We report a foodborne outbreak of the previously undetected Cryptosporidium parvum gp60 subtype IIγA11. In December 2023, notifications of cryptosporidiosis cases increased in Sweden, prompting the initiation of a national outbreak investigation, and a case–control study was performed to identify the source. We identified 60 cases between 15 December 2023 and 1 January 2024. The median age was 44 years (range: 16–81), and 73% were women. Controls were recruited from a national random pool; frequency was matched by age group and sex. Compared to controls, cases were more likely to have consumed items from salad bars in grocery stores (8% vs. 85%; adjusted odds ratios [aOR]: 58; 95% confidence interval [CI]: 22–186). In regards to food items from the salad bars, cases were more likely to have consumed kale mix salad compared to controls (62% vs. 32%; aOR: 3.6; 95%CI: 1.2–12). Trace-back investigations identified kale producers from Sweden, Belgium, and Spain, but no particular grower was identified, and no food samples were available for microbiological analysis. Our investigation indicates that leafy greens such as kale may contain Cryptosporidium spp. and cause outbreaks and it is important to understand how the contamination occurs to prevent future outbreaks and apply adequate preventive measures.

Development of gastrointestinal illness after animal contact at petting farms is well described, as are factors such as handwashing and facility design that may modify transmission risk. However, further field evidence on other behaviours and interventions in the context of Cryptosporidium outbreaks linked to animal contact events is needed. Here, we describe a large outbreak of Cryptosporidium parvum (C. parvum) associated with a multi-day lamb petting event in the south-west of England in 2023 and present findings from a cohort study undertaken to investigate factors associated with illness. Detailed exposure questionnaires were distributed to email addresses of 647 single or multiple ticket bookings, and 157 complete responses were received. The outbreak investigation identified 23 laboratory-confirmed primary C. parvum cases. Separately, the cohort study identified 83 cases of cryptosporidiosis-like illness. Associations between illness and entering a lamb petting pen (compared to observing from outside the pen; odds ratio (OR) = 2.28, 95 per cent confidence interval (95% CI) 1.17 to 4.53) and self-reported awareness of diarrhoeal and vomiting disease transmission risk on farm sites at the time of visit (OR = 0.40, 95% CI 0.19 to 0.84) were observed. In a multivariable model adjusted for household clustering, awareness of disease transmission risk remained a significant protective factor (adjusted OR (aOR) = 0.07, 95% CI 0.01 to 0.78). The study demonstrates the likely under-ascertainment of cryptosporidiosis through laboratory surveillance and provides evidence of the impact that public health messaging could have.

Cryptosporidium spp. are significant opportunistic pathogens causing diarrhoea in humans and animals. Pigs are one of the most important potential hosts for Cryptosporidium. We evaluated the prevalence of Cryptosporidium in pigs globally using published information and a random-effects model. In total, 131 datasets from 36 countries were included in the final quantitative analysis. The global prevalence of Cryptosporidium in pigs was 16.3% (8560/64 809; 95% confidence interval [CI] 15.0–17.6%). The highest prevalence of Cryptosporidium in pigs was 40.8% (478/1271) in Africa. Post-weaned pigs had a significantly higher prevalence (25.8%; 2739/11 824) than pre-weaned, fattening and adult pigs. The prevalence of Cryptosporidium was higher in pigs with no diarrhoea (12.2%; 371/3501) than in pigs that had diarrhoea (8.0%; 348/4874). Seven Cryptosporidium species (Cryptosporidium scrofarum, Cryptosporidium suis, Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium tyzzeri, Cryptosporidium andersoni and Cryptosporidium struthioni) were detected in pigs globally. The proportion of C. scrofarum was 34.3% (1491/4351); the proportion of C. suis was 31.8% (1385/4351) and the proportion of C. parvum was 2.3% (98/4351). The influence of different geographic factors (latitude, longitude, mean yearly temperature, mean yearly relative humidity and mean yearly precipitation) on the infection rate of Cryptosporidium in pigs was also analysed. The results indicate that C. suis is the dominant species in pre-weaned pigs, while C. scrofarum is the dominant species in fattening and adult pigs. The findings highlight the role of pigs as possible potential hosts of zoonotic cryptosporidiosis and the need for additional studies on the prevalence, transmission and control of Cryptosporidium in pigs.

