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Thread: The Microbiome Dimension of Koikeeping

  1. #1
    Daihonmei MikeM's Avatar
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    The Microbiome Dimension of Koikeeping

    I have given this thread a rather pretentious title. At this stage it is undeserved. However, I hope in the future to add to the thread and make it an appropriate title. (Something that cannot be done on FB.)

    Everyone has learned that human health is much influenced by the bacterial communities residing in the digestive system. It has become a matter of popular food fads of one sort or another. Some are familiar with there also being various microbe communities resident on the human skin, which can be positive contributors to health (not that I encourage bathing in active yogurt or some such thing). Not generally appreciated is that microbes reside on virtually all surfaces of all living creatures, which have evolved together over the eons, resulting in mutual benefits that are not well-understood. This mutuality is definitely true of fish and their guest microbes. These communities of microbes are referred to as the microbiome. It is a microscopic world of its own, much like the complexities of biofilm. The microbiome of fish is particularly poorly studied, despite indications that it is far richer in diversity and interactivity than the microbiomes of mammals and terrestrials in general.

    I have read various articles on the subject, but have not gained much understanding of practical use. Then, I came across a recently published study of the effects of using potassium permanganate on the microbiome of catfish in an aquaculture setting. It is worth attention. My general bias against use of PP is well-known, but I think the real value of the study is that it brings home that the little recognized microbiome of our koi deserves more attention and appreciation than our current knowledge allows. The published abstract and introduction are copied below. (If you are interested enough to read the entire report, it is available on the internet. Do a search using the title and it will show up.)


    Potassium Permanganate Elicits A Shift Of The External Fish Microbiome And Increases Host Susceptibility To Columnaris Disease
    Haitham H. Mohammed and Covadonga R. Arias*
    • * Corresponding author: Covadonga R Arias [email protected]

    Author Affiliations
    Aquatic Microbiology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, Center for Advanced Science, Innovation, and Commerce, 559 Devall Drive, Auburn 36832, AL, USA

    Veterinary Research , 46:82 doi:10.1186/s13567-015-0215-y
    The electronic version of this article is the complete one and can be found online at: Veterinary Research | Full text | Potassium permanganate elicits a shift of the external fish microbiome and increases host susceptibility to columnaris disease
    Published: 15 July
    © Mohammed and Arias
    This is an Open Access article distributed under the terms of the Creative Commons Attribution License (Creative Commons — Attribution 4.0 International — CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.

    ABSTRACT
    The external microbiome of fish is thought to benefit the host by hindering the invasion of opportunistic pathogens and/or stimulating the immune system. Disruption of those microbial communities could increase susceptibility to diseases. Traditional aquaculture practices include the use of potent surface-acting disinfectants such as potassium permanganate (PP, KMnO 4 ) to treat external infections. This study evaluated the effect of PP on the external microbiome of channel catfish and investigated if dysbiosis leads to an increase in disease susceptibility. Columnaris disease, caused by Flavobacterium columnare, was used as disease model. Four treatments were compared in the study: (I) negative control (not treated with PP nor challenged with F. columnare), (II) treated but not challenged, (III) not treated but challenged, and (IV) treated and challenged. Ribosomal intergenic spacer analysis (RISA) and pyrosequencing were used to analyze changes in the external microbiome during the experiment. Exposure to PP significantly disturbed the external microbiomes and increased catfish mortality following the experimental challenge. Analysis of similarities of RISA profiles showed statistically significant changes in the skin and gill microbiomes based on treatment and sampling time. Characterization of the microbiomes using 16S rRNA gene pyrosequencing confirmed the disruption of the skin microbiome by PP at different phylogenetic levels. Loss of diversity occurred during the study, even in the control group, but was more noticeable in fish subjected to PP than in those challenged with F. columnare. Fish treated with PP and challenged with the pathogen exhibited the least diverse microbiome at the end of the study.


