Search Results (103)

Background/Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has rapidly evolved, giving rise to multiple Variants of Concern—including Alpha, Beta, Gamma, Delta, and Omicron—which emerged independently across different regions. Licensed COVID-19 vaccines primarily target the highly mutable spike protein, resulting in reduced efficacy due to immune escape by emerging variants. Previously, we developed a live attenuated Francisella tularensis LVS ΔcapB single-vector platform COVID-19 vaccine, rLVS ΔcapB/MN, expressing the conserved membrane (M) and nucleocapsid (N) proteins from the early SARS-CoV-2 WA-01/2020 strain. In this study, we evaluate the efficacy of rLVS ΔcapB/MN and an enhanced version, rLVS ΔcapB::RdRp/MN, which additionally expresses the conserved RNA-dependent RNA polymerase (RdRp) protein from the same strain, in a hamster model. Methods: Both vaccine candidates were administered orally or intranasally to golden Syrian hamsters (equal numbers of males and females) and evaluated against intranasal challenge with SARS-CoV-2 Delta (B.1.617.2-AY.1) and Omicron (BA.5) variants. Results: Vaccinated animals developed robust, TH1-biased IgG responses specific to the nucleocapsid protein. Following SARS-CoV-2 challenge, immunized hamsters exhibited reduced weight loss, lower oropharyngeal and lung viral titers, and improved lung pathology scores compared with unvaccinated controls. Conclusion: These findings support the potential of this universal vaccine to provide broad protection against current and future SARS-CoV-2 variants, with minimal need for updating. Full article

This study aimed to evaluate microbial growth, pathogen presence, and histamine production in rainbow trout and mackerel stored on flaked ice over their shelf life. A total of 72 fish samples (rainbow trout and mackerel) were analyzed across four storage intervals (day 1, 3, 9, 12/11) on flaked ice. TVC increased from 2.59 to 5.04 log cfu/g in rainbow trout and from 3.18 to 4.88 log cfu/g in mackerel over the storage period. Significant increases were observed in Pseudomonas, Aeromonas, and Enterobacteriaceae populations, especially after the ninth day. Microbial identification revealed spoilage-associated bacteria, such as Pseudomonas fluorescens and Aeromonas salmonicida, as well as opportunistic pathogens, including Francisella tularensis, Yersinia spp., and Chromobacterium violaceum. Histamine levels rose with storage time but remained below toxic thresholds (<200 mg/kg), peaking at 1.56 mg/kg in trout and 1.87 mg/kg in mackerel. A strong positive correlation was found between TVC and histamine levels (Pearson’s r = 0.85 for trout, 0.82 for mackerel). Proper hygiene and storage are crucial, and consumption is recommended before day 9 of storage on flaked ice. Hygiene measures remain essential to minimize contamination risks and preserve product safety. Full article

A 47-year-old woman presented with fever, fatigue, night sweats and inguinal glandular swelling following a tick bite. Weeks of diagnostic uncertainty followed, and a lymph node biopsy was sent to be investigated for tularemia and pathology. An ¹⁸F-FDG PET/CT scan was performed due to a suspicion of malignant lymphoma. The scan revealed high metabolic activity in the left inguinal region, which was compatible with abscesses. The diagnosis of glandular tularemia was established on a positive PCR for Francisella tularensis (F. tularensis) and positive F. tularensis serology. This case highlights the challenges of diagnosing tularemia and illustrates the role of imaging. Full article

Background: Tularemia is a rare zoonosis caused by the bacterium Francisella tularensis. In the head and neck region, it can manifest as cervical lymphadenopathy. Despite intensive therapy with various antibiotics, there is often a prolonged medical course. Methods: In this paper, all documented cases of tularemia in the head and neck region at the Medical University of Innsbruck (Austria) are analyzed and the results compared with the literature. A retrospective analysis of all patients diagnosed with tularemia at the Department of Otorhinolaryngology-Head and Neck Surgery, Medical University of Innsbruck (Austria), was performed. Tularemia was diagnosed using a serologic agglutination antibody test. Results: Thirteen patients with tularemia presented at the Department of Otorhinolaryngology-Head and Neck Surgery, Medical University of Innsbruck (Austria), between 2010 and 2024. In 10 patients (10/13; 77%), animal contact or an insect bite was the suspected cause. The mean time from the onset of the first symptoms to diagnosis was 36 ± 15 days. The therapy took a mean of 5 ± 2 months until the last follow-up. On average, the patients were treated with 4 ± 1 different antibiotics. The median duration of hospital stay was 13 days (range: 0–36). In addition, a median of 9 (range: 2–20) further outpatient check-ups with several neck ultrasounds were carried out. Also, 10 patients (10/13; 77%) received a diagnostic and/or therapeutic surgical intervention. Conclusions: Tularemia is a rare infectious disease with a prolonged diagnostic and therapeutic course. Screening for tularemia should be performed in cases of cervical lymphadenopathy, especially if empirical antibiotic treatment has been ineffective or if there is a specific medical history. Full article

