Comparative genomics of Cryptosporidium parvum reveals the emergence of an outbreak-associated population in Europe and its spread to the United States.

The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g., IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water- and food-borne outbreaks. In this study, we generated new whole-genome sequence (WGS) data from 123 human- and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China, and the United States (n = 72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analyzed a data set from 141 isolates from the zoonotic groups IIa (n = 119) and IId (n = 22). Based on 28,047 high-quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: Isolates from China (IId) and Egypt (IIa and IId) formed population 1; a minority of European isolates (IIa and IId) formed population 2; and the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the United States (IIa) clustered in population 3. Based on analyses of the population structure, population genetics, and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the United Kingdom, reach the United States, where it also expanded. The reason(s) for the successful spread of population 3 remain elusive, although genes under selective pressure uniquely in this population were identified.

Hiding in plain sight: Genome-wide recombination and a dynamic accessory genome drive diversity in Fusarium oxysporum f.sp. ciceris.

Understanding the origins of variation in agricultural pathogens is of fundamental interest and practical importance, especially for diseases that threaten food security. Fusarium oxysporum is among the most important of soil-borne pathogens, with a global distribution and an extensive host range. The pathogen is considered to be asexual, with horizontal transfer of chromosomes providing an analog of assortment by meiotic recombination. Here, we challenge those assumptions based on the results of population genomic analyses, describing the pathogen's diversity and inferring its origins and functional consequences in the context of a single, long-standing agricultural system. We identify simultaneously low nucleotide distance among strains, and unexpectedly high levels of genetic and genomic variability. We determine that these features arise from a combination of genome-scale recombination, best explained by widespread sexual reproduction, and presence-absence variation consistent with chromosomal rearrangement. Pangenome analyses document an accessory genome more than twice the size of the core genome, with contrasting evolutionary dynamics. The core genome is stable, with low diversity and high genetic differentiation across geographic space, while the accessory genome is paradoxically more diverse and unstable but with lower genetic differentiation and hallmarks of contemporary gene flow at local scales. We suggest a model in which episodic sexual reproduction generates haplotypes that are selected and then maintained through clone-like dynamics, followed by contemporary genomic rearrangements that reassort the accessory genome among sympatric strains. Taken together, these processes contribute unique genome content, including reassortment of virulence determinants that may explain observed variation in pathogenic potential.

Global Population Genomics of Two Subspecies of Cryptosporidium hominis during 500 Years of Evolution.

Cryptosporidiosis is a major global health problem and a primary cause of diarrhea, particularly in young children in low- and middle-income countries (LMICs). The zoonotic Cryptosporidium parvum and anthroponotic Cryptosporidium hominis cause most human infections. Here, we present a comprehensive whole-genome study of C. hominis, comprising 114 isolates from 16 countries within five continents. We detect two lineages with distinct biology and demography, which diverged circa 500 years ago. We consider these lineages two subspecies and propose the names C. hominis hominis and C. hominis aquapotentis (gp60 subtype IbA10G2). In our study, C. h. hominis is almost exclusively represented by isolates from LMICs in Africa and Asia and appears to have undergone recent population contraction. In contrast, C. h. aquapotentis was found in high-income countries, mainly in Europe, North America, and Oceania, and appears to be expanding. Notably, C. h. aquapotentis is associated with high rates of direct human-to-human transmission, which may explain its success in countries with well-developed environmental sanitation infrastructure. Intriguingly, we detected genomic regions of introgression following secondary contact between the subspecies. This resulted in high diversity and divergence in genomic islands of putative virulence genes, including muc5 (CHUDEA2_430) and a hypothetical protein (CHUDEA6_5270). This diversity is maintained by balancing selection, suggesting a co-evolutionary arms race with the host. Finally, we find that recent gene flow from C. h. aquapotentis to C. h. hominis, likely associated with increased human migration, maybe driving the evolution of more virulent C. hominis variants.

Volunteer-contributed observations of flowering often correlate with airborne pollen concentrations.

