Population structure and pathogen interaction of Escherichia coli in freshwater: Implications of land-use for water quality and public health in Aotearoa New Zealand.

Freshwater samples (n = 199) were obtained from 41 sites with contrasting land-uses (avian, low impact, dairy, urban, sheep and beef, and mixed sheep, beef and dairy) and the E. coli phylotype of 3980 isolates (20 per water sample enrichment) was determined. Eight phylotypes were identified with B1 (48.04%), B2 (14.87%) and A (14.79%) the most abundant. Escherichia marmotae (n = 22), and Escherichia ruysiae (n = 1), were rare (0.68%) suggesting that these environmental strains are unlikely to confound water quality assessments. Phylotypes A and B1 were overrepresented in dairy and urban sites (p < 0.0001), whilst B2 were overrepresented in low impact sites (p < 0.0001). Pathogens ((Salmonella, Campylobacter, Cryptosporidium or Giardia) and the presence of diarrhoeagenic E. coli-associated genes (stx and eae) were detected in 89.9% (179/199) samples, including 80.5% (33/41) of samples with putative non-recent faecal inputs. Quantitative PCR to detect microbial source tracking targets from human, ruminant and avian contamination were concordant with land-use type and E. coli phylotype abundance. This study demonstrated that a potential recreational health risk remains where pathogens occurred in water samples with low E. coli concentration, potential non-recent faecal sources, low impact sites and where human, ruminant and avian faecal sources were absent.

Optimization of a DiCre recombinase system with reduced leakage for conditional genome editing of Cryptosporidium.

BACKGROUND: The dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes. METHODS: Based on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses. RESULTS: The use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35-75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies. CONCLUSIONS: A DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established.

Risk-based critical concentrations of enteric pathogens for recreational water criteria and recommended minimum sample volumes for routine water monitoring.

Concerns are rising about the contamination of recreational waters from human and animal waste, along with associated risks to public health. However, existing guidelines for managing pathogens in these environments have not yet fully integrated risk-based pathogen-specific criteria, which, along with recent advancements in indicators and markers, are essential to improve the protection of public health. This study aimed to establish risk-based critical concentration benchmarks for significant enteric pathogens, i.e., norovirus, rotavirus, adenovirus, Cryptosporidium spp., Giardia lamblia, Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7. Applying a 0.036 risk benchmark to both marine and freshwater environments, the study identified the lowest critical concentrations for children, who are the most susceptible group. Norovirus, C. jejuni, and Cryptosporidium presented lowest median critical concentrations for virus, bacteria, and protozoa, respectively: 0.74 GC, 1.73 CFU, and 0.39 viable oocysts per 100 mL in freshwater for children. These values were then used to determine minimum sample volumes corresponding to different recovery rates for culture method, digital polymerase chain reaction and quantitative PCR methods. The results indicate that for children, norovirus required the largest sample volumes of freshwater and marine water (52.08 to 178.57 L, based on the 5th percentile with a 10 % recovery rate), reflecting its low critical concentration and high potential for causing illness. In contrast, adenovirus and rotavirus required significantly smaller volumes (approximately 0.24 to 1.33 L). C. jejuni and Cryptosporidium, which required the highest sampling volumes for bacteria and protozoa, needed 1.72 to 11.09 L and 4.17 to 25.51 L, respectively. Additionally, the presented risk-based framework could provide a model for establishing pathogen thresholds, potentially guiding the creation of extensive risk-based criteria for various pathogens in recreational waters, thus aiding public health authorities in decision-making, strengthening pathogen monitoring, and improving water quality testing accuracy for enhanced health protection.

Cryptosporidium spp. in captive snakes from 26 provinces in China: Prevalence, molecular characterization, and symptoms.

