Any alternatives to Copper?

[Minh B.]

I have to echo the concerns. I came across a paper (Chen W et al, Parasitology Research 2008) where they developed a PCR assay to detect the presence of live Cryptocaryon irritans (at any stage) within the water column by amplifying its genomic DNA. They designed species specific primers to detect C. irritans and I. multifiliis (freshwater ich). I almost lost my entire fish collection last week (except for a pair of clowns) but my DT is now completely fishless. I will be PCR testing my tank water frequently before I reintroduce fish because PCR is extremely sensitive (down to the nmol range). If you're interested, I can test your water at the same time.

[Orion]

I have to echo the concerns. I came across a paper (Chen W et al, Parasitology Research 2008) where they developed a PCR assay to detect the presence of live Cryptocaryon irritans (at any stage) within the water column by amplifying its genomic DNA. They designed species specific primers to detect C. irritans and I. multifiliis (freshwater ich). I almost lost my entire fish collection last week (except for a pair of clowns) but my DT is now completely fishless. I will be PCR testing my tank water frequently before I reintroduce fish because PCR is extremely sensitive (down to the nmol range). If you're interested, I can test your water at the same time.

 

What is a PCR test? My DT is currently fallow as I just lost my fish due to an ich outbreak.

[Minh B.]

PCR is polymerase chain reaction. The scientist who invented this method received a nobel prize in chemistry in 1993. Using DNA polymerase and a pair of single stranded oligos/primers, you can specifically amplify any gene of interest from any organism. Although the author for this C. irritans paper used a very old technique of extracting DNA from the parasite, it was still successful and he proved you can specifically amplify a gene from either marine or freshwater ich. Would be a great tool to test for the presence of ich in the water column even if fishes are not symptomatic. I plan to test all fish coming into the QT tank (test the water column) to see whether copper testing is necessary and DT tank after the 1 month and 2 month period. If after 2 months I still get amplification, either the DNA in the DT is still present because there's residual parasites still living or the parasite died and released its guts and the DNA hasn't had time to be degraded.

Edited January 30, 2013 by Minh B.

[matt]

Minh, my brain is swimming but I'm glad to have you on the boards

[Orion]

Are there any other ways someone can test for the presence for ich other than leaving the display fallow and hoping that after 8-12 weeks the parasites are dead?

[Coral Hind]

Minh, You should start a PCR testing service and test people's tank water. I think there is some serious money there!

[Minh B.]

Minh, my brain is swimming but I'm glad to have you on the boards

Thanks! The study (for people interested, it's an open source research article/free at http://link.springer.com/article/10.1007%2Fs00436-008-0993-5?LI=true) showed the assay is sensitive down to 45 pg of DNA! That's a considerably small amount of DNA needed for the assay.

 

Are there any other ways someone can test for the presence for ich other than leaving the display fallow and hoping that after 8-12 weeks the parasites are dead?

Other research studies have utilized protein detection (as opposed to DNA detection) with immunoblots/ELISAs which are sensitive, reliable and accurate (but not as much as DNA detection), but such assays costs many fold more. Our laser scanner (for immunoblots/protein detection) costs $50,000 while UV imager (for DNA detection) costs $15,000. I'm sure one day, someone will produce an ELISA assay where you can add the antibody solution to a tube of your tank water, and if it turns blue, it means you have a positive result for marine ich. Making antibodies is a costly endeavor though and requires a lab full of caged rabbits where you immunized them against the ich peptide and the rabbit produces antibodies. Then you have to sacrifice the animal and purify antibodies. If only I had a million dollars...

 

Minh, You should start a PCR testing service and test people's tank water. I think there is some serious money there!

It's funny you mention this. A long time ago, I thought about creating a business of DNA sexing tarantulas (I had pet tarantulas and scorpions, some quite venomous too). A lot of people tried to breed their tarantulas but some species were (sexually) monomorphic so you couldn't distinguish male from female, and sometimes, one would kill the other (generally females would kill the males after the deed is done). Long story short, I didn't have time for it as different species require different primers for PCR. Marine ich is simple though. If it turns out it works well, then who knows. Maybe I'll do this on the side of cancer research

[smallreef]

Great way to put that degree to work helping fellow reefers!!!

