Pathogens

21 pages, 471 KiB

Open AccessReview

Role and Contribution of Serological Surveillance in Animals and Exposed Humans to the Study of Zoonotic Influenza Disease Epidemiology: A Scoping Review

by Rebecca Badra, Wenqing Zhang, John S. L. Tam, Richard Webby, Sylvie van der Werf, Sergejs Nikisins, Ann Cullinane, Saad Gharaibeh, Richard Njouom, Malik Peiris, Ghazi Kayali and Jean-Michel Heraud

Pathogens 2025, 14(8), 739; https://doi.org/10.3390/pathogens14080739 (registering DOI) - 27 Jul 2025

Abstract

Background: Zoonotic influenza viruses pose a significant and evolving public health threat. In response to the recent rise in H5N1 cross-species transmission, the World Health Organization (WHO) R&D Blueprint for Epidemics consultations have prioritized strengthening surveillance, candidate vaccines, diagnostics, and pandemic preparedness. Serological [...] Read more.

Background: Zoonotic influenza viruses pose a significant and evolving public health threat. In response to the recent rise in H5N1 cross-species transmission, the World Health Organization (WHO) R&D Blueprint for Epidemics consultations have prioritized strengthening surveillance, candidate vaccines, diagnostics, and pandemic preparedness. Serological surveillance plays a pivotal role by providing insights into the prevalence and transmission dynamics of influenza viruses. Objective: This scoping review aimed to map the global research landscape on serological surveillance of zoonotic influenza in animals and exposed humans between 2017, the date of the last WHO public health research agenda for influenza review, and 2024, as well as to identify methodological advancements. Methods: Following PRISMA-ScR guidelines, we searched PubMed for English-language peer-reviewed articles published between January 2017 and March 2024. Studies were included if they reported serological surveillance in wild or domestic animals or occupationally exposed human populations, or novel methodologies and their technical limitations and implementation challenges. Results: Out of 7490 screened records, 90 studies from 33 countries, covering 25 animal species, were included. Seroprevalence studies were in domestic poultry and swine. Surveillance in companion animals, wild mammals, and at the human–animal interface was limited. Emerging serological methods included multiplex and nanobody-based assays, though implementation barriers remain. Conclusions: The review is limited by its restriction to one database and English-language articles, lack of quality appraisal, and significant heterogeneity among the included studies. Serological surveillance is a critical but underutilized tool in zoonotic influenza monitoring. Greater integration of serological surveillance into One Health frameworks, especially in high-risk regions and populations, is needed to support early detection and pandemic preparedness. Full article

(This article belongs to the Section Emerging Pathogens)

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30 pages, 6219 KiB

Open AccessArticle

The Molecular Epidemiology of HIV-1 in Russia, 1987–2023: Subtypes, Transmission Networks and Phylogenetic Story

by Aleksey Lebedev, Dmitry Kireev, Alina Kirichenko, Ekaterina Mezhenskaya, Anastasiia Antonova, Vyacheslav Bobkov, Ilya Lapovok, Anastasia Shlykova, Alexey Lopatukhin, Andrey Shemshura, Valery Kulagin, Aleksei Kovelenov, Alexandra Cherdantseva, Natalia Filoniuk, Galina Turbina, Alexei Ermakov, Nikita Monakhov, Michael Piterskiy, Aleksandr Semenov, Sergej Shtrek, Aleksej Sannikov, Natalia Zaytseva, Olga Peksheva, Aleksandr Suladze, Dmitry Kolpakov, Valeriia Kotova, Elena Bazykina, Vasiliy Akimkin and Marina Bobkova Show full author list Hide full author list

Pathogens 2025, 14(8), 738; https://doi.org/10.3390/pathogens14080738 (registering DOI) - 26 Jul 2025

Abstract

Regional HIV-1 epidemics are evolving with distinct patterns in transmission routes, subtype distribution, and molecular transmission cluster (MTCs) characteristics. We analyzed 9500 HIV-1 cases diagnosed over 30 years using phylogenetic and network methods, integrating molecular, epidemiological, demographic, and behavioral data. Subtype A6 remains [...] Read more.

Regional HIV-1 epidemics are evolving with distinct patterns in transmission routes, subtype distribution, and molecular transmission cluster (MTCs) characteristics. We analyzed 9500 HIV-1 cases diagnosed over 30 years using phylogenetic and network methods, integrating molecular, epidemiological, demographic, and behavioral data. Subtype A6 remains dominant nationally (80.6%), followed by 63_02A6 (7.9%), subtype B (5.6%), 02_AG_FSU (1.2%), 03_A6B (0.7%), and 14/73_BG (0.6%). Non-A6 infections were more common among males (OR 1.51) and men who have sex with men (OR 7.33). Network analysis identified 421 MTCs, with 256 active clusters. Clustering was more likely among young individuals (OR: 1.31), those not receiving antiretroviral therapy (OR: 2.70), and injecting drug users (OR: 1.28). Non-A6 subtypes showed a higher likelihood of clustering. Phylogenetic analysis revealed that local clusters of the major subtypes originated between the late 1970s (subtype B) and the mid-2000s (63_02A6) with links to populations in Eastern Europe, Central Asia (subtypes A6, 63_02A6, 02_AG_FSU, 03_A6B), and Western Europe and the Americas (subtype B, 14/73_BG). These findings indicate a complex, evolving regional epidemic transitioning from subtype A6 dominance to a more diverse mix of subtypes. The ability of non-A6 subtypes to form active MTCs suggests their establishment in the local population. Full article

(This article belongs to the Special Issue HIV/AIDS: Epidemiology, Drug Resistance, Treatment and Prevention)

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15 pages, 406 KiB

Open AccessArticle

Outbreak Caused by VIM-1- and VIM-4-Positive Proteus mirabilis in a Hospital in Zagreb

by Branka Bedenić, Gernot Zarfel, Josefa Luxner, Andrea Grisold, Marina Nađ, Maja Anušić, Vladimira Tičić, Verena Dobretzberger, Ivan Barišić and Jasmina Vraneš

Pathogens 2025, 14(8), 737; https://doi.org/10.3390/pathogens14080737 (registering DOI) - 26 Jul 2025

Abstract

Background/objectives: Proteus mirabilis is a frequent causative agent of urinary and wound infections in both community and hospital settings. It develops resistance to expanded-spectrum cephalosporins (ESCs) due to the production of extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpC β-lactamases (p-AmpCs). Recently, carbapenem-resistant isolates of [...] Read more.

