Pathogens

14 pages, 633 KB

Open AccessArticle

Comparative Efficacy of Horse and Chicken Serum for the In Vitro Cultivation of Mycoplasma hyorhinis Clinical Isolates

by Yi-Chia Li, Yu-Wei Tseng, Wei-Hao Lin, Chao-Nan Lin and Ming-Tang Chiou

Pathogens 2025, 14(10), 1056; https://doi.org/10.3390/pathogens14101056 (registering DOI) - 19 Oct 2025

Abstract

Mycoplasma hyorhinis is an important respiratory pathogen in swine, yet optimal culture conditions for high-yield propagation remain undefined. This study compared horse serum (HS) and chicken serum (CS) at graded concentrations (10%, 20%, 30%) for their ability to support in vitro growth of [...] Read more.

Mycoplasma hyorhinis is an important respiratory pathogen in swine, yet optimal culture conditions for high-yield propagation remain undefined. This study compared horse serum (HS) and chicken serum (CS) at graded concentrations (10%, 20%, 30%) for their ability to support in vitro growth of four clinical M. hyorhinis isolates (strains A, B, C, and D). Cultures were prepared in modified Friis medium, and growth performance was assessed by final titer (color changing unit, CCU/mL) and time-to-detection at 10² and 10⁴ CCU/mL. All media supported growth, but HS consistently outperformed CS in both yield and growth kinetics. The highest titers (10⁹ CCU/mL) and shortest detection times (3.6–6 days) were observed in 20% HS for most strains. Increasing HS concentration to 30% reduced yield for several strains, suggesting a concentration-dependent inhibitory effect. CS demonstrated limited but strain-dependent growth support, with comparable performance to HS for strain B at lower thresholds. These findings identify 20% HS as an optimal supplement for efficient M. hyorhinis cultivation, while highlighting the potential of CS as a cost-effective alternative under certain conditions, with implications for diagnostic reagent production and vaccine development. Full article

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14 pages, 1928 KB

Open AccessArticle

Prevalence of Rotavirus in Diarrheic Piglets on RVA-Vaccinated and Non-Vaccinated Farms

by Weronika Rybkowska, Aleksandra Woźniak, Nicole Bakkegård Goecke, Lars Erik Larsen, Piotr Cybulski and Tomasz Stadejek

Pathogens 2025, 14(10), 1055; https://doi.org/10.3390/pathogens14101055 (registering DOI) - 18 Oct 2025

Abstract

Rotaviruses (RVs) are an important cause of piglet diarrhea. This study aimed to determine the prevalence of rotavirus A, B, and C (RVA, RVB and RVC) in two RVA-vaccinated (VAC) and four non-vaccinated (NON-VAC) farms, and the impact of RVA vaccination on production [...] Read more.

Rotaviruses (RVs) are an important cause of piglet diarrhea. This study aimed to determine the prevalence of rotavirus A, B, and C (RVA, RVB and RVC) in two RVA-vaccinated (VAC) and four non-vaccinated (NON-VAC) farms, and the impact of RVA vaccination on production parameters. Additionally, RVs prevalence in consecutive weekly groups from one vaccinated and one non-vaccinated farm was assessed. Diarrheic feces or ileum content were screened for RVs using real-time RT-PCR. In VAC, no RVA or RVB was detected, while RVC was found in all the samples (15/15). In NON-VAC, RVA, RVB, and RVC were detected in 10.5%, 13.2%, and 52.6% of samples, respectively. RVC was the most prevalent species in longitudinal study, while RVA was found in single samples. RVB was detected in one sample from the vaccinated farm, and in four out of five groups from the non-vaccinated farm. The pre-wean mortality and weaning weight were lower in the vaccinated than in the non-vaccinated farm. Low RVA prevalence and no noticeable improvement in weaning outcomes suggest vaccination was probably unjustified. Our study emphasizes the importance of comprehensive screening before and after vaccination and highlights the importance of including RVB and RVC in diagnostics of neonatal diarrhea. Full article

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17 pages, 2606 KB

Open AccessReview

Plasmid Genomic Dynamics and One Health: Drivers of Antibiotic Resistance and Pathogenicity

by Célia P. F. Domingues, João S. Rebelo, Francisco Dionisio and Teresa Nogueira

Pathogens 2025, 14(10), 1054; https://doi.org/10.3390/pathogens14101054 (registering DOI) - 18 Oct 2025

Abstract

Seen through a One Health perspective, plasmids act as global links, connecting human, animal, and environmental microbiomes while broadening the ecological scope of resistance and virulence. By combining knowledge about plasmid classification, mobility, resistance, virulence, and data sources, this review emphasizes their key [...] Read more.

