Pathogenicity assessment of Arcobacter butzleri isolated from Canadian agricultural surface water.

BACKGROUND: Water is considered a source for the transmission of Arcobacter species to both humans and animals. This study was conducted to assess the prevalence, distribution, and pathogenicity of A. butzleri strains, which can potentially pose health risks to humans and animals. Cultures were isolated from surface waters of a mixed-use but predominately agricultural watershed in eastern Ontario, Canada. The detection of antimicrobial resistance (AMR) and virulence-associated genes (VAGs), as well as enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays were performed on 913 A. butzleri strains isolated from 11 agricultural sampling sites. RESULTS: All strains were resistant to one or more antimicrobial agents, with a high rate of resistance to clindamycin (99%) and chloramphenicol (77%), followed by azithromycin (48%) and nalidixic acid (49%). However, isolates showed a significantly (p < 0.05) high rate of susceptibility to tetracycline (1%), gentamycin (2%), ciprofloxacin (4%), and erythromycin (5%). Of the eight VAGs tested, ciaB, mviN, tlyA, and pldA were detected at high frequency (> 85%) compared to irgA (25%), hecB (19%), hecA (15%), and cj1349 (12%) genes. Co-occurrence analysis showed A. butzleri strains resistant to clindamycin, chloramphenicol, nalidixic acid, and azithromycin were positive for ciaB, tlyA, mviN and pldA VAGs. ERIC-PCR fingerprint analysis revealed high genetic similarity among strains isolated from three sites, and the genotypes were significantly associated with AMR and VAGs results, which highlight their potential environmental ubiquity and potential as pathogenic. CONCLUSIONS: The study results show that agricultural activities likely contribute to the contamination of A. butzleri in surface water. The findings underscore the importance of farm management practices in controlling the potential spread of A. butzleri and its associated health risks to humans and animals through contaminated water.

A cost-effective RNA extraction and RT-qPCR approach to detect California serogroup viruses from pooled mosquito samples.

Mosquito-borne diseases pose ongoing global health concerns, demanding more cost-efficient methods to detect pathogens to support enhanced surveillance efforts. This study introduces an adapted TRIzol-based high-throughput RNA extraction protocol, tailored for the detection of California serogroup viruses in pooled mosquito samples in a rapid and cost-effective manner. This approach provided consistent RNA yields and sensitive viral detection relative to two commercial extraction kits (QIAGEN RNeasy Mini Kit and MACHEREY-NAGEL NucleoSpin RNA Plus Kit). The incorporation of a user-friendly and non-spiking-based RT-qPCR internal control designed for the 18S rRNA gene in mosquitoes minimizes potential false positives/negatives, improving the fidelity of viral detection outcomes. Effective RNA yields, purity, and successful target amplification across 25 mosquito species and varied pool sizes (1-50 mosquitoes per tube) affirm the reliability of our approach. The extraction method is cost-effective, with an incurred cost of $0.58 CAD per sample, in contrast to the $5.25 CAD cost per sample of the two kits, rendering it promising for mosquito-borne disease surveillance initiatives.

Future land-use change predictions using Dyna-Clue to support mosquito-borne disease risk assessment.

Mosquitoes are known vectors for viral diseases in Canada, and their distribution is driven by climate and land use. Despite that, future land-use changes have not yet been used as a driver in mosquito distribution models in North America. In this paper, we developed land-use change projections designed to address mosquito-borne disease (MBD) prediction in a 38 761 km2 area of Eastern Ontario. The landscape in the study area is marked by urbanization and intensive agriculture and hosts a diverse mosquito community. The Dyna-CLUE model was used to project land-use for three time horizons (2030, 2050, and 2070) based on historical trends (from 2014 to 2020) for water, forest, agriculture, and urban land uses. Five scenarios were generated to reflect urbanization, agricultural expansion, and natural areas. An ensemble of thirty simulations per scenario was run to account for land-use conversion uncertainty. The simulation closest to the average map generated was selected to represent the scenario. A concordance matrix generated using map pair analysis showed a good agreement between the simulated 2020 maps and 2020 observed map. By 2050, the most significant changes are predicted to occur mainly in the southeastern region's rural and forested areas. By 2070, high deforestation is expected in the central west. These results will be integrated into risk models predicting mosquito distribution to study the possibility of humans' increased exposure risk to MBDs.

