Kannika K, Pisuttharachai D, Srisapoome P, Wongtavatchai J, Kondo H, Hirono I, Unajak S, Areechon N
Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand.
Center for Advanced Studies for Agriculture and Food, Kasetsart University Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand.
J Appl Microbiol. 2017 Jun;122(6):1497-1507. doi: 10.1111/jam.13447. Epub 2017 May 9.
This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems.
Streptococcus agalactiae were isolated from diseased tilapia from different areas of Thailand. A total of 124 S. agalactiae isolates were identified by phenotypic analysis and confirmed by 16S rRNA PCR. Bacterial genotyping was conducted based on (i) molecular serotyping of the capsular polysaccharide (cps) gene cluster and (ii) virulence gene profiling using multiplex PCR analysis of 14 virulence genes (lmb, scpB, pavA, cspA, spb1, cyl, bca, rib, fbsA, fbsB, cfb, hylB, bac and pbp1A/ponA). Only serotypes Ia and III were found in this study; serotype Ia lacks the lmb, scpB and spb1 genes, whereas serotype III lacks only the bac gene. Virulence tests in juvenile Nile tilapia demonstrated a correlation between the pathogenicity of the bacteria and their virulence gene profile, with serotype III showing higher virulence than serotype Ia. Epidemiological analysis showed an almost equal distribution in all regions of Thailand, except serotype III was found predominantly in the southern areas.
Only two serotypes of S. agalactiae were isolated from diseased tilapia in Thailand. Serotype Ia showed fewer virulence genes and lower virulence than serotype III. Both serotypes showed a similar distribution throughout Thailand.
We identified two major serotypes of S. agalactiae isolates associated with the outbreak in tilapia culture in Thailand. We developed multiplex PCR assays for 14 virulence genes, which may be used to predict the pathogenicity of the isolates and track future infections. Multiplex PCR typing of the GBS virulence genes was developed and might be further used to predict the pathogenicity of S. agalactiae.
本研究旨在基于分子分型方法对从泰国罗非鱼养殖场分离出的无乳链球菌进行生物分型，并确定细菌血清型与毒力之间的相关性。除了基于cps基因多重PCR的生物分型（血清分型）技术外，本研究还开发了B族链球菌（GBS）毒力基因的多重PCR分型，以检测三簇毒力基因及其与无乳链球菌致病性的相关性。分析泰国无乳链球菌的流行病学，为罗非鱼养殖系统未来合理的疫苗策略提供细菌遗传信息。
从泰国不同地区患病罗非鱼中分离出无乳链球菌。通过表型分析鉴定出124株无乳链球菌分离株，并经16S rRNA PCR确认。基于以下方法进行细菌基因分型：（i）荚膜多糖（cps）基因簇的分子血清分型；（ii）使用14个毒力基因（lmb、scpB、pavA、cspA、spb1、cyl、bca、rib、fbsA、fbsB、cfb、hylB、bac和pbp1A/ponA）的多重PCR分析进行毒力基因谱分析。本研究仅发现血清型Ia和III；血清型Ia缺乏lmb、scpB和spb1基因，而血清型III仅缺乏bac基因。对尼罗罗非鱼幼鱼进行的毒力测试表明，细菌的致病性与其毒力基因谱之间存在相关性，血清型III的毒力高于血清型Ia。流行病学分析表明，在泰国所有地区分布几乎相等，除了血清型III主要在南部地区发现。
从泰国患病罗非鱼中仅分离出两种无乳链球菌血清型。血清型Ia的毒力基因比血清型III少，毒力也较低。两种血清型在泰国各地的分布相似。
我们鉴定出与泰国罗非鱼养殖疫情相关的两种主要无乳链球菌血清型。我们开发了针对14个毒力基因的多重PCR检测方法，可用于预测分离株的致病性并追踪未来感染情况。开发了GBS毒力基因的多重PCR分型，可能进一步用于预测无乳链球菌的致病性。
