The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, St Lucia, Qld 4067, Australia.
The University of Queensland, School of Veterinary Science, Gatton, QLD 4343, Australia.
J Microbiol Methods. 2021 Mar;182:106159. doi: 10.1016/j.mimet.2021.106159. Epub 2021 Feb 4.
Glaesserella parasuis consists of 15 serovars with some of them highly virulent and some of them avirulent. As killed vaccines do not provide crossprotection across serovars, serotyping is of importance. Serotyping, previously done by gel diffusion, is now done by multiplex PCR followed by electrophoresis. Accurately differentiating 15 serovars by electrophoresis is problematic. To overcome this problem, a Luminex microbead-based multiplex assay was used to differentiate the serovars. The assay consisted of a multiplex PCR assay followed by hybridisation to microbeads which were then analysed on a Luminex machine. The newly developed assay was compared to the multiplex serotyping PCR and the gel diffusion/indirect haemagglutination assay (GD/IHA). The microbead-based assay worked very well for the 15 reference strains but when used on the 74 Australian field strains displayed some problems. The main problems were with the eight out of nine serovar 4 field isolates and the five serovar 7 and three serovar 14 field isolates. While the microbead-based assay could differentiate between the serovar 5 and 12 reference strains, which the serovar multiplex PCR could not, all four field isolates identified by GD/IHA as serovar 12 were identified as serovar 5 by the microbead-based assay. Serovar 4 has been noted to have a high diversity especially among strains from different countries. Our work clearly shows that the diversity of strains at both the national and the international level has to be taken into account when developing diagnostic assays.
副猪嗜血杆菌包含 15 个血清型，其中一些具有高度致病性，而另一些则没有致病性。由于灭活疫苗不能在血清型之间提供交叉保护，因此血清分型很重要。以前通过凝胶扩散进行血清分型，现在通过多重 PCR 后电泳进行。通过电泳准确地区分 15 个血清型是有问题的。为了解决这个问题，使用基于 Luminex 微球的多重分析来区分血清型。该分析包括多重 PCR 分析，然后与微球杂交，然后在 Luminex 仪器上进行分析。新开发的分析与多重血清分型 PCR 和凝胶扩散/间接血凝测定 (GD/IHA) 进行了比较。该基于微球的分析对 15 个参考菌株非常有效，但在对 74 个澳大利亚现场分离株进行使用时显示出一些问题。主要问题出现在 9 个血清型 4 个野外分离株中的 8 个，以及 5 个血清型 7 和 3 个血清型 14 个野外分离株。虽然基于微球的分析可以区分血清型 5 和 12 的参考菌株，而血清型多重 PCR 不能区分，但 GD/IHA 鉴定为血清型 12 的所有 4 个野外分离株均被基于微球的分析鉴定为血清型 5。已经注意到血清型 4 在不同国家的菌株中具有高度多样性。我们的工作清楚地表明，在开发诊断分析时，必须考虑到国家和国际层面菌株的多样性。
