Development and validation of a multiple locus variable number tandem repeat analysis (MLVA) scheme for Fusobacterium necrophorum

Rachel Clifton,Laura E Green,Kevin J Purdy

doi:10.1016/j.vetmic.2017.11.017

Abstract

Fusobacterium necrophorum is associated with various diseases in humans and animals. Reservoirs (sites where the pathogen persists in the absence of disease) of F. necrophorum are believed to be present in healthy individuals e.g. tonsillar epithelium, or their environment e.g. soil, but for most diseases the reservoir sites are unknown. Strain typing of F. necrophorum would facilitate linking specific reservoirs with a specific disease. The aim of this study was to develop multiple locus variable number tandem repeat analysis (MLVA) as a strain typing technique for F. necrophorum, and to test the use of this scheme to analyse both isolates and mixed communities of bacteria. Seventy-three tandem repeat regions were identified in the F. necrophorum genome; three of these loci were suitable and developed as a MLVA scheme. The MLVA scheme was sensitive, specific, and discriminatory for both isolates and communities of F. necrophorum. The MLVA scheme strain typed 46/52F. necrophorum isolates including isolates of both subspecies and from different countries, host species and sample sites within host. There were 12 unique MLVA strain types that clustered by subspecies. The MLVA scheme characterised the F. necrophorum community in DNA from 32/49 foot- and 28/33 mouth swabs from sheep. There were 17 community types in total. In 31/32 foot swabs, single strains of F. necrophorum were detected while in the 28 mouth swabs there were up to a maximum of 8 strains of F. necrophorum detected. The results demonstrate the potential for this method to elucidate reservoirs of F. necrophorum.

Highlights

Fusobacterium necrophorum is a Gram-negative, rod-shaped, anaerobic bacterium that is associated with a variety of diseases, termed necrobacilloses, in humans and animals
Reservoirs of F. necrophorum are believed to be present in healthy individuals e.g. tonsillar epithelium, or their environment e.g. soil, but for most diseases the reservoir sites are unknown
F. necrophorum causes hepatic abscesses that occur in intensively reared beef cattle (Lechtenberg et al, 1988; Nagaraja and Chengappa, 1998; Narayanan et al, 1997) and it is associated with footrot in sheep (Egerton et al, 1969; Witcomb et al, 2014), foot infections in other ungulates (Clark et al, 1985; Edwards et al, 2001; Handeland et al, 2010), endometritis in cattle (Ruder et al, 1981), calf diphtheria (Panciera et al, 1989), respiratory disease in deer (Brooks et al, 2014) and periodontal disease in wallabies (Antiabong et al, 2013b)

Summary

Introduction

Fusobacterium necrophorum is a Gram-negative, rod-shaped, anaerobic bacterium that is associated with a variety of diseases, termed necrobacilloses, in humans and animals. F. necrophorum has been isolated from the gingiva (Bennett et al, 2009; McCourtie et al, 1990) and detected on both healthy and footrot-diseased feet (Calvo-Bado et al, 2011; Frosth et al, 2015; Maboni et al, 2016; Witcomb et al, 2014), but the significance of these sites as reservoirs is unknown. MLVA was originally developed to analyse individual isolates but it can be used to analyse samples that may contain a mixed community of strains within a species (Vranckx et al, 2011) In these cases, MLVA is used to produce a molecular “fingerprint” of the strains present and so identify similarities and differences between communities

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Conclusion