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PubMed · 42710724

A 29-plex MOL-PCR assay for simultaneous detection of selected major, non-typing, and accessory virulence genes in Clostridium perfringens.

Michaela Nesvadbova·Radka Dziedzinska·Martin Klanica·Petr Kralik
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Abstract

Clostridium perfringens is an important pathogen of humans and animals, responsible for a broad spectrum of diseases mediated by diverse toxins and virulence factors. Precise and extended toxin-gene profiling is valuable for strain characterization and molecular epidemiological surveillance. Here, we describe the development of a 29-plex Multiple Oligonucleotide Ligation PCR (MOL-PCR) assay that enables the simultaneous detection of a large and important panel of 27 C. perfringens toxin-related genes - covering major typing toxins as well as an extended panel of non-typing and accessory virulence genes - thus moving beyond the classical toxinotyping framework. The assay was evaluated in comparison with six multiplex qPCR assays. In both systems, the gene encoding the Clostridium perfringens-specific serine O-acetyltransferase (EpsC) was used as a molecular marker for species confirmation, and an internal amplification control was included to detect potentially false-negative results. Analytical specificity testing confirmed exclusive amplification in C. perfringens and sequencing confirmed the toxin-gene profiles of reference strains. Comparative analysis of 72 reference and field isolates (1,944 data points) demonstrated complete concordance for 637 positive detections, yielding 100% positive agreement and 99.7% negative agreement relative to the comparative qPCR method. The limit of detection was 100 fg/µl (approx. 3 × 101 genome equivalents; GE) for qPCR and 1  pg/µl (approx. 3 × 102 GE) for MOL-PCR. Despite its high multiplex level, MOL-PCR showed high agreement with qPCR. The developed MOL-PCR method provides a rapid, high-throughput, and cost-effective tool for expanded toxin-gene profiling of C. perfringens isolates targeting major typing toxins and selected non-typing and accessory virulence genes. Therefore, it may support advanced toxin-gene characterization, molecular epidemiology, and One Health-oriented surveillance of evolving virulence landscapes.

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Clostridium perfringensMOL-PCRMolecular epidemiologyMultiplex qPCRToxinotypingVirulence profilingJournal Article

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Michaela Nesvadbova, Radka Dziedzinska, Martin Klanica, Petr Kralik. 2026-09-08. A 29-plex MOL-PCR assay for simultaneous detection of selected major, non-typing, and accessory virulence genes in Clostridium perfringens.. https://doi.org/10.1016/j.ymeth.2026.09.001

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Host immunological response of Ross 308 broilers fed an anti-IL-10 antibody to Eimeria and Clostridium perfringens challenge.

Understanding host response mechanisms may lead to improved management of poultry diseases such as necrotic enteritis. Ross 308 broilers (20 birds/cage, 32 cages) received diets &#xb1; anti-interleukin-10 (IL-10) antibody, an intervention to improve immune activation, in three experimental replicates (R1, R2, R3). R1 and R2 received 1 &#xd7; 108 colony forming units (CFU) Salmonella Typhimurium on day (d) 1. On d 14, birds were assigned to unchallenged control or 15,000 sporulated Eimeria maxima oocyst gavage challenge, with a subset receiving E. maxima + Clostridium perfringens (1 &#xd7; 10&#x2078; CFU d 18-19) to induce necrotic enteritis. Intestinal tissue and digesta samples were collected from 6 birds/treatment at baseline (d 14), peak infection (d 21), and post-peak infection (d 25) to measure luminal IL-10 (ELISA), interferon gamma (IFN&#x3b3;) via immunohistochemistry, and IL-10, CD4, CD8, CD25, and KUL01 via RNAscope. Data were analyzed via two-way ANOVA (P &#x2264; 0.05). In R3, baseline duodenal IL-10 was 458 ng/mg protein greater in birds fed the control diet, while E. maxima + C. perfringens inoculation increased jejunal and ileal IL-10 content by 528 and 649 ng/mg protein at peak infection (P < 0.05). In R2, peak jejunal tissue % IL-10 area increased during E. maxima + C. perfringens challenge (P < 0.05). Baseline and peak jejunal R3 RNA was sequenced on an Illumina NovaSeq 6000, mapped to the chicken genome, and differential expression identified with DESeq2 (|log&#x2082; fold change| > 1; FDR &#x2264; 0.05). At peak, E. maxima challenge overrepresented genes involved in cell cycle, lipid metabolism, and B cell proliferation (P < 0.002), while E. maxima + C. perfringens overrepresented defense response and catabolic process terms (P < 0.0001). Sex altered the host immune response to challenge (P < 0.05), while E. maxima &#xb1; C. perfringens induced a similar host immune response regardless of dietary anti-IL-10. Overall, pathogenic challenge status was the primary driver of host immune response with the effect of anti-IL-10 antibody influenced by S. Typhimurium.

Clostridium perfringens↗