BAD NEWS - Velvet Strain Survives 1.75 PPM Copper!

[DarkSky]

It shouldn't matter, velvet's life cycle is 48 hours. So if it makes it through 14 days, it survived several life cycles, it seems reasonable that 30 days would not be an issue. The 14 days is only acceptable if you transfer to a completely sterile quarantine post-treatment. If you're keeping in a single QT for the duration, 30 days is required. This is more for ich than velvet, however.

Is it 48 hours at 78F? According to this paper, 14 days is the minimum to control an outbreak of the infection - but they claim its life cycle is 3-6 days @ 20c.

http://agrilife.org/fisheries/files...mportant-Parasite-of-Cultured-Marine-Fish.pdf

 

[MnFish1]

Interested to see what you hear back. I admire your attentiveness to these types of threads. Your participation and research is much appreciated!

I was able to find the full text to that article - and posted it subsequently.

Here are my comments after reading for hours (blizzard here): (and may explain what happened to your fish)

1. AO (Amyloodinium Ocellatum) or velvet may live longer on fish and most QT protocols use a 30 day as compared to your 14 day and then transfer into a new tank. Could one or more fish have still had a bit of AO (the particles on the fish are mostly resistant to copper)?
2. AO is transmitted (can be) by aerosol droplets - though the study says up to 9.84 feet that was only one study - and may be higher in higher air flow states (not to mention cross contamination). I know that you guys try to limit that extensively - but - its a possibility.
3. I dont know what concentration of bleach you use to sterilize (I assume its higher than this) but at 1:500 bleach - AO continued to multiply (that was the highest dose they tested) - is it possible another sterilization method would be better?
4. Usually (key word) - when an organism is resistant to something an antibiotic, etc changing the dose by the percentage you're suggesting MAY not fix the problem (i.e. its a very small change). In the one article they showed an example of potential copper resistance - the resistant AO was at 10x the dose you were using - and still resistant (if indeed it was AO causing the problem).
5. Chloroquine (according to the U Florida presentation) may be a better choice if copper resistance is suspected.

Again - no criticism - just information.

 

[ngoodermuth]

@4FordFamily what this article/presentation suggests - is rather than increasing the copper level using Chloroquine might be better if you suspect copper resistance. When I first read this article - I thought - it must be copper resistance because when they removed the copper chloroquine resulted in survival. But one problem chloroquine also can kill bacteria. So it's interesting.

I felt like there was a tone of doubt that chelated copper works the same as other forms also... I wonder if there is more research here too. We use chelated copper because we “know” it’s easier on fish and has a more forgiving therapeutic range.

Well, what if the same can be said for the parasites we are trying to eliminate? What if by making copper more tolerable, we have also made it less effective? I’m just speculating... but as a long time cupramine user- the statement at the end saying that further testing on chelated copper was needed, caught my attention.

 

[HotRocks]

I was able to find the full text to that article - and posted it subsequently.

Here are my comments after reading for hours (blizzard here): (and may explain what happened to your fish)

1. AO (Amyloodinium Ocellatum) or velvet may live longer on fish and most QT protocols use a 30 day as compared to your 14 day and then transfer into a new tank. Could one or more fish have still had a bit of AO (the particles on the fish are mostly resistant to copper)?
2. AO is transmitted (can be) by aerosol droplets - though the study says up to 9.84 feet that was only one study - and may be higher in higher air flow states (not to mention cross contamination). I know that you guys try to limit that extensively - but - its a possibility.
3. I dont know what concentration of bleach you use to sterilize (I assume its higher than this) but at 1:500 bleach - AO continued to multiply (that was the highest dose they tested) - is it possible another sterilization method would be better?
4. Usually (key word) - when an organism is resistant to something an antibiotic, etc changing the dose by the percentage you're suggesting MAY not fix the problem (i.e. its a very small change). In the one article the showed an example of potential copper resistance - the resistant AO was at 10x the dose you were using - and still resistant (if indeed it was AO causing the problem).
5. Chloroquine (according to the U Florida presentation) may be a better choice if copper resistance is suspected.

Again - no criticism - just information.