Snakes are sometimes regarded as pets and are used in traditional Chinese medicine. Cryptosporidium spp. are frequently identified in snakes, representing an important pathogen and causing gastrointestinal diseases. Current data indicate that risk factors for infection and patterns of clinical symptom presentation may differ among Cryptosporidium spp. To better understand the infection status by Cryptosporidium spp., fecal samples were collected from 603 asymptomatic and 147 symptomatic snakes in 26 provinces of China. These samples came from Elaphe guttata, Elaphe obsoleta, Pituophis melanoleucus, Thamnophis sirtalis, Lampropeltis getulus, and Heterodon nasicus. The partial small subunit (SSU) rRNA gene was amplified using nested polymerase chain reaction (PCR) to investigate the infection rate of Cryptosporidium spp., and to assess evolutionary relationships and genetic characterization. A prevalence of 20% was recorded in asymptomatic snakes, with age identified as a significant risk factor. In contrast, 70% of symptomatic snakes were positive for Cryptosporidium spp., with Cryptosporidium serpentis and Cryptosporidium varanii (syn. C. saurophilum). Further analysis revealed a potential association between C. serpentis and regurgitation, and C. varanii and diarrhea, while neither species was linked to flatulence. To our knowledge, this is the first study to report Cryptosporidium spp. and associated clinical signs in symptomatic snakes in China. This study aims to enhance the understanding of Cryptosporidium infections, risk factors, and clinical manifestations in snakes, providing data crucial for the control and prevention of cryptosporidiosis.

Title: Cryptosporidium spp. chez les serpents captifs de 26 provinces de Chine : prévalence, caractérisation moléculaire et symptômes. Abstract: Les serpents sont parfois considérés comme animaux de compagnie et sont utilisés en médecine traditionnelle chinoise. Des Cryptosporidium spp. sont fréquemment identifiés chez les serpents, ont un rôle d'agent pathogène important et provoquent des maladies gastro-intestinales. Les données actuelles indiquent que les facteurs de risque d'infection et les schémas de présentation des symptômes cliniques peuvent varier en fonction des espèces de Cryptosporidium. Pour mieux comprendre l'état d'infection par Cryptosporidium spp., des échantillons fécaux ont été collectés auprès de 603 serpents asymptomatiques et 147 serpents symptomatiques dans 26 provinces de Chine. Ces échantillons provenaient d'Elaphe guttata, Elaphe obsoleta, Pituophis melanoleucus, Thamnophis sirtalis, Lampropeltis getulus et Heterodon nasicus. Le gène de l'ARNr de la petite sous-unité partielle (SSU) a été amplifié à l'aide d'une réaction en chaîne par polymérase (PCR) imbriquée pour étudier le taux d'infection par Cryptosporidium spp. et évaluer les relations évolutives et la caractérisation génétique. Une prévalence de 20 % a été trouvée chez les serpents asymptomatiques, l'âge étant identifié comme un facteur de risque important. En revanche, 70 % des serpents symptomatiques étaient positifs à Cryptosporidium spp. avec Cryptosporidium serpentis et Cryptosporidium varanii (syn. C. saurophilum). Une analyse plus approfondie a révélé une association potentielle entre C. serpentis et la régurgitation, et C. varanii et la diarrhée, alors qu'aucune des deux espèces n'était liée aux flatulences. À notre connaissance, il s'agit ici de la première étude à signaler la présence de Cryptosporidium spp. et les signes cliniques associés chez des serpents symptomatiques en Chine. Cette étude vise à améliorer la compréhension des infections à Cryptosporidium, des facteurs de risque et des manifestations cliniques chez les serpents, en fournissant des données cruciales pour le contrôle et la prévention de la cryptosporidiose.

Multiplex PCR for gastrointestinal parasites in stool: Benchmarking against direct microscopy and simplex PCR.

PURPOSE: To develop and validate a multiplex conventional PCR assay to simultaneously detect Cryptosporidium spp., Entamoeba histolytica, and Giardia lamblia in diarrheal samples as a rapid, cost-effective, and sensitive diagnostic tool for prevalent co-infections for improved diagnostic accuracy and efficiency in resource-limited settings. METHODS: Stool samples collected from patients with gastrointestinal symptoms after taking written consent, processed via wet mount, iodine mount, and PCR assays. Cohen's kappa statistical analysis was done to test agreement. RESULT: Among 240 patients, 28.75% showed intestinal protozoa via Microscopy; Single-plex and multiplex PCR demonstrated 100% concordance, detecting 27.9%; confirmed by sequencing. Highest parasite positivity was observed in transplant and immunocompromised patients, with moderate to almost perfect agreement between microscopy and molecular methods. CONCLUSION: Multiplex-conventional PCR offers superior sensitivity and specificity over microscopy and 100% concordance with single-plex PCR, enabling rapid, cost-effective diagnosis of multiple parasites from single stool sample. Its adoption could revolutionize parasitic infection management in routine diagnostics.

