Genotypes of Cryptosporidium from Sydney water catchment areas

AIMS: Currently cryptosporidiosis represents the major public health concern of water utilities in developed nations and increasingly, new species and genotypes of Cryptosporidium are being identified in which the infectivity for humans is not clear. The complicated epidemiology of Cryptosporidium and the fact that the majority of species and genotypes of Cryptosporidium cannot be distinguished morphologically makes the assessment of public health risk difficult if oocysts are detected in the raw water supplies. The aim of this study was to use molecular tools to identify sources of Cryptosporidium from the Warragamba catchment area of Sydney, Australia. METHODS AND RESULTS: Both faecal and water samples from the catchment area were collected and screened using immunomagnetic separation (IMS) and immunofluorescence microscopy. Samples that contained Cryptosporidium oocysts were genotyped using sequence and phylogenetic analysis of the 18S rDNA, and the heat-shock (HSP-70) gene. Analysis identified five Cryptosporidium species/genotypes including C. parvum (cattle genotype), C. suis, pig genotype II, the cervid genotype and a novel goat genotype. CONCLUSIONS: Monitoring and characterization of the sources of oocyst contamination in watersheds will aid in the development and implementation of the most appropriate watershed management policies to protect the public from the risks of waterborne Cryptosporidium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that quantification by IMS analysis can be combined with the specificity of genotyping to provide an extremely valuable tool for assessing the human health risks from land use activities in drinking water catchments.     

Environmental inactivation of Cryptosporidium oocysts in catchment soils

AIMS: To generate field-relevant inactivation rates for Cryptosporidium oocysts in soil that may serve as parameter values in models to predict the terrestrial fate and transport of oocysts in catchments. METHODS AND RESULTS: The inactivation of Cryptosporidium oocysts in closed soil microcosms over time was monitored using fluorescence in situ hybridization (FISH) as an estimate of oocyst 'viability'. Inactivation rates for Cryptosporidium in two soils were determined under a range of temperature, moisture and biotic status regimes. Temperature and soil type emerged as significantly influential factors (P < 0.05) for Cryptosporidium inactivation. In particular, temperatures as high as 35 degrees C may result in enhanced inactivation. CONCLUSIONS: When modelling the fate of Cryptosporidium oocysts in catchment soils, the use of inactivation rates that are appropriate for the specific catchment climate and soil types is essential. FISH was considered cost-effective and appropriate for determining oocyst inactivation rates in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous models for predicting the fate of pathogens in catchments have either made nonvalidated assumptions regarding inactivation of Cryptosporidium in the terrestrial environment or have not considered it at all. Field-relevant inactivation data are presented, with significant implications for the management of catchments in warm temperate and tropical environments.     

Survival of Cryptosporidium species in environments relevant to foods and beverages

AIMS: To provide data on the survival of Cryptosporidium oocysts in a range of conditions relevant to foods and beverages. METHODS AND RESULTS: Cryptosporidium parvum and C. hominis oocysts were stored in buffered media at different pH values and with various acids. In addition, neutral solutions with high salt (4.5% w/v), glycerol (20% v/v), sucrose (50% w/v) or ethanol (9 and 40% v/v) were used to determine their effects on survival. After storage periods of between 1 h and 14 days, viability was assessed using sporozoite ratio or infection of MRC-5 cell monolayers (not previously reported for culture of this organism). With all treatments, and with both assay techniques, viable oocysts were found at the end of the storage periods. However, treatments with one of the following additions: high salt, glycerol, sucrose or ethanol showed a negative and statistically significant effect on survival. Decline was noted after 1 day or even 1 h of treatment. CONCLUSIONS: MRC-5 cells are suitable for infection by C. parvum and C. hominis. Both tissue culture and sporozoite ratio gave broadly similar survival results and the greatest effects were seen with addition of components which reduced water activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided useful additional information to the food industry when considering the risk posed by this organism.     

Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal

Aims:  Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium spp. and Giardia spp. Their presence in aquatic systems makes it essential to develop preventive strategies for water and food safety. This study was undertaken to monitor the presence of Cryptosporidium and Giardia in a total of 175 water samples, including raw and treated water from both surface and ground sources in Portugal.
Methods and Results:  The samples were processed according to USEPA Method 1623 for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts, followed by detection of oocysts/cysts by immunofluorecence (IFA) microscopy, PCR-based techniques were done on all water samples collected. Out of 175 samples, 81 (46·3%) were positive for Cryptosporidium and 67 (38·3%) for Giardia by IFA. Cryptosporidium spp. and G. duodenalis genotypes were identified by PCR in 37 (21·7%) and 9 (5·1%) water samples, respectively. C. parvum was the most common species (78·9%), followed by C. hominis (13·2%), C. andersoni (5·3%), and C. muris (2·6%). Subtype IdA15 was identified in all C. hominis -positive water samples. S ubtyping revealed the presence of C. parvum subtypes IIaA15G2R1, IIaA16G2R1 and IIdA17G1. Giardia duodenalis subtype A1 was identified.
Conclusions:  The results of the present study suggest that Cryptosporidium spp. and Giardia spp. were widely distributed in source water and treated water in Portugal. Moreover, the results obtained indicate a high occurrence of human-pathogenic Cryptosporidium genotypes and subtypes in raw and treated water samples.
Significance and Impact of the Study:  Thus, water can be a potential vehicle in the transmission of cryptosporidiosis, and giardiasis of humans and animals in Portugal.     

Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain

AIMS: To evaluate the prevalence of Cryptosporidium and Giardia in surface water supplies from the province of Alava, northern Spain, and to investigate possible associations among the presence of these pathogenic protozoa with microbiological, physicochemical and atmospheric parameters. METHODS AND RESULTS: A total of 284 samples of drinking and recreational water supplies were analysed. Cryptosporidium oocysts were found in 63.5% of river samples, 33.3% of reservoirs samples, 15.4% and 22.6% of raw water samples from conventional and small water treatment facilities (respectively), 30.8% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. Giardia cysts were found in 92.3% of river samples, 55.5% of reservoirs samples, 26.9% and 45.2% of raw water samples from conventional and small water treatment facilities (respectively), 19.2% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. The presence of Cryptosporidium and Giardia had significant Pearson's correlation coefficients (P < 0.01) with the turbidity levels of the samples, and a number of significant associations were also found with the count levels for total coliforms and Escherichia coli. The samples were positive for Cryptosporidium significantly (P < 0.05) more frequently during the autumn season than during the spring and winter seasons. No significant differences were found in the seasonal pattern of Giardia. A moderate association (r = 0.52) was found between rainfall and the presence of Cryptosporidium oocysts. CONCLUSIONS: Cryptosporidium and Giardia are consistently found at elevated concentrations in surface waters for human consumption from the province of Alava, northern Spain. SIGNIFICANCE AND IMPACT OF THE STUDY: Water treatments based on rapid filtration process and/or chlorination only are often unsatisfactory to provide safe drinking water, a situation that represents an important public health problem for the affected population because of the risk of waterborne outbreaks.     

Evaluation of an internal positive control for Cryptosporidium and Giardia testing in water samples

AIMS: An internal positive control for Cryptosporidium and Giardia monitoring was evaluated for use in routine water monitoring quality control. The control, known as ColorSeed C&G (BTF Pty Ltd, Sydney, Australia), is a suspension containing exactly 100 Cryptosporidium oocysts and 100 Giardia cysts that have been modified by attachment of Texas Red to the cell wall, allowing them to be differentiated from unmodified oocysts and cysts. The control enables recovery efficiencies to be determined for every water sample analysed. METHODS AND RESULTS: A total of 494 water samples were seeded with ColorSeed C&G and with unlabelled Cryptosporidium and Giardia and then analysed. Additionally, the robustness of the ColorSeed labelling was challenged with various chemical treatments. Recoveries were significantly lower for the ColorSeed Texas Red labelled Cryptosporidium and Giardia than recoveries of unlabelled Cryptosporidium and Giardia. However, the differences in recoveries were small. On average ColorSeed Cryptosporidium recoveries were 3.3% lower than unlabelled Cryptosporidium, and ColorSeed Giardia recoveries were 4% lower than unlabelled Giardia. CONCLUSIONS: ColorSeed C&G is suitable for use as an internal positive control for routine monitoring of both treated and raw water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The small differences in recoveries are unlikely to limit the usefulness of ColorSeed C&G as an internal positive control. The ColorSeed labelling was found to be robust after different treatments.     

