[Xenopus] Urgent request for documentation about frog health and care

[Zhou, Coral Yishan]

Dear Xenopus community,

I am a new PI trying to get my Xenopus laevis colony off the ground and have run into serious difficulties. I am writing to request your help in a conflict with my university animal care unit (ACU). If unresolved, I will be without frogs for 3 additional months (6 months total).

Short story:

After a few bloated frogs died in our brand-new colony, we discovered Mycobacterium ulcerans in our colony. After culling the frogs and completely decontaminating our Iwaki system and frog room (3 months of work), a few surfaces are testing negative for M. ulcerans but positive for M. chelonae and M. gordonae by PCR. From my understanding these are not harmful to humans or frogs unless in extreme cases. The ACU is requiring us to repeat all of our decontamination work, which will set us back by 3 additional months. When I suggested to try a less time consuming, but more targeted approach to get rid of the mycobacteria, they said no.

Longer story:

We are a PI-managed colony, with oversight from the ACU. Despite being extremely careful and clean, a few months after starting our colony of 70 frogs we had a few deaths (~1 every 2 weeks, 4-5 in total over 2 months). In each case, the frog would bloat and upon isolation in high salt water, it would bleed out and die within a few days. All other frogs appeared healthy and asymptomatic. When we sent one of these frogs for pathogen testing, the test results showed presence of Mycobacterium ulcerans. The ACU told us we needed to cull our colony and completely decontaminate our frog room.

So far we have followed Iwaki’s protocol for complete sanitization of the Iwaki system (bleach, rinse, virkon, rinse), which took 2 months. We have also wiped down every surface with Peroxigard and also fogged the entire room with hydrogen peroxide. In total the entire cleaning procedure took 3 months. Afterwards, we performed swab tests of several areas of the room and performed a complete mycobacterium PCR panel. We isolated a few external areas (outside of tanks, inside tanks) that still had signs of two species of mycobacteria: M. chelonae and M. gordonae. There were no traces of M. ulcerans, the original culprit.

Based on the test results, there is no longer contamination inside the tubing of the Iwaki unit. However, the ACU wants us to repeat every single step of our decontamination procedure (3 months of work) before repeating the PCR test. They require a completely negative PCR test in order to proceed with ordering new frogs. When I suggested doing every step of the procedure except the Iwaki sanitization, and then see if we have eliminated the bacteria, they said no.

From my understanding, in an overwhelming majority of cases, M. chelonae and M. gordonae are not alarming or harmful to frogs or humans. If they are pathogenic, they are likely secondary infections to primary stresses. We were very careful with our biofilter, and never got spikes in ammonia, nitrates, or nitrites. However, from talking to folks in the community, we think the real culprit was that we were over-cleaning the tanks. We had an agreement with ACU to clean each tank once a month, and we used virkon and ethanol. I am now understanding that nobody else uses detergents to clean their tanks.

My concerns:
- Short term: I am concerned that despite stringent cleaning, it will be impossible to get rid of all mycobacteria from an aquatic facility.
- Short term: I am concerned that introducing even more cleaning chemicals into our facility will be harmful for frogs.
- In the long term, I am concerned that the ACU will perceive any mycobacteria as a threat, regardless of strain. However, we know as a community that only a few species (M. Lifandi, M. marinum) are of serious concern.

My ask:

I am looking for documentation for any/all that address the following:
- How you deal with positive pathogen tests, especially for mycobacterium
- Pathogens that are actually harmful/alarming for frog or human health vs. those that are not of concern, especially for mycobacteria
- Detergents or other cleaning chemicals, even at residual levels, are harmful to frog health

This documentation should ideally be formalized (publications or protocols that are approved by IACUC , AALAC, veterinarians). However, personal testimony from experienced Xenopus researchers will also be useful.

This is urgent because I have students available to work full time this summer and really need to get frogs in order to capitalize on their availability.

Please send any documents you have my way, even if you aren’t sure if it’s helpful.

Thanks so much!!!

Best wishes,

Coral Y. Zhou, Ph.D. (ella)
Assistant Professor
Department of Molecular Biosciences
University of Kansas
Lab website<https://www.coralzhoulab.com> | Faculty profile<https://molecularbiosciences.ku.edu/people/coral-zhou>













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