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.

Complete sequencing of the Cryptosporidium suis gp60 gene reveals a novel type of tandem repeats-Implications for surveillance.

Cryptosporidiosis is an infectious enteric disease caused by species (some of them zoonotic) of the genus Cryptosporidium that in many countries are under surveillance. Typing assays critical to the surveillance of cryptosporidiosis typically involve characterization of Cryptosporidium glycoprotein 60 genes (gp60). Here, we characterized the gp60 of Cryptosporidium suis from two samples-a human and a porcine faecal sample-based on which a preliminary typing scheme was developed. A conspicuous feature of the C. suis gp60 was a novel type of tandem repeats located in the 5' end of the gene and that took up 777/1635 bp (48%) of the gene. The C. suis gp60 lacked the classical poly-serine repeats (TCA/TCG/TCT), which is usually subject to major genetic variation, and the length of the tandem repeat made a typing assay incorporating this region based on Sanger sequencing practically unfeasible. We therefore designed a typing assay based on the post-repeat region only and applied it to C. suis-positive samples from suid hosts from Norway, Denmark, and Spain. We were able to distinguish three different subtypes; XXVa-1, XXVa-2, and XXVa-3. Subtype XXVa-1 had a wider geographic distribution than the other subtypes and was also observed in the human sample. We think that the present data will inform future strategies to develop a C. suis typing assay that could be even more informative by including a greater part of the gene, including the tandem repeat region, e.g., by the use of long-read next-generation sequencing.

Molecular detection of Cryptosporidium in Alpine musk deer (Moschus chrysogaster) in Gansu Province, Northwest China.

Cryptosporidium spp. are protozoa commonly found in domestic and wild animals. Limited information is available on Cryptosporidium in deer worldwide. In this study, 201 fecal samples were collected from Alpine musk deer on three farms in Gansu Province, China. Detection and subtyping of Cryptosporidium were performed by PCR and sequence analysis of the SSU rRNA and gp60 genes. The prevalence of Cryptosporidium infection in Alpine musk deer was 3.9% (8/201), with infection rates of 1.0% (1/100), 2.8% (1/36), and 9.2% (6/65) in three different farms. All positive samples for Cryptosporidium were from adult deer. Two Cryptosporidium species were identified, including C. parvum (n = 2) and C. xiaoi (n = 6). The C. parvum isolates were subtyped as IIdA15G1, while the C. xiaoi isolates were subtyped as XXIIIa (n = 2) and XXIIIg (n = 4). The IIdA15G1 subtype of C. parvum was found for the first time in deer. These results provide important insights into the identity and human infectious potential of Cryptosporidium in farmed Alpine musk deer.

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.

Investigating the infection dynamics and molecular detection of Cryptosporidium in Buffaloes in Sylhet, Bangladesh.

Cryptosporidiosis, a zoonotic infection impacting both livestock and humans, is inadequately understood in terms of its prevalence and transmission dynamics involving buffaloes in Bangladesh. This research, conducted in the Sylhet division, aimed to explore the prevalence and potential risk factors influencing Cryptosporidium spp. in the faecal samples of 392 buffaloes. Detection of the parasite utilized modified Ziehl-Neelsen staining, with molecular identification achieved through nested PCR (nPCR). The comprehensive analysis revealed 9.18% (36/392) prevalence at the individual animal level and 40.48% (17/42) at the herd level. Age-based analysis revealed fluctuating infection rates of Cryptosporidium spp. in buffaloes across distinct age brackets, with rates of 22.61% in those aged 0-6 months, 5.00% in those aged 6-12 months, and 1.03% in those aged 12-18 months. Diarrheic buffaloes showed a significantly (p < 0.001) higher infection rate (26.67%; 28/105) compared to non-diarrheic buffaloes (2.79%; 8/287). In risk factor analysis, binary logistic regression revealed that buffaloes aged 0-6 months were experiencing a likelihood that is 14.84 times higher to be affected by Cryptosporidium in contrast to their older counterparts (OR = 14.85; p = 0.02). Additionally, diarrhoeic buffaloes were found to be more susceptible to Cryptosporidium compared to healthy buffaloes (OR = 17.50; p < 0.001). A higher stocking density was associated with an increased likelihood of infection in buffaloes (OR = 11.20; p = 0.01). The results of this study emphasize the necessity for targeted interventions, considering factors like diarrheic condition and stocking density, to effectively manage and control cryptosporidiosis in Bangladesh.