[LCDRDATA]

I will be PCR testing my tank water frequently before I reintroduce fish because PCR is extremely sensitive (down to the nmol range). If you're interested, I can test your water at the same time.

 

I may take you up on that. To what extent does the test confirm/deny presence versus indicate relative concentration? I haven't decided whether to try the low-dose Cupramine or not -- my wife will have significant input on that -- and if I don't, it would be rather silly to test simply whether or not the organism was present. That answer (yes) is available without the test. But if it also indicates concentration, that might be worth looking at. Besides, my wife was a zoology/wildlife management major, and my daughter is majoring in biology, so I expect they'd both think it was cool.

[Minh B.]

 

I may take you up on that. To what extent does the test confirm/deny presence versus indicate relative concentration? I haven't decided whether to try the low-dose Cupramine or not -- my wife will have significant input on that -- and if I don't, it would be rather silly to test simply whether or not the organism was present. That answer (yes) is available without the test. But if it also indicates concentration, that might be worth looking at. Besides, my wife was a zoology/wildlife management major, and my daughter is majoring in biology, so I expect they'd both think it was cool.

 

It will confirm presence if there's a band on the gel and it could also indicate 'relative' concentration by the intensity of the band. I know my DT has a high concentration bc I haven't done a water change, but you have so I anticipate a medium concentration for you and my QT has cupramine w/newly mixed salt water + the potentially infected clownfishes, so I anticipate a low intensity band. To get exact concentration readings, I would have to do Real Time PCR which accurately tracks fluorescent intensity of the PCR product after each successive cycle and plots it on a graph. However, RT-PCR is too expensive so I only plan on doing regular semi-quantitative/sqPCR like they did in the paper. I ordered primers for the assay and will test my tank water first to make sure it works in my hands. If it does, I may ask you to bring a sample of your water to the meeting.

[LCDRDATA]

It will confirm presence if there's a band on the gel and it could also indicate 'relative' concentration by the intensity of the band...I ordered primers for the assay and will test my tank water first to make sure it works in my hands. If it does, I may ask you to bring a sample of your water to the meeting.

Sounds good, just let me know.

[Origami]

It's great that you have access to such tools, Minh. You should start a new thread on the topic to make it easier to find by title.

 

Just curious, I kind of understand the gene amplification and the tagging prior to (what sounds like a gel electrophoresis) assay. However, how long do you think you'll need to leave the fish in the QT water before there's sufficient genetic material to allow a reasonable probability that a sample will have what you're looking for (a parasite DNA sample to confirm presence)? Or do you use the entire QT tank's contents for the amplification process?

[Minh B.]

Thanks. I will do that.

 

Just curious, I kind of understand the gene amplification and the tagging prior to (what sounds like a gel electrophoresis) assay. However, how long do you think you'll need to leave the fish in the QT water before there's sufficient genetic material to allow a reasonable probability that a sample will have what you're looking for (a parasite DNA sample to confirm presence)? Or do you use the entire QT tank's contents for the amplification process?

Short answer: 7 days after introducing fish to the QT is plenty! However, it's actually quite easy to spot the theronts visually with a 100X microscope. These things are like race cars...they zoom by so quickly you can't keep up! But if you look carefully enough, you'll find their surface is riddled with cilia/hair-like structures.

 

Long answer:

I was very curious about this too. Today, I took to work, 10 mL of water from my DT's return pump compartment of the sump/refugium and 10 mL of water from the QT with the 2 clownfishes that survived. I first checked on the microscope at 100X and saw the highest concentration of theronts (free-swimming infective stage), tomites (very difficult to see) and what may look to be tomonts as well in the QT. I was quite shocked to see this, but to answer your question, the QT was setup exactly a week ago today (last Thursday) and already there were so much more activity than in the return compartment of my sump. It possible the fishes that died with lots of trophonts in the QT released a large amount of protomonts after the fishes died in an attempt to find a new host. I'm not familiar with the parasites response after their host dies. If anyone knows, please chime in. However, I saw very few theronts in the sump suggesting they're starting to die off, but there were still many tomites. Tomorrow, I will grab some water from the actual DT itself where the fishes were housed. Because I saw more activity in the QT than the DT's sump, my prediction is that I should get a higher DNA yield in the QT.