Cryptosporidium spp. are important pathogens with some species causing diarrhoea in humans and animals. Sheep are one of the most common potential hosts for various Cryptosporidium spp. The prevalence of Cryptosporidium in sheep globally was evaluated from published information including molecular data via meta-analysis. In total, 126 datasets from 41 countries were included for final quantitative analysis. Sheep aged <3 months had a significantly higher prevalence (27.8%; 3284/11 938) than those at the age of 3–12 and >12 months. The prevalence of Cryptosporidium in sheep with diarrhoea of 35.4% (844/1915) was higher than in sheep that did not show diarrhoea (11.3%; 176/1691). Fourteen Cryptosporidium species/genotypes were detected in sheep globally. The proportion of subgenotype family XIIa of Cryptosporidium ubiquitum was 90.0% (216/240); the proportions of subgenotypes IIdA20G1 and IIaA15G2R1 of Cryptosporidium parvum were 15.4% (62/402) and 19.7% (79/402). The results indicate that C. parvum is the dominant species in Europe while Cryptosporidium xiaoi is the dominant species in Oceania, Asia and Africa and C. ubiquitum is the dominant species in North America and South America. Subgenotype family IIa of C. parvum is particularly widespread among sheep worldwide. The results highlight the role of sheep as a reservoir host for zoonotic cryptosporidia and the need for further study of prevalence, transmission and control of this pathogen in sheep.

Species of the genus Cryptosporidium (phylum Apicomplexa) infect the epithelium of the gastrointestinal tract of several vertebrate hosts, including humans and domestic and wild animals. In the past 20 years, several studies have focused on Cryptosporidium in fish. To date, a total of four piscine-host-specific species (Cryptosporidium molnari, Cryptosporidium huwi, Cryptosporidium bollandi and Cryptosporidium abrahamseni), nine piscine genotypes and more than 29 unnamed genotypes have been described in fish hosts. In addition, Cryptosporidium species and genotypes typical of other groups of vertebrates have also been identified. This review summarizes the history, biology, pathology and clinical manifestations, as well as the transmission, prevalence and molecular epidemiology of Cryptosporidium in wild, cultured and ornamental fish from both marine and freshwater environments. Finally, the potential role of piscine hosts as a reservoir of zoonotic Cryptosporidium species is also discussed.

Coronavirus disease-2019 (Covid-19) nonpharmaceutical interventions have proven effective control measures for a range of respiratory illnesses throughout the world. These measures, which include isolation, stringent border controls, physical distancing and improved hygiene also have effects on other human pathogens, including parasitic enteric diseases such as cryptosporidiosis. Cryptosporidium infections in humans are almost entirely caused by two species: C. hominis, which is primarily transmitted from human to human, and Cryptosporidium parvum, which is mainly zoonotic. By monitoring Cryptosporidium species and subtype families in human cases of cryptosporidiosis before and after the introduction of Covid-19 control measures in New Zealand, we found C. hominis was completely absent after the first months of 2020 and has remained so until the beginning of 2021. Nevertheless, C. parvum has followed its typical transmission pattern and continues to be widely reported. We conclude that ~7 weeks of isolation during level 3 and 4 lockdown period interrupted the human to human transmission of C. hominis leaving only the primarily zoonotic transmission pathway used by C. parvum. Secondary anthroponotic transmission of C. parvum remains possible among close contacts of zoonotic cases. Ongoing 14-day quarantine measures for new arrivals to New Zealand have likely suppressed new incursions of C. hominis from overseas. Our findings suggest that C. hominis may be controlled or even eradicated through nonpharmaceutical interventions.