Study on intestinal parasitic infections and gut microbiota in cancer patients at a tertiary teaching hospital in Malaysia.

Intestinal parasitic infections (IPIs) can lead to significant morbidity and mortality in cancer patients. While they are unlikely to cause severe disease and are self-limiting in healthy individuals, cancer patients are especially susceptible to opportunistic parasitic infections. The gut microbiota plays a crucial role in various aspects of health, including immune regulation and metabolic processes. Parasites occupy the same environment as bacteria in the gut. Recent research suggests intestinal parasites can disrupt the normal balance of the gut microbiota. However, there is limited understanding of this co-infection dynamic among cancer patients in Malaysia. A study was conducted to determine the prevalence and relationship between intestinal parasites and gut microbiota composition in cancer patients. Stool samples from 134 cancer patients undergoing active treatment or newly diagnosed were collected and examined for the presence of intestinal parasites and gut microbiota composition. The study also involved 17 healthy individuals for comparison and control. Sequencing with 16S RNA at the V3-V4 region was used to determine the gut microbial composition between infected and non-infected cancer patients and healthy control subjects. The overall prevalence of IPIs among cancer patients was found to be 32.8%. Microsporidia spp. Accounted for the highest percentage at 20.1%, followed by Entamoeba spp. (3.7%), Cryptosporidium spp. (3.0%), Cyclospora spp. (2.2%), and Ascaris lumbricoides (0.8%). None of the health control subjects tested positive for intestinal parasites. The sequencing data analysis revealed that the gut microbiota diversity and composition were significantly different in cancer patients than in healthy controls (p < 0.001). A significant dissimilarity was observed in the bacterial composition between parasite-infected and non-infected patients based on Bray-Curtis (p = 0.041) and Jaccard (p = 0.021) measurements. Bacteria from the genus Enterococcus were enriched in the parasite-infected groups, while Faecalibacterium prausnitzii reduced compared to non-infected and control groups. Further analysis between different IPIs and non-infected individuals demonstrated a noteworthy variation in Entamoeba-infected (unweighted UniFrac: p = 0.008), Cryptosporidium-infected (Bray-Curtis: p = 0.034) and microsporidia-infected (unweighted: p = 0.026; weighted: p = 0.019; Jaccard: p = 0.031) samples. No significant dissimilarity was observed between Cyclospora-infected groups and non-infected groups. Specifically, patients infected with Cryptosporidium and Entamoeba showed increased obligate anaerobic bacteria. Clostridiales were enriched with Entamoeba infections, whereas those from Coriobacteriales decreased. Bacteroidales and Clostridium were found in higher abundance in the gut microbiota with Cryptosporidium infection, while Bacillales decreased. Additionally, bacteria from the genus Enterococcus were enriched in microsporidia-infected patients. In contrast, bacteria from the Clostridiales order, Faecalibacterium, Parabacteroides, Collinsella, Ruminococcus, and Sporosarcina decreased compared to the non-infected groups. These findings underscore the importance of understanding and managing the interactions between intestinal parasites and gut microbiota for improved outcomes in cancer patients.

Cryptosporidium occultus in disguise.

As data accumulate in GenBank, the difficulties of delineating species of Cryptosporidium based on nuclear small subunit ribosomal RNA (ssu rRNA) gene information alone becomes increasingly evident. Here, we summarize currently available evidence suggesting that several ssu rDNA sequences primarily referred to as Cryptosporidium suis (some of them from non-suid hosts) should be considered Cryptosporidium occultus.

Diagnostic evaluation of nested PCR and microscopy for Cryptosporidiosis in goats: can COWP gene based qRT-PCR be useful in assessment of active Cryptosporidial infections?

