Fleas (Siphonaptera) as obligate, blood-feeding ectoparasites are, together with ticks, hosted by small mammals and can transmit causative agents of serious infections. This study aimed to determine and characterize the presence and genetic diversity of Bartonella, Rickettsia, and apicomplexan parasites (Babesia, Hepatozoon) in fleas feeding on small mammals from three different habitat types (suburban, natural, and rural) in Slovakia. The most common pathogen in the examined fleas was Bartonella spp. (33.98%; 95% CI: 30.38–37.58), followed by Rickettsia spp. (19.1%; 95% CI: 16.25–22.24) and apicomplexan parasites (4.36%; 95% CI: 2.81–5.91). Bartonella strains belonging to B. taylorii, B. grahamii, B. elizabethae, Bartonella sp. wbs11, and B. rochalimae clades were identified in Ctenophthalmus agyrtes, C. congener, C. assimilis, C. sciurorum, C. solutus, C. bisoctodentatus, Palaeopsylla similis, Megabothris turbidus, and Nosopsyllus fasciatus within all habitats. The presence of Rickettsia helvetica, R. monacensis, and rickettsiae, belonging to the R. akari and R. felis clusters, and endosymbionts with a 96–100% identity with the Rickettsia endosymbiont of Nosopsyllus laeviceps laeviceps were also revealed in C. agyrtes, C. solutus, C. assimilis, C. congener, M. turbidus, and N. fasciatus. Babesia and Hepatozoon DNA was detected in the fleas from all habitat types. Hepatozoon sp. was detected in C. agyrtes, C. assimilis, and M. turbidus, while Babesia microti was identified from C. agyrtes, C. congener, and P. similis. The present study demonstrated the presence of zoonotic pathogens in fleas, parasitizing the wild-living small mammals of southwestern and central Slovakia and widens our knowledge of the ecology and genomic diversity of Bartonella, Rickettsia, Babesia, and Hepatozoon. Full article

The Bacillus cereus (B. cereus) group is a widespread foodborne pathogen with a persistent ability to form biofilm, and with inherent resistance to traditional treatment in the food industry. Bacteriophages are a promising biocontrol agent that could be applied to prevent or eliminate biofilms formation. We have described, in this study, the isolation from sewage samples and preliminary characterization of bacteriophages that are active against the B. cereus group. The effectiveness of phage treatment for reducing B. cereus attachment and biofilms on stainless steel surfaces has been also assessed using three incubation periods at different titrations of each phage. Out of 62 phages isolated, seven showed broad-spectrum lytic action against 174 B. cereus isolates. All selected phages appeared to be of the Siphoviridae family. SDS-PAGE proved that two phages have a similar profile, while the remainder are distinct. All isolated phages have the same restriction pattern, with an estimated genome size of around 37 kb. The isolated bacteriophages have been shown to be effective in preventing biofilm formation. Reductions of up to 1.5 log₁₀ UFC/cm² have been achieved, compared to the untreated biofilms. Curative treatment reduced the bacterial density by 0.5 log₁₀ UFC/cm². These results support the prospect of using these phages as a potential alternative strategy for controlling biofilms in food systems. Full article

Bacillus cereus group bacteria containing the anthrax toxin genes can cause fatal anthrax pneumonia in welders. Two welder’s anthrax cases identified in 2020 were investigated to determine the source of each patient’s exposure. Environmental sampling was performed at locations where each patient had recent exposure to soil and dust. Samples were tested for the anthrax toxin genes by real-time PCR, and culture was performed on positive samples to identify whether any environmental isolates matched the patient’s clinical isolate. A total of 185 environmental samples were collected in investigation A for patient A and 108 samples in investigation B for patient B. All samples from investigation B were real-time PCR-negative, but 14 (8%) samples from investigation A were positive, including 10 from patient A’s worksite and 4 from his work-related clothing and gear. An isolate genetically matching the one recovered from patient A was successfully cultured from a worksite soil sample. All welder’s anthrax cases should be investigated to determine the source of exposure, which may be linked to their worksite. Welding and metalworking employers should consider conducting a workplace hazard assessment and implementing controls to reduce the risk of occupationally associated illnesses including welder’s anthrax. Full article

Anti-Spike monoclonal antibodies have been considered a promising approach to COVID-19 therapy. Unfortunately, the advent of resistant lineages jeopardized their effectiveness and prompted limitations in their clinical use. Change in the dominant variant can be fast to such an extent that, in the absence of timely medical education, prescribers can keep using these drugs for relatively long periods even in patients with resistant variants. Therefore, many patients could have been exposed to drugs with unlikely benefits and probable risks. We show here that about 20% of bamlanivimab+etesevimab, 30% of casirivimab+imdevimab, and 30% of sotrovimab courses were administered in Italy during periods in which a fully resistant variant was dominant. Additionally, for monoclonal antibody cocktails, the vast majority of usage occurred against variants for which one of the mAbs within the cocktail was ineffective. Given the high costs of these drugs and their potential side effects, it would be important to consider a frequent review of the appropriateness of these drugs and timely communication when the benefit/risk balance is no longer favorable. Full article

Candida auris is an emerging fungal pathogen that commonly causes nosocomial blood infections in the immunocompromised. Several factors make this pathogen a global threat, including its misidentification as closely related species, its ability to survive for weeks on fomites, and its resistance to commonly prescribed antifungal drugs, sometimes to all three classes of systemic antifungal drugs. These factors demonstrate a need for the development of novel therapeutic approaches to combat this pathogen. In the present study, the antifungal activities of 21 essential oils were tested against C. auris. Several essential oils were observed to inhibit the growth and kill C. auris, Candida lusitaniae, and Saccharomyces cerevisiae when in direct contact and at concentrations considered safe for topical use. The most effective essential oils were those extracted from lemongrass, clove bud, and cinnamon bark. These essential oils also elicited antifungal activity in gaseous form. The efficacies of formulations comprised of these three essential oils in combination with fluconazole, amphotericin B, flucytosine, and micafungin were explored. While synergism was neither observed with cinnamon bark oil nor any of the antifungal drugs, lemongrass oil displayed synergistic, additive, and indifferent interactions with select drugs. Formulations of clove bud oil with amphotericin B resulted in antagonistic interactions but displayed synergistic interactions with fluconazole and flucytosine. These essential oils and their combinations with antifungal drugs may provide useful options for surface disinfection, skin sanitization, and possibly even the treatment of Candida infections. Full article

Theileria orientalis causes losses to cattle producers in Eastern Asia, Oceania and, more recently, North America. One pathogenic genotype (Ikeda) has been sequenced to the chromosomal level, while only draft genomes exist for globally distributed Chitose and Buffeli genotypes. To provide an accurate comparative gene-level analysis and help further understand their pathogenicity, we sequenced isolates of the Chitose and Buffeli genotypes of T. orientalis using long-read sequencing technology. A combination of several long-read assembly methods and short reads produced chromosomal-level assemblies for both Fish Creek (Chitose) and Goon Nure (Buffeli) isolates, including the first complete and circular apicoplast genomes generated for T. orientalis. Comparison with the Shintoku (Ikeda) reference sequence showed both large and small translocations in T. orientalis Buffeli, between chromosomes 2 and 3 and chromosomes 1 and 4, respectively. Ortholog clustering showed expansion of ABC transporter genes in Chitose and Buffeli. However, differences in several genes of unknown function, including DUF529/FAINT-domain-containing proteins, were also identified and these genes were more prevalent in Ikeda and Chitose genotypes. Phylogenetics and similarity measures were consistent with previous short-read genomic analysis. The generation of chromosomal sequences for these highly prevalent T. orientalis genotypes will also support future studies of population genetics and mixed genotype infections. Full article