Seen through a One Health perspective, plasmids act as global links, connecting human, animal, and environmental microbiomes while broadening the ecological scope of resistance and virulence. By combining knowledge about plasmid classification, mobility, resistance, virulence, and data sources, this review emphasizes their key role as drivers of bacterial evolution and worldwide health risks. Recognizing plasmids as connectors across microbiomes highlights both the urgency and opportunity to address plasmid-mediated resistance with integrated strategies. Current plasmid databases, such as NCBI RefSeq, PLSDB, IMG/PR, and PlasmidScope, have already greatly advanced our understanding of these connections, and they are likely to profoundly alter how we see plasmid biology and One Health relationships. Full article

(This article belongs to the Special Issue Bacterial Pathogenesis and Antibiotic Resistance)

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14 pages, 1586 KB

Open AccessArticle

Real-Time Quaking-Induced Conversion Assay Applied to the Italian Chronic Wasting Disease Monitoring Plan: Comparison of Classical and Innovative Diagnostic Methods

by Maria Mazza, Alessandra Favole, Valentina Campia, Barbara Iulini, Romolo Nonno, Ciriaco Ligios, Davide Pintus, Simone Peletto, Cristina Casalone, Cristiano Corona, Elena Bozzetta and Pier Luigi Acutis

Pathogens 2025, 14(10), 1053; https://doi.org/10.3390/pathogens14101053 (registering DOI) - 18 Oct 2025

Abstract

CWD surveillance and diagnosis are important issues in Europe since its detection in Norway, as some of its strains, like that of classical scrapie, are contagious. In addition, there are concerns as several matters about CWD are not yet known. Although diagnostic methods [...] Read more.

CWD surveillance and diagnosis are important issues in Europe since its detection in Norway, as some of its strains, like that of classical scrapie, are contagious. In addition, there are concerns as several matters about CWD are not yet known. Although diagnostic methods for the active surveillance in bovine and small ruminants have been able to detect the European CWD strains, a retrospective study on Italian wild red deer (Cervus elaphus) samples was performed to compare the results obtained from rapid screening tests, authorized according to EU Regulation 999/2001, and the RT-QuIC, a highly sensitive method in the detection of prion disease infection. A total of one hundred brainstems and medial retropharyngeal lymph nodes were selected out of those received from the CWD Italian surveillance system. Confirmed CWD-positive and -negative samples were included in the study as controls. All of the samples were first tested with the HerdChek BSE–Scrapie Antigen Test and then using the RT-QuIC. The rapid test was negative in all brainstem and lymph node samples. RT-QuIC analyses showed only one red deer brainstem sample positive for seeding activity, while all lymph nodes were negative, including the one from this case. This positive brainstem sample was then re-extracted and retested using two different recombinant prion protein substrates (Ha90-231; BV23-231) and their different batches from the first analyses. Seeding activity was consistently confirmed across both substrates and extractions, with positive signals detected down to dilutions of 10⁻⁴ using rPrP Ha90-231 and as low as 10⁻⁶ with rPrP BV23-231. The additional diagnostic investigations performed on this red deer using the alternative rapid test (TeSeE SAP Combi), Western blot, and immunohistochemistry showed negative results both in the brainstem and lymph nodes. This study showed that overall, the results obtained with the HerdChek BSE–Scrapie Antigen Test and RT-QuIC agree except in one case. Our findings highlight the potential of the RT-QuIC method to detect very low levels of PrP^Sc-associated seeding activity that may escape detection using classical methods. While seeding activity does not always equate to infectivity, only a bioassay will confirm the real disease status of this Italian case. These findings support the integration of RT-QuIC as a powerful complementary tool within existing surveillance frameworks to strengthen early detection and diagnostic accuracy. Full article

(This article belongs to the Collection Prions and Chronic Wasting Diseases)

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21 pages, 2543 KB

Open AccessArticle

Multi-Marker Approach for the Identification of Different Heterodera Species (Nematoda: Heteroderidae)

by Maria João Camacho, Maria L. Inácio and Eugénia de Andrade

Pathogens 2025, 14(10), 1052; https://doi.org/10.3390/pathogens14101052 (registering DOI) - 18 Oct 2025

Abstract

Cyst nematodes of the genus Heterodera are important plant-parasitic nematodes that cause significant crop losses worldwide but are often overlooked due to their non-specific symptoms and complex biology. This study assessed Heterodera diversity in Portugal using an integrative molecular approach based on four [...] Read more.

Cyst nematodes of the genus Heterodera are important plant-parasitic nematodes that cause significant crop losses worldwide but are often overlooked due to their non-specific symptoms and complex biology. This study assessed Heterodera diversity in Portugal using an integrative molecular approach based on four genetic markers (mtCOI, 18S rDNA, ITS, and 28S rDNA). Five valid species were identified: Heterodera cruciferae, H. mani, H. schachtii, H. trifolii, and H. zeae, with H. mani reported for the first time in the country. A distinct taxon from Coimbra (central Portugal) may represent a new or unsequenced species, highlighting gaps in reference datasets. Among the markers, mtCOI was the most effective, though some taxa remained unresolved. These results reinforce the value of multi-marker approaches, contribute with new sequences, and improve diagnostic capability for nematode management. Full article

(This article belongs to the Section Parasitic Pathogens)

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15 pages, 974 KB

Open AccessArticle

Comparative Evaluation of Organic and Commercial Treatments Against Varroa destructor in Apis mellifera: Implications for Honey Yield in Northeastern Mexico

by Jesús Humberto Reyna-Fuentes, Oscar Vicente Vazquez-Mendoza, Mirelly Venecia Mireles-Villanueva, Daniel López-Aguirre, Juana Maria Coronado-Blanco, Ruben Alberto Muñoz-Sánchez and Francisco Reyes-Zepeda

Pathogens 2025, 14(10), 1051; https://doi.org/10.3390/pathogens14101051 (registering DOI) - 18 Oct 2025

Abstract

Infestation by Varroa destructor represents one of the major challenges for beekeeping, as it compromises both colony health and honey productivity. The objective of this study was to evaluate the efficacy of different organic treatments for the control of V. destructor and their [...] Read more.

