Confusing X. laevis disease

[kittyb]

We keep X. laevis in a large circulating system (from Marine Biotech) that contains a 3 step filtration of de-ionized water and a UV ballast for sterilization.

Over the last few months, we've seen a problem with some of the females we use for experiments. The illness seems to be limited to those that were recently injected (hCG, sterile) for induced ovulation. They develop sores on their abdomen/underside and/or back (near the back legs). The first case definitely appeared like the skin was just rotting off. Two very mild cases (only a few spots that looked like scratches or small sores resembling road rash) made a full recovery after a week in isolation. In another case, the frog showed sloughing of the abdominal skin in the early morning, and by late afternoon her darker skin was turning from dark green and black to bright green and gray. I believe there were another couple cases where we found them dead a few days after being returned to the system.

Today I came into the frog room to find one female dead with thick sloughing of the skin. A second female in the same tank was showing the same sloughing and was very weak. She was euthanized, and upon inspection both frogs were developing sores on their abdomens and the underside of their back legs. The other 3 tank mates are being monitored but appear fine.

I've sterilized (autoclaved) the container that we hold the frogs in from injection and through use. I've also sterilized the larger containers we put them in (overnight) to be returned to the system the next day. The injection bench is wiped clean before each frog, the needles/syringes/hCG are all sterile. Anyone handling a frog wears gloves. And still, we have these cases. Needless to say, I'm at a loss.

My only reasonable conclusion is a bacterial infection brought on in response to stress (ie, being used). The symptoms don't match "red leg," and I can't find another bacteria that could be the likely culprit. The symptoms don't match a typical fungal infection. We're planning on flushing the systems with antibiotics (this has apparently been done before with no ill effects) to try and curb the problem.

Has anyone experienced this type of illness before? I apologize for not having pictures (as graphic as they would be), but I don't keep a camera at work.

 

[Greatwtehunter]

I hate to say it but it sounds alot like Batrachochytrium dendrobatidis which can lead to chytridiomycosis, commonly known as chytrid fungus. Xenopus laevis have long been known to be carriers of chytrid, although they seem to have somewhat of an immunity to it, the stress of the injections probably is what triggered the outbreak. Now I can't say for sure thats what it is without the proper tests but it definitely should be looked into.

 

[herpvet]

Hello,

It sounds like you really need to get a post-mortem done, or possibly biopsies, microbiological tests etc from living affected animals. Anything else is just guessing. You need to find out what you're dealing with. A full veterinary assessment may also be useful; for example, when was the last time you introduced new animals?

Chytrid would be one top infectious possibility, although there are others; another thing to double check would be whether the HCG formulation (probably not the HCG, but possibly the carrier solution?) has changed - manufacturers will do this without warning sometimes.

Hope this helps, and you can get it sorted out.

Bruce.

 

[kittyb]

Hello,

It sounds like you really need to get a post-mortem done, or possibly biopsies, microbiological tests etc from living affected animals. Anything else is just guessing. You need to find out what you're dealing with. A full veterinary assessment may also be useful; for example, when was the last time you introduced new animals?

Chytrid would be one top infectious possibility, although there are others; another thing to double check would be whether the HCG formulation (probably not the HCG, but possibly the carrier solution?) has changed - manufacturers will do this without warning sometimes.

Hope this helps, and you can get it sorted out.

Bruce.

the carrier solution is just sterile water. i agree that a necropsy is needed, along with testing any new individuals who become infected. i've wanted to do this, but i think they were just hoping it was a fluke death here or there (with around 1000 animals, it's bound to happen occasionally). we're just instituted a policy of treating the water and buffers (that they are contained in the day before and day of use) with gentamycin. apparently the flushing of the whole system will cost us thousands of dollars, so it's a last resort. all of our new individuals all come from the same company (Nasco), though i'd have to look to see if our last shipment of males matched up with the beginning of the illness (but i don't believe it does).

thanks for all the advice. :happy:

with this new procedure in place, hopefully it will curb things. if it does, we may be able to treat the whole system down the road. university budget cuts can make things get "put off" a lot.

