Ichy assay

[Minh B.]

The PCR assay for the presence of marine ich or Cryptocaryon irritans worked well. Results using the common ciliate pair of primers shows an amplicon of the expected ~750 bp in the quarantine tank (QT). Samples from the display tank (DT) and DT sump, despite being able to extract sufficient DNA, yielded no DNA from the parasite. However, this doesn't conclude that the DT and DT sump are free of the parasite. The DT having live rock (LR) and other non-fish livestock may have likely provided the hiding place for the parasite. This test is good, however, for the detection of the parasite in a QT setup that has nothing but a heater, a pump and a pair of asymptomatic clownfishes that survived the ich war while the others did not (a PBT, foxface and a few anthiases).

 

Some background, the PBT died while in the DT on 1/22, the QT was setup 1/20 but no fishes were transferred until 1/24 (the clown pair and foxface). On 1/26 (two days after transferring to the QT), the foxface died and possibly the culprit for the release of excess parasites into the water column. Clownfish pair still remains asymptomatic to this day. 10 mL of water was collected from both DT sump and QT on 1/31 and the main DT on 2/1, and DNA extracted the same day. PCR was done using primers specfic to the ciliate's ribosomal DNA (rDNA) at 35 cycles with annealing temperature of 50C and extension of 15 sec.

[jaddc]

Very cool. How much total DNA did you extract?

[Origami]

The primers are, I assume, specific to marine ich? No chance of confounding with other organisms commonly found in our tanks? I'm surprised somewhat that you didn't detect the organism in the DT or DT sump. Were you surprised by this? Is there any evidence that they could or would "hide" vice drift / swim in the water column? I was kind of hoping that, with some relatively simple testing, you could assess the truth behind the conventional wisdom that "every tank harbors ich." However, if it's possible that the parasite hides, making testing more difficult, a negative result would be indeterminate.

[jimlin]

maybe take a sample from the sandbed in the display? only had experience with freshwater ick which can be found on the sandbed.

[Minh B.]

Very cool. How much total DNA did you extract?

The typical concentration range was 120-170 ng/uL (~120 being the return compartment of the sump).

 

The primers are, I assume, specific to marine ich? No chance of confounding with other organisms commonly found in our tanks? I'm surprised somewhat that you didn't detect the organism in the DT or DT sump. Were you surprised by this? Is there any evidence that they could or would "hide" vice drift / swim in the water column? I was kind of hoping that, with some relatively simple testing, you could assess the truth behind the conventional wisdom that "every tank harbors ich." However, if it's possible that the parasite hides, making testing more difficult, a negative result would be indeterminate.

 

I ordered two sets of primers. This PCR was done nested so I added three primers (two forwards and one common reverse). One pair was not specific to marine ich and is common for the identification of several ciliates and the one extra primer that I added to that pair mix is specific/unique only to marine ich. The PCR from the 3 primers yielded a bright band ~750 (what you see in the image) and a very faint band at ~500 bp, which is the marine ich specific-amplicon. That 500 bp was only observed in the QT but as I didn't take a picture while running the short distance, low intensity bands fade quickly the longer you run the gel to resolve the size difference. I will repeat without the common ciliate primer and only use the marine ich specific primer. But the ciliate primers are only unique to other ciliates so it won't amplify DNA from other organisms -like fish (the paper confirmed this too), snails, crabs, pods, etc., but being that there were no amplicons in the DT or DT sump, it's another testament to how specific these primers are to ciliates.

 

I was absolutely surprised at this result as I predicted the DT to have the strongest intensity. But several factors are against me being 1) the size of the DT (~90 + ~30 gallon sump) vs the QT (30 gallon) which could translate to lower concentration of the parasite in the water per 10 mL; 2) the availability of places that the parasite can adhere to (sand, LR, etc.) that it can't in a QT; 3) a fishless DT vs a QT with a potential pair of hosts. I agree...a negative test would conclude it's indeterminant. Further standardization is required especially for large tanks.

 

maybe take a sample from the sandbed in the display? only had experience with freshwater ick which can be found on the sandbed.

Great suggestion! I was just thinking of a place that I could look next to verify the presence of ich in my DT. Never thought of the sandbed. Thanks. I'll give it a try.

