A new chromosome-level genome assembly and annotation of Cryptosporidium meleagridis.

Cryptosporidium spp. are medically and scientifically relevant protozoan parasites that cause severe diarrheal illness in infants and immunosuppressed populations as well as animals. Although most human Cryptosporidium infections are caused by C. parvum and C. hominis, there are several other human-infecting species including C. meleagridis, which is commonly observed in developing countries. Here, we polished and annotated a long-read genome sequence assembly for C. meleagridis TU1867, a species which infects birds and humans. The genome sequence was generated using a combination of whole genome amplification (WGA) and long-read Oxford Nanopore Technologies sequencing. The assembly was then polished with Illumina data. The chromosome-level genome assembly is 9.2 Mbp with a contig N50 of 1.1 Mb. Annotation revealed 3,923 protein-coding genes. A BUSCO analysis indicates a completeness of 96.6% (n=446), including 430 (96.4%) single-copy and 1 (0.224%) duplicated apicomplexan conserved gene(s). The new C. meleagridis genome assembly is nearly gap-free and provides a valuable new resource for the Cryptosporidium community and future studies on evolution and host-specificity.

Changes in CpG Methylation of the Vitellogenin 1 Promoter in Adult Male Zebrafish after Exposure to 17α-Ethynylestradiol.

Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to developmental disorders and other pathologies. The synthetic estrogen 17α-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20 ng/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n ≥ 4/group/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6 h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post-EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;43:1547-1556. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Improved Equine Fecal Microbiome Characterization Using Target Enrichment by Hybridization Capture.

GITDs are among the most common causes of death in adult and young horses in the United States (US). Previous studies have indicated a connection between GITDs and the equine gut microbiome. However, the low taxonomic resolution of the current microbiome sequencing methods has hampered the identification of specific bacterial changes associated with GITDs in horses. Here, we have compared TEHC, a new approach for 16S rRNA gene selection and sequencing, with conventional 16S rRNA gene amplicon sequencing for the characterization of the equine fecal microbiome. Both sequencing approaches were used to determine the fecal microbiome of four adult horses and one commercial mock microbiome. Our results show that TEHC yielded significantly more operational taxonomic units (OTUs) than conventional 16S amplicon sequencing when the same number of reads were used in the analysis. This translated into a deeper and more accurate characterization of the fecal microbiome when the samples were sequenced with TEHC according to the relative abundance analysis. Alpha and beta diversity metrics corroborated these findings and demonstrated that the microbiome of the fecal samples was significantly richer when sequenced with TEHC compared to 16S amplicon sequencing. Altogether, our study suggests that the TEHC strategy provides a more extensive characterization of the fecal microbiome of horses than the current alternative based on the PCR amplification of a portion of the 16S rRNA gene.

"Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing".

Multiple Displacement Amplification (MDA) outperforms conventional PCR in long fragment and whole genome amplification which makes it attractive to couple with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for genome sequence assembly using Oxford Nanopore Technologies (ONT) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Cryptosporidium meleagridis, Staphylococcus aureus, Enterococcus faecium, and Escherichia coli, with the ability to generate high-quality data from samples starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size-increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.

Long-term gut colonization with ESBL-producing Escherichia coli in participants without known risk factors from the southeastern United States.

We evaluated gut carriage of extended spectrum beta lactamase producing Enterobacteriaceae (ESBL-E) in southeastern U.S. residents without recent in-patient healthcare exposure. Study enrollment was January 2021-February 2022 in Athens, Georgia, U.S. and included a diverse population of 505 adults plus 50 child participants (age 0-5). Based on culture-based screening of stool samples, 4.5% of 555 participants carried ESBL-Es. This is slightly higher than reported in studies conducted 2012-2015, which found carriage rates of 2.5-3.9% in healthy U.S. residents. All ESBL-E confirmed isolates (n=25) were identified as Escherichia coli. Isolates belonged to 11 sequence types, with 48% classified as ST131. Ninety six percent of ESBL-E isolates carried a blaCTX-M gene. Isolated ESBL-Es frequently carried virulence genes as well as multiple classes of antibiotic resistance genes. Long-term colonization was common, with 64% of ESBL-E positive participants testing positive when rescreened three months later. One participant yielded isolates belonging to two different E. coli sequence types that carried blaCTX-M-1 genes on near-identical plasmids, suggesting intra-gut plasmid transfer. Isolation of E. coli on media without antibiotics revealed that ESBL-E. coli typically made up a minor fraction of the overall gut E. coli population, although in some cases they were the dominant strain. ESBL-E carriage was not associated with a significantly different stool microbiome composition. However, some microbial taxa were differentially abundant in ESBL-E carriers. Together, these results suggest that a small subpopulation of US residents are long-term, asymptomatic carriers of ESBL-Es, and may serve as an important reservoir for community spread of these ESBL genes.