Molecular identification of sarcocysts from tissue of fallow deer (Dama dama) farmed in the open pasture system based on ssu rRNA gene

Abstract

PurposeSarcocystis spp. are protozoan parasites of livestock which also infect birds, lower vertebrates and mammals, including man. Wild and domestic ruminants such as red deer, roe deer, fallow deer, cattle, sheep and goats may act as intermediate hosts for many Sarcocystis species, some of which are significant pathogens causing sarcocystosis in livestock and humans. The purpose of the present study was to determine the prevalence of Sarcocystis species in fallow deer farmed in an open pasture system.Methods
Samples of heart and oesophagus tissue taken from five fallow deer were examined by light microscope for the presence of sarcocysts. Genomic DNA was extracted from individual sarcocysts. ssu rRNA was successfully amplified using their DNA as templates.ResultsAnalysis of the ssu rRNA identified the presence of two S. morae sarcocysts in the heart tissue; similarly, S. gracilis sarcocysts were identified in the heart and oesophagus, and Sarcocystis sp. most closely related to S. linearis and S. taeniata were detected in oseophagus.Conclusions
These findings confirm the presence of Sarcocystis spp. in farmed fallow deer in Poland; however, more molecular studies are needed.

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Acta Parasitologica
https://doi.org/10.2478/s11686-019-00159-0
ORIGINAL PAPER
Molecular identication ofsarcocysts fromtissue offallow deer (Dama
dama) farmed intheopen pasture system based onssu rRNA gene
WładysławCabaj1 · JustynaBień‑Kalinowska1· KatarzynaGoździk1· KatarzynaBasałaj1·
ŻanetaSteiner‑Bogdaszewska1· MarekBogdaszewski1· BożenaMoskwa1
Received: 23 October 2019 / Accepted: 14 December 2019
© The Author(s) 2020
Abstract
Purpose Sarcocystis spp. are protozoan parasites of livestock which also infect birds, lower vertebrates and mammals,
including man. Wild and domestic ruminants such as red deer, roe deer, fallow deer, cattle, sheep and goats may act as
intermediate hosts for many Sarcocystis species, some of which are significant pathogens causing sarcocystosis in livestock
and humans. The purpose of the present study was to determine the prevalence of Sarcocystis species in fallow deer farmed
in an open pasture system.
Methods Samples of heart and oesophagus tissue taken from five fallow deer were examined by light microscope for the
presence of sarcocysts. Genomic DNA was extracted from individual sarcocysts. ssu rRNA was successfully amplified using
their DNA as templates.
Results Analysis of the ssu rRNA identified the presence of two S. morae sarcocysts in the heart tissue; similarly, S. gracilis
sarcocysts were identified in the heart and oesophagus, and Sarcocystis sp. most closely related to S. linearis and S. taeniata
were detected in oseophagus.
Conclusions These findings confirm the presence of Sarcocystis spp. in farmed fallow deer in Poland; however, more
molecular studies are needed.
Keywords Fallow deer· Sarcocystis spp.· Tissue cysts· Sequencing
Introduction
Sarcocystis spp. are obligate heteroxenous protozoan para-
sites (Apicomplexa, Sarcocystidae): the continuation of
their life cycle requires the presence of both intermediate
and definitive hosts. The intermediate host, which is usually
a herbivore or omnivore infected by ingesting food or water
contaminated with sporocysts, hosts the asexual phase of the
life cycle. In immature cysts, further asexual reproduction
takes place by repeated endodyogeny of metrocytes. Mature
cysts contain several hundred thousand bradyzoites; these do
not divide further and represent the terminal asexual stage in
the intermediate host. Following the ingestion of their cysts
by a carnivorous definitive host, the bradyzoites initiate the
sexual phase of the life cycle (gamogony) in the cells of
the small intestine of the host, ultimately resulting in the
formation of oocysts [1]. Free sporocysts are released into
the intestinal lumen and passed into the soil or water of the
surrounding environment with the faeces.