Marked increase in cryptosporidiosis cases, Spain, 2023.

BackgroundBy mid-September 2023, several event notifications related to cryptosporidiosis had been identified from different regions in Spain. Therefore, a request for urgent notification of cryptosporidiosis cases to the National Surveillance Network was launched.AimWe aimed at assessing the extent of the increase in cases, the epidemiological characteristics and the transmission modes and compared to previous years.MethodsWe analysed data on case notifications, outbreak reports and genotypes focusing on June-October 2023 and compared the results to 2016-2022.ResultsIn 2023, 4,061 cryptosporidiosis cases were notified in Spain, which is an increase compared to 2016-2022. The cumulative incidence was 8.3 cases per 100,000 inhabitants in 2023, sixfold higher than the median of 1.4 cases per 100,000 inhabitants 2016-2022. Almost 80% of the cases were notified between June and October. The largest outbreaks were related to contaminated drinking water or swimming pools. Cryptosporidium hominis was the most common species in the characterised samples (115/122), and the C. hominis IfA12G1R5 subtype, previously unusual in Spain, was detected from 76 (62.3%) of the 122 characterised samples.ConclusionsA substantial increase in cryptosporidiosis cases was observed in 2023. Strengthening surveillance of Cryptosporidium is essential for prevention of cases, to better understand trends and subtypes circulating and the impact of adverse meteorological events.

Assessment of pathogenic protozoa in a drinking water treatment plant with UV treatment.

Controlling drinking water treatment processes is essential to address water contamination and the adaptability of certain pathogenic protozoa. Sometimes, standard treatment methods and chlorine disinfection may prove insufficient in eliminating pathogenic protozoa. However, ultraviolet (UV) radiation has proved to be more effective than chlorine. This study aims to characterize the eukaryotic community of a drinking water treatment plant that applies a final UV disinfection treatment, focusing on pathogenic protozoa. Fifty water samples (raw water, before and after UV treatment) were evaluated to comply with regulation parameters and identify relevant protozoa. Despite physicochemical and microbiological parameters meeting the regulation, some potentially pathogenic protozoa, such as Blastocystis or Cryptosporidium, were still detected in very low relative abundances in treated water. It was found for the first time in Spain the pathogenic amoebae Naegleria fowleri in one river water, which was not found after the treatment. Moreover, Blastocystis subtypes ST1-ST6 were detected in this study in raw, before and after UV water samples. Blastocystis was only found in 2 two samples after UV treatment, with a very low abundance (≤0.02%). Obtained results demonstrate the effectiveness of water treatment in reducing the prevalence of pathogenic protozoa.

The epidemiology of intestinal protozoa in the Israeli population based on molecular stool test: a nationwide study.

Stool examination using microscopy was the traditional method for the diagnosis of intestinal parasites. Recently, the use of molecular tests to identify stool protozoa has become the main tool used in most clinical laboratories in Israel. This study aimed to evaluate the prevalence of intestinal parasites in Israel and to compare this prevalence in laboratories that use molecular tests vs a laboratory that uses microscopy. Samples collected from January to October 2021 at seven laboratories were analyzed by real-time PCR (RT-PCR) or by microscopy. The multiplex panel included the following pathogens: Giardia lamblia, Entamoeba histolytica, Cryptosporidium spp., Cyclospora, Dientamoeba fragilis, and Blastocystis spp. Overall, 138,415 stool samples were tested by RT-PCR and 6,444 by microscopy. At least one protozoa species was identified in 28.4% of the PCR-tested samples compared to 4.6% of the microscopy-tested samples. D. fragilis was the most common PCR-identified species (29%). D. fragilis, G. lamblia, and Cryptosporidium spp. were mainly found in pediatric population, while Blastocystis spp. was most prevalent among adults (P < 0.001). In a sub-cohort of 21,480 samples, co-infection was found in 4,113 (19.15%) samples, with Blastocystis spp. and D. fragilis being the most common (14.9%) pair. Molecular stool testing proved more sensitive compared to microscopy. D. fragilis was the most commonly detected pathogen. The above profile was identified during the COVID pandemic when traveling was highly restricted and most likely represents the locally circulating protozoa. IMPORTANCE: This study sheds light on the prevalence of stool parasites in Israel. Additionally, this study indicates that the shift from microscope analysis to molecular tests improved protozoa diagnosis.