Infestation by Varroa destructor represents one of the major challenges for beekeeping, as it compromises both colony health and honey productivity. The objective of this study was to evaluate the efficacy of different organic treatments for the control of V. destructor and their effect on honey production in Apis mellifera colonies located on the central region of Tamaulipas, Mexico. A total of 150 colonies were assigned to five treatments: T1, oxalic acid with glycerin; T2, sublimated oxalic acid; T3, Thymol, T4; HappyVarr; and T5, an untreated control. Mite infestation (initial and final) and honey production were analyzed using a non-parametric approach and were evaluated with the Kruskal–Wallis test, and when significant differences were detected, Steel–Dwass multiple comparisons were performed. To examine the relationship between infestation reduction and honey yield, Spearman’s rank correlation was applied. No significant differences were observed in the initial infestation levels. However, final infestation levels showed highly significant differences among treatments (p < 0.0001), with T1, T2, T3, and sublimated oxalic acid (T4) significantly reducing mite infestation compared with the control. Sublimated oxalic acid represents the most effective and productive control method under the tested conditions. Honey production also differed significantly among treatments (p < 0.0001), with the highest yields recorded in T3 and T4. A strong negative correlation was detected between final infestation and honey production (p < 0.0001). In conclusion, treatments based on oxalic acid (particularly sublimated) and HappyVarr proved effective in reducing V. destructor infestation and improving honey production, highlighting their relevance as viable alternatives for sanitary management in beekeeping. Full article

(This article belongs to the Special Issue Surveillance, Detection and Control of Infectious Diseases of Bees)

16 pages, 947 KB

Open AccessArticle

Comparative Analysis of Concentration and Quantification Methods for Antibiotic Resistance Genes and Their Phage-Mediated Dissemination in Treated Wastewater and Biosolids

by Irene Falcó, Ana Allende, Francesca Cutripi, Rosa Aznar, Gloria Sánchez and Pilar Truchado

Pathogens 2025, 14(10), 1050; https://doi.org/10.3390/pathogens14101050 (registering DOI) - 18 Oct 2025

Abstract

Antimicrobial resistance poses a growing threat to public health, and integrated surveillance strategies across environmental compartments such as treated wastewater and biosolids can substantially improve monitoring efforts. A key challenge is the diversity of available protocols, which complicates comparability for the concentration and [...] Read more.

Antimicrobial resistance poses a growing threat to public health, and integrated surveillance strategies across environmental compartments such as treated wastewater and biosolids can substantially improve monitoring efforts. A key challenge is the diversity of available protocols, which complicates comparability for the concentration and detection of antibiotic resistance genes (ARGs), particularly in complex matrices. In this study, we compared two commonly used concentration methods—filtration–centrifugation (FC) and aluminum-based precipitation (AP)—and two detection techniques, quantitative PCR (qPCR) and droplet digital PCR (ddPCR), for the quantification of four clinically relevant ARGs: tet(A), bla_CTX-M group 1, qnrB, and catI. Analyses were performed in both secondary treated wastewater and biosolid samples, including their purified bacteriophage-associated DNA fractions. Results showed that the AP method provided higher ARG concentrations than FC, particularly in wastewater samples. ddPCR demonstrated greater sensitivity than qPCR in wastewater, whereas in biosolids, both methods performed similarly, although ddPCR yielded weaker detection. Importantly, ARGs were detected in the phage fraction of both matrices, with ddPCR generally offering higher detection levels. These results provide comparative insights into established methodologies and highlight the value of selecting appropriate protocols based on matrix characteristics and surveillance objectives. Full article

(This article belongs to the Special Issue Antimicrobial Resistance in Foodborne Pathogens: Prevalence, Mechanisms, and Interventions)

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9 pages, 1015 KB

Open AccessArticle

Efficacy and Safety of Voriconazole for Difficult-to-Treat Distal Lateral Subungual Onychomycosis (DLSO)

by Aditya K. Gupta, Mesbah Talukder, Elizabeth A. Cooper, Lee Magal and Avner Shemer

Pathogens 2025, 14(10), 1049; https://doi.org/10.3390/pathogens14101049 - 17 Oct 2025

Abstract

This retrospective case series evaluated off-label voriconazole for distal lateral subungual onychomycosis (DLSO) unresponsive to standard therapy. Twenty-nine culture-confirmed patients who had failed terbinafine (250 mg daily × 12 weeks) and itraconazole pulses (200 mg twice daily for 1 week/month × 3) received [...] Read more.