Sarcocystosis is a zoonotic disease presented by a wide
range of domestic ungulates, including cattle, sheep, goats or
water buffalo, or wild ones, such as camels and wild boar [2-
6]. The Sarcocystis of cervids has been intensively studied over
the last few years. At least 14 Sarcocystis species have so far
been identified in red deer (Cervus elaphus) in Europe: S. cer-
vicanis, S. elongata, S. cf. grueneri, S. hardangeri, S. hjorti, S.
cf. hofmanni, S. iberica, S. linearis, S. morae, S. ovalis, S. tae-
niata, S. tarandi, S. truncata and S. venatoria [3, 4, 7, 8]; how-
ever, little is known of their potential zoonotic or public health
significance [9, 10]. Although more than 200 valid Sarcocystis
species have been identified, only three, S. hominis, S. sui-
hominis, and S. heydorni, have so far been shown to be capa-
ble of causing intestinal disease in humans [4, 11]. Although
the intestinal forms of infection are often asymptomatic and
* Władysław Cabaj
cabajw@twarda.pan.pl
1 Witold Stefański Institute ofParasitology ofthePolish
Academy ofSciences, Twarda 51/55, 00-818Warszawa,
Poland

Acta Parasitologica
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self-limiting, a number of studies have reported symptoms
ranging from mild gastrointestinal distress to nausea, loss of
appetite, vomiting, stomach ache, bloat, diarrhea, dyspnea and
tachycardia [12]. Whether individuals remain asymptomatic
or develop some degree of disease appears to be related to the
quantity of the meat consumed, inoculum size and various
host factors [12]. Compared to red deer, less data concern on
fallow deer (Dama dama) [13-18]. The latest work [19] report
fallow deer in Spain as a new host for S. morae, originally
described from the red deer. Despite being regularly consumed
in other parts of the EU, meat from fallow deer and red deer
is just entering the market for human consumption in Poland.
It is important to note that although the numbers of wild and
farmed fallow deer have increased over the last decade in the
country, no studies have yet examined the presence of Sarco-
cystis species among them. The present paper, therefore, aims
to determine the presence of Sarcocystis species in fallow deer
farmed in an open pasture system.
Materials andmethods
Animals andsampling
The study was carried out in the Breeding Station of the
Witold Stefański Institute of Parasitology, Academy of Sci-
ences in Kosewo Górne (Region of Warmia and Mazury;
Poland; N: 53° 48′; E: 21° 71 23′). Samples of heart and
oesophagus tissue were obtained from five 1-year-old male
fallow deer slaughtered for commercial purposes. All sam-
ples (10g of weight) were gently broken up in physiological
saline solution using a blender and filtered through gauze
and the resulting sediments were collected. The basic mor-
phology (shape, size) of any cysts present was examined in
wet mounts under an inverted Olympus IX50 microscope fit-
ted with a camera. The samples were preserved for molecu-
lar investigation in sterile H2O in Eppendorf tubes and stored
at −72°C.
DNA extraction
Individual sarcocysts were detected in muscles by light
microscopy. Total DNA was extracted from 10 individ-
ual sarcocysts using the NucleoSpin®Tissue kit (Mach-
erey–Nagel, Germany) according to the manufacturers’
instructions. The DNA was eluted in 50μl of distilled water.
The extracted DNA was stored at −20°C for PCR assay.
Two dierent PCR protocols were employed
forthemolecular examination oftissue cysts
In the first protocol, DNA amplification was performed
using PCR targeted at a 18S rDNA sequence of approxi-
mately 900bp in accordance with More etal. [20]. The
following specific primers were used: SarcoFext (GGT GAT
TCA TAG TAA CCG AAC G) and SarcoRext (GAT TTC
TCA TAA GGT GCA GGA G).
Reactions were performed in a final volume of 25µl reac-
tion mixture with 1 unit of Taq DNA polymerase per reac-
tion (Thermo Fisher Scientific) under the following condi-
tions (final concentrations): 1× reaction buffer supplied with
the DNA polymerase, 1.25mM MgCl2, 200mM dNTPs,
0.2M of each primer and the following cycler program:
initial denaturation (95°C, 5min), followed by 40 cycles of
denaturation (94°C, 40s), annealing (59°C, 1min), elonga-
tion (72°C, 1min) and a final extension at 72°C (10min).
The PCR products were analyzed by electrophoresis in
1.5% agarose gel and stained with GelRed (Nucleic Acid
Gel Stain, Biotium). PCR products 900bp in size were
cut out from the gel and purified using Clean-up Product
Purification Kits (A&A Biotechnology, Poland) according
to the manufacturer’s instructions. DNA concentration was
estimated using a NanoDrop ND-1000 Spectrophotometer
(NanoDrop Technologies, USA).
