Giardia is a genus of flagellated protozoans that parasitize the gastrointestinal tract of humans and wildlife worldwide. While G. duodenalis is well-studied due to its potential to cause outbreaks of diarrheal illness in humans, other Giardia species from wildlife have been largely understudied. This study examines the occurrence, host specificity, and genotypic diversity of Giardia in wild rodents living within the New York City water supply watershed. A novel nested PCR assay targeting the 18S ssu-rDNA gene is introduced, which captures nearly the entire gene for improved species-level determination versus existing molecular typing methods. Molecular characterization of 55 Giardia specimens reveals at least seven novel lineages. Phylogenetic analysis indicates a close relationship between the newly characterized Giardia lineages and rodent hosts, suggesting rodents as important reservoirs of Giardia and its close relatives. These findings provide insights into the diversity of Giardia species and their public health potential in localities with human-wildlife interaction and further emphasizes the need for continued efforts to improve the molecular tools used to study microbial eukaryotes, especially those with zoonotic potential. © 2024

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted by hematophagous reduviid bugs within the subfamily Triatominae. These vectors take blood meals from a wide range of hosts, and their feeding behaviors have been used to investigate the ecology and epidemiology of T. cruzi. In this study we describe two PCR-based methodologies that amplify a fragment of the 16S mitochondrial rDNA, aimed to improve the identification of blood meal sources for Triatoma infestans: a.--Sequence analyses of two heminested PCRs that allow the identification of mammalian and avian species, and b.--restriction fragment length polymorphism (RFLP) analysis from the mammalian PCR to identify and differentiate multi-host blood meals. Findings from both methodologies indicate that host DNA could be detected and the host species identified in samples from laboratory reared and field collected triatomines. The implications of this study are two-fold. First, these methods can be used in areas where the fauna diversity and feeding behavior of the triatomines are unknown. Secondly, the RFLP method led to the identification of multi-host DNA from T. infestans gut contents, enhancing the information provided by this assay. These tools are important contributions for ecological and epidemiological studies of vector-borne diseases.

Complete reference genomes, including correct feature annotations, are a fundamental aspect of genomic biology. In the case of protozoan species such as Giardia duodenalis, a major human and animal parasite worldwide, accurate genome annotation can deepen our understanding of the evolution of parasitism and pathogenicity by identifying genes underlying key traits and clinically relevant cellular mechanisms, and by extension, the development of improved prevention strategies and treatments. This study used bioinformatics analyses of Giardia mRNA libraries to characterize known introns and identify new intron candidates, working towards completion of the G. duodenalis assemblage A strain 'WB' genome and further elucidating Giardia's gene expression. By using a set of experimentally validated positive control loci to calibrate our intron detection pipeline, we were able to detect evidence of previously missed candidate splice junctions directly from expressed transcript data. These intron candidates were further studied in silico using NMDS (non-metric multidimensional scaling) clustering to determine shared characteristics and their relative importance such as secondary structure, splicing efficiency and motif conservation, and thus to refine intron models. Results from this study identified 34 new intron candidates, with several potential introns showing evidence that secondary structure of the mRNA molecule might play a more significant role in splicing than previously reported eukaryotic splicing activity mediated by a reduced spliceosome present in G. duodenalis.

Cryptosporidium canis is a zoonotic species causing cryptosporidiosis in humans in addition to its natural hosts dogs and other fur animals. To understand the genetic basis for host adaptation, we sequenced the genomes of C. canis from dogs, minks, and foxes and conducted a comparative genomics analysis. While the genomes of C. canis have similar gene contents and organisations, they (~41.0 %) and C. felis (39.6 %) have GC content much higher than other Cryptosporidium spp. (24.3-32.9 %) sequenced to date. The high GC content is mostly restricted to subtelomeric regions of the eight chromosomes. Most of these GC-balanced genes encode Cryptosporidium-specific proteins that have intrinsically disordered regions and are involved in host-parasite interactions. Natural selection appears to play a more important role in the evolution of codon usage in GC-balanced C. canis, and most of the GC-balanced genes have undergone positive selection. While the identity in whole genome sequences between the mink- and dog-derived isolates is 99.9 % (9365 SNVs), it is only 96.0 % (362 894 SNVs) between them and the fox-derived isolate. In agreement with this, the fox-derived isolate possesses more subtelomeric genes encoding invasion-related protein families. Therefore, the change in subtelomeric GC content appears to be responsible for the more GC-balanced C. canis genomes, and the fox-derived isolate could represent a new Cryptosporidium species.

