Collaboration Caught Clostridium in Immunodeficient Mice

“When we have conversations, we're able to recognize patterns that we're all dealing with”

Dr. Harshan Pisharath, DVM, Director of the Animal Resources Center at St. Jude Children’s Research Hospital, had a problem. Some of his immunodeficient mice, including the strains NSG, NSG-SGM3, and NBSGW, were presenting with a mysterious illness. Over one thousand mice showed loose stool, minimal weight loss, and low mortality. Though the infection did not seem severe, there was a problem – they couldn’t find it.

Each of the strains had tested negative in the usual rounds of PCR using exhaust air dust, and pooled dirty bedding samples tested for known pathogenic microbes that could be causing this illness also proved inconclusive. This meant that the usual assays were not detecting the organism responsible – it was time to take a different approach.

How Researchers Worked Together to Solve the Mystery

“We are thankful to have great relationships with many of the clients that use the RADS diagnostic testing lab for their routine health monitoring,” said Kourtney Nickerson, PhD, Senior Scientist at Charles River. “And this was this was no exception.”

Dr. Pisharath came to her at a conference with his troubles. He knew that her lab at Charles River had the capability to phenotype, sequence and create tools to understand the isolate that his lab had identified. They had narrowed the genus to the gram-positive Clostridium bacteria, but they were having trouble tracking the microbe.

“One of the issues they had with this was that the microbe itself wasn't in any reference databases, so when they were doing some of their initial microbiome analysis, their potential ID's were something called Clostridium celatum, which wasn’t a good fit for what they were seeing,” Nickerson said. “He knew that bringing us together and combining efforts would give them a chance to overcome the outbreak. They sent us their isolates, we were able to grow it using our anaerobic tools, and we were able to sequence it.”  Sequencing led us to a molecular assay which empowered their team to understand scope of affected animals, animals at risk for disease, and formulate a plan to eliminate the microbes.

St. Jude’s was not alone in this mystery. A few other locations that work with immunodeficient mice were seeing the same issue, with the same presentation.

“The University of Pennsylvania had reported very similar issues in their immunodeficient mice down to the same kind of approach with microbiome analysis, identifying Clostridium species as being the problem, and even speciating it as Clostridium celatum,” Nickerson said. “It was all happening concurrently, and it was cool to see we're not alone in this.”

Nickerson’s lab was able to grow the isolated bacteria and run a whole genome sequence, finally identifying the species as Clostridium cuniculi. Once identified, St. Jude’s could begin the organized elimination of the offending bacteria. St. Jude’s and Charles River together developed a couple of assays to screen for the organism, and now St. Jude’s is proud to be Clostridium cuniculi free.

But what about the bigger picture? What does this mysterious outbreak tell us about immunodeficient mice?

How Can We Protect Immunodeficient Mice?

“Now when mice are coming in, C. cuniculi is part of their screening list,” Nickerson said. “Any of these specialized immunodeficient mice are now housed in their own facility using sporicidal disinfectants, and we're trying to stop the spread.” 

Through their research, the collaborative team found that C. cuniculi is susceptible to the antibiotic combination trimethoprim-sulfamethoxazole. They could also trace the bacterial outbreak to a specific colony and found probable routes for spontaneous outbreaks through irradiated food. Luckily the species was relatively easy to fight, once identified, but the bigger question remained – how can they prevent similar issues in immunodeficient mice?

“I think what we've learned a lot about immunodeficient models is that they're just so susceptible to so many things that are normal, even within their own body,” Nickerson said. “Microbes that may have been previously well tolerated or microbes that are generally found in the mouse microbiome in healthy, immunocompetent mice could actually be problematic for an immunodeficient mouse.”

In order to protect these mice better, research labs like the one at St. Jude’s have implemented or reinforced protective workflows. Having dedicated facilities and staff that only house immunodeficient models, for example, or ensuring a workflow where cleaning crews work from the most vulnerable rooms outwards, and do not go back once they have moved on to a less sterile room.

Nickerson predicts that these issues will become more important as immunodeficient models grow in research value. The team’s findings were presented at this year’s meeting of the American Association for Laboratory Animal Science (AALAS) . Nickerson says she was glad to share their data with the industry at a convention like this, since the rising tide of collaborative data lifts all boats.

“A lot of different institutions with immunodeficient mice coming together at the same time underscores the importance of these kinds of meetings, because we all do science in our own silos,” she said. “But when we have conversations, we're able to recognize themes and patterns that we're all dealing with.”