Background/objectives: Proteus mirabilis is a frequent causative agent of urinary and wound infections in both community and hospital settings. It develops resistance to expanded-spectrum cephalosporins (ESCs) due to the production of extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpC β-lactamases (p-AmpCs). Recently, carbapenem-resistant isolates of P. mirabilis emerged due to the production of carbapenemases, mostly belonging to Ambler classes B and D. Here, we report an outbreak of infections due to carbapenem-resistant P. mirabilis that were observed in a psychiatric hospital in Zagreb, Croatia. The characteristics of ESBL and carbapenemase-producing P. mirabilis isolates, associated with an outbreak, were analyzed. Materials and methods: The antibiotic susceptibility testing was performed by the disk-diffusion and broth dilution methods. The double-disk synergy test (DDST) and inhibitor-based test with clavulanic and phenylboronic acid were applied to screen for ESBLs and p-AmpCs, respectively. Carbapenemases were screened by the modified Hodge test (MHT), while carbapenem hydrolysis was investigated by the carbapenem inactivation method (CIM) and EDTA-carbapenem-inactivation method (eCIM). The nature of the ESBLs, carbapenemases, and fluoroquinolone-resistance determinants was investigated by PCR. Plasmids were characterized by PCR-based replicon typing (PBRT). Selected isolates were subjected to molecular characterization of the resistome by an Inter-Array Genotyping Kit CarbaResisit and whole-genome sequencing (WGS). Results: In total, 20 isolates were collected and analyzed. All isolates exhibited resistance to amoxicillin alone and when combined with clavulanic acid, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, cefepime, imipenem, ceftazidime–avibactam, ceftolozane–tazobactam, gentamicin, amikacin, and ciprofloxacin. There was uniform susceptibility to ertapenem, meropenem, and cefiderocol. The DDST and combined disk test with clavulanic acid were positive, indicating the production of an ESBL. The MHT was negative in all except one isolate, while the CIM showed moderate sensitivity, but only with imipenem as the indicator disk. Furthermore, eCIM tested positive in all of the CIM-positive isolates, consistent with a metallo-β-lactamase (MBL). PCR and sequencing of the selected amplicons identified VIM-1 and VIM-4. The Inter-Array Genotyping Kit CarbaResist and WGS identified β-lactam resistance genes bla_VIM, bla_CTX-M-15, and bla_TEMgenes; aminoglycoside resistance genes aac(3)-IId, aph(6)-Id, aph(3″)-Ib, aadA1, armA, and aac(6′)-IIc; as well as resistance genes for sulphonamides sul1 and sul2, trimethoprim dfr1, chloramphenicol cat, and tetracycline tetJ. Conclusions: This study revealed an epidemic spread of carbapenemase-producing P. mirabilis in two wards in a psychiatric hospital. Due to the extensively resistant phenotype (XDR), therapeutic options were limited. This is the first report of carbapenemase-producing P. mirabilis in Croatia. Full article

(This article belongs to the Special Issue Emerging and Neglected Pathogens in the Balkans)

27 pages, 11638 KiB

Open AccessArticle

Trypanosoma cruzi Growth Is Impaired by Oleoresin and Leaf Hydroalcoholic Extract from Copaifera multijuga in Human Trophoblast and Placental Explants

by Guilherme de Souza, Clara Peleteiro Teixeira, Joed Pires de Lima Júnior, Marcos Paulo Oliveira Almeida, Marina Paschoalino, Luana Carvalho Luz, Natália Carine Lima dos Santos, Rafael Martins de Oliveira, Izadora Santos Damasceno, Matheus Carvalho Barbosa, Guilherme Vieira Faria, Maria Anita Lemos Vasconcelos Ambrosio, Rodrigo Cassio Sola Veneziani, Jairo Kenupp Bastos, Angelica Oliveira Gomes, Rosiane Nascimento Alves, Carlos Henrique Gomes Martins, Samuel Cota Teixeira, Eloisa Amália Vieira Ferro and Bellisa Freitas Barbosa

Pathogens 2025, 14(8), 736; https://doi.org/10.3390/pathogens14080736 - 25 Jul 2025

Abstract

Congenital Chagas disease (CCD) is caused when Trypanosoma cruzi crosses the placental barrier during pregnancy and reaches the fetus, which can lead to serious consequences in the developing fetus. Current treatment is carried out with nifurtimox or benznidazole, but their effectiveness is limited, [...] Read more.