 

Although the paper used repetitive freeze/thaw cycles to burst open the parasite, I came up with a protocol that combines DNA extraction from both bacteria and human cells, and proceeded to extract both 10 mL samples. Briefly, samples were spun down at 10,000 rpm's for 10 min, lysed with a strong detergent and DNA precipitated with phenol (high toxic), chloroform and then ethanol. The DNA was solubilized and quantified by UV and guess what? QT's DNA concentration was 165 ng/uL and DT return sump's concentration was 117 ng/uL. The paper claims that 45 pg of DNA is the minimum for the assay, and assuming the majority of the DNA I extracted is from the parasite, I have >2000X the necessary amount of DNA for the assay from just 10 mL of sample. The real test is when I get the primers next week to run the assay.

[Origami]

Can you capture video of the theronts? It would be really interesting to see that.

 

Strong detergent to lyse the cells. Reminds me of my old Genetics professor who taught us the technique back in the late 70's. PCR came about in the early 80's, if I recall. I had just gotten out of school (and was a couple of years past securing my bio degree) back when it started to be reported.

 

It's remarkable to think that the concentration was as high as it was and so quickly (1 week). It's reasonable to question why. I assume that you did not sterilze the QT tank after the initial deaths? If the trophonts derive nutrition or utilize other processes from the host, it very well be that they bail out. I wouldn't be surprised if this were the case. I assume that you have access to research papers in this field? If not, I believe that there's at least one or two here that do and maybe they can report back on what they find.

[Minh B.]

Can you capture video of the theronts? It would be really interesting to see that.

 

Strong detergent to lyse the cells. Reminds me of my old Genetics professor who taught us the technique back in the late 70's. PCR came about in the early 80's, if I recall. I had just gotten out of school (and was a couple of years past securing my bio degree) back when it started to be reported.

 

It's remarkable to think that the concentration was as high as it was and so quickly (1 week). It's reasonable to question why. I assume that you did not sterilze the QT tank after the initial deaths? If the trophonts derive nutrition or utilize other processes from the host, it very well be that they bail out. I wouldn't be surprised if this were the case. I assume that you have access to research papers in this field? If not, I believe that there's at least one or two here that do and maybe they can report back on what they find.

 

Unfortunately, our department is not equipped with video capture for a regular light microscope. Instead, we have two $1.5M (that's right...million) fluorescent scopes that are and several regular light scopes equipped with 100X. The one scope that has a camera has a max 40X lens. I'll take a pic w/the 40X but it won't look as convincing as with a 100X and unfortunately, you won't be able to see the theronts being the road runner.

 

Yes, we still use detergents. Easy & less messy than liquid nitrogen. You're right...I did not sterilize the QT. Yes, I have access to almost all research journals as our National Library of Medicine is on campus. And NLM isn't just for medicine, but all general/basic research as well. Let me know if you can't access a paper and I'll get you a PDF.

 

There's actually many research articles on marine ich. Some, to be honest, are quite bad. I came across one that proposed an 'alternative' form of treatment but their study showed ich numbers came back with the vengeance after 3 weeks.

[zygote2k]

If you come up with an easy "ick" test, or one that requires a water sample to be sent in, I/we have lots of business to send your way.

[LCDRDATA]

If the test is sensitive down to 45 pico-grams, as stated, I wonder - how often you could find a tank that was a true negative? My understanding has been that the organism is usually endemic (in most any tank with fish) but has a difficult time infesting otherwise healthy fish in good water conditions to the point that it threatens them. Would you get too many false positives? Alternatively, perhaps the strength of the reaction would be the way to gauge it.

[Minh B.]

If you come up with an easy "ick" test, or one that requires a water sample to be sent in, I/we have lots of business to send your way.