Real-time quantitative PCR assay development and application for assessment of agricultural surface water and various fecal matter for prevalence of Aliarcobacter faecis and Aliarcobacter lanthieri.

BACKGROUND: Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL- 1 or g- 1 obtained using known quantities of spiked A. faecis and A. lanthieri reference strains in autoclaved agricultural surface water and dairy cow manure samples. RESULTS: Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of A. faecis for surface water and feces was 21 and 28%, respectively. The maximum A. faecis concentration for water and feces was 2.3 × 107 cells 100 mL- 1 and 1.2 × 107 cells g- 1, respectively. A. lanthieri was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 105 cells 100 mL- 1; fecal samples had a prevalence and maximum density of 10% and 2.0 × 106 cells g- 1, respectively. CONCLUSIONS: The results indicate that the occurrence of these species in agricultural surface water is potentially due to fecal contamination of water from livestock, human, or wildlife as both species were detected in fecal samples. The new real-time qPCR assays can facilitate rapid and accurate detection in < 3 h to quantify total numbers of A. faecis and A. lanthieri cells present in various complex environmental samples.

Towards a general framework for the assessment of interactive effects of multiple stressors on aquatic ecosystems: Results from the Making Aquatic Ecosystems Great Again (MAEGA) workshop.

A workshop was held in Wageningen, The Netherlands, in September 2017 to collate data and literature on three aquatic ecosystem types (agricultural drainage ditches, urban floodplains, and urban estuaries), and develop a general framework for the assessment of multiple stressors on the structure and functioning of these systems. An assessment framework considering multiple stressors is crucial for our understanding of ecosystem responses within a multiply stressed environment, and to inform appropriate environmental management strategies. The framework consists of two components: (i) problem identification and (ii) impact assessment. Both assessments together proceed through the following steps: 1) ecosystem selection; 2) identification of stressors and quantification of their intensity; 3) identification of receptors or sensitive groups for each stressor; 4) identification of stressor-response relationships and their potential interactions; 5) construction of an ecological model that includes relevant functional groups and endpoints; 6) prediction of impacts of multiple stressors, 7) confirmation of these predictions with experimental and monitoring data, and 8) potential adjustment of the ecological model. Steps 7 and 8 allow the assessment to be adaptive and can be repeated until a satisfactory match between model predictions and experimental and monitoring data has been obtained. This paper is the preface of the MAEGA (Making Aquatic Ecosystems Great Again) special section that includes three associated papers which are also published in this volume, which present applications of the framework for each of the three aquatic systems.

Novel virulence, antibiotic resistance and toxin gene-specific PCR-based assays for rapid pathogenicity assessment of Arcobacter faecis and Arcobacter lanthieri.

BACKGROUND: Arcobacter faecis and A. lanthieri are two newly classified species of genus Arcobacter. The prevalence and distribution of virulence, antibiotic resistance and toxin (VAT) genes in these species are required to assess their potential pathogenic health impacts to humans and animals. This study (i) developed species- and gene-specific primer pairs for the detection of six virulence, two antibiotic resistance, and three toxin genes in two target species; (ii) optimized eight single-tube multiplex and three monoplex PCR protocols using the newly developed species- and gene-specific primers; and (iii) conducted specificity and sensitivity evaluations as well as validation of eleven mono- and multiplex PCR assays by testing A. faecis (n= 29) and A. lanthieri (n= 10) strains isolated from various fecal and agricultural water sources to determine the prevalence and distribution of VAT genes and assess the degree of pathogenicity within the two species. RESULTS: Detection of all ten and eleven target VAT genes, and expression of cytolethal distending toxin (cdtA, cdtB and cdtC) genes in A. faecis and A. lanthieri reference strains with high frequency in field isolates suggest that they are potentially pathogenic strains. These findings indicate that these two species can pose a health risk to humans and animals. CONCLUSIONS: The study results show that the developed mono- and multiplex PCR (mPCR) assays are simple, rapid, reliable and sensitive for the simultaneous assessment of the potential pathogenicity and antibiotic resistance profiling of tet(O) and tet(W) genes in these two newly discovered species. Also, these assays can be useful in diagnostic and analytical laboratories to determine the pathotypes and assessment of the virulence and toxin factors associated to human and animal infections.