Occurrence of Cryptosporidium spp. oocysts in raw and treated sewage and river water in north-eastern Spain

AIMS: To determine the occurrence and levels of Cryptosporidium parvum oocysts in wastewater and surface waters in north-eastern Spain. METHODS AND RESULTS: Samples from five sewage treatment plants were taken monthly and quarterly during 2003. In addition, water was collected monthly from the River Llobregat (NE Spain) during the period from 2001 to 2003. All samples were analysed by filtration on cellulose acetate filters or through Envirocheck using EPA method 1623, followed by immunomagnetic separation and examination by laser scanning cytometry. All raw sewage, secondary effluent and river water samples tested were positive for Cryptosporidium oocysts. Of the tertiary sewage effluents tested, 71% were positive for Cryptosporidium oocysts. The proportion of viable oocysts varied according to the sample. CONCLUSIONS: Two clear maxima were observed during spring and autumn in raw sewage, showing a seasonal distribution and a correlation with the number of cryptosporidiosis cases and rainfall events. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first data on the occurrence of Cryptosporidium oocysts in natural waters in north-eastern Spain.     

An immunomagnetic separation polymerase chain reaction assay for rapid and ultra-sensitive detection of Cryptosporidium parvum in drinking water

A sensitive and rapid method was developed to detect Cryptosporidium parvum oocysts in drinking water. This molecular assay combined immunomagnetic separation with polymerase chain reaction amplification to detect very low levels of C. parvum oocysts. Magnetic beads coated with anti-cryptosporidium were used to capture oocysts directly from drinking water membrane filter concentrates, at the same time removing polymerase chain reaction inhibitory substances. The DNA was then extracted by the freeze-boil Chelex-100 treatment, followed by polymerase chain reaction. The immunomagnetic separation-polymerase chain reaction product was identified by non-radioactive hybridization using an internal oligonucleotide probe labelled with digoxigenin. This immunomagnetic separation-polymerase chain reaction assay can detect the presence of a single seeded oocyst in 5-100-1 samples of drinking water, thereby assuring the absence of C. parvum contamination in the sample under analysis.     

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.     

Phenotypic and genotypic characterization of Cryptosporidium species and isolates

Recent outbreaks of cryptosporidiosis from contaminated water supplies have led to a need for the detection of Cryptosporidium oocysts from various hosts and contaminating sources. The presence of nonpathogenic species or strains of Cryptosporidium is important for diagnostic purposes as there is a potential for false-positive detection of pathogenic parasites. The present review focuses on phenotypic differences and recent advances in genotypic analyses of the genus Cryptosporidium with an emphasis on detecting various isolates and identifying differences in Cryptosporidium parvum and other species in this genus. The information currently available demonstrates important patterns in DNA sequences of Cryptosporidium, and our understanding of macro- and microevolutionary patterns has increased in recent years. However, current knowledge of Cryptosporidium genetic diversity is far from complete, and the large amount of both phenotypic and genotypic data has led to problems in our understanding of the systematics of this genus. Journal of Industrial Microbiology & Biotechnology (2001) 26, 95–106. Received 18 March 2000/ Accepted in revised form 13 August 2000     

Detection and identification by real time PCR/RFLP analyses of Cryptosporidium species from human faeces

AIMS: To detect a wide range of Cryptosporidium species from human faeces by analysis of the Cryptosporidium oocyst wall protein gene by PCR. METHODS AND RESULTS: The nested-assay comprised an initial amplification using a conventional thermocycler followed by real time PCR using a LightCycler with SYBR Green I for the characterization of the amplicons. The technique uses four sets of primers composed of five to six oligonucleotides with one to six base differences corresponding to the inter-species sequence differences of the gene fragment. Restriction fragment length polymorphism analysis identified Cryptosporidium hominis and C. parvum. The assay was evaluated using DNA extracted from purified material and faecal specimens containing a range of potential pathogens (including Cryptosporidium). The assay was specific, sensitive, reproducible and rapid. CONCLUSIONS: This unique technique enables the rapid detection of a range of polymorphic COWP gene sequences directly from faeces using real time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a novel approach to identification of Cryptosporidium species and the identification of C. hominis and C. parvum. The technique may be especially useful for the analysis of environmental samples which are likely to contain heterogeneous mixtures of Cryptosporidium species.     

