Development of a Rapid Multiplex PCR Assay To Genotype Pasteurella multocida Strains by Use of the Lipopolysaccharide Outer Core Biosynthesis Locus

Abstract

Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the "gold standard." The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains. P asteurella multocida is the primary causative agent of a wide range of economically important diseases, including hemorrhagic septicemia in ungulates, atrophic rhinitis in pigs, fowl cholera in birds, snuffles in rabbits, and enzootic pneumonia and shipping fever in cattle, sheep, and pigs (1). P. multocida also causes opportunistic infections in humans, often following cat or dog bites, and plays a contributory role, together with other pathogens, in a range of lower respiratory tract infections and sporadic septicemias in ungulates (1).P. multocida strains have classically been differentiated using serological techniques. Strains can be classified into five capsular serogroups (A, B, D, E, and F) using an indirect hemagglutination test (2) and into 16 somatic or lipopolysaccharide (LPS) serovars (serotypes) using the Heddleston gel diffusion precipitin test (3). Both of these schemes have been widely used. Isolates are commonly assigned a combined designation, such as A:1 (capsular serogroup A and LPS serovar 1) or B:2 (capsular serogroup B and LPS serovar 2).P. multocida LPS is an immunodominant antigen critical for homologous protection stimulated by bacterin (killed-cell) vaccines (4). Furthermore, in the P. multocida strain VP161, a fulllength LPS molecule is essential for the ability to cause acute disease (5, 6). Heddleston serotyping is currently the only method used to differentiate P. multocida strains on the basis of LPS type. However, the accuracy of Heddleston serotyping has never been objectively tested, as the precise LPS structures produced by different strains have not been known. Indeed, there have been many informal as well as formal reports that the Heddleston system fails to type many isolates and lacks accuracy and reproducibilit...

A Pasteurella multocida strain affecting nulliparous heifers and calves in different ways

“…The isolates were all non-typable by the Heddleston serotyping scheme, but the LPS PCR showed all isolates to be L3, a group that includes serovars 3 and 4 (Harper et al 2015). The isolates were all the same ST -ST 79, which is associated with the clonal complex ST13 (Table 1).…”

“…As well, a PCR that targets the LPS outer core biosynthesis locus and assigns isolates to one of eight LPS types (termed L1 to L8) was performed as previously described (Harper et al, 2015) except that the genomic DNA was prepared using the PrepMan Ultra Sample Preparation Reagent according to manufacturer's protocol (Life Technology -Applied Biosystems, Warrington, UK).…”

“…The specific disease syndromes vary with the host species. There are five capsule serogroups (A, B, D, E and F), and 16 somatic serovars based on lipopolysaccharide (LPS) antigens according to the Heddleston serotyping scheme, which has recently been transferred to a polymerase chain reaction (PCR) methodology recognising eight LPS types (Harper et al, 2015).…”

Evaluation of a proposed molecular methodology for the serotyping of Avibacterium paragallinarum

“…5 For A. paragallinarum serogrouping by the Page scheme, a multiplex (m)PCR and a PCR-restriction fragment length polymorphism (RFLP) method have been developed. 8 The assays are based on a hypervariable region of the HMTp210 gene, also referred to as region 2, encoding the protective hemagglutinin (HA) protein.…”

Re-emergence of bovine haemorrhagic septicaemia in Hungary

“…With the exception of serogroup A, capsular type was identified using a multiplex PCR method as described previously (Townsend et al, 2001). The somatic serotype was determined using a multiplex PCR assay (Harper et al, 2014). Classical serotyping using agar gel diffusion precipitation test was also carried out (Heddleston et al, 1972).…”