Genomic epidemiology of porcine Lawsonia intracellularis in China revealed by metagenome-assembled genomes and global comparative analysis

Abstract

Lawsonia intracellularis, the etiological agent of porcine proliferative enteropathy, is widely distributed in swine herds and poses a significant economic burden to the global swine industry. However, its obligate intracellular lifestyle severely limits in vitro cultivation, hindering large-scale genomic surveillance and epidemiological tracking. In this study, we combined nationwide PCR-based prevalence screening with culture-independent metagenomic sequencing to characterize the molecular epidemiology and genomic features of circulating L. intracellularis strains in China. PCR screening of 1,831 clinical samples collected from 13 provinces revealed widespread circulation, with an overall individual positivity rate of 26.38%. From strongly positive specimens, nine high-quality metagenome-assembled genomes (MAGs) were successfully reconstructed. Comparative analysis against all publicly available global genomes revealed exceptionally high average nucleotide identity (> 99.9%) and limited single-nucleotide polymorphism (SNP) variation (ranging from 4 to 370). Phylogenetic reconstruction divided the strains into two major clades, within which Chinese isolates were interspersed with international strains, suggesting recent shared ancestry and potential cross-regional transmission. Pangenome analysis revealed a highly conserved core genome with restricted accessory diversity, which is consistent with the genomic architecture typical of obligate intracellular bacteria. Only a few high-confidence antimicrobial resistance determinants were identified, although several homologous loci potentially associated with resistance were detected. Notably, comparative analysis revealed four virulence-associated genes that were conserved in field isolates but highly divergent from or absent from the vaccine strain, indicating the development of a niche-specific adaptive evolution. This study establishes the first comprehensive genomic epidemiological framework for L. intracellularis in China, demonstrating the utility of metagenomics for tracking unculturable pathogens, informing regional surveillance strategies, and highlighting conserved versus variable genomic targets for future vaccine and therapeutic development.

Keywords

Lawsonia intracellularis / Genomic epidemiology / Metagenomics / Pangenome analysis / SNP variation / Antimicrobial resistance determinants / Virulence-associated genes

References

[1] Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, et al.. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024, 630(8016): 493-500

[2] Alcock BP, Huynh W, Chalil R, Smith KW, Raphenya AR, Wlodarski MA, Edalatmand A, Petkau A, Syed SA, Tsang KK, et al.. CARD 2023: Expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. Nucleic Acids Research, 2023, 51(D1): D690-D699

[3] Bengtsson RJ, Wee BA, Yebra G, Bacigalupe R, Watson E, Guedes RMC, Jacobson M, Stadejek T, Archibald AL, Fitzgerald JR, et al.. Metagenomic sequencing of clinical samples reveals a single widespread clone of Lawsoniaintracellularis responsib-le for porcine proliferative enteropathy. Microbial Genomics, 2020, 6(4 ArticleID: e000358

[4] Cantalapiedra CP, Hernández-Plaza A, Letunic I, Bork P, Huerta-Cepas J. eggNOG-mapper v2: Functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Molecular Biology and Evolution, 2021, 38(12): 5825-5829

[5] Chaumeil P, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk: A toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics (Oxford, England), 2019, 36(6): 1925-1927

[6] Chen, L., J. Yang, J. Yu, Z. Yao, L. Sun, Y. Shen, and Q. Jin. 2005. VFDB: a reference database for bacterial virulence factors. Nucleic Acids Research 33 (Database issue): D325-D328. https://doi.org/10.1093/nar/gki008.

[7] Chen, S. 2025. fastp 1.0: An ultrafast all-round tool for FASTQ data quality control and preprocessing. IMeta 4 (5): e70078. https://doi.org/10.1002/imt2.70078.