Use of Meta-Analysis to Assess the Effect of Conventional Water Treatment Methods on the Prevalence of Cryptosporidium Spp. in Drinking Water

Meta-analysis is defined as the statistical analysis of a collection of analytic results for the purpose of integrating the findings. However, its use in environmental and agricultural health protection has been minor in research to date. Few meta-analysis studies have been applied to assess factors relating to Cryptosporidium but none have examined the effect of conventional water treatment on oocyst prevalence. The objective of this study was to synthesize past scientific research and analyze the effects of conventional water treatment methods on the prevalence of Cryptosporidium in drinking water using a traditional parametric approach of meta-analysis. Twenty-three different published studies featuring 27 separate water treatment surveys were identified for inclusion in the meta-analysis. The meta-analysis indicated that general conventional water treatment methods reduced Cryptosporidium occurrence in drinking water by an average factor of ～11.82 (95% CI = 2.7–52.5). Results highlight the resistance of the pathogen to such treatment methods and emphasize the parasites standing as a waterborne pathogen of primary concern given the possibility of viable oocyst occurrence in water, post treatment. Consequently, findings accentuate the need for new approaches to the control of Cryptosporidium aimed at integrated catchment management, source protection, and also including new treatment technologies. Until such approaches are actively adopted the potential risk posed to consumers by pathogens in potable water will remain active.     

Use of multispectral satellite datasets to improve ecological understanding of the distribution of Invasive Alien Plants in a water-limited catchment,South Africa

Invasive Alien Plants (IAPs) pose major threats to biodiversity, ecosystem functioning and services. The availability of moderate resolution satellite data (e.g. Sentinel-2 Multispectral Instrument and Landsat-8 Operational Land Imager) offers an opportunity to map and monitor the occurrence and spatial distribution of IAPs. The use of two multispectral remote sensing data sets to map and monitor IAPs in the Heuningnes Catchment, South Africa, was therefore investigated using the maximum likelihood classification algorithm. It was possible to identify areas infested with IAPs using remote sensing data. Specifically, IAPs were mapped with a higher overall accuracy of 71%, using Sentinel-2 MSI as compared to using Landsat 8 OLI, which produced 63% accuracy. However, both sensors showed similar patterns in the spatial distribution of IAPs within the hillslopes and riparian zones of the catchment. This work demonstrates the utility of the two multispectral data sets in mapping and monitoring the occurrence and distribution of IAPs, which contributes to improved ecological modelling and thus to improved management of invasions and biodiversity in the catchment.     

Beyond species distribution modeling: A landscape genetics approach to investigating range shifts under future climate change

Understanding how biodiversity will respond to future climate change is a major conservation and societal challenge. Climate change is predicted to force many species to shift their ranges in pursuit of suitable conditions. This study aims to use landscape genetics, the study of the effects of environmental heterogeneity on the spatial distribution of genetic variation, as a predictive tool to assess how species will shift their ranges to track climatic changes and inform conservation measures that will facilitate movement. The approach is based on three steps: 1) using species distribution models (SDMs) to predict suitable ranges under future climate change, 2) using the landscape genetics framework to identify landscape variables that impede or facilitate movement, and 3) extrapolating the effect of landscape connectivity on range shifts in response to future climate change. I show how this approach can be implemented using the publicly available genetic dataset of the grey long-eared bat, Plecotus austriacus, in the Iberian Peninsula. Forest cover gradient was the main landscape variable affecting genetic connectivity between colonies. Forest availability is likely to limit future range shifts in response to climate change, primarily over the central plateau, but important range shift pathways have been identified along the eastern and western coasts. I provide outputs that can be directly used by conservation managers and review the viability of the approach. Using landscape genetics as a predictive tool in combination with SDMs enables the identification of potential pathways, whose loss can affect the ability of species to shift their range into future climatically suitable areas, and the appropriate conservation management measures to increase landscape connectivity and facilitate movement.     

Evaluation of water treatment plant UV reactor efficiency against Cryptosporidium parvum oocyst infectivity in immunocompetent suckling mice

Aim: To assess the efficiency of a medium‐pressure UV reactor under full‐scale water treatment plant (WTP) conditions on the infectivity of Cryptosporidium parvum oocysts in an Naval Medical Research Institute (NMRI) suckling mice infectivity model. Methods and Results: Six/seven‐day‐old mice were administered orally 2–10 × 10⁴Cryptosporidium parvum oocysts. Compared with nonirradiated oocysts, 40 mJ cm^?2 UV irradiation of ingested oocysts resulted 7 days later in a 3·4–4·0 log₁₀ reduction in the counts of small intestine oocysts, using a fluorescent flow cytometry assay. Conclusion: Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development. Significance and Impact of the study: Present results suggest that in WTP conditions, a medium‐pressure UV reactor is efficient in reducing the infectivity of Cryptosporidium parvum oocysts, one of the most resistant micro‐organisms present in environmental waters.     