The present research delved into the transmission patterns, diagnostic methods, molecular traits, and phylogenetic analysis of Cryptosporidium species. The research was undertaken to enhance comprehension of the epidemiology and the potential for zoonotic transmission. A total of 80 goat-kid samples were tested, 7 were confirmed positive by mZN microscopy and 12 by nested-PCR. By PCR, 18SSUrRNA, HSP70, and GP60 amplicons were tested for Cryptosporidium. The restriction enzymes viz., SspI, VspI and MboII were used to genotype 12 Cryptosporidium positive samples by which C. parvum and C. bovis mixed infections were detected. Quantitative reverse transcription real-time PCR was used to transcriptionally screen the COWP-subunit genes to assess the severity of the infection in goat-kids, which showed upregulation of COWP6 and COWP4, while COWP9 and COWP3 genes were downregulated. A silent mutation was found at the codon CCA→CCC, which is being reported for the first time in goat field isolates. Phylogenetic and sequencing analyses confirmed the presence of the anthropozoonotic IIe subtype.

Novel strategy to quantify the viability of oocysts of Cryptosporidium parvum and C. hominis, a risk factor of the waterborne protozoan pathogens of public health concern.

While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.

Cryptosporidium spp. in large-scale sheep farms in China: prevalence and genetic diversity.

Cryptosporidium spp. are significant zoonotic intestinal parasites that induce diarrhea and even death across most vertebrates, including humans. Previous studies showed that sheep are important hosts for Cryptosporidium and that its distribution in sheep is influenced by geography, feeding patterns, age, and season. Environmental factors also influence the transmission of Cryptosporidium. Molecular studies of Cryptosporidium in sheep have been conducted in only a few regions of China, and studies into the effect of sheep-housing environments on Cryptosporidium transmission are even rarer. To detect the prevalence of Cryptosporidium in large-scale sheep-housing farms, a total of 1241 fecal samples were collected from sheep, 727 environmental samples were taken from sheep housing, and 30 water samples were collected in six regions of China. To ascertain the existence of the parasite and identify the species of Cryptosporidium spp., we conducted nested PCR amplification of DNA extracted from all samples using the small-subunit (SSU) rRNA gene as a target. For a more in-depth analysis of Cryptosporidium spp. subtypes, C. xiaoi-and C. ubiquitum-positive samples underwent separate nested PCR amplification targeting the 60 kDa glycoprotein (gp60) gene. The amplification of the Cryptosporidium spp. SSU rRNA gene locus from the whole genomic DNA of all samples yielded a positive rate of 1.2% (20/1241) in fecal samples, 0.1% (1/727) in environmental samples, and no positive samples were found in water samples. The prevalence of Cryptosporidium spp. infection in large-scale housed sheep was 1.7%, which was higher than that in free-ranging sheep (0.0%). The highest prevalence of infection was found in weaning lambs (6.8%). Among the different seasons, the peaks were found in the fall and winter. The most prevalent species were C. xiaoi and C. ubiquitum, with the former accounting for the majority of infections. The distribution of C. xiaoi subtypes was diverse, with XXIIIc (n = 1), XXIIId (n = 2), XXIIIe (n = 2), and XXIIIl (n = 4) identified. In contrast, only one subtype, XIIa (n = 9), was found in C. ubiquitum. In this study, C. xiaoi and C. ubiquitum were found to be the predominant species, and Cryptosporidium was found to be present in the environment. These findings provide an important foundation for the comprehensive prevention and management of Cryptosporidium in intensively reared sheep. Furthermore, by elucidating the prevalence of Cryptosporidium in sheep and its potential role in environmental transmission, this study deepens our understanding of the intricate interactions between animal health, environmental contamination, and public health dynamics.

Enteric parasites Cyclospora cayetanensis and Cryptosporidium hominis in domestic and wildlife animals in Ghana.

