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Keywords:

  • fish mycobacteriosis;
  • mycolactone-producing Mycobacterium;
  • Belgium

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the past few years, a mycolactone-producing subgroup of the Mycobacterium marinum complex has been identified and analyzed. These IS2404-positive species cause pathology in frogs and fish. A recently isolated mycobacterial strain from a fish in Belgium was analyzed using a variety of molecular methods and the results were identical to those obtained from a mycolactone-producing M. marinum from Israel.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The Mycobacterium marinum complex is a group of mycobacteria that includes M. marinum, the cause of chronic systemic infections in fish (Ucko et al., 2002; Decostere et al., 2004) and other cold-blooded animals (Thoen & Schliesser, 1984), and occasionally causes superficial and self-limiting granulomatous skin lesions involving the hands, forearms, elbows and knees in humans (Huminer et al., 1986). Mycobacterium ulcerans, the cause of the human necrotizing skin disease Buruli ulcer, is a second member of the complex, having evolved recently from an M. marinum progenitor by plasmid acquisition and reductive evolution (Stinear et al., 2004, 2007). The plasmid confers on M. ulcerans the ability to elaborate an immunosuppressive and cytotoxic lipid called mycolactone, which is a major determinant of virulence for this pathogen (George et al., 1999). A third member of the M. marinum complex was recently uncovered with the discovery that other mycobacteria, not associated with Buruli ulcer, also produce mycolactone (Mve-Obiang et al., 2005; Rhodes et al., 2005; Ranger et al., 2006). These mycobacteria, recovered from diseased fish and frogs around the world, have been given various species names including M. marinum, Mycobacterium pseudoshottsii and Mycobacterium liflandii, but detailed genetic studies have confirmed that they are all highly related and have evolved from the same common M. marinum progenitor as M. ulcerans by plasmid acquisition (Yip et al., 2007). The mycolactone-producing mycobacteria (MPM) can be distinguished from M. ulcerans by the type of mycolactone they produce and the fact that they do not exhibit the same high level of genome reduction and decay as M. ulcerans (Yip et al., 2007).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation of MPM

A mycobacterial culture was obtained from a liver sample from a diseased necropsied fish from Belgium. The liver sample was homogenized in 1 mL of saline, and decontaminated using the ‘fortep’ technique (Palomino & Portaels, 1998) by adding 3 mL 1 N HCl for 20 min, neutralizing with 1 N NaOH and centrifuging for 20 min at 3500 g. The pellet (0.1 mL), suspended in 1 mL distilled water, was inoculated onto Löwenstein–Jensen medium and incubated at 32 °C.

DNA extraction and PCR

Genomic DNA was extracted from the mycobacteria on the Löwenstein–Jensen slope by boiling the harvest for 5 min in 400 μL of Tris-EDTA. DNA amplification by PCR was obtained using a HotStarTaq DNA polymerase (Qiagen, Hilden, Germany) as described previously (Stragier et al., 2005). Mycolactone was extracted and screened by LC-MS as described (Hong et al., 2003). The multilocus sequence typing analysis is based on the nucleotide sequences of partial gyrB/gyrA and rpoB genes and the full rpsL gene (P. Stragier et al., unpublished data).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Recently, three MPM isolates from Israel and Greece were analyzed using mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) (Stragier et al., 2007). Along with these isolates an unknown isolate from a fish farm in Belgium (ITM06-3844) was tested. This unknown isolate shared an identical and unique VNTR profile with the fresh water MPM from Israel (CC240299). The culture was isolated in 2006 from an Acipenser baerii–Acipenser guldenstaedtii hybrid sturgeon, which was captured after finding chronic problems in all batches of A. baerii and A. baerii–A. guldenstaedtii hybrids with mortalities and septicemia. Six sturgeons from three different tanks were necropsied and granulomas were observed in the liver and kidney of all fish as well as enlarged spleens and hemorrhages in different sites such as the peritoneal wall and the genital organs in two fish. A rapid blood stain (Hemacolor®, Merck, Darmstadt, Germany) of some liver and kidney smears revealed macrophages containing unstained rods. Histological examination of liver and/or kidney samples of all sturgeons showed multiple foci of acid-fast rods. One liver sample yielded a mycobacterial culture on Löwenstein–Jensen medium (ITM06-3844) and it was subjected to DNA extraction for further molecular analyses as described previously (Stragier et al., 2005, 2007). The results of these analyses are shown in Table 1. The positive IS2404 PCR result indicates that the isolate is possibly a MPM. Until now, IS2404 (Stinear et al., 1999) has been found only in MPM. The genes repA and mup038 are two of 81 genes that are present on the pMUM plasmid of M. ulcerans (Stinear et al., 2004), and their presence in the Belgian isolate is further confirmation that this isolate harbors pMUM and is an MPM of the M. marinum complex. The presence of esxA and esxB genes (RD1) was tested and confirmed. To better establish the taxonomic position of the Belgian isolate, the 16S rRNA gene–internal transcribed spacer region was sequenced and was found to be identical to MPM isolate CC240299.

Table 1.   Test results of MPM isolates CC240299 and ITM06-3844
TestCC240299ITM06- 3844Primer references
  1. =, identical sequence or VNTR profile; NA, not applicable.

IS2404 PCR++Stinear et al. (1999)
IS2606 PCRStinear et al. (1999)
repA/mup038++ 
esxA/esxB++Mve-Obiang et al. (2005)
MLST analysis== 
MIRU-VNTR==Stragier et al., 2007
16S rRNA gene–ITS== 
MycolactoneFFNA

Extraction of total acetone-soluble lipids and screening by LC-MS as described (Hong et al., 2003) confirmed that this isolate, like MPM isolate CC240299, produces mycolactone F.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study has identified a mycolactone-producing mycobacterium of the M. marinum complex from a fish in Belgium. Initial phenotypic and genetic characterization of the strain indicates that it is identical to MPM recovered from fish in Israel, suggesting a worldwide distribution for these mycobacteria. So far, MPM have been described in Israel, Greece, Italy and the United States, and a more thorough analysis of M. marinum isolates might identify an increasing number of isolates belonging to the MPM group (Ranger et al., 2006). Within the M. marinum group, some distinct clusters have been identified previously, consistent with their difference in virulence and pathogenic host (van der Sar et al., 2004; Ranger et al., 2006; Ostland et al., 2008). It is not clear to which cluster the MPM belong, but although probably more virulent because of mycolactone production, no MPM infection has been described in humans so far. These data also indicate that care should be taken when handling fish infected with MPM because they are associated with severe pathology (Colorni, 1992; Rhodes et al., 2005; Ranger et al., 2006), and outbreaks of fish mycobacteriosis can have a dramatic economic impact on fish agriculture and may represent a health risk for fish handlers. A higher level of awareness should be in place and it is recommended not to postpone clinical tests on diseased fish.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Hui Hong for performing the mycolactone analysis, and Dr François Lieffrig and Prof. Richard Ducatelle for the clinical and histological analysis. This work was partly supported by the European Commission, project #INCO-CT-2005-051476-BURULICO, and by FWO Vlaanderen, Belgium G.0321.07.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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