Molecular Epidemiology of Pasteurella multocida Associated with Bovine Respiratory Disease Outbreaks

Abstract

Studies that characterize bovine respiratory disease (BRD)-associated Pasteurella multocida isolates are scarce compared with research on isolates from other hosts and clinical backgrounds. In the present study, 170 P. multocida isolates from 125 BRD outbreaks were characterized by capsular and LPS typing as well as by virulotyping. Three capsular types (A, B, F) and three LPS genotypes (L2, L3, L6) were identified. Capsular and LPS typing revealed a very low genetic diversity (GD = 0.02) among P. multocida, with most isolates belonging to genotype A:L3 (97.6%). Virulotyping identified seven virulence-associated gene profiles, with two profiles including 95.9% of the isolates. A subset of isolates was further characterized by MLST and PFGE. The sequence types ST79 and ST13 were the most frequently identified and were grouped into the same clonal complex (CC13), a result that supports the clonal population structure of BRD-associated P. multocida isolates. PFGE typing also revealed a low genetic diversity (GD = 0.18), detecting a single pattern in 62.5% of the outbreaks in which multiple isolates were analyzed. Overall, 85.2% of the isolates belonged to pulsotypes with at least 80% genetic similarity, consistent with a clonal population structure observed by MLST analysis and corroborating the genetic relatedness of most P. multocida isolates associated with BRD in cattle.

Genotypic Comparison of Pasteurella multocida from Healthy Animals at Entry to the Feedlots with That and from Bovine Respiratory Disease-Affected Animals during the Fattening Period

“…Capsular, Lipopolysaccharide, and Pulsed-Field Gel Electrophoresis (PFGE) Typing of P. multocida Isolates All P. multocida isolates (n = 106; 32 from cases and 74 from apparently healthy animals) were subjected to capsular and LPS detection by PCR. Bacterial DNA was obtained as described by Calderón et al [5]. Capsular types (A, B, D, E, and F) were determined by a multiplex PCR, as described by Townsend et al [9].…”

“…All P. multocida isolates, except four isolates that did not grow after freezing, were subjected to PFGE as previously described [5]. Salmonella enterica serovar Braenderup H9812 restricted with enzyme XbaI (Thermo Fisher, Madrid, Spain) was used as a molecular weight control.…”

“…Pasteurella multocida is one of the main bacterial pathogens contributing to bovine respiratory disease (BRD) [1][2][3][4][5]. P. multocida can be found as a commensal resident in the nasopharynx of healthy animals and has been isolated from both healthy and sick cattle [2,6,7].…”

“…multocida BRD-associated isolates have shown a very low level of genetic diversity, as demonstrated in several studies using a variety of typing methods, including capsular and LPS genotyping, virulotyping, and multilocus sequence typing (MLST) [5,6,13]. P. multocida isolates from BRD clinical cases belong mainly to the A: L3 genotype and closely related sequence types ST79, ST13, or ST80, which are included in clonal complex CC13 [5,6]. Sequence types ST79 and ST13 also seem to be the most prevalent among respiratory isolates from healthy cattle [6].…”

“…Whole-genome sequencing with phylogenetic analysis of P. multocida genomes identified ST79 as the most dominant clonal lineage in BRDassociated isolates worldwide and suggested that ST79 shows host specificity to cattle but genomic heterogeneity [8,14,15]. Using PFGE, it was found that some closely related clones (pulsotypes) were consistently associated with clinical cases of BRD [5]. However, whether these clones are prevalent among healthy animals is unknown, since studies analyzing the molecular characteristics of P. multocida isolates from healthy animals are scarce [6].…”