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The host-pathogen interactions are tripartite and include the host, the pathogen, and the environment. For a long time, attention has mainly been addressed to the host's immune mechanisms and the pathogen's virulence factors while the environmental factor has received less interest. About a decade ago, studies on environmental DNA (eDNA) started to emerge as this approach is versatile for numerous research fields and interest is persistently growing. In the present study, the relationship between disease progression and pathogen water environmental DNA level was investigated. To perform this investigation, an infection model was setup using zebrafish and the gram-negative bacteria Vibrio anguillarum. Two different fish densities and bacterial concentrations were used to determine different patterns of pathogen eDNA in the fish tanks. Water sampling to collect eDNA was conducted alongside with observation of the fish Clinical Signs, mortalities, and immune responses. Water eDNA relative abundance of the pathogen could be determined directly after fish challenge, when Clinical Signs occurred, and when mortality started. Moreover, immune-relevant gene expression in zebrafish gills showed that il-1β, il-8, il-10, nf-κb and saa correlated to the pathogen eDNA signal, depending on fish density and bacterial concentration. In conclusion, this study demonstrated that water eDNA is a promising non-invasive tool to state disease progression and immune response in zebrafish infected with V. anguillarum.

Leptospirosis is a neglected zoonotic disease that is endemic in tropical regions, including Ecuador. It is caused by spirochetes of the genus Leptospira, which can infect humans through animal reservoirs such as rats and dogs, or through contact with contaminated water or soil. In March 2023, public health authorities declared a concerning outbreak of leptospirosis in Durán Cantón, located in the Coastal region of Ecuador. For the first time in the country, a multidisciplinary approach involving physicians and veterinarians was implemented for the surveillance and management of this leptospirosis outbreak. A total of 335 samples were collected, including suspected human cases, household contacts, household dogs, synanthropic rats, and water samples within the area of human cases. Samples were processed by qPCR targeting lipL32, secY, and rrs fragment genes and characterized further for Sanger sequencing. Overall, 26.2 % of human samples, 43.8 % of dog samples, 38.5 % of rat samples, and 39.4 % of water samples tested positive for Leptospira. Further, phylogenetic analysis shows that human, dog, and rat sequences are clustered within the pathogenic subclade P1, within the branch of L. kirschneri and L. interrogans. This study is the first of its kind in Ecuador, where an ongoing outbreak of leptospirosis was managed in real-time by using molecular diagnosis and not serological tools, and where the epidemiological surveillance was done following a One Health approach. This experience should inspire public and animal health authorities in Ecuador to promote a national One Health surveillance and control program for zoonotic diseases.

ABSTRACT: Growing broiler chickens of the Cobb500 strain were used to determine the effects on intestinal microbiota composition of a protein-free (PF) diet as compared to a diet based in casein (CAS) as the only protein source. CAS was formulated to contain the same amount of protein (190 g kg−1) as a commercial Maize-soy diet which was used as a practical reference. The ileal AA flow (g kg−1 dry matter intake) was significantly higher (P < 0.001) than PF in birds fed protein containing diets (CAS or Maize-soy). Taken as a whole (discriminant and ANOSIM analysis), the intestinal (ileal and caecal contents and ileal tissue) microbiota composition of PF and CAS were significantly (P < 0.001) different from Maize-soy and not different from each other in some cases. RT-qPCR and sequencing analysis of the ileal and caecal microbiota revealed significant (P < 0.05) differences in a number of bacterial groups between broilers fed PF, CAS or Maize-soy diets. The main result was that the lack of protein in the intestinal medium of PF birds resulted in a drop of Lactobacillus spp. counts (on average, 43 in PF vs 1,734 in the Maize-soy diet) and increased Enterobacteriaceae (on average, 419 in PF vs 172 in the Maize-soy diet) and other potentially pathogenic bacterial groups (in both intestinal contents and tissue). Thus, the lack of protein in the intestinal medium of PF birds resulted in a microbiota composition compatible with a pro-inflammatory state, and this effect was somewhat less marked in birds fed CAS. The results reported here suggest that the adverse effects on microbiota composition in broilers fed CAS were less marked than in those fed PF, which would be in line with a preferential use of a highly digestible protein containing diet to determine endogenous AA excretion instead of a PF diet.

Quantitative PCR (qPCR) is a technique commonly employed in laboratories and core facilities. In our previous study, we had shown the possibility to automate steps in a panel-specific gene expression workflow by pairing Mosquito HV with BioMark HD. Here we aimed to automate the full workflow and explore miniaturization capabilities. Each step of the gene expression workflow was scripted on Mosquito HV genomics software. We performed three different automated runs: i. Replicates of a Reference RNA sample (obtained by pooling RNA isolated from 10 healthy individuals) were run on an immunology gene expression panel. We tested the full reaction (FR) and three miniaturization conditions, namely: 1.5x, 2.5x and 5x; the data obtained from the automated FR replicates was compared to the data obtained from the manual processing; ii. Biological RNA samples (isolated from n = 45 individuals) were run as FR and 1.5x on the immunology gene expression panel; iii. Biological RNA samples (isolated from n = 45 individuals) were run as FR and 1.5x on a pregnancy gene expression panel. The expression of each gene was calculated using the 2(-delta Ct) method.Successful amplification was observed for the reference samples when using FR and 1.5x conditions. The 2.5x condition exhibited suboptimal amplification with a lower success rate while the 5x condition retrieved no amplification. The 2.5x and 5x miniaturization conditions were excluded from further runs. A strong significant positive correlation was observed between the manual and automated workflows for the reference RNA sample, underscoring the robustness of the gene expression assay. The automation of the immunology and pregnancy gene expression panels on the 45 individual samples retrieved a success rate >70 % for both the FR and the 1.5x miniaturization conditions. A significant positive correlation was also observed between the FR and 1.5x miniaturization conditions for both panels.Our results show that the adoption and the 1.5x miniaturization capabilities of Mosquito HV system for automating the gene expression workflow did not interfere with data quality and reproducibility.

The identification of novel therapeutic agents using nanomedicine is critical in the fight against diabetes mellitus (DM). Cholic acid (CA) has shown potential in diabetes management, but its effectiveness is limited by poor solubility and stability. To overcome these challenges, CA was encapsulated in chitosan nanoparticles (CACNPs). The CACNPs had a negative zeta potential (ZP) of −13.6 ± 5.81 mV, which is an indication of good stability and potential for enhanced uptake by diseased cells. The average particle size (PS) measured by dynamic light scattering (DLS) was 169.8 ± 84.3 nm. The polydispersity index (PDI) was 0.220, indicating uniform particle size distribution. The drug loading capacity (DL%) of the CACNPs was 60.96 ± 0.9 %, whereas, the entrapment efficiency (EE%) was 69.19 ± 1.02 %. The MTT assay on 3 T3-L1 cells revealed a concentration-dependent effect on cell viability, with an IC50 value of 766.0 ± 0.09 µg/ml. Furthermore, CACNPs demonstrated dose-dependent enhancement of glucose uptake in differentiated adipocytes, while at 500 µg/ml, they inhibited adipocyte differentiation, suggesting a potential role in adipogenesis inhibition. Quantitative PCR (qPCR) indicated positive modulation of glucose metabolism-related genes (PI3K, GLUT4, PPARg) upon treatment with CACNPs. These findings suggest that CACNPs could serve as a novel inhibitor of adipocyte differentiation and may influence key pathways in glucose metabolism, making them promising candidates for the management of DM.

Black tea is highly favored by consumers worldwide, with enzymatic reactions being recognized as a pivotal factor influencing tea quality. The role of microorganisms in shaping the composition of black tea has emerged as a focus of research due to their involvement in enzyme catalysis and metabolic processes. In this study, full-length amplicon sequencing combined with qPCR more accurately reflected microbial profile, and Pantoea, Pseudomonas, Paucibacter, and Cladosporium were identified as the main microbial genera. Moreover, by comprehensively analyzing color, aroma, and taste components over time in black tea samples, correlations were established between the dominant genus and various quality factors. Notably, peroxidase activity levels, total soluble sugar content, and tea pigments concentration exhibited significant associations with the dominant genus. Consequently, this microbiological perspective facilitated the exploration of driving factors for improving black tea quality while establishing a theoretical foundation for quality control in industrial production.