Hepatitis E is a major cause of acute liver disease in humans worldwide. The infection is caused by hepatitis E virus (HEV) which is transmitted in Europe to humans primarily through zoonotic foodborne transmission from domestic pigs, wild boar, rabbits, and deer. HEV belongs to the family Hepeviridae, and possesses a positive-sense, single stranded RNA genome. This agent usually causes an acute self-limited infection in humans, but in people with low immunity, e.g., immunosuppressive therapy or underlying liver diseases, the infection can evolve to chronicity and is able to induce a variety of extrahepatic manifestations. Pig and wild boar have been identified as the primary animal reservoir in Europe, and consumption of raw and undercooked pork is known to pose a potential risk of foodborne HEV infection. In this study, we analysed pig and wild boar liver, faeces, and muscle samples collected in 2019 in Mecklenburg-Western Pomerania, north-east Germany. A total of 393 animals of both species were investigated using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), conventional nested RT-PCR and sequence analysis of amplification products. In 33 animals, HEV RNA was detected in liver and/or faeces. In one individual, viral RNA was detected in muscle tissue. Sequence analysis of a partial open reading frame 1 region demonstrated a broad variety of genotype 3 (HEV-3) subtypes. In conclusion, the study demonstrates a high, but varying prevalence of HEV RNA in swine populations in Mecklenburg-Western Pomerania. The associated risk of foodborne HEV infection needs the establishment of sustainable surveillance and treatment strategies at the interface between humans, animals, and the environment within a One Health framework. Full article

Mycobacterium bovis (M. bovis) infection in wildlife, including lions (Panthera leo), has implications for individual and population health. Tools for the detection of infected lions are needed for diagnosis and disease surveillance. This study aimed to evaluate the Mabtech Cat interferon gamma (IFN-γ) ELISA^Basic kit for detection of native lion IFN-γ in whole blood samples stimulated using the QuantiFERON^® TB Gold Plus (QFT) platform as a potential diagnostic assay. The ELISA was able to detect lion IFN-γ in mitogen-stimulated samples, with good parallelism, linearity, and a working range of 15.6–500 pg/mL. Minimal matrix interference was observed in the recovery of domestic cat rIFN-γ in lion plasma. Both intra- and inter-assay reproducibility had a coefficient of variation less than 10%, while the limit of detection and quantification were 7.8 pg/mL and 31.2 pg/mL, respectively. The diagnostic performance of the QFT Mabtech Cat interferon gamma release assay (IGRA) was determined using mycobacterial antigen-stimulated samples from M. bovis culture-confirmed infected (n = 8) and uninfected (n = 4) lions. A lion-specific cut-off value (33 pg/mL) was calculated, and the sensitivity and specificity were determined to be 87.5% and 100%, respectively. Although additional samples should be tested, the QFT Mabtech Cat IGRA could identify M. bovis-infected African lions. Full article

There is limited information available on the Trichinella britovi (T. britovi) muscle larvae (ML) distribution in pig muscle and the humoral immune response of pigs infected with moderate doses of this parasite; therefore, this study investigated the infectivity of a Polish strain of T. britovi for pigs, the antibody response of this host to various doses of T. britovi, and the efficiency of three different commercial ELISA kits and an immunoblot assay at detecting anti-T. britovi IgG. No significant differences in terms of the infection level of particular muscles or of whole carcasses between pigs infected with 3000 and those infected with 5000 ML of T. britovi were observed. The highest intensity of T. britovi infection was reported in the diaphragm pillars. The larvae of T. britovi showed homogeneous distribution with respect to the muscle side. Statistically, specific IgG antibodies against excretory–secretory (ES) antigens of Trichinella ML were first detected by all ELISA protocols on day 36 post infection; however, individual pig results showed some differences between the three tests applied. A significant increase in the level of anti-T. britovi IgG was observed between days 36 and 41 post infection, and from day 45 until day 62 after T. britovi infection, production of these antibodies reached its plateau phase. No positive correlation was found between the anti-T. britovi IgG level and the larvae density in 15 different muscles. Sera of T. britovi-infected pigs showed reactivity with T. britovi ML ES antigens of 62, 55, and 52 kDa. The results provide novel information on spatial larvae distribution in muscles and the humoral immune response of pigs exposed to two different doses of a Polish strain of T. britovi, extend knowledge on serological diagnostic tools which may be introduced in veterinary practice for the detection of T. britovi infections in pig production, and offer practical solutions for meat hygiene inspectors in the field at sampling sites when examining pig carcasses for Trichinella. Full article

Anthrax has been feared for its high mortality in animals and humans for centuries. The etiologic agent is considered a potentially devastating bioweapon, and since 1876―when Robert Koch demonstrated that Bacillus anthracis caused anthrax―it has been considered the sole cause of the disease. Anthrax is, however, a toxin-mediated disease. The toxins edema toxin and lethal toxin are formed from protein components encoded for by the pXO1 virulence plasmid present in pathogenic B. anthracis strains. However, other members of the Bacillus cereus group, to which B. anthracis belongs, have recently been shown to harbor the pXO1 plasmid and produce anthrax toxins. Infection with these Bacillus cereus group organisms produces a disease clinically similar to anthrax. This suggests that anthrax should be defined by the exotoxins encoded for by the pXO1 plasmid rather than the bacterial species it has historically been associated with, and that the definition of anthrax should be expanded to include disease caused by any member of the B. cereus group containing the toxin-producing pXO1 plasmid or anthrax toxin genes specifically. Full article

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed. Full article

Viral recombination contributes to the emergence of novel strains with the potential for altered host range, transmissibility, virulence, and immune evasion. For foot-and-mouth disease virus (FMDV), cell culture experiments and phylogenetic analyses of field samples have demonstrated the occurrence of recombination. However, the frequency of recombination and associated virus–host interactions within an infected host have not been determined. We have previously reported the detection of interserotypic recombinant FMDVs in oropharyngeal fluid (OPF) samples of 42% (5/12) of heterologously superinfected FMDV carrier cattle. The present investigation consists of a detailed analysis of the virus populations in these samples including identification and characterization of additional interserotypic minority recombinants. In every animal in which recombination was detected, recombinant viruses were identified in the OPF at the earliest sampling point after superinfection. Some recombinants remained dominant until the end of the experiment, whereas others were outcompeted by parental strains. Genomic analysis of detected recombinants suggests host immune pressure as a major driver of recombinant emergence as all recombinants had capsid-coding regions derived from the superinfecting virus to which the animals did not have detectable antibodies at the time of infection. In vitro analysis of a plaque-purified recombinant virus demonstrated a growth rate comparable to its parental precursors, and measurement of its specific infectivity suggested that the recombinant virus incurred no penalty in packaging its new chimeric genome. These findings have important implications for the potential role of persistently infected carriers in FMDV ecology and the emergence of novel strains. Full article

The objective of this study was to evaluate the significance of jejunal mucosa-associated microbiota and its impacts on the intestinal health of pigs challenged with F18⁺ Escherichia coli. Forty-four newly-weaned pigs were allotted to two treatments in a randomized complete block design with sex as blocks. Pigs were fed common diets for 28 d. At d 7 post-weaning, pigs were orally inoculated with saline solution or F18⁺ E. coli. At d 21 post-challenge, feces and blood were collected and pigs were euthanized to collect jejunal tissue to evaluate microbiota and intestinal health parameters. The relative abundance of Firmicutes and Bacteroidetes was lower (p < 0.05) in jejunal mucosa than in feces, whereas Proteobacteria was greater (p < 0.05) in jejunal mucosa. F18⁺ E. coli increased (p < 0.05) protein carbonyl, Helicobacteraceae, Pseudomonadaceae, Xanthomonadaceae, and Peptostreptococcaceae and reduced (p < 0.05) villus height, Enterobacteriaceae, Campylobacteraceae, Brachyspiraceae, and Caulobacteraceae in jejunal mucosa, whereas it reduced (p < 0.05) Spirochaetaceae and Oscillospiraceae in feces. Collectively, jejunal mucosa-associated microbiota differed from those in feces. Compared with fecal microbiota, the change of mucosa-associated microbiota by F18⁺ E. coli was more prominent, and it was mainly correlated with increased protein carbonyl and reduced villus height in jejunal mucosa impairing the intestinal health of nursery pigs. Full article

