COUNTERMEASURES TO PREVENT THE PORCINE RESPIRATORY DISEASE COMPLEX (PRDC)
Location: Virus and Prion Research Unit
Title: A Modified Multi-Locus Sequence Typing (MLST) Scheme for Haemophilus parasuis
Submitted to: Pig Veterinary Society International Congress Proceedings
Publication Type: Proceedings
Publication Acceptance Date: March 15, 2010
Publication Date: July 20, 2010
Citation: Register, K.B., Mullins, M., Bayles, D.O. 2010. A Modified Multi-Locus Sequence Typing (MLST) Scheme for Haemophilus parasuis. In: Proceedings of the International Pig Veterinary Society Congress, July 18-21, 2010, Vancouver, Canada. p. 516.
Introduction. Haemophilus parasuis is the etiologic agent of Glässer’s disease and pneumonia in swine. Phenotypic classification systems are of assistance in epidemiologic studies but molecular methods provide numerous distinct advantages. An MLST scheme proposed by others appears promising (4). However, recently acquired genome data (3,5) reveal the primers used, based on sequences of other bacteria, are not optimal for H. parasuis. Furthermore, several different PCR mixes and cycling programs are required for amplification of all targets. Here we report modifications to enhance and simplify the original method, thereby facilitating data acquisition and increasing accuracy. The modified MLST was applied to a group of 35 diverse H. parasuis isolates and the results compared with those obtained using the original method.
Materials and Methods. Thirty-five genetically distinct strains of H. parasuis, including the 15 serovar reference strains, were evaluated by the original (4) and modified MLST methods. Amplicon sequences were trimmed, concatenated and used to construct Neighbor-Joining trees as reported (2). Target-specific alignments used to guide primer redesign included genome sequences of four H. parasuis strains (3,5). Vector NTI Advance was used for sequence editing and analysis and to assist in primer design.
Results and Discussion. Alignments of genome sequences from 7 target genes, atpD, infB, mdh, rpoB, 6pgd, g3pd and frdB, were examined for variability and quality of match within the original primer-binding regions (see Fig. 1 for an example). Ten of the 14 originally recommended primers (4) were redesigned to eliminate mismatches and avoid regions of high sequence heterogeneity.
Fig. 1. DNA sequence from the mdh gene of the H. parasuis strain indicated on the right as compared to the MLST primers originally recommended. Base mismatches with the primers are highlighted.
To simplify the method for end users, we standardized the Tm of all primers and identified a universal PCR mix and set of cycling conditions that resulted in robust and reproducible amplification of all targets.
The number and percentage of variable positions within the dataset obtained using our modifications (240 of 3154 positions; 7.6%) is slightly less than for the original method (408 of 3806 positions; 10.7%) but the grouping of strains in major clusters is nearly identical (Fig. 2). Analysis of H. parasuis genomes indicates some MLST targets are in close proximity along the chromosome (e.g., 6pgd and frdB are separated by ~10Kb), in opposition to general guidelines for MLST (1). Nonetheless, the discriminatory power of the scheme proposed here suggests it is a highly useful phylogenetic and epidemiologic tool.
Fig. 2 Neighbor-Joining tree derived from the modified MLST method. Major clusters are unaffected as compared to the original method, with the exception of strain MN-H, which originally clustered with SW124.
1. Maiden, MC. 2006. Ann Rev Microbiol 60:561-88.
2. Mullins, MA et al. 2009. J Bacteriol 191:5988-6002
3. Mullins, MA, Bayles, DO, Register, KB. unpublished
4. Olvera, A et al. 2006. Microbiol 152:3683-3690.
5. Yue, M et al. 2008. J Bacteriol 191:1359-60.