# Some questions about chytrid



## trinacliff

With all this talk about chytrid, I'd be interested in hearing a few facts about it if anyone knows the answers to some questions...these all pertain to chytrid and how to keep our collections safe. Some of these might be dumb questions, but my knowledge on chytrid is very limited to what I've briefly read on here.

1) How is it spread? i.e. only if I touch something that harbors the fungus then touch something that I use within my collection OR if something with the fungus is in the same room as another frog

2) How is it killed if we come in contact with it? (will hand washing do it? Or something more potent?)

3) How long does it usually take to show up if a frog has been infected? (another way of putting this is at what point do we know any "farm raised" frogs or newly aquired frogs that we are quarantining are clear of chytrid? I hate to worry everytime I buy a new frog if the person I'm buying from has unknowingly exposed his/her frogs to chytrid.)

4) Aside from quarantine, what are the best ways of preventing this from affecting our collections? 

My main concerns are twofold... first, my son is always wanting to bring frogs into the house, keep them for a day, then lets them go...he's into catching tadpoles and raising them until they are froglets so we can let them go. Second, I have some of the imports...they have been kept seperate since I got them, so I'd like to know when it's "safe" to allow them into the same room as my other frogs. Is this the same as general quarantine procedures or should other measures be taken before adding them to my main frog room? 

Thanks from a chytrid numbskull... 

Kristen


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## JJuchems

Chytrid is a fungus that feeds on dead tissue and causes neurological damage. Toe curling has been seen in White’s Tree Frogs along with loss of mobility. I do not have my notes on it in front of me but when I do I will make a better post. It is treatable in captivity. It has been shown to have no effect on clawed frogs and bull frogs. 
Later- Jason Juchems


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## Ed

snip "1) How is it spread? i.e. only if I touch something that harbors the fungus then touch something that I use within my collection OR if something with the fungus is in the same room as another frog "endsnip

Through infected moist/wet items, water and direct contact from amphibian to amphibian. So yes if you do not wash your hands then you can transfer it to another enclosure. 

snip "2) How is it killed if we come in contact with it? (will hand washing do it? Or something more potent?) "endsnip 

To date most disinfectants will kill the infectious stage however it is more problematic to clear infected frogs. 

snip "3) How long does it usually take to show up if a frog has been infected? (another way of putting this is at what point do we know any "farm raised" frogs or newly aquired frogs that we are quarantining are clear of chytrid? I hate to worry everytime I buy a new frog if the person I'm buying from has unknowingly exposed his/her frogs to chytrid.) "endsnip 

Off the top of my head, it can occur in as little as two weeks or the infection can last for years pending the conditions needed to cause pathnogencity. 
The current recommendations and laws in many states prohibit the collection and release of animals once they have been brought into captivity (typically covered under the rehabber permits and laws) without meeting some criteria. 

snip "4) Aside from quarantine, what are the best ways of preventing this from affecting our collections? "endsnip

Simple quarantine is insufficient as the frogs can be asymptomatic for long periods of time. 

snip "My main concerns are twofold... first, my son is always wanting to bring frogs into the house, keep them for a day, then lets them go...he's into catching tadpoles and raising them until they are froglets so we can let them go."endsnip

See above for the comments on this..

snip " Second, I have some of the imports...they have been kept seperate since I got them, so I'd like to know when it's "safe" to allow them into the same room as my other frogs. Is this the same as general quarantine procedures or should other measures be taken before adding them to my main frog room? "endsnip

Given that chytrid has been found at both importers and Zoos, I would suspect that cb, farmed and wc frogs are all at risk of being infected... 

Ed


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## Ed

snip "Chytrid is a fungus that feeds on dead tissue and causes neurological damage"endsnip

While other chytrids feed on dead tissues (mainly plants) this one so far appears to attack keratin in the skin layers and not just the outer layers of dead skin. So I wouldn't want to restrict it to only dead tissues. If it only attacked the dead layers of the skin, then it shouldn't be able to kill the amphibians.

Do you have a reference for the neurological damage? The last articles I have seen have not determined the actual manner in which chytrid kills the frogs. 

Ed


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## JJuchems

Good Post Ed. I do have to disagree with your comment on quarantine. Quarantine is always a safety net. An animal may show symptoms during this period, through the animal may not show signs for several months. I honestly believe few folks properly quarantine. 

Later and Happy Frogging,
Jason Juchems


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## Ed

With respect to chytrid, simple quarantine is not an effective method to detect chytrid because if the frog is kept outside of the lethal temperature range for chytrid, then the frog will be asymptomatic throughout quarantine but still infectious. This period can potentially last for years allowing for widespread dissemination of the fungus. If the temperature drops down into the range then a massive outbreak can occur resulting in massive mortality in a very short period of time (for example this has been documented at bullfrog farms). 
Quarantine with either at least one PCR test or in very high risk amphibians (such as Bolitoglossa dolflini, Atelopus sp) prophylatic treatment with itraconazole is required to prevent the establishment in the collection. 

