Volume 74, Issue 2\nResearch Article\nOpen Access\nErysipelothrix amsterdamensis sp.\n\nnov., associated with mortalities among endangered seabirds\nView Affiliations Hide Affiliations\nAffiliations: 1​ School of Biodiversity, One Health & Veterinary Medicine, University of Glasgow, Glasgow, G12 8QQ, UK 2​ School of Life Sciences, Westlake University, Hangzhou, Zhejiang, PR China 3​ Bioinformatics Research Technology Platform, University of Warwick, Coventry, CV4 7AL, UK 4​ CEFE, UMR 5175, Univ Montpellier, CNRS, EPHE, IRD, Montpellier, France 5​ Ceva Biovac, Beaucouzé, France 6​ École Normale Supérieure de Lyon, 69342 Lyon Cedex 07, France 7​ Vet Diagnostics, 69260 Charbonnières-les-Bains, France 8​ Department of Ecology and Evolutionary Biology, University of California, Los Angeles, USA 9​ Department of Public and Ecosystem Health, Cornell University, Ithaca, New York, USA\nAbstract\nInfectious diseases threaten endangered species, particularly in small isolated populations.\n\nSeabird populations on the remote Amsterdam Island in the Indian Ocean have been in decline for the past three decades, with avian cholera caused by Pasteurella multocida proposed as the primary driver.\n\nHowever, Erysipelothrix species have also been sporadically detected from albatrosses on Amsterdam Island and may be contributing to some of the observed mortality.\n\nIn this study, we genomically characterized 16 Erysipelothrix species isolates obtained from three Indian yellow-nosed albatross (Thalassarche carteri) chick carcasses in 2019.\n\nHistological analyses suggest that they died of bacterial septicaemia.\n\nTwo isolates were sequenced using both Illumina short-read and MinION long-read approaches, which – following hybrid assembly – resulted in closed circular genomes.\n\nMapping of Illumina reads from the remaining isolates to one of these new reference genomes revealed that all 16 isolates were closely related, with a maximum of 13 nucleotide differences distinguishing any pair of isolates.\n\nThe nucleotide diversity of isolates obtained from the same or different carcasses was similar, suggesting all three chicks were likely infected from a common source.\n\nThese genomes were compared with a global collection of genomes from Erysipelothrix rhusiopathiae and other species from the same genus.\n\nThe isolates from albatrosses were phylogenetically distinct, sharing a most recent common ancestor with E. rhusiopathiae.\n\nBased on phylogenomic analysis and standard thresholds for average nucleotide identity and digital DNA–DNA hybridization, these isolates represent a novel Erysipelothrix species, for which we propose the name Erysipelothrix amsterdamensis sp.\n\nnov.\n\nThe type strain is A18Y020dT (=CIP 112216T=DSM 115297T).\n\nThe implications of this bacterium for albatross conservation will require further study.\n\nKeyword(s): comparative genomics , conservation , Erysipelothrix and yellow-nosed albatross\nFunding\nThis study was supported by the:\n© 2024 The Authors\nArticle metrics loading...\n/content/journal/ijsem/10.1099/ijsem.0.006264\n2024-02-15\n2024-08-15\nGo to section...\nErysipelothrix amsterdamensis sp.\n\nnov., associated with mortalities among endangered seabirds\nJiadong Zhong12; Matej Medvecky13; Jérémy Tornos45; Augustin Clessin46; Rozenn Le Net7; Hubert Gantelet5; Amandine Gamble189; Taya L. Forde1*†; Thierry Boulinier4†\n1: 1​School of Biodiversity, One Health & Veterinary Medicine, University of Glasgow, Glasgow, G12 8QQ, UK; 2: 2​School of Life Sciences, Westlake University, Hangzhou, Zhejiang, PR China; 3: 3​Bioinformatics Research Technology Platform, University of Warwick, Coventry, CV4 7AL, UK; 4: 4​CEFE, UMR 5175, Univ Montpellier, CNRS, EPHE, IRD, Montpellier, France; 5: 5​Ceva Biovac, Beaucouzé, France; 6: 6​École Normale Supérieure de Lyon, 69342 Lyon Cedex 07, France; 7: 7​Vet Diagnostics, 69260 Charbonnières-les-Bains, France; 8: 8​Department of Ecology and Evolutionary Biology, University of California, Los Angeles, USA; 9: 9​Department of Public and Ecosystem Health, Cornell University, Ithaca, New York, USA\n†These authors contributed equally to this work\nFN1Reads for all samples sequenced in this study are available on European Nucleotide Archive Sequence Read Archive (SRA) under accession number PRJEB50151.\n\nComplete annotated genomes for isolates A18Y020dT (CIP 112216T=DSM 115297T) and A18Y016a are available under accession numbers GCA_940143175 and GCA_940143155, respectively.\n\nThe 16S rRNA sequence for A18Y020dT is available on GenBank as accession OQ355811.\n\nABSTRACT\nGo to section...\nInfectious diseases threaten endangered species, particularly in small isolated populations.\n\nSeabird populations on the remote Amsterdam Island in the Indian Ocean have been in decline for the past three decades, with avian cholera caused by Pasteurella multocida proposed as the primary driver.\n\nHowever, Erysipelothrix species have also been sporadically detected from albatrosses on Amsterdam Island and may be contributing to some of the observed mortality.