Whole-genome sequence data have made significant contributions to studies of the biology and transmission of Cryptosporidium spp. Well-assembled genomes, free of sequence ambiguity and structural errors, are critical for comparative genomic analysis at the species and subtype levels. The optimized method for Cryptosporidium genome assembly described here comprises several individual protocols to generate chromosome- and contig-level genomes.

The increasing amount of whole-genome sequencing (WGS) data allows us to study the evolution and transmission of Cryptosporidium spp. at the population level. Population genomics is helping us to understand the genetic structure and adaptive evolution of Cryptosporidium and the mechanism and determinants of emergence of hypertransmissible subtypes and variants. The integrated pipeline described here provides an example of the use of WGS data for evolutionary genomic analysis of Cryptosporidium.

Cryptosporidium is a significant zoonotic protozoan parasite that primarily infects intestinal epithelial cells, causing microvillus damage and other gastrointestinal malfunctions. However, the precise molecular mechanisms underlying this process have yet to be elucidated. In this study, IFN-γ knockout mice were infected with a virulent Cryptosporidium parvum IId isolate, and intestinal pathological changes and microvillus alterations were monitored throughout the infection. The results showed that C. parvum invasion induced both intestinal and microvillus damage in the host. Ileum tissues were collected at the early, peak, and late stages of infection for transcriptome sequencing and analysis. Differential expression and enrichment analyses revealed significant downregulation of genes associated with microvilli, lipid metabolism, and fatty acid metabolism during the peak and late stages of infection. A gene co-expression network constructed using the Pearson correlation coefficient further identified that two epithelial cell transcription factors, HNF4α and HNF4γ, were downregulated after C. parvum infection, along with multiple microvillus-related genes showing at least a two-fold decrease in expression, such as Vil1, Cdhr2, and Ush1c. The role of HNF4α in C. parvum-induced microvillus damage was validated in vitro. Activation of HNF4α using an agonist promoted C. parvum growth in Caco-2 cells. These findings reveal that C. parvum infection interferes with HNF4α expression, leading to reduced expression of brush border and related genes and ultimately resulting in microvillus damage. This study provides new insights and directions for further research on the interaction between C. parvum and the host. Elucidating this HNF4α mediated mechanism not only deepens our understanding of host-parasite interactions but also highlights potential therapeutic targets for alleviating Cryptosporidium-induced intestinal injury.

ABSTRACT Fatty acid and retinol binding proteins (FARs) are nematode-specific proteins that orchestrate lipid metabolism, development, and host immune response. Here, the antagonists of Nippostrongylus brasiliensis FAR-1 ( Nb FAR-1) were identified through integrating virtual screening, fluorescent ligand binding assay, and in vitro egg hatching assays. An in vivo mouse model was employed to evaluate anthelmintic efficacy against intestine-parasitized N. brasiliensis and brain-parasitized Angiostrongylus cantonensis . Forty-eight candidates were selected by virtual screening, six of them showed more than 40% antagonism to Nb FAR-1 by fluorescent ligand-competition binding assay and suppressed N. brasiliensis egg hatching by 40%–80% at 20 μM. In mice, E002-0872 and 4340-0245 reduced intestinal N. brasiliensis burdens by 61.74% and 62.15%, respectively, and ameliorated intestinal damages. 4340-0245 reduced cerebral A. cantonensis burdens by 52.73% and alleviated meningeal bleeding and neurological signs. Moreover, treatment with 4340-0245 at 15 mg/kg shortened the body length of female worms, consistent with the higher far-1 expression in females. Alanine scanning showed I95 of Nb FAR-1 as a key residue for binding fatty acid, retinol, and 4340-0245. Intraperitoneal administration of 4340-0245 at 50 mg/kg did not cause any significant toxic effects, whereas 15 mg/kg resulted in a plasma C max of 56,473 ng/mL at 10 min and a half-life of ~7 h. These data provide evidence that FAR-1 is a promising target for developing anthelmintic drugs.