This retrospective case series evaluated off-label voriconazole for distal lateral subungual onychomycosis (DLSO) unresponsive to standard therapy. Twenty-nine culture-confirmed patients who had failed terbinafine (250 mg daily × 12 weeks) and itraconazole pulses (200 mg twice daily for 1 week/month × 3) received voriconazole (200 mg twice on day 1, then 200 mg daily for 3–4 months). Assessments occurred at 2, 4, 6–9, and 12 months; the primary endpoint was combined clinical cure (≥90% nail clearance) plus mycological cure (negative KOH and culture) at 12 months. Intention-to-treat included 29 patients; per-protocol included 27 (two did not complete follow-up). In the per-protocol cohort, combined cure was 55.6% (15/27) and mycological cure 74.1% (20/27). Complete clinical cure occurred in 66.7% (18/27); 25.9% (7/27) improved markedly, 3.7% (1/27) mildly, and 3.7% (1/27) showed no improvement. Voriconazole was well tolerated and may be considered for DLSO refractory to terbinafine ± itraconazole. Antifungal stewardship remains essential. Full article

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18 pages, 2740 KB

Open AccessArticle

Campylobacter hepaticus Transcriptomics Identified Genes Involved in Spotty Liver Disease (SLD) Pathogenesis

by Varsha Bommineni, Lekshmi K. Edison, Chaitanya Gottapu, Gary D. Butcher and Subhashinie Kariyawasam

Pathogens 2025, 14(10), 1048; https://doi.org/10.3390/pathogens14101048 - 17 Oct 2025

Abstract

Campylobacter hepaticus is the etiological agent of Spotty Liver Disease (SLD), a newly emerging bacterial disease of laying hens resulting in significant mortality and production losses primarily in free-range systems. Although its economic impact continues to grow, the molecular basis of C. hepaticus [...] Read more.

Campylobacter hepaticus is the etiological agent of Spotty Liver Disease (SLD), a newly emerging bacterial disease of laying hens resulting in significant mortality and production losses primarily in free-range systems. Although its economic impact continues to grow, the molecular basis of C. hepaticus pathogenesis remains poorly understood. In this study, we conducted transcriptomic profiling of C. hepaticus in three host-relevant conditions, exposure to chicken bile, infection of a chicken liver hepatocellular carcinoma (LMH) cell line, and isolation from liver lesions of naturally infected chickens. Through RNA-seq analysis, we found unique gene expression signatures in each environment. In the bile, C. hepaticus exhibited differential expression of 412 genes, with upregulation of genes related to motility, cell envelope remodeling, glycosylation, nitrate respiration, and multidrug efflux systems, indicating a stress-adaptive, metabolically active lifestyle. In LMH, on the other hand, 125 genes were differentially expressed, primarily reflecting downregulation of motility, oxidative stress response, chaperones, and core metabolic processes, suggesting that these cells adopt a less active, intracellular dormant lifestyle. Transcriptomic analysis of C. hepaticus isolated from the liver identified 26 differentially expressed genes, featuring selective upregulation of genes associated with nitrate respiration, sulfur metabolism, and pyridoxal 5’ phosphate homeostasis, alongside downregulation of the major outer membrane porin (mompA), stress response chaperones (dnaK, groL), and genes involved in oxidative stress defense and energy production. Furthermore, the immune evasion-related gene cmeA and a glycosyltransferase gene were found to be highly upregulated. This study presents the first in-depth transcriptomic exploration of C. hepaticus in multiple host relevant niches. Our findings reveal niche-specific gene expression profiles and highlight metabolic and structural adaptations that enable C. hepaticus to survive during bile exposure, persist within host cells, and contribute to liver pathology. These insights provide a basis for identifying novel virulence determinants and may inform the development of targeted interventions, including vaccines or antimicrobial therapy, to control SLD in commercial poultry operations. Full article

(This article belongs to the Section Immunological Responses and Immune Defense Mechanisms)

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55 pages, 3869 KB

Open AccessReview

A Review of the Biology of Chikungunya Virus Highlighting the Development of Current Novel Therapeutic and Prevention Approaches

by Geovana Martelossi-Cebinelli, Jessica A. Carneiro, Kelly M. Yaekashi, Mariana M. Bertozzi, Beatriz H. S. Bianchini, Fernanda S. Rasquel-Oliveira, Camila Zanluca, Claudia N. Duarte dos Santos, Rachel Arredondo, Tiffani A. Blackburn, Rubia Casagrande and Waldiceu A. Verri

Pathogens 2025, 14(10), 1047; https://doi.org/10.3390/pathogens14101047 - 16 Oct 2025

