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Abstract Blastocystis is a common microeukaryotic intestinal parasite in humans and other animal hosts globally. However, no large-scale longitudinal study has ever been conducted for Blastocystis. To understand patterns of infection prevalence and subtype diversity and their relationship with host age, we have conducted the most comprehensive longitudinal study of Blastocystis infection ever performed. Dairy calves from a herd located in Maryland, USA, were followed from birth through 24 months of age, and 990 individual fecal samples from 30 calves were collected over the study period, representing three age groups (pre-weaned, post-weaned, and heifer). All samples were screened for Blastocystis via PCR, and subtype determination was performed using next-generation amplicon sequencing. Associations between age group and infection status were assessed using logistic regression analyses. Blastocystis infection prevalence increased with time, significant associations were observed between age groups and infection risk, and a cumulative prevalence of 100% was observed among the study population during the 24-month period. Thirteen previously reported subtypes (ST1-6, ST10, ST14, ST21, ST23–26) and one potentially novel subtype were observed. Diversity within ST10 supports the need for division of the subtype into new subtype designations. Associations between subtype and age group were explored, and relationships between subtypes and infection chronicity are described. While subtype diversity increased with age in the study population, distinct patterns of individual subtype prevalence and chronicity were observed, supporting the importance of subtype discrimination in studies of host infection and disease. The data from this study represent a significant advance in our understanding of Blastocystis infection dynamics within a single host population over time and can be used to inform future studies of Blastocystis epidemiology in both humans and other animal hosts. Graphical Abstract

Three recent studies of Blastocystis epidemiology in mammalian hosts identified four novel sequences that appeared to share B. lapemi as the most similar sequence. However, full-length ssu rRNA gene sequences were not available to confirm the validity of these new subtypes. In the present study, Nanopore MinION sequencing was used to obtain full-length reference sequences for each of the new subtypes. Additionally, phylogenetic analyses and pairwise distance comparisons were performed to confirm the validity of each of these new subtypes. We propose that the novel sequences described in this study should be assigned the subtype designations ST35-ST38. The full-length reference sequences of ST35-ST38 will assist in accurate sequence descriptions in future studies of Blastocystis epidemiology and subtype diversity.

Abstract Background The intestinal parasite Blastocystis is found in humans and animals around the world. It is spread through the consumption of contaminated food and water and has been associated with a variety of intestinal symptoms. Blastocystis is one of the most common intestinal parasites in humans, yet its prevalence and distribution in humans in North America is not well characterized. Methods Next-generation amplicon sequencing of a region of the Blastocystis SSU rRNA gene was applied to DNA extracted from fecal specimens obtained from 182 inhabitants of a rural population in Mexico to characterize Blastocystis prevalence, subtype distribution, and intra-host subtype diversity in humans. Results Of the 182 samples tested in this study, 68.1% (124) contained one or more Blastocystis subtypes. Subtype 3 was the most common subtype observed and was found in 81.5% of the positive samples. Subtype 1, 16.9% of the positive samples, and subtype 2, 17.7% of the positive samples, were also found in this population. Mixed infections were observed in 13.7% of the positive samples. In this population, the odds of having Blastocystis increased in adulthood (> 15 years; OR: 1.72, P < 0.0001), and the odds of having subtype 1 increased in the presence of farm animals (OR: 1.51, P = 0.03). The odds of having subtype 1, subtype 2, or a mixed infection decreased in the presence of cement flooring (OR: − 1.61, P = 0.005; OR: − 1.14, P = 0.03; OR: − 1.48, P = 0.02) possibly indicating socioeconomic factors are involved in the risk of acquiring one of these subtypes. Conclusions These data contribute to our understanding of the epidemiology of Blastocystis infection in humans and can be used to shape future studies which aim to better characterize the transmission pathways and health outcomes of Blastocystis infections.