Congenital Chagas disease (CCD) is caused when Trypanosoma cruzi crosses the placental barrier during pregnancy and reaches the fetus, which can lead to serious consequences in the developing fetus. Current treatment is carried out with nifurtimox or benznidazole, but their effectiveness is limited, and they cause side effects, requiring the search for new therapeutic strategies. In this sense, many studies have demonstrated the potential of different compounds of the Copaifera genus in the control of parasitic diseases. Here, we aimed to evaluate the effect of oleoresin (OR) and leaf hydroalcoholic extract (LHE) of Copaifera multijuga on Trypanosoma cruzi infection in human villous trophoblast cells (BeWo line) and human placenta explants. Treatment with both compounds reduced invasion, proliferation, and release of trypomastigotes. Furthermore, OR and LHE affected the trypomastigotes and amastigote morphology, compromising their ability to invade and proliferate in BeWo cells, respectively. Also, treatment with OR decreased ROS production in infected BeWo cells, while LHE induced an increase. In addition, both compounds induced pro-inflammatory and anti-inflammatory cytokine production. In human placental explants, both compounds also decreased T. cruzi infection, in addition to inducing the production of pro-inflammatory cytokines. Thus, both OR and LHE of C. multijuga control T. cruzi infection at the human maternal–fetal interface, highlighting the possible therapeutic potential of these compounds for the treatment of CCD. Full article

(This article belongs to the Special Issue Exploring Natural Products as Antiparasitic Agents: Efficacy Against Parasites of Veterinary and Public Health Significance)

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Graphical abstract

19 pages, 5484 KiB

Open AccessArticle

Comprehensive Molecular and Epidemiological Characterization of Staphylococcus aureus Isolated from Bovine Mastitis in Water Buffalo of the Peshawar Division, Khyber Pakhtunkhwa, Pakistan

by Salma Javed, Jo-Ann McClure, Irfan Ullah, Shahzad Ali, Mohammad Ejaz, Sadia Tabassum, Muhammad Ali Syed and Kunyan Zhang

Pathogens 2025, 14(8), 735; https://doi.org/10.3390/pathogens14080735 - 25 Jul 2025

Abstract

Water buffalo (Bubalus bubalis) are a primary source of milk in Pakistan, where bovine mastitis is a significant health issue among cattle, leading to substantial economic losses. Staphylococcus aureus is a predominant pathogen associated with mastitis; however, a detailed molecular characterization [...] Read more.

Water buffalo (Bubalus bubalis) are a primary source of milk in Pakistan, where bovine mastitis is a significant health issue among cattle, leading to substantial economic losses. Staphylococcus aureus is a predominant pathogen associated with mastitis; however, a detailed molecular characterization of the strains in the country remains limited. We previously characterized mastitis strains from the Hazara division of Khyber Pakhtunkhwa, Pakistan. In this study, we investigated mastitis cases in the Peshawar division, including samples from both animals and human farm workers for comparison. Higher rates of mastitis (67.27% of animals) and sub-clinical mastitis (91.03% of positive animals) were identified in Peshawar than for those (34.55% and 75.31%, respectively) previously observed in Hazara. Methicillin-susceptible S. aureus (MSSA) belonging to clonal complex 9 (ST2454) were predominant. Methicillin-resistant S. aureus (MRSA) belonging to ST22 and ST8 were also detected in the Nowshera district. While no S. aureus colonization was observed among animal handlers, evidence of hand contamination suggests a potential route for pathogen spread. Low levels of antibiotic resistance were noted amongst isolates, but higher rates were seen in MRSA. This study presents only the second comprehensive molecular investigation of S. aureus isolated from buffalo mastitis in Pakistan and indicates a concerning rise in mastitis within the province. Full article

(This article belongs to the Special Issue Dairy Cattle Health: Mastitis, Milk Quality, and Antimicrobial Resistance from a One Health Perspective)

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17 pages, 1873 KiB

Open AccessArticle

A Novel Amdoparvovirus of Badgers and Foxes and the Perpetuation of Aleutian Mink Disease Virus 3 in the Wildlife of Denmark

by Frederikke Juncher Høeg, Anne Sofie Vedsted Hammer, Anna Cecilie Boldt Eiersted, Joost Theo Petra Verhoeven, Lars Erik Larsen, Tim Kåre Jensen and Marta Canuti

Pathogens 2025, 14(8), 734; https://doi.org/10.3390/pathogens14080734 - 25 Jul 2025

Abstract

Amdoparvoviruses, encompassing the well-characterized Aleutian mink disease viruses (AMDV) as well as less investigated viruses infecting both captive and wild animals, are important carnivoran viruses that are significant pathogens in the mink farming industry. We investigated the molecular epidemiology of amdoparvoviruses among Danish [...] Read more.

Amdoparvoviruses, encompassing the well-characterized Aleutian mink disease viruses (AMDV) as well as less investigated viruses infecting both captive and wild animals, are important carnivoran viruses that are significant pathogens in the mink farming industry. We investigated the molecular epidemiology of amdoparvoviruses among Danish wildlife. Spleen samples from 118 animals of seven carnivoran species were screened with a pan-amdoparvovirus PCR, and the identified viruses were molecularly characterized. In one of five European badgers (Meles meles), we identified an AMDV-3 strain whose ancestors were likely of farmed mink origin. This virus was last reported on a mink farm in 2002, demonstrating how farm-derived viruses have established themselves among wildlife. We also discovered and fully characterized a novel virus found in five of 81 (6.2%) foxes (Vulpes vulpes) and one of five badgers (20.0%), which we named fox and badger amdoparvovirus 1 (FBAV-1). FBAV-1 fulfills the criteria for classification as a novel species and phylogenetically is positioned as an intermediate between the North American and Eurasian amdoparvoviral clades. This study provides baseline data and expands our understanding of amdoparvoviral ecology. Further studies including more animals across diverse geographic areas are warranted to clarify amdoparvovirus epidemiology, spread, cross-species transmission, epidemic potential, and evolutionary paths. Full article

(This article belongs to the Special Issue Molecular Detection and Characterisation of Viral Pathogens: 2nd Edition)