Toxoplasma gondii, a globally prevalent apicomplexan parasite, causes severe morbidity and mortality in immunocompromised individuals and livestock. Current therapies exhibit limited efficacy against chronic bradyzoite stages and face drug resistance. Here, we screened imidocarb and its derivatives for anti-Toxoplasma activity. Among 20 synthesized compounds, squaramide derivative 18 (SA-18) emerged as a promising candidate with nanomolar EC50 against tachyzoites, potent inhibition of bradyzoite differentiation, and > 100-fold selectivity index. Transcriptomic analysis revealed transcriptional dysregulation in parasite metabolic pathways and downregulation of bradyzoite marker BAG1. In vivo studies exhibited significant parasite load reduction in murine models, albeit with suboptimal survival rates. These findings highlight SA-18 as a novel candidate for toxoplasmosis therapy, warranting further optimization for clinical translation.

Polyunsaturated fatty acids (PUFAs) and their oxygenated metabolites (oxylipins) are mediators in regulating membrane function, larval development, and host immune response. In this review, we present that free-living nematodes maintain a relatively complete repertoire of enzymes for PUFA and oxylipin biosynthesis, while parasitic lineages exhibit extensive gene loss and plant-parasitic nematodes show expansion in some enzymes. Meanwhile, parasitic nematodes compensate by scavenging lipids from hosts through the recruitment of various lipid transport proteins. Thus, parasites have lost some enzymes in this metabolic pathway, correlating with host dependence for portions of lipid acquisition, suggesting the plasticity of lipid biosynthesis and acquisition for nematode adaptation to diverse ecological niches.

Nippostrongylus brasiliensis (rat hookworm) is a key model for studying human hookworm infection. A wild N. brasiliensis isolate from Guangzhou maintains high fecundity for 10 days and persists for 21 days in rats, which is twice as long as reported for reference strains, but survives only 10 to 11 days in mice while causing more severe pathology than that observed in rats. Comparative genomic, transcriptomic, and functional analyses were conducted to explore potential genetic factors associated with these phenotypic differences. Whole-genome sequencing revealed 92.6–93.3

SUMMARYCryptosporidiosis is a major public health concern, the extent of which has only truly been appreciated within the last decade. Cryptosporidium research has undergone a renaissance, with new insights into population structure, species diversity, and evolution of the parasite driven by the advent of genetic transformation techniques and novel models for culture in vitro and in vivo. Here, we summarize the impact of these advances on our understanding of this important parasite. In the initial section, we focus on what we have learned about host range and infectivity from comparative genomics, briefly review the public health impact of human infection, and summarize recent findings on immune control and interactions with other gut microbes that influence infection. The second half of the review is devoted to new technical advances that have uncovered novel biological findings. As research on Cryptosporidium is still in its infancy, we finish by summarizing some of the challenges and opportunities for future research.

Metabolic streamlining as a consequence of parasitism has resulted in the loss of lipid biosynthetic genes in nematodes. Angiostrongylus cantonensis, a zoonotic neurotropic parasite that causes eosinophilic meningitis in mammals, completes a complex migration through the rat brain before maturing in the pulmonary arteries. Polyunsaturated fatty acids (PUFAs) and their bioactive metabolites (oxylipins) are known to modulate nematode survival; however, their abundance and endogenous biosynthetic capacity in A. cantonensis remain elusive. We integrated comparative genomics, stage-specific transcriptomics, and lipidomics to reconstruct the expression patterns of the biosynthetic enzymes in A. cantonensis and characterize their dynamic PUFA and oxylipin profiles during the neuro-pulmonary transition from brain-residing fourth-stage (L4) to lung-migrating L5 larvae. Genomic and transcriptomic analyses revealed a streamlined biosynthetic repertoire in A. cantonensis. The worm lacks Δ6 desaturase and canonical cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes, and expresses only residual levels of ptges2 and lta4h, which are markedly lower than those in free-living Caenorhabditis elegans across developmental stages. Using liquid chromatography-tandem mass spectrometry, L4–L5 larvae residing in the rat brain maintained stable PUFA and oxylipin profiles. In contrast, L5 larvae migrating to the lungs accumulated n-6 PUFAs and anti-inflammatory hydroxy eicosatetraenoic acids, epoxy eicosatrienoic acids and hydroxy docosahexaenoic acids, concurrent with reduced levels of pro-inflammatory prostaglandins and 5-oxo-eicosatetraenoic acid. These stage- and tissue-specific lipidomic shifts correlate with distinct host microenvironments encountered during migration. These findings provide a foundation for understanding the evolutionary adaptation of lipid metabolism in parasitic nematodes.