Ed


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## JJuchems

"chytrid fungus which impacts the nervous system, causes curling of the toes and a yellowish discoloration." Reptiles Feb. 2006 

Not a great source I know. 

Later and Happy Frogging,
Jason Juchems


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## Ed

This could be a neurological sign but it could also be due to a disruption of the osmotic balance...... 

Ed


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## Dane

> Quarantine with either at least one PCR test or in very high risk amphibians (such as Bolitoglossa dolflini, Atelopus sp) prophylatic treatment with itraconazole is required to prevent the establishment in the collection.


I've tried several times to get a straight answer as to the procedure of a PCR test, as well as contact info for a lab that can run the test, but with no avail. Can anyone shed some light on either?


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## Ed

see http://www.open.ac.uk/daptf/declines/decl2.htm 


Ed


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## Rain_Frog

Yes. I have contacted Pisces Molecular and they charge 21 bucks for a PCR skin swapping and they sent me some documents how to properly collect the sample. Unfortunately, they may take a few few weeks before your results will be finished.

Dane, basically you hold the frog firmly and get a cotton swab and rub the belly of the frog multiple times and you put it in a mixture of ethanol and water. Another option, you can ship the sample with cold packs overnight or 2nd day, I forget what the guy said, but it is to prevent other molds from attacking the dead keratin and ruining the diagnosis for chytrid zoospores.

However, it has a 3 in 15 sample failure rate, so it may be best to send in two samples. Additionally, dead amphibians you can send in toe clippings or skin pieces for diagnosis.

I was wondering. What about collecting shed skin? Many types of amphibians like clawed frogs (which I will be acquiring shortly) leave dead skin to float in the tank, allowing for easy collection.


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## Dane

What about toe clippings from live frogs? I've heard of this being done for other procedures, and I don't imagine that the experience would be much more traumatic than a belly swab. 
I was also wondering, if the fungus isn't airborne, and can only be spread through moisture contact, how is it that it can threaten an entire private collection? It would seem that normal safety protocol would prevent it from becoming widespread.


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## Rain_Frog

that's what I'm debating too Dane. Should I just blow off treatment, and just be cautious, or should I take the extra step and treat them if need be? Dr. Frye told me he can order itraconazole but it is pricey. We never followed up on a price quote, so I cannot give you a ballpark figure.

I am wanting to acquire some Xenopus frogs which are known to be asymptopic. (I cannot take it anymore, I'm getting some pipidae no matter what!) Trying to restrain some of the smaller pipids is challenging, even holding onto a slippery large X. laevis is not an easy task.

Many people here on the board keep plenty of caudates, like A. tigrinum which can harbor chytrid. Michael Shrom keeps many species of pipids, caudates, etc. which can harbor the disease and not die from it. As far as I know of, he hasn't run into chytridiomycosis.

But, if you remember, Devin Edmonds in 2002 lost his entire 25+ collection of dart frogs (and I think mantellas?) within 2 weeks. It was a big mystery. He never was conclusive, but when we all started talking about chytrids (and from the symptoms of the animals on their last hours), he seemed to conclude that this was most likely the reason what killed his former collection. How it got in, we may never know.

I think there is plenty of helpful information from Xenopus labs what to do about chytrid. On a few pages, there are protocol steps for preventing the disease spread because Silurana tropicalis (another common lab xenopus) is not resistant to the disease.

However, there recommendation for treating the disease involves formalin, which Ed Kowalski told me that it never truly destroyed the disease and it is carcinogenic.


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## Ed

snip "I was also wondering, if the fungus isn't airborne, and can only be spread through moisture contact, how is it that it can threaten an entire private collection?"endsnip

It depends what you mean by airborn. If you are referring to transfer on dust motes then no it isn't transmissiable via airborn methods but if you have some thing that sprays droplets of water then it is possible for it to spread via airborn (at least until the droplet dries out). Anything that is moist or damp can transfer the zoospores. This can potentially range from unwashed hands to insects... 
If for example when adding water to an infected tank, you get some splashback that contaiminates your water container the zoospores may be able to surivive in the container and if there are sufficient nutrients reproduce. (see http://tinyurl.com/z2r3p) <In Ed's own words: snip, Catfur> This could even include a back flow from a mister nozzle (and give that the zoospores are active they could colonize a misting system... and from there multiple enclosures. 

As for the toe clippings, this is much more invasive and stressful then a simple restraint for a swab as you haven't tanken into account the stress from the toe clip and the effects of the pain and open wound... 

