De Lorenzo Cíntia, de Andrade Caroline P, Machado Verônica S L, Bianchi Matheus V, Rolim Veronica M, Cruz Raquel A S, Driemeier David
Department of Veterinary Pathology, School of Veterinary Medicine, Federal University of Rio Grande do Sul, Porto Alegre 91540-000, Brazil.
State Foundation of Livestock Research, Eldorado do Sul 92990-000, Brazil.
J Vet Sci. 2018 Jan 31;19(1):27-33. doi: 10.4142/jvs.2018.19.1.27.
Enterotoxigenic (ETEC) causes diarrhea in pigs, referred to as colibacillosis. The aim of this study was to optimize multiplex polymerase chain reaction (PCR) and immunohistochemistry (IHC) analyses of paraffin-embedded material to detect pathogenic strains causing colibacillosis in pigs. Multiplex PCR was optimized for fimbriae (F18, F4, F6, F5, and F41) and toxins (types A and B heat-stable toxins [STaP and STb], heat-labile toxin [LT], and type 2 Shiga toxin [STx]), and IHC was optimized for an anti- polyclonal antibody. Samples (132) from pigs received between 2006 and 2014 with clinical and histopathological diagnoses of colibacillosis were analyzed. was detected by IHC in 78.7%, and at least one virulence factor gene was detected in 71.2%. Pathogenic strains of ETEC with at least one fimbria and one toxin were detected in 40% of the samples in multiplex PCR. The most frequent virulence types were F18-STaP (7.5%), F18-STaP-STb (5.7%), and F4-STaP (3.8%). A statistically significant association was noted between virulence factors F4, F18, STaP, and STb and positive immunostaining results. Colibacillosis diagnosis through multiplex PCR and IHC of paraffin-embedded tissues is a practical approach, as samples can be fixed and stored for long periods before analysis.
产肠毒素大肠杆菌（ETEC）可导致猪腹泻，即仔猪大肠杆菌病。本研究的目的是优化对石蜡包埋材料的多重聚合酶链反应（PCR）和免疫组织化学（IHC）分析，以检测引起猪大肠杆菌病的致病菌株。对菌毛（F18、F4、F6、F5和F41）和毒素（A 型和 B 型耐热毒素[STaP和STb]、不耐热毒素[LT]以及2型志贺毒素[STx]）进行多重PCR优化，对抗多克隆抗体进行免疫组织化学优化。分析了2006年至2014年间接收的132份临床和组织病理学诊断为大肠杆菌病的猪样本。免疫组织化学检测阳性率为78.7%，至少检测到一种毒力因子基因的样本占71.2%。多重PCR在40%的样本中检测到至少含有一种菌毛和一种毒素的ETEC致病菌株。最常见的毒力类型为F18-STaP（7.5%）、F18-STaP-STb（5.7%）和F4-STaP（3.8%）。毒力因子F4、F18、STaP和STb与免疫染色阳性结果之间存在统计学显著关联。通过对石蜡包埋组织进行多重PCR和免疫组织化学诊断大肠杆菌病是一种实用方法，因为样本在分析前可长期固定和保存。