The PCR amplicons were then ligated into pGEM-T easy
cloning vector (Promega). Escherichia coli strain XL-1 Blue
MRF electrocompetent cells (Promega) were used for clon-
ing. Positive clones (six) were identified by colony PCR with
primers directed against vector sequences outside the multi-
cloning site. The clones found to contain inserts were used
for further examination. Positive plasmids were purified
using GeneAll Exprep Plasmid SV mini (GeneAll, Korea)
according to the manufacturer’s instructions.
The ssu rRNa gene products concentration was measured
using a NanoDrop ND-1000 Spectrophotometer (NanoDrop
Technologies, USA) and was then sequenced (Genomed,
Poland). The sequence information obtained from all the
isolated clones was assembled using Vector NTI Advance
10 software (Invitrogen, Scotland). The complete sequences
were checked against sequences published in GenBank using
BLAST (https ://www.ncbi.nlm.nih.gov/BLAST /).
In the second protocol, the ssu rRNA gene was amplified
by PCR using the primers for Sarcocystis [ERIB1 5′-ACC
TGG TTG ATC CTG CCA G-3′, Primer1L 5′-CCA TGC
ATG TCT AAG TAT AAG C-3′, Primer3H 5′-GGC AAA
TGC TTT CGC AGT AG-3′] [21] in a 25μl final reaction
volume with one unit of Taq DNA polymerase/reaction
(Thermo Fisher Scientific). The following final concentra-
tions of reagents were used: 1× reaction buffer, 1.5mM
MgCl2, 250μM dNTPs, 0.5μM of each primer. PCR was
started with initial denaturation (95°C, 5min), followed by
35 cycles of denaturation (95°C, 30s), annealing (55.5°C,
30s), elongation (72°C, 1min) and a final extension at
72°C (10min).
PCR products were analyzed and sequenced as described
above. The purified products were then sequenced
(Genomed, Poland).

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Phylogenetic analyses
The evolutionary history was inferred using the Maximum
Likelihood method based on theKimura 2-parameter model
[22]. The tree with the highest log likelihood (−2638.89)
is shown. The percentage of trees in which the associated
taxa clustered together is shown next to the branches. Initial
tree(s) for the heuristic search were obtained by applying the
Neighbor-Joining method to a matrix of pairwise distances
estimated using the Maximum Composite Likelihood (MCL)
approach. A discrete Gamma distribution was used to model
evolutionary rate differences among sites [four categories
(+G, parameter = 0.3385)]. The tree is drawn to scale, with
branch lengths measured in the number of substitutions per
site. The analysis involved 100 nucleotide sequences. All
positions with less than 95% site coverage were eliminated.
That is, fewer than 5% alignment gaps, missing data, and
ambiguous bases were allowed at any position. There were
a total of 789 positions in the final dataset. Evolutionary
analyses were conducted in MEGA7 [23].
Results
Microscopic sarcocysts were detected in all of the exam-
ined fallow deer under light microscope examination in wet
mounts. This preliminary microscopic observation revealed
the presence of at least three morphs among the isolated
sarcocysts depending on their size or shape (Fig.1a, b).
Two fragments of 18S rDNA (851bp and 776bp) were
amplified using primers specific for Sarococystis spp. The
two nucleotide sequences obtained from sarcocysts isolated
from the heart were deposited in GenBank under acces-
sion numbers KX364266.1 and KX364267.1. A BLAST-N
search found that one nucleotide sequence deposited under
accession number KX364266.1 share 99.41% similarity with
sequences deposited in GenBank under accession numbers
MK790239, MK790238, KY973379.1, KY973375.1 and
KY973374.1 and described as S. morae. Second nucleotide
sequence deposited under accession number KX364267.1
share 99.74% identity with sequences KY973379.1,
KY973375, MK790239 and MK790238—originally
described from red deer and newly described in fallow deer.
The primers specific for Sarococystis spp. used in the
second PCR protocol were then used to amplify ssu rRNA
fragments about 1kb in length. Three nucleotide sequences
of the ssu rRNA were obtained from three sarcocysts: one
isolated from fallow deer heart tissue and two isolated from
oesophagus tissue. All three sequences were deposited in
GenBank under accession numbers MH221019.1 (1064bp),
MH221020.1 (1016bp) and MH221021.1 (807bp).
