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

Abstract

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.

Keywords: Blastocystis; MinION; NGS; USA; ribosomal RNA; subtypes; white-tailed deer.

Figure 1

Relative abundance of reads (%) of each subtype present in the 71 Blastocystis-positive white-tailed deer (WTD) samples arranged by number of subtypes present from lowest to highest.

Figure 2

Phylogenetic relationships among Blastocystis full-length sequences generated in the present study (novel subtypes are represented with a black filled circle and other subtypes with an unfilled circle) and representative reference sequences of all accepted subtypes. Proteromonas lacertae was used as outgroup taxon to root the tree. Analysis was conducted by a neighbor-joining method. Genetic distances were calculated using the Kimura two-parameter model. This analysis involved 70 nucleotide sequences, and there were a total of 1950 positions in the final dataset. Bootstrap values lower than 50% are not displayed.

Figure 3

Phylogenetic relationships among Blastocystis barcoding region sequences generated in the present study (novel subtypes are represented with a black filled circle and other subtypes with an unfilled circle) and representative reference sequences of all accepted subtypes. Proteromonas lacertae was used as outgroup taxon to root the tree. Analysis was conducted by a neighbor-joining method. Genetic distances were calculated using the Kimura two-parameter model. This analysis involved 70 nucleotide sequences, and there were a total of 590 positions in the final dataset. Bootstrap values lower than 50% are not displayed.

Figure 4

Phylogenetic relationships among Blastocystis Santin region sequences generated in the present study (novel subtypes represented are with a black filled circle and other subtypes with an unfilled circle) and representative reference sequences of all accepted subtypes. Proteromonas lacertae was used as outgroup taxon to root the tree. Analysis was conducted using a neighbor-joining method. Genetic distances were calculated using the Kimura two-parameter model. This analysis involved 70 nucleotide sequences, and there were a total of 571 positions in the final dataset. Bootstrap values lower than 50% are not displayed.