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.

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.

Microbial Health Risks of Cryptosporidium parvum and Giardia lamblia in Tropical Coastal Water in Araromi, Nigeria.

Objective: Giardia and Cryptosporidium are enteric protozoa that can cause a variety of gastrointestinal diseases, especially in vulnerable people like children, the elderly, and those with impaired immune systems. In order to ascertain the microbiological quality of the recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria. This risk assessment is of great significance to human health protection against waterborne diseases. The aim of this study was to determine the microbial quality of recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria. Methods: Microscopic examination of Cryptosporidium and Giardia oocysts were done. Results: Results revealed maximum occurrence of Cryptosporidium parvum (20 oocysts/100 mL) of water sample in the month of April and maximum occurrence of Giardia lamblia (300 cysts/100 mL) of water sample in the month of June. Additionally, according to Kolmogorov-Smirnov tests for normalcy Ho =0.05, Giardia lamblia and Cryptosporidium parvum were not regularly distributed in the water samples collected from the beach throughout the study period. The average likelihood of contracting Giardia lamblia and Cryptosporidium parvum infections after consuming 100 mL of beach water was 0.96 and 0.35, respectively. The risks of infection associated with Cryptosporidium parvum was lower than those associated with Giardia lamblia in water from the beach, but were both above the acceptable risk limit of 10-4. Conclusion: The results of this study indicate that Giardia and Cryptosporidium may represent serious health hazards to people who engage in aquatic activities. Adopting a comprehensive strategy that includes regular inspections, enhanced detection techniques, and the prevention of aquatic environment pollution may provide clean and safe recreational water for all, thereby safeguarding the public's health.

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.

[Effect of oxymatrine on Cryptosporidium parvum infection in mice based on the HMGB1-TLR2/TLR4-NF-κB pathway].