Ed


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## Jeffey_Kasparek

Just a few other things I've noted...

Itraconzole was recommended a number of times when quarenteening new specimens for chytrid. Other things that my research projects have also revealed to me thus far which I am looking to explore further next semester.

1.) If temperatures exceed 31 degrees celsius constantly for five days, all the chytrid dies off. However, darts typically wouldn't be able to survive this.

2.) When frogs (and particularly salamanders) become visibily infected with chytrid, often there are dark rings of melanin that form around infected sites. Later it was discovered that this helps to keep the chytrid from spreading, or trapping it.

3.) Additionally, chytrid does well in very humid environments because the spores can survive being airborne. Another anomoly that I later managed to solve was how control groups were becoming infected with chytrid when the study subjects were in the other room. The answer: Arachnids, a single egg case can contain up to 500+ eggs that can transport hundreds of thousands of zoospores per baby when they hatch. They managed to move across the room and right into our control groups tanks because the food was available there. Also considering spiderwebs are nearly indestructible, chytrid can remain there for quite some time. 

Driving through local pond areas infected with chytrid can bring it right home with the mud on your tires, or your boots. 

I'm not trying to scare the (*&*% out of everyone, but just wanted to let you know of the transfers. Personally, I would try to get a frog from the pond that your son has been collecting from and take it in for a PCR. I work primarily with salamanders in the lab, and they don't eat their own slough so I can use that for reference, unlike most frogs. Take care and happy frogging,

Jeff


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## Dane

> They managed to move across the room and right into our control groups tanks because the food was available there. Also considering spiderwebs are nearly indestructible, chytrid can remain there for quite some time.


How is it that the spiderlings were able to get into your control groups? Did you use larger mesh on the vents? Also, what ambient humidity levels will allow Chytrid to remain viable while not in contact with a host or water source?


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## Roadrunner

i thought chytrid would die at 98 or human body temps?


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## Jeffey_Kasparek

I'm pretty sure that human body temps would be sufficient to kill of chytrid as well, but a continuous exposure of five days or longer at 30 degrees celsius wiped it out completely. I didn't check for the ambient humidity around the control groups, but yes the vent holes were apparently large enough to allow the spiderlings to get in there. I am also not sure where I remember reading this, but there was also something I remember reading (and possibly it was in a thread here on dendroboard) about someone finding chytrid spores on a preserved specimen. If I can find another study to (I might have to dig around a bit) there was also another case where I think american cockroaches helped to transfer chytrid. If I find it I will post it. 

Additionally I think that 30 degrees celcius is close to about 88 degrees farenheit. a lot lower than human body temps


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## Jeffey_Kasparek

Its going to take a little while to relocate the article that I was looking for with regard to the insect transfer, but here are some abstracts from some other articles that you guys might be interested in:

First off, from the look of things chytrid has been around for a while, so its curious to think that it would be so fatal all of a sudden, which is why I kinda go with the UVB theory and melanin concentrations as sufficient buffers. Anyhow there's clip of an abstract:



> Rates of chytrid infection in 1990 to 2001were not statistically different from those in 1960 to 1969. No chytrid infections were found in 440 specimens from 23 other countries. Chytrid infection has been present in North American amphibian population since at least 1961. The findings suggest that chytrid fungus is common, probably enzootic, and was previously unrecognized in wild amphibians. Batrachochytrium dendrobatidis is widely distributed in apparently healthy amphibian North American amphibians, suggesting that the recent devastating outbreaks of chytridiomycosis may involve complex underlying causes and predisposing factors.
> 
> Conservation Biology, vol. 19, no. 5, pp. 1431-1440, October, 2005


And then also this little tidbit (you might want to try to read the full article to understand the implications, but basically chytrid is thriving in areas with much lower relative humidity).



> The fungus is considered an aquatic pathogen because it requires water for zoospore transmission, cannot survive desiccation, and in the wild is associated only with aquatic habitats. Researchers recently reported the first case of chytridiomycosis in a terrestrial amphibian, a wild-caught Jemez Mountain salamander (Plethodon neomexicanus), a species endemic to the dry slopes of the Jemez Mountains of New Mexico. The infected gravid female had typical infection around the vent. Cytological and histological examination showed thalli and zoosporangia of B. dendrobatidis. A polymerase chain reaction (PCR) assay detected B. dendrobatidis deoxyribonucleic acid (DNA) in the skin of the ventrum and tail. This is the first report of B. dendrobatidis in a strictly terrestrial species and suggests that the fungus can survive in terrestrial habitats, although the transmission route of the pathogen in terrestrial habitats is not known. The discovery of chyridiomycosis in a wild-caught terrestrial amphibian significantly expands the potential host-range of B. dendrobatidis and increases the number of amphibians that may be at risk. Terrestrial communities may play a crucial but previously unrecognized role in the distribution, persistence, or dispersal of this pathogenic fungus.
> 
> Journal of Herpetology, vol. 36, no. 3, pp. 248-249, September, 2005