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod that has been isolated from a variety of oral infections including periodontitis, peri-implantitis, and odontogenic abscesses. As a newly emerging pathogen, its type strain has been investigated for pathogenic properties, yet little is known about its virulence variations among strains. We previously screened the whole genome of nine clinical oral isolates and a reference strain of F. alocis, and they expressed a novel RTX toxin, FtxA. In the present study, we aimed to use label-free quantification proteomics to characterize the full proteome of those ten F. alocis strains. A total of 872 proteins were quantified, and 97 among them were differentially expressed in FtxA-positive strains compared with the negative strains. In addition, 44 of these differentially expressed proteins formed 66 pairs of associations based on their predicted functions, which included clusters of proteins with DNA repair/mediated transformation and catalytic activity-related function, indicating different biosynthetic activities among strains. FtxA displayed specific interactions with another six intracellular proteins, forming a functional cluster that could discriminate between FtxA-producing and non-producing strains. Among them were FtxB and FtxD, predicted to be encoded by the same operon as FtxA. While revealing the broader qualitative and quantitative proteomic landscape of F. alocis, this study also sheds light on the deeper functional inter-relationships of FtxA, thus placing this RTX family member into context as a major virulence factor of this species. Full article

In this study, a Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) method for genetic typing of Trueperella pyogenes, an opportunistic bacterial pathogen, was designed. The method optimization was performed for 37 clinical T. pyogenes strains isolated from various infections in different animal species. Optimal conditions for reliable and reproducible DNA fingerprinting were determined according to the modified Taguchi method. The developed method was assessed regarding its typeability, reproducibility, and discriminatory power using the Hunter’s and Gatsons’ index of discrimination. A high degree of genetic diversity was shown between the studied strains, which represented 31 genotypes. The generated RAPD profiles were relatively complex and simultaneously easy to interpret due to the wide size range of amplicons. The discriminatory index of the designed method was sufficiently high; thus, only strains epidemiologically related displayed identical RAPD genotypes. In conclusion, the DNA fingerprinting of T. pyogenes by the developed RAPD-PCR method is a reliable typing tool that may allow a better understanding of the epidemiology as well as pathogenesis of infections caused by this pathogen. Full article

Highly pathogenic avian influenza viruses’ (HPAIVs) transmission from wild birds to poultry occurs globally, threatening animal and public health. To predict the HPAI outbreak risk in relation to wild bird densities and land cover variables, we performed a case-control study of 26 HPAI outbreaks (cases) on Dutch poultry farms, each matched with four comparable controls. We trained machine learning classifiers to predict outbreak risk with predictors analyzed at different spatial scales. Of the 20 best explaining predictors, 17 consisted of densities of water-associated bird species, 2 of birds of prey, and 1 represented the surrounding landscape, i.e., agricultural cover. The spatial distribution of mallard (Anas platyrhynchos) contributed most to risk prediction, followed by mute swan (Cygnus olor), common kestrel (Falco tinnunculus) and brant goose (Branta bernicla). The model successfully distinguished cases from controls, with an area under the receiver operating characteristic curve of 0.92, indicating accurate prediction of HPAI outbreak risk despite the limited numbers of cases. Different classification algorithms led to similar predictions, demonstrating robustness of the risk maps. These analyses and risk maps facilitate insights into the role of wild bird species and support prioritization of areas for surveillance, biosecurity measures and establishments of new poultry farms to reduce HPAI outbreak risks. Full article

Background: The hepatitis B and D virus (HBV/HDV) hepatocyte entry inhibitor bulevirtide (BLV) has been available in Europe since July 2020, after the registrational trial MYR202. Real-life data on the efficacy and safety of BLV are sparse. Methods: We have analysed the course of treatment with BLV (2 mg/day) plus tenofovir disoproxil fumarate (TDF) (245 mg/day) in patients with chronic hepatitis delta (CHD). Virologic (≥2 log reduction in HDV RNA or suppression of HDV RNA below the lower limit of detection) and biochemical (normalisation of serum ALT) treatment responses after 24 weeks were defined according to the MYR202 trial. Results: Seven patients were recruited (four with liver cirrhosis Child–Pugh A). After 24 weeks, a virologic response was observed in five of seven and a biochemical response was seen in three of six patients with elevated serum ALT at baseline. Extended treatment data > 48 weeks were available in three cases: two presented with continuous virologic and biochemical responses and in one individual an HDV-RNA breakthrough was observed. Adverse effects were not recorded. Conclusions: The first real-life data of the approved dosage of 2 mg of BLV in combination with TDF confirm the safety, tolerability, and efficacy of the registrational trial MYR202 for a treatment period of 24 weeks and beyond. Full article

One of the tools to contain the SARS-CoV-2 pandemic was to increase the number of performed tests and to improve the access to diagnostics. To this effect, mobile collection sites (MCSs) were established. This study was performed on samples collected at the MCS between November 2020 and March 2021. We aimed to confirm/exclude SARS-CoV-2, differentiate SARS-CoV-2 variants, and detect other respiratory pathogens. SARS-CoV-2 and other respiratory viruses were identified by RT-qPCRs. A total of 876 (46.35%) SARS-CoV-2 positive specimens in the diagnostic tests were identified. The wild-type variant was determined in 667 (76.14%) samples; the remaining 209 (23.86%) samples specimens were identified as Alpha variant. A total of 51 (5.6%) non-SARS-CoV-2 cases were detected in retrospective studies. These accounted for 33 cases of mono-infection including rhinovirus (RV), human adenovirus (HAdV), human metapneumovirus (HMPV), enterovirus (EV), and influenza virus, and 18 cases of co-infection (SARS-CoV-2 with RV or HAdV or HMPV, and RV with EV). Our research shows that the results obtained from the MCS have value in epidemiological studies, reflecting national trends on a micro scale. Although the spread of COVID-19 is a major public health concern, SARS-CoV-2 is not the only pathogen responsible for respiratory infections. Full article

While Bacillus cereus typically causes opportunistic infections in humans, within the last three decades, severe and fatal infections caused by isolates of the B. cereus group harboring anthrax toxin genes have been reported in the United States. From 1994 to 2020, seven cases of anthrax-like illness resulting from these isolates have been identified. With one exception, the cases have occurred in the Gulf States region of the United States among metalworkers. We aimed to develop an ecological niche model (ENM) to estimate a spatial area conducive to the survival of these organisms based on the presence of known human infections and environmental variables. The estimated ecological niche for B. cereus was modeled with the maximum entropy algorithm (Maxent). Environmental variables contributing most to the model were soil characteristics (cation exchange capacity, carbon content, soil pH), temperature, enhanced vegetation index (EVI), and land surface temperature (LST). Much of the suitable environments were located throughout the Gulf Coast Plain, Texas Backland Prairies, East Central Texas Plains, Edwards Plateau, Cross Timbers, Mississippi Alluvial Plain, and Central Great Plains. These findings may provide additional guidance to narrow potential risk areas to efficiently communicate messages to metalworkers and potentially identify individuals who may benefit from the anthrax vaccine. Full article

