Identification of Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia in commercial laying hens and commercial broiler breeders using fluorescence in situ hybridization (FISH) in paraffin-embedded tissues

Abstract

ABSTRACT: Bacteria of the genus Brachyspira can cause enteric diseases in birds; thus, this study evaluated the efficacy of the fluorescence in situ hybridization (FISH) technique for the identification of B. pilosicoli, B. hyodysenteriae and B. intermedia using cecum samples fixed in formaldehyde from laying hens and commercial broiler breeders. Samples were collected from 112 birds aged between 35 and 82 weeks that originated from commercial laying and broilers farms. For the initial evaluation, spirochaetes were isolated from the cloacal swabs. Positive samples were analysed using qPCR to identify pathogenic species. Formalized cecum segments of these same birds were then analyzed using the FISH technique with labelled probes specific to B. pilosicoli, B. hyodysenteriae and B. intermedia. Forty isolates characteristic of Brachyspira were obtained, of which 14 were identified as B. hyodysenteriae and seven were identified as B. intermedia by qPCR; two samples were positive for both species, and 21 were not characterized. Using the FISH technique, 52 samples were positive for Brachyspira spp., 22 were positive for B. hyodysenteriae, 28 were positive for B. intermedia, seven were positive for B. pilosicoli, and eight were positive for two species. The FISH technique was able to identify significantly more positive birds compared with bacterial isolation followed by qPCR. Thus, it is concluded that the FISH technique was effective for identifying the three Brachyspira species evaluated and can thus be used as a rapid and effective diagnostic tool.

Keywords

• avian intestinal spirochaetosis
• broiler breeders
• enteropathogens
• Fluorescence in situ hybridization
• laying hens