# Disease (Chytrid) Testing on large collections



## Roadrunner (Mar 6, 2004)

I`m devising a plan for simple disease (in particular chytrid) testing done on a large group w/ minimal capture and swabing for the tests. Last year was simple as all my tads were in one system and tests only had to be performed on a few juvis to get most of my collection. This year wasn`t so easy. With the addition of many pairs of pumilio I had to capture each animal in a medicine vial and swab them for fear of hurting them, re-gloving 20 different times and using 20 different vials, etc. Not to mention a male granuliferous who started seizing after capture who is now undergoing a daily calcium gluconate treatment, but thats another topic. Not fun but worth the peace of mind. This year I`ve ordered itraconazole and am treating everything that comes in as if it had chytrid before it goes into the collection. I also do fecals for anything new coming in. I`ll feel pretty safe with this method but would still like to get tests done yearly. So my question, mainly for Ed and others working w/ specific parasites and on the board instead of private to give others the idea if it`ll work: if I gather waste water from all my adults tanks(pumilio, tincs, terribilis, etc which is easily done w/out worry about cross contamination because of the spigots on each tank) and set up a clean tank w/ auratus froglets and add this waste water to the tank and test the froglets a month later what all can I gather from the tests? What parasites would transfer thru the waste water? Hookworm and lungworm eggs can float and may be transferred( if they hatched they would likely drown?) although not as likely as say coccidia, chytrid or protozoa. What about things like ranavirus and red lynch disease, which I believe was found present in tadpoles in virginia(although I`m not sure if this was in the pet trade or in the wild). Do they even have tests for these things or is it likely nothing would live very long if any of these nasties were contracted.
This seems as if it would save me and my collection a lot of hassle and needless stress. Sure it wouldn`t be the greatest for the 5 aurautus froglets but they could be cleaned considering I haven`t found any signs of coccidia in my collection.
It just seems w/ large collections and good quarentine procedures a lot of testing is required to keep tabs on everything once everything has been tested clean in the first place.
Obviously if something was found your on to a lot of individual testing but the initial tests could be waste water from pumilio(seperate), waste water from phyllos(seperate), waste water from tincs, azureus and auratus etc. depending on how big the collection is.


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## Dane (Aug 19, 2004)

I love and hate this topic equally. It can be maddening trying to keep tabs on the health of a large or growing collection. If you're treating for Chytrid on all new additions, running fecals, and being cautious with your feedings/waterings, I would say that you're pretty well covered as far as preventing contamination. I recently worked myself into a fervor over some frogs that I thought "might" have Chytrid, and I now know the difficulty of getting a 1/2" pumilio to sit still while you swab his belly. The process is much easier with larger frogs, but caution must be taken in either case to prevent removal of the mucous layer when taking the test. Needless to say my situation turned out fine. I think that your wastewater solution would be a fine alternative to mass-swabbing, but if it did turn out that the frogs became infected, it would be difficult to determine where it came from if you are planning on dumping water from all enclosures into the same test group.


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## Roadrunner (Mar 6, 2004)

If you get 1 clutch of 8 auratus and considering you could pool 8 to a test, you could divide your collection by 8 and put waste water from each of 8 groups to one auratus per group and label. It costs about $50 for 1 test and pooling. If that test came up positive you could then have them test each of the 8 sample($around $200) to find out each groups status. you could then go thru the group or groups to individually test or just treat everyone in said group. At $350-500 for minimal order on the meds and a 3 week waiting period for vets trying to acquire human meds it`s almost not worth testing if you think somethings got chytrid. By the time you get it in the mail it may not even be needed then. I had to buy enough to make about 5 gallons of baths. at about 4 - 6oz. per bath and the fact that it lasts about a year you think I bought too much(considering I only need it for new acquisitions and there won`t be more than 10-20 of them this year)? If I were to try to advertise, ship or distribute the rest I`d be in violation of federal law. I guess, with the circumstances, nobody but someone with a large collection who makes a living off breeding amphibians would be able to afford the meds if anything has chytrid. 
With that being said anyone who would ever be in said predicament contact me privately!
Since snap test kits have to be bought in packets of 50 I may start offering them. If anyone is interested contact me and I can order them. 
Anyone interested in testing contact Pisces Molecular 2200 Central Avenue, Suite F, Boulder, CO 80301, V: 303-546-9300, F: 303-546-9400, email: [email protected]
Note that o-ring screw cap centrifuge vials may leak w/ pressure differential between altitudes differing from boulder, co. I used plastic ziploc bags for each individual container and it was worth it.
This isn`t an issue w/ snap tests(i think).