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8 pages, 1302 KiB

Open AccessCommunication

Vaccinia and Monkeypox Virus-Neutralizing Antibodies in People Living with HIV: A Serological Study in a Orthopoxvirus-Endemic, Low-Income Region in Brazil

by Thyago José Silva, Ana Gabriella Stoffella-Dutra, Victor Lacerda Gripp, Pollyana R. C. Gorgens, Iago José da Silva Domingos, Pedro Henrique Bastos e Silva, Bruna Caroline Chaves-Garcia, Erna Geessien Kroon, Etel Rocha-Vieira, Giliane de Souza Trindade and Danilo Bretas de Oliveira

Pathogens 2025, 14(8), 733; https://doi.org/10.3390/pathogens14080733 - 25 Jul 2025

Abstract

Co-infections of Orthopoxviruses (OPVs), such as vaccinia virus (VACV) and monkeypox virus (MPXV), and the human immunodeficiency virus (HIV) can be associated with severe outcomes. Serro’s dairy region, located in Minas Gerais, southeastern Brazil, is an endemic area for VACV, where zoonotic outbreaks [...] Read more.

Co-infections of Orthopoxviruses (OPVs), such as vaccinia virus (VACV) and monkeypox virus (MPXV), and the human immunodeficiency virus (HIV) can be associated with severe outcomes. Serro’s dairy region, located in Minas Gerais, southeastern Brazil, is an endemic area for VACV, where zoonotic outbreaks affect rural communities. This epidemiological context is especially relevant for at-risk populations, such as people living with HIV (PLHIV). This study aimed to assess the presence of neutralizing antibodies (NAbs) against OPV in PLHIV in this endemic setting. Serum samples were collected from 177 PLHIV in treatment at the specialized service between December 2021 and August 2022. VACV and MPXV NAbs were measured using the plaque reduction neutralization test (PRNT) and VACV-infected cells. The overall occurrence of OPV NAbs was 27.7%. NAbs were higher in individuals born before 1980 (53.3%) than those born after 1980 (1.1%). Among anti-VACV-seropositive individuals, 40.8% also had MPXV NAbs, suggesting cross-immunity. These findings indicate the circulation of VACV in PLHIV and highlight the increased susceptibility to OPV infections among individuals born after the cessation of smallpox vaccination. The results reinforce the importance of continued surveillance of OPV, especially in endemic regions and vulnerable populations. Full article

(This article belongs to the Section Emerging Pathogens)

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18 pages, 2215 KiB

Open AccessArticle

Exploration of Phosphoproteins in Acinetobacter baumannii

by Lisa Brémard, Sébastien Massier, Emmanuelle Dé, Nicolas Nalpas and Julie Hardouin

Pathogens 2025, 14(8), 732; https://doi.org/10.3390/pathogens14080732 - 24 Jul 2025

Abstract

Acinetobacter baumannii is a multidrug-resistant bacterium that has gained significant attention in recent years due to its involvement in a growing number of hospital-acquired infections. The World Health Organization has classified it as a critical priority pathogen, underscoring the urgent need for new [...] Read more.

Acinetobacter baumannii is a multidrug-resistant bacterium that has gained significant attention in recent years due to its involvement in a growing number of hospital-acquired infections. The World Health Organization has classified it as a critical priority pathogen, underscoring the urgent need for new therapeutic strategies. Post-translational modifications (PTMs), such as phosphorylation, play essential roles in various bacterial processes, including antibiotic resistance, virulence or biofilm formation. Although proteomics has increasingly enabled their characterization, the identification of phosphorylated peptides remains challenging, primarily due to the enrichment procedures. In this study, we focused on characterizing serine, threonine, and tyrosine phosphorylation in the A. baumannii ATCC 17978 strain. We optimized three parameters for phosphopeptide enrichment using titanium dioxide (TiO₂) beads (number of enrichment fractions between the phosphopeptides and TiO₂ beads, the quantity peptides and type of loading buffer) to determine the most effective conditions for maximizing phosphopeptide identification. Using this optimized protocol, we identified 384 unique phosphorylation sites across 241 proteins, including 260 novel phosphosites previously unreported in A. baumannii. Several of these phosphorylated proteins are involved in critical bacterial processes such as antimicrobial resistance, biofilm formation or pathogenicity. We discuss these proteins, focusing on the potential functional implications of their phosphorylation. Notably, we identified 34 phosphoproteins with phosphosites localized at functional sites, such as active sites, multimer interfaces, or domains important for structural integrity. Our findings significantly expand the current phosphoproteomic landscape of A. baumannii and support the hypothesis that PTMs, particularly phosphorylation, play a central regulatory role in its physiology and pathogenic potential. Full article

(This article belongs to the Section Bacterial Pathogens)

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15 pages, 1416 KiB

Open AccessArticle

High Prevalence of Virulence and bla_OXA Genes Encoding Carbapenemases Among Acinetobacter baumannii Isolates from Hospitalised Patients in Three Regions of Poland

by Magdalena Szemraj, Małgorzata Piechota, Kamila Olszowiec, Jolanta Wicha, Agata Pruss, Monika Sienkiewicz, Małgorzata Witeska, Piotr Szweda and Barbara Kot

Pathogens 2025, 14(8), 731; https://doi.org/10.3390/pathogens14080731 - 24 Jul 2025

Abstract

Infections caused by Acinetobacter baumannii are increasing worldwide. We evaluated the antibiotic resistance profile, biofilm production, and the frequency of 12 genes encoding carbapenemases and 13 virulence factors in 90 isolates from patients of three hospitals in various regions of Poland. Antibiotic resistance [...] Read more.