    Introduction

    Fish are in intimate contact with the aquatic environment which harbors pathogenic and opportunistic organisms [1]. As a result, cutaneous diseases are more common in fish than in terrestrial vertebrates [2] and the external epithelial surfaces are often the major route of entry for infectious agents in aquatic animals [3]. Skin and gills of fish are extremely important as the first line of defense against invasion by opportunistic pathogens and subsequent infections that may result in disease. In addition to being mechanical barriers, skin and gills represent a biologically active environment [4],[5] that is colonized by a diverse, complex and dynamic microbial communities that constitutes the fish external microbiome [6]-[10]. A healthy microbiome exerts antagonistic effects against pathogens by competitive exclusion for nutrient and/or synthesis of antimicrobial compounds and promotes host homeostasis [11],[12]. Suppression of pathogenic organisms by the resident microbiota has been reported in birds, fish, crustaceans, and other aquatic organisms [10],[13],[14]. Thus, preserving the integrity of the normal protective microbiome is key for excluding potential invaders and maintaining health [15].
    Intensive production practices used in fish farms can result in environmental stressors such as low dissolved oxygen or high organic loads that favor opportunistic pathogens and are stressful to fish [16]. Moreover, the use of chemical treatments to control or prevent specific pathogens can alter the normal healthy fish microbiome making the fish more vulnerable to infections [17]. The effect of these intensive culture practices on the fish external microbiome is for the most part unknown. We hypothesized that the use of harsh chemicals as treatment against external bacterial, parasitic and fungal infections disrupts the skin and gill microbiome and increases susceptibility to opportunistic bacterial pathogens. To test our hypothesis, we chose to use PP (KMnO 4 ), a potent oxidizing agent commonly used in aquaculture to treat external infections, and Flavobacterium columnare as the causative agent of columnaris disease, a very common bacterial infection in freshwater aquaculture farms.
    Columnaris disease courses primarily as an external infection and the bacteria frequently attack the fins, skin, and gills of fish causing frayed fins, depigmented or ulcerated skin and necrotic gills [18],[19]. Skin and gills are believed to be the point of entry and the primary site of infection for F. columnare[3],[20] and bacterial competition is considered one of the factors determining the degree of the infection [21]. Previous studies have shown that survival and infectivity of F. columnare decline in presence of competitive bacteria species such as Aeromonas hydrophila (an opportunistic fish pathogen) and Citrobacter freundii (nonpathogenic to fish) [22] or when the density of F. columnare was too low relative to total bacterial counts [23]. Thus, it has been suggested that when F. columnare is present in low numbers, it may not be able to compete with other naturally occurring bacteria on the fish skin and gills [24].
    To prove if PP altered the composition of the fish external microbiome and, subsequently, increased susceptibility to columnaris disease we applied culture-independent methods to characterize and compare the channel catfish (Ictalurus puntactus) external microbiome before and after exposure to PP and challenge with F. columnare. Our model has direct implications for commercial aquaculture as channel catfish is the main aquaculture species in the U.S. and is highly susceptible to columnaris disease. In addition, PP is routinely used in freshwater fish farms around the world to control external infections.
    Last edited by MikeM; 12-15- at 01:07 PM.

  2. #2
    Oyagoi yerrag's Avatar
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    Thanks Mike for sharing. Leaving nature alone to do its work is the essence of our newfound appreciation for the microbiome. The term itself implies a community in balance supporting life on a relationship of interdependency. The use of PP has its place in disinfecting the ponds of dealers prior to receiving new shipments, but its use to "improve" the ORP of pond water fails to acknowledge and take advantage of fish's need for a healthy and thriving microbiome in the pond that is vital to the growth and full development of koi.

    The more we understanding these microscopic critters that help keep things in balance and in order, and supports the energy flow of life in all its beauty, the less we need to cling on to reductionistic concepts of caring for ourselves and for our koi.

  3. #3
    Daihonmei MikeM's Avatar
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    You are welcome, Yerrag.

    The microbiome field is a vast new area for research, making it exciting for young researchers. It is less than a hundred years ago that science gave us antibiotics to treat disease. And, 50 years earlier there was no understanding of germs as a cause of disease. It all seems so basic now. The world population would be so much smaller without such understanding and medicines. But, over that period mankind has mostly thought of microbes as 'bad'. It is only recently that the notion of 'good germs' has begun to be appreciated. There really is an explosion of research on the human microbiome. Understandably, not so much on the microbiome of fish, and what does occur is oriented to commercial aquaculture of food fish. Most of the fish research concerns the digestive system microbiome. The goal is to learn ways to maximize growth.

    Some neat stuff has been learned, but much is preliminary. It seems that there are particular bacteria species that consistently appear in the intestinal tract of certain fish species, as well as other species that do not appear universally. The population varies according to foodstuffs consumed, and due to other factors. For example, using pellet foods containing soy bean meal results in different bacteria thriving, including some not usually found in marine environments. It may turn out that long term use of pellets containing soy bean meal on a regular, consistent basis results in better digestion and absorption of nutrient than occasional or irregular use. The well-being of the bacteria directly contributes to the well-being of the fish. So, it seems logical to remember that you are feeding the bacteria as well as your koi every time you reach into that container of pellets. Using probiotics (adding beneficial bacteria to food) and prebiotics (adding ingredients to food which will cause the resident microbes to thrive) are the subject of a lot of study. It has become well-known that lactic acid bacteria in particular produce beneficial results, as Hikari makes sure we know in their Saki Hikari advertising. Many others are under study. The research goals are not limited to growth. Enhancing immune response is also a goal. But, not much is really understood about how the various mechanisms work. In another decade, I expect we will know much more and see all sorts of changes in fish feeds and recommended practices.