It had to remain on the fish.

All 6 QTs are in an unfinished basement. No tank is within 15' of one another. Even clean observation tanks have at least 10-12' between one another.

While cross contamination is always a possibility I don't think it's the case here. I literally have separate food for every tank, separate equipment, tools, etc.

I don't go anywhere near observation or clean tanks once I have been in a QT or treatment tank (until the next day). I'm a freak to an extent that would surprise most people.

I even have separate RO systems. One for QTs, one for clean tanks and DTs.

Bleach sterilization is done at 500ppm.
Just FYI the bleaching wouldn't really be a factor in this case as the last batch of fish that came out of this tank that became reinfected are all parasite free. So the fish were transferred from a copper QT to the clean tank and velvet symptoms arose within about 5 days. So the AO was still on the fish when transferred and the absence of copper in the observation tank allowed it to run wild.

It was suppressed enough while the fish were in copper that they seemed to be velvet free.

 

[MnFish1]

It had to remain on the fish.

All 6 QTs are in an unfinished basement. No tank is within 15' of one another. Even clean observation tanks have at least 10-12' between one another.

While cross contamination is always a possibility I don't think it's the case here. I literally have separate food for every tank, separate equipment, tools, etc.

I don't go anywhere near observation or clean tanks once I have been in a QT or treatment tank (until the next day). I'm a freak to an extent that would surprise most people.

I even have separate RO systems. One for QTs, one for clean tanks and DTs.

Bleach sterilization is done at 500ppm.
Just FYI the bleaching wouldn't really be a factor in this case as the last batch of fish that came out of this tank that became reinfected are all parasite free. So the fish were transferred from a copper QT to the clean tank and velvet symptoms arose within about 5 days. So the AO was still on the fish when transferred and the absence of copper in the observation tank allowed it to run wild.

EDI
It was suppressed enough while the fish were in copper that they seemed to be velvet free.

Do you know how to translate a dilution of 1:500 to PPM? ( I don't).
I meant if there could have been some AO left in the second tank from the batch before (i.e. if not adequately sterilized). I have read before how strong/excellent your system is. The only reason im asking is that I would rather find a problem with something you're doing as compared to resistant velvet

EDIT - as was mentioned - perhaps using non-chelated copper would have made a difference.

 

[Mortie31]

Do you know how to translate a dilution of 1:500 to PPM? ( I don't).
I meant if there could have been some AO left in the second tank from the batch before (i.e. if not adequately sterilized). I have read before how strong/excellent your system is. The only reason im asking is that I would rather find a problem with something you're doing as compared to resistant velvet

EDIT - as was mentioned - perhaps using non-chelated copper would have made a difference.

This might sound mad, but could the resistance be to the bleach? So the sterilisation fluid would need to be much stronger, just throwing it out there as you guys seem to have covered every other possibility..

 

[4FordFamily]

I was able to find the full text to that article - and posted it subsequently.

Here are my comments after reading for hours (blizzard here): (and may explain what happened to your fish)

1. AO (Amyloodinium Ocellatum) or velvet may live longer on fish and most QT protocols use a 30 day as compared to your 14 day and then transfer into a new tank. Could one or more fish have still had a bit of AO (the particles on the fish are mostly resistant to copper)?
2. AO is transmitted (can be) by aerosol droplets - though the study says up to 9.84 feet that was only one study - and may be higher in higher air flow states (not to mention cross contamination). I know that you guys try to limit that extensively - but - its a possibility.
3. I dont know what concentration of bleach you use to sterilize (I assume its higher than this) but at 1:500 bleach - AO continued to multiply (that was the highest dose they tested) - is it possible another sterilization method would be better?
4. Usually (key word) - when an organism is resistant to something an antibiotic, etc changing the dose by the percentage you're suggesting MAY not fix the problem (i.e. its a very small change). In the one article they showed an example of potential copper resistance - the resistant AO was at 10x the dose you were using - and still resistant (if indeed it was AO causing the problem).
5. Chloroquine (according to the U Florida presentation) may be a better choice if copper resistance is suspected.

Again - no criticism - just information.