Infections caused by Acinetobacter baumannii are increasing worldwide. We evaluated the antibiotic resistance profile, biofilm production, and the frequency of 12 genes encoding carbapenemases and 13 virulence factors in 90 isolates from patients of three hospitals in various regions of Poland. Antibiotic resistance survey was performed using the disc-diffusion method, genes encoding resistance to carbapenems and virulence factors were detected with PCR, and biofilm formation was tested using microtiter plates. A total of 52.2% of isolates were resistant to all tested antibiotic groups (penicillins with β-lactamase inhibitors, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and trimethoprim plus sulfamethoxazole). Among the genes encoding carbapenem resistance, the bla_OXA-23 (68.9%), bla_OXA-40 (83.3%), and ISAba-bla_OXA-51 (18.9%) were detected. The ompA, ata, and recA genes responsible for biofilm formation, adhesion, and stress response, respectively, occurred in all isolates. Genes responsible for the production of other adhesins (bap—94.4%, espA—4.4%, chop—37.7%), biofilm formation (pbpG—90.0%), production of siderophore (basD—97.7%), toxins (lipA—92.2%, cpaA—1.1%), glycoconjugates (bfmR—84.4%), and inducing host cell death (fhaB—71.1%, abeD—93.3%) were also found. A total of 68.8% of isolates produced biofilm. The isolates from Masovia had more virulence genes than isolates from the other regions; moreover, all isolates from Masovia and West Pomerania were multidrug-resistant (MDR), including resistance to carbapenems. Full article

(This article belongs to the Section Bacterial Pathogens)

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14 pages, 722 KiB

Open AccessArticle

When the Last Line Fails: Characterization of Colistin-Resistant Acinetobacter baumannii Reveals High Virulence and Limited Clonal Dissemination in Greek Hospitals

by Dimitrios Karakalpakidis, Theofilos Papadopoulos, Michalis Paraskeva, Michaela-Eftychia Tsitlakidou, Eleni Vagdatli, Helen Katsifa, Apostolos Beloukas, Charalampos Kotzamanidis and Christine Kottaridi

Pathogens 2025, 14(8), 730; https://doi.org/10.3390/pathogens14080730 - 24 Jul 2025

Abstract

Acinetobacter baumannii has emerged as a major pathogen responsible for healthcare-associated infections, particularly in intensive care units, contributing to significant morbidity and mortality due to its multidrug resistance and ability to persist in clinical environments. This study aimed to investigate the phenotypic and [...] Read more.

Acinetobacter baumannii has emerged as a major pathogen responsible for healthcare-associated infections, particularly in intensive care units, contributing to significant morbidity and mortality due to its multidrug resistance and ability to persist in clinical environments. This study aimed to investigate the phenotypic and genomic characteristics of all multidrug-resistant A. baumannii isolates collected between January and June 2022 from two tertiary care hospitals in Thessaloniki, Greece. A total of 40 isolates were included. All isolates exhibited resistance to colistin; however, none harbored the mcr-1 to mcr-9 genes, as confirmed by polymerase chain reaction (PCR). PCR-based screening for virulence-associated genes revealed high prevalence rates of basD (100%), pld (95%), csuE (87.5%), and bap (77.5%). In contrast, ompA and pglC were not detected. Twitching motility ranged from 2 to 50 mm, with 25% of the isolates classified as non-motile and 20% as highly motile. Swarming motility was observed in all strains. Additionally, all isolates demonstrated positive α-hemolysis, suggesting a potential virulence mechanism involving tissue damage and iron acquisition. Pulsed-field gel electrophoresis (PFGE) revealed significant genomic diversity among the isolates, indicating a low likelihood of patient-to-patient or clonal transmission within the hospital setting. These findings highlight the complex relationship between antimicrobial resistance and virulence in clinical A. baumannii isolates and emphasize the urgent need for robust infection control strategies and continued microbiological surveillance. Full article

(This article belongs to the Special Issue Acinetobacter baumannii: An Emerging Pathogen)

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10 pages, 1165 KiB

Open AccessBrief Report

Serum Amyloid A3 Expression Is Enhanced by Gram-Negative Bacterial Stimuli in Bovine Endometrial Epithelial Cells

by Kazuha Aoyagi, Keishi Owaki, Hiroki Sakai, Ayaka Okada and Yasuo Inoshima

Pathogens 2025, 14(8), 729; https://doi.org/10.3390/pathogens14080729 - 23 Jul 2025

Abstract

Bovine endometritis is a common postpartum disease that significantly impairs reproductive performance and reduces economic sustainability in dairy and beef cattle. It is primarily caused by gram-negative and -positive bacteria, triggering strong inflammatory responses in the endometrium. Serum amyloid A (SAA) is an [...] Read more.

Bovine endometritis is a common postpartum disease that significantly impairs reproductive performance and reduces economic sustainability in dairy and beef cattle. It is primarily caused by gram-negative and -positive bacteria, triggering strong inflammatory responses in the endometrium. Serum amyloid A (SAA) is an acute-phase protein and precursor of amyloid A (AA) in AA amyloidosis. In cattle, multiple SAA isoforms have been identified; however, the biological functions of SAA3 remain unclear. Hence, this study investigated the role of SAA3 in bovine endometrial epithelial cells (BEnEpCs) following stimulation with gram-negative or -positive bacterial antigens. BEnEpCs were treated with lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and, subsequently, the expression levels of SAA3 and SAA1 mRNA were compared by real-time PCR. To further investigate protein-level changes, immunocytochemistry (ICC) was performed to assess the expressions of SAA3 and SAA1. These analyses revealed that SAA3 mRNA expression was significantly enhanced by LPS and LTA, whereas SAA1 mRNA remained undetectable or showed only minimal responsiveness. Notably, only SAA3 protein expression increased in response to stimulation. These results indicate that SAA3 plays a crucial role in the innate immune response of BEnEpCs against gram-negative bacteria. Our in vitro findings may facilitate understanding of the innate immune activity in bovine uterus. Full article

