Advanced
Molecular and Histopathological Evidence of Mycobacteriosis in Paradise Fish <italic>Macropodus opercularis</italic> Imported into Korea
Molecular and Histopathological Evidence of Mycobacteriosis in Paradise Fish Macropodus opercularis Imported into Korea
Fisheries and aquatic sciences. 2013. Sep, 16(3): 165-169
Copyright ©2013, The Korean Society of Fisheries and Aquatic Science
This is an Open Access article distributed under the terms of the CreativeCommons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use,distribution, and reproduction in any medium, provided the original workis properly cited.
  • Received : February 02, 2013
  • Accepted : June 06, 2013
  • Published : September 30, 2013
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Hyun-Ja Han
Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Korea
Jeong-Ho Kim
Faculty of Marine Bioscience and Technology, Gangneung-Wonju National University, Gangneung 210-702, Korea
Chan-Hyeok Jeon
Faculty of Marine Bioscience and Technology, Gangneung-Wonju National University, Gangneung 210-702, Korea
Wi-Sik Kim
The Fisheries Science Institute, Chonnam National University, Yeosu 550-901, Korea
Do-Hyung Kim
Present address: Aquatic Life Disease Control Division, Fundamental Research Department, National Fisheries Research and Development Institute, Busan 619-705, Korea
Sung-Ju Jung
Present address: Aquatic Life Disease Control Division, Fundamental Research Department, National Fisheries Research and Development Institute, Busan 619-705, Korea
Myung-Joo Oh
Present address: Aquatic Life Disease Control Division, Fundamental Research Department, National Fisheries Research and Development Institute, Busan 619-705, Korea
ohmj@chonnam.ac.kr
Abstract
We report on mycobacteriosis in an imported tropical ornamental fish Macropodus opercularis commonly known as the paradise fish. Mass mortality occurred in paradise fish imported to Korea from Southeast Asia in 2008. The affected fish did not show any outward clinical signs, but enlargement of the spleen, kidneys, and liver was observed on dissection. Histopathological examination revealed numerous granulomas in the spleen, and acid-fast bacilli were observed in the centers of the granulomas. About 65% of spleen DNA samples were PCR positive using mycobacteria-specific primers targeting the 16S rRNA and hsp65 genes. The nucleotide identities of the 16S rRNA and hsp65 genes with those of Mycobacterium marinum were 99.5% and 99.4%, respectively. Although the bacterium was not cultured, the molecular diagnosis and histopathological findings were consistent with mycobacteriosis in paradise fish.
Keywords
Introduction
Mycobacteria are pleomorphic, aerobic, Gram-positive, acid-fast, non-motile rods, 0.2-0.6 μm in diameter and 1-10 μ m long in the genus Mycobacterium , the only genus in the family Mycobacteriaceae (Gauthier and Rhodes, 2009). The Mycobacterium comprises obligate pathogens that cause serious human and animal diseases, opportunistic pathogens, and saprophytic species (Bland et al., 2005).
Piscine mycobacteriosis is a systemic infectious disease that is typically a subacute to chronically progressive condition caused by different Mycobacterium species, including Mycobacterium marinum , M. fortuitum , M. chelonae , and M. abscessus (Kusunoki and Ezaki, 1992; Decostere et al., 2004). More recently, additional mycobacterial species have been implicated as the causes of mycobacterial infections, including M. shottsii , M. montefiorense , and M. haemophilum (Levi et al., 2003; Rhodes et al., 2003; Whipps et al., 2007). Although mycobacteriosis is usually a chronic disease and may not produce external lesions, the internal lesions typically include enlargement and typical grey or white nodules of the spleen, kidneys, and liver (Chinabut, 1999; Gauthier and Rhodes, 2009).
This study reports on mycobacteriosis in an imported tropical ornamental fish, Macropodus opercularis , commonly known as the paradise fish.
Materials and Methods
- Disease outbreak and fish sampling
Forty paradise fish (body weight, 1.8 ± 0.4 g) were bought from an ornamental fish wholesaler in Korea in June 2008. After 2 days of acclimation, the first fish death was recorded, and all of the fish died by July (cumulative mortality rate, approximately 100% per month). Few of the diseased fish showed any external signs, but enlargement of the spleen and discoloration of the liver were observed after postmortem examination. Moribund and freshly killed fish were examined using histopathology and microbial cultures after euthanasia with excessive 2-phenoxyethanol (Sigma, St Louis, MO, USA). The gill and body surfaces were examined microscopically for the presence of parasites.
- Histopathology
The spleen was collected from five moribund fish and fixed immediately in 10% neutral buffered formalin. After fixation, standard histological procedures were used for tissue dehydration and paraffin embedding. Tissue sections were stained with hematoxylin and eosin (H&E) and Ziehl-Neelsen (ZN).