\n\nIn this study, we genomically characterized 16 Erysipelothrix species isolates obtained from three Indian yellow-nosed albatross (Thalassarche carteri) chick carcasses in 2019.\n\nHistological analyses suggest that they died of bacterial septicaemia.\n\nTwo isolates were sequenced using both Illumina short-read and MinION long-read approaches, which – following hybrid assembly – resulted in closed circular genomes.\n\nMapping of Illumina reads from the remaining isolates to one of these new reference genomes revealed that all 16 isolates were closely related, with a maximum of 13 nucleotide differences distinguishing any pair of isolates.\n\nThe nucleotide diversity of isolates obtained from the same or different carcasses was similar, suggesting all three chicks were likely infected from a common source.\n\nThese genomes were compared with a global collection of genomes from Erysipelothrix rhusiopathiae and other species from the same genus.\n\nThe isolates from albatrosses were phylogenetically distinct, sharing a most recent common ancestor with E. rhusiopathiae.\n\nBased on phylogenomic analysis and standard thresholds for average nucleotide identity and digital DNA–DNA hybridization, these isolates represent a novel Erysipelothrix species, for which we propose the name Erysipelothrix amsterdamensis sp.\n\nnov.\n\nThe type strain is A18Y020dT (=CIP 112216T=DSM 115297T).\n\nThe implications of this bacterium for albatross conservation will require further study.\n\nKeywords\nAuthor Notes\n†These authors contributed equally to this work\nReads for all samples sequenced in this study are available on European Nucleotide Archive Sequence Read Archive (SRA) under accession number PRJEB50151.\n\nComplete annotated genomes for isolates A18Y020dT (CIP 112216T=DSM 115297T) and A18Y016a are available under accession numbers GCA_940143175 and GCA_940143155, respectively.\n\nThe 16S rRNA sequence for A18Y020dT is available on GenBank as accession OQ355811.\n\nAbbreviations\nGo to section...\nANI, average nucleotide identity; CDS, coding sequences; dDDH, digital DNA-DNA hybridization; GWAS, genome-wide association study; kb, kilobases; SNP, single nucleotide polymorphism.\n\nIntroduction\nGo to section...\nInfectious diseases are among the major threats to the conservation of endangered species, especially at local scales [1].\n\nThis is particularly true for populations in remote areas, where anthropogenic activity has historically been limited but is increasing, such as with commercial tourists to the Antarctic [2, 3].\n\nConcerns have been raised about disease-associated declines among albatross populations and how this could influence their long-term sustainability [4].\n\nAvian cholera, caused by the bacterium Pasteurella multocida, has been causing recurrent chick mortalities among two endangered albatross species on the remote Amsterdam Island in the southern Indian Ocean since at least the mid-1990s when it was first detected [5–7].\n\nBoth the Indian yellow-nosed albatross (Thalassarche carteri) and the sooty albatross (Phoebetria fusca) have been affected, and the endemic endangered Amsterdam albatross (Diomedea amsterdamensis) is also at risk [5].\n\nThe bacterium Erysipelothrix rhusiopathiae has also been detected by PCR in swabs from live birds of these three albatross species and from the endangered northern rockhopper penguin (Eudyptes moseleyi) on Amsterdam Island [6], and it has been isolated concomitantly from mortalities among the Indian yellow-nosed albatross [5].\n\nDuring ongoing population health monitoring conducted to infer transmission dynamics and management opportunities [8–10], our team isolated Erysipelothrix species (identified at the time as E. rhusiopathiae) in 2019 from the carcasses of three Indian yellow-nosed albatross chicks on the island, strengthening the hypothesis that this bacterium could also be contributing to albatross mortalities and decreased breeding success.\n\nErysipelothrix rhusiopathiae is a Gram-positive rod-shaped bacterium.\n\nWhile best known as an opportunistic pathogen of pigs and poultry [11, 12], it affects a wide range of species [13] and in recent years it has been implicated in large-scale wildlife mortalities [14].\n\nIt was also responsible for jeopardising the translocation success of several critically endangered kakapo – a flightless parrot – between New Zealand and offshore islands [15], and has caused the death of other endangered birds in that area [16].\n\nErysipelas in wild birds is a septicaemic disease [12], and deaths have been reported in a range of wild terrestrial birds and seabirds worldwide [15].\n\nMortalities occur sporadically, and tend to be limited to a small number of individuals, although cases are likely under-recognised and under-reported [17].\n\nRare mass mortality events in wild birds have also been observed [18].