# Betta splendens, Graphite Disease, and Mycobacteria



## Zhylis (Nov 4, 2014)

*General fact sheets on fish-related mycobacteria:*
Prevention and Disinfection of Mycobacterium sp. in Aquaculture
Mycobacterial Infections in Fish
Mycobacteriosis in Fish

*A scientific review on fish-related mycobacteria:*
Mycobacteriosis in fishes: A review

*Chronic and acute mycobacterial infections*
So far, two types of mycobacterial infections have been observed in _Betta splendens_. The first type is a relatively slow, chronic infection with symptoms similar to those described for _M. marinum_ (refer to links above). The major defining symptom is the accumulation of internal granulomas, which can eventually cause the fish to appear lumpy/misshapen and swollen and are observable upon necropsy (see link here).

A second type has been observed on these forums and by others but has not been well documented officially. This type of mycobacterium produces an acute infection that rapidly causes death within the first 24 hours of external symptoms. A change in coloration - usually on the fins, occasionally on the body - is the first visible sign of an infection. The region initially turns a grey/graphite color that changes to black or complete color loss as the infected cells die. Fin rays and webbing disintegrate while scales lose partial to all pigmentation. 

These symptoms are typical of a new classification of mycobacterium named mycolactone-producing mycobacteria (MPM) that evolved from _M. marinum_ (1). The initial member of the MPM group was _M. ulcerans_, the causative agent of a skin necrosis disease in humans (2); however, additional members have since been discovered, including several strains that infect fish (3, 4, 5). It is currently unknown if these fish-related strains are zoonotic. In addition to the normal genomic DNA, these bacteria contain an extra piece of circular DNA (the pMUM plasmid), which encodes the only known mycobacterial virulence factor, mycolactone (6). MPMs can also be identified by the presence of multiple copies of the insertion sequence element IS2404_._ At this time, although mycobacteria were identified by laboratory testing by two breeders, no tests have been performed to determine if the mycobacteria that is associated with "graphite disease" also expresses mycolactone, as suggested by the rapid fin necrosis.
*
Virulence factor: Mycolactone* (7)
Mycolactone is a fatty-acid-like metabolite produced only by MPMs that causes the necrotic effect in host cells. Injection of this metabolite alone in the absence of any bacteria is sufficient to cause necrosis. Anchorage-dependent cells (like skin and gill cells) are the most sensitive to the effects of mycolactone. This metabolite enters the cell by passive diffusion through the plasma membrane, after which it kills the cell by disrupting the cytoskeleton and blocking the synthesis of cell surface proteins. Mycolactone can also supress the host immune response, preventing the normal signs of infection including swelling and inflammation, which is why fish appear normal except for the discoloration up until death occurs.

...to be continued.


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## logisticsguy (Sep 7, 2012)

Good info. This is why I started wearing gloves in the fish room. This may not be for everyone but I have a compromised immune system and these bacteria have become more common and they are getting resistant to antibiotics. Sadly these bacteria do not get destroyed by salinity or change in salinity making salt baths or adding aq salt to the tank useless in prevention.


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## hrutan (Jun 25, 2014)

When you choose to research and cite something, your posts are a joy to read, even about such a heavy topic. Thank you.


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## Hallyx (Jun 11, 2011)

Terrific article, Zhylis. Easy to understand with quality references. I appreciate the time, talent and understanding of the topic that this kind of presentation requires. 

Thank you.


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## Zhylis (Nov 4, 2014)

Thank you and you're welcome; it's a fortunate side effect of an absolutely ridiculous amount of time spent on higher education and laboratory research. >.< Publish or perish! 

This will probably be a 3-5 segment post. Updates will be slow, since there's a lot of information to assemble. I'll try to keep to publicly available references, but a lot of these papers are behind a paywall. (A soapbox rant for another day.) I'll link the NCBI abstract for the ones that aren't available. If it's a US publisher, it'll become open access 1-2 years later.

If anyone has any questions, feel free to PM them to me, and I'll add a Q&A section or post as needed. I may not be able to find answers for everything, but I can probably point in the right direction.


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## Zhylis (Nov 4, 2014)

Or it could be a quick update due to a really slow Friday. Never start an experiment on a Friday; that way leads to madness. Sundays are OK though, that just makes the following week more efficient.
_______________________
...continued from above._
Currently, five different types of mycolactones have been identified (8, 9). Mycolactone A/B is associated with human infections with _M. ulcerans_, while Mycolactone F was recently discovered in fish infections (10). All have similar abilities to cause immunosuppression, tissue necrosis, and cell death although with varying efficiency. These functions are attributed to the conserved lactone core (11). The unsaturated fatty acid side chain varies in form but shares the same function in enhancing the uptake of mycolactone into cells.

Fig. 1. Structure of the mycolactones identified to date (8)









*Considerations for developing a treatment for mycobacteriosis*
Currently, there is no treatment available for mycobacteriosis in infected fish. However, when faced with the loss of valuable breeding lines or rare fish, there are several treatments that could be tested (alone or in combination) based on the following rationale. A successful treatment would require a two pronged approach: 1. neutralization of mycolactone and 2. clearance of the bacterial population and followed by disinfection of the fish room and equipment (discussed later).

_M. ulcerans_ requires a break in the skin barrier in order to infect an organism (12). Upon entering a wound, _M. ulcerans _causes an extracellular infection (13), which is vastly different from the intracellular infections typically seen during mycobacteriosis. Interestingly, this phenotype to dependent on the presence of mycolactone. Bacteria that lose the pMUM plasmid or cannot express mycolactone revert from an acute, cytotoxic, extracellular infection to a chronic, granuloma-forming intracellular infection typical of mycobacteria (14). 

This would need to be confirmed for the B. splendens "graphite disease". The observation that this mycobacterial infection typically occurs on the fins and then spreads toward the body is highly suggestive that the bacteria share a similar pathology with _M. ulcerans_, which would mean that the initial infection is both localized and extracellular. Therefore, in early infections, surgical remove of the infected region (and part of the surrounding healthy tissue) would be sufficient to diminish the cytotoxic effect of the mycolactone and reduce the bacterial load on the fish. Following surgery, a dip in formalin/Seachem Paraguard and temporary housing in pure RO water (lacking in essential multivalent ions) would add environmental stress to the remaining bacteria, while leaving the fish less affected. Finally, although mycobacteria are antibiotic resistant, combinational treatment with rifampin/streptomycin or rifampin/amikacin has been effective in curing early infections of _M. ulcerans_ in mice (15).


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