The parasite Cryptosporidium hominis is a leading cause of the diarrheal disease cryptosporidiosis, whose incidence in the United States has increased since 2005. Here, we show that the newly emerged and hyper-transmissible subtype IfA12G1R5 is now dominant in the United States. In a comparative analysis of 127 newly sequenced and 95 published C. hominis genomes, IfA12G1R5 isolates from the United States place into three of the 14 clusters (Pop6, Pop13, and Pop14), indicating that this subtype has multiple ancestral origins. Pop6 (IfA12G1R5a) has an East Africa origin and has recombined with autochthonous subtypes after its arrival. Pop13 (IfA12G1R5b) is imported from Europe, where it has recombined with the prevalent local subtype, whereas Pop14 (IfA12G1R5c) is a progeny of secondary recombination between Pop6 and Pop13. Selective sweeps in invasion-associated genes have accompanied the emergence of the dominant Pop14. These observations offer insights into the emergence and evolution of hyper-transmissible pathogens.

Giardia duodenalis is a parasitic flagellated protozoan which infects a wide range of mammalian hosts, including humans, and is subdivided into at least eight genetic assemblages commonly thought to represent cryptic species. Molecular studies have shown that G. duodenalis assemblage A, which parasitizes humans and animals, contains several phylogenetically distinct groupings known as sub-assemblages. Molecular studies employing poor phylogenetic-resolution markers routinely recover these sub-assemblages, implying that they represent evolutionarily distinct clades and possibly cryptic species, a hypothesis which is supported by epidemiologic trends. Here, we further tested this hypothesis by using available data from 41 whole genomes to characterize sub-assemblages and coalescent techniques for statistical estimation of species boundaries coupled to functional gene content analysis, thereby assessing the stability and distinctiveness of clades. Our analysis revealed two new sub-assemblage clades as well as novel signatures of gene content geared toward differential host adaptation and population structuring via vertical inheritance rather than recombination or panmixia. We formally propose sub-assemblage AII as a new species, Giardia hominis, while preserving the name Giardia duodenalis for sub-assemblage AI. Additionally, our bioinformatic methods broadly address the challenges of identifying cryptic microbial species to advance our understanding of emerging disease epidemiology, which should be broadly applicable to other lower eukaryotic taxa of interest. Giardia hominis n. sp. Zoobank LSID: urn:lsid: zoobank.org:pub:4298F3E1-E3EF-4977-B9DD-5CC59378C80E.

Genetic recombination plays a critical role in the emergence of pathogens with phenotypes such as drug resistance, virulence, and host adaptation. Here, we tested the hypothesis that recombination between sympatric ancestral populations leads to the emergence of divergent variants of the zoonotic parasite Cryptosporidium parvum with modified host ranges. Comparative genomic analyses of 101 isolates have identified seven subpopulations isolated by distance. They appear to be descendants of two ancestral populations, IIa in northwestern Europe and IId from southwestern Asia. Sympatric recombination in areas with both ancestral subtypes and subsequent selective sweeps have led to the emergence of new subpopulations with mosaic genomes and modified host preference. Subtelomeric genes could be involved in the adaptive selection of subpopulations, while copy number variations of genes encoding invasion-associated proteins are potentially associated with modified host ranges. These observations reveal ancestral origins of zoonotic C. parvum and suggest that pathogen import through modern animal farming might promote the emergence of divergent subpopulations of C. parvum with modified host preference.

Enterocytozoon bieneusi is the most common microsporidia in humans worldwide, in addition to infecting a wide range of animals. However, there is limited information about this pathogen in children in Egypt. Here, we carried out a molecular epidemiological study of E. bieneusi in child care centers in three provinces in Egypt. Altogether, 585 fresh fecal samples were collected from children attending 18 child care centers in El-Dakahlia, El-Gharbia, and Damietta provinces in Northeast Egypt during March 2015 to April 2016. PCR and sequence analyses of the ribosomal internal transcribed spacer (ITS) were used to detect and genotype E. bieneusi. Twenty-seven fecal samples (4.6%, 27/585) were positive for E. bieneusi. Five genotypes were identified, including type IV (n = 13), Peru8 (n = 9), Peru6 (n = 2), Peru11 (n = 2), and D (n = 1). Phylogenetic analysis indicated that the five genotypes of E. bieneusi detected in this study were clustered into zoonotic group 1. These data provide important information on the prevalence and genetic diversity of E. bieneusi in children in this country. Further epidemiological studies should be conducted to elucidate the role of zoonotic transmission in human E. bieneusi infections.