\n\nIn this study, we sought to genomically characterize the Erysipelothrix strains isolated from Indian yellow-nosed albatross on Amsterdam Island to identify potential sources, and to assess the relationship between the multiple observed cases on the island.\n\nMethods\nGo to section...\n\nBetween December 2018 and March 2019, 27 chick and two adult carcasses of Indian yellow-nosed albatross were necropsied in the field on Amsterdam Island (−37.797135, 77.571521) as part of long-term population health monitoring (Appendix S1, Fig.\n\nS1, available in the online version of this article, Table S1).\n\nSamples from the heart, lung, liver, kidney, spleen, brain and bursa of Fabricius in all three birds, of the adrenal glands in one bird (AMS18-NEC-019), and of the thymus in one bird (AMS18-NEC-016) were collected and fixed in 4 % neutral buffered formalin at room temperature.\n\nSamples were routinely processed as formalin-fixed paraffin-embedded blocks, and sectioned at 4 µm thickness.\n\nTissue sections were routinely stained with haematoxylin and eosin for examination by light microscopy.\n\nSamples from multiple organs (liver, lung, brain and heart) were collected from the carcasses for bacteriological analysis (Appendix S1).\n\nIsolates of Erysipelothrix species were identified from three of the chick carcasses, all sampled between the 13 and 16 January 2019.\n\nThe bacterial growth in the field (Appendix S1) showed Erysipelothrix species exclusively, suggesting that this bacterium could have been the cause of death.\n\nBetween four and six isolates were obtained from each carcass (n=16 total; Table S1).\n\nThese were initially identified in the laboratory as E. rhusiopathiae based on colony morphology on horse blood agar and MALDI-TOF, with the exception of strain A18Y019b, which was classified as an Erysipelothrix species.\n\nThe following phenotypic analyses of isolate A18Y020d were carried out by DSMZ Services, Leibniz-Institut (Braunschweig, Germany).\n\nBiochemical characterization was performed using the API Rapid ID32 Strep test (bioMérieux) according to instructions of the manufacturer; this was done in two replicates, starting from individual precultures.\n\npH (tested at increments of one) and temperature range (tested at increments of 5 °C) were determined.\n\nGenomic DNA was extracted from sub-cultured isolates (loop of colonies) at the University of Glasgow using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich) as per the manufacturer’s instructions.\n\nExtracted DNA was tested with a probe-based qPCR targeting E. rhusiopathiae [19], then sequenced at MicrobesNG (Birmingham, UK).\n\nLibraries were prepared using the Nextera XT v2 kit (Illumina) and sequenced on the Illumina HiSeq platform, generating 250 base pair paired-end reads.\n\nLong-read MinION sequencing (Oxford Nanopore) was performed at the University of Glasgow for two isolates (A18Y016a and A18Y020d), with library preparation done using the rapid barcoding kit (SQK-RBK004).\n\nIllumina reads were adapter and quality trimmed using Trimmomatic version 0.30 [20] and assembled de novo using SPAdes version 3.7 [21] by MicrobesNG.\n\nAssembled genomes were evaluated using the online version of Quality Assessment Tool for Genome Assemblies (http://cab.cc.spbu.ru) [22].\n\nUnless otherwise indicated, the remaining bioinformatics analyses were performed within the climb computing platform for microbial genomics [23].\n\nTo generate circularized (closed) reference genomes, the Illumina and MinION sequence data were jointly assembled by Unicycler version 0.4.4 [24] applying the ‘normal’ (default) hybrid mode.\n\nFor isolate A18Y016a, this did not result in a closed genome, so subsequently Canu version 1.7 [25] was used to perform an initial assembly using the long-read MinION data, and Pilon version 1.22 [26] applied to polish the assembly using Illumina reads, including three iterations.\n\nTablet version 1.21.02.08 [27] was used to visually check the distance between forward and reverse reads across the start and end positions of the linearized chromosome to confirm the genomes’ circular nature.\n\nProkka version 1.13 [28] and blast2GO [29] were used to predict coding sequences (CDS), tRNA, rRNA and tmRNA.\n\nThe general feature format (.gff) files obtained by the two programs were merged to build a complete .gff annotation file.\n\nThe phaster server (https://phaster.ca/) [30] was used to identify and annotate prophage sequences.\n\nplasmidFinder server version 2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) [31] was used to search for plasmid sequence data in the genomes against the replicon database.\n\nAntimicrobial resistance genes were searched for in the hybrid assembly of A18Y020d using both CARD (database downloaded on 28 April 2023) [32] and ResFinder-4.4.1 [33], using a detection threshold of 80 % over a minimum length of 60 %.\n\nblast searches for 16 genes that have been putatively linked with virulence in E. rhusiopathiae [34] were conducted against the hybrid assembly of A18Y020d.