Quantitative real-time PCR-based assessment of tile drainage management influences on bacterial pathogens in tile drainage and groundwater.

This study compared the impact of controlled tile drainage (CD) and freely draining (FD) systems on the prevalence and quantitative real-time PCR-based enumeration of four major pathogens including Arcobacter butzleri, Campylobacter jejuni, Campylobacter coli, and Helicobacter pylori in tile- and groundwater following a fall liquid swine manure (LSM) application on clay loam field plots. Although the prevalence of all target pathogens were detected in CD and FD systems, the loads of A. butzleri, C. jejuni, and C. coli were significantly lower in CD tile-water (p<0.05), in relation to FD tile-water. However, concentrations of A. butzleri were significantly greater in CD than FD tile-water (p<0.05). In shallow groundwater (1.2m depth), concentrations of A. butzleri, C. coli, and H. pylori showed no significant difference between CD and FD plots, while C. jejuni concentrations were significantly higher in FD plots (p<0.05). No impact of CD on the H. pylori was observed since quantitative detection in tile- and groundwater was scarce. Although speculative, H. pylori occurrence may have been related to the application of municipal biosolids four years prior to the LSM experiment. Overall, CD can be used to help minimize off-field export of pathogens into surface waters following manure applications to land, thereby reducing waterborne pathogen exposure risks to humans.

Assessment of a new Bacteroidales marker targeting North American beaver (Castor canadensis) fecal pollution by real-time PCR.

In many settings wildlife can be a significant source of fecal pathogen input into surface water. The North American beaver (Castor canadensis) is a zoonotic reservoir for several human pathogens including Cryptosporidium spp. and Giardia spp. In order to specifically detect fecal pollution by beavers, we have developed and validated a beaver-specific Bacteroidales marker, designated Beapol01, based on the 16S rRNA gene. The marker is suitable for quantifying pollution using real-time PCR. The specificity and sensitivity of the marker was excellent, Beaver signal was detected in water of a mixed-activity watershed harbouring this rodent. Overall, Beapol01 will be useful for a better understanding of fecal source inputs in drainage basins inhabited by the beaver.

Environmental risk assessment of human pharmaceuticals in the European Union: A case study with the ß-blocker atenolol.

ß-Adrenergic receptor blockers (ß-blockers) are applied to treat high blood pressure, ischemic heart disease, and heart rhythm disturbances. Due to their widespread use and limited human metabolism, ß-blockers are widely detected in sewage effluents and surface waters. ß-Adrenergic receptors have been characterized in fish and other aquatic animals, so it can be expected that physiological processes regulated by these receptors in wild animals may be affected by the presence of ß-blockers. Because ecotoxicological data on ß-blockers are scarce, it was decided to choose the ß-blocker atenolol as a case study pharmaceutical within the project ERAPharm. A starting point for the assessment of potential environmental risks was the European guideline on the environmental risk assessment of medicinal products for human use. In Phase I of the risk assessment, the initial predicted environmental concentration (PEC) of atenolol in surface water (500 ng L−1) exceeded the action limit of 10 ng L−1. Thus, a Phase II risk assessment was conducted showing acceptable risks for surface water, for groundwater, and for aquatic microorganisms. Furthermore, atenolol showed a low potential for bioaccumulation as indicated by its low lipophilicity (log KOW = 0.16), a low potential for exposure of the terrestrial compartment via sludge (log KOC = 2.17), and a low affinity for sorption to the sediment. Thus, the risk assessment according to Phase II-Tier A did not reveal any unacceptable risk for atenolol. Beyond the requirements of the guideline, additional data on effects and fate were generated within ERAPharm. A 2-generation reproduction test with the waterflea Daphnia magna resulted in the most sensitive no-observed-effect concentration (NOEC) of 1.8 mg L−1. However, even with this NOEC, a risk quotient of 0.003 was calculated, which is still well below the risk threshold limit of 1. Additional studies confirm the outcome of the environmental risk assessment according to EMEA/CHMP (2006). However, atenolol should not be considered as representative for other ß-blockers, such as metoprolol, oxprenolol, and propranolol, some of which show significantly different physicochemical characteristics and varying toxicological profiles in mammalian studies.

Tracking host sources of Cryptosporidium spp. in raw water for improved health risk assessment.

Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.