The production of biofilms is a critical factor in facilitating the survival of Staphylococcus spp. in vivo and in protecting against various environmental noxa. The possible relationship between the antibiotic-resistant phenotype and biofilm-forming capacity has raised considerable interest. The purpose of the study was to assess the interdependence between biofilm-forming capacity and the antibiotic-resistant phenotype in 299 Staphylococcus spp. (S. aureus n = 143, non-aureus staphylococci [NAS] n = 156) of environmental origin. Antimicrobial susceptibility testing and detection of methicillin resistance (MR) was performed. The capacity of isolates to produce biofilms was assessed using Congo red agar (CRA) plates and a crystal violet microtiter-plate-based (CV-MTP) method. MR was identified in 46.9% of S. aureus and 53.8% of NAS isolates (p > 0.05), with resistance to most commonly used drugs being significantly higher in MR isolates compared to methicillin-susceptible isolates. Resistance rates were highest for clindamycin (57.9%), erythromycin (52.2%) and trimethoprim-sulfamethoxazole (51.1%), while susceptibility was retained for most last-resort drugs. Based on the CRA plates, biofilm was produced by 30.8% of S. aureus and 44.9% of NAS (p = 0.014), while based on the CV-MTP method, 51.7% of S. aureus and 62.8% of NAS were identified as strong biofilm producers, respectively (mean OD₅₇₀ values: S. aureus: 0.779±0.471 vs. NAS: 1.053±0.551; p < 0.001). No significant differences in biofilm formation were observed based on MR (susceptible: 0.824 ± 0.325 vs. resistant: 0.896 ± 0.367; p = 0.101). However, pronounced differences in biofilm formation were identified based on rifampicin susceptibility (S: 0.784 ± 0.281 vs. R: 1.239 ± 0.286; p = 0.011). The mechanistic understanding of the mechanisms Staphylococcus spp. use to withstand harsh environmental and in vivo conditions is crucial to appropriately address the therapy and eradication of these pathogens. Full article

Bovine respiratory disease (BRD) is the leading cause of morbidity in feedlot cattle. The ability to accurately identify the expected BRD risk of cattle would allow managers to detect high-risk animals more frequently. Five classification models were built and evaluated towards predicting the expected BRD risk (high/low) of feedlot cattle within the first 45 days on feed (DOF) and incorporate an economic analysis to determine the potential health cost advantage when using a predictive model compared with standard methods. Retrospective data from 10 U.S. feedlots containing 1733 cohorts representing 188,188 cattle with known health outcomes were classified into high- (≥15% BRD morbidity) or low- (<15%) BRD risk in the first 45 DOF. Area under the curve was calculated from the test dataset for each model and ranged from 0.682 to 0.789. The economic performance for each model was dependent on the true proportion of high-risk cohorts in the population. The decision tree model displayed a greater potential economic advantage compared with standard procedures when the proportion of high-risk cohorts was ≤45%. Results illustrate that predictive models may be useful at delineating cattle as high or low risk for disease and may provide economic value relative to standard methods. Full article

Acute kidney injury (AKI) is a life-threatening complication. Malaria and sepsis are leading causes of AKI in low-and-middle-income countries, but its etiology and pathogenesis are poorly understood. A prospective observational cohort study was conducted to evaluate pathways of immune and endothelial activation in children hospitalized with an acute febrile illness in Uganda. The relationship between clinical outcome and AKI, defined using the Kidney Disease: Improving Global Outcomes criteria, was investigated. The study included 967 participants (mean age 1.67 years, 44.7% female) with 687 (71.0%) positive for malaria by rapid diagnostic test and 280 (29.1%) children had a non-malarial febrile illness (NMFI). The frequency of AKI was higher in children with NMFI compared to malaria (AKI, 55.0% vs. 46.7%, p = 0.02). However, the frequency of severe AKI (stage 2 or 3 AKI) was comparable (12.1% vs. 10.5%, p = 0.45). Circulating markers of both immune and endothelial activation were associated with severe AKI. Children who had malaria and AKI had increased mortality (no AKI, 0.8% vs. AKI, 4.1%, p = 0.005), while there was no difference in mortality among children with NMFI (no AKI, 4.0% vs. AKI, 4.6%, p = 0.81). AKI is a common complication in children hospitalized with acute infections. Immune and endothelial activation appear to play central roles in the pathogenesis of AKI. Full article

Background: Schistosomiasis is a major poverty-related disease caused by dioecious parasitic flatworms of the genus Schistosoma with a health impact on both humans and animals. Hybrids of human urogenital schistosome and bovine intestinal schistosome have been reported in humans in several of Nigeria’s neighboring West African countries. No empirical studies have been carried out on the genomic diversity of Schistosoma haematobium in Nigeria. Here, we present novel data on the presence and prevalence of hybrids and the population genetic structure of S. haematobium. Methods: 165 Schistosoma-positive urine samples were obtained from 12 sampling sites in Nigeria. Schistosoma haematobium eggs from each sample were hatched and each individual miracidium was picked and preserved in Whatman^® FTA cards for genomic analysis. Approximately 1364 parasites were molecularly characterized by rapid diagnostic multiplex polymerase chain reaction (RD-PCR) for mitochondrial DNA gene (Cox1 mtDNA) and a subset of 1136 miracidia were genotyped using a panel of 18 microsatellite markers. Results: No significant difference was observed in the population genetic diversity (p > 0.05), though a significant difference was observed in the allelic richness of the sites except sites 7, 8, and 9 (p < 0.05). Moreover, we observed two clusters of populations: west (populations 1–4) and east (populations 7–12). Of the 1364 miracidia genotyped, 1212 (89%) showed an S. bovis Cox1 profile and 152 (11%) showed an S. haematobium cox1 profile. All parasites showed an S. bovis Cox1 profile except for some at sites 3 and 4. Schistosoma miracidia full genotyping showed 59.3% of the S. bovis ITS2 allele. Conclusions: This study provides novel insight into hybridization and population genetic structure of S. haematobium in Nigeria. Our findings suggest that S. haematobium x S. bovis hybrids are common in Nigeria. More genomic studies on both human- and animal-infecting parasites are needed to ascertain the role of animals in schistosome transmission. Full article

Sicily was the first Italian region to introduce rotavirus (RV) vaccination with the monovalent G1P[8] vaccine Rotarix^® in May 2012. In this study, the seasonal distribution and molecular characterization of RV strains detected over 19 years were compared to understand the effect of Rotarix^® on the evolutionary dynamics of human RVs. A total of 7846 stool samples collected from children < 5 years of age, hospitalized with acute gastroenteritis, were tested for RV detection and genotyping. Since 2013, vaccine coverage has progressively increased, while the RV prevalence decreased from 36.1% to 13.3% with a loss of seasonality. The local distribution of RV genotypes changed over the time possibly due to vaccine introduction, with a drastic reduction in G1P[8] strains replaced by common and novel emerging RV strains, such as equine-like G3P[8] in the 2018–2019 season. Comparison of VP7 and VP4 amino acid (aa) sequences with the cognate genes of Rotarix^® and RotaTeq^® vaccine strains showed specific aa changes in the antigenic epitopes of VP7 and of the VP8* portion of VP4 of the Italian RV strains. Molecular epidemiological surveillance data are required to monitor the emergence of novel RV strains and ascertain if these strains may affect the efficacy of RV vaccines. Full article

Phosphodiesterases are essential regulators of cyclic nucleotide signaling with diverse physiological functions. Two phosphodiesterases, PdeH and PdeL, have been identified from yeast and filamentous fungi. Here, the orthologs of PdeH and PdeL were characterized in a typical nematode-trapping fungus Arthrobotrys oligospora by gene disruption and phenotypic comparison. Deletion of AopdeH caused serious defects in mycelial growth, conidiation, stress response, trap formation, and nematicidal efficiency compared to the wild-type strain. In contrast, these phenotypes have no significant difference in the absence of AopdeL. In addition, deletion of AopdeH and AopdeL resulted in a remarkable increase in cAMP level during vegetative growth and trap formation, and the number of autophagosomes was decreased in ΔAopdeH and ΔAopdeL mutants, whereas their volumes considerably increased. Moreover, metabolomic analyses revealed that many metabolites were downregulated in ΔAopdeH mutant compared to their expression in the wild-type strain. Our results indicate that AoPdeH plays a crucial role in mycelial growth, conidiation, stress response, secondary metabolism, and trap formation. In contrast, AoPdeL only plays a minor role in hyphal and conidial morphology, autophagy, and trap formation in A. oligospora. This work expands the roles of phosphodiesterases and deepens the understanding of the regulation of trap formation in nematode-trapping fungi. Full article