This pilot study investigated the presence of potentially zoonotic microorganisms in bat species from Kopaonik National Park, Serbia. A total of 40 individuals from 12 bat species were sampled and screened using microbiological and molecular methods. Salmonella spp., Chlamydia spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Lyssavirus, Filoviridae, henipaviruses, and SARS-CoV-2 were not detected in any bats. Coronavirus genomes were confirmed in four bats—one Myotis brandtii, two Myotis daubentonii, and one Myotis cf. mystacinus. Sequence analysis identified the presence of alphacoronavirus genomes with high similarity to strains previously found in Europe. Mycoplasma spp. genomes were found in 18 bats (45%), and Rickettsia spp. were detected in five bats (12.5%), although species-level identification was not possible. The findings highlight the presence of certain bacteria and viruses in bats that could have implications for public health, especially in areas with close human–wildlife interaction. Although no direct evidence of high-risk pathogens was found, the results support the importance of continued surveillance and ecological studies on bats, given their role as potential reservoirs. Monitoring bat-associated microorganisms is essential to better understand possible transmission routes and improve the prevention of emerging zoonotic diseases. Full article

Wild ruminants often harbor pathogens transmissible to other animals and humans, but their epidemiological role is not always defined for all microorganisms. In this survey, spleens, kidneys, and hearts sampled from 162 fallow deer (Dama dama) were subjected to molecular analyses to detect bacterial (Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Brucella spp., Chlamydia abortus, Coxiella burnetii, Francisella tularensis, Leptospira spp.) and protozoan (piroplasms, Neospora caninum, Toxoplasma gondii) pathogens. Five (3.08%) spleens were positive for A. phagocytophilum, and twelve (7.40%) spleens were positive for Theileria cervi. The remaining pathogens investigated were not detected, and no coinfections were found. The analyzed animals do not seem to have a relevant role in the spreading of these pathogens; however, monitoring is pivotal to understand the epidemiological scenarios and take appropriate preventive measures in areas frequently visited by people. Full article

North American beavers (Castor canadensis) are semi-aquatic rodents recognized as keystone species because they increase the diversity of freshwater ecosystems. This study aimed to characterize the mortality and pathological findings in free-ranging beavers in California and, based on these results, identify potential threats to freshwater ecosystems. This study included 18 beavers submitted for postmortem examination at the California Animal Health and Food Safety Laboratory, UC Davis, between 2008 and 2024. Gross and microscopic examinations, and bacteriological, parasitological, immunohistochemical, and molecular techniques, were used as tools to diagnose the cause of death/reason for euthanasia and comorbidities in the beavers. Baylisascaris spp.-associated or -suspected encephalitis was the most prevalent (9/18, 50%) cause of mortality/reason for euthanasia, followed by bacterial infections in six individuals. In these six animals, bacterial bronchopneumonia was diagnosed in two (Staphylococcus aureus and a mix of Gram-negative and -positive bacterial infection) and Listeria monocytogenes encephalitis, bacterial myofascitis (Aeromonas bestiarum and Pasteurella multocida), bacterial encephalitis (Acinetobacter towneri), and tularemia (Francisella tularensis) were diagnosed in one beaver each. Three animals died or were euthanized due to non-infectious causes, including motor vehicle trauma, squamous cell carcinoma, and capture cardiomyopathy. Endoparasitism was the main comorbidity, including granulomatous hepatitis caused by a suspected capillarid species, cerebral toxoplasmosis, Giardia infection, gastric nematodiasis, and cecal trematodiasis. In California, beavers are exposed to various pathogens that represent threats to humans, domestic animals, and wildlife. Since the interspecies transmission of these pathogens occurs in rivers, streams, lakes, and ponds, we suggest that studying beaver health can reflect freshwater ecosystem health. This study also indicates that the translocation of beavers to new areas without consideration and/or mitigation represents a potential risk of pathogen introduction. Full article