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## Ed

I thought it took 8 days at 30 C to kill 50% of chytrid in cultures? (see http://www.mycologia.org/cgi/content/full/96/1/9?ck=nck) not a 100% mortalityof the fungus. Does your research refute this? 
In the published literature, it took temperatures of 37C or higher to clear chytrid in Litoria (see Woodhams, D. C.; Alford, R.A.; Marantelli, G. June 2003. Emerging disease of amphibianscured by elevated body temperature. Diseases of Aquatic Organisms 55: 65-67). 

I would expect a 50% reduction in a culture to be significantly different than if this was applied to the amphibian as a potential cure. 

At this time it looks like there is informatiom coming out that indicates that an adult population maybe able to be infected with the fungus but it kills 100% of the froglets when they metamorph. 

With respect to the conservation biology article, they are assuming that because there hasn't been a documented decline in the affected amphibians that hasn't been one. This is a potential flaw in the article when you consider that the status of most non-game amphibians has been unknown for decades in most states (data deficient) and has only really gotten good attention since the inception of the DAPTF.

Chytrid has long been considered able to survive in moist or damp soil for potentially long periods of time. 

Ed


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## Jeffey_Kasparek

> With respect to the conservation biology article, they are assuming that because there hasn't been a documented decline in the affected amphibians that hasn't been one. This is a potential flaw in the article when you consider that the status of most non-game amphibians has been unknown for decades in most states (data deficient) and has only really gotten good attention since the inception of the DAPTF.


- Good point :roll: .

Great find on that article! Boy this should definitely help stir some things up! Let me know if you find anything on UVB exposure later. Anyhow, I had trouble locating this article, perhaps you will have better luck than I will, but it has been referenced in a few articles I've looked at. I think there have been a few misconceptions which might have unfortunately been passed on and I apologize if I contributed. According to 


> Longcore, J.E., Pessier, A.P, and Nichols, D.K. (1999) Batrachochytrium dendrobatidis gen. et. sp. nov., a chytrid pathogenic to amphibians. Mycologia 91:219-227.


 there is a cessation of growth at 30 C, which I've seen other studies infer to mean that *all *the chytrid died off, including the spores. We haven't gone in to entirely validating that claim as when exposing the salamanders to a heat bath of 30 C they were able to clear themselves of the infection without any reoccurance. I'll try to get you some more links when I can. Thanks for you help,

Jeff


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## Jeffey_Kasparek

> At this time it looks like there is informatiom coming out that indicates that an adult population maybe able to be infected with the fungus but it kills 100% of the froglets when they metamorph.


I remember hearing something about that too. I believe someone that got an initial look at an abstract was telling me that the damage to the gills and mouth from infection made respiration exceedingly difficult but I wasn't aware of the morality rate being that high :shock: . 

I like the fact that I get to work with some great scientists and participate in this global endeavor, but at the same time I think I'm nuts to be working with chytrid.


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## Ed

snip "I remember hearing something about that too. I believe someone that got an initial look at an abstract was telling me that the damage to the gills and mouth from infection made respiration exceedingly difficult but I wasn't aware of the morality rate being that high "endsnip

In a couple of the documented cases most/all metamorphs were dead within a few days of metamorphosis (I can't find the reference off hand). 

Tadpoles can be carriers for chytrid and can be unaffected by the fungus. The fungus infects the keratinized mouthparts and is harbored there until it metamorphs when it affects the skin (I believe the bilbliography in this article covers it, http://socrates.berkeley.edu/~es196/pro ... /lyser.pdf). 

I have a copy of the article you are referencing somewhere but I can't lay my hands on it as I am redoing my library area but that is why I cited the 2004 article whose test results are as follows snip "Exposure of cultures to 30 C for 8 d killed 50% of the replicates. "endsnip. If I remember the article you reference correctly, there is a cessation of growth but the fungus doesn't die. It remains static and resumes growth as soon as the temperatures cool down again. 

At least some caudates are apparently more resistant to chytrid than the anurans found in the same reason so this may be interfering with the tests.. Do a literature search for Ambystoma and chytrid it should pop up. I'd do it but my computer is acting weird at the moment. 