Heavy metal (HM) contamination affects the composition and structure of soil microbial communities, but there are few studies on the assembly process and co-occurrence network of soil microbial community succession driven by Cd in volcanic ecosystem. To address this gap in knowledge, we collected and analyzed soil samples from the Nvshan Volcanic area to understand the microbial characteristics in primary succession soil (PS) and secondary succession soil (SS). We found that the soil was contaminated with different levels of Cd (PS > SS), resulting in obvious heterogeneity of microorganisms. The absolute abundance of bacteria (16S rRNA gene copies) varied significantly between the two successions (P < 0.0001). The co-occurrence networks analysis showed that the number of nodes in bacterial communities was lower in PS compared to SS (1002 vs. 1004), indicating that heavy metal contamination would reduce the number of soil microbial communities. Compared with PS, bacterial communities exhibited stronger competitiveness in SS (positive: negative, P/N: 25.69 vs. 64.22), whereas fungal communities were closer symbiotic relationships (positive/negative, P/N: 15.85 vs. 14.29). The neutral community model (NCM) analysis revealed that stochastic processes predominantly governed the microbial assembly process (bacterial R2: 0.657, fungal R2: 0.686). The Mantel test analysis revealed that Cd was negatively associated with cbbLR, amoA, and phoD. The results of the Sankey diagram showed that fungi were more resistant than bacteria (27 vs. 13). This study contributes to understanding the process of soil microbial succession under Cd stress and identifying microbial strains with potential for Cd remediation.

ABSTRACT: The source of infection of digital dermatitis (DD), an infectious lameness condition, is still uncertain. In this cross-sectional study, we aimed to identify potential reservoirs of DD bacteria in dairy cattle body sites with different stages of the disease and farm environments. We collected skin swabs from 85 dairy cows from 5 herds, 3 with and 2 without DD, from foot, hock, and udder cleft skin (with lesions or not), saliva, urine, and feces. We also obtained environmental samples. Real-time quantitative PCR targeted Treponema phagedenis, Treponema medium, Treponema pedis, Porphyromonas levii, Bacteroides pyogenes, Fusobacterium necrophorum, and Fusobacterium mortiferum. Digital dermatitis-associated Treponema spp. were exclusively detected in DD-affected herds in DD-foot and other skin lesions, healthy skin, saliva, and environmental samples. In contrast, the non-Treponema spp. were found in samples from both DD-negative and affected herds. As expected, DD lesions had higher bacterial loads than healthy skin. Interestingly, similar counts were observed in udder cleft lesions, indicating a potential opportunistic behavior on compromised skin. None of the targeted species were detected in fecal samples, but P. levii, B. pyogenes, and F. necrophorum were detected in urine. All 7 species were detected in saliva, although in low quantities. No associations were observed between the presence of each bacterial species in DD lesions and urine; however, there was an association between the presence of DD-Treponema spp. in lesions and saliva, hock, and udder skin. Feces and urine do not seem to be a DD bacteria primary source, but saliva and other skin lesions may play a role. Longitudinal studies would improve our understanding of DD-associated bacteria's transient or persistent presence in these sites. Investigating the sources of DD-associated bacteria will guide future interventions to minimize bacterial shedding and transmission, ultimately more effectively reducing bacterial load, transmission, and sources of infection in dairy herds.

Objectives: This study aimed to investigate the prevalence of mpox virus (MPXV) infections in the general population consulting for routine sexually transmitted infections (STIs) in our Marseille public hospital. In fact, the recent worldwide MPXV outbreak mainly impacted men who have sex with men and the prevalence of MPXV infections in the general population remains poorly defined. Methods: All samples addressed routinely to our microbiological laboratory for STIs between July 1 and October 15, 2022 were screened with MPXV-specific quantitative polymerase chain reaction. Results: A total of 2688 samples from 1896 patients suspected of having STIs were tested and eight (0.4%) patients were incidentally diagnosed with MPXV infection, including six men and two women. MPXV was detected in rectal swabs (n = 2), urine (n = 2), vaginal swabs (n = 2), a urethral swab (n = 1), and a skin swab (n = 1). Conclusions: This study suggests that some MPXV infections are likely to be underdiagnosed because of their non-specific clinical presentation and/or insufficient clinical knowledge of the disease. Our data showed that systematic screening was particularly useful for detecting MPXV in patients without classic lesions or cases of asymptomatic carriage in patients reporting recent high-risk exposure and in patients presenting no obvious risk factor.

Insects, such as black soldier flies (BSF), are sustainable alternatives to fishmeal and fish oil that have been shown maintain fish growth performance, especially at low dietary inclusions. However, BSF is commonly processed to remove chitin and oil that may have additional benefits to the immune response and disease resistance of fish. The aim of this study was to investigate the hepatic gene expression and plasma biochemistry of rainbow trout (Oncorhynchus mykiss) fed BSF meals and BSF components. A three-month feeding trial was performed where rainbow trout (90.0 ± 4.0g; mean ± SD) were fed either a control diet or diets that contained: 10 % full-fat BSF, 10 % defatted BSF, 4 % BSF oil, and 1 % BSF chitin. Liver was analyzed for gene expression via qPCR and plasma was analyzed for 21 biochemical markers. The 4 % oil and 1 % chitin diets had the highest effect on growth performance, i.e. final weight (p=0.043) and feed intake (p=0.017), compared to the control diet, while no effects of these diets were found on hepatic gene expression. The 10 % full-fat BSF diet affected gene expression, specifically upregulating the pro-inflammatory cytokine IL-8 (p=0.033), which may be due to the higher content of saturated fatty acids (SFA) and subsequently immune cell activity in the fish or the combination of the SFA and antimicrobial peptides in BSF that stimulate the innate immune system. No other immune and oxidative stress genes, or 21 plasma biochemical markers were found to be significantly affected (p>0.05). This research provides support to include and not remove oil and chitin from BSF since these components can improve growth performance and immune response of rainbow trout.

Harmful Algal Blooms (HAB) have the potential to impact human health primarily through their possible cyanotoxins production. While conventional water treatments can result in the removal of unlysed cyanobacterial cells and low levels of cyanotoxins, during severe HAB events, cyanotoxins can break through and can be present in the treated water due to a lack of adequate toxin treatment. The objectives of this study were to assess the HAB conditions in drinking water sources in New Jersey and investigate relationships between environmental variables and cyanobacterial communities in these drinking water sources. Source water samples were collected monthly from May to October 2019 and analyzed for phytoplankton and cyanobacterial cell densities, microcystins, cylindrospermopsin, Microcystis 16S rRNA gene, microcystin-producing mcyB gene, Raphidiopsis raciborskii-specific rpoC1 gene, and cylindrospermopsin-producing pks gene. Water quality parameters included water temperature, pH, dissolved oxygen, specific conductance, fluorescence of phycocyanin and chlorophyll, chlorophyll-a, total suspended solids, total dissolved solids, dissolved organic carbon, total nitrogen, ammonia, and total phosphorus. In addition to source waters, microcystins and cylindrospermopsin were analyzed for treated waters. The results showed all five selected New Jersey source waters had high total phosphorus concentrations that exceeded the established New Jersey Surface Water Quality Standards for lakes and rivers. Commonly found cyanobacteria were identified, such as Microcystis and Dolichospermum. Site E was the site most susceptible to HABs with significantly greater HAB variables, such as extracted phycocyanin, fluorescence of phycocyanin, cyanobacterial cell density, microcystins, and Microcystis 16S rRNA gene. All treated waters were undetected with microcystins, indicating treatment processes were effective at removing toxins from source waters. Results also showed that phycocyanin values had a significantly positive relationship with microcystin concentration, copies of Microcystis 16S rRNA and microcystin-producing mcyB genes, suggesting these values can be used as a proxy for HAB monitoring. This study suggests that drinking water sources in New Jersey are vulnerable to forthcoming HAB. Monitoring and management of source waters is crucial to help safeguard public health.

The last decade, smoke and smokeless products claiming to be tobacco-free, including herbal cigarettes and herbal shisha, became available on the European market and gained popularity. This study proposes a new digital droplet PCR (ddPCR) method, designed based on a previously developed real-time PCR (qPCR) method being currently used by the U.S. Food and Drug Administration (FDA) to specifically detect the presence of tobacco DNA in targeting a sequence from the Nicotiana tabacum nia-1 gene. To ensure a harmonized and reliable control by enforcement laboratories, both of these qPCR and ddPCR methods were then evaluated and validated for their compliance to an international standard. First, the performance of these PCR-based methods was successfully assessed as specific and sensitive, and in line with minimum performance requirements from international standard. Secondly, the transferability to external laboratory was confirmed for these PCR-based methods. Finally, the applicability of these PCR-based methods was demonstrated using 7 ground tobacco reference materials from the Tobacco Research Center (TRC) Toronto University as well as 6 commercial smokeless and tobacco-free smoke and smokeless products. Based on this study, the previously developed qPCR method was confirmed as complying with international standard, ensuring a efficient and harmonize use by enforcement laboratories for tobacco control on the European market. Moreover, this study proposed to enforcement laboratories the possibility to use a ddPCR method, enabling the simultaneous detection and absolute quantification of tobacco DNA as well as a limited impact of PCR inhibitors.