 

[herpvet]

Hi Kittyb,

A personal view, I must stress, but I have to say that gentamicin would not be my first choice of antimicrobial. I would say there are four main possible pathogens involved here (chytrid fungus, mycobacteria, chlamydophila, gram negative bacteria, roughly in that order of likelihood I think). Others are possible (other bacteria, other fungi) but less likely.

Of the four main ones, gentamicin might actually make things worse if it's chytrid (by removing bacterial competition), have some effect on mycobacteria, have no effect on Chlamydophila and be probably (depending on resistance) effective against gram negative bacteria. Quinolones such as enrofloxacin would be a better choice in my opinion, preferably along with baths in itraconazole to cover against chytrid, if testing really isn't an option.

I would also tend to suspect that flushing the whole system might well not solve the problem; the animal themselves may well be the reservoirs of any pathogen.

Just my thoughts on it!

Bruce.

 

[Jennewt]

With 1000 animals, it would seem to be "penny wise but pound foolish" to delay getting a good diagnosis of the problem.

What type of gloves are used? You've seen the recent article about toxicity of gloves to amphibians? I'm thinking of a possible correlation between the injected (handled) females and the symptoms.

Good luck to you!

 

[Jan]

kittyb - A few additional thoughts in that the animals who seem to be affected are the ones that have been injected:

I would follow-up on Bruce's suggestion to look at the formulation of the hCG product you are using...and has it changed. The vehicle you are using for dilution is sterile water, however, Bruce was addressing the buffers and preservatives in the product. If you are purchasing a commercially available hCG and using the diluent provided to reconstitute the product...this is where the additives may change from vendor to vendor or even within the same vendor. hCG injection is a dry mixture of the lyophilized drug with suitable diluents and buffers. Following reconstitution with the sterile diluent provided, the commercially available injections have a pH of 6–8 and may contain phenol and/or benzyl alcohol as preservatives, and lactose or mannitol; sodium hydroxide, hydrochloric acid, or monobasic or dibasic sodium phosphate may be added during manufacture of the powder for injection to adjust the pH.

If you are not using the diluent provided, but instead using SW as the product diluent....is this the protocol that has historically been used? Just from the standpoint of the drug - this could induce pH swings and without the preservative (unless the vial is being used as a single dose) could present operator induced contamination. Have you used multiple vials and seen this problem....or has the same vial been used? Very basic or acidic injections can produce skin sloughing. In people I have seen this to the point of requiring skin grafts.

Jen refers to gloves and the potential for induced toxicity. If you haven't seen this research, you may find it interesting:

Lethal Effect of Latex, Nitrile and Vinyl Gloves on Tadpoles
http://www.parcplace.org/Cashins_etal_2008_glovesandtads .pdf

I would also agree with Bruce that gentamicin would not be a top choice antimicrobial for the application you described.

Best of luck

 

[kittyb]

We use sterile water (from Sigma, I believe) when reconstituting our hCG, and this has been the protocol for years. The vial is used multiple times (tx is with 500 U (0.5 ml), leaving roughly 20 injections per bottle) with new needles and syringes for every use. The symptoms have occurred after different vials (but obviously not with many frogs from the same vial), though the vials may all be from the same batch. Not all vials from this batch have resulted in sick or dead frogs, and the wounds are not consistently near the area of injection.

We use powder-free nitrile gloves (N-GEN or X-GEN), and have completely removed the use of latex gloves from the lab. To my knowledge, these nitrile gloves have been used exclusively for AT LEAST 6 months and only within the last couple months have frogs been showing illness. We've gone through different boxes and batches, obviously. But this lab has always used latex or nitrile gloves, and my PI doesn't recall a situation like this ever arising.

I agree that a different antimicrobial would be a better choice, but in the end it isn't my call. It may take another loss under this new protocol before the PI is willing to try something else. I do plan on discussing it with him today and seeing where we can go from here.