    I had some stuff on slime coat bacteria tucked away. That's the area I am most interested in, perhaps because it is not as popular as the probiotics? I'll look for it and perhaps post some more in a day or three.

  4. #4
    Daihonmei MikeM's Avatar
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    Getting back to this subject, one of things to understand about the immune system of animals is that immunities develop in tandem with the microbiota living on and in all animals, including fish. The mechanisms of how it all works are the subject of much study, but the big picture is gaining focus. A common study subject is the tropical zebrafish. It has become for fish studies what mice are for mammal studies. They are cheap, easily produced and the small size makes experiments more manageable and financially feasible. After something is learned from zebrafish, replication with commercial food fish often occurs. Much of the time what has been learned from zebrafish holds true for food fish, although some of the bacteria involved may be different. As mentioned in the introduction to the study initially posted, the external microbiota of fish has a competitive exclusion effect. Every fish species appears to have a core group of bacteria that dominate the colonization of the external surfaces of the fish, especially including the mucus coat. Folks in the koi hobby have long talked about the mucus coat being the first line of defense against pathogens. However, how the mucus coat works as a defense has not been much discussed. There is a physical aspect in that any pathogen has to get through the slime itself. But, that may not be all that much of a challenge. The mucus coat houses multiple species of bacteria that work at keeping their territory safe for themselves. Fish house some bacteria that seem to have evolved specifically to thrive on the mucus coat of that species of fish, and also bacteria that are commonly found on many fish species. There is a high degree of similarity in the commensal bacteria communities on wild fish and domesticated fish of the same species, indicating that there is a core group of bacteria intimately tied to a fish species (or multiple fish species). Additional bacteria species show up from place to place, seemingly based on variations in the water environment and undetermined factors. There is reason to think the commensal bacteria use the slime coat not just as a habitat, but also as a major food source, while also gaining nutrient from the surrounding waters. That is, the fish is feeding its microbiome. Remember, they have evolved together to be mutually supportive.

    A healthy population of commensal bacteria also results in the exclusion of pathogens both by their physical presence and by the release of substances that adversely affect bacteria which have not evolved to tolerate the substances. Just what these substances are needs much more study. Based on what I have read, I would not necessarily lump them all together as "antibiotics", but it may not be incorrect to say there is a sort of antibiotic effect in the sense that foreign microbes are repelled or retarded... and killed, by the chemical warfare waged by the commensal bacteria.

    There is also evidence that the commensal bacteria positively facilitate the development of the adaptive immune system. You might say that the commensal bacteria teach the immune system how to work to change with changes in bacteria encountered. So, without the commensal bacteria, the the immune system itself would not be as ready to respond to a pathogen's attack. The study of the microbiome of the slime coat has found that it is common for there to be some normally pathogenic bacteria living on the host, usually in very small proportion to the total microbiome. These normally pathogenic bacteria do not appear to cause any harm when incorporated in these commensal communities. It is suggested that disruptions disturbing the microbiome, such as environmental changes and other stresses, can give the opportunity for these pathogenic bacteria to attack the host fish. That is, pathogens that were kept controlled get out of control.

    An interesting aspect of how the host fish and its microbiome are mutually dependent is what happens when the fish is stressed. The proteins of the mucus coat actually shift in response, which in turn affects the relative proportions of the various bacteria making up the microbiome of the host fish. Just what occurs is unclear, but it seems the success of each of the various commensal bacteria is linked to particular proteins in the mucus coat. [It makes me wonder if there has been an evolutionary adaptation by which the host fish increases production of a particular protein which encourages a particular microbe to have a population explosion, and that microbe releases a toxin that happens to repel some pathogen that thrives in the particular environmental condition the fish finds stressful. I hope someone studies that subject.]

    As a last curious tidbit for the day, when eggs are released, a set of commensal bacteria immediately begin colonizing the surface and quickly explode to be a population estimated to be millions of bacteria on a single egg. It would seem the fertilized egg has food for favored bacteria on its surface and keeps on producing it. So, when you see fertile eggs developing well, you can thank those well-fed bacteria. The fungused infertile eggs were not feeding their bacteria friends, allowing fungus an easy time.

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    Daihonmei MikeM's Avatar
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    I was asked, "So, if that study is right, why don't all the koi get sick in the ponds of people who regularly use PP to remove organics from their pond?"

    I think the answer is relatively simple. The PP wipes out just about everything to the same degree. With some beneficial bacteria remaining within the slime coat, they can re-colonize the fish before pathogens re-populate to a level that infects. That a koi survives repeated use of PP, or any other medication, does not mean its immune response has not been impacted.