黄土高原西部旱农区不同品种马铃薯集雨限灌效应

通过大田试验研究了集雨限灌对同薯23号和大西洋两个马铃薯品种的补灌效应.结果表明：集蓄雨水在苗期补灌能显著提高旱作马铃薯的产量,且大西洋增产幅度高于同薯23号;在薯块膨大期补灌同薯23号的产量增加不显著,而大西洋减产.低额补灌（补灌45 mm）有利于同薯23号水分利用效率（WUE）和灌水利用效率（IWUE）的提高.大西洋苗期补灌的WUE和IWUE均高于薯块膨大期.补灌提高了同薯23号的大薯率、中薯率和单株薯产量,降低了小薯率, 但绿薯率、烂薯率有所增加;大西洋的大薯率和小薯率增加,中薯率稍有下降,烂薯率增加.     

Effects of hydrogeomorphic region, catchment storage and mature forest on baseflow and snowmelt stream water quality in second-order Lake Superior Basin tributaries

1. In this study we predict stream sensitivity to non‐point source pollution based on the non‐linear responses of hydrological regimes and associated loadings of non‐point source pollutants to catchment properties. We assessed two hydrologically based thresholds of impairment, one for catchment storage (5–10%) and one for mature forest (<50% versus >60% of catchment in mature forest cover) across two different hydrogeomorphic regions within the Northern Lakes and Forest (NLF) ecoregion: the North Shore [predominantly within the North Shore Highlands Ecological Unit] and the South Shore (predominantly within the Lake Superior Clay Plain Ecological Unit). Water quality samples were collected and analysed during peak snowmelt and baseflow conditions from 24 second‐order streams grouped as follows: three in each region × catchment storage × mature forest class. 2. Water quality was affected by a combination of regional influences, catchment storage and mature forest. Regional differences were significant for suspended solids, phosphorus, nitrogen: phosphorus ratios, dissolved organic carbon (DOC) and alkalinity. Catchment storage was significantly correlated with dissolved silica during the early to mid‐growing season, and with DOC, specific conductance and alkalinity during all seasons. Total nitrogen and dissolved nitrogen were consistently less in low mature forest than in high mature forest catchments. Catchment storage interacted with the influence of mature forest for only two metrics: colour and the soluble inorganic nitrogen : phosphorus ratio. 3. Significant interaction terms (region by mature forest or region by storage) suggest differences in regional sensitivity for conductance, alkalinity, total organic carbon, and colour, as well as possible shifts in thresholds of impact across region or mature forest class. 4. Use of the NLF Ecoregion alone as a basis for setting regional water quality criteria would lead to the misinterpretation of reference condition and assessment of condition. There were pronounced differences in background water quality between the North and South Shore streams, particularly for parameters related to differences in soil parent material and glacial history. A stratified random sampling design for baseflow and snowmelt stream water quality based on both hydrogeomorphic region and catchment attributes improves assessments of both reference condition and differences in regional sensitivity.     

基于MaxEnt模型的延河流域草本植物适生分布与功能性状分析

明确延河流域常见草本植物的潜在适生区分布,是植被恢复工作持续推进的基础。本研究收集了延河流域8种常见草本植物的地理分布信息和13个环境变量,采用MaxEnt和ArcGIS模拟了延河流域常见草本植物在当前气候下的潜在适生性分布,进而研究这8种不同草本植物适生性分布与功能性状变异特征之间的相关关系。研究结果显示：根据物种-性状排序图的分布格局判断,本研究选择的七个功能性状在植物所属科之间发生了明显趋异分化现象,在PC1右侧为禾本科植物,PC1左侧为菊科、豆科和唇形科植物。对物种适生性分布模拟结果表明,达乌里胡枝子在研究区内的适生性最高,百里香的适生性最低,表明达乌里胡枝子比其他常见草本物种更适合被选择为该流域的植被恢复的先锋物种。在功能性状变异特征相关性分析中,物种适生区大小与比叶面积变异系数呈显著正相关,与其他植物功能性状变异特征不显著。因此,比叶面积的变异系数更适合作为指示延河流域草本植物适生区大小的性状。