Ed


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## Jeffey_Kasparek

Yeah, Dr. EW Davidson is heading a few projects down at ASU with some intermittent help from Dr. Collins, though he's not currently at the university. This is what I am planning to explore next semester but I'll definitely need to do a literatue search first to make sure Blaustein hasn't beaten me to it. At least with ambystoma tigrinum, there are dark circles surrounding chytrid. If UVA increases the amount of melanin present and UVB intensifies it, could varying amounts of exposure to UV act as an immunization for these amphibians, or are the concentrations of melanin secondary effects, wherein the UV exposure would only compound the problem. I plan to exposue the salamanders to UV for four weeks before actually attempting to innoculate them, and study the difference between them, and the other control groups. Hopefully it should be done next year. Be really nice to have you aboard on this Ed


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## Ed

snip "If UVA increases the amount of melanin present and UVB intensifies it, could varying amounts of exposure to UV act as an immunization for these amphibians, or are the concentrations of melanin secondary effects,"endsnip

I think that the increase in pigmentation is an artifact of the infection and not an indication of an immune response but given that we know so little about the acutal effects on the physiology of the amphibians its still worth investigating. I would suspect that the caudates are able to better resist the infection possibly due to differences in thier ability to osmoregulate. 

snip " wherein the UV exposure would only compound the problem. I plan to exposue the salamanders to UV for four weeks before actually attempting to innoculate them, and study the difference between them, and the other control groups."endsnip

Given that excess exposure to UV can also potentially immunosuppress the salamanders, this may increase susceptiablity to chytrid. I would suspect that at least some Ambystomids may either have a more effective immunoresponse at the temperatures where chytrid is most active and/or peptides that are effective against the fungus. 

snip "Hopefully it should be done next year. Be really nice to have you aboard on this Ed"endsnip

Thanks for the offer, given that I'm just a Zookeeper, I'm not sure how I would have any value. I just try to keep up on the info and share it.... 

Ed


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## steelcube

Whimzikal, Ed, and others,

After reading a few articles about chytrid, I am just wondering, how sure are they about chytrid being the primary cause? 

What if the chytrid infection is the aftermath, a secondary infection...

How about viruses? say attaching themselves to the spores? 

Have there ever been a test to eliminate viruses from being the primary infection?

just wondering...


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## r90s

For some more information there is a "mp3" radio show on this subject.
It can be played with a normal player (not just itunes).










Direct Link

Clyde


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## Ed

snip "What if the chytrid infection is the aftermath, a secondary infection... "endsnip


They have isolated chytrid and infected amphibians with it from pure cultures of the specific fungus and had it kill the amphibians. 

snip "How about viruses? say attaching themselves to the spores?"endsnip

There hasn't been any signs of this on electron microscopy of the zoospores nor signs of viral infection in the amphibians upon histopathology of the amphibians in question (and I believe that several new viruses have been detected due this sort of vigilance. 

snip "Have there ever been a test to eliminate viruses from being the primary infection? "endsnip

I have not seen all of the literature as of yet but in what I have read there aren't the signs of viral infection on histopathology of the affected amphibians (and see above). 

It also (as I understand it) doesn't really fit a viral etiology. 

Ed


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## sports_doc

Ed said:


> snip
> 
> I think that the increase in pigmentation is an artifact of the infection and not an indication of an immune response but given that we know so little about the acutal effects on the physiology of the amphibians its still worth investigating.
> .
> .
> .Ed


Ed,
Is this a common feature of infection? a change in pigmentation? I am curious.
Any photos of infected animals available?

Shawn


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## Ed

Hi Shawn,

I don't this is that common in anurans but dark pigmented patches have been noted in infected Ambystomids and some infected Plethodontids ( If I remember correctly this article has some good pictures of the pigmentation changes in the ventral surface of the salamander. Cummer, Michelle R.; Green, D. Earl; O'Neill. Eric M.; 2005; Aquatic chytrid pathogen detected in terrestrial plethodontid salamander, Herpetological Review 36(3):248-249)


Ed


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## r90s

Please Note The Links Avaliable In "Science & Conservation Section"

Endemic Infection of the Amphibian Chytrid Fungus in a Frog

Clyde


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## fishdoc

*sampling and shed skin*

Just a couple of additional notes:

we work with the san diego zoological society on chytrid, and the current recommendation is a small tipped plastic swab, shipped directly. no alcohol. It appears that air drying is preferred. 

Sloughed skin can be deceptive, at least in frogs. Often the part that is sloughed contains empty thalli, with the zoospores already released, meaning very little DNA remain for detection. 

Toe clips are not good, often the fungus is diffuse, and most often inhabits the interdigit webbing, so a stomach swab, or a lone toe clip can miss an infected animal.

Their are two types of PCRs for chytrid, one of which is a 'standard' PCR, that determines presence or absence, and the other is a quantitative PCR that actually determines the infection intensity, and can amplify one zoospore.


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## Beau

Is there any guidance on making plants safe? From what I've read, they can pretty much keep the fungus forever, and baking your plants would kill them. So... quarantine is pretty much out of the question. Is there any way to make the plants that are in your yard safe? I have lots of moss that grows around the house and they'd go great in the viv but I'm kinda scared.