    I think one lesson to take from the study is that the introduction of new koi is particularly risky when a pond has been recently treated with PP or other medications having an anti-bacterial impact (such as MG/Formalin). Another lesson is that it can be worthwhile to know about a dealer's treatment of koi put on sale. Some dealers regularly use PP or mg/f on batches of newly imported koi and place those koi up for sale very quickly. Such a koi placed directly in an established pond has a heightened risk of resident pathogens
    attacking it. In time we may learn that one of the main benefits of quarantine tanks is allowing the microbiome of the new koi to become fully established in the water conditions of its new home.
    Last edited by MikeM; 12-31- at 09:17 AM. Reason: typo
    coolwon and yerrag like this.

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    Sansai
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    [QUOTE=MikeM;219653]I was asked, "So, if that study is right, why don't all the koi get sick in the ponds of people who regularly use PP to remove organics from their pond?"

    What is an ORGANIC?

    Garfield

    PS I thought people used PP to get rid of irritating GOGGA's

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    Daihonmei MikeM's Avatar
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    [QUOTE=coolwon;219654]
    Quote Originally Posted by MikeM View Post
    I was asked, "So, if that study is right, why don't all the koi get sick in the ponds of people who regularly use PP to remove organics from their pond?"

    What is an ORGANIC?

    Garfield

    PS I thought people used PP to get rid of irritating GOGGA's
    About 15 years ago, a fellow (Roddy Conrad) who had very poor source water began using PP dosing to reduce the need/frequency/ amount of water changes. His idea was that by oxidizing organics in the pond (dissolved wastes were the target, but all living tissue and formerly living material is oxidized by PP) he could rejuvenate the water. He began posting about his practice and promoted it as a way to eliminate (or reduce) the work of performing water changes. The practice became quite controversial, leading to many heated exchanges on various forums. The fundamental objection to the practice was that everything got oxidized, not just contaminants... the mucus coat of the fish, the gill filaments, the biofilm, etc. The counter-argument was that koi exposed to the practice did not show permanent damage, any harm to gill filaments healed, and koi with reduced mucus coat exhibited brighter colors. Over time, Roddy drew back from much that he originally promoted and moved to using a lowered dose of PP and suggesting it's benefit was for those with poor source water or inadequate filtration or limited ability to perform water changes (as opposed to recommending it broadly). But, even this modified thinking continued to draw much opposition. The controversy raged for years, but pretty much ended when Roddy stopped posting about it. However, some number of pondkeepers adopted the practice along the way in lieu of improving filtration and performing water changes. These continue to recommend it, but I am not aware of any who are on a crusade to promote it. It is not a practice followed by many who would be considered serious koikeepers. My impression is that most who use PP as a water purification treatment are on the watergardening side of the spectrum. However, the record breaking drought in California with the attendant water use restrictions has caused some increased interest. In such a situation where water changes simply are not possible, the use of PP or ozone may well be necessary. But, like with ozone, I would strongly recommend that the treatment occur in a separate facility/tank, not in the pond, to avoid impacts on the koi and the biofilm, with water being returned to the pond only after complete neutralization of the oxidizer.

    I have tried to explain the practice as neutrally as I can, although my opposition to it surely came through.
    coolwon likes this.

  8. #8
    Sansai
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    PP treatment. Very much like gambling, people are mum about the losses.

    Garfield

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    Oyagoi yerrag's Avatar
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    I agree. PP as a disinfectant as a topical solution for applying to wounds would work in the absence of betadine. But I'm not keen on its use regularly in ponds, especially in its use to increase ORP. However, it would be hard for me to get koi from dealers who do not use PP to disinfect their ponds, then draining it, and refilling with fresh water, before stocking the pond with new koi shipment. In place of PP, some salt in these quarantine ponds would be a great help in getting these stressed koi recover from shipping stress and adapt to a new environment.

    Sent from my XT1068 using Tapatalk

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    Daihonmei MikeM's Avatar
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    Quote Originally Posted by yerrag View Post
    ****it would be hard for me to get koi from dealers who do not use PP to disinfect their ponds, then draining it, and refilling with fresh water, before stocking the pond with new koi shipment.
    That sort of procedure would not affect the fish, although it might affect filtration, etc. depending on how it was done. Treatment of the fish is often necessary. Nothing wrong about eliminating parasites. It could make a difference, however, whether the fish were treated a few days before purchase, or a month before. Some dealers treat shortly following arrival and then hold the koi in quarantine for several weeks before selling/shipping to customers. It has been known that koi quarantined for several weeks (or even a couple of months) generally do better. Re-establishment of the microbiome may prove to be an important aspect of that.

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