Cryptosporidium parvum is a parasitic zoonotic pathogen responsible for diarrheal illness in humans and animals worldwide. We report an investigation of a cryptosporidiosis outbreak in raccoons and wildlife rehabilitation workers at a Virginia facility. Fifteen (31%) of 49 facility personnel experienced symptoms meeting the case definition, including four laboratory-confirmed cases. Seven juvenile raccoons were reported to have diarrhoea; six had laboratory-confirmed cryptosporidiosis. Cryptosporidium parvum of the same molecular subtype (IIaA16G3R2) was identified in two human cases and six raccoons. Raccoon illness preceded human illness by 11days, suggesting possible zoonotic transmission from raccoons to humans. This appears to be the first report of a human cryptosporidiosis outbreak associated with exposure to raccoons infected with C.parvum. Raccoons might be an under-recognized reservoir for human C.parvum infections. Further study is needed to explore the prevalence of cryptosporidial species in raccoons and their role as a wildlife reservoir.

Cryptosporidium spp. are important enteric pathogens in a wide range of vertebrates including humans. Previous comparative analysis revealed conservation in genome composition, gene content, and gene organization among Cryptosporidium spp., with a progressive reductive evolution in metabolic pathways and invasion-related proteins. In this study, we sequenced the genome of zoonotic pathogen Cryptosporidium felis and conducted a comparative genomic analysis. While most intestinal Cryptosporidium species have similar genomic characteristics and almost complete genome synteny, fewer protein-coding genes and some sequence inversions and translocations were found in the C. felis genome. The C. felis genome exhibits much higher GC content (39.6 %) than other Cryptosporidium species (24.3-32.9 %), especially at the third codon position (GC3) of protein-coding genes. Thus, C. felis has a different codon usage, which increases the use of less energy costly amino acids (Gly and Ala) encoded by GC-rich codons. While the tRNA usage is conserved among Cryptosporidium species, consistent with its higher GC content, C. felis uses a unique tRNA for GTG for valine instead of GTA in other Cryptosporidium species. Both mutational pressures and natural selection are associated with the evolution of the codon usage in Cryptosporidium spp., while natural selection seems to drive the codon usage in C. felis. Other unique features of the C. felis genome include the loss of the entire traditional and alternative electron transport systems and several invasion-related proteins. Thus, the preference for the use of some less energy costly amino acids in C. felis may lead to a more harmonious parasite-host interaction, and the strengthened host-adaptation is reflected by the further reductive evolution of metabolism and host invasion-related proteins.

Anthropogenic activities, such as human population expansion and land-use change, create ecological overlap between humans, domesticated animals, and wildlife and can exacerbate the zoonotic transmission of parasites. To improve our understanding of this dynamic, we employed multi-locus genotyping to conduct a cross-sectional study of the potential for zoonotic transmission of the protozoan parasite Giardia duodenalis among humans, household associated livestock and dogs, and black and gold howler monkeys (Alouatta caraya) in the Corrientes Province of Argentina. We found Giardia prevalence to be highest in howler monkeys (90.3% (47/52)), followed by humans (61.1% (22/36)), dogs (44.4% (16/36)), and cattle (41.9% (18/43)). We further established that howler monkeys exclusively harbored strains of assemblage B (100%) while humans were infected with either assemblage A (13.3%) or B (80%) or A and B (6.7%), and cattle and dogs were infected with either assemblage A (cattle, 94.1%; dogs, 80%)), A and C (10%), or their host-adapted assemblage (cattle, 5.9%; dogs, 10%). Our finding of G. duodenalis in both humans and domesticated animals (assemblage A) and humans and wild primates (assemblage B) suggests that cross-species transmission of multiple assemblages of G. duodenalis may occur in rural complexes such as northern Argentina where people, domesticated animals, and wildlife overlap. We further highlight the need to investigate the implications of these results for human health, the economics of livestock production, and wildlife conservation in this and similar systems. This article is protected by copyright. All rights reserved.

Rabbits are increasingly farmed in Egypt for meat. They are, however, known reservoirs of infectious pathogens. Currently, no information is available on the genetic characteristics of Cryptosporidium spp. in rabbits in Egypt. To understand the prevalence and genetic identity of Cryptosporidium spp. in these animals, 235 fecal samples were collected from rabbits of different ages on nine farms in El-Dakahlia, El-Gharbia, and Damietta Provinces, Egypt during the period from July 2015 to April 2016. PCR-RFLP analysis of the small subunit rRNA gene was used to detect and genotype Cryptosporidium spp. The overall detection rate was 11.9% (28/235). All 28 samples were identified as Cryptosporidium cuniculus. The 16 samples successfully subtyped by the sequence analysis of the partial 60 kDa glycoprotein gene belonged to two subtypes, VbA19 (n = 1) and VbA33 (n = 15). As C. cuniculus is increasingly recognized as a cause of human cryptosporidiosis, Cryptosporidium spp. in rabbits from Egypt have zoonotic potential.