LncRNA Nostrill promotes interferon-γ-stimulated gene transcription and facilitates intestinal epithelial cell-intrinsic anti-Cryptosporidium defense.

Intestinal epithelial cells possess the requisite molecular machinery to initiate cell-intrinsic defensive responses against intracellular pathogens, including intracellular parasites. Interferons(IFNs) have been identified as cornerstones of epithelial cell-intrinsic defense against such pathogens in the gastrointestinal tract. Long non-coding RNAs (lncRNAs) are RNA transcripts (>200 nt) not translated into protein and represent a critical regulatory component of mucosal defense. We report here that lncRNA Nostrill facilitates IFN-γ-stimulated intestinal epithelial cell-intrinsic defense against infection by Cryptosporidium, an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children. Nostrill promotes transcription of a panel of genes controlled by IFN-γ through facilitating Stat1 chromatin recruitment and thus, enhances expression of several genes associated with cell-intrinsic defense in intestinal epithelial cells in response to IFN-γ stimulation, including Igtp, iNos, and Gadd45g. Induction of Nostrill enhances IFN-γ-stimulated intestinal epithelial defense against Cryptosporidium infection, which is associated with an enhanced autophagy in intestinal epithelial cells. Our findings reveal that Nostrill enhances the transcription of a set of genes regulated by IFN-γ in intestinal epithelial cells. Moreover, induction of Nostrill facilitates the IFN-γ-mediated epithelial cell-intrinsic defense against cryptosporidial infections.

Quantifying Replication Slippage Error in Cryptosporidium Metabarcoding Studies.

Genetic variation in Cryptosporidium, a common protozoan gut parasite in humans, is often based on marker genes containing trinucleotide repeats, which differentiate subtypes and track outbreaks. However, repeat regions have high replication slippage rates, making it difficult to discern biological diversity from error. Here, we synthesized Cryptosporidium DNA in clonal plasmid vectors, amplified them in different mock community ratios, and sequenced them using next-generation sequencing to determine the rate of replication slippage with dada2. Our results indicate that slippage rates increase with the length of the repeat region and can contribute to error rates of up to 20%.

Molecular characterizations of Cryptosporidium spp. in brown rat (Rattus norvegicus) from an animal feedlot in Xinjiang, China.

Cryptosporidium infection is a common occurrence in rodents worldwide. In this study, 435 wild brown rats were captured from an animal feedlot in Xinjiang, China, with a fecal sample obtained directly from the rectal contents of each rat. The DNA extracted from these fecal samples was analyzed for Cryptosporidium spp. using PCR targeting the SSU rRNA gene. The prevalence of Cryptosporidium infection in brown rats was found to be 5.5% (24 out of 435). Interestingly, the infection rates varied among different animal enclosures, with rates of 0% in the chicken coop (0/51), cowshed (0/3), and varying rates in other areas including the sheepfold (6.1%, 6/98), the pigsty (7.6%, 10/132), the dovecote (7.0%, 5/71), and outdoor environments (3.8%, 3/80). The study identified three species and one genotype of Cryptosporidium, namely C. occultus (n = 10), C. parvum (n = 4), C. ditrichi (n = 1), and Cryptosporidium rat genotype IV (n = 9). Additionally, two of the C. parvum isolates were successfully subtyped as IIdA19G1 (n = 2) at the gp60 gene. These results offer valuable insights into the prevalence and genetic diversity of Cryptosporidium in brown rats within the region.