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19 pages, 1094 KiB

Open AccessReview

Global Perspectives on Rabies Control and Elimination: A Scoping Review of Dog Owners’ Knowledge, Attitudes, and Practices

by Moumita Das, Valeriia Yustyniuk, Andres M. Perez and Maria Sol Perez Aguirreburualde

Pathogens 2025, 14(8), 728; https://doi.org/10.3390/pathogens14080728 - 23 Jul 2025

Abstract

Rabies is a fatal but entirely vaccine-preventable disease, with the highest risk in areas where free-roaming domestic dogs are prevalent. Understanding dog owners’ knowledge, attitudes, and practices (KAP) is crucial for shaping effective rabies control strategies. This scoping review aimed to synthesize global [...] Read more.

Rabies is a fatal but entirely vaccine-preventable disease, with the highest risk in areas where free-roaming domestic dogs are prevalent. Understanding dog owners’ knowledge, attitudes, and practices (KAP) is crucial for shaping effective rabies control strategies. This scoping review aimed to synthesize global evidence from studies evaluating dog owners’ KAP to identify behavioral factors relevant to rabies prevention and control. A systematic literature search was conducted using PubMed, Web of Science, and Scopus, covering the period from 2012 to 2025. Seventy full-text articles were included based on predefined criteria. The findings reveal substantial gaps in dog owners’ knowledge, beliefs, and behaviors regarding rabies prevention. While general awareness of rabies is high among dog owners, their knowledge about transmission, clinical signs, and the fatal nature of the disease is inconsistent, with significant variability across studies. The vaccination uptake also varied widely across studies, ranging from less than 1% to over 90%, with no study reporting full coverage. Furthermore, a strong positive correlation was found between vaccination practice and the awareness of vaccine benefits (r = 0.69, p = 0.004). Common barriers to vaccination include lack of information, vaccine accessibility, distance to clinics, and personal constraints. These insights underscore the importance of early and targeted communication about vaccination campaigns. Future research should focus on periodically evaluating KAP before and after interventions to better inform rabies control efforts. Full article

(This article belongs to the Special Issue Current Challenges in Veterinary Virology)

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14 pages, 5085 KiB

Open AccessCommunication

Development and Validation of a Histologic Respiratory Index (HRI) in Poultry

by Tamer A. Sharafeldin, Mohamed Selim, Noreen Bashir and Sunil K. Mor

Pathogens 2025, 14(8), 727; https://doi.org/10.3390/pathogens14080727 - 23 Jul 2025

Abstract

Respiratory viral diseases infecting poultry lead to variable lesions in the respiratory organs, including nasal sinuses, trachea, lungs, and air sacs. Additional involvement of eyelids/conjunctiva was reported. The distribution and the intensity of lesions depend on multiple factors, including virulence, the host’s immunity, [...] Read more.

Respiratory viral diseases infecting poultry lead to variable lesions in the respiratory organs, including nasal sinuses, trachea, lungs, and air sacs. Additional involvement of eyelids/conjunctiva was reported. The distribution and the intensity of lesions depend on multiple factors, including virulence, the host’s immunity, and secondary or concurrent infections. It may be challenging to detect remarkable lesions during experimental infections conducted in a controlled environment because some viruses fail to produce the intense lesions seen in field cases. This creates a challenge in developing a reliable model to study pathogenicity or vaccine efficacy experimentally. The development of the proposed histologic respiratory index (HRI) aims to help monitor the least microscopic changes that can be scored, thereby creating an objective and accurate grading of lesions in experimentally infected birds. HRI scores the changes in eyelids/conjunctiva and respiratory mucosa, including hyperplasia, metaplasia, inflammatory cellular infiltration in the submucosa, including lymphocytes and heterophils, and vascular changes (vasculitis) in nasal sinuses, trachea, and lungs. The score was validated in birds infected experimentally with avian metapneumovirus (aMPV) and low pathogenic avian influenza (LPAI-H4N6). The HRI reliably graded higher scores in the respiratory organs of experimentally infected birds compared with non-infected control ones. The HRI is the first of its type with poultry viral respiratory pathogens and it was initially proven to be a reliable in pathogenicity and vaccine trials of certain poultry respiratory viral diseases. Full article

(This article belongs to the Section Viral Pathogens)

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16 pages, 3400 KiB

Open AccessArticle

Antimicrobial Resistance Genotypes and Mobile Genetic Elements of Poultry-Derived Escherichia coli: A Retrospective Genomic Study from the United States

by Sohyun Cho, Hazem Ramadan, Lari M. Hiott, Jonathan G. Frye and Charlene R. Jackson

Pathogens 2025, 14(8), 726; https://doi.org/10.3390/pathogens14080726 - 23 Jul 2025

Abstract

The presence of antibiotic resistance in commensal bacteria may be an influential factor in the persistence of resistance in pathogens. This is especially critical for Escherichia coli that consumers may be exposed to through the consumption of uncooked meat. In this study, E. [...] Read more.