Octomitus is a diplomonad genus known to inhabit the intestinal tracts of rodents. Ultrastructural morphology and 18S rDNA gene sequence analysis support the placement of Octomitus as the closest sister lineage to Giardia, a parasite which causes diarrheal disease in humans and animals worldwide. However, further information on the ecology and diversity of Octomitus is currently scarce. Expanding the available database of characterized sequences for this organism would therefore be helpful to studies of Diplomonad ecology, evolution, and epidemiology, particularly related to the evolution of parasitism in Giardia and Spironucleus, another related Diplomonad common in commercial fish farming. In order to study the prevalence and genotypic diversity of Octomitus, we developed a nested PCR assay specific to Octomitus and optimized to detect genotypes in fecal samples collected from wildlife in a New York watershed, and sequenced a portion of the small subunit ribosomal DNA (18S rDNA) gene to identify samples to species level. Molecular evidence suggested that Octomitus genotypes display similar prevalence to Cryptosporidium and microsporidian pathogens in wildlife as well as strong host preference for rodent and opossum hosts. Phylogenetic analysis showed strong support for 14 Octomitus genotypes, 13 of these novel, and patterns of host-parasite co-evolution. © 2021

Giardia duodenalis (syn. G. lamblia, G. intestinalis) is the causative agent of giardiasis, one of the most common diarrheal infections in humans. Evolutionary relationships among G. duodenalis genotypes (or subtypes) of assemblage B, one of two genetic assemblages causing the majority of human infections, remain unclear due to poor phylogenetic resolution of current typing methods. Here, we devised a methodology to identify new markers for a streamlined multi-locus sequence typing (MLST) scheme based on comparisons of all core genes against the phylogeny of whole-genome sequences (WGS). Our analysis identified three markers with comparable resolution to WGS data. Using newly designed PCR primers for our novel MLST loci, we typed an additional 68 strains of assemblage B. Analyses of these strains and previously determined genome sequences showed that genomes of this assemblage can be assigned to 16 clonal complexes, each with unique gene content that is apparently tuned to differential virulence and ecology. Obtaining new genomes of Giardia spp. and other eukaryotic microbial pathogens remains challenging due to difficulties in culturing the parasites in the laboratory. Hence, the methods described here are expected to be widely applicable to other pathogens of interest and advance understanding of their ecology and evolution.IMPORTANCE Giardia duodenalis assemblage B is a major waterborne pathogen and the most commonly identified genotype causing human giardiasis worldwide. The lack of morphological characters for classification requires the use of molecular techniques for strain differentiation, however, the absence of scalable and affordable NGS-based typing methods has prevented meaningful advancements in high resolution molecular typing for further understanding of the evolution and epidemiology of Assemblage B. Prior studies have reported high sequence diversity but low phylogenetic resolution at standard loci in Assemblage B, highlighting the necessity of identifying new markers for accurate and robust molecular typing. Data from comparative analyses of available genomes in this study identified three loci that together form a novel high-resolution typing scheme with high concordance to whole-genome-based phylogenomics and which should aid in future public health endeavors related to this parasite. In addition, data from newly characterized strains suggest evidence of biogeographic and ecologic endemism.

Habitat disturbance, a common consequence of anthropogenic land use practices, creates human–animal interfaces where humans, wildlife, and domestic species can interact. These altered habitats can influence host–microbe dynamics, leading to potential downstream effects on host physiology and health. Here, we explored the effect of ecological overlap with humans and domestic species and infection with the protozoan parasite Giardia duodenalis on the bacteria of black and gold howler monkeys (Alouatta caraya), a key sentinel species, in northeastern Argentina. Fecal samples were screened for Giardia duodenalis infection using a nested PCR reaction, and the gut bacterial community was characterized using 16S rRNA gene amplicon sequencing. Habitat type was correlated with variation in A. caraya gut bacterial community composition but did not affect gut bacterial diversity. Giardia presence did not have a universal effect on A. caraya gut bacteria across habitats, perhaps due to the high infection prevalence across all habitats. However, some bacterial taxa were found to vary with Giardia infection. While A. caraya's behavioral plasticity and dietary flexibility allow them to exploit a range of habitat conditions, habitats are generally becoming more anthropogenically disturbed and, thus, less hospitable. Alterations in gut bacterial community dynamics are one possible indicator of negative health outcomes for A. caraya in these environments, since changes in host–microbe relationships due to stressors from habitat disturbance may lead to negative repercussions for host health. These dynamics are likely relevant for understanding organism responses to environmental change in other mammals.