Abstract

Chikungunya virus (CHIKV) is an arthritogenic alphavirus transmitted primarily via Aedes aegypti and Aedes albopictus mosquitoes. Since its identification, CHIKV remained confined to parts of Africa and Asia until the early 2000s, when it expanded to other continents, causing epidemics. Structurally, it is [...] Read more.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus transmitted primarily via Aedes aegypti and Aedes albopictus mosquitoes. Since its identification, CHIKV remained confined to parts of Africa and Asia until the early 2000s, when it expanded to other continents, causing epidemics. Structurally, it is an enveloped virus with a positive-single-stranded RNA genome, which encodes four non-structural proteins (nsP1-nsP4), responsible for viral replication, and five structural proteins (C, E3, E2, 6K, and E1), which form the capsid and envelope. Of these proteins, glycoproteins E1 and E2 are essential for cell recognition and membrane fusion, determining infectivity and viral tropism. CHIKV replication occurs in the cytosol of different cell types, triggering an intense inflammatory and immune response, which manifests clinically as Chikungunya fever (CHIKF). Despite its epidemiological impact, current treatment is limited to symptomatic approaches, including the use of analgesics and anti-inflammatories, as no specific antiviral therapies are available. In response, promising advances are being made, including the development of vaccines, targeted antivirals, and immunotherapies. This article aims to review the main aspects of viral biology, epidemiology, and immunopathogenesis of CHIKV infection, in addition to discussing the main advances in the development of new therapeutic approaches for its control. Full article

(This article belongs to the Special Issue Virus–Host Cell Interactions and Research of New Antivirals)

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20 pages, 4163 KB

Open AccessArticle

From Patterns to Projections: A Spatiotemporal Distribution of Drug-Resistant Tuberculosis in Paraná, Brazil (2012–2023)

by Laiz Mangini Cicchelero, Gustavo Cezar Wagner Leandro, Luciano de Andrade, Jean Eduardo Meneguello, Katiany Rizzieri Caleffi-Ferracioli, Rosilene Fressatti Cardoso and Regiane Bertin de Lima Scodro

Pathogens 2025, 14(10), 1046; https://doi.org/10.3390/pathogens14101046 - 16 Oct 2025

Abstract

Drug-resistant tuberculosis represents a challenge with high potential for spread. This ecological study aimed to describe the prevalence and incidence of drug-resistant tuberculosis and analyze its spatial, temporal, and spatiotemporal patterns in Paraná, Brazil, 2012–2023, and forecast trends through 2030. National surveillance data [...] Read more.

Drug-resistant tuberculosis represents a challenge with high potential for spread. This ecological study aimed to describe the prevalence and incidence of drug-resistant tuberculosis and analyze its spatial, temporal, and spatiotemporal patterns in Paraná, Brazil, 2012–2023, and forecast trends through 2030. National surveillance data were analyzed using descriptive statistics, Mann–Kendall trend tests, Global and Local Moran’s I, Kulldorff’s spatial scan statistic, and Seasonal AutoRegressive Integrated Moving Average modeling. A total of 576 cases were identified, corresponding to an incidence of 5.08 per 100,000 inhabitants, with an increasing trend (p < 0.001). After peaking in 2019, incidence declined during the pandemic and rose in 2023. Isoniazid monoresistance was the most frequent profile. Prevalence was higher among males, young adults (15–34 years), and Asian and Black individuals. Spatial distribution showed expansion over time, with early circulation in the West. The North and Northwest exhibited an initial high incidence. Spatial and spatiotemporal analyses identified persistent high-risk clusters in these regions (p < 0.05). Forecasting suggests that, if current conditions persist, the incidence may continue to rise through 2030. The findings highlight the need for surveillance to ensure treatment adherence and interrupt transmission of resistant bacilli, supporting progress toward the global goal of tuberculosis elimination. Full article

(This article belongs to the Section Bacterial Pathogens)

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25 pages, 1246 KB

Open AccessReview

Mitochondrial Dysfunction in Aging, HIV, and Long COVID: Mechanisms and Therapeutic Opportunities

by María Victoria Delpino and Jorge Quarleri

Pathogens 2025, 14(10), 1045; https://doi.org/10.3390/pathogens14101045 - 16 Oct 2025

Abstract

We hypothesize that a unified mitochondrial perspective on aging, HIV, and long COVID reveals shared pathogenic mechanisms and specific therapeutic vulnerabilities that are overlooked when these conditions are treated independently. Mitochondrial dysfunction is increasingly recognized as a common factor driving aging, HIV, and [...] Read more.

We hypothesize that a unified mitochondrial perspective on aging, HIV, and long COVID reveals shared pathogenic mechanisms and specific therapeutic vulnerabilities that are overlooked when these conditions are treated independently. Mitochondrial dysfunction is increasingly recognized as a common factor driving aging, HIV, and long COVID. Shared mechanisms—including oxidative stress, impaired mitophagy and dynamics, mtDNA damage, and metabolic reprogramming—contribute to ongoing energy failure and chronic inflammation. Recent advancements highlight new therapeutic strategies such as mitochondrial transfer, transplantation, and genome-level correction of mtDNA variants, with early preclinical and clinical studies providing proof-of-concept. This review summarizes current evidence on mitochondrial changes across aging and post-viral syndromes, examines emerging organelle-based therapies, and discusses key challenges related to safety, durability, and translation. Full article