\n\nBWA-MEM [35] with default settings was used to map Illumina reads of each albatross isolate against the A18Y020d closed genome, and the mpileup command in SAMtools version 1.13 [36] was used to generate summary text files of coverages across the reference genome.\n\nThe pileup2snp command in Varscan version 2.4.4 [37] was used to generate a list of high-quality single nucleotide polymorphisms (SNPs) that were filtered by applying parameters ‘--min-coverage’ of 10, ‘--min-var-freq’ of 0.8 and ‘--min-avg-qual’ of 20.\n\nA custom R script was developed to generate a multi-fasta alignment file based on concatenated SNPs.\n\nPairwise SNP differences between isolates were calculated using the snp-dists package version 0.8.2.\n\nRoary version 3.12.0 [38], with default parameters, was used to construct a core gene alignment for a wider phylogenetic analysis, i.e.\n\nto place the albatross isolates within the context of global Erysipelothrix genomes.\n\nThis included 106 genomes: (a) the 16 isolates from this study; (b) 86 previously sequenced E. rhusiopathiae isolates, representing a diversity of geographic and host origins and spanning the clades described for this species [clade 1 (n=7), clade 2 (n=14), intermediate clade (n=8), clade 3 (n=57)] [39]; (c) two genomes of Erysipelothrix piscisicarius (formerly referred to as E. sp.\n\nstrain 2) [40, 41]: the type strain 15TAL0474T (GCA_003931795.1) [42] and EsS2-7-Brazil (GCA_016617655.1)–E.\n\npiscisicarius is the most closely related species to E. rhusiopathiae currently known; (d) a recently deposited Erysipelothrix genome, reported as a new species, Erysipelothrix sp.\n\nPoltava (GCA_023221615.1); and (e) Erysipelothrix tonsillarum DSM14972T (GCA_000373785.1), which was used as an outgroup, since this was previously reported to be ancestral to these other species [39].\n\nThe SNP alignment and core gene alignment files were used to estimate phylogenetic trees for the 16 albatross isolates and the full set of Erysipelothrix genomes, respectively, using RAxML version 8.2.4 [43] with the maximum-likelihood method implementing a GTR-GAMMA nucleotide substitution model, selected after using MrModeltest version 2.4 [44].\n\nTo determine the relatedness of the albatross isolates from this study with other less well-described Erysipelothrix species available in the Genome Taxonomy Database (https://gtdb.ecogenomic.org/), a separate phylogenetic tree based on the rpoB gene (beta subunit of RNA polymerase) was estimated.\n\nThis gene has been found to be a more suitable marker for Erysipelothrix species delineation than the widely used 16S rRNA gene [40].\n\nNucleotide sequences of rpoB genes were extracted from representative whole-genome assemblies (one per species, n=15 total), and aligned using the L-INS-i method as implemented in mafft version 7.313 [45].\n\nTree topology was then inferred by RAxML version 8.2.4, implementing the GTR model of nucleotide substitution; robustness of the tree was assessed by performing 1000 bootstrap replicates.\n\nFor completeness, the 16S rRNA gene sequence of the newly sequenced strain A18Y020d was extracted through blastn alignment and compared with 16S gene sequences from E. rhusiopathiae ATCC 19414T (NR_040837.1), E. piscisicarius 15TAL0474T (NR_170392.1) and E. tonsillarum DSM 14972T (NR_040871.1).\n\nTwo different genome relatedness indices were calculated in order to compare the Erysipelothrix species isolated from albatrosses with previously characterized Erysipelothrix species and determine the species assignment.\n\nPairwise nucleotide-level comparisons were made using average nucleotide identity (ANI) Calculator for OrthoANIu [46] (www.ezbiocloud.net/tools/ani) and digital DNA–DNA hybridization (dDDH) was performed using GGDC [47, 48] (https://ggdc.dsmz.de), using Formula 2.\n\nThis formula is independent of genome length and is thus robust to the use of incomplete draft genome assemblies, as well as differences in gene content.\n\nThe output of Roary was used in Scoary version 1.6.16 [49] to conduct a pan-GWAS (genome-wide association study) in order to identify any genes that might be unique to the albatross isolates, where the binary trait considered was ‘albatross origin’ yes/no.\n\nNCBI blastn searches were conducted on the Scoary results (i.e.\n\ngenes identified as unique to the albatross isolates that were absent in other Erysipelothrix species) to verify their absence in other bacterial species.\n\nPrimer sets typically used to amplify Erysipelothrix species [50] and E. rhusiopathiae specifically [19] were evaluated in silico against the complete assemblies using Geneious version 11.0.5 [51] to check for any mismatches.\n\nFinally, to further characterize the Erysipelothrix isolates from albatross, blastn searches of the de novo Illumina assemblies were conducted to determine in silico the serotype [52] and whether any spa genes (A, B or C) [53] were present, using methods described previously [54].