This epidemiological study assesses the occurrence of enteric parasites in 4303 patients attended at two public hospitals in Ankara (Turkey) during 2018–2019. Microscopy was used as a screening test. Giardia duodenalis was also identified using a commercial ELISA for the detection of parasite-specific coproantigens. Giardia-positive samples by microscopy/ELISA were confirmed by real-time PCR and characterized using a multilocus genotyping scheme. Blastocystis sp. was genotyped in a sample subset. Blastocystis sp. (11.1%, 95% CI 11.4‒14.8%) and G. duodenalis (1.56%, 95% CI 1.22‒1.96) were the most prevalent pathogens found. Cryptosporidium spp., Entamoeba histolytica and intestinal helminths were only sporadically (<0.5%) found. For G. duodenalis, sequence (n = 30) analyses revealed the presence of sub-assemblages AII (23.3%), discordant AII/AIII (23.3%) and mixed AII + AIII (6.7%) within assemblage A, and BIII (10.0%), BIV (3.3%) and discordant BIII/BIV (23.3%) within assemblage B. Two additional sequences (6.7%) were assigned to the latter assemblage but sub-assemblage information was unknown. No associations between G. duodenalis assemblages/sub-assemblages and sociodemographic and clinical variables could be demonstrated. For Blastocystis sp., sequence (n = 6) analyses identified subtypes ST1, ST2 and ST3 at equal proportions. This is the first molecular characterization of G. duodenalis based on MLG conducted in Turkey to date.

One of the ways of human parasitic infection is the accidental ingestion of vegetables contaminated with parasites, which represents a major human health hazard. This non-exhaustive review aims to evaluate studies carried out on five types of vegetables (lettuce, parsley, coriander, carrot and radish) since 2000, particularly the methods used for recovery, concentration, detection and identification of protozoan parasites such as Toxoplasma gondii, Giardia duodenalis and Cryptosporidium spp., and the results of each work. Various studies have determined the presence of pathogenic parasites in fresh vegetables with different rates; this variation in rate depends particularly on the detection method used which is related to each parasite and each vegetable type. The variation in parasitic prevalence in food could be due to different factors such as the geographical location, the size of analysed samples and the methods used for parasite detection.

Understanding endemic infectious disease risk factors through traditional epidemiological tools is challenging. Population-based case–control studies are costly and time-consuming. A case–case analyses using surveillance data addresses these limitations by using resources more efficiently. We conducted a case–case analyses using routine surveillance data reported by 16 U.S. states (2005–2015), wherein reported cases of salmonellosis were used as a comparison group to identify exposure associations with reported cases of cryptosporidiosis and giardiasis. Odds ratios adjusted for age and reporting state (aOR) and 95% confidence intervals (95% CI) were calculated. A total of 10 704 cryptosporidiosis cases, 17 544 giardiasis cases, and 106 351 salmonellosis cases were included in this analyses. When compared with cases of salmonellosis, exposure to treated recreational water (aOR 4.7, 95% CI 4.3–5.0) and livestock (aOR: 3.2; 95% CI: 2.9–3.5) were significantly associated with cryptosporidiosis and exposure to untreated drinking (aOR 4.1, 95% CI 3.6–4.7) and recreational water (aOR 4.1, 95% CI 3.7–4.5) were associated with giardiasis. Our analyses shows that routine surveillance data with standardised exposure information can be used to identify associations of interest for cryptosporidiosis and giardiasis.

In the autumn of 2018, an outbreak of cryptosporidiosis affected adult employees from the same company in Western Norway. The organism was Cryptosporidium parvum, GP60 subtype IIaA14G1R1. All those infected had drunk from the same container of self-pressed apple juice. Incubation period (1 week) and clinical signs were similar among those infected, although some experienced a more prolonged duration of symptoms (up to 2–3 weeks) than others. The infections resulted after consumption from only one of 40 containers of juice and not from any of the other containers. It seems that although Cryptosporidium oocysts were detected in a sample from another container, the contamination did not affect the whole batch. This is perhaps indicative of a restricted contamination event, either from contaminated ground in the orchard, or during collection of the fruit, or during processing. Although outbreaks of food-borne cryptosporidiosis have previously been associated with consumption of contaminated apple juice, most of the more recent outbreaks of food-borne cryptosporidiosis have been associated with salad vegetables or herbs. This outbreak, the first outside USA reported to be associated with apple juice, is a timely reminder that such juice is a suitable transmission vehicle for Cryptosporidium oocysts, and that appropriate hygienic measures are essential in the production of such juice, including artisanal (non-commercial) production.