(This article belongs to the Special Issue Pathogen–Host Interactions: Death, Defense, and Disease)

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36 pages, 3640 KB

Open AccessArticle

Bioinformatic Identification of CRISPR–Cas Systems in Leptospira Genus: An Update on Their Distribution Across 77 Species

by Ronald Guillermo Peláez Sánchez, Juanita González Restrepo, Santiago Pineda, Alexandra Milena Cuartas-López, Juliana María Martínez Garro, Marco Torres-Castro, Rodrigo Urrego, Luis Ernesto López-Rojas, Jorge Emilio Salazar Florez and Fernando P. Monroy

Pathogens 2025, 14(10), 1044; https://doi.org/10.3390/pathogens14101044 - 16 Oct 2025

Abstract

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the [...] Read more.

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify these arrays across the genomes of all described Leptospira species using bioinformatics tools. Methods: a bioinformatics workflow was followed: genomes were downloaded from the NCBI database; Cas protein detection was carried out using the CRISPR-CasFinder and RAST web servers; functional analyses of Cas proteins were performed with InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol; conservation pattern detection was conducted using MEGA12, and Seqlogos; spacer identification was carried out with the Actinobacteriophages database and BLAST version 1.4.0; and bacteriophage detection was performed using PHASTER, and PHASTEST. Results: Cas proteins were detected in 36 out of the 77 species of the Leptospira species, including Cas1 to Cas9 and Cas12. These proteins were classified into Class 1 and Class 2 systems, corresponding to types I, II, and V. Direct repeats and spacers were detected in 19 species, with the direct repeats exhibiting two conserved nucleotide motifs. Analysis of spacer sequences revealed 323 distinct bacteriophages. Additionally, three intact bacteriophages were detected in the genomes of four Leptospira species. Notably, two saprophytic species have complete CRISPR-Cas systems. Conclusions: The presence of Cas proteins, direct repeats, and spacer sequences with homology to bacteriophage genomes provides evidence for a functional CRISPR-Cas system in at least 19 species. Full article

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17 pages, 462 KB

Open AccessReview

Human Papillomavirus: An Old New History

by Nicole West, Valentina Boz, Nunzia Zanotta, Carolina Cason, Giuseppina Campisciano, Alessandra Casuccio, Daniele Gianfrilli, Teresa Maria Assunta Fasciana, Giuseppina Capra, Maria Cristina Salfa, Franz Sesti, Barbara Suligoi, Francesca Valent, Laura Brunelli and Manola Comar

Pathogens 2025, 14(10), 1043; https://doi.org/10.3390/pathogens14101043 - 14 Oct 2025

Abstract

Human papillomavirus (HPV) represents the most common sexually transmitted infection worldwide and a major public health challenge. Nearly all sexually active individuals will acquire HPV during their lifetime, with the highest prevalence observed in adolescents and young adults shortly after sexual debut. More [...] Read more.

Human papillomavirus (HPV) represents the most common sexually transmitted infection worldwide and a major public health challenge. Nearly all sexually active individuals will acquire HPV during their lifetime, with the highest prevalence observed in adolescents and young adults shortly after sexual debut. More than 200 genotypes have been described, ranging from low-risk types, mainly responsible for benign lesions, to high-risk types, which are associated with cervical, anogenital, and head and neck cancers. While most infections are transient and spontaneously cleared by the immune system, persistent high-risk HPV can lead to precancerous lesions and malignant transformation, often in synergy with other sexually transmitted pathogens or in the context of microbiome imbalance. The introduction of vaccines and advanced screening technologies has substantially modified prevention strategies. Vaccination coverage remains heterogeneous, with persistent gaps particularly among males due to cultural, social, and educational barriers. Schools are increasingly recognized as strategic environments to promote awareness, sex education, and gender-neutral vaccination. Innovative approaches such as microbiome modulation, therapeutic vaccines, and liquid biopsy biomarkers are emerging as promising perspectives. This review aims to provide an updated overview of HPV epidemiology, clinical impact, prevention strategies, and future frontiers, with special attention to adolescents as a priority target group. Full article

(This article belongs to the Special Issue Focus on Cervicovaginal Health: Microbiome, Sexually Transmitted Infections, and Beneficial Microbes: 2nd Edition)

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19 pages, 6927 KB

Open AccessArticle

Serum Proteomics Reveals Systemic Responses in Didelphis aurita Naturally Infected with Hepatozoon sp.

by Andrés Mauricio Ortega Orozco, Camilo Jose Ramirez-Lopez, Lucas Drumond Bento, Pollyanna Cordeiro Souto, Fabrícia Modolo Girardi, Veronica Rodrigues Castro, Edvaldo Barros, Joao Vitor Gonçalves de Oliveira, Renner Philipe Rodrigues Carvalho, Artur Kanadani Campos and Leandro Abreu da Fonseca

Pathogens 2025, 14(10), 1042; https://doi.org/10.3390/pathogens14101042 - 14 Oct 2025

Abstract

Didelphis aurita is a widely distributed neotropical marsupial frequently found in peri-urban environments and known to harbor various pathogens, including hemoparasites of the genus Hepatozoon. However, the systemic physiological responses of naturally infected individuals remain poorly understood. This study aimed to characterize the [...] Read more.

