文献   

12.
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.  相似文献   

13.
Fish iridovirus causes systemic disease with high morbidity and mortality in various species of wild and farm‐raised fish, resulting in severe economic losses. Recently, frequent outbreaks of iridovirus infection have occurred among cultured fish in many Asian countries, emphasizing the need for a protective vaccine programme or the development of a suitable therapy. In this study, we expressed a recombinant major capsid protein (rMCP) of rock bream iridovirus (RBIV) from yeast using codon optimization. The rMCP in yeast was added to feed in an attempt to induce intestinal mucosal immunity for protection against and/or to reduce the severity of fish iridovirus infection. We found that fish immunized orally with rMCP underwent a successful induction of antibodies (P < 0.05) and were protected (P = 0.0001) against viral challenge. Based upon these results, oral administration of immunogenic protein as an antigen can be considered a useful method for implementation of vaccine programmes against iridovirus as well as other marine viral diseases.  相似文献   

14.
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.  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
Weissella ceti is an emerging bacterial pathogen that affects rainbow trout, Oncorhynchus mykiss (Walbaum), farms. The aims of this study were to genotype W. ceti strains isolated from distinct geographical origins and to determine the efficacy of an oil-adjuvanted vaccine against the disease. Between 2010 and 2012, outbreaks were recorded in five Brazilian farms, and 34 W. ceti isolates were genetically characterized by repetitive extragenic palindromic PCR, enterobacterial repetitive intergenic consensus sequences PCR and pulsed-field gel electrophoresis. Two different W. ceti vaccines were tested: an aqueous-based whole-cell inactivated vaccine (bacterin) and oil-adjuvanted vaccine. Their efficacy was evaluated in rainbow trout at 30 and 60 days post-vaccination (d.p.v.). W. ceti was found to be a highly homogeneous population in Brazil, with clonally related genotypes. Oil-adjuvanted vaccine exhibited the best (P < 0.05) protection against disease, reaching relative percentage survival (RPS)values of 92% at 30 and 60 d.p.v. Bacterin resulted in RPS values of 67% and 58% at day 30 and 60, respectively. The oil-adjuvanted vaccine provided effective protection against W. ceti infection in rainbow trout.  相似文献   

20.
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.  相似文献   

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