Wastewater discharge and agricultural activities may pose microbial risks to natural water sources. The impact of different sources can be assessed by water quality modelling. The aim of this study was to use hydrological and hydrodynamic models to illustrate the risk of exposing grazing animals to faecal pollutants in natural water sources, using three zoonotic faecal pathogens as model microbes and fictitious pastures in Sweden as examples. Microbial contamination by manure from fertilisation and grazing was modelled by use of a hydrological model (HYPE) and a hydrodynamic model (MIKE 3 FM), and microbial contamination from human wastewater was modelled by application of both models in a backwards process. The faecal pathogens Salmonella spp., verotoxin-producing Escherichia coli O157:H7 (VTEC) and Cryptosporidium parvum were chosen as model organisms. The pathogen loads on arable land and pastures were estimated based on pathogen concentration in cattle faeces, herd prevalence and within-herd prevalence. Contamination from human wastewater discharge was simulated by estimating the number of pathogens required from a fictitious wastewater discharge to reach a concentration high enough to cause infection in cattle using the points on the fictitious pastures as their primary source of drinking water. In the scenarios for pathogens from animal sources, none of the simulated concentrations of salmonella exceeded the concentrations needed to infect adult cattle. For VTEC, most of the simulated concentrations exceeded the concentration needed to infect calves. For C. parvum, all the simulated concentrations exceeded the concentration needed to infect calves. The pathogen loads needed at the release points for human wastewater to achieve infectious doses for cattle were mostly above the potential loads of salmonella and VTEC estimated to be present in a 24-h overflow from a medium-size Swedish wastewater treatment plant, while the required pathogen loads of C. parvum at the release points were below the potential loads of C. parvum in a 24-h wastewater overflow. Most estimates in this study assume a worst-case scenario. Controlling zoonotic infections at herd level prevents environmental contamination and subsequent human exposure. The potential for infection of grazing animals with faecal pathogens has implications for keeping animals on pastures with access to natural water sources. As the infectious dose for most pathogens is more easily reached for calves than for adult animals, and young calves are also the main shedders of C. parvum, keeping young calves on pastures adjacent to natural water sources is best avoided.

Although the impact of diarrhoeal disease on paediatric health in Nigeria has decreased in recent years, it remains an important cause of morbidity and mortality in children under 5 years. Rotavirus is recognised as an important aetiological agent, but information on the contribution of intestinal protozoa to watery diarrhoea in this age group in Nigeria is scarce. In this cross-sectional study, faecal samples from children admitted to healthcare centres in Abakaliki, Nigeria with acute watery diarrhoea (N = 199) and faecal samples from age-matched controls (N = 37) were examined for Cryptosporidium and Giardia using immunofluorescent antibody testing and molecular methods. Cryptosporidium was identified in 13 case samples (6.5%) and no control samples. For three samples, molecular characterisation indicated C. hominis, GP60 subtypes IaA30R3, IaA14R3 and IdA11. Giardia was not detected in any samples. This contrast in prevalence between the two intestinal protozoa may reflect their variable epidemiologies and probably differing routes of infection. Given that these two parasitic infections are often bracketed together, it is key to realise that they not only have differing clinical spectra but also that the importance of each parasite is not the same in different age groups and/or settings.

In June 2015, an outbreak of cryptosporidiosis with 35 cases (23 probable and 12 laboratory-confirmed) occurred among 191 attendees of a residential rehabilitation holiday for paediatric organ transplant patients (n = 49) and their families at a hotel in Somogy county, Hungary. The overall attack rate was 18%. Most of the cases were transplanted children who experienced severe acute disease and required adjustment to their tacrolimus immunosuppression. A retrospective case-control study suggested an association between recreational water exposures and illness: cases were seven times more likely than controls to have swum in the children's pool (odds ratio 7.17; 95% confidence interval 2.9–17.2; P < 0.0001) and five times more likely to have used the jetted whirlpool (odds ratio 5.25; 95% confidence interval 2.1–13.1; P < 0.0001). This was the first outbreak of cryptosporidiosis in Hungary and it is especially unfortunate that it affected vulnerable children who experienced severe symptoms. Cryptosporidium presents specific infection control difficulties in treated recreational water venues; the link to a whirlpool is unusual and highlights the importance of the age-appropriate use of these facilities and reminding users not to immerse their heads or swallow the water. Cryptosporidiosis is more commonly linked to children’ pools where improved bather hygiene and promoting exclusion of diarrhoea cases could help to avoid similar outbreaks.

