Molecular Characterization of Blastocystis in Captive and Free-Ranging New World Primates, Platyrrhini

Abstract

Purpose:
Blastocystis species are widely distributed micro-eukaryote parasites found in both human and nonhuman primates. Despite having a global distribution, descriptions of Blastocystis subtype diversity in neotropical primates is largely limited to captive animals. The aim of this study was to molecularly characterize the presence of Blastocystis in free-ranging black-headed night monkeys, Aotus nigriceps, and to analyze Blastocystis heterogeneity in primates of the Parvorder Platyrrhini.

Methods:
We analyzed Blastocystis small sub-unit ribosomal DNA (SSUrDNA) from both A. nigriceps and Azara's night monkey, A. azarae boliviensis, in Southeastern Peru. We also included additional Blastocystis sequence from other neotropical primate studies to explore the distribution and host specificity of Blastocystis subtypes (ST) throughout the neotropics.

Results:
Thirteen percent of A. nigriceps samples were Blastocystis positive. Only ST8 was amplified in A. nigriceps and this partial DNA sequence was highly similar to ST8 sequence previously obtained from a human in Brazil. In our analysis of all available Blastocystis SSU sequences from primates of the Parvorder Platyrrhini, we found 15 monophyletic lineages corresponding to previously described subtypes ST1-ST10, ST12-15, and ST17.

Conclusions:
Blastocystis SSU sequences amplified from A. nigriceps fecal samples shared high sequence similarity to isolates found in several other neotropical primates, Alouatta palliata, A. caraya, Ateles fusciceps, and Lagothrix. Similar subtypes have been found in human and captive primates which supports the possibility of transmission when in close contact. Expanded sampling of sympatric neotropical primates in the wild will establish whether subtypes and clades are limited to taxonomic group or whether transmission occurs between overlapping species.

Full text

This is a post-peer-review, pre-copyedit version of an article published in Acta Parasitologica. The final
authenticated version is available online at: https://doi.org/10.1007/s11686-021-00397-1
Molecular characterization of Blastocystis in captive and free-ranging New World Primates, Platyrrhini
William D. Helenbrook1,2 *; Christopher M. Whipps2
1 Tropical Conservation Fund, Marietta, GA 30064, USA; 2Fish and Wildlife Disease Laboratory, State University
of New York College of Environmental Science and Forestry, Syracuse, NY, USA
*Corresponding author, wdhelenb@syr.edu, https://orcid.org/0000-0002-2706-3525
Abstract
Purpose
Blastocystis species are widely distributed micro-eukaryote parasites found in both human and nonhuman primates.
Despite having a global distribution, descriptions of Blastocystis subtype diversity in neotropical primates is largely
limited to captive animals. The aim of this study was to molecularly characterize the presence of Blastocystis in free-
ranging black-headed night monkeys, Aotus nigriceps, and to analyze Blastocystis heterogeneity in primates of the
Parvorder Platyrrhini.
Methods
We analyzed Blastocystis small sub-unit ribosomal DNA (SSUrDNA) from both A. nigriceps and Azara’s night
monkey, A. azarae boliviensis, in Southeastern Peru. We also included additional Blastocystis sequence from other
neotropical primate studies to explore the distribution and host specificity of Blastocystis subtypes (ST) throughout
the neotropics.
Results
Thirteen percent of A. nigriceps samples were Blastocystis positive. Only ST8 was amplified in A. nigriceps and this
partial DNA sequence was highly similar to ST8 sequence previously obtained from a human in Brazil. In our
analysis of all available Blastocystis SSU sequences from primates of the Parvorder Platyrrhini, we found fifteen
monophyletic lineages corresponding to previously described subtypes ST1-ST10, ST12-15, and ST17.