This study proposes a risk analysis model based on the principles of ISO 31000 and decision theory for biological agents with potential for offensive use in Brazil. Bibliographic research was conducted on the main models already published on the subject. The German risk classification system was adopted as the main reference because it is adjustable and adopts a semiquantitative approach. After translating and adapting this model to the Brazilian context, the analytic hierarchy process (AHP) was applied to generate a hierarchical tree with criteria and subcriteria to be considered in the risk assessment. A questionnaire was administered to a group of experts to evaluate the relevant criteria and subcriteria and the risks related to three biological agents (Bacillus anthracis, Yersinia pestis, and Francisella tularensis), which were selected in an exploratory manner to exemplify the application of the model. According to the results, the criterion of impact of a biological attack was given greater importance when compared with the probability of occurrence of this event. According to the subcriteria, the greatest weight was attributed to human health when compared with agriculture, the environment, or the economy. Finally, in the evaluation of the three pathogens, B. anthracis was identified as having the highest risk for offensive use in Brazil. This research is focused on a practical approach and can be implemented by competent agencies to foster their capacity for biological defense by determining which pathogens represent the greatest risk to society. Full article

The COVID-19 and mpox crisis has reminded the world of the potentially catastrophic consequences of biological agents. Aside from the natural risk, biological agents can also be weaponized or used for bioterrorism. Dissemination in a population or among livestock could be used to destabilize a nation by creating a climate of terror, by negatively impacting the economy and undermining institutions. The Centers for Disease Control and Prevention (CDC) classify biological agents into three categories (A or Tier 1, B and C) according to the risk they pose to the public and national security. Category A or Tier 1 consists of the six pathogens with the highest risk to the population (Bacillus anthracis, Yersinia pestis, Francisella tularensis, botulinum neurotoxins, smallpox and viral hemorrhagic fevers). Several medical countermeasures, such as vaccines, antibodies and chemical drugs, have been developed to prevent or cure the diseases induced by these pathogens. This review presents an overview of the primary medical countermeasures, and in particular, of the antibodies available against the six pathogens on the CDC’s Tier 1 agents list, as well as against ricin. Full article

Rodents play a significant role in the transmission of zoonotic diseases; anthropization has increased human contact with these animals, vectors of infectious agents. However, the processes driving parasitism of hosts remains poorly understood. Yersinia pestis, Rickettsia spp., and Francisella tularensis are three infectious agents transmitted to humans through ectoparasites, with rodents serving as the primary reservoirs. To explore the relationship between both intrinsic and extrinsic factors on host pathogen status, we evaluated heteromyid rodents in the Chihuahuan desert (ChD). From December 2022 to May 2023, we sampled 213 rodents at three locations with different anthropization levels. A total of 103 rodent blood samples, 84 organ samples, and 204 collected ectoparasites were analyzed for molecular detection of infectious agents (Y. pestis, Rickettsia spp., and F. tularensis) with PCR. We captured seven species of rodents (Dipodomys ordii, D. merriami, D. spectabilis, Chaetodipus hispidus, Ch. eremicus, Perognathus flavus, and P. flavescens) and identified one tick (Rhipicephalus sanguineus), two fleas (Meringis altipecten and M. dipodomys) and one louse (Fahrenholzia spp.). Molecular analyses yielded positive for Y. pestis, Rickettsia spp., and negative for F. tularensis. We then modelled the pathogen status as a function of intrinsic (body condition and sex) and extrinsic factors (locality, anthropization level, season, sample type, and parasite-infestation status). We found that non-parasite-infested individuals with better body condition have a higher probability of pathogen infection. Furthermore, we observed that blood samples had a higher probability of detecting pathogen-infected individuals, as compared to spleen or liver samples. Our results offer important insights into host–pathogen interactions and the role of body condition in the pathogen status. Full article

Francisella tularensis is a vector-borne zoonotic bacterium that causes tularemia, a disease of great importance for animal and public health. Although wild lagomorphs are considered one of the major reservoirs of this bacterium, information about the circulation of F. tularensis in European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) populations in Europe is still very limited. In Spain, F. tularensis is present in northern central regions, with recurrent outbreaks occurring annually. However, southern Spain has been neglected in terms of monitoring and research. Thus, the aim of this study was to investigate the occurrence of F. tularensis in wild lagomorphs in the Mediterranean ecosystems of southern Spain through using molecular techniques. Spleen samples from 774 European wild rabbits and 178 Iberian hares were collected on 135 hunting grounds between the 2017/2018 and 2022/2023 hunting seasons. A primer set targeting the 16S rRNA gene was used for the detection of F. tularensis DNA. None of the 952 (0.0%; 95%CI: 0.0–0.4) wild lagomorphs sampled showed presence of F. tularensis DNA. Our finding indicates the absence or very low circulation of the bacterium in wild rabbit and Iberian hare populations in the Mediterranean ecosystems of southern Spain, denoting a limited risk of transmission of this pathogen from wild lagomorphs to other sympatric species, including human beings, in this Iberian region. Full article