Background The determinants of differences in host infectivity among Cryptosporidium species and subtypes are poorly understood. Results from recent comparative genomic studies suggest that gains and losses of multicopy subtelomeric genes encoding insulinase-like proteases (INS-19 and INS-20 in Cryptosporidium parvum and their orthologs in closely related species) may potentially contribute to these differences. Methodology/Principal findings In this study, we investigated the expression and biological function of the INS-19 and INS-20 of C. parvum. CRISPR/Cas9 was used to endogenously tag both genes with the hemagglutinin epitope. Immunofluorescence analysis revealed that INS-19 and INS-20 are expressed at different developmental stages of the pathogen. Although knockout of either had no detectable effect on the in vitro growth of C. parvum, knockout of INS-20, deletion of its multiple domains, or mutation of the active motif in the functional domain reduced the intensity of C. parvum infection in IFN-γ knockout mice. Consistent with this, mice infected with the INS-20-deleted mutant had reduced intestinal damage and parasite burden. Conclusions/Significance These results suggest that INS-19 and INS-20 have stage-specific expression with distinct biological functions, and that the presence of the INS-20 in zoonotic C. parvum contributes to its infectivity and fitness in mice.

In addition to the ribosomal internal transcribed spacer (ITS) locus, four loci (MS1, MS3, MS4, and MS7) have been identified to develop multilocus sequence typing tools for high-resolution genotyping of Enterocytozoon bieneusi in previous studies. However, the use of only five loci was insufficient for population genetic analysis of E. bieneusi from diverse hosts. In this study, comparison of a clinical genome sequence (C44566) with the whole genome sequence of an E. bieneusi isolate (H348) in GenBank led to the selection of the hypothetical protein 1 (hp1) and tubulin 1 (tub1) loci. Further analysis of the two loci with 156 E. bieneusi-positive samples showed high sequence polymorphisms in ITS Groups 1–6 and 10. Altogether, 30 and 23 sequence types were identified at hp1 and tub1, respectively. Genotyping based on the two loci confirmed the lack of genetic differentiation between Group 1 and Group 2 genotypes, as previously reported. Moreover, the genotypes in Groups 4 and 5 are more divergent from other genotypes within Groups 1–10. However, isolates in Group 11 and 12 could not be amplified at the hp1 and tub1 loci, supporting the previous conclusion of genetic uniqueness of the two genotype groups. The identified genetic markers and generated data could be used to develop a multilocus sequence typing tool for high-resolution genotyping of E. bieneusi, which would also have implications for understanding the taxonomy of Enterocytozoon spp., the public health significance of E. bieneusi in animals, and sources of E. bieneusi infections in humans.

Giardia is the most common protozoan cause of diarrhoeal illness in humans worldwide. Despite this, our understanding of the zoonotic transmission of Giardia, and in particular the role of cattle as a zoonotic reservoir, is not well understood, due to the limitations of current typing systems and a recent taxonomic revision of the genus. Newly improved multilocus sequencing typing tools are not yet widely used and are not applicable to all species. However, data generated to date suggest that zoonotic transmission of Giardia of bovine origin is limited. Carefully designed epidemiological investigations using improved typing tools are essential to understand the extent of zoonotic transmission from cattle. Improved on-farm biosecurity measures are also needed to control the transmission of zoonotic Giardia in cattle.