Background: SARS-CoV-2 enters the body through inhalation or self-inoculation to mucosal surfaces. The kinetics of the ocular and nasal mucosal-specific-immunoglobulin A(IgA) responses remain under-studied. Methods: Conjunctival fluid (CF, n = 140) and nasal epithelial lining fluid (NELF, n = 424) obtained by paper strips and plasma (n = 153) were collected longitudinally from SARS-CoV-2 paediatric (n = 34) and adult (n = 47) patients. The SARS-CoV-2 spike protein 1(S1)-specific mucosal antibody levels in COVID-19 patients, from hospital admission to six months post-diagnosis, were assessed. Results: The mucosal antibody was IgA-predominant. In the NELF of asymptomatic paediatric patients, S1-specific IgA was induced as early as the first four days post-diagnosis. Their plasma S1-specific IgG levels were higher than in symptomatic patients in the second week after diagnosis. The IgA and IgG levels correlated positively with the surrogate neutralization readout. The detectable NELF “receptor-blocking” S1-specific IgA in the first week after diagnosis correlated with a rapid decline in viral load. Conclusions: Early and intense nasal S1-specific IgA levels link to a rapid decrease in viral load. Our results provide insights into the role of mucosal immunity in SARS-CoV-2 exposure and protection. There may be a role of NELF IgA in the screening and diagnosis of SARS-CoV-2 infection. Full article

Salmonellosis is a zoonosis of major relevance to global public health. Here we present the assessment of Salmonella enterica contamination in pork and poultry meat sold at retail markets in São Paulo, Brazil. A total of 780 meat samples (386 poultry meat and 394 pork samples) were collected from 132 markets. From these, 57 samples (7.3%) were positive for S. enterica isolation, including 32 (8.3%) poultry meat and 25 (6.3%) pork samples. S. enterica isolates were further characterized for serotyping, antimicrobial resistance and genotyping by amplified fragment length polymorphism and pulsed field gel electrophoresis. Antimicrobial resistance analysis demonstrated two main profiles: pork isolates were more resistant to macrolides, β-lactams, tetracycline, phenicols, and fluoroquinolones, and poultry meat isolates presented higher resistance to fluoroquinolones, sulfonamides, tetracycline, and β-lactams. A total of 72.4% of poultry meat isolates were identified as S. Heidelberg, while most of pork isolates were S. Typhimurium (31.7%) and S. Give (16.7%). Genotyping resulted in most clusters consisting exclusively of pork or poultry meat, no cross-contamination was detected, and a tendency to differentiate isolates according to their serotypes and markets of origin. High resistance rates to critically important antimicrobials reinforce the importance of controlling Salmonella contamination in meat production chains. Full article

The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has forced the scientific community to acquire knowledge in real-time, when total lockdowns and the interruption of flights severely limited access to reagents as the global pandemic became established. This unique reality made researchers aware of the importance of designing efficient in vitro set-ups to evaluate infectious kinetics. Here, we propose a histology-based method to evaluate infection kinetics grounded in cell microarray (CMA) construction, immunocytochemistry and in situ hybridization techniques. We demonstrate that the chip-like organization of the InfectionCMA has several advantages, allowing side-by-side comparisons between diverse cell lines, infection time points, and biomarker expression and cytolocalization evaluation in the same slide. In addition, this methodology has the potential to be easily adapted for drug screening. Full article

The appearance of the severe acute respiratory syndrome virus-2 (SARS-CoV-2) has had a significant impact on the balance of public health and social life. The data available so far show that newborns and young children do not develop severe forms of COVID-19, but a small proportion of them will still need hospitalization. Even though young children represent an important vector of the infection, vaccination at such a young age was not yet considered. Thus, the question of whether potentially protective antibodies against SARS-CoV-2 could be provided to them via breast milk or across the placenta, as “passive immunity”, still stands. Materials and Methods: Between January–July 2021, we have conducted a prospective study that aimed to measure the immunoglobulin (Ig) A and IgG anti-SARS-CoV-2 titers in the breast milk of 28 vaccinated lactating mothers, sampled at 30 and 60 days after the second dose of the anti-SARS-CoV-2 Pfizer or Moderna mRNA vaccines. Anti-RBD reactive IgA and IgG antibodies were detected and quantified by a sandwich enzyme-linked immunosorbent assay. Results: Anti-RBD IgA and IgG were present in all breast milk samples, both in the first and in the second specimens, without a significant difference between those two. The anti-RBD IgA titers were approximately five-times higher than the anti-RBD IgG ones. The anti-RBD IgA and IgG titers were correlated with the infants’ age, but they were not correlated with the vaccine type or mother’s age. The anti-RBD IgA excreted in milk were inversely correlated with the parity number. Conclusions: Anti-SARS-CoV-2 IgA and IgG can be found in the milk secretion of mothers vaccinated with mRNA vaccines and, presumably, these antibodies should offer protection to the newborn, considering that the antibodies’ titers did not decrease after 60 days. The antibody response is directly proportional to the breastfed child’s age, but the amount of anti-RBD IgA decreases with the baby’s rank. The antibody response did not depend on the vaccine type, or on the mother’s age. Full article

Cultivation of resistant varieties is an environmentally friendly and inexpensive method of crop protection. Numerous alleles of specific disease resistance occur in cereals and other crops, and knowledge of their presence in individual varieties has wide utilization in research and practice. Postulation based on phenotyping host—pathogen interactions and the gene-for-gene model is a common way of identifying these genes. The same technique and design of tests are used for postulating virulence when pathogen populations are studied. Powdery mildews caused by different formae speciales of Blumeria graminis (Bg) are important cereal diseases. In this contribution, experimental methods are described that use a model organism Bg f. sp. hordei, which can be employed for other cereal mildews and possibly rusts. It includes illustrations and a summary of our long-term practical experience. It also critically evaluates the benefits of leaf segment tests compared with screening whole plants. Full article

Many pathogens can cause cancer, but cancer itself does not normally act as an infectious agent. However, transmissible cancers have been found in a few cases in nature: in Tasmanian devils, dogs, and several bivalve species. The transmissible cancers in dogs and devils are known to spread through direct physical contact, but the exact route of transmission of bivalve transmissible neoplasia (BTN) has not yet been confirmed. It has been hypothesized that cancer cells from bivalves could be released by diseased animals and spread through the water column to infect/engraft into other animals. To test the feasibility of this proposed mechanism of transmission, we tested the ability of BTN cells from the soft-shell clam (Mya arenaria BTN, or MarBTN) to survive in artificial seawater. We found that MarBTN cells are highly sensitive to salinity, with acute toxicity at salinity levels lower than those found in the native marine environment. BTN cells also survive longer at lower temperatures, with 50% of cells surviving greater than 12 days in seawater at 10 °C, and more than 19 days at 4 °C. With one clam donor, living cells were observed for more than eight weeks at 4 °C. We also used qPCR of environmental DNA (eDNA) to detect the presence of MarBTN-specific DNA in the environment. We observed release of MarBTN-specific DNA into the water of laboratory aquaria containing highly MarBTN-diseased clams, and we detected MarBTN-specific DNA in seawater samples collected from MarBTN-endemic areas in Maine, although the copy numbers detected in environmental samples were much lower than those found in aquaria. Overall, these data show that MarBTN cells can survive well in seawater, and they are released into the water by diseased animals. These findings support the hypothesis that BTN is spread from animal-to-animal by free cells through seawater. Full article

Transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) have been extensively used to investigate the pathogenesis and tissue tropism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Neuroinvasion and the replication of SARS-CoV-2 within the central nervous system (CNS) of K18-hACE2 mice is associated with increased mortality; although, the mechanisms by which this occurs remain unclear. In this study, we generated primary neuronal cultures from K18-hACE2 mice to investigate the effects of a SARS-CoV-2 infection. We also evaluated the immunological response to SARS-CoV-2 infection in the CNS of K18-hACE2 mice and mouse neuronal cultures. Our data show that neuronal cultures obtained from K18-hACE2 mice are permissive to SARS-CoV-2 infection and support productive virus replication. Furthermore, SARS-CoV-2 infection upregulated the expression of genes involved in innate immunity and inflammation, including IFN-α, ISG-15, CXCL10, CCL2, IL-6 and TNF-α, in the neurons and mouse brains. In addition, we found that SARS-CoV-2 infection of neurons and mouse brains activates the ZBP1/pMLKL-regulated necroptosis pathway. Together, our data provide insights into the neuropathogenesis of SARS-CoV-2 infection in K18-hACE2 mice. Full article

Human alveolar echinococcosis (AE), which is caused by the cestode Echinococcus (E.) multilocularis, is an epidemiologically relevant issue in modern medicine and still poses a diagnostic and therapeutic challenge. Since diagnosis mainly relies on imaging procedures and serological testing, we retrospectively and comparatively analyzed the performance of an Echinococcus IgG screening ELISA, whole serum IgE, and two specific confirmatory ELISA platforms using the defined E. multilocularis antigens Em2-Em18 (Em2+) and recombinant Em18 (recEm18). With special emphasis on the clinical usefulness of recEm18, we correlated the laboratory results with clinical characteristics and imaging findings in a large and well-characterized cohort of N = 124 AE patients, who were followed over several years after either surgical plus subsequent pharmacological treatment or pharmacotherapy alone. All patients had routinely received PET-CTI every two years. Our data reveal strong correlations for both Echinococcus IgG and recEm18 with tracer uptake in PET-CTI and parasitic lesion size and number, suggesting additional clinical usefulness of recEm18 for certain constellations only, while IgG and Em2+ still appear reasonable and sensitive screening methods for initial diagnosis of AE. With this study, we aim to contribute to further optimizing medical care of AE patients. For instance, it might be reasonable to consider the replacement of some PET-CTI follow-ups by imaging procedures with less radiation exposure or serological means alone. Further studies that clarify the correlation of serological markers with ultrasound criteria might be particularly useful, and further retrospective as well as prospective investigations are justified in this context. Full article

Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. Such discrimination is necessary for quarantine pathogens, whose management is strictly dependent on genotype identification. To address these needs, we have established and evaluated a novel all-in-one diagnostic assay based on nanopore sequencing for the detection and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa as a case study. The assay proved to be at least as sensitive as standard diagnostic tests and the quantitative results agreed closely with qPCR-based analysis. The same sequencing results also allowed discrimination between subspecies when present either individually or in combination. Pathogen detection and typing were achieved within 13 min of sequencing owing to the use of an internal control that allowed to stop sequencing when sufficient data had accumulated. These advantages, combined with the use of portable equipment, will facilitate the development of next-generation diagnostic assays for the efficient monitoring of other plant pathogens. Full article

Capsicum, an important vegetable crop in Queensland, Australia, is vulnerable to both elevated temperatures and capsicum chlorosis virus (CaCV). Thus, it is imperative to understand the genetic responses of capsicum plants (Capsicum annuum) to CaCV under elevated temperature conditions. Here, we challenged susceptible plants (cv. Yolo Wonder) with CaCV and investigated the effects of elevated temperature on symptom expression, the accumulation of virus-derived short interfering RNA (vsiRNA) and viral RNA, and the expression of plant defense-associated genes. CaCV-inoculated plants initially showed more severe symptoms and higher viral concentrations at a higher temperature (HT, 35 °C) than at ambient temperature (AT, 25 °C). However, symptom recovery and reduced viral RNA accumulation were seen in the CaCV-infected plants grown at HT at later stages of infection. We also observed that HT enhanced the accumulation of vsiRNAs and that, concurrently, RNA interference (RNAi)-related genes, including Dicer-like2 (DCL2), DCL4, RNA-dependent RNA polymerase 1 (RdRp1), RdRp6, and Argonaute2 (AGO2), were upregulated early during infection. Moreover, continuous high levels of vsiRNAs were observed during later stages of CaCV infection at HT. Overall, our investigation suggests that HT facilitates CaCV replication during early infection stages. However, this appears to lead to an early onset of antiviral RNA silencing, resulting in a subsequent recovery from CaCV in systemic leaves. Full article

The cultivar Plavac Mali (Vitis vinifera L.), the most important indigenous red grapevine cultivar in Croatia, was tested for the presence of 16 grapevine viruses. Thirty-five samples from the collection vineyard were tested for the presence of grapevine leafroll-associated viruses-1, -2, and -3 (GLRaV-1, GLRaV-2 and GLRaV-3, respectively), grapevine fanleaf virus (GFLV), arabis mosaic virus (ArMV), grapevine virus-A (GVA), -B (GVB), -G (GVG), -H (GVH), -I (GVI), -J (GVJ), grapevine fleck virus (GFkV), grapevine rupestris stem pitting associated virus (GRSPaV), and grapevine pinot gris virus (GPGV) by reverse transcription–polymerase chain reaction (RT-PCR). Furthermore, standard PCR was conducted for grapevine badnavirus 1 (GBV-1) and grapevine red blotch virus (GRBV). Mixed infections were most common and GLRaV-3, the most abundant virus found in 85.71% of the vines tested, was further molecularly characterised. Different genomic variants of the heat shock protein homologue (HSP70h) were separated by cloning, detected by single-strand conformation polymorphism (SSCP) analysis, sequenced, and phylogenetically analysed. The presence of phylogenetic groups I and II was only confirmed. This study demonstrates the high virus infection rate of Plavac Mali vines and the heterogeneity of GLRaV-3 present nowadays in a collection vineyard. Full article

African swine fever viruses (ASFV), currently a serious threat to the global pig industry, primarily target porcine macrophages. Macrophages are characterized by their remarkable plasticity, being able to modify their phenotype and functions in response to diverse stimuli. Since IL-10 and TGF-β polarize macrophages toward an anti-inflammatory phenotype, we analyzed their impact on porcine monocyte-derived macrophages’ (moMΦ) susceptibility to infection and their responses to two genotype I ASFV strains, virulent 26544/OG10 and attenuated NH/P68. At a low multiplicity of infection (MOI), NH/P68, but not 26544/OG10, presented a higher ability to infect moM(IL-10) compared to moMΦ and moM(TGF-β), but no differences were appreciated at a higher MOI. Both strains replicated efficiently in all moMΦ subsets, with no differences at later times post-infection. Both strains downregulated CD14 and CD16 expression on moMΦ, irrespective of the activation status. ASFV’s modulation of CD163 and MHC II DR expression and cytokine responses to NH/P68 or 26544/OG10 ASFV were not affected by either IL-10 or TGF-β pre-treatment. Our results revealed little impact of these anti-inflammatory cytokines on moMΦ interaction with ASFV, which likely reflects the ability of the virus to effectively modulate macrophage responses. Full article

In this study, the vaccination coverage, serological sampling and infection rate of sheep and goats were evaluated as predictors for the modeling of human brucellosis in Greece. The human brucellosis disease frequency per local regional unit (RU) varied significantly (RR90) among consecutive years. The notification rate was higher (p < 0.001) in the RUs with implementation of vaccination in sheep and goats (vaccination zone—VZ) with a median of 1.4 (IQR 0.0–3.1) compared with the RUs of the eradication zone (EZ) with a median of 0.0 (IQR 0.0–0.0). In VZ, the increased frequency of human cases was associated with delayed vaccine administration (estimate: 0.14 (0.04; 0.29), p = 0.03) and higher vaccination coverage of the animals (estimate: −0.349 (−0.72; −0.07), p < 0.01). However, the flock sampling rate was highly heterogenous among RUs (IQR 10.56–52.93), and inconsistent within RUs throughout the period of the study 2013–2017 (p = 0.001), limiting the reliable estimation of the infection rate in livestock and the design of an integrated One Health model for human disease. Full article