Conclusions
Blastocystis SSU sequences amplified from A. nigriceps fecal samples shared high sequence similarity to isolates
found in several other neotropical primates, Alouatta palliata, A. caraya, Ateles fusciceps, and Lagothrix. Similar
subtypes have been found in human and captive primates which supports the possibility of transmission when in
close contact. Expanded sampling of sympatric neotropical primates in the wild will establish whether subtypes and
clades are limited to taxonomic group or whether transmission occurs between overlapping species.
Keywords: Blastocystis; zoonotic disease; transmission; primates; Aotus
Acknowledgements
We would like to thank the following organizations for hosting us: Asociación para la Conservación de la Cuenca
Amazónica, Crees, ARCAmazon, and Taricaya Eco Reserve. And thank you to Jessica Suarez for her field support.
Declarations
Funding
Logistical support was provided by the Tropical Conservation Fund.
Conflicts of interest
Both authors state that there is no conflict of interest.
Authors’ contributions
Study was conceived by WDH. All field and laboratory research was conducted by WDH. First draft was written by
WDH. Figure construction and revisions contributed by CMW. Both authors read and approved the final
manuscript.
Ethics approval
We carried out data collection and analysis in accordance with the legal requirements of Peru, and with permission
of Villa Carmen and Los Amigos Biological Field Stations, MLC, ArcAmazon, and Taricaya.
Availability of data
The data that support the findings of this study are openly available in NCBI at https://www.ncbi.nlm.nih.gov.

Introduction
Blastocystis species hereafter referred to simply as Blastocystis are some of the most common protists
found in vertebrate hosts [1-2]. Molecular characterization of these parasites has uncovered a complex assortment of
subtypes (STs) representing unique phylogenetic lineages [3]. To date, there are 17 officially accepted subtypes and
another five which are likely valid. Several others are presumably invalid, including ST18-20 and ST22 [4]. The
presence of this parasite in wildlife is of great interest because the impact on host health is still poorly understood
and the potential for zoonotic transmission is still debated [5-6].
There is continued debate on the pathogenicity of Blastocystis, particularly in humans [7-8]. Subtype may
be important, with some evidence to suggest that specific parasite proteins and associated proteases may be
responsible for pathogenicity [9-10]. Certain subtypes are also more often associated with disease in humans than
others [11]. Blastocystis in wildlife and captive animals is often characterized as asymptomatic [12]. Yet researchers
continue to be interested in Blastocystis infections and genetic heterogeneity because it is considered a possible
reservoir for human infections [1]. All but one human subtype (ST9) has been found in other animals [13]. And,
ST1, ST2, and ST3 are widely found in both human and nonhuman primates [6]. We have previously characterized
the widespread occurrence of Blastocystis in sympatric human and a non-human neotropical primates but found no
evidence of zoonotic transmission [5]. For example, only ST8 was found in howler monkeys, Alouatta palliata, and
ST1-ST3 were found in humans living nearby.
There are currently eleven described night monkey species Aotus spp. that are distributed from Panama to
Argentina. Several studies have morphologically examined Blastocystis in Aotus species, but none of them have
molecularly characterized Blastocystis in any free-ranging night monkey species [13-15]. Therefore, the purpose of
our study was to i) characterize Blastocystis sequence data from free-ranging black-headed night monkeys Aotus
nigriceps and assess subtype, ii) examine the relationship of Blastocystis subtypes with other neotropical primates in
the Parvorder Platyrrhini, and iii) examine whether Blastocystis subtype is restricted to taxon or by geography.
Methods
We collected twenty-three fecal samples from free-ranging black-headed night monkeys, Aotus nigriceps,
in Southeastern Peru and a single sample from the Azara’s night monkey, Aotus azarae boliviensis. All samples
were molecularly characterized previously to confirm host species. We used primer sets modified from Babb et al.