Previous investigations have explored the involvement of wolves in parasitic and viral diseases, but data on the zoonotic bacteria are limited. The aim of this study was to assess the occurrence of bacterial zoonotic agents in 16 wolf (Canis lupus italicus) fecal samples collected in a protected area in Central Italy. Campylobacter spp., Salmonella spp., Yersinia spp., Listeria monocytogenes, and Shiga Toxin-Producing Escherichia coli (STEC) were investigated by culture, while polymerase chain reaction (PCR) was employed to detect Coxiella burnetii, Mycobacterium spp., Brucella spp., and Francisella tularensis. The presence of Extended Spectrum β-Lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae was also evaluated, using selective isolation media and detection of antimicrobial resistance genes. All samples were negative for Campylobacter spp., Salmonella spp., C. burnetii, Mycobacterium spp., Brucella spp., F. tularensis, and carbapenemase-producing Enterobacteriaceae. One sample tested positive for Yersinia aldovae and three for Yersinia enterocolitica BT1A. One L. monocytogenes (serogroup IIa) and one STEC, carrying the stx1 gene, were isolated. Two ESBL isolates were detected: one Serratia fonticola, carrying bla_FONA-3/6 gene, and one Escherichia coli, carrying bla_CTX-M-1 gene. Both ESBL isolates were resistant to different antimicrobials and therefore classified as multi-drug-resistant. Our data suggest that wolves are potential carriers of zoonotic bacteria and may contribute to the environmental contamination through their feces. Full article

Tularemia is an acute infectious disease classified as a natural focal infection, requiring continuous monitoring of both human and animal morbidity, as well as tracking of pathogen circulation in natural reservoirs and vectors. These efforts are essential for a comprehensive prevention and containment strategy. The causative agent, Francisella tularensis, comprises three subspecies—tularensis, holarctica, and mediasiatica—which differ in their geographic distribution and virulence. The ability to directly detect the pathogen and differentiate between subspecies has enhanced diagnostics and allowed a more accurate identification of circulation areas. Real-time PCR protocols for identification of F. tularensis subspecies tularensis and holarctica have been developed, utilizing specific primers and probes that target unique genomic regions. In this study, we present the development of a new real-time PCR assay for the detection of Francisella spp. and differentiation of F. tularensis subsp. mediasiatica. The specificity of the assay was tested on DNA from 86 bacterial species across 31 families unrelated to Francisella spp., as well as on DNA collections of F. tularensis subsp. mediasiatica and F. tularensis subsp. holarctica. The limit of detection (LOD95%) for real-time PCR in detecting Francisella spp. was 0.297 fg (0.145 genomic equivalents, GE) for holarctica DNA and 0.733 fg (0.358 GE) for mediasiatica DNA. The LOD95% for subspecies differential identification of mediasiatica was 8.156 fg (3.979, GE). The high sensitivity and specificity of these developed protocols enable direct detection of pathogens in biological and environmental samples, thereby improving the efficiency of tularemia surveillance in Kazakhstan. Full article

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π−π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two–five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 10⁵ to 0.01 nM and has good specificity. The biosensor’s detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R² = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs. Full article

Tularemia is an acute febrile disease caused by the Gram-negative bacillus Francisella tularensis. Based on genetic and phenotypic characteristics, three subspecies are distinguished: tularensis, holarctica, and mediasiatica. F. tularensis subsp. mediasiatica remains the least studied subspecies. Over the past decade, new foci of distribution of F. tularensis subsp. mediasiatica have been discovered in Russia (Siberia), expanding the possible distribution area by thousands of kilometers. This article provides whole genome single nucleotide polymorphism (wgSNP) and polymorphic tandem repeats (MLVA) analyses of 28 mediasiatica strains isolated between 1965 and 2004 in Kazakhstan. Despite high genetic homogeneity, MLVA with eleven loci (MLVA11) demonstrates a high discriminatory ability (diversity index, 0.9497). The topological structure of the trees based on wgSNP and MLVA is not comparable; however, clustering remains congruent for most outbreaks, with the exception of two strains from one outbreak that are identical in terms of wgSNP but differ at three tandem repeat loci. Based on wgSNP, the strains are assigned to one of the three currently known mediasiatica sublineages, lineage M.I, together with other historical strains maintained in collections in Russia and Sweden. wgSNP shows limited previously unknown genetic diversity, with the M.I lineage size being only 118 SNPs. The wgSNP genotype is not strongly correlated with year and place of isolation. Full article