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## Roadrunner (Mar 6, 2004)

O.K. maybe I figured this wrong. I was told the baths made from the liquid Itraconazole were reuseable and good indefinately(not forever but at least thru one treatment(10 days)). I was told the suspension made from one pill and orange juice was only good for a limited time. 
If a $400/ 5oz. bottle of liquid makes 500oz. and a bath is about 10oz or more it`s about $10/bath. If you have to make a new bath every day for 10 days it`s $80? for treatment as opposed to $20/test or $50/8 tests.
If the liquid suspension diluted is good for 10 days or more and you can use one bath for 5 individuals(one at a time) then it`s only $10 to profalacticly treat 5 animals and $500 would be good to treat 50 groups or more(the reason I bought it thinking treating new acquisitions instead of testing was cheaper).
I know the shelf life of the liquid is a year or more but how long does it last when it`s mixed w/ water.


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## Ed (Sep 19, 2004)

Some comments,

At work all incoming animals are tested via three fecals for any intestional parasites. If they originated in a group (say siblings) then group fecals are fine as opposed to individual fecals. Then the animals have thier fecals checked twice a year after that (although if they are then housed in a group a single fecal is used to check the enclosure) unless there are health issues noted when multiple fecal submissions maybe required. 

the water would not drown the hookworm or lungworm larva so yes if either of these are present in the collection it would facilitate thier dispersal into the wider community and lungworms can be very difficult to clear... 

Yes you could easily spread ranaviruses, iridioviruses etc through the collection. Red leg is a little different as this is typically caused by one or more species of bacteria that are always found in the enclosures we use to house amphibians. This typically only shows up when the amphibian is immunosuppressed for some reason. 

When I last looked into the testing of chytrid they were working on a PCR test on water the frog was soaked/washed in but hadn't gotten it to the point where it was reliable. 

We automatically test/treat all suspected amphibians for chytrid. Species that are very sensitive such as Atelopus ssp and Bufo baxteri are prophylactically treated upon arrival. 

The problem with itraconazole is that it is an acidic solution which is why it caused problems in the initial testing of its effectiveness and needs to be buffered to a neutral pH (hence the use of ARS) so I am at a loss why you would want to mix it with orange juice which is already a low pH.... 

We make the baths up daily from the liquid itraconazole suspension and discard after treatment. You can treat animals in groups in each bath (1 liter is a typical bath amount for us) although I would use different baths for each group to prevent the spread of coccidia, lungworms etc... 
The liquid suspension as long as it is kept out of the light (dark containers) and cool is supposed to have a reasonable shelf life. 


Ed


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## Roadrunner (Mar 6, 2004)

Holy shcrap, you know how expensive that is? 1 liter of suspension is about $10, that`s $110 to treat a group for 11 days. I guess testing new arrivals would be cheaper. Guess I should`ve got my facts straight before I ordered the stuff. But then again if I wait for a positive I have to wait another 3 weeks for the drugs. What a pain in the ass. 
I know it was long and drawn out but you kinda missed some of the gist of that Ed. Red Lynch is the name of a disease first documented in Red Lynch Australia which has also shown up in tadpoles in Virginia. I wasn`t worried about treating multiple groups from one bath, just multiple individuals(same group) from the same bath over the course of treatment. If they had hookworm or lungworm that could be taken care of after treating the chytrid(possible chytrid).
My whole idea is to reduce the total # of fecals and chytrid tests by culturing anything present in the collection in one frog from all the tanks wastewater. I see that can be done easily from what you suggested w/ parasite transfer from sharing itraconazole baths. 
The Itraconazole pills are supposed to be crushed and suspended in an oz of orange juice or something similar then it`s supposed to be diluted 1oz to 100 oz. of water. The liquid is already suspended and just has to be diluted. I was told the stability of the orange juice suspension is limited while the liquid is more stable. I thought this meant that the bath made form the liquid, not the pill, was ok to use for a while too.


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## Ed (Sep 19, 2004)

Do you have a reference for the appearence of RedLynch virus in tadpoles in the USA I can't find any references on a quick search although there are plenty of references for iridio and rana virus outbreaks... 

But dissolving the tablet in the OJ still doesn't resolve the issues around the low pH (around 3-4)(see http://www.cfsan.fda.gov/~comm/lacf-phs.html) which can be a problem for the frogs. 

