Identification of Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia in commercial laying hens and commercial broiler breeders using fluorescence in situ hybridization em tecidos incluidos em parafina

• Published in: Ciência rural Vol. 54; no. 10; p. 1
• Main Authors: Gruchouskei, Leonardo, Piccoli, Ronaldo Jose, Streauslin, Jessica Santos, Cavasin, Joao Pedro, de Matos, Monica Regina, Faccin, Mayane, Viott, Aline de Marco
• Format: Journal Article
• Language: English
• Published: Universidade Federal de Santa Maria 01-10-2024
• Subjects: Brasil
• ISSN: 0103-8478
• DOI: 10.1590/0103-8478cr20230445

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. Key words: avian intestinal spirochaetosis, broiler breeders, enteropathogens, Fluorescence in situ hybridization, laying hens. Bacterias do genero Brachyspira podem ocasionar enfermidades entericas em aves, assim o objetivo deste estudo foi avaliar a eficacia da tecnica de hibridizacao fluorescente in situ (FISH) para a identificacao de Brachyspira pilosicoli, Brachyspira hyodysenteriae e Brachyspira intermedia utilizando amostras de ceco de aves de postura e matrizes comerciais fixados em formol. Foram coletadas amostras de 112 aves entre granjas de postura comercial e granjas de matrizes de corte com idade entre 35 e 82 semanas. Para avaliacao, primeiramente procedeu-se ao isolamento bacteriano a partir das fezes. As amostras positivas foram submetidas a qPCR para identificacao de especies patogenicas. Posteriormente, segmentos formolizados de ceco dessas mesmas aves, foram processados pela tecnica de FISH utilizando sondas marcadas de Brachyspira pilosicoli, Brachyspira hyodysenteriae e Brachyspira intermedia. Das 112 amostras foram obtidos 40 isolamentos caracteristicos de Brachyspira e destes, na qPCR, 14 identificadas como B. hyodysenteriae e sete como B. intermedia, duas amostras foram positivas para ambas e 21 nao foram caracterizadas. Na tecnica de FISH, 52 amostras foram positivas para Brachyspira sp., 22 para B. hyodysenteriae, 28 para B. intermedia, sete para B. pilosicoli e oito amostras foram positivas para duas especies. A avaliacao estatistica revelou que a tecnica de FISH foi capaz de indicar maior n mero de aves positivas quando comparada ao isolamento bacteriano seguido por qPCR, inclusive revelando aves positivas para B. pilosicoli que nao tinham sido identificados anteriormente. Dessa forma, conclui-se que a tecnica de FISH apresentou-se eficiente para identificacao das Brachyspiras avaliadas, servindo assim como uma ferramenta rapida e eficiente para o diagnostico. Palavras-chave: espiroquetose intestinal aviaria, matriz de corte, enteropatogenos, hibridizacao fluorescente in situ, aves de postura.