Identification of Multiple Blastocystis Subtypes in Domestic Animals From Colombia Using Amplicon-Based Next Generation Sequencing
Fecha de publicación: 24/08/2021

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.

Genetic diversity and population structure of Rhipicephalus sanguineus sensu lato across different regions of Colombia
Fecha de publicación: 23/08/2021

There has been a long-standing debate over the taxonomic status of Rhipicephalus sanguineus sensu lato. Different studies worldwide have reported the occurrence of different well-defined lineages, in addition to Rhipicephalus sanguineus sensu stricto. To date, there are very few studies examining the diverse aspects of this tick in Colombia. We assessed the population structure and genetic diversity of R. sanguineus s.l. in eight departmental regions across Colombia.

Identifying HLA DRB1-DQB1 alleles associated with Chlamydia trachomatis infection and in silico prediction of potentially-related peptides
Fecha de publicación: 18/06/2021

HLA class II (HLA-II) genes’ polymorphism influences the immune response to Chlamydia trachomatis (Ct), it is considered a sexually transmitted infection. However, associations between HLA-II alleles and Ct-infection have been little explored in humans; this study was thus aimed at determining HLA-DRB1-DQB1 alleles/haplotypes’ effect on Ct-infection outcome in a cohort of Colombian women. Cervical sample DNA was used as template for detecting Ct by PCR and typing HLA-DRB1-DQB1 alleles/haplotypes by Illumina MiSeq sequencing. Survival models were adjusted for identifying the alleles/haplotypes’ effect on Ct-outcome; bioinformatics tools were used for predicting secreted bacterial protein T- and B-cell epitopes. Sixteen HLA-DRB1 alleles having a significant effect on Ct-outcome were identified in the 262 women analysed. DRB1*08:02:01G and DRB1*12:01:01G were related to infection-promoting events. Only the DQB1*05:03:01G allele related to clearance/persistence events was found for HLA-DQB1. HLA-DRB1 allele homozygous women were associated with events having a lower probability of clearance and/or early occurrence of persistence. Twenty-seven peptides predicted in silico were associated with protective immunity against Ct; outer membrane and polymorphic membrane protein-derived peptides had regions having dual potential for being T- or B-cell epitopes. This article describes HLA-DRB1-DQB1 alleles/haplotypes related to Ct-infection resolution and the peptides predicted in silico which might probably be involved in host immune response. The data provides base information for developing future studies leading to the development of effective prevention measures against Ct-infection.

Two 20-Residue-Long Peptides Derived from Plasmodium vivax Merozoite Surface Protein 10 EGF-Like Domains Are Involved in Binding to Human Reticulocytes
Fecha de publicación: 01/02/2021

Plasmodium parasites’ invasion of their target cells is a complex, multi-step process involving many protein-protein interactions. Little is known about how complex the interaction with target cells is in Plasmodium vivax and few surface molecules related to reticulocytes’ adhesion have been described to date. Natural selection, functional and structural analysis were carried out on the previously described vaccine candidate P. vivax merozoite surface protein 10 (PvMSP10) for evaluating its role during initial contact with target cells. It has been shown here that the recombinant carboxyl terminal region (rPvMSP10-C) bound to adult human reticulocytes but not to normocytes, as validated by two different protein-cell interaction assays. Particularly interesting was the fact that two 20-residue-long regions (³⁸⁸DKEECRCRANYMPDDSVDYF⁴⁰⁷ and ⁴¹⁵KDCSKENGNCDVNAECSIDK⁴³⁴) were able to inhibit rPvMSP10-C binding to reticulocytes and rosette formation using enriched target cells. These peptides were derived from PvMSP10 epidermal growth factor (EGF)-like domains (precisely, from a well-defined electrostatic zone) and consisted of regions having the potential of being B- or T-cell epitopes. These findings provide evidence, for the first time, about the fragments governing PvMSP10 binding to its target cells, thus highlighting the importance of studying them for inclusion in a P. vivax antimalarial vaccine.

Babesia Bovis Ligand-Receptor Interaction: AMA-1 Contains Small Regions Governing Bovine Erythrocyte Binding
Fecha de publicación: 13/01/2021

Apical membrane antigen 1 is a microneme protein which plays an indispensable role during Apicomplexa parasite invasion. The detailed mechanism of AMA-1 molecular interaction with its receptor on bovine erythrocytes has not been completely defined in Babesia bovis. This study was focused on identifying the minimum B. bovis AMA-1-derived regions governing specific and high-affinity binding to its target cells. Different approaches were used for detecting ama-1 locus genetic variability and natural selection signatures. The binding properties of twelve highly conserved 20-residue-long peptides were evaluated using a sensitive and specific binding assay based on radio-iodination. B. bovis AMA-1 ectodomain structure was modelled and refined using molecular modelling software. NetMHCIIpan software was used for calculating B- and T-cell epitopes. The B. bovis ama-1 gene had regions under functional constraint, having the highest negative selective pressure intensity in the Domain I encoding region. Interestingly, B. bovis AMA-1-DI (¹⁰⁰YMQKFDIPRNHGSGIYVDLG¹¹⁹ and ¹²⁰GYESVGSKSYRMPVGKCPVV¹³⁹) and DII (³⁰²CPMHPVRDAIFGKWSGGSCV³²¹)-derived peptides had high specificity interaction with erythrocytes and bound to a chymotrypsin and neuraminidase-treatment sensitive receptor. DI-derived peptides appear to be exposed on the protein’s surface and contain predicted B- and T-cell epitopes. These findings provide data (for the first-time) concerning B. bovis AMA-1 functional subunits which are important for establishing receptor-ligand interactions which could be used in synthetic vaccine development.