Murine Pathogen Used in Research Surfaces in Majority of Vivaria

A collaborative investigation between Memorial Sloan Kettering Cancer Center, Rockefeller University and Charles River began with unexpected clinical symptoms and histological lesions in genetically modified mice.

Microbes can lead a Jekyll and Hyde life. Consider the murine bacterial pathogen Chlamydia muridarum. As the only natural chlamydial pathogen of mice, it is uniquely useful for modeling sexually transmitted Chlamydia trachomatis infection of humans, the most common STD reported today.

But Chlamydia muridarum (Cm) can also occur naturally in mice, causing respiratory and intestinal distress. Cases of Cm in laboratory mice had not been seen since the 1940's until two years ago, when comparative pathologists at Memorial Sloan Kettering Cancer Center (MSKCC) in collaboration with Charles River's Research Animal Diagnostics and Rockefeller University reported in 2022 of natural Cm infections in the mouse colonies of numerous academic institutions. Specifically, Cm was detected in 33% of incoming mouse shipments from 39 academic institutions and 63% of soiled bedding sentinels from three institutions; 14% of 120 institutions submitting microbiota samples also reported finding Cm as did 16% of ~ 1,000 random field rodent and environmental samples from a multi-institutional cohort.

These findings immediately raised concerns for the health of severely immunocompromised NSG mice, who are highly susceptible to opportunistic bacterial infections. (NSG mice are the primary model for creating humanized mice, increasingly important in the development of new treatments, particularly for cancer.)

More recent histopathology investigation in NSG mice produced stunning images reported in August's edition of Veterinary Pathology, for which the journal singled out the paper as one of their top three observational papers published this year.

Eureka connected with Charles River's Senior Director of Laboratory Services, Ken Henderson, PhD-one of the co-authors on the Vet. Pathol. paper-to talk about this important research and what it could mean for future directions in the environmental monitoring of laboratory mice. This Q&A is part of an occasional series, Research Notes, where we highlight recently published work by our scientists.

When I think of Memorial Sloan Kettering Cancer Center I think of, well, cancer? Why are they screening for bacterial pathogens and how did Charles River become involved in this study?

Ken: Sloan Kettering has been a longtime collaborator of ours. It is also one of the few research institutions in the US that is still pursuing infectious disease research and understanding of emerging agents in laboratory mice. We have worked with Neil Lipman [Animal Resource Center Director at MSKCC) on quite a few agents, including murine chapparovirus and murine astrovirus 2.

The interest in chlamydia came about after Noah Mishkin, a post-doc with Sloan, published a paper highlighting two case studies in their facility. The animals were showing signs of lethargy. They were not acting right. They did some histology, and they found bronchial pneumonia in these animals. Next, they looked at a worst-case scenario and inoculated NSG mice-which are helpless for most agents-with Cm to see how it might progress in that model. It basically kind of wiped them out and caused bronchial pneumonia. So, it is now an agent we need to consider. We are not at the point yet where it will be excluded by vendors, but it probably will be based on what they're saying.

Cm is rarely found in research animal facilities, so why did it start showing up in so many vivariums?

Ken: It probably dates back to the 1990s and the early generation of genetically engineered mice. At that time, people were still using open-caging, and the Cm microbe might have gotten away from a study into other mice. Or it might have just been a natural infection in mice. It spread through GEMs animals, and so about 16% became infected with chlamydia. Nobody knew it though until Noah discovered those case studies.

Is it in any of Charles River's colonies?

Ken: We have screened our animals, and we are negative for it.

How did Charles River contribute to this study?

Ken: We confirmed the disease first reported by Noah Mishkin by providing both quantitative PCR and sequence conformation of Cm presence in tissues. The pathology is important because if a veterinarian sees bronchial pneumonia, is it a result of the study treatment or an infectious agent? Knowing the pathology gives the veterinary pathologist more details on what to look for when identifying the disease caused by the agent.

How do the mice acquire Cm and how can we rid colonies of it?

Ken: Mice catch it through fecal oral route, meaning fecal matter infected with Cm is ingested. One way you can get rid of it is through embryo transfer. You can also eliminate it by Caesarean, and you can also cross-foster them - when you take very young animals and place them on chlamydia-free mothers-though this approach is still questionable. As far as treatment, Sloan Kettering reported recently on the use of antibiotics, such as sulfamethoxazole in combination with trimethoprim, or doxycycline.

Does Cm in a mouse colony impact research?

Ken: In immunocompetent animals, there is likely an immune impact because it has infected the animal. So, you will see some kind of immune modulation that will take place. I do not think there has been data out there yet showing it is causing detrimental disease in animal health in immunocompetent mice. If we did, we would know about it more by now, right? Remember, this is an intracellular bacterium, so this thing replicates. It has to go inside of a cell to replicate. So that means it is impacting the cells that it replicates, which is also the gut. So, there could be some issues with the gut, too.

A lot of this stuff is just unknown, which again, is why maybe people are not quick to say, "I don't want it in my facility." But if it causes bronchial pneumonia in at least severely immunodeficient lines and it replicates in the gut, it is going to have some research implications that are yet to be further uncovered in immunocompetent animals, even the ones that do not show a disease. As always, lack of clinical signs does not mean lack of infection, or more importantly, a research impact.

So, to wrap up, when do you think institutions might add Cm to their list of excluded pathogens?

Ken: The vendors are not screening it or formally excluding it now. One of the reasons is cost. Also, these GEMs animals back in the 1990s that had chlamydia probably had other agents that have not yet been eliminated from many of the GEM colonies. A lot of institutions do have a room or rooms that are designated SPF, but they have a lot of other agents that they do not eliminate because of the cost. We call these tolerated agents. … Chlamydia is in that wait-and-see area. But I have a feeling, in the next two or three years that eventually it will be excluded, because many institutions house immunodeficient animals alongside of immunocompetent animals.

To learn more about pathogen screening in rodents, check out Ken's 2022 blog post.

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