Ideally what you want to get is the 1% solution (Sporanox, Janssen Pharmaceutica, Inc) and then make the solution as needed by buffering in Amphibian Ringers. 

Ed


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## Roadrunner (Mar 6, 2004)

your dissolving 1 tablet in 1 oz. o.j. and diluting it 100 fold with water. there is only 1oz of acidic liquid and when it`s diluted I imagine the ph comes back up a couple points depending on your water source(mines around 8). that`s wierd, the smiley is supposed to be an eight

The article about the red lynch disease was in reptiles magazine(I`ll have to find the article when I get back).


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## Grassypeak (Jun 14, 2005)

Aaron, diluting an acidic solution with neutral water by 100 fold should bring the pH up by exactly 2. As you probably know pH is a logarithmic scale, with each point going down representing 10X the [H+] (hydrogen ion concentration). Sorry if that is exactly what you meant by bringing the pH up by a couple of points.

To Ed’s point if the pH of the orange juice + medication solution is down around 3 the dilution will only bring the pH up to 5. I’m not sure of the point at which [H+] begins to bother the frogs though. Maybe Ed can shed some more light on this one.

On another note, as one of your customers, I’m glad to see that you are doing so much to mitigate parasite infestation. I hope that you are able to find a happy medium between parasite problems and driving your self nuts with prophylactic treatments.


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## Ed (Sep 19, 2004)

There appear to be differences in different genera with thier ability to tolerate the low pHs, the problem is that most of the good data has been done with tadpoles and only with some notations when adults died from pH issues (like in the original treatment article). 

The orange juice is from bird treaments and is used in birds that do not have a sufficiently acidic stomach to dissolve the itraconazole pill (itraconazole doesn't dissolve unless its in an acidic pH). 
This is why using the 1% solution gets around that issue. 

The reason I questioned the Red Lynch is that to my understanding and publications is that this is an uncharacterized herpes virus. Given that it is uncharacterized I find it hard to believe that the same virus is suddenly showing up in Virginia given that Australia doesn't export tadpoles... I would not be surprised if it was a similar herpes virus but I would be very surprised if it was the same virus.. 


Ed


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## Roadrunner (Mar 6, 2004)

Ahh, my fault. They jumped from Red Lynch in the article to something they just called a "respiratory disease"(also from australia) which was likely caused by a soil fungus which produces a mycotoxin. It was said to probably affect snakes also. It turned up in oklahoma in a white`s treefrog and in virginia where infected tadpoles were sold.
I get confused w/ log scales. I just figured 1 oz w/ a ph of 8 added to 1 oz w/ a ph of 3 would get you 2 oz w/ a ph of about 4. I guess your right. Although it doesn`t seem 1 oz of oj would keep it that acidic your working w/ an ounce that is 10,000 times more acidic than the water is basic, thats what it comes out to.


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## Rain_Frog (Apr 27, 2004)

not trying to hijack, but have there been other anti-fungal medications being tested? From the vets and people I've talked to, this medicine is extremely expensive for the average hobbyist. You might as well just try and be fastidious to prevent contamination in case some of your animals (like clawed frogs or certain salamanders) are asymptompic.

Ed, you once told me in a PM that ketaconazole will not work (which is for animals). Have there been any new drugs tested that are already used for animals? (therefore, the medication *should be less expensive).


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## Ed (Sep 19, 2004)

I think ketoconazole will work fine for chytrid but I do not think the frogs will tolerate it. Unlike itraconazole which is still effective at higher pHs (once dissolved and stabilized at the lower pH), ketoconazole decreases in effectiveness as the pH increases so achieving a theraputic dose without killing the frogs is the problem (unless you are going to administer the doses orally and the stomach of the frog is sufficiently acidic to activate the ketoconazole (although the Merk manual makes it look like oral treatments may be ineffective (see http://www.merckvetmanual.com/mvm/index ... 170104.htm)). 

I would suspect that any of the azole group of antifungals would work provided that they are effective at higher pHs (also ketoconazole can have reproductive disruption side effects in mammals so it is also possible in frogs) and a safe dosage can be worked out (may require testing, treatment and retesting of the frogs... 

Since the discovery of the effectiveness of itraconazole solution, I suspect that there is less study to see what else is effective as a treatment option as it is safe and effective and simple sterilization methods will control the Zoospores.. 

Ed


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