BLAST-N search found nucleotide sequence
MH221019.1 obtained from the heart tissue to share
99.91% identity with those from the diaphragm of Capre-
olus capreolus and deposited in GenBank as S. gracilis
Fig. 1 The appearance of unstained sarcocysts isolated from fallow deer in Kosewo, Poland, under the light microscope: lens 10 × 25PhC; eye-
piece 10× WH10 × 22; magnification 10 × 10. a Sarcocysts from heart tissue, b Sarcocysts from oesophagus tissue

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under accession numbers KY019031.1 and KY019030.1.
Of the nucleotide sequences isolated from the oesophagus,
the first (MH221020.1) demonstrated 100% similarity with
sequences KY019031.1 and JN256131.1, previously isolated
from the diaphragm of Capreolus capreolus and described
as S. gracilis. The second sequence (MH221021.1.) repre-
sent Sarcocystis sp. and had 96.58%-99.50% identity with S.
linearis and 97.09–99.01% identity with S. taeniata.
The phylogenetic analysis placed two new sequences,
MH221019.1 and MH221020.1, within the same clade as S.
gracilis sequences KY019031.1, KY019030.1, KF880741.1,
JN256131.1 and JN226126.1 (Fig.2). Sequence Sarco-
cystis sp. (MH221021.1) is closely related to S. linearis
(KY973359.1) and S. taeniata (KF831286.1). Further
investigations using cox1 are needed for the conclusive
identification of MH221021.1. Sequences KX364266.1
and KX364267.1 were placed in the same clade as Sarco-
cystis sp. sequence KT873778.1 and S. morae sequences
(MK790239.1 and MK790238.1) both isolated form sarco-
cysts from the tongue of fallow deer as well as KY973373.1
and KY973378.1 sequences: these were identified from
bradyzoites isolated from the oesophagus of Cervus elaphus.
Discussion
Over the last decades, an increasing amount of research has
been devoted to the life cycle, morphology and pathogenic-
ity of Sarcocystis species. Although Sarcocystis infection is
common in many domestic and wild animal species, little is
known about its occurrence in Poland, with only a handful
of studies having been performed. One study based on trichi-
noscopy and the compression method identified the presence
of large-scale invasions of cysts in muscle tissue among 166
wild boars, 53 deer and 53 roe deer in the years 1997–99
[2]. A later study of four Polish roe deer based on light
microscopy and sequence analysis of small subunit riboso-
mal RNA (ssu rRNA) and subunit I of cytochrome oxidase
(cox 1) identified the presence of S. gracilis, S. oviformis
and S. silva sarcocysts [24]. S. tenella has been detected in
Tatra chamois (Rupicapra rupicapra tatrica) previously only
reported in sheep [25]. A light microscopy study by Pyziel
and Demiaszkiewicz (2009) [26] identified S.cruzi in heart,
oesophagus and diaphragm muscle from one European bison
from the Białowieża Forest.
Fig. 2 Phylogenetic tree of Sarcocystidae based on ssu rRNA gene
sequences and inferred using the maximum likelihood (ML) method.
The ssu rRNA tree was constructed based on the alignment of nearly
complete ssu rRNA gene sequences of five Polish Sarcocystis spp.
isolates and available ssu rRNA gene sequences of related species
deposited in GenBank. The trees were rooted with Eimeria adeneo-
dei and Neospora caninum ssu rRNA sequences. Polish isolates are
marked in green (color figure online)
▸

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Although Sarcocystis spp. have been identified in fallow
deer in previous studies [13, 14, 17], the precise species
was unknown. Only Hernandez-Rodriquez etal. (1992) [15]
described S. jorrini sp. nov. as a new species in fallow deer:
macroscopic cysts were detected in the skin, and virtually all
striated muscles, including those of the esophagus and heart.
