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Piglet colibacillosis diagnosis by multiplex PCR and immunohistochemistry of paraffin-embedded tissues
Cíntia De Lorenzo*, Caroline P. de Andrade, Verônica S. L. Machado, Matheus V. Bianchi, Veronica M. Rolim, Raquel A. S. Cruz, David Driemeier
Department of Veterinary Pathology, School of Veterinary Medicine, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil (Lorenzo, Andrade, Machado, Bianchi, Rolim, Sales and Driemeier); and State Foundation of Livestock Research, Eldorado do Sul, Rio Grande do Sul, Brazil (Lorenzo and Driemeier)
Correspondence to: Tel: +555133086107, E-mail: cintiadelorenzobr@gmail.com
Received: February 10, 2017; Revised: May 10, 2017; Accepted: June 8, 2017; Published online: July 10, 2017.
Abstract
Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs, a condition termed colibacillosis, in which these bacteria should adhere and produce enterotoxins. 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 of E. coli causing colibacillosis in pigs, which requires the expression of at least one fimbria and one toxin of the agent. 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 (STx2e)], and IHC was optimized for an anti-E. coli polyclonal antibody. A total of 132 samples from pigs received by the laboratory between 2006 and 2014 with a clinical and histopathological diagnosis of colibacillosis were analyzed. E. coli was detected by IHC in 78.7%, and at least one virulence factor gene was detected in 71.2% of the cases. Pathogenic strains of ETEC were detected in 40% of the samples, with at least one fimbria and one toxin, 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. Colibacillosis diagnosis through multiplex PCR and IHC of paraffin-embedded tissues is a suitable approach, as the samples can be fixed in 10% formalin and stored for longer periods before analysis.
Keywords: Enteritis, enterotoxigenic Escherichia coli, swine diseases, virulence factors


© 2017 The Korean Society of Veterinary Science.