Molecular characterizations of the microsporidian pathogen Enterocytozoon bieneusi at the ribosomal internal transcribed spacer (ITS) locus have identified nearly 500 genotypes in 11 phylogenetic groups with different host ranges. Among those, one unique group of genotypes, Group 11, is commonly found in dogs. Genetic characterizations of those and many divergent E. bieneusi genotypes at other genetic loci are thus far impossible. In this study, we sequenced 151 E. bieneusi isolates from several ITS genotype groups at the 16S rRNA locus and two new semi-conservative genetic markers (casein kinase 1 (ck1) and spore wall protein 1 (swp1)). Comparison of the near full (~1,200 bp) 16S rRNA sequences showed mostly two to three nucleotide substitutions between Group 1 and Group 2 genotypes, while Group 11 isolates differed from those by 26 (2.2%) nucleotides. Sequence analyses of the ck1 and swp1 loci confirmed the genetic uniqueness of Group 11 genotypes, which produced sequences very divergent from other groups. In contrast, genotypes in Groups 1 and 2 produced similar nucleotide sequences at these genetic loci, and there was discordant placement of ITS genotypes among loci in phylogenetic analyses of sequences. These results suggest that the canine-adapted Group 11 genotypes are genetically divergent from other genotype groups of E. bieneusi, possibly representing a different Enterocytozoon sp. They also indicate that there is no clear genetic differentiation of ITS Groups 1 and 2 at other genetic loci, supporting the conclusion on the lack of strict host specificity in both groups. Data and genetic markers from the study should facilitate population genetic characterizations of E. bieneusi isolates and improve our understanding of the zoonotic potential of E. bieneusi in domestic animals.

Cryptosporidium canis is an important cause of cryptosporidiosis in canines and humans. Studies of the transmission characteristics of C. canis are currently hampered by lack of suitable subtyping tools. In this study, we conducted a genomic survey of the pathogen and developed a subtyping tool targeting the partial 60-kDa glycoprotein (gp60) gene. Seventy-six isolates previously identified as C. canis were analyzed using the new subtyping tool. Amplicons of expected size were obtained from 49 isolates, and phylogenetic analysis identified 10 subtypes clustered in five distinct groups (XXa-XXe). The largest group XXa contained 43 isolates from four subtypes that differed slightly from each other at the nucleotide level, while groups XXb-XXe contain one to three isolates each. The similar distribution of subtypes in humans and canines suggests that zoonotic transmission might play an important role in the epidemiology of C. canis In addition, a suspected zoonotic transmission of C. canis between dogs and humans in a household was confirmed using the subtyping tool. The subtyping tool and data generated in this study might improve our understanding of the transmission of this zoonotic pathogen.

Cryptosporidium felis is the major etiologic agent of cryptosporidiosis in felines and has been reported in numerous human cryptosporidiosis cases. Sequence analysis of the 60-kDa glycoprotein (gp60) gene has been developed for subtyping C. felis recently. In this study, 66 C. felis isolates from the United States, Jamaica, Peru, Portugal, Slovakia, Nigeria, Ethiopia, Kenya, China, India and Australia were subtyped using the newly established tool. Forty-four specimens yielded gp60 sequences, generating 23 subtypes clustered in 4 subtype families (XIXa, XIXc, XIXd and XIXe) with high bootstrap support in a phylogenetic analysis of sequence data. Among them, XIXa showed high genetic diversity at the nucleotide level, with the formation of 18 subtypes from both cats and humans with different geographic distribution. In contrast, all 11 XIXd isolates derived from humans from various countries had identical sequences. Results of this study improve our understanding of the genetic diversity, host specificity and transmission dynamics of C. felis.

We assessed the potential contribution of hospitals to contaminations of wastewater by enteric protists, including Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in raw wastewater. Wastewater samples were collected from storage tanks in two hospitals and one associated wastewater treatment plant in Shanghai, China, from March to November 2009. Enteric pathogens were detected and identified using PCR and DNA sequencing techniques. Among a total of 164 samples analyzed, 31 (18.9%), 45 (27.4%), and 122 (74.4%) were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. Altogether, three Cryptosporidium species, four G. duodenalis assemblages, and 12 E. bieneusi genotypes were detected. Cryptosporidium hominis, G. duodenalis sub-assemblage AII, and E. bieneusi genotype D were the dominant ones in wastewater from both hospitals and the wastewater treatment plant. A similar distribution in genotypes of enteric pathogens was seen between samples from hospitals and the wastewater treatment plant, suggesting that humans are one of the major sources for these pathogens and hospitals are important contributors of enteric parasites in urban wastewater. Data from this study might be useful in the formulation of preventive measures against environmental contamination of waterborne pathogens.