- Bacterial examination
The liver, kidney, and spleen of moribund and freshly killed fish were inoculated into tryptone soy agar (TSA; Difco, Sparks, MD, USA) and incubated at 30℃ for 72 h. Frozen kidney and spleen tissues were homogenized in sterile 0.85% saline (w/v), decontaminated with 0.35% hexadecylpyridinium chloride for 30 min, and then centrifuged at 2,800 g for 30 min (Corner and Trajstman, 1988). The supernatant was removed, and the sediment was washed twice with 1 mL of sterile phosphate-buffered saline (PBS). The sediments were inoculated on Middlebrook 7H10 (Difco), and the medium was incubated aerobically at 30℃ and checked daily for 2 months. Before decontamination, samples were also cultured in TSA (Difco) to detect common bacterial fish pathogens and incubated for 72 h at 30℃.
- Molecular diagnosis
For PCR, genomic DNA was extracted from frozen spleen stored at –80℃ using an AccuPrep Genomic DNA purification kit (Bioneer, Daejeon, Korea). Molecular detection of Mycobacterium species was based on 16S ribosomal DNA (16S rRNA) and 65-kDa heat-shock protein-encoding gene ( hsp65 ) sequences. The primers for the 16S rRNA (T39 and T13; PCR product size, 924 bp) (Talaat et al., 1997) and hsp65 (Tb11 and Tb12; PCR product size, 441 bp) (Aranaz et al., 2008) genes were used for amplification and sequencing. The DNA was amplified with an initial 5-min denaturation at 95℃, followed 166by 35 cycles of 94℃ for 30 s, 55℃ (for 16S rRNA) or 58℃ (for hsp65 ) for 30 s, and 72℃ for 1 min, with a final 5-min extension at 72℃. The sequencing reactions were carried out with a BigDye terminator ver.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA), and the products were sequenced on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). 16S rRNA and hsp65 DNA sequences for Mycobacterium species available from GenBank plus our sequences were aligned using ClustalX ver. 1.83 (Thompson et al., 1997). Phylogenetic trees were obtained using the neighbor-joining method with Kimura’s two-parameter distance correction model with 1,000 bootstrap replications using MEGA ver. 4 (Tamura et al., 2007).
Results
- Histopathology
Granulomas 20-57 μm in diameter were observed in the H&E-stained spleen sections of all individuals examined ( Fig. 1 A). Numerous granulomas appeared in the splenic pulp and sheathed tissue ( Fig. 1 A and 1 B). Severe necrosis was observed in the central eosinophilic area of the granulomas, surrounded by extensive fibrous infiltration containing several layers of epithelioid cells ( Fig. 1 B). When stained with ZN, numerous acid-fast bacilli were found in the centers of the granulomas in the spleen ( Fig. 2 ).
- Bacterial examination
Two fast-growing bacteria, Citrobacter freundii and Pseudomonas sp., grew on TSA, but they were isolated from one fish only. Histopathological examination led us to suspect mycobacteriosis, so we attempted bacterial isolation using Middlebrook 7H10 from frozen paradise fish tissue. However, no mycobacterial growth was observed after incubation for 2 months.
- Molecular diagnosis
Thirteen (62%) and 14 (67%) of 21 fish were positive for mycobacteriosis, determined by PCR targeting the 16S rRNA and hsp65 genes, respectively. All of the samples positive for the 16S rRNA gene were also positive for the hsp65 gene. Analysis of the 16S rRNA (838-bp product) and hsp65 (380-bp product) DNA sequences revealed the greatest similarity with those of Mycobacterium marinum retrieved from the NCBI database (GenBank accession nos. AF456239 [16S rRNA] and AF456471 [ hsp65 ]). Phylogenetic trees based on the 16S rRNA and hsp65 genes showed that the strains isolated in this study were related most closely to M. marinum ( Fig. 3 ). The nucleotide identities of the 16S rRNA and hsp65 genes with those of M. marinum ( M. shottsii ) were 99.5% (99.4%)
PPT Slide
Lager Image
Histopathology of mycobacteriosis in Macropodus opercularis. (A) Numerous granulomas are present in spleen of diseased fish. G, granulomas. (B) Higher magnification of a square in (A) showing granuloma morphology. Fibrosis (FI) of the granulation tissue containing epithelioid cells. N, necrotic core; L, lymphocyte. Haematoxylin-eosin stain. Scale bars: A = 200 μm, B = 50 μm.
PPT Slide
Lager Image
Mycobacteria infecting Macropodus opercularis. Ziehl-Neelsen stain of a section from spleen of diseased fish. Numerous acid-fast bacilli are present in the center of granuloma. Scale bar = 10 μm.
and 99.4% (99.1%), respectively.
