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Identification of Multiple Blastocystis Subtypes in Domestic Animals From Colombia Using Amplicon-Based Next Generation Sequencing.

ABSTRACT: Blastocystis is frequently reported in fecal samples from animals and humans worldwide, and a variety of subtypes (STs) have been observed in wild and domestic animals. In Colombia, few studies have focused on the transmission dynamics and epidemiological importance of Blastocystis in animals. In this study, we characterized the frequency and subtypes of Blastocystis in fecal samples of domestic animals including pigs, minipigs, cows, dogs, horses, goats, sheep, and llama from three departments of Colombia. Of the 118 fecal samples included in this study 81.4% (n = 96) were positive for Blastocystis using a PCR that amplifies a fragment of the small subunit ribosomal RNA (SSU rRNA) gene. PCR positive samples were sequenced by next generation amplicon sequencing (NGS) to determine subtypes. Eleven subtypes were detected, ten previously reported, ST5 (50.7%), ST10 (47.8%), ST25 (34.3%), ST26 (29.8%), ST21 (22.4%), ST23 (22.4%), ST1 (17.9%), ST14 (16.4%), ST24 (14.9%), ST3 (7.5%), and a novel subtype, named ST32 (3.0%). Mixed infection and/or intra -subtype variations were identified in most of the samples. Novel ST32 was observed in two samples from a goat and a cow. To support novel subtype designation, a MinION based sequencing strategy was used to generate the full-length of the SSU rRNA gene. Comparison of full-length nucleotide sequences with those from current valid subtypes supported the designation of ST32. This is the first study in Colombia using NGS to molecularly characterize subtypes of Blastocystis in farm animals. A great diversity of subtypes was observed in domestic animals including subtypes previously identified in humans. Additionally, subtype overlap between the different hosts examined in this study were observed. These findings highlight the presence of Blastocystis subtypes with zoonotic potential in farm animals indicating that farm animals could play a role in transmission to humans.

SUBMITTER: Higuera A

PROVIDER: S-EPMC8421793 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundApplication of next-generation sequencing (NGS) to genomic DNA extracted from sewage offers a unique and cost-effective opportunity to study the genetic diversity of intestinal parasites. In this study, we used amplicon-based NGS to reveal and differentiate several common luminal intestinal parasitic protists, specifically Entamoeba, Endolimax, Iodamoeba, and Blastocystis, in sewage samples from Swedish treatment plants.Materials and methodsInfluent sewage samples were subject to gradient centrifugation, DNA extraction and PCR-based amplification using three primer pairs designed for amplification of eukaryotic nuclear 18S ribosomal DNA. PCR products were sequenced using ILLUMINA® technology, and resulting sequences were annotated to species and subtype level using the in-house BION software, sequence clustering, and phylogenetic analysis.ResultsA total of 26 samples from eight treatment plants in central/southern Sweden were analysed. Blastocystis sp. and Entamoeba moshkovskii were detected in all samples, and most samples (n = 20) were positive for Entamoeba coli. Moreover, we detected Entamoeba histolytica, Entamoeba dispar, Entamoeba hartmanni, Endolimax nana, and Iodamoeba bütschlii in 1, 11, 4, 10, and 7 samples, respectively. The level of genetic divergence observed within E. nana and E. moshkovskii was 20.2% and 7.7%, respectively, across the ~400-bp region studied, and two clades of E. moshkovskii were found. As expected, Blastocystis sp. subtypes 1-4 were present in almost all samples; however, ST8 was present in 10 samples and was the only subtype not commonly found in humans that was present in multiple samples.ConclusionsEntamoeba and Blastocystis were identified as universal members of the "sewage microbiome". Blastocystis sp. ST8, which has been rarely reported in humans, was a very common finding, indicating that a hitherto unidentified but common host of ST8 contributed to the sewage influent. The study also provided substantial new insight into the intra-generic diversity of Entamoeba and Endolimax.

Project description:COVID-19 is caused by SARS-CoV-2, several virulent variants of which have emerged since 2019. More than 529 million people have been infected, and at least 6 million have died. Our aim was to develop a fast, accurate, low-cost method for detecting and identifying newly emerging variants of concern (VOCs) that could pose a global threat. The 341-bp DNA sequence of a specific region of the SARS-CoV-2's spike protein was amplified by a one-step PCR on RNA samples from 46 patients. The product was sequenced using next-generation sequencing (NGS). DNA sequences from seven genomes, the original Wuhan isolate and six different representative variants obtained from the GISAID website, were used as references. Complete whole-genome sequences from local isolates were also obtained from the GISAID website, and their RNA was used for comparison. We used an amplicon-based NGS method (termed VOC-NGS) for genotyping and successfully identified all 46 samples. Fifteen (32.6%) were like the original isolate. Twenty-seven were VOCs: nine (19.5%) Alpha, eight (19%) Delta, six (14%) Beta, and four (8.7%) Omicron. Two were variants of interest (VOI): one (2%) Kappa and one (2%) Zeta. Two samples were mixtures of two variants, one of Alpha and Beta and one of Alpha and Delta. The Spearman correlation between whole-genome sequencing (WGS) and VOC-NGS was significant (P < 0.001) with perfect agreement (Kappa = 0.916) for 36/38 (94.7%) samples with VOC-NGS detecting all the known VOCs. Genotyping by VOC-NGS enables rapid screening of high-throughput clinical samples that includes the identification of VOCs and mixtures of variants, at lower cost than WGS. IMPORTANCE The manuscript described SARS-Cov-2 genotyping by VOC-NGS, which presents an ideal balance of accuracy, rapidity, and cost for detecting and globally tracking VOCs and some VOI of SARS-CoV-2. A large number of clinical samples can be tested together. Rapid introduction of new mutations at a specific site of the spike protein necessitates efficient strain detection and identification to enable choice of treatment and the application of vaccination, as well as planning public health policy.

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Identification of Multiple Blastocystis Subtypes in Domestic Animals From Colombia Using Amplicon-Based Next Generation Sequencing.

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