OBJECTIVE: To investigate the involvement of the high mobility group box protein B1 (HMGB1)-Toll-like receptor 2 (TLR2)/TLR4-nuclear factor κB (NF-κB) pathway in the intestinal mucosal injury induced by Cryptosporidium parvum infection, and to examine the effect of oxymatrine (OMT) on C. parvum infection in mice. METHODS: Forty SPF 4-week-old BALB/c mice were randomly divided into four groups, including the control group, infection group, glycyrrhizin (GA) group and OMT group. Each mouse was orally administered with 1 × 105 C. parvum oocysts one week in the infection, GA and OMT groups following dexamethasone-induced immunosuppression to model C. parvum intestinal infections in mice. Upon successful modeling, mice in the GA group were intraperitoneally injected with GA at a daily dose of 25.9 mL/kg for successive two weeks, and animals in the OMT group were orally administered OMT at a daily dose of 50 mg/kg for successive two weeks, while mice in the control group were given normal food and water. All mice were sacrificed two weeks post-treatment, and proximal jejunal tissues were sampled. The pathological changes of mouse intestinal mucosal specimens were observed using hematoxylin-eosin (HE) staining, and the mouse intestinal villous height, intestinal crypt depth and the ratio of intestinal villous height to intestinal crypt depth were measured. The occludin and zonula occludens protein 1 (ZO1) expression was determined in mouse intestinal epithelial cells using immunohistochemistry, and the relative expression of HMGB1, TLR2, TLR4, myeloid differentiation primary response gene 88 (MyD88) and NF-κB p65 mRNA was quantified in mouse jejunal tissues using quantitative real-time PCR (qPCR) assay. RESULTS: HE staining showed that the mouse intestinal villi were obviously atrophic, shortened, and detached, and the submucosal layer of the mouse intestine was edematous in the infection group as compared with the control group, while the mouse intestinal villi tended to be structurally intact and neatly arranged in the GA and OMT groups. There were significant differences among the four groups in terms of the mouse intestinal villous height (F = 6.207, P = 0.000 5), intestinal crypt depth (F = 6.903, P = 0.000 3) and the ratio of intestinal villous height to intestinal crypt depth (F = 37.190, P < 0.000 1). The mouse intestinal villous height was lower in the infection group than in the control group [(321.9 ± 41.1) µm vs. (399.5 ± 30.9) µm; t = 4.178, P < 0.01] and the GA group [(321.9 ± 41.1) µm vs. (383.7 ± 42.7) µm; t = 3.130, P < 0.01], and the mouse intestinal crypt depth was greater in the infection group [(185.0 ± 35.9) µm] than in the control group [(128.4 ± 23.6) µm] (t = 3.877, P < 0.01) and GA group [(143.3 ± 24.7) µm] (t = 2.710, P < 0.05). The mouse intestinal villous height was greater in the OMT group [(375.3 ± 22.9) µm] than in the infection group (t = 3.888, P < 0.01), and there was no significant difference in mouse intestinal villous height between the OMT group and the control group (t = 1.989, P > 0.05). The mouse intestinal crypt depth was significantly lower in the OMT group [(121.5 ± 27.3) µm] than in the infection group (t = 4.133, P < 0.01), and there was no significant difference in mouse intestinal crypt depth between the OMT group and the control group (t = 0.575, P > 0.05). The ratio of the mouse intestinal villous height to intestinal crypt depth was significantly lower in the infection group (1.8 ± 0.2) than in the control group (3.1 ± 0.3) (t = 10.540, P < 0.01) and the GA group (2.7 ± 0.3) (t = 7.370, P < 0.01), and the ratio of the mouse intestinal villous height to intestinal crypt depth was significantly higher in the OMT group (3.1 ± 0.2) than in the infection group (t = 15.020, P < 0.01); however, there was no significant difference in the ratio of the mouse intestinal villous height to intestinal crypt depth between the OMT group and the control group (t = 0.404, P > 0.05). Immunohistochemical staining showed significant differences among the four groups in terms of occludin (F = 28.031, P < 0.000 1) and ZO1 expression (F = 14.122, P < 0.000 1) in mouse intestinal epithelial cells. The proportion of positive occluding expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.3 ± 4.5)% vs. (28.3 ± 0.5)%; t = 3.810, P < 0.01], and the proportions of positive occluding expression were significantly higher in mouse intestinal epithelial cells in the GA group [(30.3 ± 1.3)%] and OMT group [(25.8 ± 1.5)%] than in the infection group (t = 7.620 and 5.391, both P values < 0.01); however, there was no significant differences in the proportion of positive occluding expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 1.791 and 2.033, both P values > 0.05). The proportion of positive ZO1 expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.4 ± 1.8)% vs. (24.2 ± 2.8)%; t = 4.485, P < 0.01], and the proportions of positive ZO1 expression were significantly higher in mouse intestinal epithelial cells in the GA group [(24.1 ± 2.3)%] (t = 5.159, P < 0.01) and OMT group than in the infection group [(22.5 ± 1.9)%] (t = 4.441, P < 0.05); however, there were no significant differences in the proportion of positive ZO1 expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 0.037 and 0.742, both P values > 0.05). qPCR assay showed significant differences among the four groups in terms of HMGB1 (F = 21.980, P < 0.000 1), TLR2 (F = 20.630, P < 0.000 1), TLR4 (F = 17.000, P = 0.000 6), MyD88 (F = 8.907, P = 0.000 5) and NF-κB p65 mRNA expression in mouse jejunal tissues (F = 8.889, P = 0.000 7). The relative expression of HMGB1 [(5.97 ± 1.07) vs. (1.05 ± 0.07); t = 6.482, P < 0.05] 、TLR2 [(5.92 ± 1.29) vs. (1.10 ± 0.14); t = 5.272, P < 0.05] 、TLR4 [(5.96 ± 1.50) vs. (1.02 ± 0.03); t = 4.644, P < 0.05] 、MyD88 [(3.00 ± 1.26) vs. (1.02 ± 0.05); t = 2.734, P < 0.05] and NF-κB p65 mRNA [(2.33 ± 0.72) vs. (1.04 ± 0.06); t = 2.665, P < 0.05] was all significantly higher in mouse jejunal tissues in the infection group than in the control group. A significant reduction was detected in the relative expression of HMGB1 (0.63 ± 0.01), TLR2 (0.42 ± 0.10), TLR4 (0.35 ± 0.07), MyD88 (0.70 ± 0.11) and NF-κB p65 mRNA (0.75 ± 0.01) in mouse jejunal tissues in the GA group relative to the control group (t = 8.629, 5.830, 11.500, 4.729 and 6.898, all P values < 0.05), and the relative expression of HMGB1, TLR2, TLR4, MyD88 and NF-κB p65 mRNA significantly reduced in mouse jejunal tissues in the GA group as compared to the infection group (t = 7.052, 6.035, 4.084, 3.165 and 3.274, all P values < 0.05). In addition, the relative expression of HMGB1 (1.14 ± 0.60), TLR2 (1.00 ± 0.24), TLR4 (1.14 ± 0.07), MyD88 (0.96 ± 0.25) and NF-κ B p65 mRNA (1.12 ± 0.17) was significantly lower in mouse jejunal tissues in the OMT group than in the infection group (t = 7.059, 5.320, 3.510, 3.466 and 3.273, all P values < 0.05); however, there were no significant differences between the OMT and control groups in terms of relative expression of HMGB1, TLR2, TLR4, MyD88 or NF-κB p65 mRNA in mouse jejunal tissues (t = 0.239, 0.518, 1.887, 0.427 and 0.641, all P values > 0.05). CONCLUSIONS: C. parvum infection causes intestinal inflammatory responses and destruction of intestinal mucosal barrier through up-regulating of the HMGB1-TLR2/TLR4-NF-κB pathway. OMT may suppress the intestinal inflammation and repair the intestinal mucosal barrier through inhibiting the activity of the HMGB1-TLR2/TLR4-NF-κB pathway.