Genetic characterization of Cryptosporidium spp. in the North African hedgehog (Atelerix algirus) in the Canary Islands, Spain.

The North African hedgehog (Atelerix algirus) is an introduced species from Northwest Africa and is currently distributed in the Canary Islands. This species of hedgehog has been studied as a reservoir of enteropathogens, including Cryptosporidium spp. However, there are no data at species level. Therefore, the aim of the present study was to identify the Cryptosporidium species present in a population of hedgehogs (n = 36) in the Canary Islands. Molecular screening was performed using conventional polymerase chain reaction (PCR) targeting the small subunit ribosomal RNA (18S rRNA) gene of Cryptosporidium spp. Seven of the 36 fecal samples (19.45%) were positive and confirmed by nested PCR targeting the 18S rRNA gene and Sanger sequencing. Cryptosporidium parvum and Cryptosporidium muris were identified in 11.1% (4/36) and 5.6% (2/36) of the samples, respectively, while one sample could only be identified at the genus level. The zoonotic subtypes IIdA15G1 (n = 1), IIdA16G1b (n = 1), and IIdA22G1 (n = 1) of C. parvum were identified by nested PCR followed by analysis of the 60 kDa glycoprotein (gp60) gene sequence. This study is the first genetic characterization of Cryptosporidium spp. in A. algirus, identifying zoonotic species and subtypes of the parasite.

Cross-species transmission of Cryptosporidium in wild rodents from the southern region of Zhejiang Province of China and its possible impact on public health.

Wild rodents serve as reservoirs for Cryptosporidium and are overpopulated globally. However, genetic data regarding Cryptosporidium in these animals from China are limited. Here, we have determined the prevalence and genetic characteristics of Cryptosporidium among 370 wild rodents captured from three distinct locations in the southern region of Zhejiang Province, China. Fresh feces were collected from the rectum of each rodent, and DNA was extracted from them. The rodent species was identified by PCR amplifying the vertebrate cytochrome b gene. Cryptosporidium was detected by PCR amplification and amplicon sequencing the small subunit of ribosomal RNA gene. Positive samples of C. viatorum and C. parvum were further subtyped by analyzing the 60-kDa glycoprotein gene. A positive Cryptosporidium result was found in 7% (26/370) of samples, involving five rodent species: Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155), and R. tanezumi (86). Their respective Cryptosporidium positive rates were 8.3%, 5.3%, 11.1%, 7.1%, and 7.0%. Sequence analysis confirmed the presence of three Cryptosporidium species: C. parvum (4), C. viatorum (1), and C. muris (1), and two genotypes: Cryptosporidium rat genotype IV (16) and C. mortiferum-like (4). Additionally, two subtypes of C. parvum (IIdA15G1 and IIpA19) and one subtype of C. viatorum (XVdA3) were detected. These results demonstrate that various wild rodent species in Zhejiang were concurrently infected with rodent-adapted and zoonotic species/genotypes of Cryptosporidium, indicating that these rodents can play a role in maintaining and dispersing this parasite into the environment and other hosts, including humans.