It's all very frustrating for me. Granted, it's been maybe half a dozen cases within the use of well over a hundred females, but I don't like anyone dying on my watch. And while I may want to try different avenues of diagnosis and treatment, I have to convince the "higher up" first. Fortunately, he respects my opinion and previous experience.

 

[John]

I would pay Bruce a lot of attention. He doesn't like to toot his own horn but he is a rare commodity: a herpetile savvy veterinarian and he's interested in amphibians. Jan and Jen are probably the most knowledgeable people on our forum and both have professional scientific backgrounds. Good luck convincing that PI. They can often be immovable objects.

 

[kittyb]

I would pay Bruce a lot of attention. He doesn't like to toot his own horn but he is a rare commodity: a herpetile savvy veterinarian and he's interested in amphibians. Jan and Jen are probably the most knowledgeable people on our forum and both have professional scientific backgrounds. Good luck convincing that PI. They can often be immovable objects.

Please don't misunderstand me. I truly appreciate all the advice I can get. I've only become involved in amphibians earlier this year (when I took this job as research associate/lab animal coordinator/lab manager), so a lot of this is still new to me.

I spoke to the PI, and he doesn't suspect our hCG to be the culprit. He only implemented the gentamycin to prevent a secondary infection, but agrees that it could make the problem worse. He's asked that I contact IACUC next time a frog shows symptoms so that we can get a full necropy/biopsy/culture.

 

[froggy]

It could be a ranavirus; depending on individuals and mode of infection it can be ulcerating or haemorrhagic.

 

[kittyb]

Update

Today I noticed another female laevis with a small open sore on her chest. She was not recently used for egg collection. I've quarantined her and called the DLAM veterinarian to arrange for a necropsy. Since finding her this morning, she's started to develop another sore (skin is pulling away but still intact) on the other side of her chest. I'm keeping her alive so that the examination can be as fresh as possible (since she is not showing signs of distress yet).

OF COURSE this had to happen the day before Thanksgiving, when everyone is skipping town early and no one will be back until Monday. Hopefully she's just out to lunch and will call me back soon.
:uhoh:

 

[erman]

Re: Update

Hi

I have the same problem
I have two old frogs (Mom and Dad - x. laevis albino) and a separate tank with six froglets (two months old)
Couple of weeks ago I put some new stones in the tank of Mom and Dad and after a few days I noticed excessive shedding but I didn't pay attention to that. Three days ago I saw brown spots on their abdomens growing in size and number. I took a detailed look on the froglets and established that the situation is the same. Very scared I read everything that could be read around the net and came to the conclusion that the cause is probably chytridiomycosis. I began treating with 0,1 mg/L malachite green for 24 hours every other day, and on the next day began with itraconazole baths 0,01 % for 5 min. I also inject eartworm (Lumbricus terrestris) with 1 mg itraconazole oral suspension and give one to every frog every day. On the third day, after two days in malachite green solution and after one itraconazole bath and worm, the spots become faded, many of them disappeared, no shedding of the skin. Today is the last day (fourth) in malachite green solution. I'm thinking of stopping it and continuing only with the itraconazole procedures.
The froglets are exposed only to malachite green and they are completely clean of spots and no shedding is observed.

 

[John]

Re: Update

That's fantastic, Erman. Thank you for contributing this information.

 

[Jennewt]

Re: Update

But don't Xenopus carry chytrid without any symptoms? I'm skeptical of this diagnosis. Also, symptoms like these can be caused by multiple organisms.

I have a PDF of the following article. If anyone would like a copy, send me an e-mail or PM.
Concurrent Infection with Ranavirus, Batrachochytrium dendrobatis, and Aeromonas in a Captive Anuran Colony. Journal of Zoo and Wildlife Medicine 39(3):445-449.