The presence of antibiotic resistance in commensal bacteria may be an influential factor in the persistence of resistance in pathogens. This is especially critical for Escherichia coli that consumers may be exposed to through the consumption of uncooked meat. In this study, E. coli isolates previously recovered from poultry in the US between 2001 and 2012 were whole-genome sequenced to identify their antibiotic resistance genes and mobile genetic elements. The genomes of 98 E. coli isolates from poultry carcass rinsates and 2 isolates from poultry diagnostic samples with multidrug resistance or potential extended-spectrum β-lactam (ESBL)-producing phenotypes as well as the genetic variabilities among the E. coli were assessed. All E. coli isolates were positive for at least one antibiotic resistance gene and plasmid replicon, with 37 resistance genes and 27 plasmid replicons detected among the isolates. While no ESBL genes were detected, bla_CMY-2 was the most common β-lactamase gene, and bla_TEM and bla_CARB-2 were also identified. Most isolates (95%) harbored at least one intact phage, and as many as seven intact phages were identified in one isolate. These results show the occurrence of antibiotic resistance genes and mobile genetic elements in these 100 poultry-associated E. coli isolates, which may be responsible for the resistance phenotypes exhibited by the isolates. This retrospective study also enables comparisons of resistance genes and mobile genetic elements from more recent E. coli isolates associated with poultry to aid in understanding the trends of both antibiotic resistance phenotypes and genotypes in the poultry setting over time. Full article

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12 pages, 1531 KiB

Open AccessArticle

Challenges and Achievements in the In Vitro Culture of Balantioides coli: Insights into the Excystation Process

by Alexandra Ibañez-Escribano, Lorena Esteban-Sánchez, Cristina Fonseca-Berzal, Francisco Ponce-Gordo and Juan José García-Rodríguez

Pathogens 2025, 14(8), 725; https://doi.org/10.3390/pathogens14080725 - 23 Jul 2025

Abstract

Balantioides coli is the only ciliate currently described as an intestinal parasite of humans, although it can also infect other animals, particularly pigs. Its in vitro cultivation remains challenging, and no axenic culture system is currently available. Cultures are initiated by adding small [...] Read more.

Balantioides coli is the only ciliate currently described as an intestinal parasite of humans, although it can also infect other animals, particularly pigs. Its in vitro cultivation remains challenging, and no axenic culture system is currently available. Cultures are initiated by adding small amounts of feces containing cysts or trophozoites to the culture medium. Implantation success is lower when starting from cysts, and the mechanisms and early events of excystation remain poorly understood. In this study, we describe the sequence of events involved in excystation and identify factors potentially important for culture establishment. Cysts were obtained from orangutan feces and genetically confirmed as B. coli. Only viable cysts, determined by trypan blue or methylene blue exclusion, were used. After artificial digestion with pepsin and trypsin, cysts were incubated at 28 °C for up to 72 h in DMEM supplemented with L-glutamine, yeast extract, fetal bovine serum, and starch granules. Excystation began with a fissure in the cyst wall, allowing for bacterial entry. This appeared to stimulate the trophozoites, the increased motility of which progressively weakened and ruptured the wall, allowing for their emergence. Wall rupture and bacterial entry were critical for activation., whereas starch type had no apparent influence. Excystation occurred within the first hours; otherwise, cysts degenerated. Full article

(This article belongs to the Section Parasitic Pathogens)

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13 pages, 901 KiB

Open AccessArticle

Efficacy and Safety of an Oxalic Acid and Glycerin Formulation for Varroa destructor Control in Honey Bee Colonies During Summer in a Northern Climate

by Daniel Thurston, Les Eccles, Melanie Kempers, Daniel Borges, Kelsey Ducsharm, Lynae Ovinge, Dave Stotesbury, Rod Scarlett, Paul Kozak, Tatiana Petukhova, Ernesto Guzman-Novoa and Nuria Morfin

Pathogens 2025, 14(8), 724; https://doi.org/10.3390/pathogens14080724 - 22 Jul 2025

Abstract

Effective control of the parasitic mite Varroa destructor in honey bee (Apis mellifera) colonies relies on integrated pest management (IPM) strategies to prevent mite populations from reaching economic injury levels. Formulations of oxalic acid combined with glycerin may provide a viable [...] Read more.

Effective control of the parasitic mite Varroa destructor in honey bee (Apis mellifera) colonies relies on integrated pest management (IPM) strategies to prevent mite populations from reaching economic injury levels. Formulations of oxalic acid combined with glycerin may provide a viable summer treatment option in continental Northern climates. This study evaluated the efficacy of oxalic acid and glycerin strips compared to oxalic acid dribble and 65% formic acid when applied in mid-August. Mite levels and colony health parameters were assessed, and honey samples from oxalic acid-treated colonies were analyzed for residue levels. Results showed that the oxalic acid and glycerin strips had a moderate acaricidal efficacy (55.8 ± 3.2%), which was significantly higher than those of 65% formic acid (42.6 ± 3.2%) and oxalic acid dribble (39.5 ± 4.3%), which did not differ between them, suggesting potential for summer mite control. No significant adverse effects on cluster size, worker mortality, queen status, or colony survival were observed. Oxalic acid and glycerin increased the proportion of spotty brood patterns at early timepoints after treatment, but recovery was noted after 45 days of starting the treatment. Similar effects on brood were observed with 65% formic acid 14 days after starting the treatment, with recovery by 28 and 45 days after starting the treatment. No significant differences in oxalic acid residues in honey from the control and treatment colonies were found. Oxalic acid and glycerin strips might help control varroa mite populations, delaying their exponential growth and helping reduce economic losses for beekeepers, but this treatment should be considered as part of an IPM strategy and not a stand-alone method for V. destructor control. Full article

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

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14 pages, 2694 KiB

Open AccessArticle

Functional Amyloids in Adhesion of Non-albicans Candida Species

by Melissa C. Garcia-Sherman, Safraz A. Hamid, Desmond N. Jackson, James Thomas and Peter N. Lipke

Pathogens 2025, 14(8), 723; https://doi.org/10.3390/pathogens14080723 - 22 Jul 2025

Abstract

Candida fungal species are the most common fungal opportunistic pathogens. Their ability to form antifungal resistant biofilms contributes to their increasing clinical frequency. These fungi express surface-anchored adhesins including members of the Als family. These adhesins mediate epithelial adhesion, aggregation, and biofilm formation. [...] Read more.

