Pathogenic and genomic characterization of rabbit-sourced Pasteurella multocida serogroup F isolates recovered from dead rabbits with respiratory disease

Abstract

Pasteurella multocida serogroup F can infect a number of animals. However, the pathogenicity and genomic features of this serogroup are still largely unknown. In the present study, the pathogenicity and genomic sequences of 19 rabbit-sourced P. multocida serogroup F isolates were determined. The 19 isolates were highly pathogenic for rabbits causing severe pathologic lesions and high mortality in inoculated rabbits. Nevertheless, the pathologic lesions in rabbits caused by the 19 isolates were distinct from those caused by the previously reported high-virulent serogroup F strains J-4103 (rabbit), P-4218 (turkey), and C21724H3km7 (chicken). Moreover, the 19 isolates were avirulent to white feather broilers. The genomes of the 19 isolates were determined to understand the pathogenicity of these isolates. The finding of a number of functional genes in the 19 isolates by comparison with the low-virulent rabbit-sourced serogroup F strain s4 might contribute to the high virulence of these isolates. Notably, polymorphisms were determined in the lipopolysaccharide outer core biosynthetic genes natC and gatF among the serogroup F strains of different hosts. However, the sequences of natC and gatF from rabbit-sourced strains (except for SD11) were identical, which might be responsible for the host specific of the 19 isolates. The observations and findings in this study would be helpful for the understanding of the pathogenicity variation and host predilection of P. multocida.

Importance: The 19 rabbit-sourced Pasteurella multocida serogroup F isolates showing high virulence to rabbits were avirulent to the broilers. Notably, polymorphisms were determined in the lipopolysaccharide outer core biosynthetic genes natC and gatF among all serogroup F strains of different hosts. However, the sequences of natC and gatF from rabbit-sourced strains (except for SD11) were identical, which might be responsible for the host specific of the 19 isolates.

Keywords: Pasteurella multocida serogroup F; pathogenicity; rabbit; whole-genome sequence.

Fig 1

Neighbor-joining tree indicating the positions of the 19 rabbit-sourced P. multocida isolates (PF1–PF19) based on the housekeeping genes of multi-host MLST and RIRDC MLST schemes. The multi-host MLST and RIRDC MLST housekeeping genes of the isolate were concatenated, and then the neighbor-joining phylogenetic tree (1,000 bootstrap replications) was constructed using MEGA 5.0. The multi-host MLST and RIRDC MLST housekeeping genes of the strains were freely obtained from the NCBI database under the accession numbers: Pm70 (AE004439), HN07 (CP007040), s4 (CP084165), CIRMBP-0873 (CP020347), CIRMBP-0884 (CP020345), 9N (CP028927), AH09 (CP090521), SD11 (CP090520), and 36502 (CP097792).

Fig 2

Pathologic lesions of the rabbits subcutaneously inoculated with the 19 isolates. Rabbits were inoculated with 6.0 × 10⁴ CFU of the isolate. Diffuse hemorrhagic pneumonia (A) and diffuse subcutaneous hemorrhage (B) were observed in the rabbits that were euthanized within 24 h post-inoculation. Fibrino-hemorrhagic pleuropneumonia (C) and diffuse subcutaneous abscess (D) were observed in the rabbits that were euthanized after 24 h post-inoculation. Hemorrhagic pneumonia (E) and local subcutaneous abscess (F) were observed in the rabbits that survived the experiment. No pathologic lesions were observed in the lungs of the subcutaneous control rabbits (G).

Fig 3

Pathologic and histological lesions of the rabbits intranasally inoculated with the 19 isolates. Rabbits were inoculated with 6.0 × 10⁴ CFU of the isolate. Fibrino pleuropneumonia (A) or fibrinopurulent pleuropneumonia (B) was observed in the rabbits that were euthanized during the 15-day experiment period. Pulmonary consolidation with hemorrhagic pneumonia (C) was observed in the rabbits that survived the experiment. No pathologic lesions were observed in the lungs of the intranasal control rabbits (D). Inflammatory exudates in the bronchiole and alveoli, penetration of red blood cells in the bronchiole and alveoli as well as degeneration of the alveolar epithelial cells (E) were observed in the lungs with fibrino pleuropneumonia or fibrinopurulent pleuropneumonia. Inflammatory exudates in bronchiole and alveoli and proliferation of alveolar epithelial cells (F) were observed in the lungs with pulmonary consolidation and hemorrhagic pneumonia. No histopathological lesions (G) were observed in the lungs of the intranasal control rabbits.

Fig 4

Comparison between the genome of PF13 and those of s4, CIRMBP-0884, HN07, HN06, CQ2, and Pm70. From the outside to the inside, circle 1 (gray): plasmid of PF13; circle 2 (gray): chromosome of PF13; circle 3 (purple): plasmid of s4; circle 4 (purple): chromosome of s4; circle 5 (blue): plasmid of CIRMBP-0884; circle 6 (blue): chromosome of CIRMBP-0884; circle 7 (cyan-blue): HN07; circle 8 (red): HN06; circle 9 (yellow): CQ2; circle 10 (green): Pm70; circles 11 and 12 represent the G + C content and GC skew, respectively; the innermost circle represents DNA base position.

Fig 5

Comparative analyses of the entire cap locus between the 19 isolates and other P. mltocida strains. The entire cap locus of the 19 isolates was compared with those of Pm70 (type F), CQ2 (type A), and HN06 (type D). The color code represents the BLASTn identity.

Fig 6

Comparative analyses of the LPS outer core locus between the 19 isolates and other P. multocida serogroup F:L3 strains. The LPS outer core biosynthetic genes of the 19 isolates were compared with those of s4, CIRMBP-0873, CIRMBP-0884, AH09, Pm70, 9N, SD11, HN07, F, and 36502.

Fig 7

Comparative analyses of the LPS outer core biosynthetic glycosyltransferases NatC and GatF between the 19 isolates and other P. multocida serogroup F:L3 strains. (A) Pm70 and 9N produce the full-length NatC of 392 amino acids in length (1). PF1–PF19, s4, CIRMBP-0873, CIRMBP-0884, and AH09 produce the truncated NatC of 331 amino acids in length because of the in-frame 61 amino acid truncation (yellow) (2). SD11, HN07, F, and 36502 produce two hypothetical proteins that were fully matched with the N-terminal (red) and C-terminal (blue) of the NatC of Pm70 (3). (B) Pm70 produces the shortest GatF of 208 amino acids in length (1). F produces the GatF of 260 amino acids in length with 52 amino acids redundancy at the N-terminal by comparison with that of Pm70 (red) (2). PF1–PF19, s4, AH09, SD11, and 36502 produce the GatF of 278 amino acids in length with 70 amino acids redundancy at the N-terminal by comparison with that of Pm70 (yellow) (3). CIRMBP-0873, HN07, and 9N produce the longest GatF of 280 amino acids in length with 72 amino acids redundancy at the N-terminal by comparison with that of Pm70 (green) (4). CIRMBP-0884 (rabbit) produced the GatF of 268 amino acids in length with 70 amino acids redundancy at the N-terminal (blue) and a truncated C-terminal of MQK (cyan-blue) [the GatF C-terminal of the other P. multocida serogroup F strains were NAKMKLKCIVKFE (purple)] by comparison with that of Pm70 (5).