I cannot rule anything out, especially with so many "unknowns". While we KNOW a lot more than we did, say 10 years ago. Kyle and I have probably invested more of our own money and time as a hobbyist than most people doing actual studies/scientific research anymore. I don't see much out there. It has a dual purpose, for me I want a tank full of disease-free fish. Thank goodness for observation periods post-treatment.

Perhaps the 14 days component is an issue, but I struggle a lot with that for velvet. If this were ich, I'd be much more inclined to feel that's the likely culprit.

And by the way, I'd love to be proven wrong. I am just cautioning others, and my personal belief is that velvet is surviving copper levels we previously thought it wouldn't. I do wish we had @Humblefish around to discuss further.

 

[HotRocks]

Do you know how to translate a dilution of 1:500 to PPM? ( I don't).
I meant if there could have been some AO left in the second tank from the batch before (i.e. if not adequately sterilized). I have read before how strong/excellent your system is. The only reason im asking is that I would rather find a problem with something you're doing as compared to resistant velvet

EDIT - as was mentioned - perhaps using non-chelated copper would have made a difference.

I wish what I was doing was the problem! Trust me! It would be such an easy fix.

The tank wasn't sterilized between batches. Meaning it's a clean observation tank, so the last fish that were in that tank reside in both our DTs now. Velvet was introduced with the new fish. I know this because there were three fish in this tank for weeks (Moorish idol, mag fox, and a thalassoma Wrasse) from the last batch that were clean and waiting on permanent homes.

The velvet symptoms became aparent 5 days after the newly treated fish were combined with the existing fish. Had it been cross contamination or something of the like, the idol should have shown symptoms well before the newly treated fish joined them.

Bleach sanitation;
Stock household bleach is 5.25% sodium hypochlorite equal to approximately 50,000 ppm available chlorine; therefore, it must be diluted to with water (or expressed as a ratio, 9 parts water to 1 part bleach) to arrive at the 5,000 ppm strength or 99 parts water to arrive at 500 ppm bleach.

FYI 200ppm is probably enough to kill anything living in saltwater.

 

[puffy127]

So did 2.0 ppm copper kill off the 1.75 ppm copper-resistant velvet?

 

[DarkSky]

So did 2.0 ppm copper kill off the 1.75 ppm copper-resistant velvet?

A good question - further, I wonder if another attempt @ 1.75ppm would have killed it off.

I'm not blaming or making any assumptions here, so please don't take my comments in a negative way. But I'd feel much more safe placing the issue on a lapse in propper QT procedure than copper resistant velvet.

 

[MnFish1]

This might sound mad, but could the resistance be to the bleach? So the sterilisation fluid would need to be much stronger, just throwing it out there as you guys seem to have covered every other possibility..

I mentioned it - velvet can continue to multiply even in 1:500 bleach solution that was the highest dose (I believe it was up to an hour). @HotRocks said he uses 500 ppm bleach - I dont know how to translate that to 1;500 - I will try to find the article...

 

[4FordFamily]

So did 2.0 ppm copper kill off the 1.75 ppm copper-resistant velvet?

So far, yes. More testing necessary.

 

[MnFish1]

I wish what I was doing was the problem! Trust me! It would be such an easy fix.

The tank wasn't sterilized between batches. Meaning it's a clean observation tank, so the last fish that were in that tank reside in both our DTs now. Velvet was introduced with the new fish. I know this because there were three fish in this tank for weeks (Moorish idol, mag fox, and a thalassoma Wrasse) from the last batch that were clean and waiting on permanent homes.

The velvet symptoms became aparent 5 days after the newly treated fish were combined with the existing fish. Had it been cross contamination or something of the like, the idol should have shown symptoms well before the newly treated fish joined them.

Bleach sanitation;
Stock household bleach is 5.25% sodium hypochlorite equal to approximately 50,000 ppm available chlorine; therefore, it must be diluted to with water (or expressed as a ratio, 9 parts water to 1 part bleach) to arrive at the 5,000 ppm strength or 99 parts water to arrive at 500 ppm bleach.

FYI 200ppm is probably enough to kill anything living in saltwater.