BACKGROUND: Enteric parasitic infections remain a major public health problem globally. Cryptosporidium spp., Cyclospora spp. and Giardia spp. are parasites that cause diarrhea in the general populations of both developed and developing countries. Information from molecular genetic studies on the speciation of these parasites and on the role of animals as vectors in disease transmission is lacking in Ghana. This study therefore investigated these diarrhea-causing parasites in humans, domestic rats and wildlife animals in Ghana using molecular tools. METHODS: Fecal samples were collected from asymptomatic school children aged 9-12 years living around the Shai Hills Resource Reserve (tourist site), from wildlife (zebras, kobs, baboons, ostriches, bush rats and bush bucks) at the same site, from warthogs at the Mole National Park (tourist site) and from rats at the Madina Market (a popular vegetable market in Accra, Ghana. The 18S rRNA gene (18S rRNA) and 60-kDa glycoprotein gene (gp60) for Cryptosporidium spp., the glutamate dehydrogenase gene (gdh) for Giardia spp. and the 18S rDNA for Cyclospora spp. were analyzed in all samples by PCR and Sanger sequencing as markers of speciation and genetic diversity. RESULTS: The parasite species identified in the fecal samples collected from humans and animals included the Cryptosporidium species C. hominis, C. muris, C. parvum, C. tyzzeri, C. meleagridis and C. andersoni; the Cyclopora species C. cayetanensis; and the Gardia species, G. lamblia and G. muris. For Cryptosporidium, the presence of the gp60 gene confirmed the finding of C. parvum (41%, 35/85 samples) and C. hominis (29%, 27/85 samples) in animal samples. Cyclospora cayetanensis was found in animal samples for the first time in Ghana. Only one human sample (5%, 1/20) but the majority of animal samples (58%, 51/88) had all three parasite species in the samples tested. CONCLUSIONS: Based on these results of fecal sample testing for parasites, we conclude that animals and human share species of the three genera (Cryptosporidium, Cyclospora, Giardia), with the parasitic species mostly found in animals also found in human samples, and vice-versa. The presence of enteric parasites as mixed infections in asymptomatic humans and animal species indicates that they are reservoirs of infections. This is the first study to report the presence of C. cayetanensis and C. hominis in animals from Ghana. Our findings highlight the need for a detailed description of these parasites using high-throughput genetic tools to further understand these parasites and the neglected tropical diseases they cause in Ghana where such information is scanty.

Prevalence and antimicrobial resistance of major foodborne pathogens isolated from pangas and tilapia fish sold in retail markets of Dhaka city, Bangladesh.

Fish sold at retail markets are often contaminated with harmful bacterial pathogens, posing significant health risks. Despite the growing aquaculture industry in Bangladesh to meet high demand, little attention has been paid to ensuring the safety of fish. The objective of this study was to evaluate the microbiological quality of tilapia and pangas fish sold in retail markets across Dhaka city, Bangladesh. Specifically, the study aimed to compare the quality of fish from traditional wet markets and modern supermarkets, as well as fish samples collected during morning and evening hours. A total of 500 raw cut-fish samples (250 tilapia and 250 pangas) were collected at the point of sale from 32 wet markets and 25 supermarkets. All samples were tested for Escherichia coli, extended-spectrum ß-lactamase-producing E. coli (ESBL-Ec), along with the foodborne pathogens Salmonella, Shigella, Vibrio, and Cryptosporidium spp. Bacterial isolates were characterized using antibiotic susceptibility tests (AST) and the presence of common virulence and antibiotic-resistant genes. Fish samples from retail markets had higher prevalence of tested bacteria including E. coli (92 %), V. cholerae (62 %), ESBL-Ec (48 %), and Salmonella spp. (24 %). There was a significant difference in the prevalence of E. coli (97 % vs. 71 %), ESBL-Ec (58 % vs. 8 %) and Salmonella spp. (28 % vs. 8 %) on the wet market samples compared to supermarket samples (p < 0.005). The mean concentration of E. coli on fish from the wet market was 3.0 ± 0.9 log10 CFU/g, while that from supermarkets was 1.6 ± 0.9 log10 CFU/g. The mean concentration of ESBL-Ec in fish from wet markets and supermarkets were 2.3 ± 0.8 log10 CFU/g and 1.6 ± 0.5 log10 CFU/g, respectively. AST revealed that 46 % of E. coli isolates were multi-drug resistant (MDR), while 4 %, 2 % and 5 % of E. coli, Salmonella spp. and Vibrio spp. isolates, respectively, were resistant to carbapenems. At least 3 % of total E. coli isolates were found to be diarrheagenic, while 40 % of Salmonella isolates harbored pathogenic genes (stn, bcfC, ssaQ, avrA and sodC1), and none of the V. cholerae isolates harbored ctxA and tcpA. Our research shows that raw-cut fish samples from retail markets are contaminated with pathogenic and antibiotic-resistant bacteria, which could be a significant food safety concern. Public health interventions should be implemented to improve food safety and hygiene practices in the retail fish markets.