Title: Transmission interspécifique de Cryptosporidium chez les rongeurs sauvages de la région sud de la province chinoise du Zhejiang et son impact possible sur la santé publique. Abstract: Les rongeurs sauvages servent de réservoirs à Cryptosporidium et ont des grandes populations à l'échelle mondiale. Cependant, les données génétiques concernant Cryptosporidium chez ces animaux en Chine sont limitées. Ici, nous avons déterminé la prévalence et les caractéristiques génétiques de Cryptosporidium parmi 370 rongeurs sauvages capturés dans trois endroits distincts de la région sud de la province du Zhejiang, en Chine. Des excréments frais ont été collectés dans le rectum de chaque rongeur et l'ADN en a été extrait. L'espèce de rongeur a été identifiée par amplification par PCR du gène du cytochrome b des vertébrés. Cryptosporidium a été détecté par amplification PCR et séquençage d'amplicons de la petite sous-unité du gène de l'ARN ribosomal. Les échantillons positifs de C. viatorum et C. parvum ont ensuite été sous-typés en analysant le gène de la glycoprotéine de 60 kDa. Un résultat positif pour Cryptosporidium a été trouvé dans 7 % (26/370) des échantillons, impliquant cinq espèces de rongeurs : Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155) et R. tanezumi (86). Leurs taux respectifs de positivité pour Cryptosporidium étaient de 8,3 %, 5,3 %, 11,1 %, 7,1 % et 7,0 %. L'analyse des séquences a confirmé la présence de trois espèces de Cryptosporidium : C. parvum (4), C. viatorum (1) et C. muris (1), et de deux génotypes : Cryptosporidium génotype IV de rat (16) et C. mortiferum-like (4). De plus, deux sous-types de C. parvum (IIdA15G1 et IIpA19) et un sous-type de C. viatorum (XVdA3) ont été détectés. Ces résultats démontrent que diverses espèces de rongeurs sauvages du Zhejiang sont simultanément infectées par des espèces/génotypes de Cryptosporidium zoonotiques et adaptés aux rongeurs, ce qui indique que ces rongeurs peuvent jouer un rôle dans le maintien et la dispersion de ce parasite dans l'environnement et d'autres hôtes, y compris les humains.

Unveiling risks in healthy food: Vegetables and fruits are linked to the distribution chain of protozoan parasites.

Vegetable and fruit contamination is recognized as a significant parasite transmission route. This review presents the current state of vegetables ad fruits contamination with food-borne parasitic protozoa worldwide. We consider the methodologies and strategies for detecting parasitic stages developed in the last decade and the contamination data. Asia had the highest number of reports (94 studies), followed by Africa (74 studies). At the country level, with 41 studies, Iran had the most reports among other countries, followed by Nigeria (28 studies). According to the studies included in the current review, 41.22% of vegetables and fruits were contaminated with different species of protozoan parasites. Among different continents, Asia accounted for the highest contamination rate of protozoan parasites (57.12%). Giardia spp. (10%) had the highest contamination rate in vegetables and fruits, followed by Entamoeba coli (8%), E. histolytica/dispar (7%), and Cryptosporidium spp. (6%). This study provides essential data for health authorities to develop food safety programs. The presence of protozoan parasites in fruits and vegetables highlights the critical need for maintaining rigorous food safety measures across the entire production and distribution process, particularly in countries that are major producers and distributors of these food items.

Machine learning and explainable artificial intelligence for the prevention of waterborne cryptosporidiosis and giardiosis.

Cryptosporidium and Giardia are important parasitic protozoa due to their zoonotic potential and impact on human health, and have often caused waterborne outbreaks of disease. Detection of (oo)cysts in water matrices is challenging and extremely costly, thus only few countries have legislated for regular monitoring of drinking water for their presence. Several attempts have been made trying to investigate the association between the presence of such (oo)cysts in waters with other biotic or abiotic factors, with inconclusive findings. In this regard, the aim of this study was the development of an holistic approach leveraging Machine Learning (ML) and eXplainable Artificial Intelligence (XAI) techniques, in order to provide empirical evidence related to the presence and prediction of Cryptosporidium oocysts and Giardia cysts in water samples. To meet this objective, we initially modelled the complex relationship between Cryptosporidium and Giardia (oo)cysts and a set of parasitological, microbiological, physicochemical and meteorological parameters via a model-agnostic meta-learner algorithm that provides flexibility regarding the selection of the ML model executing the fitting task. Based on this generic approach, a set of four well-known ML candidates were, empirically, evaluated in terms of their predictive capabilities. Then, the best-performed algorithms, were further examined through XAI techniques for gaining meaningful insights related to the explainability and interpretability of the derived solutions. The findings reveal that the Random Forest achieves the highest prediction performance when the objective is the prediction of both contamination and contamination intensity with Cryptosporidium oocysts in a given water sample, with meteorological/physicochemical and microbiological markers being informative, respectively. For the prediction of contamination with Giardia, the eXtreme Gradient Boosting with physicochemical parameters was the most efficient algorithm, while, the Support Vector Regression that takes into consideration both microbiological and meteorological markers was more efficient for evaluating the contamination intensity with cysts. The results of the study designate that the adoption of ML and XAI approaches can be considered as a valuable tool for unveiling the complicated correlation of the presence and contamination intensity with these zoonotic parasites that could constitute, in turn, a basis for the development of monitoring platforms and early warning systems for the prevention of waterborne disease outbreaks.