So the article I read (and I'm trying to find it) that at a 1:500 dilution it still survived - I would assume you are using a 1:10 solution - which should have killed it many times lol.

I'm interested in your opinion on this - and I will try to re-find both articles in the next post - but - if hypo salinity (and I dont think its much) improves copper efficiency against velvet (i.e. supposedly you can use a lower dose) - would it make more sense rather than increasing the dose to 2 - to decrease the salinity - and leave the copper where it is? Just a question.

And - I will say - if you have resistant velvet - I cant see how increasing the dose from 1.75 - 2.00 ppm will be enough.

PS - what do you think about the 'non-chelated copper' idea

 

[4FordFamily]

A good question - further, I wonder if another attempt @ 1.75ppm would have killed it off.

I'm not blaming or making any assumptions here, so please don't take my comments in a negative way. But I'd feel much more safe placing the issue on a lapse in propper QT procedure than copper resistant velvet.

No negativity received, friend. I cannot answer your question, but I suspect no, based on the short life cycle of velvet. I’d prefer to subject fish to higher levels of copper for 14 days than the same level for 30, but that’s based again solely on anecdote and advice from those that do delve in to the research, such as @Humblefish.

I mentioned it - velvet can continue to multiply even in 1:500 bleach solution that was the highest dose (I believe it was up to an hour). @HotRocks said he uses 500 ppm bleach - I dont know how to translate that to 1;500 - I will try to find the article...

I can actually answer this, the tanks are completely run in bleach solution for 24-72 hours. Filters, powerheads, everything.

The “clean tank” was clean for the three fish he mentioned, no discernible issue. Within 3 days of the other fish that went through copper were added, color loss in the Acanthurus tangs and scratching with everyone was noted. By day 4 you could see velvet. Since the fish had been in meds, their immune response was likely much weaker and they were sitting ducks as velvet took over with a vengeance.

These are all things we know about copper/medication treatment. A necessary evil IMO, but an evil nonetheless.

 

[dwest]

One thing I know is that the procedure provided by hotrocks and 4fordfamily have saved the lives of a lot of my fish in quarantine. Thanks again!

 

[4FordFamily]

One thing I know is that the procedure provided by hotrocks and 4fordfamily have saved the lives of a lot of my fish in quarantine. Thanks again!

Glad to help, it makes all the money and time worth it, even if it kicks me in the teeth!

 

[MnFish1]

Here are a couple quotes from articles:

Treatment with copper-containing compounds should be continued for at least 2 to 3 weeks to control an outbreak of A. ocellatum, with the goal of maintaining a continuous concentration of 0.15 to 0.2 mg/L free copper ion. When treatment fails it is often because monitoring was inadequate and additional copper sulfate was not added to maintain the proper concentration.

Source: http://agrilife.org/fisheries/files...mportant-Parasite-of-Cultured-Marine-Fish.pdf

We achieved a marked improvement in the health status of fish following the 3 treatments, albeit with
different efficiencies as revealed by relative differences in prevalence rates and cumulative mortalities at the end of each treatment (Table 3). In T3
(copper sulfate), ~12% cumulative mortality was registered. This was slightly better than in T4 (hyposalination/freshwater bath), for which a higher
cumulative mortality rate of 15% was recorded. Nevertheless, and despite controlling mortalities in both cases, parasite stages could still be detected in tissue biopsies of some fish samples, though with much lower intensities when compared with pre-treatmentsamples (Table 3). The use of the dual hyposalination
+ copper sulfate (T5) was more efficient when compared to previous regimens as shown by both the lowrate of cumulative mortality (12%), as well as the
absence of parasite stages in the examined tissue biopsies of both skin and gill smears of fish (Table 3).

