The first Cyclospora cayetanensis lineage A genome from an isolate from Mexico.

BACKGROUND: Cyclospora cayetanensis is a protozoan parasite that causes intestinal illness in humans worldwide. Despite its global distribution, most genomic data for C. cayetanensis has been obtained from isolates collected in the United States, leaving genetic variability among globally distributed isolates underexplored. RESULTS: In the present study, the genome of an isolate of C. cayetanensis obtained from a child with diarrhea living in Mexico was sequenced and assembled. Evaluation of the assembly using a lineage typing system recently developed by the Centers for Disease Control and Prevention revealed that this isolate is lineage A. CONCLUSIONS: Given that the only other whole genome assembly available from Mexico was classified as lineage B, the data presented here represent an important step in expanding our knowledge of the diversity of C. cayetanensis isolates from Mexico at the genomic level.

Division of Blastocystis ST10 into three new subtypes: ST42-ST44.

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.

Exploring Blastocystis genetic diversity in rural schoolchildren from Colombia using next-generation amplicon sequencing reveals significant associations between contact with animals and infection risk.

Blastocystis is a common intestinal protist with a global distribution in humans and many other animals. Yet, the status of Blastocystis as a pathogen, the risk factors associated with its transmission, and its zoonotic potential remain ill-defined. Here, we explored subtype (ST) diversity and potential risk factors for Blastocystis infection in 98 children from Apulo, Colombia. Samples were screened for Blastocystis via PCR, and ST identification was performed through next-generation amplicon sequencing (NGS). Associations between the presence of Blastocystis and individual STs and sociodemographic variables were assessed via logistic regression analyses. Seventy-one samples (72.4%) were Blastocystis-positive, and NGS revealed the presence of five STs (ST1-ST5). ST1, ST2, and ST3 were common and observed in nearly equal proportions (~ 40%), while samples with ST4 (1.4%) and ST5 (5.6%) were comparatively rare. The presence of mixed STs in the same sample was also common (28.2%). Comparisons among children within the same household identified that shared ST profiles were common, but diversity within family units was also observed. Logistic regression analyses returned significant associations between the presence of Blastocystis, individual subtypes, or mixed subtypes for several variables. Intriguingly, the presence of animals was one of the most common significant associations. Taken together, these data represent an important step forward in understanding both the potential routes and risk factors that may influence Blastocystis transmission and will be useful in shaping future studies which seek to clarify the relationships between STs, pathogenicity, and zoonotic transmission.

Next-generation sequencing reveals wide genetic diversity of Blastocystis subtypes in chickens including potentially zoonotic subtypes.

Blastocystis sp. is an intestinal protist parasite commonly found in the feces of humans and animals worldwide. Blastocystis exhibits extensive genetic diversity and has been identified in humans and a variety of animals including other mammals and birds. Blastocystis subtypes do not exhibit strict host specificity which raises the possibility of zoonotic transmission through either direct contact or fecal contamination of food or water. However, reports detailing the subtypes and prevalence of Blastocystis in avian species are limited. Therefore, this study investigated the presence of Blastocystis in chickens by molecular characterization of the small subunit rRNA (SSU rRNA) gene. Fecal samples from 130 chickens were collected from local markets in Uberlândia and Belo Horizonte in the state of Minas Gerais, Brazil. To detect and identify subtypes of Blastocystis, a next-generation amplicon sequencing protocol was used. Forty-four of the 130 (33.8%) chickens examined were positive for Blastocystis. Blastocystis subtypes ST6 (23/130; 17.7%), ST7 (43/130; 33.1%), ST10 (1/130; 0.8%), ST14 (5/130; 3.8%), ST25 (1/130; 0.8%), and a novel subtype (ST29) (2/130; 1.5%) were observed. A nanopore sequencing strategy was used to obtain the near full-length SSU rRNA gene nucleotide sequence and validate novel subtype ST29. Mixed infections containing multiple subtypes were common and identified in 63.6% of Blastocystis-positive chickens. All positive samples contained one or both potentially zoonotic subtypes ST6 and ST7. The prevalence of Blastocystis in chickens was high, and molecular characterization mostly identified subtypes previously found in humans. Thus, chickens may be a source of human infection and environmental contamination.