Genetic characterizations of Cryptosporidium spp. from children with or without diarrhea in Wenzhou, China: high probability of zoonotic transmission.

BACKGROUND: Cryptosporidium is a highly pathogenic parasite responsible for diarrhea in children worldwide. Here, the epidemiological status and genetic characteristics of Cryptosporidium in children with or without diarrhea were investigated with tracking of potential sources in Wenzhou City, China. METHODS: A total of 1032 children were recruited, 684 of whom had diarrhea and 348 without, from Yuying Children's Hospital in Wenzhou, China. Samples of stool were collected from each participant, followed by extraction of DNA, genotyping, and molecular identification of Cryptosporidium species and subtypes. RESULTS: Twenty-two of the 1032 (2.1%) children were infected with Cryptosporidium spp. with 2.5% (17/684) and 1.4% (5/348) in diarrhoeic and asymptomatic children, respectively. Four Cryptosporidium species were identified, including C. parvum (68.2%; 15/22), C. felis (13.6%; 3/22), C. viatorum (9.1%; 2/22), and C. baileyi (9.1%; 2/22). Two C. parvum subtypes named IIdA19G1 (n = 14) and IInA10 (n = 1), and one each of C. felis (XIXa) and C. viatorum (XVaA3g) subtype was found as well. CONCLUSIONS: This is the first research that identified Cryptosporidium in children of Wenzhou, China, using PCR. Identification of zoonotic C. parvum, C. felis, C. viatorum, and their subtypes indicate potential cross-species transmission of Cryptosporidium between children and animals. Additionally, the presence of C. baileyi in children suggests that this species has a wider host range than previously believed and that it possesses the capacity to infect humans.

[Prevalence and molecular detection of human Cryptosporidium infections: a review].

Cryptosporidium is an important intestinal parasite that is mainly transmitted through the fecal-oral route. Human infection may occur following ingestion of water and food contaminated by Cryptosporidium oocysts, and children and immunocompromised individuals are at a high risk of infections. The main symptoms of Cryptosporidium infections include diarrhea, vomiting, malnutrition, and even death. Because of high sensitivity and rapid procedures, molecular tests are helpful for the diagnosis of cryptosporidiosis and may reduce the public health risk of cryptosporidiosis. This review summarizes the advances in the latest prevalence and molecular detection of human Cryptosporidium infections during recent years.

Cryptosporidium life cycle small molecule probing implicates translational repression and an Apetala 2 transcription factor in macrogamont differentiation.

The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation.

Molecular characterization and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in humans and domestic animals in Heilongjiang Province, China.

