A specific polymerase chain reaction based on the gyrB gene sequence and subsequent restriction fragment length polymorphism analysis of Pasteurella pueumotropica isolates from laboratory mice
JOURNAL OF THE AMERICAN ASSOCIATION FOR LABORATORY ANIMAL SCIENCE
Authors: Hayashimoto, Nobuhito; Ueno, Masami; Takakura, Akira; Ltoh, Toshio
Abstract
For a molecular identification and typing tool, we developed a specific polymerase chain reaction (PCR) based on the gyrB gene sequence and a subsequent restriction fragment length polymorphism (RFLP) analysis using the products amplified from the specific PCR to facilitate discrimination of biotypes of Pasteurella pneumotropica from laboratory mice. Appropriate PCR products, a 1039-basepair fragment for biotype Jawetz and a 1033-basepair fragment for biotype Heyl, were amplified by use of the primers CZO-1 and NJO-2 from all 105 P. pneumotropica isolates tested and the 2 reference strains but not from other bacterial species tested. MspI digestion of PCR-generated products showed 3 RFLP patterns among the 105 isolates, and these patterns correlated with the biotype of the isolate (RFLP pattern 1, biotype Jawetz; RFLP pattern 2, biotype Heyl; and RFLP pattern 3, biotype Jawetz with beta-hemolytic activity). Our procedure identifies and biotypes isolates of P. pneumotropica from laboratory mice, using simple PCR and enzymatic restriction techniques. Therefore, this procedure is useful as a rapid identification and biotyping tool for isolates of P. pneumotropica from laboratory mice.
PCR Prevalence of Murine Opportunistic Microbes and their Mitigation by Using Vaporized Hydrogen Peroxide
JOURNAL OF THE AMERICAN ASSOCIATION FOR LABORATORY ANIMAL SCIENCE
Authors: Ragland, Natalie H.; Miedel, Emily L.; Engelman, Robert W.
Abstract
Exposing immunodeficient mice to opportunistic microbes introduces risks of data variability, morbidity, mortality, and the invalidation of studies involving unique human reagents, including the loss of primary human hematopoietic cells, patient-derived xenografts, and experimental therapeutics. The prevalence of 15 opportunistic microbes in a murine research facility was determined by yearlong PCR-based murine and IVC equipment surveillance comprising 1738 specimens. Of the 8 microbes detected, 3 organisms-Staphylococcus xylosus, Proteus mirabilis, and Pasteurella pneumotropica biotype Heyl-were most prevalent in both murine and IVC exhaust plenum specimens. Overall, the 8 detectable microbes were more readily PCR-detectable in IVC exhaust airways than in murine specimens, supporting the utility of PCR testing of IVC exhaust airways as a component of immunodeficient murine health surveillance. Vaporized hydrogen peroxide (VHP) exposure of IVC equipment left unassembled (that is, in a 'static-open' configuration) did not eliminate PCR detectable evidence of microbes. In contrast, VHP exposure of IVC equipment assembled 'active-closed' eliminated PCR-detectable evidence of all microbes. Ensuring data integrity and maintaining a topographically complex immunodeficient murine research environment is facilitated by knowing the prevalent opportunistic microbes to be monitored and by implementing a PCR-validated method of facility decontamination that mitigates opportunistic microbes and the risk of invalidation of studies involving immunodeficient mice.