Multi-locus sequence typing of Salmonella enterica serovar Typhimurium isolates from wild birds in northern England suggests host-adapted strain

Authors


Laura A. Hughes, National Centre for Zoonosis Research, University of Liverpool, Leahurst, Chester High Road, Neston CH64 7TE, UK. E-mail: laurahughes03@gmail.com

Abstract

Aims:  Recent studies have suggested that Salmonella Typhimurium strains associated with mortality in UK garden birds are significantly different from strains that cause disease in humans and livestock and that wild bird strains may be host adapted. However, without further genomic characterization of these strains, it is not possible to determine whether they are host adapted. The aim of this study was to characterize a representative sample of Salm. Typhimurium strains detected in wild garden birds using multi-locus sequence typing (MLST) to investigate evolutionary relationships between them.

Methods and Results:  Multi-locus sequence typing was performed on nine Salm. Typhimurium strains isolated from wild garden birds. Two sequence types were identified, the most common of which was ST568. Examination of the public Salmonella enterica MLST database revealed that only three other ST568 isolates had been cultured from a human in Scotland. Two further isolates of Salm. Typhimurium were determined to be ST19.

Conclusions:  Results of MLST analysis suggest that there is a predominant strain of Salm. Typhimurium circulating among garden bird populations in the United Kingdom, which is rarely detected in other species, supporting the hypothesis that this strain is host adapted.

Significance and Impact of the Study:  Host–pathogen evolution is often assumed to lead to pathogens becoming less virulent to avoid the death of their host; however, infection with ST568 led to high mortality rates among the wild birds examined, which were all found dead at wild bird-feeding stations. We hypothesize that by attracting unnaturally high densities of birds, wild bird-feeding stations may facilitate the transmission of ST568 between wild birds, therefore reducing the evolutionary cost of this pathogen killing its host, resulting in a host-adapted strain with increased virulence.

Salmonella enterica subsp. enterica serovar Typhimurium (Salm. Typhimurium) has been associated with winter ‘die-off’ episodes in UK garden bird populations, particularly among members of the Fringillidae (finch) and Passeridae (sparrow) families (Hughes et al. 2008). It has also been suggested that wild birds transmit salmonellae to humans and livestock (Kapperud et al. 1998). This hypothesis was tested recently in a study investigating the molecular epidemiology of Salm. Typhimurium isolates from wild birds found dead at garden bird-feeding stations across northern England (Hughes et al. 2008). A collection of 30 isolates were characterized through a combination of phage typing, macro-restriction pulsed-field gel electrophoresis, PCR virulotyping, macrophage cell invasion assays and antimicrobial susceptibility testing. Salm. Typhimurium phage-type DT56 was most commonly detected (23 isolates). Phage types DT40 (2 isolates), DT41 (2 isolates) and PT U277 (1 isolate) were detected less frequently, and one isolate could not be phage typed. Interestingly, in previous studies, a variant of DT56 has been frequently recovered from wild finches and sparrows (Pennycott et al. 2006); however, in this study, DT56 was isolated rather than the variant strain. After characterization, it was concluded that these wild bird strains were significantly different to those commonly associated with disease in humans and livestock, and it was hypothesized that they may be host adapted (Hughes et al. 2008). However, to investigate this hypothesis further, it was necessary to characterize isolates using genome-wide approaches to determine genetic differences between them. Therefore, in the current study, we carried out multi-locus sequence typing (MLST) on a representative sample of the Salm. Typhimurium strains to investigate evolutionary relationships between them.

MLST is a sequence-based method. This gives it the advantage of being objective and allows for both the comparison of isolates between laboratories and the evolutionary relationships between isolates from diverse sources to be inferred (Feil et al. 2004). An MLST scheme for Salm. enterica has recently been developed (Kidgell et al. 2002), and the public database (http://mlst.ucc.ie/mlst/dbs/Senterica) currently contains approximately 3000 isolates that have been characterized using this scheme, allowing all Salm. Typhimurium isolates to be compared.

Chromosomal DNA was extracted from nine freshly grown Salm. Typhimurium strains (Table 1) using a Wizard Genomic DNA Purification Kit (Promega, Hampshire, United Kingdom) according to the manufacturer’s instructions. Segments of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA and thrA) were amplified by PCR and sequenced as described previously (Kidgell et al. 2002). Allele numbers and sequence types (STs) were assigned by comparing sequences with those held on the public MLST profile database. All STs generated by this study were submitted to the database. Relationships between these strains and those on held on the MLST database were explored using the burst algorithm (based upon related STs) in the START software package (Jolley et al. 2001; Feil et al. 2004).

Table 1.   Multi-locus sequence typing (MLST) allelic profiles and sequence types (ST) of nine Salmonella Typhimurium isolates detected in the faecal samples of nine wild birds. Allele numbers were assigned by comparing sequences with those held on the public MLST profile database (http://mlst.ucc.ie/mlst/dbs/Senterica). Sequence types were also assigned by comparison with those held on the MLST database. The source of the isolates is also given
IsolateSourcePhage typeAllelic profileSequence type
Scientific nameCommon nameFamilyaroCdnaNhemDhisDpurEsucAthrA
WB190Passer domesticusHouse sparrowPasseridaeDT561837129592ST568
WB238Carduelis chlorisGreenfinchFringillidaeDT40107129592ST19
WB244Carduelis chlorisGreenfinchFringillidaeDT561837129592ST568
WB245Carduelis carduelisGoldfinchFringillidaeDT561837129592ST568
WB246Passer domesticusHouse sparrowPasseridaePT U2771837129592ST568
WB249Carduelis spinusSiskinFringillidaeDT561837129592ST568
WB254Streptopelia decaoctoCollared doveColumbidaeDT561837129592ST568
WB256Columba palumbusWood pigeonColumbidaeDT561837129592ST568
WB273Carduelis chlorisGreenfinchFringillidaeDT41107129592ST19

Two Salm. enterica sequence types (STs) were identified among the nine Salm. Typhimurium strains examined (Table 1). The most common ST was ST568, represented by seven isolates, including all DT56 isolates and an isolate of phage type PT U277. Examination of the Salm. enterica MLST database revealed that at the time of writing only three other ST568 isolates had been cultured from a human in Scotland. ST568 is a single-locus variant of ST19, which is currently predicted to be the founder strain of all Salm. Typhimurium sequence types and to date is the most commonly reported Salm. Typhimurium ST from a wide range of hosts (Cooke et al. 2008). One isolate each of Salm. Typhimurium DT40 and DT41, which occur at lower prevalences in garden birds, was determined to be ST19.

Results of MLST analysis suggest that there is a predominant strain of Salm. Typhimurium circulating among garden bird populations in the United Kingdom, in particular, finches and sparrows. ST568 may represent a host-adapted strain of Salm. Typhimurium. However, this strain led to systemic disease and mortality in an unusually high proportion of the wild birds examined. It is often assumed that host–pathogen evolution leads to pathogens becoming less virulent as it is an evolutionary disadvantage for a pathogen to kill its host (Anderson and May 1982). However, it has also been demonstrated that pathogen virulence can increase when host death has little impact on pathogen fitness (Day 2002). Indeed, host adaptation in S. enterica appears to be associated with systemic rather than enteric infection. For example, Salm. enterica serovar Gallinarum, the host-restricted agent of fowl typhoid, which causes a severe systemic infection of galliform birds, appears to have evolved from Salm. enterica serovar Enteritidis associated with enteric infection in a wide range of hosts (Thompson et al. 2008). Recently, a highly invasive clone of Salm. Typhimurium, MLST sequence type 313, has emerged as a major public health problem in people in sub-Saharan Africa, which is a single-locus variant of ST19 (Kingsley et al. 2009). The current study and that carried out by Kingsley et al. (2009) suggest that Salm. Typhimurium strains that lie outside ST19 may have increased host specificity; however, further work is needed to confirm this hypothesis.

All wild birds infected with Salm. Typhimurium ST568 were found dead at wild bird-feeding stations. Such feeding stations can attract un-naturally high densities of birds, which may facilitate the transmission of ST568 between wild birds. It is possible that this strain of Salm. Typhimurium has evolved to persist in the environment enabling it to be transmitted from the environment to wild bird hosts at garden feeding stations, therefore reducing the evolutionary cost of this pathogen killing its host, resulting in a virulent strain. We aim to investigate this latter hypothesis by investigating whether such strains are able to persist in the environment compared to other strains.

Acknowledgements

The authors thank the Merseyside Ringing Group for collecting field samples, RSPCA Stapeley Grange Wildlife Centre for providing dead bird carcases and members of the public who submitted dead birds for postmortem examination. This work was partially funded through the Defra/Hefce VTRI programme and partly through the Garden Bird Health Initiative (British Veterinary Association Animal Welfare Foundation, CJ Wildbird Foods Ltd, Cranswick Pet Products, Gardman Ltd, RSPB, The Birdcare Standards Association, Universities Federation for Animal Welfare).

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