BACKGROUND: Cryptosporidiosis and giardiasis are significant parasitic diseases shared between humans and domestic animals. Due to the close contact between humans and domestic animals in rural areas, it is important to consider the potential transmission of zoonotic parasites from infected domestic animals to humans. This investigation aimed to determine the prevalence and molecular characteristics of Cryptosporidium spp. and Giardia duodenalis in domestic animals and villagers. METHODS: A total of 116 fecal samples from villagers and 686 fecal samples from domestic animals in Heilongjiang Province, China, were analyzed for two parasites using nested polymerase chain reaction (PCR) targeting various genetic loci and DNA sequence analysis of the PCR products. RESULTS: By sequence analysis of the SSU rRNA gene, the prevalence of Cryptosporidium in humans was 0.9% (1/116), with one species of C. parvum (n = 1) detected; among domestic animals, the prevalence was 2.6% (18/686), with five species identified: C. suis (n = 7) and C. scrofarum (n = 7) in pigs, C. meleagridis (n = 1) in chickens, C. andersoni (n = 1) in cattle, and C. canis (n = 2) in foxes. C. parvum and C. canis were further subtyped as IIdA19G1 and XXa4 on the basis of gp60 gene. Regarding G. duodenalis, based on the SSU rRNA, bg, gdh, and tpi genes, the prevalence in domestic animals was 5.1% (31/608), with three assemblages identified: A (n = 1) in pigs, D (n = 1) in foxes, and E (n = 27) in geese, cattle, pigs, ducks, and sheep, along with mixed infection of A + E (n = 1) in one pig and B + E (n = 1) in one sheep. No G. duodenalis was detected in humans (0/116). CONCLUSIONS: The present results show that no overlap of subtypes between animals and villagers was found in Cryptosporidium spp. and G. duodenalis, indicating a minor role of domestic animals in infecting humans in this population. However, the presence of zoonotic protozoa in domestic animals highlights the need for special attention to high-risk individuals during close contact with domestic animals.

Comparative genomics analysis reveals sequence characteristics potentially related to host preference in Cryptosporidium xiaoi.

Cryptosporidium spp. are important diarrhea-associated pathogens in humans and livestock. Among the known species, Cryptosporidium xiaoi, which causes cryptosporidiosis in sheep and goats, was previously recognized as a genotype of the bovine-specific Cryptosporidium bovis based on their high sequence identity in the ssrRNA gene. However, the lack of genomic data has limited characterization of the genetic differences between the two closely related species. In this study, we sequenced the genomes of two C. xiaoi isolates and performed comparative genomic analysis to identify the sequence uniqueness of this ovine-adapted species compared with other Cryptosporidium spp. Our results showed that C. xiaoi is genetically related to C. bovis as shown by their 95.8% genomic identity and similar gene content. Consistent with this, both C. xiaoi and C. bovis appear to have fewer genes encoding mitochondrial metabolic enzymes and invasion-related protein families. However, they appear to possess several species-specific genes. Further analysis indicates that the sequence differences between these two Cryptosporidium spp. are mainly in 24 highly polymorphic genes, half of which are located in the subtelomeric regions. Some of these subtelomeric genes encode secretory proteins that have undergone positive selection. In addition, the genomes of two C. xiaoi isolates, identified as subtypes XXIIIf and XXIIIh, share 99.9% nucleotide sequence identity, with six highly divergent genes encoding putative secretory proteins. Therefore, these species-specific genes and sequence polymorphism in subtelomeric genes probably contribute to the different host preference of C. xiaoi and C. bovis.

Enterocytozoon bieneusi and Cryptosporidium bat genotype XXI and bat genotype XXII in fruit bats (Rousettus leschenaultii) inhabiting a tropical park in Hainan Province, China.

Bats stand as one of the most diverse groups in the animal kingdom and are key players in the global transmission of emerging pathogens. However, their role in transmitting Enterocytozoon bieneusi and Cryptosporidium spp. remains unclear. This study aimed to evaluate the occurrence and genetic diversity of the two pathogens in fruit bats (Rousettus leschenaultii) in Hainan, China. Ten fresh fecal specimens of fruit bats were collected from Wanlvyuan Gardens, Haikou, China. The fecal samples were tested for E. bieneusi and Cryptosporidium spp. using Polymerase Chain Reaction (PCR) analysis and sequencing the internal transcribed spacer (ITS) region and partial small subunit of ribosomal RNA (SSU rRNA) gene, respectively. Genetic heterogeneity across Cryptosporidium spp. isolates was assessed by sequencing 4 microsatellite/minisatellite loci (MS1, MS2, MS3, and MS16). The findings showed that out of the ten specimens analyzed, 2 (20 %) and seven (70.0 %) were tested positive for E. bieneusi and Cryptosporidium spp., respectively. DNA sequence analysis revealed the presence of two novel Cryptosporidium genotypes with 94.4 to 98.6 % sequence similarity to C. andersoni, named as Cryptosporidium bat-genotype-XXI and bat-genotype-XXII. Three novel sequences of MS1, MS2 and MS16 loci identified here had 95.4 to 96.9 % similarity to the known sequences, which were deposited in the GenBank. Two genotypes of E. bieneusi were identified, including a novel genotype named HNB-I and a zoonotic genotype PigEbITS7. The discovery of these novel sequences provides meaningful data for epidemiological studies of the both pathogens. Meanwhile our results are also presented that the fruit bats infected with E. bieneusi, but not with Cryptosporidium, should be considered potential public health threats.