Cryptosporidium is an enteric pathogen that is transmitted through animal-to-person or person-to-person contact or through ingestion of contaminated water or food. In the United States, Cryptosporidium affects an estimated 750,000 persons each year; however, only approximately 11,000 cases are reported nationally (1,2). Persons infected with Cryptosporidium typically develop symptoms within 2 to 10 days after exposure. Common symptoms include watery diarrhea, abdominal cramps, nausea, vomiting, or fever, which can last 1 to 2 weeks. Cryptosporidiosis is a nationally notifiable disease in the United States. Nebraska presents a unique setting for the evaluation of this pathogen because, compared with other states, Nebraska has a greater reliance on agriculture and a higher proportion of the population residing and working in rural communities. Cryptosporidium species and subtypes are generally indistinguishable using conventional diagnostic methods. Using molecular characterization, Nebraska evaluated the genetic diversity of Cryptosporidium and found a dichotomy in the distribution of cases of cryptosporidiosis caused by Cryptosporidium parvum and Cryptosporidium hominis among rural and urban settings. Characterizing clusters of C. hominis cases revealed that several child care facilities were affected by the same subtype, suggesting community-wide transmission and indicating a need for effective exclusion policies. Several cases of cryptosporidiosis caused by non-C. parvum or non-C. hominis species and genotypes indicated unique animal exposures that were previously unidentified. This study enhanced epidemiologic data by validating known Cryptosporidium sources, confirming outbreaks, and, through repeat interviews, providing additional information to inform cryptosporidiosis prevention and control efforts.

Cryptosporidium genotyping has made significant contributions to the species structure and population genetics of Cryptosporidium spp. In addition, the standardized method has shown utility in epidemiologic investigations identifying case linkages and contamination sources that could not be determined with traditional epidemiologic tools. The standardized method for Cryptosporidium genotyping from stool specimens described here comprises multiple individual protocols to amplify and sequence regions of the SSU rRNA and gp60 genes of Cryptosporidium.

BACKGROUND: Cryptosporidiosis is a major cause of gastrointestinal diseases in humans and other vertebrates. Previous analyses of invasion-related proteins revealed that Cryptosporidium parvum, Cryptosporidium hominis, and Cryptosporidium ubiquitum mainly differed in copy numbers of secreted MEDLE proteins and insulinase-like proteases and sequences of mucin-type glycoproteins. Recently, Cryptosporidium chipmunk genotype I was identified as a novel zoonotic pathogen in humans. In this study, we sequenced its genome and conducted a comparative genomic analysis. RESULTS: The genome of Cryptosporidium chipmunk genotype I has gene content and organization similar to C. parvum and other intestinal Cryptosporidium species sequenced to date. A total of 3783 putative protein-encoding genes were identified in the genome, 3525 of which are shared by Cryptosporidium chipmunk genotype I and three major human-pathogenic Cryptosporidium species, C. parvum, C. hominis, and Cryptosporidium meleagridis. The metabolic pathways are almost identical among these four Cryptosporidium species. Compared with C. parvum, a major reduction in gene content in Cryptosporidium chipmunk genotype I is in the number of telomeric genes encoding MEDLE proteins (two instead of six) and insulinase-like proteases (one instead of two). Highly polymorphic genes between the two species are mostly subtelomeric ones encoding secretory proteins, most of which have higher dN/dS ratios and half are members of multiple gene families. In particular, two subtelomeric ABC transporters are under strong positive selection. CONCLUSIONS: Cryptosporidium chipmunk genotype I possesses genome organization, gene content, metabolic pathways and invasion-related proteins similar to the common human-pathogenic Cryptosporidium species, reaffirming its human-pathogenic nature. The loss of some subtelomeric genes encoding insulinase-like proteases and secreted MEDLE proteins and high sequence divergence in secreted pathogenesis determinants could contribute to the biological differences among human-pathogenic Cryptosporidium species.

Little is known of the occurrence and age patterns of species/genotypes and subtypes of Cryptosporidium spp. and Giardia duodenalis in calves in Egypt. In this study, 248 fecal specimens were collected from dairy calves aged 1day to 6months on eight farms in three provinces during March 2015 to April 2016. Cryptosporidium spp. were detected and genotyped by using PCR-RFLP analysis of the small subunit rRNA (SSU rRNA) gene, while G. duodenalis was detected and genotyped by using PCR and sequence analyses of the triose phosphate isomerase (tpi), glutamate dehydrogenase (gdh) and beta-giardin (bg) genes. The overall infection rates of Cryptosporidium spp. and G. duodenalis were 9.7 and 13.3%, respectively. The highest Cryptosporidium infection rate (26.7%) was in calves of age</=1month while the highest G. duodenalis infection rate (44.4%) was in calves of 2months. Three Cryptosporidium spp. were identified, including C. parvum (n=16), C. bovis (n=5) and C. ryanae (n=3), with the former being almost exclusively found in calves of </=3months of age and the latter two being only found in calves of over 3months. Subtyping of C. parvum by PCR-sequence analysis of the 60kDa glycoprotein gene identified subtypes IIaA15G1R1 (n=15) and IIaA15G2R1 (n=1). The G. duodenalis identified included both assemblages E (n=32) and A (n=1), with the latter belonging to the anthroponotic subtype A2. These data provide new insights into the genetic diversity and age patterns of Cryptosporidium spp. and G. duodenalis in calves in Egypt.