Background: The Grand Magal of Touba (GMT) associates with risks of infection, but no study on the circulation of resistant bacteria has yet been conducted. Materials and methods: qPCR was performed on rectal samples from GMT pilgrims between 2018 and 2021, before and after their participation in the gathering. Rectal samples from between 2018 and 2020 were also cultured on specific media, and antibiotic susceptibility testing was performed. Results: Forty-one of the 296 (13.8%) pilgrims had at least one gastrointestinal symptom and 91/290 (31.4%) acquired pathogenic bacteria, mostly Escherichia coli. A total of 54.7% of pilgrims reported washing their hands more frequently than usual and 89.2% used soap. One hundred and five (36.2%) acquired at least one resistance gene, notably CTX-M A (21.0%), SHV (16.5%) and TEM (8.2%). The strains isolated by culture were mostly E. coli. These bacteria were found to be sensitive to carbapenems and resistant to amoxicillin and amoxicillin-clavulanic acid. The acquisition of enteroaggregative E. coli was independently associated with CTX-M A and TEM acquisition. Conclusion: Pilgrims presented a risk for acquisition of CTX-M A after the GMT. Surveillance of the prevalence of resistant bacteria and the occurrence of associated clinical infections among pilgrims are necessary in the future.

Antibiotic resistance (AR) is a major global health concern, but current surveillance efforts primarily focus on healthcare settings, leaving a lack of understanding about AR across all sectors of the One Health approach. To bridge this gap, wastewater surveillance provides a cost-effective and efficient method for monitoring AR within a population. In this study, we implemented a surveillance program by monitoring the wastewater effluent from two large-scale municipal treatment plants situated in Isfahan, a central region of Iran. These treatment plants covered distinct catchment regions and served a combined population about two million of residents. Furthermore, the effect of physicochemical and microbial characteristics of wastewater effluent including biological oxygen demand (BOD), chemical oxygen demand (COD), total suspended solids (TSS), temperature, total coliforms and Escherichia coli concentration on the abundance of ARGs (blaCTX-M, tetW, sul1, cmlA, and ermB) and class 1 integron-integrase gene (intI1) were investigated. Sul1 and blaCTX-M were the most and least abundant ARGs in the two WWTPs, respectively. Principal Component Analysis showed that in both of the WWTPs all ARGs and intI1 gene abundance were positively correlated with effluent temperature, but all other effluent characteristics (BOD, COD, TSS, total coliforms and E. coli) showed no significant correlation with ARGs abundance. Temperature could affect the performance of conventional activated sludge process, which in turn could affect the abundance of ARGs. The results of this study suggest that other factors than BOD, COD and TSS may affect the ARGs abundance. The predicted AR could lead to development of effective interventions and policies to combat AR in the clinical settings. However, further research is needed to determine the relationship between the AR in wastewater and clinical settings as well as the effect of other influential factors on ARGs abundance.

Surveillance data for Ancylostoma spp. and the A. caninum benzimidazole treatment resistance associated F167Y polymorphism using molecular diagnostics was obtained in a large population of dogs from the United States and Canada. Real-time PCR (qPCR) for Ancylostoma spp. and allele-specific qPCR detecting a single nucleotide polymorphism (SNP) F167Y was used in 262,872 canine stool samples collected between March and December of 2022. Ancylostoma spp. was found at an overall prevalence of 2.5% (6538/262,872), with the highest prevalence in the Southern US, 4.4% (4490/103,095), and the lowest prevalence in Canada 0.6% (101/15,829). The A. caninum F167Y polymorphism was found with the highest prevalence (13.4%, n = 46/343) in the Western US and the lowest in Canada at 4.1% (4/97). The F167Y polymorphism was detected every month over the 10-month collection period. Seasonal distribution showed a peak in June for both Ancylostoma spp. (3.08%, 547/17,775) and A. caninum F167Y (12.25%, 67/547). However, the A. caninum F167Y polymorphism prevalence was highest in September (13.9%, 119/856). Age analysis indicates a higher prevalence of both hookworm infections and occurrence of resistant isolates in puppies. The breeds with the highest F167Y polymorphism prevalence in Ancylostoma spp. detected samples were poodles (28.9%), followed by Bernese Mountain dogs (25%), Cocker spaniels (23.1%), and greyhounds (22.4%). Our data set describes widespread geographic distribution of the A. caninum benzimidazole resistance associated F167Y polymorphism in the United States and Canada, with no clear seasonality compared to the Ancylostoma spp. prevalence patterns. The F167 polymorphism was present in all geographic areas with detected hookworms, including Canada. Our study highlights that the F167Y polymorphism is represented in many dog breeds, including greyhounds.

Apis mellifera yemenitica is the native honeybee of the Arabian Peninsula. It demonstrates unique morphological, behavioral, and molecular aspects to survive extreme summer temperatures dominating the region. In this study, relative expression of Apis mellifera lethal(2)-essential-for-life-like (l(2)efl) gene family (ID: 724405; 724488; 234274) were measured in A. m. yemenitica and A. m. carnica under desert (Riyadh) and semiarid (Baha) conditions of Saudi Arabia. Results demonstrated significantly higher day-long expression levels of l(2)efl mRNAs in A. m. yemenitica than in A. m. carnica under the same conditions. Under desert conditions of Riyadh, fold changes in expression levels of l(2)efl mRNAs were ∼100× higher in A. m. yemenitica than the calibrator. In Baha (semiarid conditions), l(2)efl expression levels were very modest compared to those in Riyadh with significant interaction between location and subspecies. In conclusion, expression of l(2)efl (mRNAs) (724405; 724488; 234274) can be considered as a key component of A. m. yemenitica response to extreme desert temperatures characterizing the Riyadh region. On the other hand, the semi-arid conditions (Baha) are a more suitable habitat for A. m. carnica compared to Riyadh.

Drought is one of the important abiotic factors that affect faba bean growth and productivity in the Mediterranean region. In order to study the response of faba bean plant to water-deficit stress, a physiological and proteomic analysis was carried out in leaf tissue. All physiological parameters were affected by drought. The physiological mechanism underlying the response of faba bean leaves to water-deficit was therefore attributed to the alleviation of oxidative stress via the accumulation of proline and to the synergistic action of the antioxidant enzyme system (CAT, SOD, APX and GPOX). Proteomic analysis identified 2000 proteins from faba bean leaves, of which were 81 differentially expressed. Of those, 36 were downregulated and 45 were upregulated under water-deficit treatment. KEGG and GO enrichments indicated differentially abundant proteins (DAPs) related to photosynthesis, antioxidants and ROS detoxifying enzymes, biosynthesis of amino acids and secondary metabolites, molecular chaperones, signal transduction, energy and carbohydrate metabolism and metabolic enzymes. The current results provide evidence for a complex synergetic pathway, in which ROS detoxification mechanisms and photoprotection constituted the major aspect of water-deficit tolerance in faba bean leaves. These results offer a foundational basis regarding the molecular mechanism involved in drought resistance within the faba bean species.

Large bone defects after trauma demand for adequate bone substitutes. Bone void fillers should be antibacterial and pro-angiogenic. One viable option is the use of composite materials like the combination of PLGA and amorphous calcium phosphate (aCaP).Copper stimulates angiogenesis and has antibacterial qualities. Either copper oxide (CuO) nanoparticles (NPs) were therefore added to PLGA/aCaP/CuO in different concentrations (1, 5 and 10 w/w %) or copper-doped tricalcium phosphate NPs (TCP with 2% of copper) were electrospun into PLGA/CuTCP nanocomposites.Bi-layered nanocomposites of PLGA/aCaP with different copper NPs (CuO or TCP) and a second layer of pristine PLGA were fabricated. Two clinical bacterial isolates (Staphylococcus aureus and Staphylococcus epidermidis) were used to assess antibacterial properties of the copper-containing materials. For angiogenesis, the chorioallantoic membrane (CAM) assay of the chicken embryo was performed.The higher the CuO content, the higher were the antibacterial properties, with 10 % CuO reducing bacterial adhesion most effectively. Vessel and cell densities were highest in the 5 % CuO containing scaffolds, while tissue integration was more pronounced at lower CuO content. The PLGA/aCaP/CuO (1 % CuO) behaved similar like PLGA/CuTCP in all angiogenic and antibacterial readouts, based on the same copper fraction.We conclude that CuO NPs or CuTCP NPs are useful components to increase angiogenic properties of nanocomposites and at the same time exhibiting antibacterial characteristics.