Carriers of highly leukotoxic genotypes of Aggregatibacter actinomycetemcomitans are at high risk for rapid degradation of tooth-supporting tissues. The leukotoxin (LtxA) expressed by this bacterium induces a rapid pro-inflammatory response in leukocytes that results in cell death. The aim of the present study was to increase the understanding of LtxA-induced leukocyte activation mechanisms and of possible associated osteoclast differentiation. The effect of LtxA on activation of the inflammasome complex was studied in THP-1 wild type and in NLRP3- and ASC knockout cells. Cell-to-cell communication was assessed by fluorescent parachute assays, and THP-1 differentiation into osteoclast-like cells was investigated microscopically. The results showed that LtxA induced inflammatory cell death, which involved activation of the NLRP3 inflammasome and gap junction cell-to-cell communication. THP-1 cells treated with lipopolysaccharide (LPS) and LtxA together differentiated into an osteoclast-like phenotype. Here, LPS prevented LtxA-mediated cell death but failed to induce osteoclast differentiation on its own. However, pit formation was not significantly enhanced by LtxA. We conclude that A. actinomycetemcomitans leukotoxicity mediates activation of the NLRP3 inflammasome and cell-to-cell communication in the induced pro-inflammatory cell death. In addition, LtxA stimulated differentiation towards osteoclasts-like cells in LPS-treated THP-1 cells. Full article

A higher expression of human endogenous retroviruses (HERVs) has been associated with several malignancies, including prostate cancer, implying a possible use as a diagnostic or prognostic cancer biomarker. For this reason, we examined the humoral response against different epitopes obtained from the envelope protein of HERV-K (HERV-K env-su_19–37, HERV-K env-su_109–126), HERV-H (HERV-H env-su_229–241, HERV-H env_387–399) and HERV-W (HERV-W env-su_93–108, HERV-W env-su_248–262) in the plasma of patients affected by prostate cancer (PCa), and compared to that of benign prostate hyperplasia (BPH) and a borderline group of patients with atypical small acinar proliferation (ASAP) and prostate intraepithelial neoplasia (PIN) and healthy controls. A significant antibody response was observed against HERV-K env-su_109–126 (p = 0.004) and HERV-H env-su_229–241 (p < 0.0001) in PCa patients compared to HCs, BPH and borderline cohorts, whilst no significance difference was found in the antibodies against HERV-W env-su_93–108 and HERV-W env-su_248–262 in patients with PCa. Our results provided further proof of the association between HERV-K and PCa and added new evidence about the possible involvement of HERV-H in PCa pathogenesis, highlighting their possibility of being used as biomarkers of the disease. Full article

The genus Fusarium is considered to be one of the most pathogenic, phytotoxic and toxin-producing group of microorganisms in the world. Plants infected by these fungi are characterized by a reduced consumer and commercial value, mainly due to the contamination of crops with mycotoxins. Therefore, effective methods of reducing fungi of the genus Fusarium must be implemented already in the field before harvesting, especially with alternative methods to pesticides such as biocontrol. In this study we identified yeasts that inhibit the growth of the pathogenic fungi Fusarium culmorum, F. graminearum and F. poae. Tested yeasts came from different culture collections, or were obtained from organic and conventional cereals. The greater number of yeast isolates from organic cereals showed antagonistic activity against fungi of the genus Fusarium compared to isolates from the conventional cultivation system. Cryptococcus carnescens (E22) isolated from organic wheat was the only isolate that limited the mycelial growth of all three tested fungi and was the best antagonist against F. poae. Selected yeasts showed various mechanisms of action against fungi, including competition for nutrients and space, production of volatile metabolites, reduction of spore germination, production of siderophores or production of extracellular lytic enzymes: chitinase and β-1,3-glucanase. Of all the investigated mechanisms of yeast antagonism against Fusarium, competition for nutrients and the ability to inhibit spore germination prevailed. Full article

Hemodialysis patients (HDP) and kidney transplant recipients (KTR) have a high risk of infection with SARS-CoV-2 with poor clinical outcomes. Because of this, vaccination of these groups of patients against SARS-CoV-2 is particularly important. However, immune responses may be impaired in immunosuppressed and chronically ill patients. Here, our aim was to compare the efficacy of an mRNA-based vaccine in HDP, KTR, and healthy subjects. Design: In this prospective observational cohort study, the humoral and cellular response of prevalent 192 HDP, 50 KTR, and 28 healthy controls (HC) was assessed 1, 2, and 6 months after the first immunization with the BNT162b2 mRNA vaccine. Results: After 6 months, 97.5% of HDP, 37.9% of KTR, and 100% of HC had an antibody response. Median antibody levels were 1539.7 (±3355.8), 178.5 (±369.5), and 2657.8 (±2965.8) AU/mL in HDP, KTR, and HC, respectively (p ≤ 0.05). A SARS-CoV-2 antigen-specific cell response to vaccination was found in 68.8% of HDP, 64.5% of KTR, and 90% of HC. Conclusion: The humoral response rates to mRNA-based vaccination of HDPs are comparable to HCs, but antibody titers are lower. Furthermore, HDPs have weaker T-cell response to vaccination than HCs. KTRs have very low humoral and antigen-specific cellular response rates and antibody titers, which requires other vaccination strategies in addition to booster vaccination. Full article

Candida albicans is maintained as a commensal by immune mechanisms at the oral epithelia. Oral antifungal peptide Histatin 5 (Hst 5) may function in innate immunity, but the specific role Hst 5 plays in C. albicans commensalism is unclear. Since Zn-binding potentiates the candidacidal activity of Hst 5, we hypothesized that Hst 5+Zn would elicit a unique fungal stress response to shape interactions between C. albicans and oral epithelial cells (OECs). We found that Hst 5+Zn but not Hst 5 alone resulted in the activation of cell wall integrity (CWI) signaling, and deletion mutants were then used to determine that CWI-mediated chitin synthesis was protective against killing. Using flow cytometry, we confirmed that Hst 5+Zn-treated cells had significantly elevated levels of cell-wall chitin, mannan and β-1,3 glucan compared to Hst 5-treated cells. We then tested the activation of host signaling components involved in C. albicans cell-wall recognition. The immunoblot assay of C. albicans-exposed oral epithelial cells showed increased activation of EphA2 and NF-κB but not EGFR. Interestingly, C. albicans treated with Hst 5+Zn induced the global suppression of pro-inflammatory cytokine release from OECs, but an increase in negative regulator IL-10. Hst 5+Zn-treated cells were more adherent but ultimately less invasive to OECs than control cells, thus indicating lowered virulence. Therefore, Hst 5+Zn-treated C. albicans cells are discerned by epithelial monolayers, but are less virulent and promote anti-inflammatory signaling, suggesting that Hst 5+Zn in combination could play a role in regulating commensalism of oral C. albicans through cell wall reorganization. Full article