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## Ed

You would need to rinse the plants with a disinfectant... 

At the last AZA Amphibian TAG, there was some discussion that there is some indication that some strains of chytrid are already becoming reistant to itraconazole.. In addition, some colonies of anurans that were maintained in isolation once cleared of chytrid, suddenly developed chytrid and had a significant mortality... 
There isn't any publications on this yet (that I am aware of as this is current data..)

Ed


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## Dendrobait

http://flippersnfins.yuku.com/topic/993

Claims benzaklonium chloride worms


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## Ed

the benzalkonium treatment has been floating around for awhile now as it was originally used in Australia but if I remember correctly (I don't have time to dig up the following references) it works on the zoospores but not on the actual infection in the skin and if the idea that there is a resting spore pans out it won't affect it. 

Ed


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## Ben_C

Chloramphenicol works as well...


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## Ed

Ben_C said:


> Chloramphenicol works as well...


Has the actual paper been published yet? All I have seen so far is the media release and when some of the vets I know went to the chytrid conference the poster wasn't manned the couple of times they stopped by... (Dr. Wright was one of them...) 

Ed


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## Ben_C

Hi Ed,
As far as I know, it has not been published. However, I was at the conference and had a chance to discuss the work in addition to actually using chloramphenicol to treat an animal which was quite infected and experienced a recovery.
However, still not peer reviewed...[as far as i know]
~B


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## Ed

Ben_C said:


> Hi Ed,
> As far as I know, it has not been published. However, I was at the conference and had a chance to discuss the work in addition to actually using chloramphenicol to treat an animal which was quite infected and experienced a recovery.
> However, still not peer reviewed...[as far as i know]
> ~B


Did you swab it to see if it actually cleared it or was the fungus still present? The articles I have seen have been unclear on this aspect of it. 

Ed


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## Ben_C

> Did you swab it to see if it actually cleared it or was the fungus still present? The articles I have seen have been unclear on this aspect of it.


Swabbed, yes but only to check for the presence of zoospores. I did not do any PCR. However, it is still alive, 60 days later where the expected mortality in this species is approx. 40 days.
~B


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## dan

Just a mention of the current protocol used at the National Aquarium in Baltimore from a conversation with Kat Hadfield, MA VetMb MRCVS:
To Control Chytrid in Collections:
Amphibians:
1)Itraconazole 0.01% TO 5 mins SID 14d for all in-contacts
2)Chloramphenicol is effective, but may cause aplastic anemia
3)increase temp to 30-37 C
4)alternatives (unfortunately I don't have more info) involve the use of cutaneous antibmicrobial peptides/bactera with antifungal peptides in brooders
Fomites:
- disinfect with 1% bleach and rinse/dry/freeze
- Chytrid survives 4-7wks in water + 3mo in leaf litter/soil

With regards to quarantine:
- test animals (cyto/histo/PCR) and treat while maintaining quaratine
- test, heat/sterilize water, sterilize/withhold organic material

Good Luck!
- Dan


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## Ben_C

> - disinfect with 1% bleach and rinse/dry/freeze


Do you mean 10% bleach?


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## NCStateHerps

hmm...in regards to elevating temperatures, I wonder how _Phyllobates_ sp. cope?


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## slaytonp

Ben_C said:


> - disinfect with 1% bleach and rinse/dry/freeze
> 
> 
> 
> Do you mean 10% bleach?
Click to expand...

1% to 2% bleach solution is more reactive than stronger solutions, and thus more effective. (Stronger isn't always better.) So I'm sure this recommendation is correct. 

However, I'm also curious about how a raise in temperature to 30 to 37C or 86 to 98.6F. would affect the Phyllobates. I lost a group of them when the tank temperatures got to nearly 90 for several days in a row one summer.


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## bickell

Does anyone have the protocol for prophylactic treatment of frogs with itraconazole? If so would you please post it or a link to it?

Tks 
Al


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## elscotto

Al-
Here is a link with info regarding treatment of captives with chytridiomycosis:
http://www.open.ac.uk/daptf/froglog/FROGLOG-46-1.html
There is also some useful information from Amphibian Ark on detection protocol (which also links to the treatment page):
http://www.amphibianark.org/chytrid.htm
-Scott


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## krharmut

I know there have been tons of replies to questions about chytrid. Here is a nice article about it with info on labs who will test for it.

http://www.amphibianark.org/chytrid.htm


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## earthfrog

*Re:*



frogfarm said:


> i thought chytrid would die at 98 or human body temps?