Discussion
Several Mycobacterium species have been identified as ornamental fish pathogens (Lescenko et al., 2003). The paradise fish is also susceptible to Mycobacterium infection. For example, Gómez (2008) found granulomatous inflammation and numerous acid-fast bacteria in tissue sections of granulomas from a diseased paradise fish, but the causative agent was not confirmed at the species level. Additionally, Marcì et al. (2008) reported M. fortuitum infection in severely emaciated paradise fish, with ascites and granulomas of various sizes in the internal organs. In our study, the 16S rRNA and hsp65 gene analysis show that the amplified genes were related most closely to M. marinum . However, it is difficult to distinguish among M. marinum , M. shottsii , M. pseudoshottsii , and M. ulcerans using 16S rRNA and hsp65 gene sequences (Adekambi and Drancourt, 2004). Additional research to identify the Mycobacterium sp. infecting the paradise fish is needed.
We could not culture Mycobacterium sp. from paradise fish, as freezing the spleens and kidneys affected the viability of the mycobacteria, resulting in negative cultures on Middlebrook 7H10. Although bacterial isolation is considered the gold standard for diagnosing bacterial disease, the isolation of mycobacteria from field samples is generally problematic and sometimes not even attempted because mycobacteria are slow-growing fastidious organisms, and isolation can be adversely affected by other microbial contamination (Pate et al., 2005). Instead, mycobacteria have been detected by amplifying various target genes, such as 16S rRNA, IS 986 , IS 611 , and hsp65 (Kolk et al., 1992; Telenti et al., 1993; Kox et al., 1995; Noordhoek et al., 1995), and PCR has been used to detect mycobacterial infection in several studies (Frevel et al., 1999; Astrofsky et al., 2000). In our study, PCR amplification targeting the 16S rRNA and hsp65 genes was carried out, and more than 60% of the examined fish showed positive reactions for both genes. However, the sensitivities of the 16S rRNA and hsp65 PCR primers may differ, and the hsp65 PCR primers may have been more sensitive than the 16S rRNA PCR primers in this study.
Although mycobacteriosis was confirmed in imported paradise fish, the etiological agent of the mass mortality is unclear, as Mycobacterium sp. was detected only in about 65% of the fish by PCR, and megalocytivirus (10%, four of 40 fishes) and reovirus (30%, three of 10 fishes) were also detected in the diseased paradise fish by PCR (data not shown). Further studies of the pathogenicity in paradise fish are necessary. However, our results suggest that mass mortality in paradise fish was caused by multiple infections with Mycobacterium sp., megalocytivirus, and reovirus.
Over 1 billion ornamental fish are traded internationally each year (Whittington and Chong, 2007). Paradise fish are
PPT Slide
Lager Image
Neighbor-joining analysis of Mycobacterium species 16S rRNA (A) and hsp65 (B) sequences from GenBank.
small freshwater labyrinth fish found in ditches and paddy fields in Southeast Asia (Man and Hodgkiss, 1981). Paradise fish are imported into Korea from Indonesia, Malaysia, and Vietnam (National Fisheries Products Quality Inspection Service, personal communication), but there are no clear guidelines or checks on paradise fish suspected of having mycobacteriosis. This is made more difficult given the lack of obvious signs of sickness in diseased fish. In this study, it is likely that the paradise fish monitored were infected before importation to Korea, as mortality occurred during the acclimation period, and mycobacteriosis is generally a chronic, slowly developing disease.
In conclusion, we confirmed the presence of mycobacteriosis in paradise fish imported from Southeast Asia into Korea. Considering the broad host spectrum of Mycobacterium sp. and increasing global international trade in ornamental fish, stricter quarantine inspection guidelines are necessary to reduce the risk of mycobacteriosis in fish in Korea.