[16] to amplify partial sequence from the cytochrome c oxidase subunit II (COII) gene of each monkey sample.
Twelve of the A. nigriceps samples were obtained from a single group at the Villa Carmen Biological Research
Station (VC: 12°53'39"S, 71°24'16"W), three samples from a single group at the Los Amigos Biological Station
(LA: 12°34'2"S, 70° 5'40.39"W), one sample from Crees - Manu Learning Centre, MLC (12°47′22″S 71°23′32″W)
in the Manu National Park Cultural Zone, and six samples from four groups at ARCAmazon - Las Piedras Amazon
Center (LPAC: 12°04′12″S 69°29′37″W). We collected a single sample from an A. azarae boliviensis group at
Taricaya Eco Reserve (12°31′08″S 68°58′48″W). Field sites are dispersed across the Madre de Dios region, with the
most distant sites approximately 266km apart. All samples are recovered from cotton or plastic netting placed below
nesting sites or under areas where groups were observed passing overhead in order to avoid soil contamination. Nets
were checked within twelve hours, at least twice a day. All samples were preserved in RNAlater™ Stabilization
Solution Qiagen Inc. Valencia, CA.
We used Quick-DNA Fecal/Soil Microbe Kits to extract parasite DNA following the protocol provided by
the manufacturer Zymo Research, Corp, Irvine, CA. Blastocystis small sub-unit ribosomal DNA SSUrDNA was
amplified from A. nigriceps fecal samples using previously published primers BH1F [17] and BHRDr [18]. DNA
was amplified by PCR in 50 μL reaction volumes in Taq 2× Master Mix New England Biolabs, Ipswich, MA, 0.25
μM of each primer, and 3 μL of template DNA using an C1000 Thermal Cycler BioRad for 40 cycles at 95 °C for 30
s, 53 °C for 60 s, and 68 °C for 60 s, preceded by an initial denaturation at 95 °C for 3 min, and followed by a final
extension at 68 °C for 7 min. Product amplification was initially evaluated by agarose gel electrophoresis. Negative
samples were run twice more with the same PCR regime.
Amplified products (356bp) were purified with E.Z.N.A. Cycle Pure Kit Omega Bio-Tek, Inc. Norcross,
Georgia, and purified PCR products sequenced in both directions using BH1F and BHRDr. The DNA Analysis
Facility at Yale ran sequences using the ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1, on the
3730xl DNA Analyzer Applied Biosystems, Thermo Fischer Scientific, Inc. with a 96-capillary 50cm array.
Sequences were screened in FinchTV 1.4.0 and edited in JalView to identify base-calling errors. We compared
forward and reverse sequence to ensure maximum sequence length and integrity. Blastocystis subtypes (ST) were
assigned using both BLASTn analysis of National Center for Biotechnology Information NCBI and the Blastocystis
18S and Sequence Typing (PubMLST) database (http://pubmlst.org/blastocystis/).

We analyzed thirty-two Blastocystis SSUrDNA sequences in our phylogenetic analysis, including three A.
nigriceps isolates which were amplified in this study and other homologous Blastocystis sequences described in
neotropical primates (Table 1; Fig. 1). Blastocystis SSUrDNA sequences were obtained from the National Center for
Biotechnology Information (NCBI), deposited by nine other studies and representing eleven known primate species.
We also included sequence from several other unrepresented subtypes for reference purposes, including from hosts
not found in South America: Pan troglodytes, Gorilla gorilla, Macaca nemestrina, Cercopithecus aethiops, Canus
lupus, and Ctenodactylus gundi. Homologous sequence was not available for ST11. Some Blastocystis sequences
from neotropical primates were not included in our analysis either because they represented non-overlapping regions
of SSUrDNA, the overlapping sequence was insufficiently small, or they were identical to other sequences from the
same host species. Most samples were from captive individuals with the exception of two wild samples (e.g.
JQ74945 and MH784444: Table 1). We used MAFFT (version 7.407_1) for sequence alignment and curated with
trimAI (version 1.41) [19]. The phylogenetic tree was built using distance-based inference of phylogenetic trees with
combined PhyML + SMS programs (version 1.8.1_1). Phylogenetic trees were arranged in TreeGraph2 [20], and
final editing was carried out using Adobe Illustrator Adobe Systems Inc. San Jose, California.
Results
Of 23 A. nigriceps samples that we screened, three were positive for Blastocystis 13.0% (Table 1). We
found a single infection at three sampled sites (VC, MLC, LA), and no infections were found at LPAC or Taricaya
(Fig. 1). A single A. azarae boliviensis sample we obtained was negative for Blastocystis. Prevalence varied from
8.3% at VC (1/12), to 100% at MLC (1/1), 33.3% at LA (1/3), 0% at LPAC (0/6) and 0% at Taricaya (0/1). All three
amplified Blastocystis sequences (MT509449-51) were identical across overlapping sequence (i.e. 451 bp in length)
and shared 99-100% identity with several other host species, including humans, dogs, and goats. A total of 28 Aotus
samples have been tested for Blastocystis – including the 24 from our study, and 4 from Valenca-Barbosa et al. [13]
– and only five of these were positive (17.9%), of which 20% were ST1 and 80% were ST8.
Combining our Aotus findings with nine previously published reports from neotropical primates, 160 out of
360 (47.5%) samples were Blastocystis positive (Table 1). This calculation does not include studies which fail to
specify overall sample size. Alouatta spp. were the mostly widely sampled neotropical primates (N=328) of which
one hundred fifty-one samples (46.0%) were positive with Blastocystis. Three percent (2.6%) of positive Alouatta