Parvoviruses under the genus Chaphamaparvovirus (subfamily Hamaparvovirinae) are highly divergent and have recently been identified in many animals. However, the detection and characterisation of parvoviruses in psittacine birds are limited. Therefore, this study reports a novel parvovirus, tentatively named psittaciform chaphamaparvovirus 2 (PsChPV-2) under the genus Chaphamaparvovirus, which was identified in Australian Neophema birds. The PsChPV-2 genome is 4371 bp in length and encompasses four predicted open-reading frames, including two major genes, a nonstructural replicase gene (NS1), and a structural capsid gene (VP1). The NS1 and VP1 genes showed the closest amino acid identities of 56.2% and 47.7%, respectively, with a recently sequenced psittaciform chaphamaparvovirus 1 from a rainbow lorikeet (Trichoglossus moluccanus). Subsequent phylogenetic analyses exhibited that the novel PsChPV-2 is most closely related to other chaphamaparvoviruses of avian origin and has the greatest sequence identity with PsChPV-1 (60.6%). Further systematic investigation is warranted to explore the diversity with many avian-associated parvoviruses likely to be discovered. Full article

Chlamydia pecorum, an obligate intracellular pathogen, causes significant morbidity and mortality in livestock and the koala (Phascolarctos cinereus). A variety of C. pecorum gene-centric molecular studies have revealed important observations about infection dynamics and genetic diversity in both koala and livestock hosts. In contrast to a variety of C. pecorum molecular studies, to date, only four complete and 16 draft genomes have been published. Of those, only five draft genomes are from koalas. Here, using whole-genome sequencing and a comparative genomics approach, we describe the first two complete C. pecorum genomes collected from diseased koalas. A de novo assembly of DBDeUG_2018 and MC/MarsBar_2018 resolved the chromosomes and chlamydial plasmids each as single, circular contigs. Robust phylogenomic analyses indicate biogeographical separation between strains from northern and southern koala populations, and between strains infecting koala and livestock hosts. Comparative genomics between koala strains identified new, unique, and shared loci that accumulate single-nucleotide polymorphisms and separate between northern and southern, and within northern koala strains. Furthermore, we predicted novel type III secretion system effectors. This investigation constitutes a comprehensive genome-wide comparison between C. pecorum from koalas and provides improvements to annotations of a C. pecorum reference genome. These findings lay the foundations for identifying and understanding host specificity and adaptation behind chlamydial infections affecting koalas. Full article

Sexual reproduction of Plasmodium parasites takes place in anopheline mosquitoes, where male and female gametes fuse to form zygotes and then ookinetes. These processes are orchestrated by stage-specific protein expression, which is mediated in part by translational repression. Accumulating evidence shows that RNA binding proteins (RBPs) play crucial roles in these processes. Here, we report the characterization of P. berghei 103 (Pb103), which encodes a protein possessing double zinc finger domains (ZFs), an RBP. Reporter parasites expressing azami green fluorescent protein (AGFP) under the endogenous Pb103 gene promoter (Pb103-AGFP reporter) showed that the AGFP fluorescent signal was detected from gametes to ookinetes, while AGFP mRNA was translationally repressed in female gametocytes. The Pb103-disrupted parasites (Pb103(−)) grew and produced gametocytes with similar efficiencies to those of wild-type parasites. However, no oocysts were formed in mosquitoes fed Pb103(−). An in vitro fertilization assay showed abortion at the zygote stage in Pb103(−), suggesting that Pb103 plays a critical role in zygote/ookinete development. Cross-fertilization assays with Pb103(−) and male- or female-sterile parasites revealed that Pb103 was essential exclusively for female gametes. To identify the domains critical for zygote/ookinete development, transgenic parasites expressing partially deleted Pb103 were generated and assayed for ookinete maturation. As a result, deleting either of two ZFs but not the C-terminal region abolished zygote/ookinete development, highlighting the indispensable roles of ZFs in parasite sexual development, most likely via translational repression. Full article

Several species of avian schistosomes are known to cause dermatitis in humans worldwide. In Europe, this applies above all to species of the genus Trichobilharzia. For Austria, a lot of data are available on cercarial dermatitis and on the occurrence of Trichobilharzia, yet species identification of trematodes in most cases is doubtful due to the challenging morphological determination of cercariae. During a survey of trematodes in freshwater snails, we were able to detect a species in the snail Physella acuta (Draparnaud, 1805) hitherto unknown for Austria, Trichobilharzia physellae; this is also the first time this species has been reported in Europe. Species identification was performed by integrative taxonomy combining morphological investigations with molecular genetic analyses. The results show a very close relationship between the parasite found in Austria and North American specimens (similarity found in CO1 ≥99.57%). Therefore, a recent introduction of T. physellae into Europe can be assumed. Full article

A phase III clinical trial in treatment-naïve patients with chronic hepatitis B (CHB) revealed the safety and considerable therapeutic efficacy of a vaccine containing both hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) (NASVAC) at the end of treatment (EOT) and 24 weeks after EOT. Two years after EOT, we checked HBV DNA, alanine aminotransferase (ALT), and hepatitis B e antigen (HBeAg). The data reveal that 33 of 66 NASVAC-recipient CHB patients became negative for HBV DNA in the blood two years after EOT. The ALT levels were within the upper limit of normal (ULN) in 37 patients, although all 66 CHB patients had elevated ALT (above ULN) before the start of therapy. Out of the total twelve HBeAg-positive patients, eight patients became negative for HBeAg. None of the patients developed cirrhosis of the liver within this period. NASVAC is a finite treatment regimen with sustained antiviral and liver-protecting properties. This study is the first to report follow-up data of immune therapy for CHB. NASVAC, an immune therapy of finite duration, is endowed with sustained antiviral and liver protection properties in CHB patients. Full article

Chlamydia pecorum is a common gastrointestinal inhabitant of livestock but infections can manifest in a broad array of clinical presentations and in a range of host species. While C. pecorum is a known cause of ovine abortion, clinical cases have only recently been described in detail. Here, the prevalence and sequence types (STs) of C. pecorum in ewes from a property experiencing high levels of perinatal mortality (PNM) in New South Wales (NSW), Australia, were investigated using serological and molecular methods. Ewes that were PNM+ were statistically more likely to test seropositive compared to PNM− ewes and displayed higher antibody titres; however, an increase in chlamydial shedding from either the rectum, vagina or conjunctiva of PNM+ ewes was not observed. Multilocus sequence typing (MLST) indicated that C. pecorum ST23 was the major ST shed by ewes in the flock, was the only ST identified from the vaginal site, and was the same ST detected within aborted foetal tissues. Whole genome sequencing of C. pecorum isolated from one abortion case revealed that the C. pecorum plasmid (pCpec) contained a unique deletion in coding sequence 1 (CDS1) that was also present in C. pecorum ST23 shed from the ewes. A further unique deletion was noted in a polymorphic membrane protein gene (pmpG) of the C. pecorum chromosome, which warrants further investigation given the role of PmpG in host cell adherence and tissue tropism.This study describes novel infection parameters in a sheep flock experiencing C. pecorum-associated perinatal mortality, provides the first genomic data from an abortigenic C. pecorum strain, and raises questions about possible links between unique genetic features of this strain and C. pecorum abortion. Full article

Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) is an increasingly recognized complication of COVID-19 and is associated with significant over-mortality. We performed a retrospective monocentric study in patients admitted to the intensive care unit (ICU) for respiratory insufficiency due to COVID-19 from March to December 2020, in order to evaluate the incidence of CAPA and the associated risk factors. We also analysed the diagnostic approach used in our medical centre for CAPA diagnosis. We defined CAPA using recently proposed consensus definitions based on clinical, radiological and microbiological criteria. Probable cases of CAPA occurred in 9 out of 141 patients included in the analysis (6.4%). All cases were diagnosed during the second wave of the pandemic. We observed a significantly higher realization rate of bronchoalveolar lavage (BAL) (51.1% vs. 28.6%, p = 0.01) and Aspergillus testing (through galactomannan, culture, PCR) on BAL samples during the second wave (p < 0.0001). The testing for Aspergillus in patients meeting the clinical and radiological criteria of CAPA increased between the two waves (p < 0.0001). In conclusion, we reported a low but likely underestimated incidence of CAPA in our population. A greater awareness and more systematic testing for Aspergillus are necessary to assess the real incidence and characteristics of CAPA. Full article