Blastocystis is a common intestinal protist in humans and animals worldwide. Wild and domestic animals are thought to be reservoirs of Blastocystis subtypes that also infect humans. There are limited studies on the prevalence and subtype distribution of Blastocystis in horses. In this study, 185 fecal samples were collected from horses (1 month to 17 years of age) in four regions of Colombia (Sabana de Bogotá, Costa Atlántica, Llanos Orientales, and Bogotá D.C.). Blastocystis presence and subtypes were determined by PCR and next generation amplicon sequencing. Eighty-one (43.8%) horses were positive for Blastocystis, with positive horses in all four regions. Molecular characterization identified 12 Blastocystis subtypes, 10 known subtypes (ST1, ST3–ST6, ST10, ST14, ST25, ST26), and 2 novel subtypes (ST33 and ST34). The validity of the novel subtypes was confirmed via phylogenetic and pairwise distance analyses of the full-length SSU rRNA gene sequences. Mixed subtype infections were common (55.6% of Blastocystis-positive horses). ST10 was the most prevalent subtype, present in 82.8% of Blastocystis-positive horses. Potentially zoonotic subtypes were identified in 88.9% of the Blastocystis-positive horses. This constitutes the most comprehensive study of Blastocystis in horses. Our findings indicate that horses harbor potentially zoonotic subtypes and could contribute to the transmission of Blastocystis to humans.

A targeted metabolomic analysis was performed on tissues derived from pigs fed diets supplemented with white button mushrooms (WBM) to determine the effect on the liver and brain metabolome. Thirty-one pigs were fed a grower diet alone or supplemented with either three or six servings of freeze-dried WBM for six weeks. Tissue metabolomes were analyzed using targeted liquid chromatography-mass spectrometry (LC-MS) combined with chemical similarity enrichment analysis (ChemRICH) and correlated to WBM-induced changes in fecal microbiome composition. Results indicated that WBM can differentially modulate metabolites in liver, brain cortex and hippocampus of healthy pigs. Within the glycero-phospholipids, there was an increase in alkyl-acyl-phosphatidyl-cholines (PC-O 40:3) in the hippocampus of pigs fed six servings of WBM. A broader change in glycerophospholipids and sphingolipids was detected in the liver with a reduction in several lipid species in pigs fed both WBM diets but with an increase in amino acids known as precursors of neurotransmitters in the cortex of pigs fed six servings of WBM. Metabolomic changes were positively correlated with increased abundance of Cryomorphaceae, Lachnospiraceae, Flammeovirgaceae and Ruminococcaceae in the microbiome suggesting that WBM can also positively impact tissue metabolite composition.

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims to estimate the prevalence and molecular diversity of Blastocystis sp. and E. bieneusi in cattle faecal samples (n = 336) in the province of Álava, Northern Spain. Initial detection of Blastocystis and E. bieneusi was carried out by polymerase chain reaction (PCR) and Sanger sequencing of the small subunit (ssu) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host Blastocystis subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the ssu rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with Blastocystis sp. and E. bieneusi were observed in 32.1% (108/336, 95% CI: 27.2–37.4%) and 0.6% (2/336, 95% CI: 0.0–1.4%) of the cattle faecal samples examined, respectively. Sanger sequencing produced ambiguous/unreadable sequence data for most of the Blastocystis isolates sequenced. NGS allowed the identification of 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates involved mixed infections of 2–8 STs in a total of 31 different combinations. The two E. bieneusi sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that Blastocystis mixed subtype infections are extremely frequent in cattle in the study area. NGS was particularly suited to discern underrepresented subtypes or mixed subtype infections that were undetectable or unreadable by Sanger sequencing. The presence of zoonotic Blastocystis ST1, ST3, and ST5, and E. bieneusi BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.

Blastocystis is a gastrointestinal protist frequently reported in humans and animals worldwide. Wildlife populations, including deer, may serve as reservoirs of parasitic diseases for both humans and domestic animals, either through direct contact or through contamination of food or water resources. However, no studies of the occurrence and subtype distribution of Blastocystis in wildlife populations have been conducted in the United States. PCR and next generation amplicon sequencing were used to determine the occurrence and subtypes of Blastocystis in white-tailed deer (Odocoileus virginianus). Blastocystis was common, with 88.8% (71/80) of samples found to be positive. Twelve subtypes were identified, ten previously reported (ST1, ST3, ST4, ST10, ST14, ST21, and ST23–ST26) and two novel subtypes (ST30 and ST31). To confirm the validity of ST30 and ST31, MinION sequencing was used to obtain full-length SSU rRNA gene sequences, and phylogenetic and pairwise distance analyses were performed. ST10, ST14, and ST24 were the most commonly observed subtypes. Potentially zoonotic subtypes ST1, ST3, or ST4 were present in 8.5% of Blastocystis-positives. Mixed subtype infections were common (90.1% of Blastocystis-positives). This study is the first to subtype Blastocystis in white-tailed deer. White-tailed deer were found to be commonly infected/colonized with a wide diversity of subtypes, including two novel subtypes, zoonotic subtypes, and subtypes frequently reported in domestic animals. More studies in wildlife are needed to better understand their role in the transmission of Blastocystis.

