Reemergence of the Murine Bacterial Pathogen Chlamydia muridarum in Research Mouse Colonies

Abstract

Chlamydia muridarum (Cm) was detected in 2 colonies of mice with lymphoplasmacytic pulmonary infiltrates by using PCR and immunohistochemistry. This discovery was unexpected, as Cm infection had not been reported in laboratory mice since the 1940s. A Cm specific PCR assay was developed and testing implemented for the resident colonies of 8 vivaria from 3 academic institutions, 58 incoming mouse shipments from 39 academic institutions, and mice received from 55 commercial breeding colonies (4 vendors). To estimate Cm’s global prevalence in research colonies, a database containing 11,387 metagenomic fecal microbiota samples from 120 institutions and a cohort of 900 diagnostic samples from 96 institutions were examined. Results indicate significant prevalence among academic institutions, with Cm detected in 63% of soiled bedding sentinels from 3 institutions; 33% of incoming mouse shipments from 39 academic institutions; 14% of 120 institutions submitting microbiota samples; and 16% of the diagnostic sample cohort. All samples from commercial breeding colonies were negative. In addition, naïve NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice exposed to Cm-shedding mice and/or their soiled bedding developed clinical disease at 21 to 28 d after exposure. These mice had a moderate-to-severe histiocytic and neutrophilic bronchointerstitial pneumonia, with their respiratory epithelium demonstrating inclusions, chlamydial major outer membrane protein immunostaining, and hybridization with a Cm reference sequence (GenBank accession no. U68436). Cm was isolated from lungs, cecum, and feces of a Cm-infected NSG mouse by using HeLa 229 cells. The considerable prevalence of Cm is likely due to widespread global interinstitutional distribution of unique mouse strains and failure to recognize that some of these mice were from enzootically infected colonies. Given that experimental Cm colonization of mice results in a robust immune response and, on occasion, pathology, natural infection may confound experimental results. Therefore, Cm should be excluded and eradicated from enzootically infected mouse colonies.

Chlamydia muridarum infection causes bronchointerstitial pneumonia in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice

“…A proprietary real-time fluorogenic 5’ nuclease PCR assay specifically targeting a 425 bp fragment of Cm 23S rRNA was used to determine the presence of Chlamydia DNA in samples as previously described. 14 The double-stranded nucleotide sequences of selected purified PCR products were determined using Sanger dye-termination sequencing (Genomics Core, Tufts University Medical School, Boston, Massachusetts).…”

“…The diet consisted of a natural ingredient, closed source, flash-autoclaved, γ-irradiated feed that was autoclaved reverse osmosis acidified water. 14 Additional husbandry details are found in Mishkin et al 14 Following 3 to 4 weeks of initial exposure, mice were euthanized and a complete postmortem examination was performed.…”

“…14 Further investigation revealed a significant prevalence of Cm among global academic laboratory mouse colonies, with Cm detected in 33% of incoming mouse shipment received from 39 academic institutions, 63% of soiled bedding sentinels from 3 institutions, 14% of 120 institutions submitting microbiota samples, and in 16% of biological and environmental samples from a multi-institutional cohort. 14 Because natural infection by Cm had not been described in laboratory mice since the 1940s and NSG mice are highly susceptible to opportunistic bacterial infections, we performed a pilot experiment in which 4 NSG mice were cohoused with recently imported naturally infected immunocompetent mice shedding Cm in their feces, principally to facilitate isolation of the bacterium. 14 Development of severe clinical disease and pulmonary lesions reported in 2 mice prompted further investigation.…”

“…14 Because natural infection by Cm had not been described in laboratory mice since the 1940s and NSG mice are highly susceptible to opportunistic bacterial infections, we performed a pilot experiment in which 4 NSG mice were cohoused with recently imported naturally infected immunocompetent mice shedding Cm in their feces, principally to facilitate isolation of the bacterium. 14 Development of severe clinical disease and pulmonary lesions reported in 2 mice prompted further investigation. 14 The aim of this investigation was to characterize the pathology and determine the extent of disease associated with natural Cm infection in male and female NSG mice.…”

“…14 Development of severe clinical disease and pulmonary lesions reported in 2 mice prompted further investigation. 14 The aim of this investigation was to characterize the pathology and determine the extent of disease associated with natural Cm infection in male and female NSG mice.…”