Sequence introgression from exogenous lineages underlies genomic and biological differences among Cryptosporidium parvum IOWA lines.

The IOWA strain of Cryptosporidium parvum is widely used in studies of the biology and detection of the waterborne pathogens Cryptosporidium spp. While several lines of the strain have been sequenced, IOWA-II, the only reference of the original subtype (IIaA15G2R1), exhibits significant assembly errors. Here we generated a fully assembled genome of IOWA-CDC of this subtype using PacBio and Illumina technologies. In comparative analyses of seven IOWA lines maintained in different laboratories (including two sequenced in this study) and 56 field isolates, IOWA lines (IIaA17G2R1) with less virulence had mixed genomes closely related to IOWA-CDC but with multiple sequence introgressions from IOWA-II and unknown lineages. In addition, the IOWA-IIaA17G2R1 lines showed unique nucleotide substitutions and loss of a gene associated with host infectivity, which were not observed in other isolates analyzed. These genomic differences among IOWA lines could be the genetic determinants of phenotypic traits in C. parvum. These data provide a new reference for comparative genomic analyses of Cryptosporidium spp. and rich targets for the development of advanced source tracking tools.

Spliceosomal introns in the diplomonad parasite Giardia duodenalis revisited.

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.

High subtelomeric GC content in the genome of a zoonotic Cryptosporidium species.

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.

Multiple introductions and recombination events underlie the emergence of a hyper-transmissible Cryptosporidium hominis subtype in the USA.

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.

Genomic comparisons confirm Giardia duodenalis sub-assemblage AII as a unique species.

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.

Benchmark datasets for SARS-CoV-2 surveillance bioinformatics.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has spread globally and is being surveilled with an international genome sequencing effort. Surveillance consists of sample acquisition, library preparation, and whole genome sequencing. This has necessitated a classification scheme detailing Variants of Concern (VOC) and Variants of Interest (VOI), and the rapid expansion of bioinformatics tools for sequence analysis. These bioinformatic tools are means for major actionable results: maintaining quality assurance and checks, defining population structure, performing genomic epidemiology, and inferring lineage to allow reliable and actionable identification and classification. Additionally, the pandemic has required public health laboratories to reach high throughput proficiency in sequencing library preparation and downstream data analysis rapidly. However, both processes can be limited by a lack of a standardized sequence dataset. Methods: We identified six SARS-CoV-2 sequence datasets from recent publications, public databases and internal resources. In addition, we created a method to mine public databases to identify representative genomes for these datasets. Using this novel method, we identified several genomes as either VOI/VOC representatives or non-VOI/VOC representatives. To describe each dataset, we utilized a previously published datasets format, which describes accession information and whole dataset information. Additionally, a script from the same publication has been enhanced to download and verify all data from this study. Results: The benchmark datasets focus on the two most widely used sequencing platforms: long read sequencing data from the Oxford Nanopore Technologies platform and short read sequencing data from the Illumina platform. There are six datasets: three were derived from recent publications; two were derived from data mining public databases to answer common questions not covered by published datasets; one unique dataset representing common sequence failures was obtained by rigorously scrutinizing data that did not pass quality checks. The dataset summary table, data mining script and quality control (QC) values for all sequence data are publicly available on GitHub: https://github.com/CDCgov/datasets-sars-cov-2. Discussion: The datasets presented here were generated to help public health laboratories build sequencing and bioinformatics capacity, benchmark different workflows and pipelines, and calibrate QC thresholds to ensure sequencing quality. Together, improvements in these areas support accurate and timely outbreak investigation and surveillance, providing actionable data for pandemic management. Furthermore, these publicly available and standardized benchmark data will facilitate the development and adjudication of new pipelines.

Sympatric Recombination in Zoonotic Cryptosporidium Leads to Emergence of Populations with Modified Host Preference.

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.

Prevalence and genetic characterization of Enterocytozoon bieneusi in children in Northeast Egypt.

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 outbreak associated with Raccoons at a Wildlife Facility-Virginia, May-June 2019.

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 11 days, 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.

Molecular characterization of Giardia duodenalis and evidence for cross-species transmission in Northern Argentina.

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.

Cryptosporidium felis differs from other Cryptosporidium spp. in codon usage.

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.

Genetic Characterization of Cryptosporidium cuniculus from Rabbits in Egypt.

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.

Prevalence and molecular characterization of novel species of the Diplomonad genus Octomitus (Diplomonadida: Giardiinae) from wildlife in a New York watershed.

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.

Hidden Diversity within Common Protozoan Parasites as Revealed by a Novel Genomotyping Scheme.

Giardia duodenalis (syn. Giardia lamblia, Giardia 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. In this study, we devised a methodology to identify new markers for a streamlined multilocus 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 resolution comparable to that of 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 our understanding of their ecology and evolution.IMPORTANCEGiardia 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 next-generation sequencing (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.

Effects of anthropogenic habitat disturbance and Giardia duodenalis infection on a sentinel species' gut bacteria.

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.

Characterizations of Enterocytozoon bieneusi at new genetic loci reveal a lack of strict host specificity among common genotypes and the existence of a canine-adapted Enterocytozoon species.

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.

Development of a Subtyping Tool for Zoonotic Pathogen Cryptosporidium canis.

Cryptosporidium canis is an important cause of cryptosporidiosis in canines and humans. Studies of the transmission characteristics of C. canis are currently hampered by the 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 gene (gp60). Seventy-six isolates previously identified as C. canis were analyzed using the new subtyping tool. Amplicons of the expected size were obtained from 49 isolates, and phylogenetic analysis identified 10 subtypes clustered into five distinct groups (XXa to XXe). The largest group, XXa, contained 43 isolates from four subtypes that differed slightly from each other at the nucleotide level, while groups XXb to XXe contain one to three isolates each. The similar distributions of subtypes in humans and canines suggest that zoonotic transmission might play an important role in the epidemiology of C. canis In addition, 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.

Subtype distribution of zoonotic pathogen Cryptosporidium felis in humans and animals in several countries.

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.

Contribution of hospitals to the occurrence of enteric protists in urban wastewater.

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.

Occurrence and molecular characterization of Giardia duodenalis in lambs in Djelfa, the central steppe of Algeria.

Little is known of the prevalence and genetic identity of Giardia duodenalis in sheep in Algeria. The present study aimed at characterizing G. duodenalis in lambs up to 6 months of age in Djelfa, Algeria. A total of 346 fecal specimens were collected from 28 farms and screened for G. duodenalis cysts by zinc sulfate flotation microscopy, and positive specimens were confirmed using a direct immunofluorescence assay. Microscopy-positive specimens were analyzed by PCR and sequence analysis of the triosephosphate isomerase and glutamate dehydrogenase genes to determine G. duodenalis assemblages. Coprological examination indicated that the overall infection rate was 7.0% (24/346). Lambs under 3 months of age had higher infection rate (18/197, 9.0%) than older (6/149, 4.0%) animals, and animals with diarrhea (7/44, 16.0%) had higher infection rate than animals without diarrhea (17/302, 5.6%). PCR sequence analyses of the 15 G. duodenalis isolates revealed the presence of assemblages A in 6 isolates, assemblage E in 7 isolates, and both in 2 isolates. Assemblage A was only found in pre-weaned lambs with diarrhea, while assemblage E was mostly found in post-weaned lambs without diarrhea. The assemblage E isolates from sheep were genetically related to those from cattle in Algeria, while assemblage A isolates were from a well-known subtype prevalent in humans. Data generated from the study improve our understanding of the transmission of G. duodenalis in Algeria.