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.

Molecular Characterization of Blastocystis sp. in Camelus bactrianus in Northwestern China

“…ST24 was a less common subtype with the prevalence of 10.4% in this study, and it has also been found in cattle and deer in the United States [ 16 , 38 ], and in birds in Brazil [ 14 ]. Another less common subtype ST25 is identified in 6.0% of samples, which has also been identified in llama in Colombia [ 17 ], and in cattle in Spain [ 63 ], and chickens in Brazil [ 18 ]. In our study, both ST26 and ST21 were found as the lowest frequency (0.8%).…”

“…In our study, both ST26 and ST21 were found as the lowest frequency (0.8%). ST26, previously identified in dairy cattle [ 38 ] and deer [ 16 ] in the United States, and in goat, sheep, and cattle in Colombia [ 17 ], was found in one camel under two years in Inner Mongolia. ST21, which has been reported in dairy cattle and white-tailed deer in the United States [ 16 , 38 ], and in goats, llama and sheep in Colombia [ 17 ], and waterbuck in China [ 39 ], was also identified in one camel under two years in Inner Mongolia.…”

“…ST26, previously identified in dairy cattle [ 38 ] and deer [ 16 ] in the United States, and in goat, sheep, and cattle in Colombia [ 17 ], was found in one camel under two years in Inner Mongolia. ST21, which has been reported in dairy cattle and white-tailed deer in the United States [ 16 , 38 ], and in goats, llama and sheep in Colombia [ 17 ], and waterbuck in China [ 39 ], was also identified in one camel under two years in Inner Mongolia. Notably, the potentially novel subtype identified in our study was commonly identified in all geographic areas and age groups ( Table 1 and Table 2 ).…”

“…Currently, Blastocystis sp. was classified into 32 subtypes (STs) based on intra-variations within sequences of the SSU rRNA gene [ 14 , 15 , 16 , 17 , 18 ]. By reviewing subtypes ST1–ST26, it is recognized that ST18–ST20 and ST22 are not recommended in future analysis due to the possible existence of molecular chimaera within these subtypes [ 15 ].…”

Presence and genetic diversity of enteric protists in captive and semi-captive non-human primates in côte d’Ivoire, Sierra Leone, and Peru

“…At least 28 subtypes (ST) have been identified within Blastocystis sp. ( Higuera et al, 2021 ; Maloney and Santin, 2021 ; Maloney et al, 2021 ) of which ST1‒ST5, ST7‒ST11, ST13, ST15, and ST19 have been previously reported in NHP ( Hublin et al, 2021 ). More than 500 E. bieneusi genotypes have been defined to date grouped in 11 phylogenetic groups ( Li et al, 2019 ).…”