Molecular Typing of Pasteurella pneumotropica Isolated from Rodents by Amplified 16S Ribosomal DNA Restriction Analysis and Pulsed‐Field Gel Electrophoresis

Abstract

Pasteurella pneumotropica, a ubiquitous Gram-negative bacterium, is an opportunistic pathogen for rodents (28). This agent can be frequently isolated from the upper respiratory tract, lungs, vagina, trachea and other digestive organs. P. pneumotropica does not significantly affect the health of immunocompetent animals. However, immunodeficient animals infected with P. pneumotropica develop severe or lethal pneumonia (1,8,13,25). Chapes et al. (8) reported that P. pneumotropica induced lethal pneumonia in mice lacking alleles for MHCII, Tlr4, and Nramp1. Furthermore, Hart et al. (13) reported that Toll-like receptor 4 positive macrophages were indispensable for defense against the P. pneumotropica infections in mice. Therefore, control and monitoring of P. pneumotropica infections are requisites for managing the immunodeficient animals.P. pneumotropica was first characterized by Jawetz (17). Heyl (15) then proposed the biotype of P. pneumotropica based on utilization of carbon sources, and P. pneumotropica was reclassified as P. pneumotropica biotypes Jawetz and Heyl (27). Subsequently, Boot and Bisgaard (6) reported the detailed biochemical variations of P. pneumotropica including wild type strains. Further, Boot et al. (7) reported that the P. pneumotropica isolates could be differentiated by haemagglutinating properties of the isolates. However, host variation and the some phenotypic characteristics of the P. pneu- Abstract: A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed-field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with HaeIII revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty-two percent of the 23 isolates identified as a-1 were derived from mice, whereas all the isolates identified as a-3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a-2 and a-4, respectively. By restriction analysis of genomic DNA, ApaI and NotI digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a-2 and a-4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with HaeIII.

Identification and Characterization of Hemolysin-Like Proteins Similar to RTX Toxin in Pasteurella pneumotropica

“…S1c in the supplemental material). Furthermore, several studies have revealed hemolysis in wild-type strains of P. pneumotropica (16,34); these results suggest that reference strain ATCC 35149, as well as several strains of P. pneumotropica, are weak hemolytic bacteria. Further, both of the two identified RTX toxins were involved with the putative hemolysin-type Ca 2ϩ -binding regions, and as each of the RTX toxins expressed in the E. coli cells, both the crude lysates showed weak hemolytic activity in the erythrocytes.…”

“…However, its taxonomical classification is uncertain, and the exact name of the genus has not been formally given (28). Therefore, the genetic and biochemical characteristics of wild-type strains of P. pneumotropica were heterogenous and diversified (34). A method based on the detection and identification of certain virulence factors in isolates should be developed.…”

Pathogenicity of Pasteurella pneumotropica in Immunodeficient NOD/ShiJic-scid/Jcl and Immunocompetent Crlj:CD1 (ICR) Mice

“…Based on its biochemical characteristics, P. pneumotropica is classified as biovars Jawetz or Heyl. Based on restriction analysis of 16S ribosomal DNA, we divided P. pneumotropica into 4 groups, a-1, a-2, a-3, and a-4 [20]. Data from principal component analysis of biochemical and genetic characteristics were used to discriminate 3 host-dependent and host-independent groups [22].…”

“…The strain possessed the outermost surface materials on the bacterial cells [11] and was identified as a-1 by amplified 16S ribosomal DNA restriction analysis [20]. The strain was thawed from frozen stock (-80°C) and grown overnight on 5% horse blood agar plates (Climedia, Sankyo Junyaku Co., Ltd., Tokyo, Japan) and checked for purity.…”

Experimental Infection Studies of Pasteurella pneumotropica and V-factor Dependent Pasteurellaceae for F344-rnu Rats

“…Recently, P. pneumotropica isolates were reported as genetically different by its host animal species [9,18]. Although limited numbers of strains were used in this study, the pathogenicity of P. pneumotropica derived from rats may be extremely low in rats.…”