Cryptosporidium causes severe diarrhea in humans and animals. Mucin-like glycoproteins play a critical role in parasite attachment and invasion and therefore serve as potential protective antigens against reinfection. Muc25 is a highly polymorphic mucin that has been associated with differences in host infectivity in comparative genomic analyses. To study the function of Muc25, we determined its localization and secretion in Cryptosporidium parvum by genome editing. Endogenous gene tagging revealed that Muc25 is stored in small granules of sporozoites and secreted into host microvilli after invasion. Deletion of the signal peptide affected Muc25 localization and secretion, as the Muc25ΔSP protein was localized to the membrane in sporozoites and remained within intracellular life stages. In addition, a Muc25 knockout strain (ΔMuc25) was easily generated, indicating that Muc25 is not critical for parasite survival. However, the ΔMuc25 strain showed reduced growth in HCT-8 cells, and the survival time was prolonged in GKO mice infected with ΔMuc25 compared to those infected with Muc25-3 HA. Transcriptome analysis revealed that ΔMuc25 parasites caused less damage to host cells in vitro than Muc25-3 HA parasites. Taken together, these data provide evidence for the export of a Cryptosporidium protein to host microvilli and demonstrate that such manipulation of the host cell response may be involved in parasite pathogenesis.

Eimeria spp. are common coccidian parasites of a wide range of vertebrates, causing diarrhoea, poor weight gain and significant mortality in domestic animals and birds. However, there is a paucity of genomic data on these important pathogens. Of the 11 common Eimeria species in rabbits, only Eimeria stiedae invades biliary epithelial cells rather than the intestine, and the determinants of coccidian tissue tropism remain unclear. In this study, we sequenced the genomes of five common rabbit Eimeria species, including E. stiedae , Eimeria flavescens , Eimeria intestinalis , Eimeria magna and Eimeria media . Comparative genomic analysis reveals that the genome of E. stiedae appears more compact than the genomes of intestinal Eimeria species. It shows reductions in the numbers of rhoptry proteins, dense granule proteins, microneme adhesive repeats and TA4 surface antigens, suggesting that surface and invasion-associated proteins may be involved in the tissue tropism of Eimeria spp. In addition, E. stiedae -specific motifs are identified in a cluster of hypothetical surface antigens. These data provide not only new insights into the biological characteristics of coccidia but also valuable resources for functional research and drug and vaccine development.

Zoonotic cryptosporidiosis is caused primarily by Cryptosporidium parvum. Within C. parvum, the IIa and IId zoonotic subtype families are the most prevalent. The IId subtype family has emerged in China in recent years, causing outbreaks of cryptosporidiosis in dairy calves. The majority of infection studies have been conducted with IIa subtypes, while the pathogenicity of IId subtypes remains poorly understood. In this study, two IId isolates (IIdA20G1-HLJ and IIdA20G1-HB) from dairy farms in China were used to infect neonatal dairy calves, with a IIa isolate (IIaA17G2R1-Waterborne) from the USA employed as a control. The present study investigated the clinical, parasitological, and pathological characteristics of infected calves. The results demonstrated significantly greater intensity and duration of oocyst shedding in IId-infected calves than in control calves. In addition, the IIdA20G1-HLJ isolate induced peak oocyst shedding of 4.3×107 oocysts per gram of feces (OPG) in calves, with oocyst shedding over 106 OPGs at 5–9 d post-infection. In contrast, the IIaA17G2R1-Waterbrone isolate induced a lower peak oocyst shedding with 8.7×106 OPGs, and oocyst shedding with over 106 OPGs occurred at 2–4 d post-infection. Furthermore, calves infected with the two IId isolates presented more severe clinical signs and 20–25

Cryptosporidium serpentis is a host-adapted Cryptosporidium species that infects ectothermic reptiles, including snakes and lizards. In addition, C. serpentis is one of the few Cryptosporidium species that parasitize the stomach of the hosts. To date, the genomic data for gastric Cryptosporidium species are exclusively available for Cryptosporidium andersoni and Cryptosporidium muris, both of which infect warm-blooded hosts. To enhance our understanding of genomic features of C. serpentis, we undertook the Illumina-based whole genome sequencing of four C. serpentis isolates, and generated the first C. serpentis draft genome of 9.11 Mb in 292 scaffolds and with an N50 of 102,002 bp. Comparative genomic analyses revealed that C. serpentis shares high similarity in genomic characteristics with C. andersoni and C. muris, including genomic identity, gene content, and gene organization. We observed aerobic metabolism and a partial conventional electron transport chain in the three gastric species, which are absent in the intestinal Cryptosporidium species. This divergent metabolism of the three gastric species is likely associated with their parasitism in the stomach of hosts. However, genes involved in purine salvage pathway in C. parvum are lost in C. serpentis as well as in other gastric Cryptosporidium species. These specific gene losses may provide more insights into the anabolic capabilities of Cryptosporidium. A significant reduction in the number of multi-copy genes potentially involving the secretory pathogenesis determinants was observed in C. serpentis, which was also found in the other species with a narrow host range. Compared with the other gastric species, 19 unique genes and 67 divergent orthogroups with low identity were identified in C. serpentis. These genes/orthogroups could provide potential insights into investigating the host preference of C. serpentis and further biological studies should be performed on these genes.