Didelphis aurita is a widely distributed neotropical marsupial frequently found in peri-urban environments and known to harbor various pathogens, including hemoparasites of the genus Hepatozoon. However, the systemic physiological responses of naturally infected individuals remain poorly understood. This study aimed to characterize the serum proteomic profile of Didelphis aurita naturally infected with Hepatozoon sp., providing insights into host–parasite interactions and potential biomarkers of infection. Serum samples were analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS), followed by functional annotation based on Gene Ontology and KEGG pathway enrichment. A total of 67 proteins were identified, 33 of which were exclusive to infected animals. The most abundant proteins included albumin, hemoglobin subunits, and venom metalloproteinase inhibitors (DM43 and DM64). Functional enrichment revealed significant involvement in complement and coagulation cascades, protease inhibition, antioxidant defense, and extracellular vesicle localization. Key proteins such as fibrinogen, plasminogen, antithrombin, SERPIN family members, vitronectin, and fibronectin suggest an integrated host response involving hemostasis, inflammation control, and tissue remodeling. This is the first report of the serum proteome of Didelphis aurita naturally infected with Hepatozoon sp. Despite the absence of protein validation, the findings provide novel insights into marsupial immunophysiology and offer a foundation for future biomarker research and ecoimmunological surveillance in synanthropic species. Full article

(This article belongs to the Special Issue Proteomic Approaches in Different Animal Pathogens)

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20 pages, 3708 KB

Open AccessArticle

Bacillus anthracis Phylogeography: Origin of the East Asian Polytomy and Impact of International Trade for Its near Global Dispersal

by Gilles Vergnaud, Markus H. Antwerpen and Gregor Grass

Pathogens 2025, 14(10), 1041; https://doi.org/10.3390/pathogens14101041 - 14 Oct 2025

Abstract

Bacillus anthracis is the etiological agent of the zoonotic disease anthrax. The pathogen has colonized many regions of all inhabited continents. Increasing evidence points to a strong contribution of anthropogenic activities (trade) in this almost global spread. This article contributes further genomic data [...] Read more.

Bacillus anthracis is the etiological agent of the zoonotic disease anthrax. The pathogen has colonized many regions of all inhabited continents. Increasing evidence points to a strong contribution of anthropogenic activities (trade) in this almost global spread. This article contributes further genomic data from 21 B. anthracis strains, including 19 isolated in Germany, aiming to support and detail the human role in anthrax dispersal. The newly sequenced genomes belong to the B. anthracis lineage predominant in China. This lineage is remarkable because of its phylogenetic structure. A polytomy with nine branches radiating from a central node was identified by whole-genome single-nucleotide polymorphism (wgSNP) analysis. Strains from Germany populate two among the nine branches. Detailed analysis of the polytomy indicates that it most likely emerged in China. We propose that the polytomy is the result of the import of contaminated animal products in a limited spatiotemporal frame, followed by the distribution of these products to different locations within China, where new B. anthracis lineages then became independently established. Currently available data point to Bengal as a likely geographic source of the original contamination, and the history of trade exchanges between Bengal and China agrees with the early fifteenth century as a likely time period. The subsequent exports to Germany would have occurred during the 19th century according to German trade history. Notably, Germany has been experiencing localized anthrax outbreaks from this trade heritage up into the 21st century. Full article

(This article belongs to the Special Issue Current Research on Bacillus anthracis Infection)

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16 pages, 2782 KB

Open AccessArticle

Genetic Diversity and Antibiotic Resistance Paradigm of Enterobacterales in Animal-Derived Food Sources: A One Health Disquiet

by Ayesha Sarwar, Bilal Aslam, Muhammad Hidayat Rasool, Muhammad Shafique, Mohsin Khurshid, James Jacob Sasanya and Sulaiman F. Aljasir

Pathogens 2025, 14(10), 1040; https://doi.org/10.3390/pathogens14101040 - 13 Oct 2025

Abstract

The indiscriminate use of antibiotics in food-producing animals serves as a major catalyst for the emergence of antibiotic-resistant infections. This study aimed to assess the genetic diversity and antibiotic resistance of Enterobacterales in animal-derived foods. A total of 905 animal-derived food samples, including [...] Read more.