Cryptosporidium impacts epithelial turnover and is resistant to induced death of the host cell.

Infection with the apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease. Cryptosporidiosis is of particular importance in infants and shows a strong association with malnutrition, both as a risk factor and as a consequence. Cryptosporidium invades and replicates within the small intestine epithelial cells. This is a highly dynamic tissue that is developmentally stratified along the villus axis. New cells emerge from a stem cell niche in the crypt and differentiate into mature epithelial cells while moving toward the villus tip, where they are ultimately shed. Here, we studied the impact of Cryptosporidium infection on this dynamic architecture. Tracing DNA synthesis in pulse-chase experiments in vivo, we quantified the genesis and migration of epithelial cells along the villus. We found proliferation and epithelial migration to be elevated in response to Cryptosporidium infection. Infection also resulted in significant cell loss documented by imaging and molecular assays. Consistent with these observations, single-cell RNA sequencing of infected intestines showed a gain of young and a loss of mature cells. Interestingly, enhanced epithelial cell loss was not a function of enhanced apoptosis of infected cells. To the contrary, Cryptosporidium-infected cells were less likely to be apoptotic than bystanders, and experiments in tissue culture demonstrated that infection provided enhanced resistance to chemically induced apoptosis to the host but not bystander cells. Overall, this study suggests that Cryptosporidium may modulate cell apoptosis and documents pronounced changes in tissue homeostasis due to parasite infection, which may contribute to its long-term impact on the developmental and nutritional state of children. IMPORTANCE: The intestine must balance its roles in digestion and nutrient absorption with the maintenance of an effective barrier to colonization and breach by numerous potential pathogens. An important component of this balance is its constant turnover, which is modulated by a gain of cells due to proliferation and loss due to death or extrusion. Here, we report that Cryptosporidium infection changes the dynamics of this process increasing both gain and loss of enterocytes speeding up the villus elevator. This leads to a much more immature epithelium and a reduction of the number of those cells typically found toward the villus apex best equipped to take up key nutrients including carbohydrates and lipids. These changes in the cellular architecture and physiology of the small intestine may be linked to the profound association between cryptosporidiosis and malnutrition.

Comprehensive molecular epidemiology of Cryptosporidium species in Japan.

Cryptosporidium species, causing diarrheal illnesses in humans and animals worldwide, are under investigation for their molecular epidemiology in Japan. The study focuses on detecting Cryptosporidium species in humans, animals, water, and the environment, revealing three species in people: C. parvum, C. meleagridis, and C. hominis. Subtype IIa of the C. parvum gp60 gene is prevalent, indicating potential zoonotic transmission. Animal studies identified sixteen species, mainly cattle and pets, with C. parvum (subtype IIa) common in cattle and C. canis and C. felis prevalent in pets. Additionally, C. bovis and C. ryanae were found in cattle and sika deer. Knowledge gaps exist, particularly in water and environmental source typing, with limited research revealing five species and five genotypes, suggesting a significant role of water in transmission. Further research is needed to understand the molecular diversity and transmission dynamics across humans, animals, water, and the environment in Japan.

Physiological and epidemiological study of some parasitic and viral enteric infections in dromedary camels in Al-Muthanna province.