Edited February 7, 2013 by Minh B.

[Origami]

Cool work. Nice to have the right tools to do this.

[Jon Lazar]

Minh,

 

Another way to normalize would be to take baseline samples from a reef tank that has been fish-free for 45 days or so; longer than the total cryptocaryon life cycle.

 

Jon

[AlanM]

Why wouldn't DNA from dead ich parasites from a now clean tank that had once had an outbreak show up in the sample?

[jimlin]

it depends on where the sample was taken.

[Sharkey18]

DNA from dead parasites may or may not show up depending on how long the parasites are dead, the amount of filtration in the tank and the level of degradation of the DNA. (I am guessing).

 

I suspect that the reason for not seeing the ick in the display is a matter of concentration. The sick fish (high ick content) were removed and put into a smaller volume of water, while whatever parasite remained (presumably less than what was on the fish) was diluted in a greater quantity of water. One option would be to remove possible ick hiding places: A piece of rock, a sample of sand bed, etc etc, rinse them in a small volume of water and concentrate. Depending on the size of the tank you could collect maybe 10 to 20 samples and see where ( if anywhere) ick shows up.

 

Cool experiment!

[jimlin]

i just read something on marine ick. seems a little similar to freshwater ick, lays eggs in sandbed, need host to survive, short life cycle. i wonder how long the eggs can remain dormant if there are no hosts present. if the dt had ick, it is likely in the sandbed. http://www.chucksaddiction.com/ich.html

[Minh B.]

Minh,

 

Another way to normalize would be to take baseline samples from a reef tank that has been fish-free for 45 days or so; longer than the total cryptocaryon life cycle.

 

Jon

Unfortunately, being that I could not get ciliate rDNA amplification in the first test from my infected DT, I expect using water from a 45-day fish-free tank would be a bigger challenge as I wouldn't be able to normalize against a sample with no bands/PCR.

 

 

Why wouldn't DNA from dead ich parasites from a now clean tank that had once had an outbreak show up in the sample?

As parasites or living things in general die, one of the first things that occur is the cell and nuclear membranes start to quickly break-down, releasing its contents which will also include DNA in to the surroundings. Once this occurs, bacteria and other opportunistic organisms will start to consume this excess phosphates (from DNA), proteins, etc. A healthy system should have enough good bacteria to break-down all this stuff quickly.

 

DNA from dead parasites may or may not show up depending on how long the parasites are dead, the amount of filtration in the tank and the level of degradation of the DNA. (I am guessing).

 

I suspect that the reason for not seeing the ick in the display is a matter of concentration. The sick fish (high ick content) were removed and put into a smaller volume of water, while whatever parasite remained (presumably less than what was on the fish) was diluted in a greater quantity of water. One option would be to remove possible ick hiding places: A piece of rock, a sample of sand bed, etc etc, rinse them in a small volume of water and concentrate. Depending on the size of the tank you could collect maybe 10 to 20 samples and see where ( if anywhere) ick shows up.

 

Cool experiment!

 

Concentration indeed. Please see the new experiment below with DNA extracted from sandbed as well.

 

i just read something on marine ick. seems a little similar to freshwater ick, lays eggs in sandbed, need host to survive, short life cycle. i wonder how long the eggs can remain dormant if there are no hosts present. if the dt had ick, it is likely in the sandbed. http://www.chucksadd...on.com/ich.html

 

I extracted DNA from the sandbed, but keep in mind it's been over two weeks since the tank has remained fishless. I took the sand from the front of the tank where it was easily accessible (I'm only 5'2", almost fell in myself), but this is still a great idea.

 

Results below. I used the C. irritans-specific primers this time and I used 5X more template DNA in the PCR reaction than the previous test for broad ciliates. Notice also the amplicon/band is not the expected 550 bp, but much smaller (~75 bp). I assembled the gene databases from two independent labs and I have reason to believe that this strain/variant of C. irritans in my tank had a translocation or moved ~480 bp downstream of the end/reverse primer. I was shocked. Two aligned databases showed single base pair mutations and now I see a large translocation. This parasite is rearranging/mutating within the last 5 years. However, the results do confirm that the DT still had ich a week after being fishless. The second image is the incomplete gene assembly from the two gene databases and the yellow region (consensus sequence with the single bp mutation) is basically missing in this PCR. The blue arrows are the location of the two marine ich-specific primers.