BACKGROUND: Cryptosporidium is a leading cause of moderate-to-severe diarrhea (MSD) in young children in Africa. We examined factors associated with Cryptosporidium infection in MSD cases enrolled at the rural western Kenya Global Enteric Multicenter Study (GEMS) site from 2008-2012. METHODOLOGY/PRINCIPAL FINDINGS: At health facility enrollment, stool samples were tested for enteric pathogens and data on clinical, environmental, and behavioral characteristics collected. Each child's health status was recorded at 60-day follow-up. Data were analyzed using logistic regression. Of the 1,778 children with MSD enrolled as cases in the GEMS-Kenya case-control study, 11% had Cryptosporidium detected in stool by enzyme immunoassay; in a genotyped subset, 81% were C. hominis. Among MSD cases, being an infant, having mucus in stool, and having prolonged/persistent duration diarrhea were associated with being Cryptosporidium-positive. Both boiling drinking water and using rainwater as the main drinking water source were protective factors for being Cryptosporidium-positive. At follow-up, Cryptosporidium-positive cases had increased odds of being stunted (adjusted odds ratio [aOR] = 1.65, 95% CI: 1.06-2.57), underweight (aOR = 2.08, 95% CI: 1.34-3.22), or wasted (aOR = 2.04, 95% CI: 1.21-3.43), and had significantly larger negative changes in height- and weight-for-age z-scores from enrollment. CONCLUSIONS/SIGNIFICANCE: Cryptosporidium contributes significantly to diarrheal illness in young children in western Kenya. Advances in point of care detection, prevention/control approaches, effective water treatment technologies, and clinical management options for children with cryptosporidiosis are needed.

Outbreaks associated with exposure to treated recreational water can be caused by pathogens or chemicals in venues such as pools, hot tubs/spas, and interactive water play venues (i.e., water playgrounds). During 2000-2014, public health officials from 46 states and Puerto Rico reported 493 outbreaks associated with treated recreational water. These outbreaks resulted in at least 27,219 cases and eight deaths. Among the 363 outbreaks with a confirmed infectious etiology, 212 (58%) were caused by Cryptosporidium (which causes predominantly gastrointestinal illness), 57 (16%) by Legionella (which causes Legionnaires' disease, a severe pneumonia, and Pontiac fever, a milder illness with flu-like symptoms), and 47 (13%) by Pseudomonas (which causes folliculitis ["hot tub rash"] and otitis externa ["swimmers' ear"]). Investigations of the 363 outbreaks identified 24,453 cases; 21,766 (89%) were caused by Cryptosporidium, 920 (4%) by Pseudomonas, and 624 (3%) by Legionella. At least six of the eight reported deaths occurred in persons affected by outbreaks caused by Legionella. Hotels were the leading setting, associated with 157 (32%) of the 493 outbreaks. Overall, the outbreaks had a bimodal temporal distribution: 275 (56%) outbreaks started during June-August and 46 (9%) in March. Assessment of trends in the annual counts of outbreaks caused by Cryptosporidium, Legionella, or Pseudomonas indicate mixed progress in preventing transmission. Pathogens able to evade chlorine inactivation have become leading outbreak etiologies. The consequent outbreak and case counts and mortality underscore the utility of CDC's Model Aquatic Health Code (https://www.cdc.gov/mahc) to prevent outbreaks associated with treated recreational water.

Little information is available on the occurrence of the zoonotic protists Cryptosporidium spp. and none on Enterocytozoon bieneusi in camels. This preliminary study was conducted to examine the identity of Cryptosporidium subtypes and E. bieneusi genotypes in dromedary camels in Algeria. A total of 39 fecal specimens were collected from young camels. PCR-sequence analysis of the small subunit rRNA was used to detect and genotype Cryptosporidium spp. Cryptosporidium parvum present was further subtyped by sequence analysis of the 60 kDa glycoprotein gene. PCR-sequence analysis of the ribosomal internal transcribed spacer gene was used to detect and genotype E. bieneusi. Altogether, two and eight of the specimens analyzed were positive for C. parvum and E. bieneusi, respectively. The former was identified as a new subtype that is genetically related to the C. hominis If subtype family, whereas the latter was identified as two related genotypes (Macaque1 and a novel genotype) in the newly assigned E. bieneusi genotype group 8. Although they are not known hosts for C. parvum and E. bieneusi, camels are apparently infected with genetically distinct variants of these pathogens.

