Identification of multiple Blastocystis subtypes in domestic animals from Colombia using amplicon-based next generation sequencing

Authors: HIGUERA, ADRIANA - Universidad Del Rosario, Columbia; HERRERA, GIOVANNY - Universidad Del Rosario, Columbia; JIMENEZ, PAULA - Universidad Del Rosario, Columbia; GARCIA, DIEGO - Universidad Del Rosario, Columbia; PULIDO, MARTIN - Collaborator; BULLA, DIANA - Collaborator; PINILLA, JUAN - Collaborator; MORENO, ANDRES - Collaborator; Maloney, Jenny; Santin-Duran, Monica; RAMIREZ, JUAN - Universidad Del Rosario, Columbia

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/22/2021
Publication Date: 8/24/2021
Citation: Higuera, A., Herrera, G., Jimenez, P., Garcia, D.J., Pulido, M., Bulla, D.M., Pinilla, J.C., Moreno, A., Maloney, J.G., Santin, M., Ramirez, J.D. 2021. Identification of multiple Blastocystis subtypes in domestic animals from Colombia using amplicon-based next generation sequencing. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2021.732129.
DOI: https://doi.org/10.3389/fvets.2021.732129

Interpretive Summary: Blastocystis, a common enteric protist parasite of humans and many other animals, has been divided into subtypes based on variability within the small subunit ribosomal RNA (SSU rRNA) gene. Few studies that include molecular characterization in animal hosts in the Americas have been conducted. However, such information is crucial to understand the epidemiology, animal reservoirs of zoonotic subtypes, and transmission dynamics of Blastocystis infections. In this study, PCR and next generation amplicon sequencing were used to determine the occurrence and subtypes of Blastocystis in farm and companion animals from Colombia. Of the 118 fecal samples included in this study 81.4% were Blastocystis-positive. Molecular characterization detected eleven subtypes, 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 samples. Novel subtype 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 constitutes the first study in Colombia using NGS to molecularly characterized subtypes of Blastocystis in farm animals. A great diversity of subtypes was observed in domestic animals including subtypes previously identified in humans indicating that farm animals could play a role in transmission to humans. This information will be useful to other scientists, veterinarians, and public health agencies in understanding the current status of taxonomy, epidemiology, zoonotic potential, and public health importance of Blastocystis.

Technical 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 pig, minipig, cow, dog, horse, goat, 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 samples. Novel subtype 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 characterized 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.