\n\nResults\nGo to section...\nSixteen isolates identified as Erysipelothrix species were recovered from the carcasses of three albatross chicks.\n\nFifteen of these were initially identified as E. rhusiopathiae based on colony morphology and MALDI-TOF with 99.9 % confidence, and the other strain was identified as an Erysipelothrix species.\n\nHistological analyses revealed intravascular bacteria in various tissues (notably liver and heart; see for instance Figs S2–3) and occasional thrombosis.\n\nThese bacteria were associated with interstitial pneumonia and pulmonary congestion, multifocal necrotizing hepatitis and/or splenitis, and/or multicentric haemorrhages (liver, heart, kidneys, spleen, meninges) (Appendix S2).\n\nFindings are consistent with bacterial septicaemia as the cause of death of these individuals.\n\nPhenotypic characteristics determined during bacterial strain isolation are given in the species description below and in Appendix S3.\n\nAll 16 isolates were genome sequenced on the Illumina platform, which yielded a high average depth of coverage of at least 58× for all genomes (Table S1).\n\nDe novo genome assemblies comprised between 52 and 58 contigs per genome, with a total assembly length ranging from 1.80 to 1.83 megabases.\n\nComplete circular genomes were obtained for isolates A18Y020dT (Fig.\n\nS4; assembly GCA_940143175) – designated the type strain – and A18Y016a (assembly GCA_940143155), with lengths of 1 908 712 bp and 1 910 750 bp, respectively.\n\nThis is approximately 120 kilobases (kb) longer than the Fujisawa E. rhusiopathiae reference genome, corresponding to ~100 additional CDS (Table S2).\n\nLike the Fujisawa E. rhusiopathiae genome, both genomes from albatrosses had a G+C content of 36.6 mol%.\n\nPreliminary annotation of the two albatross-derived genomes using Prokka showed that this method predicted a large number of hypothetical proteins, amounting to approximately one third of predicted CDS.\n\nBlast2GO facilitated the annotation of just over 1500 further CDS in each genome.\n\nUsing phaster, an intact prophage region comprising 48 genes was detected in both genomes (Fig.\n\nS5, Table S2).\n\nBased on global alignment using muscle, this showed 43.6 % nucleotide identity with the incomplete phage sequence in the E. rhusiopathiae Fujisawa reference genome (gene loci ERH_0581 to ERH_0629).\n\nNo plasmids were detected, nor any resistance genes predicted.\n\nblast hits were obtained for all 16 putative virulence genes, all with >85 % sequence similarity.\n\nPairwise identity of the 16S rRNA gene sequence from strain A18Y020dT with that of E. rhusiopathiae ATCC 19414T was 99.9 %; only two SNP differences were present across 1479 nucleotides (at positions 472 and 473; TC instead of CT).\n\nOnly three further nucleotide differences distinguished these sequences from the 16S sequence of E. tonsillarum DSM 14972T (99.8 % pairwise identity).\n\nEight gaps were found in the alignment of the 16S sequence from E. piscisicarius 15TAL0474T compared with the others, which also shared one of the SNPs from A18Y020dT at position 472, and had one additional nucleotide difference.\n\nThe phylogenetic tree (Fig.\n\n1a), based on 427 conserved core genes, showed that the 16 Erysipelothrix isolates from albatrosses form a monophyletic clade distinct from previously characterized Erysipelothrix genomes, which shares its most recent common ancestor with E. rhusiopathiae.\n\nBased on the rpoB gene phylogeny, other Erysipelothrix species discovered to date (E. anatis, E. aquatica, E. inopinata, E. larvae, E. urinaevulpis and five other yet-to-be-named species deposited in the Genome Taxonomy Database) are more phylogenetically distant (Fig.\n\n1b), with the exception of the recently deposited sequence for Erysipelothrix sp.\n\nPoltava; based on rpoB analysis, ANI/dDDH (Fig.\n\nS6) and phylogenomic analyses (Fig.\n\nS7), strain Poltava is concluded to belong to E. rhusiopathiae clade 2.\n\nFig.\n\n1.Phylogenetic relatedness of Erysipelothrix species from Indian yellow-nosed albatross to other isolates within the genus.\n\n(a) Maximum likelihood phylogenetic tree comparing Erysipelothrix isolates (n=16) albatross chicks on Amsterdam Island (highlighted in pink) with representative genomes from the most closely related Erysipelothrix species.\n\nThe tree was estimated in RAxML using a multiple sequence alignment of conserved (core) genes identified using Roary, implementing a GTR-GAMMA model.\n\nBootstrap values for all major nodes were 100 %.\n\nE. tonsillarum DSM14972T was used as an outgroup for rooting.\n\n(b) Phylogenetic tree of Erysipelothrix species based on rpoB gene sequences.\n\nThis confirms that E. rhusiopathiae, E. piscisicarius and E. tonsillarum are the most closely related species to the Erysipelothrix species isolated from albatrosses in this study.\n\nTrees were plotted with Interactive Tree Of Life version 6.3.2 [64] and edited using Inkscape (www.inkscape.org).