Characterizing airborne pollen concentrations is crucial for supporting allergy and asthma management; however, pollen monitoring is labor intensive and, in the USA, geographically limited. The USA National Phenology Network (USA-NPN) engages thousands of volunteer observers in regularly documenting the developmental and reproductive status of plants. The reports of flower and pollen cone status contributed to the USA-NPN's platform, Nature's Notebook, have the potential to help address gaps in pollen monitoring by providing real-time, spatially explicit information from across the country. In this study, we assessed whether observations of flower and pollen cone status contributed to Nature's Notebook can serve as effective proxies for airborne pollen concentrations. We compared daily pollen concentrations from 36 National Allergy Bureau (NAB) stations in the USA with flowering and pollen cone status observations collected within 200 km of each NAB station in each year, 2009-2021, for 15 common tree taxa using Spearman's correlations. Of 350 comparisons, 58% of correlations were significant (p < 0.05). Comparisons could be made at the largest numbers of sites for Acer and Quercus. Quercus demonstrated a comparatively high proportion of tests with significant agreement (median ρ = 0.49). Juglans demonstrated the strongest overall coherence between the two datasets (median ρ = 0.79), though comparisons were made at only a small number of sites. For particular taxa, volunteer-contributed flowering status observations demonstrate promise to indicate seasonal patterns in airborne pollen concentrations. The quantity of observations, and therefore, their utility for supporting pollen alerts, could be substantially increased through a formal observation campaign.

Preliminary investigation of a significant national Cryptosporidium exceedance in the United Kingdom, August 2023 and ongoing.

Routine laboratory surveillance has identified an unprecedented and ongoing exceedance of Cryptosporidium spp. across the United Kingdom, notably driven by C. hominis transmission, since 14 August 2023. Information from 477 reported cases in England and Wales, followed up with a standardised exposure questionnaire as of 25 September 2023, identified foreign travel in 250 (54%) of 463 respondents and swimming in 234 (66%) of 353 cases. A significant, common exposure has not yet been identified in first analyses.

The adoption of climate-smart agriculture to address wildfires in the Maya Golden Landscape of Belize: Smallholder farmers' perceptions.

Ecosystems around the globe are enduring wildfires with greater frequency, intensity, and severity and this trend is projected to continue as a result of climate change. Climate-smart agriculture (CSA) has been proposed as a strategy to prevent wildfires and mitigate climate change impacts; however, it remains poorly understood as a strategy to prevent wildfires. Therefore, the authors propose a multimethod approach that combines mapping of wildfire susceptibility and social surveys to identify priority areas, main factors influencing the adoption of CSA practices, barriers to their implementation, and the best CSA practices that can be implemented to mitigate wildfires in Belize's Maya Golden Landscape (MGL). Farmers ranked slash and mulch, crop diversification, and agroforestry as the main CSA practices that can be implemented to address wildfires caused by agriculture in the MGL. In order to reduce wildfire risk, these practices should, be implemented in agricultural areas near wildlands with high wildfire susceptibility and during the fire season (February-May), in the case of slash and mulch. However, socio-demographic and economic characteristics, together with a lack of training and extension services support, inadequate consultation by agencies, and limited financial resources, hinder the broader adoption of CSA practices in the MGL. Our research produced actionable and valuable information that can be used to design policies and programs to mitigate the impacts of climate change and wildfire risk in the MGL. This approach can also be used in other regions where wildfires are caused by agricultural practices to identify priority areas, barriers and suitable CSA practices that can be implemented to mitigate wildfires.

TIDE Analysis of Cryptosporidium Infections by gp60 Typing Reveals Obscured Mixed Infections.

BACKGROUND: Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. METHODS: Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. RESULTS: For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. CONCLUSIONS: We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

An outbreak of Cryptosporidium parvum linked to pasteurised milk from a vending machine in England: a descriptive study, March 2021.

We describe the investigations and management of a Cryptosporidium parvum outbreak of linked to consumption of pasteurised milk from a vending machine. Multiple locus variable number of tandem repeats analysis was newly used, confirming that C. parvum detected in human cases was indistinguishable from that in a calf on the farm. This strengthened the evidence for milk from an on-farm vending machine as the source of the outbreak because of post-pasteurisation contamination. Bacteriological indicators of post-pasteurisation contamination persisted after the initial hygiene improvement notice. We propose that on-farm milk vending machines may represent an emerging public health risk.

Development and evaluation of a real-time PCR for genotyping of Cryptosporidium spp. from water monitoring slides.