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.

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.

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.

MiR-199a-3p regulates HCT-8 cell autophagy and apoptosis in response to Cryptosporidium parvum infection by targeting MTOR.

The microRNAs (miRNAs) of their hosts play an important role in regulating both the innate and adaptive immune responses to Cryptosporidium parvum infection. The mechanisms of autophagy and apoptosis are important components of the defense system against C. parvum infection. In this study, we investigate the role of miRNA-199a-3p in regulating MTOR-mediated autophagy and apoptosis in HCT-8 cells induced by C. parvum. The expression of miR-199a-3p increased at 3, 6 and 12 hours postinfection (hpi) but decreased at 24 and 48 hpi. The upregulation of miR-199a-3p promoted autophagy and apoptosis and limited the parasite burden in HCT-8 cells after C. parvum infection. The downregulation of miR-199a-3p inhibited the autophagy and apoptosis induced by C. parvum and enhanced the parasite burden in HCT-8 cells. A luciferase reporter showed that MTOR was a target gene of miR-199a-3p. Suppressed expression of MTOR by small interfering RNA (siRNA) promoted autophagy and apoptosis and limited C. parvum burden in HCT-8 cells. Co-transfection with miR-199a-3p inhibitor or si-mTOR revealed that miR-199a-3p regulates autophagy and apoptosis in HCT-8 cells through MTOR, to resist C. parvum infection. In conclusion, intestinal epithelial cells defend against C. parvum infection by regulating their autophagy and apoptosis through the miR-199a-3p-MTOR axis.

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%.

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.

Clostridium and Cryptosporidium outbreak linked to a splash pad.

BACKGROUND: . Splash pads for recreational purposes are widespread. Using these pads can pose a health risk if they lack installation regulation and water quality supervision. Our aim was to describe a waterborne disease outbreak caused by Clostridium perfringens and Cryptosporidium spp. in a Barcelona district and the measures taken for its control. METHODS: . On August 2018, 71 cases of acute gastroenteritis were detected, affecting people who used a splash pad or were in contact with a user. Microbiological and environmental investigations were carried out. A descriptive analysis of the sample and Poisson regression models adjusted for age and sex were performed, obtaining frequencies, median values, and adjusted prevalence ratios with their 95% confidence intervals. RESULTS: The median age of the cases was 6.7 years, 27 (38%) required medical care, and three (4.2%) were hospitalized. The greater the number of times a person entered the area, the greater the number of symptoms and their severity. Nineteen (76%) of the 25 stool samples collected from cases showed the presence of one or both pathogens. Environmental investigations showed deficiencies in the facilities and identified the presence of both species in the splash pad. Health education and hygiene measures were carried out, and 14 days after the closure of the facilities, no more cases related to the pad were recorded. CONCLUSIONS: . Specific regulations are needed on the use of splash pads for recreational purposes. Until these regulations are in place, these types of facility should comply with the regulations that apply to swimming pools and spas, including those related to the design of the tanks, water recirculation systems, and adequate disinfection systems.