Cyclospora cayetanensis is an emerging pathogen that is endemic in developing countries and responsible for many large foodborne cyclosporiasis outbreaks in North America since 1990s. Because of the lack of typing targets, the genetic diversity and population genetics of C. cayetanensis have not been investigated. In this study, we undertook a population genetic analysis of multilocus sequence typing data we recently collected from 64 C. cayetanensis specimens. Despite the extensive genetic heterogeneity in the overall C. cayetanensis population, there were significant intra- and inter-genic linkage disequilibria (LD). A disappearance of LD was observed when only multilocus genotypes were included in the population genetic analysis, indicative of an epidemic nature of C. cayetanensis. Geographical segregation-associated sub-structuring was observed between specimens from China and those from Peru and the United States. The two subpopulations had reduced LD, indicating the likely occurrence of genetic exchange among isolates in endemic areas. Further analyses of specimens from other geographical regions are necessary to fully understand the population genetics of C. cayetanensis.

Cryptosporidium skunk genotype is a zoonotic pathogen commonly identified in surface water. Thus far, no subtyping tool exists for characterizing its transmission in humans and animals and transport in environment. In this study, a subtyping tool based on the 60kDa glycoprotein (gp60) gene previously developed for Cryptosporidium chipmunk genotype I was used in the characterization of Cryptosporidium skunk genotype in animal and storm runoff samples from a watershed in New York. Altogether, 17 positive samples from this watershed and 5 human and animal specimens from other areas were analyzed. We identified 14 subtypes of Cryptosporidium skunk genotype, 11 of which were seen in the watershed. In phylogenetic analysis, these subtypes belonged to 4 subtype families (XVIa, XVIb, XVIc, and XVId). No host-adapted subtypes were identified and the two subtypes in humans were genetically similar to some in raccoons, otters, and storm runoff samples from the watershed. The characteristics of gp60 protein sequences of the Cryptosporidium skunk genotype are similar to those of other Cryptosporidium species, but only its XVIb subtype family has a putative furin cleavage site. This subtyping tool might be useful in characterizing Cryptosporidium skunk genotype in clinical and environmental samples.

We investigated a gastrointestinal illness cluster among persons who attended a baseball tournament (>200 teams) during July 2015. We interviewed representatives of 19 teams; illness was reported among only the 9 (47%) teams that stayed at Hotel A (p<.01). We identified 55 primary cases. A case-control study demonstrated that pool exposure at Hotel A was significantly associated with illness (odds ratio: 7.3; 95% confidence interval: 3.6, 15.2). Eight out of nine (89%) stool specimens tested were positive for Cryptosporidium, with C. hominis IfA12 G1 subtype identified in two specimens. The environmental health assessment detected a low free available chlorine level, and pool water tested positive for E. coli and total coliforms. A possible diarrheal contamination event, substantial hotel pool use, and use of cyanuric acid might have contributed to this outbreak and magnitude. Aquatic facilities practicing proper operation and maintenance (e.g., following the Centers for Disease Control and Prevention's Model Aquatic Health Code) can protect the public's health.

Cryptosporidiosis is a nationally notifiable gastrointestinal illness caused by parasitic protozoa of the genus Cryptosporidium, which can cause profuse, watery diarrhea that can last up to 2-3 weeks in immunocompetent patients and can lead to life-threatening wasting and malabsorption in immunocompromised patients. Fecal-oral transmission of Cryptosporidium oocysts, the parasite's infectious life stage, occurs via ingestion of contaminated recreational water, drinking water, or food, or following contact with infected persons or animals, particularly preweaned bovine calves (1). The typical incubation period is 2-10 days. Since 2004, the annual incidence of nationally notified cryptosporidiosis has risen approximately threefold in the United States (1). Cryptosporidium also has emerged as the leading etiology of nationally notified recreational water-associated outbreaks, particularly those associated with aquatic facilities (i.e., physical places that contain one or more aquatic venues [e.g., pools] and support infrastructure) (2). As of February 24, 2017, a total of 13 (54%) of 24 states reporting provisional data detected at least 32 aquatic facility-associated cryptosporidiosis outbreaks in 2016. In comparison, 20 such outbreaks were voluntarily reported to CDC via the National Outbreak Reporting System for 2011, 16 for 2012, 13 for 2013, and 16 for 2014. This report highlights cryptosporidiosis outbreaks associated with aquatic facilities in three states (Alabama, Arizona, and Ohio) in 2016. This report also illustrates the use of CryptoNet, the first U.S. molecularly based surveillance system for a parasitic disease, to further elucidate Cryptosporidium chains of transmission and cryptosporidiosis epidemiology. CryptoNet data can be used to optimize evidence-based prevention strategies. Not swimming when ill with diarrhea is key to preventing and controlling aquatic facility-associated cryptosporidiosis outbreaks (https://www.cdc.gov/healthywater/swimming/swimmers/steps-healthy-swimming.html).