Development of Serotype-Specific PCR Assays for Typing of Haemophilus parasuis Isolates Circulating in Southern China

Abstract

The bacterium Haemophilus parasuis is the specific pathogenic cause of Glässer's disease in swine. Fifteen serotypes of H. parasuis have been reported. A method to serotype H. parasuis isolates accurately would help to prevent and control Glässer's disease outbreaks through appropriate vaccination and to understand the epidemiology in specific geographic areas. However, according to traditional serotyping, the rate of nontypeable (NT) strains is 10 to 40%, which gives low accuracy. In the present study, we developed a set of PCR assays that are able to identify all the currently known H. parasuis serotypes, with a detection limit of 5 CFU. This PCR method is particularly useful to distinguish serotype 5 from serotype 12. We then surveyed the serotype prevalence of H. parasuis isolates from southern China using both the traditional indirect hemagglutination (IHA) and current PCR methods. Of the 298 isolates tested, 228 (76.51%) and 281 (94.30%) were serotyped by the IHA and PCR tests, respectively, with a concordance rate of 80.87% (241/298). The most prevalent serotypes obtained by PCR were 4, 5, 12, 13, NT, and 2, and the most prevalent obtained by IHA were NT, 5, 4, 12, 13, and 2. In conclusion, the PCR assays developed in this study provide a rapid and specific method for the molecular serotyping of H. parasuis.

Keywords: Haemophilus parasuis; indirect hemagglutination assay; serotype-specific PCR; serotyping.

FIG 1

Band patterns of serotyping PCR for all 15 serovars of H. parasuis reference strains. Lanes S1 to S15, 15 serovars of H. parasuis reference strains; lane M, DL2000 DNA molecular weight marker; H₂O, negative control.

FIG 2

Serotype distribution of 298 Chinese isolates as determined by IHA and PCR.

FIG 3

Serotype distribution of H. parasuis isolated from diseased pigs in southern China between 2007 and 2015.