RETROSPECTIVE STUDY OF MORBIDITY AND MORTALITY IN A CAPTIVE POPULATION OF CRYPTOSPORIDIUM-POSITIVE LOUISIANA PINE SNAKES (PITUOPHIS RUTHVENI).

The Louisiana pine snake (Pituophis ruthveni) is a diurnal colubrid species native to Louisiana and eastern Texas whose free-ranging populations have been declining over at least the past 30 yr. The creation and maintenance of sustainable captive breeding programs of P. ruthveni to restore native populations has also provided ample opportunity for research into this species and for P. ruthveni to serve as a research model for other colubrid snakes. However, no investigation into prevalent causes of morbidity and mortality in captive populations of this species has been described. A research population of P. ruthveni was maintained at Louisiana State University (LSU) for over 4 yr due to unsuitability for breeding after testing positive for Cryptosporidium serpentis. Since arrival at LSU, the snakes were under close veterinary surveillance. Complete postmortem examinations were performed on 12 snakes that died or were euthanized. The aim of this study was to further understanding of common factors influencing morbidity and mortality in captive P. ruthveni infected with C. serpentis, by retrospectively reviewing postmortem exam findings from the 12 deceased members of the population at LSU. A predominant finding across individuals included bacterial infections, which were responsible for major illness or death in 37.5% of the animals. Fifty percent of snakes tested positive for Cryptosporidium sp. based on PCR performed from postmortem samples; it was directly implicated as cause of death or morbidity in 83.3% of positive cases. Although infectious disease represented the most common pathologic postmortem finding, several noninfectious disease processes were identified, including gout, goiter, and neoplasia. These findings mirror those of other retrospective investigations of reptile collections at various institutions and highlight the need for appropriate emphasis on the identification, treatment, and prevention of infectious disease as part of routine veterinary care.

Protocol for the cryopreservation of Cryptosporidium isolates using enteroids.

A major bottleneck in the progress of Cryptosporidium research is the lack of accessible cryopreservation of Cryptosporidium oocysts. Here, we present a protocol for the cryopreservation of Cryptosporidium isolates using enteroids. We describe the steps for the establishment of enteroid cultures and cryopreservation of C. parvum-infected HCT-8 cultures. We then detail procedures for the recovery and propagation of frozen parasites using enteroids. For complete details on the use and execution of this protocol, please refer to Deng et al.1.

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.

Investigating the relationship between extreme weather and cryptosporidiosis and giardiasis in Colorado: A multi-decade study using distributed-lag nonlinear models.

Environmentally-mediated protozoan diseases like cryptosporidiosis and giardiasis are likely to be highly impacted by extreme weather, as climate-related conditions like temperature and precipitation have been linked to their survival, distribution, and overall transmission success. Our aim was to investigate the relationship between extreme temperature and precipitation and cryptosporidiosis and giardiasis infection using monthly weather data and case reports from Colorado counties over a twenty-one year period. Data on reportable diseases and weather among Colorado counties were collected using the Colorado Electronic Disease Reporting System (CEDRS) and the Daily Surface Weather and Climatological Summaries (Daymet) Version 3 dataset, respectively. We used a conditional Poisson distributed-lag nonlinear modeling approach to estimate the lagged association (between 0 and 12-months) between relative temperature and precipitation extremes and the risk of cryptosporidiosis and giardiasis infection in Colorado counties between 1997 and 2017, relative to the risk found at average values of temperature and precipitation for a given county and month. We found distinctly different patterns in the associations between temperature extremes and cryptosporidiosis, versus temperature extremes and giardiasis. When maximum or minimum temperatures were high (90th percentile) or very high (95th percentile), we found a significant increase in cryptosporidiosis risk, but a significant decrease in giardiasis risk, relative to risk at the county and calendar-month mean. Conversely, we found very similar relationships between precipitation extremes and both cryptosporidiosis and giardiasis, which highlighted the prominent role of long-term (>8 months) lags. Our study presents novel insights on the influence that extreme temperature and precipitation can have on parasitic disease transmission in real-world settings. Additionally, we present preliminary evidence that the standard lag periods that are typically used in epidemiological studies to assess the impacts of extreme weather on cryptosporidiosis and giardiasis may not be capturing the entire relevant period.

Intestinal cDC1s provide cues required for CD4+ T cell-mediated resistance to Cryptosporidium.

Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine.

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.