The Ossabaw Pig Is a Suitable Translational Model to Evaluate Dietary Patterns and Coronary Artery Disease Risk.

Background: Animal models that mimic diet-induced human pathogenesis of chronic diseases are of increasing importance in preclinical studies. The Ossabaw pig is an established model for obesity-related metabolic disorders when fed extreme diets in caloric excess. Objective: To increase the translational nature of this model, we evaluated the effect of diets resembling 2 human dietary patterns, the Western diet (WD) and the Heart Healthy Diet (HHD), without or with atorvastatin (-S or +S) therapy, on cardiometabolic risk factors and atherosclerosis development. Methods: Ossabaw pigs (n = 32; 16 boars and 16 gilts, aged 5-8 wk) were randomized according to a 2 × 2 factorial design into 4 groups (WD-S, WD+S, HHD-S, and HHD+S) and were fed the respective diets for 6 mo. The WD (high in saturated fat, cholesterol, and refined grain) and the HHD (high in unsaturated fat, whole grain, and fruit and vegetables) were isocaloric [38% of energy (%E) from fat, 47%E from carbohydrate, and 15%E from protein]. Body composition was determined by using dual-energy X-ray absorptiometry, serum fatty acid (FA) profiles by gas chromatography, cardiometabolic risk profile by standard procedures, and degree of atherosclerosis by histopathology. Results: Serum FA profiles reflected the predominant dietary FA. Pigs fed the WD had 1- to 4-fold higher concentrations of LDL cholesterol, non-HDL cholesterol, HDL cholesterol, high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor α (TNF-α), alkaline phosphatase (ALP), and alanine aminotransferase (ALT) compared with HHD-fed pigs (all P-diet < 0.05). Statin therapy significantly lowered concentrations of LDL cholesterol (-39%), non-HDL cholesterol (-38%), and triglycerides (-6%) (P-statin < 0.02). A greater degree of atheromatous changes (macrophage infiltration, foam cells, fatty streaks) and lesion incidence was documented in the coronary arteries (P-diet < 0.05), as well as 2- to 3-fold higher lipid deposition in the aortic arch or thoracic aorta of WD- compared with HHD-fed pigs (P-diet < 0.001). Conclusions: Ossabaw pigs manifested a dyslipidemic and inflammatory profile accompanied by early-stage atherosclerosis when fed a WD compared with an HHD, which was moderately reduced by atorvastatin therapy. This phenotype presents a translational model to examine mechanistic pathways of whole food-based dietary patterns on atherosclerosis development.

Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay.

A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.

Flavanol-Enriched Cocoa Powder Alters the Intestinal Microbiota, Tissue and Fluid Metabolite Profiles, and Intestinal Gene Expression in Pigs.

BACKGROUND: Consumption of cocoa-derived polyphenols has been associated with several health benefits; however, their effects on the intestinal microbiome and related features of host intestinal health are not adequately understood. OBJECTIVE: The objective of this study was to determine the effects of eating flavanol-enriched cocoa powder on the composition of the gut microbiota, tissue metabolite profiles, and intestinal immune status. METHODS: Male pigs (5 mo old, 28 kg mean body weight) were supplemented with 0, 2.5, 10, or 20 g flavanol-enriched cocoa powder/d for 27 d. Metabolites in serum, urine, the proximal colon contents, liver, and adipose tissue; bacterial abundance in the intestinal contents and feces; and intestinal tissue gene expression of inflammatory markers and Toll-like receptors (TLRs) were then determined. RESULTS: O-methyl-epicatechin-glucuronide conjugates dose-dependently increased (P< 0.01) in the urine (35- to 204-fold), serum (6- to 186-fold), and adipose tissue (34- to 1144-fold) of pigs fed cocoa powder. The concentration of 3-hydroxyphenylpropionic acid isomers in urine decreased as the dose of cocoa powder fed to pigs increased (75-85%,P< 0.05). Compared with the unsupplemented pigs, the abundance ofLactobacillusspecies was greater in the feces (7-fold,P= 0.005) and that ofBifidobacteriumspecies was greater in the proximal colon contents (9-fold,P= 0.01) in pigs fed only 20 or 10 g cocoa powder/d, respectively. Moreover, consumption of cocoa powder reducedTLR9gene expression in ileal Peyer's patches (67-80%,P< 0.05) and mesenteric lymph nodes (43-71%,P< 0.05) of pigs fed 2.5-20 g cocoa powder/d compared with pigs not supplemented with cocoa powder. CONCLUSION: This study demonstrates that consumption of cocoa powder by pigs can contribute to gut health by enhancing the abundance ofLactobacillusandBifidobacteriumspecies and modulating markers of localized intestinal immunity.