Candida fungal species are the most common fungal opportunistic pathogens. Their ability to form antifungal resistant biofilms contributes to their increasing clinical frequency. These fungi express surface-anchored adhesins including members of the Als family. These adhesins mediate epithelial adhesion, aggregation, and biofilm formation. Many of the adhesins contain cross-β core sequences that form amyloid-like protein aggregates on the fungal surface. The aggregates mediate high-avidity bonding that contributes to biofilm establishment and persistence. Accordingly, autopsy sections from individuals with candidiasis and other mycoses have amyloids within abscesses. An amyloid-forming peptide containing a sequence from Candida albicans Als5 bound to C. albicans, C. tropicalis, and C. parapsilosis. C. albicans and C. tropicalis aggregated with beads coated with serum albumin, and the aggregates stained with the amyloid-binding dye thioflavin T. Additionally, an Als5-derived amyloid-inhibiting peptide blocked cell aggregation. The amyloid-inhibiting peptide also blocked C. albicans, C. tropicalis, and C. parapsilosis adhesion to monolayers of FaDu epithelial cells. These results show the involvement of amyloid-like interactions in pathogenesis in several Candida species. Full article

(This article belongs to the Special Issue The Dark Side of Fungi: Exploring Pathogenesis in Candida and Other Fungal Pathogens)

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29 pages, 4944 KiB

Open AccessArticle

Distinct Immunological Landscapes of HCMV-Specific T Cells in Bone Marrow and Peripheral Blood

by Sarah E. Jackson, Rosie Fairclough, Veronika Romashova, Georgina Okecha and Mark R. Wills

Pathogens 2025, 14(8), 722; https://doi.org/10.3390/pathogens14080722 - 22 Jul 2025

Abstract

Human cytomegalovirus (HCMV) establishes lifelong latency in the host, with the bone marrow (BM) CD34+ cells serving as a key reservoir. To investigate tissue-specific immune responses to CMV, we analysed paired peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMNCs) from [...] Read more.

Human cytomegalovirus (HCMV) establishes lifelong latency in the host, with the bone marrow (BM) CD34+ cells serving as a key reservoir. To investigate tissue-specific immune responses to CMV, we analysed paired peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMNCs) from HCMV-seropositive donors using multiparametric flow cytometry and cytokine FluroSpot assays. We assessed immune cell composition, memory T cell subsets, cytokine production, cytotoxic potential, activation marker expression, and checkpoint inhibitory receptor (CIR) profiles, both ex vivo and following stimulation with lytic and latent HCMV antigens. BMMNCs were enriched in CD34+ progenitor cells and exhibited distinct T cell memory subset distributions. HCMV-specific responses were compartmentalised: IFN-γ responses predominated in PBMCs following lytic antigen stimulation, while IL-10 and TNF-α responses were more prominent in BMMNCs, particularly in response to latent antigens. US28-specific T cells in the BM showed elevated expression of CD39, PD-1, BTLA, CTLA-4, ICOS, and LAG-3 on CD4+ T cells and increased expression of PD-1, CD39, BTLA, TIGIT, LAG-3, and ICOS on CD8+ T cell populations, suggesting a more immunoregulatory phenotype. These findings highlight functional and phenotypic differences in HCMV-specific T cell responses between blood and bone marrow, underscoring the role of the BM niche in shaping antiviral immunity and maintaining viral latency. Full article

(This article belongs to the Special Issue Understanding Human Cytomegalovirus Pathogenesis: Evidence from the Clinic and Laboratory Models)

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9 pages, 403 KiB

Open AccessBrief Report

Persistence of Infectivity of Different Enteroviruses on a Surrogate Fomite: Correlation with Clinical Case Incidence

by Charles P. Gerba, M. Khalid Ijaz, Raymond W. Nims and Stephanie A. Boone

Pathogens 2025, 14(8), 721; https://doi.org/10.3390/pathogens14080721 - 22 Jul 2025

Abstract

Enteroviruses of the Picornaviridae family are transmitted primarily by the fecal–oral route. Transmission may occur following hand contact with contaminated fomites and subsequent ingestion of virus conveyed to the mouth by the contaminated hand. The persistence of these viruses on fomites likely plays [...] Read more.

Enteroviruses of the Picornaviridae family are transmitted primarily by the fecal–oral route. Transmission may occur following hand contact with contaminated fomites and subsequent ingestion of virus conveyed to the mouth by the contaminated hand. The persistence of these viruses on fomites likely plays a role in this transmission scenario. Six echoviruses (1, 2, 3, 5, 6, and 7) that cause frequently reported clinical cases in the United States were studied, along with poliovirus type 1 vaccine strain LSc-2ab. The infectivity half-lives of the enteroviruses deposited on vinyl tile coupons in a 10% fecal solution ranged from 1.7 to 12.6 h. The echovirus serotypes most commonly associated with reported infections persisted longer on the vinyl tiles than the less commonly reported types. This increased persistence on surfaces may favor the transmission of these echoviruses through the fecal–oral route. These results inform the future selection of appropriate model enteroviruses for challenging newly formulated and eco-friendly disinfectants or other strategies in infection prevention and control for enteroviruses. Full article

(This article belongs to the Special Issue Enteroviruses: From Host Interactions to Molecular Epidemiology and Clinical Impacts)

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