The easiest test is to see the theronts under a 100X mag microscope. But after barely seeing theronts in my infected sump and the tomites being extremely hard to see and recognize, this is unreliable. I still think WAMAS should have a microscope for free swimming theronts and strange looking water (algae/bacterial blooms), which I'd be happy to contribute too. It could be a member-only service like the par meter and nowadays, a decent school microscope cost <$400 and I can look into making dyes that stain cell walls.

 

A DNA test is the most conclusive, especially for asymptomatic fish (I feel like I'm Maury Povich offering paternity tests). My clowns have never displayed symptoms on their skin but I'm confident they must have it on their gills. Plus I want to be confident when it's safe to reintroduce fish to the DT and when the parasites are dead in the QT.

 

I need two more samples to test this assay. One from a DT who religiously quarantines all new fish for the ~month period and the fishes have not shown any signs of disease. Another sample would have to come from a fishless DT (no fish at all) for >2 months but still has LR and inverts. I know the second sample may be a challenge to find. I could get the samples at the meeting.

[Minh B.]

If the test is sensitive down to 45 pico-grams, as stated, I wonder - how often you could find a tank that was a true negative? My understanding has been that the organism is usually endemic (in most any tank with fish) but has a difficult time infesting otherwise healthy fish in good water conditions to the point that it threatens them. Would you get too many false positives? Alternatively, perhaps the strength of the reaction would be the way to gauge it.

 

Ha, I was thinking the same thing as I was typing the last post. That's why I want to test water from a tank that came from someone who religiously quarantines and their fish show no signs or symptoms. It may be that we are looking for DNA intensity and not the presence of its DNA.

[Minh B.]

Oh, but to answer your questions, I can adjust the sensitivity by decreasing the number of PCR cycles if it turns out the healthy tanks are getting a positive result so that it would be just a very faint band. In which case, this test would be like our pH/alkalinity etc. tests that we do routinely but has different degrees. Sorry, I, like many others I'm sure, are in the binary set of mind where we either have ich or we don't, instead of realizing and seeing your point that perhaps healthy systems have ich but are better equipped to deal with it.

 

Oh, samples I test will remain confidential. I'm not going to blast anyone on the forums if the test turns out positive. I'll treat it like how clinics treat their patients and their personal test results. I'd still show the result for this study but won't say who it came from. So please get me those two samples if anyone has them.

[Origami]

Minh, anything special about the microscope? Polarized light, color correction/filtration? I've got a couple of scopes at home (one's a decent B&L, the other's considerably cheaper) and a dissecting stage. Did you use stain when viewing the theronts or were they easily distinguished (sounds like the latter)?

 

Did you get an estimate of how large a theront was, typically? A micron? Several?

[Minh B.]

Minh, anything special about the microscope? Polarized light, color correction/filtration? I've got a couple of scopes at home (one's a decent B&L, the other's considerably cheaper) and a dissecting stage. Did you use stain when viewing the theronts or were they easily distinguished (sounds like the latter)?

 

Did you get an estimate of how large a theront was, typically? A micron? Several?

 

I took water from my DT and was disappointed to not find any theronts. I proceeded to extract DNA and achieved 170 ng/uL, about the same concentration as the QT. Why a lower concentration in the sump, not quite sure. Perhaps skimmer?

 

But to answer your questions, I used a scope equipped w/polarized light or filters without stain. This works well if there's a high number of speedy theronts that glide on by and catches your attention. I may have to make a stain to see these buggers better. My theronts were on the smaller side, ~15 x 40 um. But I've read their size can vary from one strain to the next, their host species & water temp. Are your scopes equipped with 100x? Maybe test drive your scopes with a drop of pods. They should look like giants on your scopes.

[Origami]

I'll have to look. I haven't had either out in a couple of years now. A few years ago, I'd look at cells of various algae, various yeasts and such. I don't know enough about staining to do much there, though would like to learn more about staining and fixing techniques someday.

 

You don't happen to have a fine filter sock going into your sump, do you? If so, maybe it's catching some. On the other hand, it would be notable if their cilia somehow interacted on the air-water interface in a skimmer to make them more susceptible to skimming.