 It was said that chytrid dies at a temperature of 95 degrees or greater of 6 hours duration. I would isolate the frogs, treat them with the lamisil spray method of 10 sprays per 200 mL of water for five minutes a day lasting one week. If you can manage it during the summer and you live in the southern US, simply set your FROGLESS tank out in the shade (covered with black paper to help prevent plants getting burned by the sun) and just let the chytrid die off over the course of the day, then return it to its original location. Otherwise, order Dr. Frye's itraconazole and skip the risky Lamisil treatment, but still treat the tank and DO NOT use the current insect cultures in the new tank. Discard them and order new ones. Sterilize all tank instruments with 1 gallon of water/2 ounces bleach/2 ounces vinegar. Let sit in solution for 10 minutes, then rinse very well. 

Always use hand sanitizer before and after handling frogs and/or tank-related equipment or food.


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## Ed

elscotto said:


> Al-
> Here is a link with info regarding treatment of captives with chytridiomycosis:
> http://www.open.ac.uk/daptf/froglog/FROGLOG-46-1.html
> There is also some useful information from Amphibian Ark on detection protocol (which also links to the treatment page):
> http://www.amphibianark.org/chytrid.htm
> -Scott



When using the itraconazole solutions referenced above they should be buffered as itraconazole has a low pH and this has been shown to be a problem with a number of species. 

Ed


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## sports_doc

*Re:*

<original post 2006>


Rain_Frog said:


> I have contacted Pisces Molecular and they charge 21 bucks for a PCR skin swapping and they sent me some documents how to properly collect the sample.


these folks still around I wonder? their web page is basically dead...

S


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## earthfrog

Ed said:


> elscotto said:
> 
> 
> 
> Al-
> Here is a link with info regarding treatment of captives with chytridiomycosis:
> http://www.open.ac.uk/daptf/froglog/FROGLOG-46-1.html
> There is also some useful information from Amphibian Ark on detection protocol (which also links to the treatment page):
> http://www.amphibianark.org/chytrid.htm
> -Scott
> 
> 
> 
> 
> When using the itraconazole solutions referenced above they should be buffered as itraconazole has a low pH and this has been shown to be a problem with a number of species.
> 
> Ed
Click to expand...

What would you add to it to buffer it for giving to a dart frog?


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## earthfrog

*Re: Re:*



sports_doc said:


> <original post 2006>
> [quote="Rain_Frog":120eplmy]I have contacted Pisces Molecular and they charge 21 bucks for a PCR skin swapping and they sent me some documents how to properly collect the sample.


these folks still around I wonder? their web page is basically dead...

S[/quote:120eplmy]

They're definitely still around---they're one of the fastest growing companies of their kind. Their website is under maintenance right now, but their phone number is 303-546-9300 and they still run the qPCR test for Batrachochytrium Dendrobatidis on dart frogs.


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## sports_doc

So I emailed to see about the 'proceedure' and indeed they responded rapidly [2hours].
The response I am posting here b/c I think it has some good info for all those interested.

Dear Shawn 

We would be pleased to PCR test amphibian samples for Batrachochytrium dendrobaditis for you; we test samples for many private individuals.

Attached are two files which answer most of the common questions about collecting and preserving samples for B.d. PCR testing. The first is a protocol for collecting amphibian toe clip, skin scrape or skin swab samples for PCR testing from Lauren Livo whom we work with at the Colorado Division of Wildlife. The second file from Pisces continues on from Lauren’s with instructions and information about packaging and sending samples for B.d PCR to us. 

Several additional notes beyond what is covered in these two files: 

Because our experience with the 2 ml screw-capped microfuge tubes with a captive O-ring gasket recommended in the instruction files has been very positive - nearly zero leaking tubes in thousands of samples - we strongly recommend using these tubes for your samples. In response to requests from many customers who don't want to buy 500 vials at a time, or don't want to spend the time to be sure they're getting the right tubes or ethanol, we are now offering "swab sample collection kits" consisting of 50 2ml screw-cap tubes filled with 1 ml of 70% ethanol; 50 swabs; ziploc bags for everything; and a box that can be used for return shipping to Pisces. The cost of the 50-sample kit is $50 plus shipping. 

The "general instruction ..." file specifies the materials for pooling samples, but doesn't yet explain the two alternative sample pooling strategies we have developed and offer. We can pool up to 8 swab samples into one DNA prep+PCR sample. Either you can do the pooling, or we can do it here. Although there is an extra charge if we do the pooling (see below), we generally recommend that new customers let us do it, at least for the first pass of sampling at new sites where nothing is known about the prevalence or severity of B.d. The chytrid PCR assay can detect less than 1 zoospore, so it is extremely sensitive to sample cross-contamination; we have procedures in place to pool samples efficiently without cross-contamination. Also, if we do the pooling, we can pool 1/2 (by volume) of each individual sample. Then if a given pooled sample is PCR positive, we can go back and prep and PCR test the remaining half samples individually. This works out well when one wants to know a true B.d. incidence level without spending lots of money on a large batch of samples that all turn out to be PCR negative.