BACKGROUND:Cryptosporidium parvum is a protozoan pathogen that causes moderate to severe diarrhea in both humans and animals. Calcium-dependent protein kinases (CDPKs) are attractive drug targets against cryptosporidiosis given their critical role in the life cycle of Cryptosporidium spp. and their absence in human and animal hosts. METHODOLOGY/PRINCIPAL FINDINGS:We used CRISPR-Cas9 technology to endogenously tag the CpCDPK2A gene in C. parvum IIdA20G1-HLJ strain with the hemagglutinin (HA) epitope and to delete the CpCDPK2A gene. An immunofluorescence assay was performed to localize the CpCDPK2A expression in the tagged strain and a luciferase assay was performed to compare growth rates of the tagged and deletion strains in vitro. Oocyst shedding, parasite load, villus length/crypt height ratio and survival of infected mice were used to evaluate the function of CpCDPK2A in vivo. The results revealed that CpCDPK2A was expressed in all the intracellular developmental stages, especially in the motile stages of sporozoites and merozoites. While CpCDPK2A is dispensable, deletion of the gene significantly reduced the growth of late asexual and sexual stages in vitro. In an interferon-γ knockout mouse model, gene deletion of CpCDPK2A reduced oocyst shedding by 25-fold and increased survival of infected mice. CONCLUSIONS/SIGNIFICANCE:These observations suggest that CpCDPK2A may contribute to both asexual and sexual replication of C. parvum and may be a potential target to block the transmission of this important zoonotic pathogen.

Giardia duodenalis is a common enteric pathogen in humans and animals, with the disease giardiasis being a zoonosis. Currently, little is known about the occurrence and age patterns of G. duodenalis genotypes and subtypes in calves. To examine the infection dynamics of G. duodenalis in dairy calves, cross-sectional and longitudinal studies were conducted using PCR and DNA sequencing tools. In the cross-sectional study, 467 fecal samples were obtained from dairy farms in Guangdong Province, China, and age-associated differences in the infection rate of G. duodenalis were observed. In the longitudinal cohort study, 47 calves on Farm 5 were followed from birth to nine months of age. The shedding of G. duodenalis cysts began on day four, peaked at five weeks of age, and maintained at high levels until three months of age. Most calves continued to excrete low numbers of cysts intermittently after three months. Based on the bg locus, assemblages E (n = 486), A (n = 13), B (n = 5) and D (n = 2) were identified. Overall, there were two infection peaks of assemblage E at 3-13 weeks and 20-23 weeks of age, leading to a cumulative incidence of 100% (47/47) for this dominant assemblage. The average duration of cyst shedding for assemblage E in the cohort study was 4.0 ± 2.1 weeks for the initial infection and 2.1 ± 0.5 weeks for the subsequent one. The intensity of cyst shedding was markedly high during the initial infection but was subsequently lower in the second infection. Within assemblage E, high genetic diversity was observed, with E3 (234/486) and E5 (113/486) being the dominant subtypes. In addition, zoonotic assemblages A and B were predominantly identified in calves during the second peak of infection. Among the assemblage A-positive samples, subtypes A5, A8 and A1 were found at the bg, gdh, and tpi loci, respectively, all belonging to the AI sub-assemblage. This is the first longitudinal study of the natural history of G. duodenalis in dairy calves using genotyping and subtyping tools, and we established a standardized qPCR curve to assess the intensity of G. duodenalis infection. The results provide new perspectives on the complexity and dynamics of G. duodenalis infection in these animals.