A Single Meal Containing Raw, Crushed Garlic Influences Expression of Immunity- and Cancer-Related Genes in Whole Blood of Humans.

BACKGROUND: Preclinical and epidemiologic studies suggest that garlic intake is inversely associated with the progression of cancer and cardiovascular disease. OBJECTIVE: We designed a study to probe the mechanisms of garlic action in humans. METHODS: We conducted a randomized crossover feeding trial in which 17 volunteers consumed a garlic-containing meal (100 g white bread, 15 g butter, and 5 g raw, crushed garlic) or a garlic-free control meal (100 g white bread and 15 g butter) after 10 d of consuming a controlled, garlic-free diet. Blood was collected before and 3 h after test meal consumption for gene expression analysis in whole blood. Illumina BeadArray was used to screen for genes of interest, followed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on selected genes. To augment human study findings, Mono Mac 6 cells were treated with a purified garlic extract (0.5 µL/mL), and mRNA was measured by qRT-PCR at 0, 3, 6, and 24 h. RESULTS: The following 7 genes were found to be upregulated by garlic intake: aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT), hypoxia-inducible factor 1α (HIF1A), proto-oncogene c-Jun (JUN), nuclear factor of activated T cells (NFAT) activating protein with immunoreceptor tyrosine-based activation motif 1 (NFAM1), oncostatin M (OSM), and V-rel avian reticuloendotheliosis viral oncogene homolog (REL). Fold-increases in mRNA transcripts ranged from 1.6 (HIF1A) to 3.0 (NFAM1) (P < 0.05). The mRNA levels of 5 of the 7 genes that were upregulated in the human trial were also upregulated in cell culture at 3 and 6 h: AHR, HIF1A, JUN, OSM, and REL. Fold-increases in mRNA transcripts in cell culture ranged from 1.7 (HIF1A) to 12.1 (JUN) (P < 0.01). OSM protein was measured by ELISA and was significantly higher than the control at 3, 6, and 24 h (24 h: 19.5 ± 1.4 and 74.8 ± 1.4 pg/mL for control and garlic, respectively). OSM is a pleiotropic cytokine that inhibits several tumor cell lines in culture. CONCLUSION: These data indicate that the bioactivity of garlic is multifaceted and includes activation of genes related to immunity, apoptosis, and xenobiotic metabolism in humans and Mono Mac 6 cells. This trial is registered at clinicaltrials.gov as NCT01293591.

Characterization of fecal microbiota of children with diarrhea in 2 locations in Colombia.

OBJECTIVES: Diarrhea is a leading cause of mortality and morbidity in children younger than 5 years in impoverished regions of the world. Our aim was to compare the fecal microbiota of healthy children with that of children with clinical diarrhea in a population from a tropical highland in Colombia, South America. Our hypothesis was that a reduced prevalence of inherent Lactobacillus and Bifidobacterium species would be associated with enteric viral and bacterial pathogens. METHODS: Children between 1 and 5 years of age from 2 different locations were evaluated for presence of clinical diarrhea. Nucleic acid, isolated from fecal samples, was used to determine by molecular protocols the abundance of inherent bacterial species and presence of enteric pathogens compared with clinically healthy children. The effect of host demographic factors on incidence of diarrhea was also analyzed. RESULTS: : The composition of the fecal microbiota was affected by host demographic factors: age, health status, location, and sex. In partial support of our hypothesis, the relative abundance of commensal Bifidobacterium and Lactobacillus species was inversely correlated with incidence of diarrhea regardless of location. CONCLUSIONS: Our results suggested that changes in fecal microbiota composition of children with clinical diarrhea are associated with certain demographic factors that should be considered before designing a prophylactic intervention. Delivery of certain Lactobacillus species and Bifidobacterium species or a diet rich in bifidogenic components that promote growth of Bifidobacterium species could provide a prophylactic effect to ameliorate the effect of diarrhea in children at risk.