\n\nFig.\n\n1.\n\nClick to view\nFew SNP differences were observed among the albatross isolates.\n\nA total of 53 high-quality unique SNPs were detected across all 16 genomes when mapping against the closed reference genome (A18Y020dT; Fig.\n\n2).\n\nAll genomes were unique, including where multiple isolates were sequenced from the same carcass (Table S1).\n\nThe maximum pairwise SNP difference separating two isolates was 13.\n\nIn silico serotyping based on a genomic polysaccharide biosynthetic locus [49] determined these isolates belong to serotype 1b.\n\nAll genomes had identical sequences of the spaA gene, belonging to Group 2 [54].\n\nThese all had one unique amino acid residue at position 288 in comparison with any previously described sequences (serine instead of alanine).\n\nFig.\n\n2.Unrooted maximum likelihood tree of 16 Erysipelothrix isolates from Indian yellow-nosed albatross from Amsterdam Island.\n\nThe tree was based on high-quality concatenated SNPs detected through mapping to the circularised genome of A18Y020dT.\n\nIsolates originated from three different carcasses, which are distinguished by different branch colours (as shown in the legend).\n\nTree was plotted with Interactive Tree Of Life version 6.3.2 [64].\n\nFig.\n\n2.\n\nClick to view\nThe ANI scores comparing the representative Erysipelothrix species type strain from albatross against E. rhusiopathiae were 95.0 % with E. rhusiopathiae clade 1 (Table S3), and just above 94 % for representatives from the other clades, whereas they were 86.9 % with E. piscisicarius and 80.9 % with E. tonsillarum (Fig.\n\n3).\n\nThe dDDH scores with E. rhusiopathiae clades 1, 2 and 3 were 60.0, 56.3 and 55.5 %, respectively, while they were 32.7 % with E. piscisicarius and 22.8 % with E. tonsillarum.\n\nThe accepted values for categorizing isolates as the same species are >95 % similarity for ANI [55, 56] and >70 % for dDDH [48].\n\nThus, based on these standard metrics for genomic comparison, and the phylogenomic analysis, we conclude that these isolates belong to a novel species of the genus Erysipelothrix – part of the family Erysipelotrichaceae.\n\nFig.\n\n3.Genome relatedness of Erysipelothrix species from albatross to previously described Erysipelothrix species.\n\n(a) The ANI scores comparing the Erysipelothrix species from albatross (represented by A18Y020dT) against other Erysipelothrix species were all below the threshold for being considered a common species (>95 %).\n\n(b) The dDDH scores comparing the Erysipelothrix species from albatross against other Erysipelothrix species were also well below the accepted threshold for being considered a single species (>70 %).\n\nGenomes included for comparison: E. rhusiopathiae clade 1 (P-92), clade 2 (P-190), intermediate (Fujisawa) and clade 3 (F793); E. piscisicarius (15TAL0474T); and E. tonsillarum (DSM14972T).\n\nFig.\n\n3.\n\nClick to view\nTwo hundred and two genes unique to the albatross-derived Erysipelothrix genomes (present in all 16 vs. absent in all 90 other genomes) were found using Scoary.\n\nHowever, after conducting blastn searches, the majority of these genes had significant hits to genes in E. rhusiopathiae or bacteria within the family Erysipelotrichaceae, or more rarely, with other bacteria.\n\nOnly six genes were found with no hits or hits with low query cover (≤12 %); a further six genes had <75 % nucleotide identity with the highest scoring blastn hit (Table S4).\n\nThe Erysipelothrix species DNA from all albatross isolates produced typical amplification curves using the probe-based qPCR by Pal et al.\n\n[19] designed to detect E. rhusiopathiae.\n\nThis is despite the fact that the forward primer and probe sequences had two mismatches each (Fig.\n\nS8); based on in silico alignment, the 165 bp segment being amplified corresponded to positions 1 211 721 to 1 221 885 in the A18Y020dT genome.\n\nThe primers designed by Makino et al.\n\n[50] for Erysipelothrix species more broadly were a perfect match, and based on in silico alignment would be expected to amplify the targeted 407 bp segment of the 16S rRNA gene (positions 1 221 445–1 221 851 in the A18Y020dT genome).\n\nDiscussion\nGo to section...\n\nThrough genomic comparisons, we have determined that the Erysipelothrix strains isolated from Indian yellow-nosed albatross chick carcasses on Amsterdam Island belong to a previously undescribed species, which we propose be named Erysipelothrix amsterdamensis sp.\n\nnov.\n\nThis reflects its geographic origin, as well as the co-occurrence of the iconic and endemic Amsterdam albatross, D. amsterdamensis.\n\nAnthropogenic sources are often suspected when novel pathogens are detected in new locations.\n\nWhile livestock was originally suggested as a possible source of the Erysipelothrix species found on Amsterdam Island [5], we were unable to find such a link, since this particular Erysipelothrix species has not been previously documented elsewhere.