The indiscriminate use of antibiotics in food-producing animals serves as a major catalyst for the emergence of antibiotic-resistant infections. This study aimed to assess the genetic diversity and antibiotic resistance of Enterobacterales in animal-derived foods. A total of 905 animal-derived food samples, including meat, dairy, poultry, fish, and environmental sources, were collected from various locations in Pakistan. Isolates were confirmed through selective subculturing, morphological, biochemical, and MALDI-TOF analysis, followed by antibiotic susceptibility testing. Subsequently, PCR-based detection of antibiotic resistance genes and virulence-associated genes. Overall, a total of 263 (29.06%) Enterobacterales were identified, as follows: 58.55% (154/263) E. coli, 6.84% (18/263) K. pneumoniae, 21.29% (56/263) P. mirabilis, and 13.30% (35/263) Salmonella spp. Isolates showed a varying resistance pattern against different studied antibiotics, e.g., beta-lactams and inhibitors, ciprofloxacin, and tetracycline, while colistin and tigecycline remained most effective. All the isolates displayed an array of antibiotic resistance and virulence-associated genes. Particularly significant (<0.05) co-existence of bla_NDM and mcr-1 was observed among the Enterobacterales isolated from various animal-derived foods. This study underscores the need to monitor Enterobacterales in animal-derived foods, especially in developing countries, to curb the spread of resistant pathogens and ensure effective food safety measures. Full article

(This article belongs to the Special Issue Epidemiology and Molecular Pathogenesis of Antimicrobial Resistance and Virulence for Foodborne Pathogens: 2nd Edition)

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14 pages, 1301 KB

Open AccessArticle

Deciphering Escherichia coli ESBL/pAmpC Plasmids Through High-Throughput Third-Generation Sequencing and Hybrid Assembly

by Andrea Laconi, Enea Ovedani, Roberta Tolosi, Ilias Apostolakos and Alessandra Piccirillo

Pathogens 2025, 14(10), 1039; https://doi.org/10.3390/pathogens14101039 - 13 Oct 2025

Abstract

Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC (pAmpC) β-lactamases represent a threat for public health. Their dissemination is often mediated by mobile genetic elements (MGEs), but plasmid identification and characterization could be hindered by sequencing limitations. Hybrid assembly may overcome these barriers. Eight ESBL/pAmpC-producing [...] Read more.

Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC (pAmpC) β-lactamases represent a threat for public health. Their dissemination is often mediated by mobile genetic elements (MGEs), but plasmid identification and characterization could be hindered by sequencing limitations. Hybrid assembly may overcome these barriers. Eight ESBL/pAmpC-producing E. coli isolates from broilers were sequenced using Illumina (short-read) and Oxford Nanopore MinION (long-read). Assemblies were generated individually and using a hybrid approach. Plasmids were typed, annotated, and screened for antimicrobial resistance genes (ARGs), MGEs, and virulence factors. Short-read assemblies were highly fragmented, while long reads improved contiguity but showed typing errors. Hybrid assemblies produced the most accurate and complete plasmids, including more circularized plasmids. Long and hybrid assemblies detected IS26 associated with ESBL genes and additional virulence genes not identified by short reads. ARG profiles were consistent across methods, but structural resolution and contextualization of resistance loci were superior in hybrid assembly. Hybrid assembly integrates the strengths of short- and long-read sequencing, enabling accurate plasmid reconstruction and improved detection of resistance-associated MGEs. This approach may enhance genomic surveillance of ESBL/pAmpC plasmids and support strategies to mitigate antimicrobial resistance. Full article

(This article belongs to the Special Issue One Health Perspectives on Foodborne Pathogens: Evolution, Resistance, and Prevention)

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13 pages, 2230 KB

Open AccessArticle

Therapeutic Efficacy of an Anti-P116-661 Polyclonal Antibody Against Mycoplasma pneumoniae Infection

by Yiting Zhang, Xinqi Geng, Yan Liu, Wenli Li, Feng Shao, Mengmeng Jin, Jinzhi Wang and Linding Wang

Pathogens 2025, 14(10), 1038; https://doi.org/10.3390/pathogens14101038 - 13 Oct 2025

Abstract

The aim of this study was to investigate the therapeutic potential of a polyclonal antibody against the Mycoplasma pneumoniae (MP) P116-661 protein. A polyclonal antibody against the P116-661 protein was obtained by immunizing New Zealand white rabbits, and its therapeutic effects were systematically [...] Read more.

The aim of this study was to investigate the therapeutic potential of a polyclonal antibody against the Mycoplasma pneumoniae (MP) P116-661 protein. A polyclonal antibody against the P116-661 protein was obtained by immunizing New Zealand white rabbits, and its therapeutic effects were systematically evaluated by various experimental methods. An immunofluorescence assay was used to detect the inhibitory effect of the P116-661 polyclonal antibody on the adhesion of MP cells to A549 cells. ELISAs and Western blotting were used to analyze the expression levels of inflammatory factors, such as IL-6 and TNF-α, in Beas-2b cells and model mice after MP infection. HE staining was used to observe pathological changes in the lung tissue of the infected mice. The results showed that the P116-661 polyclonal antibody effectively inhibited the adhesion of MP cells to A549 cells. It significantly reduced the secretion levels of inflammatory factors, such as IL-6 and TNF-α, in Beas-2b cells and mice after MP infection. Moreover, the antibody significantly improved the pathological damage to the lungs that was caused by MP infection in mice. This study confirms that the P116-661 polyclonal antibody has good therapeutic effects in vitro and in vivo, providing a new experimental basis for immunotherapy against MP infection. Full article

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