[8] Chklovski A, Parks DH, Woodcroft BJ, Tyson GW. CheckM2: A rapid, scalable and accurate tool for assessing microbial genome quality using machine learning. Nature Methods, 2023, 20(8): 1203-1212

[9] Didelot X, Walker AS, Peto TE, Crook DW, Wilson DJ. Within-host evol-ution of bacterial pathogens. Nature Reviews. Microbiology, 2016, 14(3): 150-162

[10] Gomathinayagam S, Kanagalingam S, Chandrasekaran S, Krishnan T, Muthukaliannan GK. Millennial-scale microbiome analysis reveals ancient antimicrobial resistance conserved despite modern selection pressures. Environmental Microbiome, 2024, 19(1): 110

[11] Gómez-Osorio L, Penagos-Tabares F, Bosnjak-Neumuller J, Guedes RMC, Vasiljevic M, Steiner T, McOrist S. Porcine proliferative enteropathy: Overview of disease dynamics and nonantibiotic alternatives for prevention and control strategies. Frontiers in Veterinary Science, 2025, 12: 1596316

[12] Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, Lago BA, Dave BM, Pereira S, Sharma AN, et al.. CARD 2017: Expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Research, 2016, 45(D1): D566-D573

[13] Kaas RS, Leekitcharoenphon P, Aarestrup FM, Lund O. Solving the problem of comparing whole bacterial genomes across different sequencing platforms. PLoS ONE, 2014, 9(8 ArticleID: e104984

[14] Kang DD, Li F, Kirton E, Thomas A, Egan R, An H, Wang Z. MetaBAT 2: An adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies. PeerJ, 2019, 7 ArticleID: e7359

[15] Kroll JJ, Roof MB, McOrist S. Evaluation of protective immunity in pigs following oral administration of an avirulent live vaccine of Lawsonia intracellularis. American Journal of Veterinary Research, 2004, 65(5): 559-565

[16] Letunic I, Bork P. Interactive Tree of Life (iTOL) v6: Recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Research, 2024, 52(W1): W78-W82

[17] Luo Y, Chen Z, Lian S, Ji X, Zhu C, Zhu G, Xia P. The love and hate relationship between T5SS and other secretion systems in bacteria. International Journal of Molecular Sciences, 2023, 25(1): 281

[18] McCutcheon JP, Moran NA. Extreme genome reduction in symbiotic bacteria. Nature Reviews. Microbiology, 2011, 10(1): 13-26

[19] Melnyk AH, Wong A, Kassen R. The fitness costs of antibiotic resistance mutations. Evolutionary Applications, 2014, 8(3): 273-283

[20] Nathues, H., C. J. B. Oliveira, M. Wurm, E. G. Beilage, and P. E. N. Givisiez. 2007. Simult-aneous detection of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in porcine feces and tissue samples by multiplex-PCR. Journal of Veterinary Medicine. A, Physiology, Pathology, Clinical Medicine 54 (9): 532–538. https://doi.org/10.1111/j.1439-0442.2007.00995.x.

[21] National Center for Biotechnology Information (NCBI). n.d. NCBI Genome Database: Lawsonia intracellularis. https://www.ncbi.nlm.nih.gov/datasets/genome/?taxon=29546. Accessed Jan 1, 2026.

[22] Nurk S, Meleshko D, Korobeynikov A, Pevzner PA. metaSPAdes: A new versatile metagenomic assembler. Genome Research, 2017, 27(5): 824-834

[23] Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MTG, Fookes M, Falush D, Keane JA, Parkhill J. Roary: Rapid large-scale prokaryote pan genome analysis. Bioinformatics (Oxford, England), 2015, 31(22): 3691-3693

[24] Pritchard L, Glover RH, Humphris S, Elphinstoneb JG, Tothc IK. Genomics and taxonomy in diagnostics for food security: Soft-rotting enterobacterial plant pathogens. Analytical Methods, 2016, 8(1): 12-24

[25] Pruitt, K. D., T. Tatusova, and D. R. Maglott. 2007. NCBI reference sequences (RefSeq): a curated nonredundant sequence database of genomes, transcripts and proteins. Nucleic Acids Research 35 (Database issue): D61-D65. https://doi.org/10.1093/nar/gkl842.