Source:
Amyloodinium ocellatum disease outbreak in cultured Dicentrarchus labrax: parasitological and molecular diagnosis, and a modified treatment protocol

• June 2018. Diseases of Aquatic Organisms 129(1)

About bleach and other chemicals (concerning dinospores):

Drug and Chemical Effects on the Behavior and Survival In Vitro of the Dinoflagellate Fish Parasite (Amyloodinium ocellatum)
IAAAM Archive
Carol E. Bower; David T. Turner

Abstract

Amyloodinium ocellatum, a dinoflagellate ectoparasitic on the gills and skin of marine and brackish-water fishes, is highly prolific and destructive in closed culture systems. The parasite reproduces successfully over a wide range of temperatures and salinities, and is extremely resistant to treatment with common fishery chemicals. The survival of Amyloodinium tomonts (encysted, reproductive stage) after prolonged exposure to fresh water, low temperature, and chlorine bleach, and the effectiveness of numerous antimicrobial and antiparasitic compounds in vitro against tomonts and dinospores (free-swimming, infective stage) were the subjects of the present study.

Tomonts divided and produced dinospores even after 6 wk of storage in fresh water; percent viability after storage was much greater in tap water than in distilled water alone or with added calcium or sodium salts. Tomonts also survived storage in seawater at 10 C for 9 wk and at 15 C for at least 12 wk. Tomonts were unaffected by immersion in a 1:2,500 dilution of liquid chlorine bleach for 24 h or a 1: 1,000 dilution for I h; a few tomonts divided and sporulated even after a 1-h exposure to 1:500 bleach.

Dinospores were immobilized within 24 h by quinine, quinacrine, chloroquine, primaquine, chiniofon, emetine, dehydroemetine, acriflavine neutral, nifurpirinol, chloramphenicol, sulfathiazole, pentamidine, amphotericin B, malachite green, methylene blue, benzalkonium chloride, Lugol's iodine, hydrogen peroxide, and formalin. They were not killed by copper sulfate alone or complexed with citric acid or EDTA, nor by furazolidone, nitrofurazone, sulfamethazine, chlortetracycline, ipronidazole, metronidazole, ivermectin, suramin, or difluoromethylomithine. Although several substances, including all three copper compounds, prevented or inhibited tomont division or sporulation during the 7-day treatment period, only five--formalin, chiniofon, pentamidine, ipronidazole, and malachite green--impaired reproduction permanently. The effectiveness of chemotherapeutic agents against the parasite in vivo, their toxicity to fish and nitrifying bacteria, and their persistence in culture water are still being studied.

 

[MnFish1]

IMHO - the reason I researched this is because this is really pretty groundbreaking if there (are) documented strains of resistant velvet. Its too bad - as someone suggested that you didn't try to re-treat with 1.75 - because a bit contrary to what you say - the higher levels will probably not effect the attached parasites (i.e. 2.00 vs 1.75) - but a longer time may very well have. I only think this because if the velvet were truly resistant - the vast likelyh0od is that they would not respond to 2 vs 1.75. But very well could respond if the issue were merely the parasites were hanging onto the fish longer than the QT time....

This might suggest that the paper quoted above 'at least 2-3 weeks' treatment is recommended. So - rather than there being resistant velvet out there - I propose that some fish need a longer QT time than your protocol - and thus - when placed into the 14 day - if they do ok - you're all good - but you cant say its 'resistant velvet' just because they develop it after 14 days - and then into the clean tank.

Please tell me if im incorrect - but - my interpretation was not that changing the dose of your protocol from 1.75 to 2 is warranted - but rather stating that even with 1.75 copper for 2 weeks - some fish may slip through (thats why you do the QT the way you do anyway - correct)?

 

[HotRocks]

A good question - further, I wonder if another attempt @ 1.75ppm would have killed it off.

I'm not blaming or making any assumptions here, so please don't take my comments in a negative way. But I'd feel much more safe placing the issue on a lapse in propper QT procedure than copper resistant velvet.

I really wish it was the case my friend. No offense taken. We are all humans here. Mistakes are always possible. When you have repeated the same process 20+ times with success and changed nothing I don't know what else to shift the blame upon...

 

[shred5]

One thing I know is that the procedure provided by hotrocks and 4fordfamily have saved the lives of a lot of my fish in quarantine. Thanks again!

Agreed.. Even though I have been in the reef hobby 30 + years and even longer saltwater fish, diseases are my weak point so I appreciate everything all these guys do in this thread.

If I ever meet you guys I owe ya a beer or what ever ya drink...