\n\nErysipelothrix species have a very wide host range, including marine mammals, fish and seabirds [17, 57, 58]; any number of different hosts could conceivably have roles as vectors and/or reservoirs of this novel Erysipelothrix species.\n\nWhile no earlier Erysipelothrix isolates from this island were sequenced, using traditional phenotypic methods, initial investigations found that they belonged to serotype 1b [5].\n\nOur finding of the same serotype using in silico approaches may suggest that the same species/strain has been in circulation since at least the mid-1990s.\n\nPrevious PCR amplification of Erysipelothrix isolates from Amsterdam Island was performed using the primers by Makino et al.\n\n[50] targeting the 16S rRNA gene and which should amplify sequence from all members of the genus Erysipelothrix.\n\nWe also found that the primers and probe described by Pal et al.\n\n[19] targeting a noncoding region 3′ to the 5S rRNA gene for detection of E. rhusiopathiae were able to amplify DNA from E. amsterdamensis; care should therefore be taken when interpreting results using this primer/probe set on Erysipelothrix species isolated from rare sources.\n\nDevelopment of a PCR protocol that distinguishes between these two species would be valuable.\n\nThe genes found to be unique to E. amsterdamensis in this study (Table S4) would be good initial candidate targets.\n\nMoreover, the two closed genomes generated during this study will facilitate further comparative genomic studies within the genus Erysipelothrix.\n\nOur comparisons of the 16S rRNA and rpoB gene sequences from different Erysipelothrix species highlight the limited nucleotide diversity in the 16S rRNA gene, and confirm previous suggestions that the rpoB gene is a more suitable marker for Erysipelothrix species delineation [40].\n\nWhen comparing the genomes of Erysipelothrix species from albatross to those of other Erysipelothrix species, ANI and dDDH scores were below the accepted thresholds for considering genomes to belong to the same species (95 and 70 %, respectively).\n\nThe one exception was that ANI values of close to, or slightly above 95 % were obtained when comparing with genomes belonging to the less common clade 1 of E. rhusiopathiae (Table S3).\n\nOur choice of a 95 % cut-off value for ANI was conservative, given that others have proposed a cut-off of 96 % [59].\n\nTaking the combined results of both ANI and dDDH analyses, and the overall differences between Erysipelothrix species from albatross and E. rhusiopathiae, there is compelling justification for the designation of the new species, E. amsterdamensis.\n\nSNP analysis of 16 E. amsterdamensis isolates from the three albatross chicks showed that they are highly related.\n\nGiven that the number of SNPs distinguishing isolates of the same carcass was similar to that among isolates from different carcasses – as well as the close proximity in space and time of the mortality events – this strongly suggests that the three chicks were infected from a common source.\n\nWhether this small number of SNPs arose during infection within the host, or was already present in the environment, remains unknown; a molecular clock for Erysipelothrix has yet to be established [39].\n\nSimilar levels of within-host genomic diversity of E. rhusiopathiae have been previously observed in other wildlife species [60].\n\nWhile we cannot rule out the possibility that mutations occurred while the strains were maintained in conservation medium (several months unfrozen), we feel it is unlikely that sufficient replication occurred to explain the number of variants observed within and among these isolates.\n\nFurther sampling of the albatross colony would help to determine whether this Erysipelothrix species was a new introduction to Amsterdam Island, and whether other strains are in circulation.\n\nMoreover, sampling from islands at comparable latitudes would help to elucidate its geographic distribution.\n\nInterestingly, E. amsterdamensis harbours a spaA gene, which codes for what is commonly described as one of the most critical proteins for immunogenicity and virulence of E. rhusiopathiae [61–63].\n\nThis gene has been found to occur in all clade 2, clade 3 and intermediate clade E. rhusiopathiae genomes described to date [54], whereas E. rhusiopathiae clade 1 carries a spaB gene [39], and E. piscisicarius carries a spaC gene [42].\n\nThat E. amsterdamensis was isolated in association with chick mortalities in the absence of other bacteria suggests it was the likely cause of death, and the presence of the spaA gene, in addition to the presence of several putative virulence genes of E. rhusiopathiae that are typically among its core genes [34], lends further support to the likely virulence of this species.\n\nHowever, further investigation, including challenge studies, will be necessary for such characterization.\n\nErysipelothrix rhusiopathiae is typically considered an opportunistic pathogen, often manifesting clinically in stressed individuals or populations, for example following translocation of kakapos [15].