Environmental oligotrophic bacteria are suspected to be highly relevant carriers of antimicrobial resistance (AMR). However, there is a lack of validated methods for monitoring in the aquatic environment. Since extended-spectrum β-lactamases (ESBLs) play a particularly important role in the clinical sector, a culturing method based on R2A-medium spiked with different combinations of β-lactams was applied to quantify β-lactamase-producing environmental bacteria from surface waters. In German surface water samples (n = 28), oligotrophic bacteria ranging from 4.0 × 103 to 1.7 × 104 CFU per 100 mL were detected on the nutrient-poor medium spiked with 3rd generation cephalosporins and carbapenems. These numbers were 3 log10 higher compared to ESBL-producing Enterobacteriales of clinical relevance from the same water samples. A MALDI-TOF MS identification of the isolates demonstrated, that the method leads to the isolation of environmentally relevant strains with Pseudomonas, Flavobacterium, and Janthinobacterium being predominant β-lactam resistant genera. Subsequent micro-dilution antibiotic susceptibility tests (Micronaut-S test) confirmed the expression of β-lactamases. The qPCR analysis of surface waters DNA extracts showed the presence of β-lactamase genes (blaTEM, blaCMY-2, blaOXA-48, blaVIM-2, blaSHV, and blaNDM-1) at concentrations of 3.7 (±1.2) to 1.0 (±1.9) log10 gene copies per 100 mL. Overall, the results demonstrate a widespread distribution of cephalosporinase and carbapenemase enzymes in oligotrophic environmental bacteria that have to be considered as a reservoir of ARGs and contribute to the spread of antibiotic resistance.

Cassava (Manihot esculenta Crantz) is a crop of global economic and food safety importance, used for human consumption and in various industrial applications. The genebank of the Genetic Resources Program of the Alliance of Bioversity International and CIAT currently holds the world's largest cassava collection, with 5965 in vitro accessions from 28 countries. Managing this extensive collection involves indexing quarantine pathogens as a phytosanitary certification requirement for safely distributing cassava germplasm. The study therefore aimed to optimize a quantitative diagnostic protocol to detect cassava common mosaic virus (CsCMV) using quantitative PCR (qPCR) as a better alternative to other molecular techniques. This was done through designing primers and a probe in the RdRP region of CsCMV, and optimizing the qPCR conditions of the diagnostic protocol using primer concentration assays, and reaction amplification conditions such as volume and reaction time. We also evaluated the qPCR protocol by comparing the results of 140 cassava accession evaluations using three diagnostic methodologies (DAS-ELISA, end-point PCR, and qPCR) for CsCMV. Our protocol established that qPCR technique analysis is ten-times more sensitive in detecting CsCMV compared to end-point PCR, showing a maximum detection level of 77.97 copies/μL of plasmid, with 76 min of reaction time. The comparison allowed us to verify the level of CsCMV detection through the techniques evaluated, concluding that qPCR was more sensitive and allowed the quantification of viral concentration. The optimized qPCR protocol will be used to accelerate diagnostic screening of cassava germplasm for the presence or absence of CsCMV to ensure safe movement and distribution of disease-free germplasm.

Cutaneous leishmaniasis (CL) is the most important neglected disease reported in North Africa, Algeria ranks second in the world with more than 5000 cases per year. In Algeria, two rodent species Psammomys obesus and Meriones shawi, are so far known as proven reservoirs of Leishmania major, however, they are absent in several endemic localities. In this study, we experimentally infected Gerbillus rodents trapped around human dwellings in Illizi, Algeria to assess their susceptibility to L. major. Seven gerbils, morphologically and molecularly identified as Gerbillus amoenus, were intradermally inoculated with 104 parasites derived from culture, monitored for six months and their infectiousness for sand flies was tested by xenodiagnosis. The study revealed that G. amoenus was susceptible to L. major and was able to maintain and transmit the parasites to sand flies tested six months after infection, suggesting the role of this gerbil as a potential reservoir for L. major.

Toxoplasma gondii is a successful coccidian parasite able to infect all warm-blooded animals and humans, causing one of the most common zoonoses worldwide. The Eurasian lynx (Lynx lynx) is one of the feline potential hosts of T. gondii in Switzerland, but little is known about its epidemiological role as a definitive or intermediate host. Serum samples from 183 Eurasian lynx collected from 2002 to 2021 were tested for antibodies to T. gondii by ELISA, IFAT and in case of inconclusive results, immunoblot. Antibodies to T. gondii were found in 150 of 183 (82%) Eurasian lynx. Older age, good health status and a low-altitude habitat were found to be significant predictors for seropositivity. T. gondii oocysts were detected in 3 of 176 (1.7%) faecal samples, indicating the Eurasian lynx as a definitive host. In addition, T. gondii DNA was detected in skeletal muscle (7/88), heart muscle (2/26) and/or brain tissue (2/36) from 10 different lynx by real-time PCR. In one animal, a T. gondii-like tissue cyst was observed in heart muscle and confirmed as T. gondii by immunohistochemistry (1/20) and real-time PCR. With an adapted nested-PCR-multilocus-sequence typing (MLST) and in silico restriction-fragment-length-polymorphism analysis (RFLP) approach two different T. gondii genotypes were detected: a lineage II variant (ToxoDB #3) in three animals (two oocyst samples and one heart muscle sample) and a novel genotype exhibiting both type II and III alleles in a further animal (skeletal muscle). The present results indicate that T. gondii infection is widespread in the Swiss lynx population. The Eurasian lynx may contribute to environmental contamination with oocysts and is able to harbour the parasite in different tissues. Genotyping revealed the presence of both a common T. gondii lineage in Europe and a previously unknown genotype and thus shedding more light on the complex molecular epidemiology of T. gondii.

ABSTRACT: Objectives: Antibiotic resistance genes (ARGs) are causing increasing problems, especially in clinical settings. Nowadays, they are considered important environmental contaminants, but little is known about their fate in the environment or how they affect natural microbial populations. In the environment, especially in water affected by anthropic activities such as discharge of hospital, urban, and industrial wastewater treatment plant (WWTP) and agricultural runoff, antibiotic determinants may become part of the environmental gene pool, spread horizontally, and be ingested by humans and animals via contaminated food and drinking water.The aim of this work was to monitor long-term the presence of antibiotic resistance determinants in water samples collected from a subalpine lake and some tributary rivers located in the southern part of Switzerland, and to assess if anthropic activities could influence the distribution of antibiotic resistance genes present in water environments. Methods: We analysed water samples by qPCR to quantify five antibiotic resistance genes that confer resistance to the major classes of antibiotics used in clinical and veterinary settings (β-lactams, macrolides, tetracycline, quinolones, and sulphonamides). Water samples were collected from January 2016 to December 2021, from three rivers located in south Switzerland and from five different sites of Lugano Lake. Results: The most abundant genes were sulII, followed by ermB, qnrS, and tetA; they were found especially in the river influenced by wastewater treatment plants and in the lake near the potable water uptake plant. We observed an overall decrease in the number of resistance genes during the three years. Conclusions: Our results indicate that the aquatic ecosystems monitored in this study are a reservoir of ARGs and could potentially be a setting for the transmission of resistance from the environment to humans.

Ostreid herpesvirus 1 (OsHV-1) is one of the most economically important pathogens of Pacific oysters. Understanding the pathogenesis of this virus is critical to developing tools to control outbreaks on shellfish farms. OsHV-1 is genetically related to vertebrate herpesviruses, which have a lytic and a latent stage, with the latent stage capable of being reactivated to the lytic stage. Here, OsHV-1 latency in Pacific oysters was investigated in experimentally and naturally infected oysters. Lytic infection in one-year-old oysters injected with the Tomales Bay strain of OsHV-1 was detectable between 1 and 4 days post-injection (dpi) but was not detectable after 5 dpi. The injected oysters shed 1 × 102 to 1 × 104 DNA copies/ml into the water during the 4-day acute phase. Lytic shedding was not detectable in two-year-old oysters injected similarly with the same strain of OsHV-1; however, the OsHV-1 genome was detectable by qPCR in the adductor muscle, gill, mantle, and hemocytes within the first 3 dpi, after which it became undetectable. No OsHV-1 was detectable in the adductor muscle, gill, or mantle from experimentally infected oysters on days 15 and 21 post-injection or from oysters sampled 9 months after surviving an OsHV-1 mortality event; however, OsHV-1 DNA could be detected in hemocytes of both experimentally infected oysters at 21 dpi and naturally infected oysters using nested PCR. In addition, lytic viral gene transcription was detectable in hemocytes of experimentally infected oysters between 1 and 21 dpi and in hemocytes of naturally infected oysters. Furthermore, OsHV-1 reactivation from latency was induced in experimentally infected oysters at 21 dpi and in naturally infected oysters 12 months after an OsHV-1 outbreak.