samples were ST1, 53.6% were positive for ST2, 1.3% were ST4 positive, 41.7% were positive with ST8 and a
single individual had ST3. Three studies recovered ten positive samples from Lagothrix lagotricha and described
ST1, ST2, ST3, and ST8. There were eight samples from Ateles – all of which were positive for Blastocystis (i.e.
ST1, ST2, ST3, ST8), and a sample with multiple subtypes. Of all studies included in our analysis, only four others
have assessed Blastocystis subtypes in free-ranging neotropical primates (Table 1).
The rooted phylogenetic tree resulted in fifteen monophyletic lineages corresponding to previously
described subtypes ST1-ST10, ST12-15, and ST17 (Fig. 1). Isolates from the same subtype clustered with each other
with good bootstrap support. The variability within subtype suggests inter-species variation with one exception –
ST8 sequence recovered from Alouatta palliata more closely resembled that from Lagothrix sp. than from Alouatta
caraya. Lagothrix sp. samples were recovered from laboratory animals and origin is unknown. Our newly amplified
samples clustered together with other ST8 samples, though the closest sequence similarity with a human sample
recovered in Brazil (MN585875).
Discussion
We molecularly characterized three Blastocystis positive samples in A. nigriceps which were all ST8 - a
subtype quite common in neotropical primates and relatively rare in humans [1; 21]. No other studies have
molecularly characterized Blastocystis in free-ranging Aotus species, though a single zoo study of an unnamed Aotus
species found both ST1 and ST8 [13]. Other studies have analyzed the presence of Blastocystis morphologically in
Aotus species [22]; yet expanded molecular analysis of the eleven currently recognized night monkey species is
needed to determine whether our results, along with those from Valenca-Barbosa et al. (2019) [13], are broadly
applicable to other members of the genus.
Small sub-unit ribosomal Blastocystis sequence amplified from A. nigriceps fecal samples shared high
sequence similarity to isolates found in several other neotropical primates, Alouatta palliata, A. caraya, Ateles
fusciceps, and Lagothrix (Fig. 1). Similar subtypes have been found in human and captive primates which support
the possibility of transmission when in close contact [e.g. 1, 23]. However, to our knowledge, there is no evidence to
suggest that free-ranging nonhuman primates are acting as a reservoir for Blastocystis in nearby communities. All of
the subtypes described in neotropical primates ST1, ST2, ST3, ST8 have been reported in both Old World primates
Catarrhini and in humans [1]. However, distinct subtype strains are likely found in people and nonhuman primates

indicating cryptic host specificity [5, 24]. For example, ST8 is rare in people but quite common in neotropical
primates. And yet, we previously found people living sympatrically with another nonhuman primate species
(Alouatta palliata) did not share the same subtypes [5].
The presence of Blastocystis subtypes in captive animals may not represent those found in the wild for
several reasons. First, only three subtypes have been described from free-ranging neotropical primates ST1, ST2 and
ST8, whereas ST1, ST2, ST3, ST4, and ST8 have been described in captive individuals. Four different primate
species housed at the Rio Zoo all shared the same subtype (ST8) with 100% sequence similarity MG280767-68 and
MG280770-71 [13]. These sequences are most similar to those recovered from other captive neotropical primates.
Similarly, sequence characterized in a captive Aotus individual MG280729 shared 100% similarity to ST1 sequence
obtained from other captive primates and people (GenBank accession numbers KY438944 and MN396274
respectively). Similar or identical Blastocystis sequence in primates housed near each other suggests the possibility
of transmission. ST1 is found in people, though molecular testing of animals in captivity – along with caretakers –
would be necessary to confirm whether these sequences are identical.
Reports in humans suggest an association of Blastocystis with irritable bowel syndrome and other
gastrointestinal problems such as diarrhea [25]. However, we are not aware of any evidence of this in wild
nonhuman primates and a single report in an Old World monkey species describes the pathogenic potential of
Blastocystis [26]. None of the samples that we collected in this study – particularly those that were Blastocystis
positive - would suggest gastrointestinal symptoms such as watery or loose stool. Continued monitoring and
expansion of sampling in free-ranging nonhuman primates along with general health examinations, when possible,
would help determine the pathogenic potential of this microparasite.
Conclusions
This study found low prevalence of Blastocystis ST8 in free-ranging black-headed night monkeys
compared to several other neotropical primate species. ST1-ST3, and ST8 varied according to host species and
whether in captivity or free-ranging. SSUrDNA sequence provided sufficient variability to distinguish phylogenetic
relationships between Blastocystis subtypes. ST8 in A. nigriceps shared high sequence similarity to other free-
ranging neotropical primate species. However, none of these primates are sympatric with A. nigriceps. Instead, ST8
is widespread, and clustering of isolates appears largely correlated to taxonomic group. Expanded sampling of