Cryptosporidium is an important cause of gastroenteritis globally and the main agent of waterborne outbreaks caused by protozoan parasites. Water monitoring for Cryptosporidium oocysts is by detection and enumeration using stained slide microscopy. Species identification (known as genotyping) may be undertaken post hoc and remains a specialist test, only undertaken in some laboratories. The benchmark method is nested PCR-sequencing of part of the SSU rRNA gene, but not all slides are typable and the workflow is cumbersome. We report the development, in-house validation and application of a real-time PCR-sequencing assay based on that gene, using a hydrolysis probe, for the detection and genotyping of all Cryptosporidium spp. The assay was investigated in two formats; a high volume DNA template for analysing all the DNA extracted from Cryptosporidium-positive water monitoring slides with <5 oocysts seen, and a lower volume DNA template permitting several technical replicates from slides with ≥5 oocysts seen where multiple species are more likely to be present. Each format conformed to the MIQE guidelines for amplification dynamics and was specific for Cryptosporidium spp. With high sensitivity, being capable of detecting and genotyping single oocysts by sequencing of a 435 bp amplicon. When 65 water monitoring slides with <5 oocysts seen were tested, slide typeability varied by sending laboratory (n = 9), and ranged from 22 to 60%. Typeability was 75% for slides with ≥5 oocysts seen that were submitted by a single laboratory. The laboratory workflow was improved by using real-time PCR, and decreased the time to result compared with nested PCR-sequencing. In practical application, there was no loss of typeability when the ≥5 oocysts assay was applied to all slides, irrespective of the number of oocysts present.

Allele-Specific Detection Methods for QoI Fungicide-Resistant Erysiphe necator in Vineyards.

Grapevine powdery mildew (GPM), caused by the fungus Erysiphe necator, is a constant threat to worldwide production of grape berries, requiring repeated use of fungicides for management. The frequent fungicide applications have resulted in resistance to commonly used quinone outside inhibitor (QoI) fungicides and the resistance is associated with single-nucleotide polymorphisms (SNPs) in the mitochondrial cytochrome b gene (cytb). In this study, we attempted to detect the most common SNP causing a glycine to alanine substitution at amino acid position 143 (i.e., G143A) in the cytb protein, to track this resistance using allele-specific TaqMan probe and digital-droplet PCR-based assays. Specificity and sensitivity of these assays showed that these two assays could discriminate SNPs and were effective on mixed samples. These diagnostic assays were implemented to survey E. necator samples collected from leaf and air samples from California and Oregon grape-growing regions. Sequencing of PCR amplicons and phenotyping of isolates also revealed that these assays accurately detected each allele (100% agreement), and there was an absolute agreement between the presence or absence of the G143A mutation and resistance to QoIs in the E. necator sampled. These results indicate that the developed diagnostic tools will help growers make informed decisions about fungicide selections and applications which, in turn, will facilitate GPM disease management and improve grape production systems.

Wildfire, Environmental Risk and Deliberative Planning in the Locarnese Region of Switzerland.

A survey of residents in the Locarnese region of Canton Ticino, Switzerland was used to examine perceptions of exposure to environmental risk in the context of the deliberative Swiss planning system. There is a growing risk of wildfire in the region, and unless residents' risk perceptions are understood and effectively integrated into decision making, confidence in environmental planning processes could erode. The research analyses how peri-urban residents conceptualise risk, place and environment, and how they perceive their influence over local planning outcomes. Descriptive and inferential statistics reveal high appreciation of lifestyle and amenity values, and support for firefighting services. While respondents recognise the increasing exposure to wildfires and landslides, it was the current level of urban expansion that was seen to be heightening risk at the interface between forests and settlements. Although Swiss deliberative governance arrangements offer citizens opportunities to be involved in decision making through official channels, respondents who were younger, of local background or who were less educated were particularly dissatisfied with their influence over planning. We discuss the implications of these findings for the distinctive Swiss planning system in the context of other countries' ambitions to develop more effective, democratic environmental planning. In particular, the relative ease offered by popular referenda may be creating a disproportionate sense of citizen entitlement to be heard on local planning issues. Dialogues of risk reduction must continue to evolve between the population and government actors to encourage residents to engage more fully with relevant topics of risk for their region.

Genetic Diversity of Cryptosporidium hominis in a Bangladeshi Community as Revealed by Whole-Genome Sequencing.