Sponges play important role within aquatic ecosystems due to their diverse abilities including filter-based feeding mechanisms. Hence, this study evaluated the potential use of sponges as ecological biomonitors for water safety surveillance, especially in the presence of Waterborne protozoan pathogens WBPP. Sponge specimens were collected from different Qatari marine ecosystems and subjected to gDNA extraction and real-time PCR using specific primer sets for the most common WBPP. Two sponges from the coastal marine ecosystems were found to be positive for Blastocystis sp., and one sponge was positive for Dientamoeba fragilis within offshore site. No Cryptosporidium spp., Giardia duodenalis, nor Toxoplasma gondii were detected. Further genotyping analysis revealed that the Blastocystis sp. positive samples were subtype ST3 (allele 34), which matched local clinical isolates and D. fragilis specimen was unambiguously clustering with Genotype 2. In conclusion, this study demonstrates the role of marine sponges as ecological biomonitors for WBPP screening and provide insights into these pathogens widespread and their potential transmission to marine and terrestrial organisms including human.

Acid mine drainage (AMD) is widely acknowledged as a substantial threat to the biodiversity of aquatic ecosystems. The present study aimed to study the toxicological effects of Cu-rich AMD from the Sulitjelma mine in zebrafish larvae. The AMD from this mine was found to contain elevated levels of dissolved metals including Mg (46.7 mg/L), Al (20.2 mg/L), Cu (18.3 mg/L), Fe (19.8 mg/L) and Zn (10.6 mg/L). To investigate the toxicological effects, the study commenced by exposing zebrafish embryos to various concentrations of AMD (ranging from 0.75% to 9%) to determine the median lethal concentration (LC50). Results showed that 96 h LC50 for zebrafish larvae following AMD exposure was 2.86% (95% CI: 2.32–3.52%). Based on acute toxicity results, zebrafish embryos (

Vanadium (V) has garnered attention due to its pharmacological properties; however, its toxic effects have also been documented. Among the vanadium compounds that are found in the environment, vanadium trioxide (V2O3) has attracted interest because of its impact on biomolecules such as DNA, RNA, and proteins. However, its precise mechanism of action remains unclear, although it is suspected to be related to oxidative stress. Therefore, this study aimed to determine the mechanisms involved in DNA damage and the associated cellular response pathways. Primary cultures of human lymphocytes were exposed to 2, 4, 8, or 16 µg/mL V2O3. DNA damage due to oxidized bases was evaluated via a comet assay. The expression levels of sensor proteins (ATM and ATR) involved in DNA damage were determined via Western blotting, and the mRNA expression levels of metallothionein 1 (Mt-1) and genes involved in DNA repair (OGG1, APE1, XPB, XPD, MRE11, RAD50, Ku70, and Ku80) were estimated via RT-PCR and qPCR. The results showed that V2O3 is an oxidant that is responsible for DNA damage through oxidized bases, as demonstrated by increased DNA migration in the presence of the FPG enzyme. At the molecular level, V2O3 treatment also increased ATM protein expression. In terms of mRNA expression, the overexpression of Mt-1, OGG1, APE1, Ku70, and Ku80 was observed. This finding suggests that DNA damage is primarily repaired via two mechanisms: base excision repair (BER) and nonhomologous end joining (NHEJ). In conclusion, one mechanism of action of V2O3 involves the oxidation of nitrogenous bases in DNA, the activation of damage sensors (such as ATMs), and the overexpression of Mt-1 as part of the antioxidant response to mitigate the effects of V and facilitate DNA repair pathways (including BER and NHEJ).

Adeno-associated virus (AAV) vectors are among the most prominent viral vectors for in vivo gene therapy, and their investigation and development using high-throughput techniques have gained increasing interest. However, sample throughput remains a bottleneck in most analytical assays. In this study, we compared commonly used analytical methods for AAV genome titer, capsid titer, and transducing titer determination with advanced methods using AAV2, AAV5, and AAV8 as representative examples. For the determination of genomic titers, we evaluated the suitability of qPCR and four different digital PCR methods and assessed the respective advantages and limitations of each method. We found that both ELISA and bio-layer interferometry provide comparable capsid titers, with bio-layer interferometry reducing the workload and having a 2.8-fold higher linear measurement range. Determination of the transducing titer demonstrated that live-cell analysis required less manual effort compared with flow cytometry. Both techniques had a similar linear range of detection, and no statistically significant differences in transducing titers were observed. This study demonstrated that the use of advanced analytical methods provides faster and more robust results while simultaneously increasing sample throughput and reducing active bench work time.

Studies are lacking that investigate the dietary nutrient requirements of lake whitefish (Coregonus clupeaformis), a newly farmed fish species in Ontario, Canada. Dietary levels of protein and lipid must be optimized to ensure high growth performance for the commercial success of this species. Additionally, the inclusion of insect meal in the diet may improve growth and immune response. The objective of this study was to evaluate the effects of dietary protein:lipid ratios and insect meal as a feed additive on the growth performance and hepatic immune function of juvenile lake whitefish (301 ± 10 g). A 16-week (112 day) trial was performed with five diets including a commercial control diet (BCC), and four experimental diets with high or low levels of protein (54 and 48%, respectively) and lipid (18 and 12%, respectively). The high protein dietary groups contained 5% of full-fat black soldier fly larvae (Hermetia illucens). Fish weights, viscera, liver, and blood were collected for further analysis. Specific growth rate, thermal growth coefficient and weight gain were significantly higher in fish fed with the BCC and high protein high lipid (HPHL) diets. However, viscerosomatic index was found to be significantly higher in fish fed the BCC diet, thus HPHL is more optimal for non-visceral weight gain. Higher levels of plasma phosphorus, aspartate aminotransferase and potassium indicated poor growth and stress in fish fed low lipid diets. Relative expression of HSP70, involved in cellular repair, was significantly downregulated in fish fed high lipid diets, and no effects were found on the expression of innate immune and oxidative stress genes. Also, IL8 (CXCL8) and catalase were upregulated (non-significant) in fish fed the HPHL diet with the largest weight gain. No effects of insects were found on growth, plasma biochemistry or gene expression, which suggests 5% dietary inclusion was too low. Overall, we recommend a HPHL diet for the cultivation of lake whitefish based on improved growth performance, low viscera weight, improved plasma biochemistry and downregulation of cellular repair genes.

Background: Colorectal cancer (CRC) is one of the primary causes of cancer-associated deaths worldwide, and growing evidence shows that alteration in the gut microbiota may be a contributing factor to the development and progression of the disease. This study investigates the correlation between CRC and specific intestinal microbiota abundance, including Firmicutes, Lactobacillus, Enterococcus, Clostridium, and Bifidobacterium. Material and methods: In this study, 100 CRC samples and adjacent normal tissues were obtained from Iranian patients. Afterward, we assessed the abundance of the mentioned bacteria in matched tumor and normal tissue samples from 100 CRC patients, by TaqMan quantitative real-time polymerase chain reaction (qPCR). Results: Most of the patients (55 %) had grade II cancer (moderately differentiated), followed by grade III (poorly Differentiated) in 19 %, and the distribution of the tumor location was 65 % in the colon and 35 % in the rectum. Our research showed a substantial difference in the relative abundance of specific bacteria in tumors and healthy tissues. To this end, four genera of bacteria, including Bifidobacterium, Lactobacillus, Clostridium, and Firmicutes, exhibited statistically significant reductions in tumor tissues compared to adjacent normal tissue (p

Here, a method using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER) for miRNAs quantification was established. The strategy has two steps: (1) ligation of two DNA probes specifically hybridize to target miRNA and (2) qPCR amplifying the ligated probe. The miRNA-binding regions of the probes are stem-looped, a motif significantly reduces nonspecific ligation at high ligation temperature (65°C). The ends of the probes are designed complementary to form a paired probe, facilitating the recognition of target miRNAs with low concentrations. RNase H proved to be able to stabilize the heteroduplex formed by the probe and target miRNA, contributing to enhanced sensitivity (limit of detection = 60 copies). High specificity (discriminating homology miRNAs differing only one nucleotide), wide dynamic range (seven orders of magnitude) and ability to accurately detect plant miRNAs (immune to hindrance of 2′-O-methyl moiety) enable SPLICER comparable with the commercially available TaqMan and miRCURY assays. SYBR green I, rather than expensive hydrolysis or locked nucleic acid probes indispensable to TaqMan and miRCURY assays, is adequate for SPLICER. The method was efficient (