 

[taherman]

I too would be highly skeptical of implicating B.d. in these cases. People are much too quick to jump on the chytrid bandwagon, and there are many other infections that might be cleared up with malachite green baths. Without histopathology or at least a positive PCR test I would not assume it is B.d., particularly in a species which is widely regarded as resistant. You should submit at least a couple of animals showing symptoms to a lab experienced in amphibian histopathology.

-Tim

 

[erman]

Yes, you are all right - there's no clinical evidence for chytridiomycosis. I just wanted to share my experience with malachite green and itraconazole, not to recommend diagnosis. By the way, last night (the forth night) the abdomen skin of the female frog peeled off and now all the frogs and froglets are completely clean. I changed the water and this time I added only Sera Ectopur (sodium perborate) along with the water hardeners that I usually add.

Concerning the symptoms:

Clinical diagnosis and treatment of epidermal chytridiomycosis in African clawed frogs (Xenopus tropicalis).

Parker JM, Mikaelian I, Hahn N, Diggs HE.
Office of Laboratory Animal Care, University of California, Berkeley 94720, USA.
An investigation was conducted to determine the cause of morbidity and mortality in a collection of 55 adult male Xenopus (Silurana) tropicalis at the University of California, Berkeley. More than 80% of affected frogs died during the epizootic. All frogs were anorectic and lethargic, had dark pigmentation and excess skin sloughing, and lacked a slime layer. Histologic examination revealed severe hyperplastic and spongiotic dermatitis associated with colonization of the stratum corneum by large numbers of zoosporangia diagnostic of Batrachochytrium dendrobatidis. Treatment with a commercial formalin/malachite green solution at a dilution of 0.007 ml/L of tank water for 24 h, repeated every other day for four treatments, eliminated the organism and was curative. These findings are indicative of epidermal chytridiomycosis as a primary cause of death in this collection of X. tropicalis.

 

[kittyb]

The drama continues...

The female survived the Thanksgiving break and is nearly healed (thought still quarantined). The vet is arranging for swabs to be taken for culture, but by the time they get to it she may not have much to offer.

Our laevis system appeared to be failing over Thanksgiving break and the water was turning green-yellow. I've changed the filter and checked to make sure the quartz sleeve of the UV sterilization system is clean. I spent 4-5 hours yesterday scrubbing all 32 tanks, draining the majority of the water and allowing them to refill. I ran the system overnight with the new filter, scrubbed tanks, and UV. Still a bit green today, but the other lab coordinator thinks it's showing improvement. Once the system recovers, we're going to reinitiate the standard pH/conductivity tests to keep a better eye on things.

Whether it's related or not, a female removed from that system and primed on Monday had developed bloat very rapidly (very severe and almost overnight). It's difficult to say if it was a consequence of infection (injection wound then exposed to the poor water) or if her lymph sac was damaged during injection and she leaked into her body cavity. When I found her this morning in the "overnight return" container, she was like a little beach ball. She was humanely euthanized.

 

[kittyb]

We finally have an answer!

I saw another X. laevis female who started showing symptoms (open sore, raw belly) and contacted our DLAM facility. A necropsy/culture showed Aeromonas hydrophila. While it's resistant to several antibiotics: ampicillin, cephalexin, erythromycin, tetracycline, and amoxicillin + clavulonic acid), it IS sensitive to enrofloxacin (Baytril), amikacin, chloramphenicol, gentamicin, piperacillin, and trimethoprim + sulfa.

Given that a good number of these medications involve oral or SQ administration, we've opted for 20 mg/L chloramphenicol in a 0.5% saline bath, changed every 24 hours for 5-7 days.

We started administering this treatment to a small group of X. tropicalis that are most likely infected with Red Leg (16 out of 22 have died of the disease with obvious symptoms, the remaining 6 appear healthy), and we have not have any more deaths.

While we're getting the handle on the disease (we haven't opted to treat the entire system yet, as it's ~700 gallons total), we're also administering Koizyme, though we're almost done with that regimen.

Fingers crossed!

 

[John]

It's good to have a follow up on this. Thank you Kitty and the best of luck to you.