We studied the genetic diversity of Cryptosporidium hominis infections in slum-dwelling infants from Dhaka over a 2-year period. Cryptosporidium hominis infections were common during the monsoon, and were genetically diverse as measured by gp60 genotyping and whole-genome resequencing. Recombination in the parasite was evidenced by the decay of linkage disequilibrium in the genome over <300 bp. Regions of the genome with high levels of polymorphism were also identified. Yet to be determined is if genomic diversity is responsible in part for the high rate of reinfection, seasonality, and varied clinical presentations of cryptosporidiosis in this population.

Nurse managers and the sandwich support model.

AIM: To explore the interplay between the work of nurse managers and the support they receive and provide. BACKGROUND: Support is the cornerstone of management practices and is pivotal in employees feeling committed to an organisation. Support for nurse managers is integral to effective health sector management; its characteristics merit more attention. METHODS: The experiences of 15 nurse managers in rural health institutions in South Australia were explored using structured interviews, observation and document review. RESULTS: Effective decision making requires adequate support, which influences the perceptions and performance of nurse managers, creating an environment in which they feel appreciated and valued. An ideal support system is proposed, the "sandwich support model," to promote effective functioning and desirable patient outcomes via support "from above" and "from below." IMPLICATIONS FOR NURSING MANAGEMENT: The need to support nurse managers effectively is crucial to how they function. The sandwich support model can improve management practices, more effectively assisting nurse managers. Organisations should revisit and strengthen support processes for nurse managers to maximize efficiencies. CONCLUSION: This paper contributes to understanding the importance of supporting nurse managers, identifying the processes used and the type of support offered. It highlights challenges and issues affecting support practices within the health sector.

Correlation Between Aeroallergen Levels and New Diagnosis of Eosinophilic Esophagitis in New York City.

OBJECTIVE: The relation between food allergies and eosinophilic esophagitis (EoE) is well established. Aeroallergens may also contribute to the development of EoE; however, there are limited data to support or refute this hypothesis. The objectives of this pilot study were to determine whether there is a seasonal variation in the onset of symptoms and/or diagnosis of EoE and whether these variations correlate with a specific pollen concentration within New York City. METHODS: We performed a retrospective chart review to identify all pediatric patients at New York Presbyterian Weill Cornell Medical Center diagnosed with EoE between 2002 and 2012. Sixty-six patients were identified and 28 were excluded. Cases were classified by both date of initial symptoms and date of histologic diagnosis. Pollen counts from a certified New York City counting station and the percentage of EoE cases were collated monthly and seasonally and compared. RESULTS: There was a seasonal variation in onset of symptoms and diagnosis of EoE, with the highest number of patients reporting onset of symptoms of EoE in July to September, and those being diagnosed with EoE in October to December. There was a seasonal correlation between peak levels of grass pollen and peak onset of EoE symptoms, which were both highest in July to September. The diagnosis of EoE peaked one season later. CONCLUSIONS: The study findings suggest that there is a correlation between specific aeroallergens and both the onset of symptoms and time of diagnosis of patients with EoE.

Suitability of loci for multiple-locus variable-number of tandem-repeats analysis of Cryptosporidium parvum for inter-laboratory surveillance and outbreak investigations.

Cryptosporidium parvum is the major cause of livestock and zoonotically-acquired human cryptosporidiosis. The ability to track sources of contamination and routes of transmission by further differentiation of isolates would assist risk assessment and outbreak investigations. Multiple-locus variable-number of tandem-repeats (VNTR) analysis provides a means for rapid characterization by fragment sizing and estimation of copy numbers, but structured, harmonized development has been lacking for Cryptosporidium spp. To investigate potential for application in C. parvum surveillance and outbreak investigations, we studied nine commonly used VNTR loci (MSA, MSD, MSF, MM5, MM18, MM19, MS9-Mallon, GP60 and TP14) for chromosome distribution, repeat unit length and heterogeneity, and flanking region proximity and conservation. To investigate performance in vitro, we compared these loci in 14 C. parvum samples by capillary electrophoresis in three laboratories. We found that many loci did not contain simple repeat units but were more complex, hindering calculations of repeat unit copy number for standardized reporting nomenclature. However, sequenced reference DNA enabled reproducible fragment sizing and inter-laboratory allele assignation based on size normalized to that of the sequenced fragments by both single round and nested polymerase chain reactions. Additional Cryptosporidium loci need to be identified and validated for robust inter-laboratory surveillance and outbreak investigations.