In addition to fulfilling many breeders’ curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

Arctic tundra fires have been increasing in extent, frequency and intensity and are likely impacting both soil nitrogen (N) and phosphorus (P) cycling and, thus, permafrost ecosystem functioning. However, little is known on the underlying microbial mechanisms, and different fire intensities were neglected so far. To better understand immediate influences of different fire intensities on the soil microbiome involved in nutrient cycling in permafrost-affected soil, we deployed experimental fires with low and high intensity on an Arctic tundra soil on Disko Island, Greenland. Soil sampling took place three days postfire and included an unburned control. Using quantitative real-time PCR, copy numbers of 16S and ITS as well as of 17 genes coding for functional microbial groups catalyzing major steps of N and P turnover were assessed.We show that fires change the abundance of microbial groups already after three days with fire intensity as key mediating factor. Specifically, low-intensity fire significantly enhanced the abundance of chiA mineralizers and ammonia-oxidizing archaea, while other groups were not affected. On the contrary, high-intensity fire decreased the abundance of chiA mineralizers and of microbes that fix dinitrogen, indicating a dampening effect on N cycling. Only high-intensity fires enhanced ammonium concentrations (by an order of magnitude). This can be explained by burned plant material and the absence of plant uptake, together with impaired further N processing. Fire with high intensity also decreased nirK-type denitrifiers. In contrast, after fire with low intensity there was a trend for a decreased nosZ : (nirK+nirS) ratio, indicating – together with increased nitrate concentrations – an enhanced potential for nitric oxide and nitrous oxide emissions. Concerning P transformation, only gcd was affected in the short term which is important for P solubilization.Changes in gene numbers consistently showed the same contrasting pattern of elevated abundance with low fire intensity and decreased abundance with high fire intensity. Differentiating fire intensities is therefore crucial for further, longer-term studies of fire-induced changes in N and P transformations and potential nutrient-climate feedbacks of permafrost-affected soils.

In this study, the presence of Acanthamoeba spp., Blastocystis sp., Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia sp., Toxoplasma gondii and Vermamoeba vermiformis was assessed in organic leafy green vegetables (lettuce, spinach, cabbage) and fruits (strawberry), which are usually consumed raw. A total of 110 organic samples were collected in Valencia (Spain). Protozoa were concentrated before detection by immunofluorescence (Cryptosporidium spp. and Giardia sp.) or real-time qPCR (Acanthamoeba spp., Blastocystis sp., C. cayetanensis, E. histolytica, T. gondii and V. vermiformis). The most abundant protozoa in organic vegetables and berry fruits were Acanthamoeba (65.5%), followed by T. gondii (37.2%), V. vermiformis (17.3%), C. cayetanensis (12.7%), Cryptosporidium spp. (6.8%), Blastocystis sp. (1.8%) and Giardia sp. (1.7%). E. histolytica was not found in any of the organic samples. Thus, results showed that consumers can be exposed to protozoan parasites by consuming organic vegetables and berry fruits. This is the first report in Spain describing the presence of the protozoan pathogens Acanthamoeba spp., Blastocystis sp., C. cayetanensis, T. gondii and V. vermiformis, Cryptosporidium spp. and Giardia sp. in organic fresh produce. The results of this research will help determine the risk of foodborne protozoan parasites on organic leafy greens and strawberries that are available at local markets.

The environment is an important component in the emergence and transmission of antimicrobial resistance (AMR). Despite that, little effort has been made to monitor AMR outside of clinical and veterinary settings. Partially, this is caused by a lack of comprehensive reference data for the vast majority of environments. To enable monitoring to detect deviations from the normal background resistance levels in the environment, it is necessary to establish a baseline of AMR in a variety of settings. In an attempt to establish this baseline level, we here performed a comprehensive literature survey, identifying 150 scientific papers containing relevant qPCR data on antimicrobial resistance genes (ARGs) in environments associated with potential routes for AMR dissemination. The collected data included 1594 samples distributed across 30 different countries and 12 sample types, in a time span from 2001 to 2020. We found that for most ARGs, the typically reported abundances in human impacted environments fell in an interval from 10-5 to 10-3 copies per 16S rRNA, roughly corresponding to one ARG copy in a thousand bacteria. Altogether these data represent a comprehensive overview of the occurrence and levels of ARGs in different environments, providing background data for risk assessment models within current and future AMR monitoring frameworks.

The aim of this study was to estimate the occurrence of Bartonella spp. per household in cats and the risk factors for Bartonella spp. positivity in cats and their owners from Valdivia, Chile. A total of 464 cats (distributed within 324 households) and 326 humans (control group [n = 112] and cat owner [n = 214]) distributed in 262 households were sampled. From the cat owners (n = 214), 128 humans were in households where the cat was also sampled, totaling 84 households with dual sampling. Real-time PCR (qPCR) was used for Bartonella spp. detection in blood from cats and humans, and immunofluorescent immunoassay (IFA) anti-Bartonella henselae was performed in human serum samples. Out of the total of 324 households, 20.43% presented at least one Bartonella positive cat. From the households with dual sampling, 29.7% (25/84) presented at least one qPCR-Bartonella spp. positive cat. However, Bartonella DNA was not amplified in humans, and in 7.3% (6/82) of the households was found at least one of the cat's owners exposed to B. henselae. Cats younger than one year (Odds Ratio (OR) = 5.3), non-neutered (OR 3.46), sampled at home (OR 5.82), and with improper application of tick/flea control products (OR 3.13) showed a higher risk for Bartonella spp. presence. Humans with occupational exposure involving animal contact, were more likely to exhibit B. henselae seropositivity (OR 7.5). Bartonella spp. was present in the cats a moderate number of households, but Bartonella DNA was not detected in owners' blood, inferring that there is a low risk of recent human infection in the studied population.

One third of the world population has a history of exposure to the hepatitis B virus (HBV), and two billion people are infected with latent tuberculosis (TB). Occult hepatitis B infection (OBI) is defined as the presence of replicative-competent HBV DNA in the liver with detectable or undetectable HBV DNA in the serum of individuals testing negative for the HBV surface antigen (HBsAg). Screening with HBV DNA could identify OBI and significantly reduce carriers and complications of chronic hepatitis B (CHB). This study aims to assess HBV serological markers and OBI molecular diagnosis among people with TB in Mashhad, northeastern Iran. We have performed HBV serological markers (HBsAg, HBc antibodies (Ab) and HBs Ab) in 175 participants. Fourteen HBsAg+ sera were excluded for further analysis. The presence of HBV DNA (C, S, and X gene regions) was assessed by the qualitative real-time PCR (qPCR) method. Frequencies of HBsAg, HBc, and HBs Ab were 8% (14/175), 36.6% (64/175), and 49.1% (86/175), respectively. Among these 42.9% (69/161) were negative for all HBV serological markers. The S, C, and X gene regions were positive in 10.3% (16/156), 15.4% (24/156), and 22.4% (35/156) of participants, respectively. The total OBI frequency was estimated at 33.3% (52/156) when based on detecting one HBV genomic region. Twenty-two and 30 participants had a seronegative and seropositive OBI, respectively. Thorough screening of high-risk groups with reliable and sensitive molecular methods could lead to OBI identification and decrease CHB long-term complications. Mass immunization remains critical in preventing, reducing, and potentially eliminating HBV complications.

Comparisons among a qPCR assay, VIDAS® assays and a conventional agar streaking method following the same enrichment for the detection of Listeria monocytogenes were performed under two challenging conditions. In the first comparison, L. innocua and L. monocytogenes were coinoculated into sausages at ratios (L. innocua-to-L. monocytogenes) of 10, 100, 1000, and 10 000. qPCR provided the most sensitive detection at all ratios after both 24-h and 48-h enrichments. A modified VIDAS® LMO2 assay (i.e., replacement of the kit-specified enrichment scheme with the enrichment scheme used in this study) and agar streaking yielded equivalent results when the ratio was 10 and 100; agar streaking was more sensitive when the ratio was 1000; neither method could detect L. monocytogenes at the ratio of 10 000. Enrichment duration of 48 h was needed for modified VIDAS® to detect L. monocytogenes when the ratio was 1000. Agar streaking after 24-h enrichment isolated L. monocytogenes better than after 48-h enrichment when the ratio was 100 and 1000. In the second comparison, we followed the validation guidelines of AOAC International and inoculated L. monocytogenes, without any L. innocua, onto lettuce and stainless-steel surfaces at low levels. The numbers of positive samples detected by qPCR, VIDAS® LIS assay, modified VIDAS® LMO2 assay, and agar streaking after 48-h enrichment were not statistically different. Our data showed that qPCR was the most sensitive method, while agar streaking and VIDAS® performed reasonably well. Streaking after 24-h enrichment was needed when background flora could overgrow L. monocytogenes during prolonged enrichment, and this is critical for confirming rapid screening assays. Appropriate selection of enrichment duration and rapid assays will enhance the testing of L. monocytogenes in food and environmental samples.