A longitudinal study of Blastocystis in dairy calves from birth through 24 months demonstrates dynamic shifts in infection rates and subtype prevalence and diversity by age.

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.

Molecular characterization of Blastocystis and Entamoeba of muskoxen and sheep in Greenland.

Molecular characterisation of endobionts that are shared among human and non-human hosts can help shed light on the epidemiology and inform studies that aim to unravel the role of these organisms in health and disease. Two of the most common of shared endobionts include the single-celled intestinal protists Blastocystis and Entamoeba. Here, we present the first known data on genetic diversity and host specificity of these two genera in Greenland. Faecal DNA samples from 243 muskoxen and 44 sheep were submitted to metabarcoding of nuclear small subunit ribosomal DNA. Entamoeba- and Blastocystis-specific sequences were clustered, and consensus sequences were subjected to taxonomic query. Using MinION-based sequencing, near-complete nuclear small subunit ribosomal DNA sequences were obtained from four faecal samples. Of the 243 muskox samples, 180 (74%) and 19 (8%) were positive for Blastocystis and Entamoeba, respectively. Forty (91%) and six (14%) of the 44 sheep samples were positive for Blastocystis and Entamoeba, respectively. Blastocystis subtypes (ST) 10, 14, 21, 24-26, and a novel subtype (ST40) were identified. Colonisation by more than one subtype was common. ST40 was common in muskoxen but limited to Northeast Greenland. Entamoeba bovis and the E. bovis-associated ribosomal lineages (RL) 1 and 8 were found, and three conditional lineages (CL) 3, 4, and 10 were confirmed; CL10 was promoted to RL12. Several novel lineages were identified, all of which were linked to the E. bovis complex. In conclusion, Blastocystis was far more common than Entamoeba and found in approximately three of every four animals; both can be considered common colonisers of large herbivorous mammals in Greenland. Multiple subtypes/lineages of both genera were commonly observed, some of which were novel, but most of which are seen in many other parts of the world.

A hybrid sequencing and assembly strategy for generating culture free Giardia genomes.

Giardia duodenalis is a pathogenic intestinal protozoan parasite of humans and many other animals. Giardia duodenalis is found throughout the world, and infection is known to have adverse health consequences for human and other mammalian hosts. Yet, many aspects of the biology of this ubiquitous parasite remain unresolved. Whole genome sequencing and comparative genomics can provide insight into the biology of G. duodenalis by helping to reveal traits that are shared by all G. duodenalis assemblages or unique to an individual assemblage or strain. However, these types of analyses are currently hindered by the lack of available G. duodenalis genomes, due, in part, to the difficulty in obtaining the genetic material needed to perform whole genome sequencing. In this study, a novel approach using a multistep cleaning procedure coupled with a hybrid sequencing and assembly strategy was assessed for use in producing high quality G. duodenalis genomes directly from cysts obtained from feces of two naturally infected hosts, a cat and dog infected with assemblage A and D, respectively. Cysts were cleaned and concentrated using cesium chloride gradient centrifugation followed by immunomagnetic separation. Whole genome sequencing was performed using both Illumina MiSeq and Oxford Nanopore MinION platforms. A hybrid assembly strategy was found to produce higher quality genomes than assemblies from either platform alone. The hybrid G. duodenalis genomes obtained from fecal isolates (cysts) in this study compare favorably for quality and completeness against reference genomes of G. duodenalis from cultured isolates. The whole genome assembly for assemblage D is the most contiguous genome available for this assemblage and is an important reference genome for future comparative studies. The data presented here support a hybrid sequencing and assembly strategy as a suitable method to produce whole genome sequences from DNA obtained from G. duodenalis cysts which can be used to produce novel reference genomes necessary to perform comparative genomics studies of this parasite.

Diversity of Blastocystis Subtypes in Horses in Colombia and Identification of Two New Subtypes.

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.

Enhanced detection of Giardia duodenalis mixed assemblage infections in pre-weaned dairy calves using next generation sequencing.