The Blastocystis subtype ST10 has been recognized to contain a great deal of diversity at the sequence level, potentially indicating the presence of multiple new STs within the clade. However, the data needed to validate these new STs were not available. To help resolve this diversity, full‐length small subunit (SSU) rRNA gene reference sequences were generated using Oxford Nanopore MinION long‐read sequencing from 21 samples representing multiple domestic and wild hosts and geographic regions and covering the sequence diversity previously described using fragments of the SSU rRNA gene. Phylogenetic and pairwise distance analyses were used to compare full‐length sequences of the SSU rRNA gene generated in this study with all other valid STs of Blastocystis. We present data supporting the division of ST10/ST23 cluster into five subtypes, ST10, ST23, and three new subtypes with the proposed ST designations of ST42, ST43, and ST44. As the host range of Blastocystis continues to expand with new subtypes and new hosts being frequently identified, the reference sequences provided in this study will assist in accurate sequence classification and help to clarify the epidemiology of this common intestinal microeukaryote. [ABSTRACT FROM AUTHOR]

Blastocystis is a common microeukaryotic intestinal parasite in humans and other animal hosts globally. However, no large-scale longitudinal study has ever been conducted for Blastocystis. To understand patterns of infection prevalence and subtype diversity and their relationship with host age, we have conducted the most comprehensive longitudinal study of Blastocystis infection ever performed. Dairy calves from a herd located in Maryland, USA, were followed from birth through 24 months of age, and 990 individual fecal samples from 30 calves were collected over the study period, representing three age groups (pre-weaned, post-weaned, and heifer). All samples were screened for Blastocystis via PCR, and subtype determination was performed using next-generation amplicon sequencing. Associations between age group and infection status were assessed using logistic regression analyses. Blastocystis infection prevalence increased with time, significant associations were observed between age groups and infection risk, and a cumulative prevalence of 100% was observed among the study population during the 24-month period. Thirteen previously reported subtypes (ST1-6, ST10, ST14, ST21, ST23–26) and one potentially novel subtype were observed. Diversity within ST10 supports the need for division of the subtype into new subtype designations. Associations between subtype and age group were explored, and relationships between subtypes and infection chronicity are described. While subtype diversity increased with age in the study population, distinct patterns of individual subtype prevalence and chronicity were observed, supporting the importance of subtype discrimination in studies of host infection and disease. The data from this study represent a significant advance in our understanding of Blastocystis infection dynamics within a single host population over time and can be used to inform future studies of Blastocystis epidemiology in both humans and other animal hosts. [ABSTRACT FROM AUTHOR]

We examined gene expression of whole blood cells (WBC) from 11 healthy elderly volunteers participating on a Phase I open label study before and after oral treatment with Lactobacillus rhamnosus GG-ATCC 53103 (LGG)) using RNA-sequencing (RNA-Seq). Elderly patients (65-80 yrs) completed a clinical assessment for health status and had blood drawn for cellular RNA extraction at study admission (Baseline), after 28 days of daily LGG treatment (Day 28) and at the end of the study (Day 56) after LGG treatment had been suspended for 28 days. Treatment compliance was verified by measuring LGG-DNA copy levels detected in host fecal samples. Normalized gene expression levels in WBC RNA were analyzed using a paired design built within three analysis platforms (edgeR, DESeq2 and TSPM) commonly used for gene count data analysis. From the 25,990 transcripts detected, 95 differentially expressed genes (DEGs) were detected in common by all analysis platforms with a nominal significant difference in gene expression at Day 28 following LGG treatment (FDR

Three recent studies of Blastocystis epidemiology in mammalian hosts identified four novel sequences that appeared to share B. lapemi as the most similar sequence. However, full-length ssu rRNA gene sequences were not available to confirm the validity of these new subtypes. In the present study, Nanopore MinION sequencing was used to obtain full-length reference sequences for each of the new subtypes. Additionally, phylogenetic analyses and pairwise distance comparisons were performed to confirm the validity of each of these new subtypes. We propose that the novel sequences described in this study should be assigned the subtype designations ST35-ST38. The full-length reference sequences of ST35-ST38 will assist in accurate sequence descriptions in future studies of Blastocystis epidemiology and subtype diversity. [ABSTRACT FROM AUTHOR]