 

Could the mutation(s) be a result of growing fitness or selection (i.e. resistance to drugs used to counter the parasite)? Possibly. Has anyone encountered increased drug resistance when treating their fishes?

[Sharkey18]

Hmmm, Something is weird. What happened to the 550 band? Even the QT is showing the 75bp band now, not the 550. Did you use the same DNA for the QT as before or did you take a new sample?

 

A large translocation mutation like that seems like a big leap. Also you used different primers and got different results.

[Origami]

Minh, this seems substantially different than is expected. Are you sure this is C. irritans? What are the chances that the DNA segment comes from a different organism? I suppose that you don't have a quick way to sequence, say, about 75 base pairs now, do you? It would be very interesting to confirm that the 75 bps match the sequences from the database.

[Origami]

Hmmm, Something is weird. What happened to the 550 band? Even the QT is showing the 75bp band now, not the 550. Did you use the same DNA for the QT as before or did you take a new sample?

 

A large translocation mutation like that seems like a big leap. Also you used different primers and got different results.

 

There wasn't a 550 band in the first run. There was one closer to 750 but it used a different primer for common cilliates.

[Minh B.]

Hmmm, Something is weird. What happened to the 550 band? Even the QT is showing the 75bp band now, not the 550. Did you use the same DNA for the QT as before or did you take a new sample?

 

A large translocation mutation like that seems like a big leap. Also you used different primers and got different results.

 

Actually translocations (common in cancers) or transposable elements of this size and even bigger (common in many organisms including humans) are quite frequent in eukaryotes. I was just shocked as it generally occurs under selective pressure (i.e. bacteria will 'jump genes' to favor antibiotic resistance, humans will jump genetic material to create new antibodies to fight disease) or a consequence of disease (like cancer). This transposable element phenomena was first reported by Barbara McClintock in maize and she later received a nobel prize for her discovery. The primers part is half true. For the common ciliate, I used for primer #1 and primer #2. For the C. irritans specific set of primers, I used primer #3 and primer #2 because primer #2 is a common primer found in C. irritans as well as all ciliates. Primer #3 is what gives the specificity of C. irrtans.

 

As for the DT result, the difference is also that I used 10 uL of DNA for the PCR instead of 2 uL. Because the DT was so much larger than the QT, I figured I would compensate for the decreased concentration of ich DNA (if there was any) by using 5 times more DNA for PCR...enough to get PCR to work for the DT. If I retry this test using the 5X more DNA from the DT and the same primers as the first test (primers #1 + #2), I should see a 750 bp UNLESS the primer recognition site was abolished due to a mutation/translocation. Yes, this is the same DNA I extracted and used for the first test, and kept frozen until this second test (DNA can be kept indefinitely if frozen and will not go bad).

 

Minh, this seems substantially different than is expected. Are you sure this is C. irritans? What are the chances that the DNA segment comes from a different organism? I suppose that you don't have a quick way to sequence, say, about 75 base pairs now, do you? It would be very interesting to confirm that the 75 bps match the sequences from the database.

I blasted (http://blast.ncbi.nl...YPE=BlastSearch) primer #3 against the entire genome database (which includes the genome from every sequenced organism) and primer #3 only reported it as C. irritans (with full length, 100% identity). Yes, I can get it sequenced but I plan to redo the nested PCR using triple primers 1 + 2 + 3 so we should get a band at 750 and 75 bp (a double confirmation if you will). I can then cut out the bands of interest and get them sequenced. Would be interesting to see if the ich has 'evolved'.

 

There wasn't a 550 band in the first run. There was one closer to 750 but it used a different primer for common cilliates.

Exactly. There wasn't because the first primer pair gave the expected 750 bp band as the paper reported. The 550 band was supposed to be in this 2nd test with their reported primers, but instead of 550, I got 75.

Edited February 9, 2013 by Minh B.

[Sharkey18]

BTW, thanks so much for doing this. Awesome idea.

 

I've run a PCR or two in my time so if you would like some help I would be happy to assist.

 

Also what is the original paper that you are referring to?

[Origami]

Great stuff. I'm really looking forward to the next run.