Acknowledgements
The current work was supported by the Technology Development Program for Fisheries, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
References
Adékambi T , Drancourt M 2004 Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int J Syst Evol Microbiol 54 2095 - 2105
Aranaz A , Gibello A , Álvarez J , Mata AI , Rodríguez A , Fallola C , Fernández-Garayzábal JF , Domínguez L 2008 Mycobacteriumperegrinum infection in farmed European tench (Tinca tincaL.). Vet Microbiol 131 393 - 399
Astrofsky KM , Schrenzel MD , Bullis RA , Smolowitz RM , Fox JG 2000 Diagnosis and management of atypical Mycobacterium spp.infections in established laboratory zebrafish (Brachydanio rerio)facilities. Comp Med 50 666 - 672
Bland CS , Ireland JM , Lozano M , Alvarez ME , Primm TP. 2005 Mycobacterial ecology of the Rio Grande. Appl Environ Microbiol 71 5719 - 5727
Chinabut S 1999 Mycobacteriosis and nocardiosis. In: Fish Diseasesand Disorders. Vol. 3. Viral, Bacterial and Fungal Infections. WooPTK and Bruno DW, eds. CABI Publishing Wallingford, GB 319 - 340
Corner LA , Trajstman AC 1988 An evaluation of 1-hexadecylpyridiniumchloride as decontaminant in the primary isolation ofMycobacterium bovis from bovine lesions. Vet Microbiol 18 127 - 134
Decostere A , Hermans K , Haesebrouck F 2004 Piscine mycobacteriosis:a literature review covering the agent and the diseaseit causes in fish and humans. Vet Microbiol 99 159 - 166
Frevel T , Schäfer KL , Tötsch M , Böcker W , Dockhorn-Dworniczak B 1999 PCR based detection of mycobacteria in paraffinwax embedded material routinely processed for morphologicalexamination. Mol Pathol 52 283 - 288
Gauthier DT , Rhodes MW 2009 Mycobacteriosis in fishes: a review. Vet J 180 33 - 47
Gómez S 2008 Prevalence of microscopic tubercular lesions infreshwater ornamental fish exhibiting clinical signs of non-specificchronic disease. Dis Aquat Org 80 167 - 171
Kolk AHJ , Schuitema ARJ , Kuijper S , van Leeuwen J , Hermans PWM , van Embden JDA , Hartskeerl RA 1992 Detection of Mycobacteriumtuberculosis in clinical samples by using polymerasechain reaction and a nonradioactive detection system. J Clin Microbiol 30 2567 - 2575
Kox LFF , van Leeuwen J , Kuijper S , Jansen HM , Kolk AHJ 1995 PCR assay based on DNA coding for 16S rRNA for detection andidentification of mycobacteria in clinical samples. J Clin Microbiol 33 3225 - 3233
Kusunoki S , Ezaki T 1992 Proposal of Mycobacterium peregrinumsp. nov., nom. rev., and elevation of Mycobacterium chelonaesubsp. abscessus (Kubica et al.) to species status: Mycobacteriumabscessus comb. nov. Int J Syst Evol Microbiol 42 240 - 245
Lescenko P , Matlova L , Dvorska L , Bartos M , Vavra O , Navratil S , Novotny L , Pavlik I 2003 Mycobacterial infection in aquariumfish. Vet Med 48 71 - 78
Levi MH , Bartell J , Gandolfo L , Smole SC , Costa SF , Weiss LM , Johnson LK , Osterhout G , Herbst LH 2003 Characterization ofMycobacterium montefiorense sp. nov., a novel pathogenic Mycobacteriumfrom moray eels that is related to Mycobacterium triplex. J Clin Microbiol 41 2147 - 2152
Man SH , Hodgkiss IJ 1981 Hong Kong Freshwater Fishes. Urban Council, Wishing Printing Company Hong Kong, CH.
Marcì D , Lo Verde V , Mancuso I , Reale S , Passantino A , Marino F 2008 Mycobacteriosis in ornamental fish: case reports in Sicilyand medical-legal considerations. Vet Res Commun 32 S215 - S217
Noordhoek GT , Kaan JA , Mulder S , Wilke H , Kolk AHJ 1995 Routine application of the polymerase chain reaction for detectionMycobacterium tuberculosis in clinical samples. J Clin Pathol 48 810 - 814
Pate M , Jencic V , Žolnir-Dovc M , Ocepek M 2005 Detection ofmycobacteria in aquarium fish in Slovenia by culture and molecularmethods. Dis Aquat Org 64 29 - 35
Rhodes MW , Kator H , Kotob S , van Berkum P , Kaattari I , Vogelbein W , Quinn F , Floyd MM , Butler WR , Ottinger CA 2003 Mycobacteriumshottsii sp. nov., a slowly growing species isolated fromChesapeake Bay striped bass (Morone saxatilis). Int J Syst Evol Microbiol 53 421 - 424
Talaat AM , Reimschuessel R , Trucksis M 1997 Identification ofmycobacteria infecting fish to the species level using polymerasechain reaction and restriction enzyme analysis. Vet Microbiol 58 229 - 237
Tamura K , Dudley J , Nei M , Kumar S 2007 MEGA4: MolecularEvolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24 1596 - 1599
Telenti A , Marchesi F , Balz M , Bally F , Böttger EC , Bodmer T 1993 Rapid identification of Mycobacteria to the species level bypolymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 31 175 - 178
Thompson JD , Gibson TJ , Plewniak F , Jeanmougin F , Higgins DG 1997 The CLUSTAL_X windows interface: flexible strategiesfor multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25 4876 - 4882
Whipps CM , Dougan ST , Kent ML 2007 Mycobacterium haemophiluminfections of zebrafish (Danio rerio) in research facilities. FEMS Microbiol Lett 270 21 - 26
Whittington RJ , Chong R 2007 Global trade in ornamental fishfrom an Australian perspective: the case for revised import riskanalysis and management strategies. Prev Vet Med 81 92 - 116