To determine the prevalence of Cryptosporidium in dairy cattle in Guangdong Province, South China, 1440 fecal samples were collected from 10 farms and screened for Cryptosporidium with PCR. The overall prevalence of Cryptosporidium was 4.38% (63/1440), and the infection rates in preweaned calves, postweaned calves, heifers and adults were 6.4% (19/297), 6.19% (33/533), 1.48% (4/271) and 2.06% (7/339), respectively. Three Cryptosporidium species, Cryptosporidium andersoni (n = 33), Cryptosporidium bovis (n = 22) and Cryptosporidium ryanae (n = 8) were detected by DNA sequence analysis of the 63 positive samples, and C. andersoni was identified as the most common species on the dairy cattle farms. In preweaned calves, C. bovis was the most prevalent species (9/19, 47.4%). In contrast, C. andersoni was the predominant species (19/33, 57.6%) in postweaned calves and the only species found in heifers and adults. The zoonotic species Cryptosporidium parvum was not detected in this study. Twenty-four C. andersoni isolates were successfully classified into three multilocus sequence typing (MLST) subtypes. MLST subtype A4,A4,A4,A1 was the predominant subtype, and MLST subtype A2,A5,A2,A1, previously found in sheep, was detected in cattle for the first time. A linkage disequilibrium analysis showed that the C. andersoni isolates had a clonal genetic population structure. However, further molecular studies are required to better understand the epidemiology of Cryptosporidium in Guangdong.

Cryptosporidium is a protozoan parasite that causes the diarrhoeal disease, cryptosporidiosis. Although many species have been identified, the majority of human disease worldwide is caused by two species; Cryptosporidium parvum and Cryptosporidium hominis. In Australia, data from the National Notifiable Diseases Surveillance System (NNDSS) show that cryptosporidiosis outbreaks occur every few years. To better understand the transmission, trends and nature of cryptosporidiosis outbreaks in Western Australia, epidemiological and genomic data from three cryptosporidiosis outbreaks in 2003, 2007 and 2011 were reviewed. The 2007 outbreak was the largest (n = 607) compared with the outbreaks in 2003 (n = 404) and 2011 (n = 355). All three outbreaks appeared to have occurred predominantly in the urban metropolitan area (Perth), which reported the highest number of case notifications; increases in case notifications were also observed in rural and remote areas. Children aged 0–4 years and non-Aboriginal people comprised the majority of notifications in all outbreaks. However, in the 2003 and 2007 outbreaks, a higher proportion of cases from Aboriginal people was observed in the remote areas. Molecular data were only available for the 2007 (n = 126) and 2011 (n = 42) outbreaks, with C. hominis the main species identified in both outbreaks. Subtyping at the glycoprotein 60 (gp60) locus identified subtype IbA10G2 in 46.3% and 89.5% of C. hominis isolates typed, respectively, in the 2007 and 2011 outbreaks, with the IdA15G1 subtype was identified in 33.3% of C. hominis isolates typed in the 2007 outbreak. The clustering of cases with the IdA15G1 subtype in the remote areas suggests the occurrence of a concurrent outbreak in remote areas during the 2007 outbreak, which primarily affected Aboriginal people. Both the C. hominis IbA10G2 and IdA15G1 subtypes have been implicated in cryptosporidiosis outbreaks worldwide; its occurrence indicates that the mode of transmission in both the 2007 and 2011 outbreaks was anthroponotic. To better understand the epidemiology, sources and transmission of cryptosporidiosis in Australia, genotyping data should routinely be incorporated into national surveillance programmes.