\n\nFurther studies should explore whether Erysipelothrix-associated mortalities detected in other subantarctic islands could have been caused by the new species described here, or other novel taxa, and which set of potential host species of those relatively simple island communities may be involved in the epidemiological dynamics.\n\nDescription of Erysipelothrix amsterdamensis sp.\n\nnov.\nGo to section...\nErysipelothrix amsterdamensis (am.ster.dam.en’sis.\n\nN.L.\n\nfem.\n\nadj.\n\namsterdamensis, referring to Amsterdam island, from which the first strains to be characterized were isolated).\n\nCells are Gram-positive, rod-shaped and non-motile.\n\nSmall pin-point sized, round, flat grey colonies with smooth contours are observed on 5 % horse blood agar after 24 h growth at 37 °C at normal atmospheric conditions.\n\nPresence of alpha haemolysis.\n\nNon-spore-forming.\n\nCatalase- and oxidase-negative.\n\npH tolerance 6–8, with weak growth at pH 9.\n\nNormal growth between 20–40 °C.\n\nThe G+C content of the genomic DNA of the type strain is 36.6 mol%.\n\nWhole genome sequencing indicates isolates carry the Erysipelothrix spaA gene and belong to Erysipelothrix serotype 1b.\n\nMembers of the species can be distinguished from other members of the genus Erysipelothrix based on phylogenomic analysis and genomic metrics.\n\nThe type strain, A18Y020dT (=CIP 112216T=DSM 115297T), was isolated from an Indian yellow-nosed albatross chick carcass on Amsterdam Island.\n\nThe whole genome of the type strain A18Y020dT is available in GenBank (GCA_940143175).\n\nThe type strain has been deposited with the collections held by Institut Pasteur, France (CIP 112216T) and with DSMZ, Germany (115297T).\n\nThe 16S rRNA sequence for A18Y020dT is available on GenBank as accession OQ355811.\n\nThe complete annotated genome for isolate A18Y016a is also available under accession number GCA_940143155.\n\nReads for all 16 samples sequenced in this study are available on European Nucleotide Archive Sequence Read Archive (SRA) under accession number PRJEB50151.\n\nFunding information\nGo to section...\nJ.T.\n\nreceived support from CEVA and ANRT for a CIFRE PhD fellowship and project France relance TVACALBA.\n\nA.G. was supported by the US National Science Foundation (DEB-1557022) and the Strategic Environmental Research and Development Program (RC2635).\n\nThe research was also supported by the French Polar Institute (IPEV ECOPATH-1151), ANR ECOPATHS (ANR-21-CE35-0016), Zone Atelier Antarctique et Terres Australes (ZATA) and OSU OREME ECOPOP.\n\nThe funders had no role in study design, data collection and analysis, or preparation of the manuscript.\n\nAcknowledgements\nGo to section...\nWe thank Nicolas Keck, Karin Lemberger, Tristan Bralet, Christophe Barbraud, Karine Delord and Iain Sutcliffe for discussions related to the work.\n\nSupport is acknowledged from Réserve Naturelle Nationale des Terres Australes Françaises, notably from Célia Lesage.\n\nWe thank technical support agents at Ceva Biovac for help with laboratory work.\n\nThis paper is a contribution of IPEV project ECOPATH-1151 to the Plan National d’Action Albatros d’Amsterdam.\n\nAuthor contributions\nGo to section...\n\nFormal analysis: J.Z., M.M.\n\nInvestigation: J.T., A.C., H.G., A.G., T.L.F., T.B.\n\nMethodology (Field): T.B., A.C., J.T., A.G.; (Laboratory): H.G., T.B., A.C., J.T., A.G., T.L.F.\n\n; (Bioinformatics): J.Z., M.M., T.L.F.\n\n; (Histology): R.L.N.\n\nSupervision: M.M., T.L.F., T.B.\n\nVisualization: J.Z., M.M.\n\nWriting – original draft: J.Z., T.L.F.\n\nWriting – review and editing: all authors.\n\nConflicts of interest\nGo to section...\n\nThe authors declare that there are no conflicts of interest.\n\nEthical statement\nGo to section...\n\nThe experimental design was approved by the French Regional Animal Experimentation Ethical Committee No.\n\n036 (Ministry of Research permit No.\n\n10257–2018011712301381) and by the Comité de l’Environnement Polaire (A-2018–123 and A 2018–139 for 2018–2019).\n\n----------\nAs a professional microbiologist, create a concise and comprehensive Chinese scientific report on Erysipelothrix Spp. research based on the provided text, whether it's an article, post, conversation, or passage. Follow these guidelines:\n\n 1. Produce a detailed and in-depth report that remains clear and concise.\n 2. Focus on main ideas and essential information, removing any extraneous language.\n 3. Strictly rely on the provided text without adding external information.\n 4. Format the report in paragraphs for easy understanding.\n 5. Conclude with “[End of Notes, Message #X],” where “X” represents the total number of messages I’ve sent. Increment the message counter with each new message.\n 6. Use English for technical terms, industry terminology, proper nouns, and species names. After you finish, add the following words: {{{ I_finished_papa!!! }}} to the end of you answer