Detection of Pasteurellaceae in Laboratory Mice by Fecal PCR

Abstract

The use of microbiologically defined laboratory animals in biomedical research has become standard practice in the last few decades. Microbiological standardization, based upon routine testing of the animals at regular intervals, has contributed to the 3Rs policy (Refinement, Reduction and Replacement). It allows the reduction of the number of animals used as it decreases the variation within and between test groups, and it plays an important role in the refinement of the overall health of laboratory animals thus improving their welfare. Additionally, it has reduced human health risks due to zoonotic diseases. Specific pathogen free (SPF) health status was developed to guarantee the absence of specific pathogens in laboratory animals whereas Specific Opportunistic and Pathogen-Free (SOPF) health status guarantees the absence of the major interfering opportunistic agents in addition to the specific pathogens. These negative definitions are based on a detailed exclusion list of agents which are likely to infect laboratory rodents and confound results from animal experiments, such as members of the Pasteurellaceae family. Among these, Pasteurella pneumotropica is considered to be the most frequently occurring opportunistic pathogen in laboratory rodents and it is usually isolated from the upper respiratory tract, lungs, genital and gastrointestinal tracts. The pathogenicity of this organism in immunocompetent laboratory mice and rats is regarded as low, but in immunodeficient animals it may lead to pneumonia, conjunctivitis, and respiratory and genital tract infections. However, due to the unsettled taxonomic structure of the Pasteurellaceae family and the occurrence of taxa other than Pasteurella pneumotropica, FELASA recommends the monitoring of SPF rodents for all Pasteurellaceae. The available diagnostic techniques for Pasteurellaceae screening traditionally include bacteriological methods, immunological and biochemical characterization. These procedures are time-consuming and sometimes yield indeterminate results due to the phenotypical diversity of this bacterial family. PCR assays based on the 16S rRNA gene sequence have recently been reported as alternatives to biochemical and culture methods for Pasteurellaceae detection. However, the protocols used are based on invasive sampling methods that require the sacrifice of animals. In this study we developed a simple, non invasive and specific PCR assay to detect Pasteurellaceae by using DNA isolated from mice feces. Furthermore we discuss the impact of this non-invasive technique in assessing the prevalence of Pasteurellaceae on laboratory rodents.

Date of Award	2011
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Isabel Fidalgo Carvalho (Supervisor) & Marta Vaz Mendes (Co-Supervisor)