Giardia duodenalis is one of the most common parasitic causes of gastrointestinal illness in humans worldwide with widespread infections in mammalian hosts. It frequently infects cattle, producing a high number of cysts. Cattle can harbor both host-adapted assemblage E and human pathogenic assemblages A and B. Previous studies have demonstrated that conventional molecular methods lack the sensitivity required for detecting mixed infections and that the occurrence of mixed infections in cattle are likely underestimated. To investigate the presence of mixed assemblage infections in cattle, 2539 pre-weaned dairy calves from the United Stated were screened for the presence of G. duodenalis using molecular tools. Next generation amplicon sequencing (NGS) was then performed for a subset of around 30% of positive samples (n = 314) and the ability of NGS and Sanger sequencing to detect mixed assemblage infections was compared. The overall prevalence of G. duodenalis in pre-weaned dairy calves in the sample using PCR was high (1013/2539; 39.9%). Molecular genotyping identified only assemblage A and E, with assemblage E as the predominant assemblage. Out of the 314 samples examined by both Sanger and NGS, 9 samples (2.9%) were identified as mixed A/E infections by Sanger while NGS identified 56 samples (17.8%), which was six-times more mixed infections compared with Sanger sequencing. NGS demonstrated superior sensitivity to Sanger in detecting assemblages present in low abundances. The percentage of mixed A/E infections found in the sampled dairy calves was higher than was hypothesized using values from the literature. This underestimation could be present in the wider cattle population as well, though further exploration would be needed to verify that claim. These findings highlight the advantages of NGS application in molecular epidemiological studies of Giardia. To better understand Giardia epidemiology, establish routes of transmission, and assess the potential role of cattle and other animals as a source of environmental contamination with zoonotic assemblages it is necessary to uncover mixed assemblage infections.

Correction: Solano-Aguilar et al. Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention. Nutrients 2021, 13, 4350.

There was an error in the original publication [...].

Identification and Molecular Characterization of Four New Blastocystis Subtypes Designated ST35-ST38.

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.

An Illumina MiSeq-Based Amplicon Sequencing Method for the Detection of Mixed Parasite Infections Using the Blastocystis SSU rRNA Gene as an Example.

Parasite mixed infections remain a relatively unexplored field in part due to the difficulties of unraveling complex mixtures of parasite DNA using classical methods of sequencing. Next-generation amplicon sequencing (NGS) is a powerful tool for exploring mixed infections of multiple genetic variants of the same parasite in clinical, environmental (water or soil), or food samples. Here, we provide a method for NGS-based detection of mixed parasite infections which uses the Blastocystis SSU rRNA gene as an example and includes steps for parasite concentration, DNA extraction, sequencing library preparation, and bioinformatic analysis.

Wide Genetic Diversity of Blastocystis in White-Tailed Deer (Odocoileus virginianus) from Maryland, USA.

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 Effects of Consuming White Button Mushroom Agaricus bisporus on the Brain and Liver Metabolome Using a Targeted Metabolomic Analysis.

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.

Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention.

A study was conducted to determine the effects of a diet supplemented with fruits and vegetables (FV) on the host whole blood cell (WBC) transcriptome and the composition and function of the intestinal microbiome. Nine six-week-old pigs were fed a pig grower diet alone or supplemented with lyophilized FV equivalent to half the daily recommended amount prescribed for humans by the Dietary Guideline for Americans (DGA) for two weeks. Host transcriptome changes in the WBC were evaluated by RNA sequencing. Isolated DNA from the fecal microbiome was used for 16S rDNA taxonomic analysis and prediction of metabolomic function. Feeding an FV-supplemented diet to pigs induced differential expression of several genes associated with an increase in B-cell development and differentiation and the regulation of cellular movement, inflammatory response, and cell-to-cell signaling. Linear discriminant analysis effect size (LEfSe) in fecal microbiome samples showed differential increases in genera from Lachnospiraceae and Ruminococcaceae families within the order Clostridiales and Erysipelotrichaceae family with a predicted reduction in rgpE-glucosyltransferase protein associated with lipopolysaccharide biosynthesis in pigs fed the FV-supplemented diet. These results suggest that feeding an FV-supplemented diet for two weeks modulated markers of cellular inflammatory and immune function in the WBC transcriptome and the composition of the intestinal microbiome by increasing the abundance of bacterial taxa that have been associated with improved intestinal health.