High-Risk Human Papillomavirus (HR-HPV) types 16 and 18 are estimated to be responsible for 72.4% of all HPV-related cancers worldwide in both men and women, including cervical, anal, penile, vulval, vaginal and head and neck cancers [1]. Important efforts worldwide have devoted to the study of these genotypes, throughout epidemiology and basic science approaches. Of particular interest are the genes from the early region (E), coding non-structural proteins. Early genes E1 and E2 products are involved in replication and transcription regulation, while E6 and E7 proteins are recognised for their oncogenic potential. In this data report, we described a set of primers based on reference sequences from HPV16 and HPV18 designed to cover the early region of these oncogenic genotypes. The design was based on multiple sequences alignment to identify the less conserved regions along the open reading frames (ORFs) E6, E7, E1 and E2. The design allows a highly stringent real time PCR essay ranged from 123 to 598 bp overlapping products for HPV16 (12 products in total) and from 183 to 526 bp for HPV18 (11 products in total), both spanning the early genomic region. The high annealing temperatures (Ta) and regions selected for primer bind were intended for genotypic specificity, without compromising the qPCR amplification efficiency (≥ 90%). Evaluation of qPCR conditions for primer set was performed using DNA standards as controls, generated from the HPV16 and 18 genomes clones. This provides relevant information for further multiple quantitative real-time PCR analysis (qPCR), using the SYBR green chemistry, which is is more affordable than generating multiple fluorescently labeled probes.

Background: Prostate Specific Membrane Antigen (PSMA) – positron emission tomography (PET) guides metastasis-directed radiotherapy (MDRT) in prostate cancer (PrCa). However, its value as a treatment response assessment tool after MDRT remains unclear. Importantly, there is limited understanding of the potential of radiotherapy (RT) to alter PSMA gene (folate hydrolase 1; FOLH1) expression. Methodology: We reviewed a series of 11 men with oligo-metastatic PrCa (25 metastasis sites) treated with MDRT before re-staging with 18F-DCFPyL (PSMA) PET upon secondary recurrence. Acute effects of RT on PSMA protein and mRNA levels were examined with qPCR and immunoblotting in human wild-type androgen-sensitive (LNCap), castrate-resistant (22RV1) and castrate-resistant neuroendocrine (PC3 and DU145) PrCa cell lines. Xenograft tumors were analyzed with immunohistochemistry. Further, we examined PSMA expression in untreated and irradiated radio-resistant (RR) 22RV1 (22RV1-RR) and DU145 (DU145-RR) cells and xenografts selected for survival after high-dose RT. Results: The majority of MDRT-treated lesions showed lack of PSMA-PET/CT avidity, suggesting treatment response even after low biological effective dose (BED) MDRT. We observed similar high degree of heterogeneity of PSMA expression in both human specimens and in xenograft tumors. PSMA was highly expressed in LNCap and 22RV1 cells and tumors but not in the neuroendocrine PC3 and DU145 models. Single fraction RT caused detectable reduction in PSMA protein but not in mRNA levels in LNCap cells and did not significantly alter PSMA protein or mRNA levels in tissue culture or xenografts of the other cell lines. However, radio-resistant 22RV1-RR cells and tumors demonstrated marked decrease of PSMA transcript and protein expression over their parental counterparts. Conclusions: PSMA-PET may be a promising tool to assess RT response in oligo-metastatic PrCa. However, future systematic investigation of this concept should recognize the high degree of heterogeneity of PSMA expression within prostate tumors and the risk for loss of PSMA expression in tumor surviving curative courses of RT.

Foodborne outbreaks are often associated with the consumption of salads. However, published studies on the detection of foodborne pathogens in ready-to-eat salads are scarce. The aim of this study was to detect Giardia duodenalis and Cryptosporidium DNA in ready-to-eat salads, by applying techniques of molecular biology to study the frequency of contamination in salads. A total of 100 packages of ready-to-eat salads containing assorted leafy green vegetables were randomly purchased from hypermarkets located in central regions of Portugal (Coimbra and Viseu). Nested-PCR and qPCR methods were used to detect G. duodenalis and Cryptosporidium DNA. Species and assemblages of the parasites were identified by sequence analysis and PCR. Eighteen of the 100 samples (18%) were positive for G. duodenalis and twelve were sequenced and identified as assemblage A. Cryptosporidium spp. were not detected in any salads. Overall, pre-harvest and post-harvest preventive measures may be need for G. duodenalis control throughout the food production industry, from the field to consumers.

Lentils (Lens culinaris) cannot withstand prolonged flooding. This project aimed to study molecular and physiological responses in L. culinaris under flood stress to understand this plant's key mechanisms for flood tolerance. Six- to eight-week-old lentil seedlings were flooded in a growth chamber for four days. On the fourth day, molecular and physiological experiments were conducted on the flooded and control lentil plants. In addition, a morphological study of the stems and leaves using a scanning electron microscope and volatile compound studies (serving as potential biomarkers for flood stress) using GC–MS were conducted. Under flooded conditions in most of the lentil plants, the expressions of the following genes were upregulated: acetyl-CoA carboxylase carboxyltransferase beta subunit (accD), aldolase, and petD-cytochrome b6/f complex subunit IV (PetD). On the other hand, chloroplast copper/zinc superoxide dismutase (SD) and PBA flash photosystem II D1 precursor (psbA) constituted two downregulated genes among most of the plants. Additionally, higher levels of absorbance for soluble sugar, surface roughness, and surface wax deposition were observed in lentils under flooded conditions. Higher wax accumulation leads to higher leaf hydrophobicity, which is a flood-related adaptation. On the contrary, chlorophyll content, catalase activity, and volatile emissions were reduced among flooded lentils, probably due to cell death and reduced photosynthesis. Oxidative damage occurs when cells’ catalase activity is low, meaning cells are unable to detoxify reactive oxygen species. In conclusion, flooding can cause a notable difference in lentils at anatomical, physiological, and molecular levels.

This study aimed to determine if exercise increases the frequency of selected respiratory viruses/bacteria shedding in non-clinical horses using nose wipes. Nose wipes were taken pre- and post- exercise from 133 healthy horses of various ages and disciplines. Wipes were assayed for the presence of selected common (equine herpesvirus-1 (EHV), EHV-4, S. equi spp equi) and commensal (EHV-2, EHV-5, S. equi spp zooepidemicus) respiratory pathogens. The frequency of detection increased significantly (p < 0.05) in the post-exercise wipes for EHV-1, EHV-2, EHV-5, and S. zooepidemicus. EHV-4 and S. equi were only detected in a small number of horses post-exercise. The absolute quantitation of pathogens was significantly lower post-exercise (p < 0.05) for EHV-2, EHV-5, and S. zooepidemicus. While exercise increased the detection rate of EHV-1, EHV-2, EHV-5 and S. zooepidemicus in nose wipes, absolute quantitation of the respective microbes was lower. The use of nose wipes post-exercise to assess the shedding status of selected respiratory viruses/bacteria in non-clinical horses may be an alternative to the more invasive nasal swabs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Inc.)