[26] Resende, T. P., C. E. R. Pereira, A. G. de S. Daniel, E. Vasquez, M. Saqui-Salces, F. A. Vannucci, and C. J. Gebhart. 2019. Effects of Lawsonia intracellularis infection in the prolifer-ation of different mammalian cell lines. Veterinary Microbiology 228: 157–164. https://doi.org/10.1016/j.vetmic.2018.11.029.

[27] Sanchez-Garrido J, Ruano-Gallego D, Choudhary JS, Frankel G. The type III secretion system effector network hypothesis. Trends in Microbiology, 2021, 30(6): 524-533

[28] Schwengers, O., L. Jelonek, M. A. Dieckmann, S. Beyvers, J. Blom, and A. Goesmann. 2021. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microbial Genomics 7 (11): 000685.https://doi.org/10.1099/mgen.0.000685.

[29] Steed DB, Koundakjian D, Harris AD, Rosato AE, Konstantinidis KT, Woodworth MH. Leveraging strain competition to address antimicrobial resistance with microbiota therapies. Gut Microbes, 2025, 17(1): 2488046

[30] Stevens, E. J., J. D. Li, T. E. Hector, G. C. Drew, K. Hoang, S. T. E. Greenrod, S. Paterson, and K. C. King. 2025. Within-host competition causes pathogen molecular evolution and perpetual microbiota dysbiosis. The ISME Journal 19 (1): wraf071. https://doi.org/10.1093/ismejo/wraf071.

[31] Streit WR, Schmitz RA. Metagenomics–the key to the uncultured microbes. Current Opinion in Microbiology, 2004, 7(5): 492-498

[32] Szabó I, Makkai I, Máté P, Molnár T, Swam H, von Berg S, Holtkamp DJ, Glávits R, Szabó I, Ózsvári L, et al.. Slaughterhouse visual and palpation method for estimating the economic damage of porcine proliferative enteropathy (PPE). Animals : An Open Access Journal from MDPI, 2023, 13(3): 542

[33] Tonkin-Hill G, MacAlasdair N, Ruis C, Weimann A, Horesh G, Lees JA, Gladstone RA, Lo S, Beaudoin C, Floto RA, et al.. Producing polished prokaryotic pangenomes with the Panaroo pipeline. Genome Biology, 2020, 21(1): 180

[34] Van Kempen M, Kim SS, Tumescheit C, Mirdita M, Lee J, Gilchrist CLM, Söding J, Steinegger M. Fast and accurate protein structure search with Foldseek. Nature Biotechnology, 2023, 42(2): 243-246

[35] Vannucci FA, Kelley MR, Gebhart CJ. Comparative genome sequencing identifies a prophage-associated genomic island linked to host adaptation of Lawsonia intracellularis infections. Veterinary Research, 2013, 44(1): 49

[36] Wang, L., W. Wu , L. Zhao , Z. Zhu, X. Yao, J. Fan, H. Chen, Song W, Huang X, Hua L, Qian P, Chen H, Peng Z, Wu B. Fecal PCR survey and genome analysis of Lawsonia intracellularis in China. Front Vet Sci. 2024; 11: 1324768. https://doi.org/10.3389/fvets.2024.1324768.

[37] Wernegreen JJ. For better or worse: Genomic consequences of intracellular mutualism and parasitism. Current Opinion in Genetics & Development, 2005, 15(6): 572-583

[38] Xiao, N., J. LI, M. LI, H. Zhou, H. Lin, H. Fan. Isolation and In vitro cultivation of Lawsonia intracellularis from China. Vet Microbiol. 2022; 270: 109457. https://doi.org/10.1016/j.vetmic.2022.109457.

[39] Zhou S, Liu B, Zheng D, Chen L, Yang J. VFDB 2025: An integrated resource for exploring anti-virulence compounds. Nucleic Acids Research, 2025, 53(D1): D871-D877

Funding

National Natural Science Foundation of China(32225048)

Natural Science Foundation of Beijing Municipality(6262013)

RIGHTS & PERMISSIONS

The Author(s)