sympatric neotropical primates will establish whether subtypes and clades are limited to taxonomic group or whether
transmission occurs between species living in proximity to one another in the wild.
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Fig. 1 Phylogenetic analysis of Blastocystis amplified sequence from SSU-rDNA gene. GenBank accession numbers
and host names are shown at terminal nodes. Values at nodes represent aLRT SH-like supports. *Zoological
specimens

Table 1 Comprehensive description of Blastocystis small sub-unit ribosomal DNA molecular sequence described in Platyrrhini species. Number of each subtype
out of total positive samples
Neotropical
primate host
NCBI Locus
Location
Free-
ranging
Sample
size N
Positive
samples
ST1
N
ST2
N
ST3
N
ST4
N
ST8
N
Mixed
References
Alouatta caraya
MH784454
Brazil
Yes
1
1
1
[27]
Al. carayaa
Zoo
No
Unknown
5
5
[1]
Al. palliata
KM374608-9;
KM374610
Ecuador
Yes
96
58
58
[5]
Al. palliata
KT591768; KT591824-
30; KT591835;
KT591837; KT591840-
47; KT591849; KT591854
Mexico
Yes
59
23
1
21
1
[29]
Al. pigra
KT591769-75; KT591777-
KT591809; KT591811-23;
KT591832-34; KT591836;
KT591838-39; KT591848;
KT591850-53
Mexico
Yes
166
64
3
59
2
[29]
Al. seniculus
MH784444; MH784446
Zoo
No
2
2
1
1
[27]
Al. sp.
MG280771
Zoo
2
1
1
[13]
Al. sp.
KF002511; KF002512
Colombia
Yes
2
2
2
[28]
Aotus sp.
MG280729; MG280767
Zoo
No
4
2
1
1
[13]
Ao. nigriceps
MT509449-51
Peru
Yes
23
3
3
Present study
Ao. azarae
boliviensis
Not applicable
Peru
Yes
1
0
Present study
Ateles sp.
MG280768
Zoo
1
1
1
[13]

Ateles spp.a
Zoo
No
Unknown
6
3
2
1
[1]
At. belzebuth
MH784443
Zoo
No
1
1
1
[27]
At. fusciceps
MH784453
Zoo
No
1
1
1
[27]
Callitrix
jacchus
HQ909921b
Zoo
No
Unknown
1
1
[24]
Lagothrix sp.
DQ232783c; DQ232785;
DQ232795 c -96 c;
DQ232799-800;
DQ232806-7
Laboratory
No
Unknown
7
2
2
1
3†
[18]
L. lagotricha
HQ909858b; HQ909943b;
HQ910018b
Zoo
No
Unknown
3
3
[24]
L. lagotricha
MG280770
Zoo
2
1
1
[13]
Leontopithecus
chrysomelas
KR259427- KR259429
Zoo
No
Unknown
3
3
[6]
Pithecia sp.a
Zoo
No
Unknown
1
1
[1]
Pithecia
pithecia
KR259491
Zoo
No
Unknown
1
1
[6]
Saguinus sp.a
Zoo
No
Unknown
2
1
1
[1]
aSequence not available in NCBI
bNon-homologous sequence excluded from phylogenetic analysis
cSequences originally misrepresented as ST4 DQ232795-96, DQ232783, DQ232842 but recharacterized as ST8