We do not yet require or have specific sample submission forms; all we require are: 1) The name of the sample submitter; 2) A hard copy list of the number of samples submitted and the individual sample IDs; and 3) To whom the report and the invoice should be sent.

The current price for carrying out the DNA extraction and the chytrid PCR test for samples sent to us is US$27 per sample. For samples that you send as individual samples and we do the pooling, there is additional $35 pooling charge for each group pooled (for example, 8 individual samples pooled into one DNA prep + one PCR test would cost:$35+$27 = $62. For samples sent to us already pooled, the charge is $37/pooled sample. Samples sent in tubes or vials other than those recommended are subject to a 20% surcharge. Samples which leak in transit – and therefore require extensive additional anti-cross contamination measures at Pisces - will be subject to a $250 per batch leak surcharge. If we discover a leak in a sample batch, we will immediately email or call you to report the leak and allow you to decide whether we should proceed, or let you resample and submit another (hopefully) non-leaky batch of samples. Individual swab sample collection kits are $50 per 50 samples, plus shipping. Kits for customer pooled samples (50 ml tubes with 70% ethanol and 8 swabs per tube, instructions) are $80 for 10 pooled samples.

Our terms are: Payment is due upon receipt of the sample test results report. All we require from new customers is a name and address to send the invoice to, and a P.O. number if required by the customer. Unfortunately, we are not able to accept credit card payments at this time. Samples should be packed per the instructions in the "General instructions" and shipped FedEx, DHL or UPS; 2nd day delivery is fine. 

Our normal turn-around time from receipt is two to four weeks. However during the summer field season, or in the fall when all the field season samples have been sent in, our turn-around can slow to four to six weeks. If you have a need for faster results, for animals in quarantine or a report deadline, please call us. Although we haven’t completely formalized our rush sample times and charges, we can very likely work something out that meets your needs – our fastest sample turnaround time to date was 9 hours (FedEx Priority delivery of the samples in the morning; PCR results emailed by 6 PM!) (currently we have been quoting < 1 week turnaround for $54/sample and < 48 hr turnaround for $108/sample). We will email and/or send out a signed hard copy of the test results report, whichever is needed by the sample submitter. Test results are kept confidential and not shared with anyone other than the sample submitter or their organization without express permission from the submitter.


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## krharmut

Thanks for posting that. I've got a bunch of new frogs coming in that I need to test. 

Katie


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## elscotto

Is anyone aware of any papers discussing the treatment of tadpoles for chytrid. 
Thanks -Scott


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## intelsuit

I started to do a little research about chytrid. It seems that one cure was to raise the tank temp to 78 to 79 for a month then to 85 or 86 degrees for 10 days. It seems extreme to me but if it works then it might be an option.

Inter Research » DAO » v94 » n3 » p235-238


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## Dane

You would probably see more losses in some species with that method as opposed to the Lamisil & Itraconazole treatments.



intelsuit said:


> I started to do a little research about chytrid. It seems that one cure was to raise the tank temp to 78 to 79 for a month then to 85 or 86 degrees for 10 days. It seems extreme to me but if it works then it might be an option.
> 
> Inter Research*»*DAO*»*v94*»*n3*»*p235-238


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## Ed

intelsuit said:


> I started to do a little research about chytrid. It seems that one cure was to raise the tank temp to 78 to 79 for a month then to 85 or 86 degrees for 10 days. It seems extreme to me but if it works then it might be an option.
> 
> Inter Research*»*DAO*»*v94*»*n3*»*p235-238


No, raising the temperature to 78-79 has nothing to do with the cure. That actually is just to acclimate the frogs to warmer temperatures so there is less risk of mortality when the temperature is spiked to 85 F. In cases like this, make sure you understand the paper before suggesting it as a treatment option. 
As a treatment option, it was originally tested out on Litoria chloris in Australia. see http://www.int-res.com/articles/dao2003/55/d055p065.pdf..... 

The conditions have to be conducive to the frog being able to end up with a skin temperature at the temperature that is lethal for the fungus but not the frog...given that there is a huge variation in anurans for not only tolerances of different temperatures but how fast those temperatures change (which are also impacted by condition, stress...). That means you also have to be able to account for the frog to cool itself via evaporative cooling (which would also cool the skin and fungus) as a method to control it's body temperature. It isn't as simple as you are making it out to be... and as such would require testing to demonstrate that the frogs were actually free of chytrid (since the frog can change it's body temperature)... when compared to treatments using buffered intraconazole or even Lamasil... 

Ed


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