Recreational waters are routinely assessed by enumeration of fecal indicator bacteria, such as enterococci, but research suggests viral indicators may be better suited for recreational water quality applications. Somatic and F+ coliphage were proposed as predictors of viral pathogens in recreational waters, but concurrent measurements of enterococci and coliphages and comparisons across different risk-based beach action values (BAVs) are limited. We collected paired measurements of enterococci, E. coli, somatic and F+ coliphage from three Gulf of Mexico beaches. Enterococci (colony forming units: CFU) were enumerated on mEI agar and by qPCR (Entero1a: target sequence [TS]). E. coli was enumerated by qPCR (EC23S587, TS). Coliphages (plaque forming units: PFU) were enumerated using dead-end hollowfiber ultrafiltration and single agar layer assay. The largest correlation observed was between Entero1a and cultured enterococci (r ≥ 0.51, p ≤ 0.0001) and the smallest (r ≤ 0.01, p = 1.0) between F+ coliphage and cultured enterococci. Applying equivalent BAVs for culturable enterococci (60 CFU/100 mL), Entero1a (9658 TS per 100 mL) and suggested somatic (14 PFU/100 mL) and F+ (3 PFU/100 mL) thresholds resulted in the overall greatest agreement in beach advisory status between coliphages and Entero1a (74-82 %) and lowest between cultured enterococci and somatic coliphage (65 %). This rich data set not only provides valuable insights on the incidence of coliphage and FIB in the Gulf of Mexico but will also provide a foundation for future research on fecal source identification and water quality forecast modeling in sub-tropical marine waters., Competing Interests: Declaration of competing interest Authors have no competing interests to declare., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Background: Human papillomavirus (HPV) is the main pathogen responsible of cervical cancer. The characterization of HPV genotypes in preneoplastic lesions and cervical cancer could establishes the effectiveness of the vaccination plan in the Chilean population. The aim of this study was to determine the frequency of HPV in women in the Coquimbo region., Methods: A total of 1235 cervical samples from women aged 20-64 years old who attended gynecological check-ups from April 2023 to July 2024 were analyzed to detect HPV genotypes using qPCR., Results: The overall prevalence rate of HPV infection was 26.56 %, while the prevalence of HR-HPV in age groups G1 (20-29 years) and G2 (30-64 years) was 47.69 % and 24.07 %, respectively. The most prevalent genotypes of HPV infection among our entire population were HPV16, HPV51 and HPV 31. Single infection (74.70 %) was the main pattern of HPV infection observed in the entire group, followed by double infection (16.46 %) and multiple infection (8.84 %), which was similar in Group 2, with percentiles of 76.07 %, 15.79 % and 7.14 %, respectively. However, in Group 1 a higher frequency of multiple HPV infections was observed, with 16.13 %., Conclusion: This HPV prevalence infections among women in the Coquimbo region appears to be higher than the reported in Chile. In the rest of country, HPV prevalence is likely underestimated. Molecular detection of 14 HR-HPV genotypes is important because it will not only help women avoid cervical cancer but could also inform the introduction of new vaccines targeting a broader spectrum of HR-HPV., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest for this work., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Anatoxins (ATX) are globally occurring toxins produced by some species of cyanobacteria in aquatic habitats. They can cause acute poisoning in animals, leading to muscle paralysis and respiratory failure, and might also pose a long-term health risk to humans. Thanks to advances in molecular methods and genomic knowledge, it is now possible to rapidly detect and quantify the genes associated with cyanotoxin production for most major groups of cyanotoxins except ATX. The aim of this study was to develop and validate a new quantitative PCR (qPCR) assay for general detection of all potential ATX producers in the environment. After specificity testing in silico and in vitro with 16 cyanobacterial strains (endpoint PCR, amplicon sequencing and qPCR), two assays targeting the anaC gene were thoroughly validated for linearity, amplification efficiency, sensitivity, dynamic range, inter-assay and intra-assay variability, and the influence of background DNA. The assays were then applied to 144 environmental samples of plankton and biofilm from lakes and rivers whose ATX content had previously been measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Amplification efficiency of the two designed assays was between 94 % and 103 %, and the limits of quantification and detection were up to, but mostly below, 322 and 32 cells/mL, respectively. Both assays showed better or equal specificity in cyanobacterial cultures than currently available PCR assays and were able to predict the presence of ATX detected by LC-MS/MS in most environmental samples (83 % in plankton and 52-62 % in biofilm). A higher number of discrepancies between qPCR and LC-MS/MS results in biofilm than in plankton samples indicates limited knowledge and sparse genomic data on benthic cyanobacteria. These assays are the first published general qPCR assays targeting all ATX producers and could provide water managers with a rapid and cost-effective risk assessment to better protect human and animal health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The excessive growth of harmful cyanobacteria, including Dolichospermum (formerly known as Anabaena), in freshwater bodies has become a pressing global concern. However, detailed information about the role of Dolichospermum in shaping bloom dynamics and producing cyanotoxins is limited. In this study, a bloom event dominated by Dolichospermum spp. at Detroit Lake (Oregon, USA) was examined from 2019 to 2021. In 2019, early summer cyanobacterial community succession reached up to 8.7 % of total phytoplankton abundance. Dolichospermum was the major microcystin (MC)-producing genus, with peak MC levels of 7.34 μg L -1 . The presence of MCs was strongly correlated with the abundance of Dolichospermum (r = 0.84, p < 0.05) and MC synthetase gene, mcyE-Ana (r = 0.63, p < 0.05). Metabolic analyses further showed that the presence of nif/pst genes linked to nitrogen and phosphorus metabolism was dominated by Dolichospermum from the bloom onset until September. In addition, the abundance of Dolichospermum was significantly correlated with the abundance of nitrogen-fixing nif-Ana gene (r = 0.62, p < 0.05). As the lake experienced a longer N and P scarcity period (May to September), the N 2 -fixing Dolichospermum was able to dominate over other non-fixing cyanobacteria present, including Microcystis and Planktothrix. Overall, our results facilitate a better understanding of the organism and will help working toward managing/predicting future blooms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

MicroRNAs (miRNAs) are promising biomarkers for forensic body fluid identification owing to their small size, stability against degradation, and differential expression patterns. However, the expression of most body fluid-miRNAs is relative (differentially expressed in certain body fluids) rather than absolute (exclusively expressed in a specific body fluid). Moreover, different body fluids contain heterogeneous cell types, complicating their identification. Therefore, appropriate normalization strategies to eliminate non-biological variations and robust models to interpret expression levels accurately are necessary prerequisites for applying miRNAs in body fluid identification. In this study, the expression stability of six candidate reference genes (RGs) across five body fluids was validated using geNorm, NormFinder, BestKeeper and RankAggreg, and the most suitable combination of RGs (hsa-miR-484 and hsa-miR-191-5p) was identified under our experimental conditions. Subsequently, we systematically evaluated the expression patterns of the 28 most promising body fluid-specific miRNA markers using TaqMan RT-qPCR and selected the optimal combination of markers (12 miRNAs) to establish a multi-class support vector machine (MSVM) classification model. An independent test set (60 samples) was used to validate the accuracy of the proposed classification model, while an additional 30 casework samples were used to assess its robustness. The MSVM model accurately predicted the body fluid origin for almost all (59/60) single-source samples. Moreover, this model demonstrated the capability to identify aged forensic samples and to predict the primary components of mixed stains to a certain extent. In summary, this study presented a miRNA-based MSVM classification model for forensic body fluid identification using the qPCR platform. However, extensive validation, especially inter-laboratory collaborative exercises, is necessary before miRNA can be routinely applied in forensic identification practice., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Antibiotic resistance genes (ARGs) have long served as adaptive defensive mechanisms among bacteria, enabling their survival and propagation in challenging environments. The consequences of inefficient wastewater treatment have culminated the emergence of untreatable and lethal extensively drug-resistant. To understand the relationship between wastewater effluent and marine ecosystems, we conducted a study to monitor the diversity and prevalence of common ARGs in Hong Kong's urban coastal areas at different seasons. Our findings revealed that sul 1 was the most abundant resistance gene, with an average relative abundance of 4.45 × 10 -2 per 16s rRNA gene copy. Moreover, temperature, dissolved oxygen, and salinity were key factors influencing seasonal variations in total ARGs abundance. The influence of environmental factors varied based on ARGs' association with Intl1, with Intl1-associated ARGs strongly correlating with temperature and dissolved oxygen. Notably, despite their abundance, sul1 and mphA exhibited similar correlations with both Intl1 and key environmental factors, suggesting these ARGs share a common dissemination mechanism. Moreover, the robust association between resistance genes and mobile genetic elements (MGE) could potentially act as a valuable indicator for assessing the efficacy of removing ARGs in wastewater treatment methods when operating under carefully optimized environmental parameters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The Persian Gulf hosts the second-largest population of Dugongs. The International Union for the Conservation of Nature (IUCN) lists them as Vulnerable to Extinction species. Incidental fishing, vessel strikes, environmental pollution, habitat degradation, and infectious diseases currently threaten these sirenians. While research focused on the abovementioned threats, little is known about pathogen infections, particularly waterborne parasites. The current study aimed to investigate the prevalence of the waterborne parasites, namely Blastocystis, Cryptosporidium, Dientamoeba fragilis, Giardia duodenalis, and Toxoplasma gondii in the Qatar Dugongs population. Hence, twenty-three tissues from stranded Dugongs' carcasses were collected postmortem and subjected to DNA extraction and parasite qPCR screening. Only Blastocystis and Cryptosporidium species were detected in different tissues across the tested animals harboring genotypes ST1 and Cryptosporidium hominis IbA9G3, respectively, with the first-time report of Blastocystis sp. within sirenian hosts. Continuous monitoring should be provided to shed lights about the contamination sources of these endangered species and clarify the zoonotic potential transmission routes between wildlife and humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)