Understanding the normal physiology of the body is the key to study the changes that occur due to any infection. It is known that enteric infections play a considerable role in affecting normal body status. Thus, this study was designed for investigating the enteric infections in Arabian camels in Al-Muthanna Province. In this investigation, 588 fecal and blood serum samples (for diarrheic camels only) were collected from the camels in different areas of Al-Muthanna Province, Iraq from both sexes of different ages during the period from October 2020 up to the end of August 2021. The samples were examined using routine microscopic examination techniques, hematological techniques, and ELISA for parasitic and viral identification. Eimeria rajasthani, Isospora orlovi were recorded for the first time in Iraqi camels with clinical signs of diarrhea, dehydration, and emaciation. The study recorded four types of protozoa: Eimeria spp., Isospora, Cryptosporidium and Balantidium coli. The recorded types of Eimeria were E. dromedarii, E. cameli, and E. rajasthani. There was a significant effect of age on infection rates with Eimeria spp. as the highest Eimeria ratio was in ages of less than two years animals. The infection rates were also affected with months which reached the highest ratios of Eimeria in October while the lowest ratio of Eimeria was recorded in July. BVDV infection rate was found in camels that suffered from diarrhea. There is no significant effect of sex on the onset of the viral disease in camels. For hematological parameters, there were significant differences in RBCs, WBCs, Hb, and PCV values in protozoal and BVDV infections. In conclusion, different kinds of protozoal and viral infections were recorded. Some of the recorded infections were associated with acute clinical signs and have zoonotic importance.

Occurrence and factors associated with Cryptosporidium infection in livestock in three districts of Zambia.

Cryptosporidium is one of the most important enteric diarrhoeal parasites that infect humans and animals worldwide. The current study investigated the occurrence and risk factors associated with Cryptosporidium infection in ruminants aged ≤6 months in Monze, Mumbwa, and Lusaka districts of Zambia. Faecal samples were collected from 328 calves, 190 lambs, and 245 goat kids and analysed for Cryptosporidium oocysts using modified Ziehl Neelsen staining. A closed structured questionnaire was used to obtain epidemiological characteristics and potential risk factors for Cryptosporidium infection. The overall occurrence of Cryptosporidium was 7.9% (60/763), while that in calves, lambs and goat kids was 14.5% (47/328), 5.3% (10/190), and 1.2% (3/245) respectively. Watery/pasty stool and sampling during the rainy season were independently associated with increased risk of infection. In calves, the odds of infection increased during the rainy season, while daily kraal cleaning reduced the infection risk. Lambs showed increased odds of infection with pasty/watery stool and male sex, whereas the wearing of protective clothing by handlers significantly reduced the risk. There were district variations in infection occurrence with Mumbwa district having higher prevalence. The findings of this study show that livestock in Zambia continue to be frequently infected with Cryptosporidium. Protective measures and appropriate farm cleanliness should be implemented in control of this infection. Regional and host-species-specific variations emphasize the need for targeted interventions. These findings, therefore, contribute to effective strategies for Cryptosporidium control, promoting good livestock health and management.

Diversity of gastrointestinal parasites and molecular characterization of Giardia duodenalis in free-living and captive howler monkeys (Alouatta guariba clamitans) in southern Brazil.

Non-human primates (NHPs) are the group that most share infectious agents with humans due to their close taxonomic relationship. The southern brown howler monkeys (Alouatta guariba clamitans) are endemic primates from Brazil and Argentina's Atlantic Forest. This study aimed to investigate the presence of intestinal parasites in free-living (FL) and captive (CA) southern brown howler monkeys. Thirty-nine stool samples were collected in two areas in southern Brazil, 15 FL and 24 CA. Stool sediments obtained by centrifugal sedimentation technique were used for microscopic analysis and direct immunofluorescence assay and evaluated by molecular analysis through amplification and sequencing of TPI fragments. Intestinal parasites Giardia duodenalis, Cryptosporidium spp., and Trypanoxyuris minutus were detected at coproparasitological analysis. This is the first report of the presence of Cryptosporidium spp. in free-living howlers. The molecular characterization of G. duodenalis isolates indicated assemblage B for the first time found in free-living A. guariba clamitans. The high prevalence of G. duodenalis transmission in CA howler monkeys can be explained by direct contact with humans and frequent soil contact. The presence of a potentially zoonotic assemblage in these animals indicates that the process of fragmentation and cohabitation with humans and livestock affects the wildlife, thus indicating a need for eco-health measures.