Accurate knowledge of the health status of experimental animals is pivotal to high scientific and ethical standards in biomedical research. Individually ventilated cages (IVCs) are becoming the predominant system for housing laboratory mice, as they prevent cage-to-cage infections. However, this feature constitutes a major drawback for hygienic monitoring of mouse colonies, as traditional screening programs build on reliable transmission of infectious agents from experimental animals to sentinel mice commonly tested as representatives for the mouse colonies. In recent years, the laboratory animal community has realized that sentinels are ineffectual for screening mouse colonies in IVC systems because infections are often not transmitted to sentinels and therefore remain undetected. Furthermore, sentinel monitoring results in high numbers of used animals. In contrast, environmental monitoring provides a more reliable approach to identify and exclude pathogens in rodent colonies. In recent studies we provided evidence that polymerase chain reaction analysis of exhaust air particles is superior to soiled bedding sentinels for different agents. In this study, we show that testing pooled environmental samples generates more meaningful information compared to soiled bedding sentinels during routine hygienic monitoring in different barriers.

Since decades, model organisms have provided an important approach for understanding the mechanistic basis of human diseases. The German Mouse Clinic (GMC) was the first phenotyping facility that established a collaboration-based platform for phenotype characterization of mouse lines. In order to address individual projects by a tailor-made phenotyping strategy, the GMC advanced in developing a series of pipelines with tests for the analysis of specific disease areas. For a general broad analysis, there is a screening pipeline that covers the key parameters for the most relevant disease areas. For hypothesis-driven phenotypic analyses, there are thirteen additional pipelines with focus on neurological and behavioral disorders, metabolic dysfunction, respiratory system malfunctions, immune-system disorders and imaging techniques. In this article, we give an overview of the pipelines and describe the scientific rationale behind the different test combinations.

One limitation to housing rodents in individually ventilated cages (IVCs) is the ineffectiveness of traditional health monitoring programs that test soiled bedding sentinels every quarter. Aerogen transmission does not occur with this method. Moreover, the transmission of numerous pathogens in bedding is uncertain, and sentinel susceptibility to various pathogens varies. A novel method using particle collection from samples of exhaust air was developed in this study which was also systematically compared with routine health monitoring using soiled bedding sentinels. We used our method to screen these samples for the presence of murine norovirus (MNV), a mouse pathogen highly prevalent in laboratory animal facilities. Exhaust air particles from prefilters of IVC racks with known MNV prevalence were tested by quantitative reverse transcription polymerase chain reaction (RT-qPCR). MNV was detected in exhaust air as early as one week with one MNV-positive cage per rack, while sentinels discharged MNV RNA without seroconverting. MNV was reliably and repeatedly detected in particles collected from samples of exhaust air in all seven of the three-month sampling rounds, with increasing MNV prevalence, while sentinels only seroconverted in one round. Under field conditions, routine soiled bedding sentinel health monitoring in our animal facility failed to identify 67% ( n = 85) of positive samples by RT-qPCR of exhaust air particles. Thus, this method proved to be highly sensitive and superior to soiled bedding sentinels in the reliable detection of MNV. These results represent a major breakthrough in hygiene monitoring of rodent IVC systems and contribute to the 3R principles by reducing the number of animals used and by improving experimental conditions.

Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.

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