Metagenomics Tracks China’s Unculturable Pig Pathogen Across Global Strains

Lawsonia intracellularis, the bacterium behind porcine proliferative enteropathy, has long frustrated researchers because it refuses to grow in ordinary laboratory culture. Its obligate intracellular lifestyle demands specialized cell culture systems and prolonged incubation, which has kept large-scale genomic surveillance of the pathogen largely out of reach. A new nationwide study in China has now sidestepped that barrier entirely, using metagenomic sequencing to reconstruct bacterial genomes directly from clinical samples and to place Chinese strains within a global phylogenetic picture for the first time.

The research team screened 1,831 clinical samples collected between December 2022 and January 2026 from pig farms and slaughterhouses across 13 provinces or municipalities. The collection included 1,452 pig ileum tissue samples and 379 fecal samples, all tested by a conventional PCR assay targeting the aspA gene. Overall, 26.38 percent of samples tested positive, with tissue samples at 26.52 percent and fecal samples at 25.85 percent. Positivity rates varied dramatically by province, ranging from 0 to 55.56 percent, and farm-level variation spanned from 0 to 100 percent, a pattern the authors interpret as potentially intense within-herd transmission.

Temporal patterns also emerged within the cross-sectional data. In Shandong, the positivity of tissue samples fell from 79.00 percent in 2022 and 60.39 percent in 2023 to 14.76 percent in 2025. The authors note that this decline did not appear closely tied to sample size, since larger sample sizes in Shandong and Shanxi in 2025 coincided with lower positivity, while both sample size and positivity rose in Hunan and Shaanxi over the same period. They caution that differences in detection methods and sample types make direct comparisons with earlier Chinese surveys less rigorous, and that the actual national epidemiological situation requires larger-scale, long-term surveillance to clarify.

From strongly PCR-positive specimens, the team reconstructed nine high-quality metagenome-assembled genomes, or MAGs: seven from fecal samples, one from an ileum sample, and one from the live attenuated vaccine strain B3903. Together with eight publicly available genomes evaluated by CheckM2, the bins showed estimated completeness of 99.13 to 99.88 percent and contamination below 5 percent, qualifying them as draft genomes for downstream analysis. The final dataset of 17 genomes included isolates from China, Japan, the United States, the United Kingdom, Denmark and South Korea.

Comparative genomics revealed an extraordinarily conserved species. Average nucleotide identity across all 17 genomes exceeded 99.9 percent, and pairwise SNP distances ranged from just 4 to 370. Within China, the isolates fell into three clusters separated by a maximum of 50 SNPs. One cluster of three isolates sat relatively close to the vaccine strain, differing by 55 to 60 SNPs, and to isolates from the United States, South Korea and Japan by 18 to 60 SNPs. The other two Chinese clusters differed far more from international isolates, at 274 to 366 SNPs, and from the vaccine strain at 268 to 367 SNPs. Isolates from the United Kingdom and remaining Japanese strains differed from all Chinese isolates by more than 100 SNPs.

Phylogenetic reconstruction based on core-genome SNPs, using an equine strain as an outgroup, divided the porcine isolates into two major sublineages. Chinese isolates appeared in both lineages and were interspersed among global strains rather than forming a distinct monophyletic group, a pattern the authors read as evidence of recent shared ancestry and potential cross-regional transmission, likely driven by modern livestock trade, shared genetic lines and standardized production practices. No clear association emerged between phylogenetic clustering and sampling year. Within individual Chinese clusters, SNP variation was generally low, at 5 to 32 SNPs, though the Guangxi isolate PPE-GX01-2022 differed from other domestic clusters by more than 100 SNPs.

Pangenome analysis of the 17 genomes identified 1,428 gene clusters, of which 1,277, or 89.43 percent, were core genes and 151, or 10.57 percent, accessory genes, a highly conserved architecture consistent with the genomic streamlining typical of obligate intracellular bacteria. Functional annotation showed the core genome dominated by translation, ribosomal structure and biogenesis, and cell wall, membrane and envelope biogenesis, while the accessory genome was enriched for signal transduction mechanisms and membrane-related functions likely tied to environmental adaptation and host interaction. Notably, 26.74 percent of all gene clusters could not be functionally annotated, and 38.18 percent of accessory genes remained unannotated, leaving open the possibility that these conserved but uncharacterized genes harbor unrecognized adaptive or virulence functions.

Antimicrobial resistance profiling against the CARD database returned a sparse picture. Only two strict hits appeared across all genomes: an efflux pump membrane transporter, ArcB, present in every strain but judged more likely a conserved efflux-related gene than a confirmed resistance determinant, and the tetracycline resistance ribosomal protection protein Tet(W), found in two US field isolates and one isolate from Liaoning, China. Its absence from all other Chinese isolates suggests acquired tetracycline resistance may be uncommon and geographically restricted. Twelve loose hits, homologous loci with potential associations with resistance to fluoroquinolones, tetracyclines and several other antimicrobial classes, were ubiquitous, but the authors stress that the presence of these homologs does not equate to functional resistance and warrants experimental investigation.

The most striking finding concerned virulence-associated genes. Comparing the B3903 vaccine strain with the 16 field isolates, the team identified 20 genes that were highly divergent or absent from the vaccine strain. Four of these, including the autotransporter-annotated LIC052 and LIC058, the hypothetical protein LIC061 within a Type V secretion system cluster on plasmid 3, and LI1155, located near a major Type III secretion system cluster, were conserved in more than 75 percent of field isolates. LI1155 showed structural similarity to secreted effector proteins, hinting at a Type III secretion-related role, though its precise function remains undetermined. A prophage-associated genomic island appeared only in two US isolates and two Liaoning isolates within the same phylogenetic group. The authors suggest that vaccine strain-based immunogens may lack these field-adapted loci, a divergence with potential implications for cross-protection, though phenotypic validation through infection assays will be needed.

The study’s limitations are clearly acknowledged: MAG recovery depends on sequencing depth and binning accuracy and may underestimate low-abundance strain diversity; in silico predictions of resistance and virulence require laboratory confirmation; antimicrobial usage data were unavailable; and the cross-sectional design cannot capture longitudinal transmission networks. Even so, the work establishes the first nationwide genomic epidemiological framework for L. intracellularis in China and demonstrates that culture-independent metagenomics can track fastidious pathogens directly from clinical material, offering a scalable pipeline that could extend to other unculturable veterinary pathogens and inform future vaccine and therapeutic targets.

Subject of Research: Genomic epidemiology of the swine pathogen Lawsonia intracellularis in China using metagenome-assembled genomes

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

Article References: Jiang, X., Cui, Q., Yang, T., Li, B., Wang, M., Wang, Y., Ying, L., Hu, C., Wang, Y., Liu, Z., Xu, S., Shi, Y., Wang, J., Shen, Z., & Fu, M. (2026). Genomic epidemiology of porcine Lawsonia intracellularis in China revealed by metagenome-assembled genomes and global comparative analysis. Animal Diseases, 6(1), Article 48. https://doi.org/10.1186/s44149-026-00256-4

Image Credits: AI Generated

DOI: 10.1186/s44149-026-00256-4

Keywords: Lawsonia intracellularis, porcine proliferative enteropathy, metagenomics, metagenome-assembled genomes, genomic epidemiology, swine, China, phylogenomics, antimicrobial resistance, virulence genes, pangenome, vaccine strain divergence