Epidemiological investigation of Cryptosporidium in children with diarrhea in middle Inner Mongolia, China.

Cryptosporidia (Cryptosporidium) is a protozoan that is widely parasitic in the intestinal cells of humans and animals, and it is also an important zoonotic parasite. However, there is no epidemiological investigation on Cryptosporidium spp. infection in infants with diarrhea of Inner Mongolia, the largest livestock region in China. To investigate the prevalence of Cryptosporidium, 2435 fresh fecal samples were collected from children with diarrhea in Inner Mongolia Maternal and Child Health Care Hospital. Molecular characterization of Cryptosporidium was carried out based on its 18S rRNA and gp60 gene sequences. The overall prevalence was 12.85% (313/2435), and in Hohhot (12.15%), it was lower than that in the surrounding city (14.87%) (P < 0.05). Moreover, Cryptosporidium was detected in different seasons and sexes. Concerning the age of children with diarrhea, the prevalence of those age groups between 0 and 1 was obviously lower than others, and there were significant differences in the prevalence at different ages (P < 0.001). Analysis of the 18S rRNA gene sequence revealed that all the positive samples were Cryptosporidium parvum, and there were 5 subtypes (IIdA23G3, IIdA24G3, IIdA24G4, IIdA25G3, and IIdA25G4). To the best of our knowledge, the above subtypes have not been reported. Our results provide a relevant basis for control and education on food safety and foodborne illness prevention.

Molecular characterization of Cryptosporidium spp. and Giardia duodenalis in pet cats in Henan Province, central China.

Cryptosporidium spp. and G. duodenalis often infect humans, cats, and other mammals, causing diarrhea and being responsible for numerous outbreaks of waterborne and foodborne infections worldwide. The rapid increase in the number of pet cats poses a substantial public health risk. However, there were few reports about the infection of Cryptosporidium spp. and G. duodenalis infections in pet cats in Henan Province, central China. Thus, to understand the prevalence and genetic distribution of Cryptosporidium spp. and G. duodenalis in pet cats, and to evaluate the zoonotic potential, possible transmission routes and public health implications of isolates, fecal samples (n = 898) were randomly collected from pet cats in 11 cities in Henan Province, central China. Nested PCR based on the SSU rRNA gene and bg gene was used to the prevalence of Cryptosporidium spp. and G. duodenalis, respectively. The prevalence was 0.8 % (7/898) and 2.0 % (18/898) for Cryptosporidium spp. and G. duodenalis respectively. Additionally, the Cryptosporidium spp. positive isolates were identified as C. parvum subtype IIdA19G1 by gp60 gene. In the present study, the IIdA19G1 subtype was discovered in pet cats for the first time in China, enriching the information on the host type and geographical distribution of Cryptosporidium spp. in China. For G. duodenalis, a total of 18 G. duodenalis positive samples were identified, belonging to four assemblages: a zoonotic assemblage A1 (4/898), three host-specific assemblages C (8/898), D (5/898), and F (1/898). Interestingly, we found that pet cats infected with Cryptosporidium spp. and G. duodenalis are more likely to experience emaciation symptoms compared to the negative group. More importantly, the prevalence of Cryptosporidium spp. and G. duodenalis detected in the present study were low, but the subtype IIdA19G1 of Cryptosporidium spp. and the assemblages A1, C, D, and F of G. duodenalis have the potential for zoonotic transmission. Thus, we should focus on preventing and controlling the risk of cross-species transmission that may occur in pet cats in Henan Province.