Cyclosporiasis in travellers returning to the United Kingdom from Mexico in 2017: lessons from the recent past to inform the future.

During the summers of 2015 and 2016, the United Kingdom experienced large outbreaks of cyclosporiasis in travellers returning from Mexico. As the source of the outbreaks was not identified, there is the potential for a similar outbreak to occur in 2017; indeed 78 cases had already been reported as at 27 July 2017. Early communication and international collaboration is essential to provide a better understanding of the source and extent of this recurring situation.

Discovery of new variable number tandem repeat loci in multiple Cryptosporidium parvum genomes for the surveillance and investigation of outbreaks of cryptosporidiosis.

Cryptosporidium parvum is a protozoan parasite causing gastro-intestinal disease (cryptosporidiosis) in humans and animals. The ability to investigate sources of contamination and routes of transmission by characterization and comparison of isolates in a cost- and time-efficient manner will help surveillance and epidemiological investigations, but as yet there is no standardised multi-locus typing scheme. To systematically identify variable number tandem repeat (VNTR) loci, which have been shown to provide differentiation in moderately conserved species, we interrogated the reference C. parvum Iowa II genome and seven other C. parvum genomes using a tandem repeat finder software. We identified 28 loci that met criteria defined previously for robust typing schemes for inter-laboratory surveillance, that had potential for generating PCR amplicons analysable on most fragment sizing platforms: repeats ≥6 bp, occurring in tandem in a single repeat region, and providing a total amplicon size of <300 bp including 50 bp for the location of the forward and reverse primers. The qualifying loci will be further investigated in vitro for consideration as preferred loci in the development of a robust VNTR scheme.

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples.

BACKGROUND: Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. RESULTS: The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing. CONCLUSION: This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.

Outbreak of Cryptosporidium hominis following river flooding in the city of Halle (Saale), Germany, August 2013.

BACKGROUND: During weeks 32-33, 2013, 24 cases of cryptosporidiosis were notified in the city of Halle (annual mean 2008-2012: 9 cases). We investigated the outbreak to identify the source and recommend control measures, considering that between weeks 23-25 the river Saale which flows through the city centre overflowed the floodplain, parts of the city centre and damaged sewage systems. METHODS: We defined a case as a resident of Halle with gastroenteritis, Cryptosporidium-positive stool and disease onset weeks 27 through 47. In a case-control study among kindergarten children, we compared cases and controls regarding environmental exposure, use of swimming pools, zoo visits and tap water consumption 14 days pre-onset or a corresponding 14-days-period (controls) and adjusted for residence. Stool specimens were tested by microscopy and PCR, and Cryptosporidium DNA was sequenced. Samples from public water system, swimming pools and river Saale were examined for Cryptosporidium oocysts (microscopy and PCR). RESULTS: Overall, 167 cases were detected, 40/167 (24%) were classified as secondary cases. First disease onsets occurred during week 29, numbers peaked in week 34 and started to decrease in week 36. Median age was 8 years (range: 0-77). Compared to controls (n = 61), cases (n = 20) were more likely to report visits to previously flooded areas (OR: 4.9; 95%-CI: 1.4-18) and the zoo (OR: 2.6; 95%-CI: 0.9-7.6). In multivariable analysis visits to the floodplain remained the sole risk factor (OR: 5.5; 95%-CI: 1.4-22). Only C.hominis of a single genotype (IbA9G2) was detected in stools. Oocysts were detected in samples from the river, two local lakes and three public swimming pools by microscopy, but not in the public water supply. CONCLUSIONS: Evidence suggests that activities in the dried out floodplain led to infection among children. Secondary transmissions may be involved. Consequently, authorities recommended to avoid playing, swimming and having picnics in the flood-affected area. Health authorities should consider the potential health risks of long-term surviving parasites persisting on flooded grounds and in open waters even several weeks after the flooding and of bathing places close to sewage spill-overs. Preventive measures comprise water sampling (involving parasites), information of the public and prolonged closures of potentially contaminated sites.