Genome sequencing has greatly contributed to our understanding of parasitic protozoa. This is particularly the case for Cryptosporidium species (phylum Apicomplexa) which are difficult to propagate. Because of their polymorphic nature, simple sequence repeats have been used extensively as genotypic markers to differentiate between isolates, but no global analysis of amino acid repeats in Cryptosporidium genomes has been reported. Taking advantage of several newly sequenced Cryptosporidium genomes, a comparative analysis of single-amino-acid repeats (SAARs) in seven species was undertaken. This analysis revealed a striking difference between the SAAR profile of the gastric and intestinal species which infect mammals and one species which infects birds. In average, total SAAR length in gastric species is only 25% of the cumulative SAAR length in the genome of Cryptosporidium parvum, Cryptosporidium hominis and Cryptosporidium meleagridis, species infectious to humans. The SAAR profile in the avian parasite Cryptosporidium baileyi stands out due to the presence of long asparagine repeats. Cryptosporidium baileyi proteins with repeats ⩾20 residues are significantly enriched in regulatory functions. As postulated for the related apicomplexan species Plasmodium falciparum, these observations suggest that Cryptosporidium SAARs evolve in response to selective pressure. The putative selective mechanisms driving SAAR evolution in Cryptosporidium species are unknown.

Cryptosporidium research has focused on the development of infection control, and effective therapy that has thus far been hampered by the inability to culture Cryptosporidium in vitro. Other limitations include inadequate animal models, cumbersome screening procedures for chemotherapeutic approaches and a lack of tools for genetic manipulation. These limitations can, however, be eased by the improvement and focused development of in vitro cultivation. The ability to culture relevant Cryptosporidium isolates in vitro and to propagate the life cycle stages that are responsible for causing disease in an infected host is still a critical link. This ability will facilitate other relevant approaches, e.g., the ability to knockout genes and the application of broader screening for drug discoveries and vaccine developments, in combination with new discoveries on the parasite's basic biology, genetic manipulation and new life cycle stages. Success in this effort represents an essential step towards significant progress in the control of cryptosporidiosis.

Cryptosporidium spp. (Apicomplexa) causing cryptosporidiosis are of medical and veterinary significance. The genus Cryptosporidium has benefited from the application of what is considered a DNA-barcoding approach, even before the term ‘DNA barcoding’ was formally coined. Here, the objective to define the DNA barcode diversity of Cryptosporidium infecting mammals is reviewed and considered to be accomplished. Within the Cryptosporidium literature, the distinction between DNA barcoding and DNA taxonomy is indistinct. DNA barcoding and DNA taxonomy are examined using the latest additions to the growing spectrum of named Cryptosporidium species and within-species and between-species identity is revisited. Ease and availability of whole-genome DNA sequencing of the relatively small Cryptosporidium genome offer an initial perspective on the intra-host diversity. The opportunity emerges to apply a metagenomic approach to purified field/clinical Cryptosporidum isolates. The outstanding question remains a reliable definition of Cryptosporidium phenotype. The complementary experimental infections and metagenome approach will need to be applied simultaneously to address Cryptosporidium phenotype with carefully chosen clinical evaluations enabling identification of virulence factors.

We undertook a study on Cryptosporidium spp. in wild cricetid rodents. Fecal samples were collected from meadow voles (Microtus pennsylvanicus), southern red-backed voles (Myodes gapperi), woodland voles (Microtus pinetorum), muskrats (Ondatra zibethicus) and Peromyscus spp. mice in North America, and from bank voles (Myodes glareolus) and common voles (Microtus arvalis) in Europe. Isolates were characterized by sequence and phylogenetic analyses of the small subunit ribosomal RNA (SSU) and actin genes. Overall, 33·2% (362/1089) of cricetids tested positive for Cryptosporidium, with a greater prevalence in cricetids from North America (50·7%; 302/596) than Europe (12·1%; 60/493). Principal Coordinate analysis separated SSU sequences into three major groups (G1-G3), each represented by sequences from North American and European cricetids. A maximum likelihood tree of SSU sequences had low bootstrap support and showed G1 to be more heterogeneous than G2 or G3. Actin and concatenated actin-SSU trees, which were better resolved and had higher bootstrap support than the SSU phylogeny, showed that closely related cricetid hosts in Europe and North America are infected with closely related Cryptosporidium genotypes. Cricetids were not major reservoirs of human pathogenic Cryptosporidium spp.

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections.