Original research paperVirulence gene profiling and ompA sequence analysis of Pasteurella multocida and their correlation with host species
Introduction
Pasteurella multocida is a Gram-negative opportunistic bacterial pathogen that causes various diseases in a wide range of host species including humans worldwide (Wilkie et al., 2012; Wilson and Ho, 2013). The organism causes fowl cholera (Rhoades and Rimler, 1989), atrophic rhinitis in pigs (Magyar and Lax, 2002), haemorrhagic septicaemia in buffalo and cattle (De Alwis, 1992), pneumonia in ruminants and swine, and respiratory tract diseases in rabbits and rodents (Boyce et al., 2010).
P. multocida is a diverse bacterial species, consisting of five capsular serogroups (Carter, 1955; Rimler and Rhoades, 1987), sixteen serovars based on the lipopolysaccharide (LPS) antigens (Heddleston et al., 1972; Harper et al., 2011) and eight LPS genotypes (Harper et al., 2015). It has a complex set of virulence associated genes (Harper et al., 2006) and several putative virulence factors have been identified that contribute to adhesion, colonization and invasion of the host. These include adhesins, the P. multocida toxin (PMT), iron-acquisition proteins, extracellular enzymes (neuraminidases) and outer membrane proteins (Harper et al., 2006). Molecular characterisation based on the detection of different virulence associated genes has proven to be useful for typing P. multocida isolates, and these virulence factors are thought to be important epidemiological markers for recognition of the pathogenic potential of the isolates (Ewers et al., 2006; Atashpaz et al., 2009; García-Alvarez et al., 2017).
For this reason, the aim of this study was to screen 180 Hungarian P. multocida isolates recovered from different host species (including humans) for the prevalence of capsular serovars, LPS genotypes and virulence-associated genes, and in addition the gene encoding outer membrane protein A (ompA), and correlate the carriage of these virulence traits to host species.
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Bacterial isolates
A total of 180 isolates of P. multocida isolated from different host species were used in this study (Supplementary Table 1). These strains were isolated from cattle, small ruminants (sheep and goat), pigs, cats and humans in Hungary from 1988 to 2018. The isolates were cultured on Columbia agar (LAB M Ltd., Bury, UK) plates supplemented with 5% sheep blood under aerobic conditions at 37 °C for 24 h. Their identity was confirmed by a species-specific PCR assay (Townsend et al., 1998).
Results
The isolates exhibited colony morphology typical of P. multocida, and their identity was confirmed by the species-specific PCR assay. Capsular typing detected two capsular genotypes (A and D) for the bovine, ovine, caprine and porcine strains of P. multocida. The isolates from human cases were capsular serogroup A or F, and all strains from cats belonged to capsular type A. Type A was the most common capsular serovar for P. multocida strains from bovine, ovine, porcine, human and feline
Discussion
P. multocida is a widespread veterinary pathogen of mammals and birds with zoonotic potential (Wilson and Ho, 2013). In the present study, 180 isolates of P. multocida from a broad range of host species were characterised comprehensively by determining their capsular type, LPS genotype. In addition, a virulence gene profile (VGP) scheme has been established analysing nine virulence-associated genes. Furthermore, ompA sequence analysis of 94 selected strains revealed an association between host
Conclusion
Bacterial virulence is determined by genetic markers (Harper et al., 2006), that include colonisation factors, outer membrane proteins and iron acquisition factors. The typing scheme we have described in this study is likely to be useful for epidemiological studies. Interestingly our results demonstrate a high level of diversity in virulence gene content among P. multocida strains from different host species, which is likely to be important in determining differences in bacterial gene
Conflict of interest
None of the authors has any financial or personal relationships that could inappropriately influence the content of the paper.
Acknowledgements
This work was supported by the National Research, Development and Innovation Fund, Hungary – Grant NKFIH K124457. The authors thank Dr. A. J. Lax for the useful comments on the manuscript. The outstanding technical assistance of É. Hegedűs and K. Oryszcsák is highly appreciated.
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