Differentiation of Brachyspira spp. isolated from laying hens using PCR-based methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

Avian intestinal spirochetosis (AIS), an important but neglected disease in laying hens, is caused by Brachyspira pilosicoli, B. intermedia, and B. alvinipulli. Poultry are also frequently colonized by putatively nonpathogenic species such as B. murdochii and B. innocens. We evaluated the differentiation of Brachyspira species by 3 methods: sequencing of the reduced nicotinamide adenine dinucleotide (NADH) oxidase gene ( nox), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a new multiplex (m)PCR targeting genes such as the tryptophanase A gene ( tnaA) and the p-aminobenzoyl-glutamate hydrolase subunit B gene ( abgB). Sequencing of 414 bp of the nox PCR amplification products generated from 41 pure cultures of avian Brachyspira isolates allowed presumptive species identification in 33 isolates with at least 99% identity in basic local alignment search tool analysis, including B. pilosicoli, B. intermedia, B. murdochii, B. innocens, and " B. pulli". MALDI-TOF MS analysis was found to be a reliable tool for differentiation after extension of the manufacturer's database. In the mPCR, all isolates identified as B. pilosicoli and B. intermedia were positive for abgB and tnaA, respectively. The mPCR might be very useful in detecting Brachyspira species in mixed cultures including not only nonpathogenic species, such as B. innocens, but also one of the AIS pathogens. We found that MALDI-TOF MS analysis combined with the mPCR targeting tnaA and abgB was suitable for the identification of avian isolates of B. pilosicoli and B. intermedia, 2 important agents of AIS.

Brachyspira in dogs: risk factors of shedding in central Germany and longitudinal study of an infected kennel

“… *criterion or criteria of the acute hemorrhagic diarrhea index (AHDi; [ 24 ]); ‡includes diarrhea (and/or hematochezia/melena, mucus, other material), vomiting, weight loss, abdominal pain, and/or flatulence …”

“…Mass spectrometry-based identification of Brachyspira pilosicoli – Identification of B. pilosicoli by MALDI-TOF MS was performed after extraction of samples following a previously published protocol [ 24 ]. Importantly, “ B. canis” and “ B. pulli ” are not represented in the commercial database of the MALDI-TOF MS spectrometer (Microflex LRF, Bruker Daltonics, Bremen, Germany).…”

“…4 ), while the abgB gene is specific for B. pilosicoli . The tnaA amplification product is found in B. intermedia, B. suanatina , and B. hyodysenteriae [ 24 ]. Sequencing of the nox gene served to differentiate Brachyspira as described [ 24 ].…”

“…The tnaA amplification product is found in B. intermedia, B. suanatina , and B. hyodysenteriae [ 24 ]. Sequencing of the nox gene served to differentiate Brachyspira as described [ 24 ]. The following reference strains were used as positive controls in every MP-PCR analysis: B. pilosicoli isolate 102/06 and B. hyodysenteriae isolate 404/06 – both from Germany [ 24 ], B. intermedia isolate AN26/93 from Sweden [ 37 ], and “ B. canis” isolate S2017 from Germany, which was isolated as part of this study.…”

“…Sequencing of the nox gene served to differentiate Brachyspira as described [ 24 ]. The following reference strains were used as positive controls in every MP-PCR analysis: B. pilosicoli isolate 102/06 and B. hyodysenteriae isolate 404/06 – both from Germany [ 24 ], B. intermedia isolate AN26/93 from Sweden [ 37 ], and “ B. canis” isolate S2017 from Germany, which was isolated as part of this study. After sequencing the nox amplicon, nucleotides 512–926 of the open reading frame were analyzed using the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information (NCBI; conducted between November 2021 and August 2022).…”