The cysts themselves were white and spindle shaped, and
could clearly be distinguished from the surrounding muscle
tissue. Wesemeier and Sedlaczek (1995a) [16] describe the
transmission electron microscope identification of sarcocysts
in two free-ranging indigenous fallow deer, Dama d. dama
(L.), from northeast Germany, and one in captive Persian
fallow deer (Dama dama mesopotamica) from the Berlin-
Friedrichsfelde Zoo. In the free-ranging D. d. dama, two
Sarcocystis species were found to be present: one known and
the other new to the host. The known species was also found
in D. dama mesopotamica. Based on a subsequent review of
previous studies and their own research on roe deer, red deer
and wapiti, Wesemeier and Sedlaczek [27], propose that the
two Sarcocystis species should be designated S. cf. grueneri
(known in D. dama but not named) and S. cf. hofmanni (new
for D. dama).
Our results confirm the presence of Sarcocystis spp. in all
tested young fallow deer and suggest that a single year of
stay in the pastures was sufficient for the animals to become
infected. It is important to note that identified Sarcocystis
species (S. morae, S. gracilis and Sarcocystis sp.) have not
previously been reported in fallow deer in Poland. Lately, S.
morae was reported in fallow deer as a new host in Spain by
De las Cuevas etal. [19]. Sarcocystis gracilis is considered
to be parasites of roe deer (Capreolus capreolus) [28] while
S. morae occur in red deer (Cervus elaphus) [8] and fallow
deer (Dama dama) [19]. The Sarcocystis isolates obtained
in the present study shared 96.58–100% identity with those
taken previously from different tissues of various hosts from
other geographical localities.
Although Sarcocystis spp. can be recognized using sim-
ple and inexpensive methods, such as naked eye examina-
tion or light microscopy, a variety of molecular methods
have been developed and implemented to identify Sarco-
cystis spp. and to assess the genetic diversity among these
parasite species. The slowly-evolving small subunit (ssu)
rRNA gene, commonly used in phylogenetic studies, is also
well suited for studying the phylogeny of Sarcocystis spe-
cies [21, 24, 29, 30]. The phylogenetic tree created from
the S. gracilis, S. linearis and Sarcocystis sp. sequences
from the fallow deer in the present analysis is in agreement
with those described in other studies. Two new S. gracilis
sequences (MH221019.1 and MH221020.1), two new S.
morae sequences (KX364266.1 and KX364267.1) and one
Sarcocystis sp. sequence (MH221021.1) were identified and
positioned within clades corresponding to other sequences
of the same species. BLAST searches and phylogenetic
analyses based on ssu rRNA gene sequences confirmed that
our isolates closely matched the sequences of those depos-
ited in GenBank and demonstrated a low level of intraspe-
cific sequence variation. Although Januškevičius etal. [31]
conclude that no statistically significant differences in infec-
tion prevalence exist between muscle groups in the Cervi-
dae, e.g. sika deer, elk, red deer and roe deer, Malakauskas
and Grikienienė [32] found infection intensity to be higher in
samples of oesophagus and heart muscle than those from the
diaphragm. In the present study, samples were only obtained
from oesophagus and heart tissue. Therefore, further studies
are needed to determine the presence and the intensity of
Sarcocystis spp. infection in other groups of muscles.
Although approximately 200 species of Sarcocystis are
recognized, the definitive and intermediate hosts are known
for only 62 of them [4]. In the case of S. gracilis, the defini-
tive hosts are known to be dogs, red foxes (Vulpes vulpes)
or blue foxes (V. lagopus); the parasite must be consumed
by these to complete its life cycle [4, 28]. Domestic dogs,
wild dogs and red foxes (Vulpes vulpes) are also very com-
mon in the region covered by the present study, and these
may also serve as definitive hosts; foxes, in particular, have
unlimited access to farm animals, unlike dogs. Of the 13
wild carnivorous species known to carry unnamed Sarcocys-
tis species [4] four of them, viz. raccoons (Procyon lotor),
European badgers (Meles meles), otters (Lutra lutra) and
mink (Mustella vision), are common in the area where the
study was carried out.
The definitive hosts of S. morae and S. linearis are
unclear. However, phylogenetic data indicate canids as most
likely definitive host of these two species [18, 28]. It is pos-
sible that some Sarcocystis species in red deer may use other
cervids as their principal intermediate hosts, and this may
also indicate that many species of Sarcocystis are not as
specific to their intermediate host as once believed.
Conclusion
Our study confirms the presence of Sarcocystis spp. in
farmed fallow deer and is the first to identify three species
of the genus Sarcocystis in them. Additionally, our results
indicate that a wide range of mammals in different areas may
harbour Sarcocystis species.
Funding This study was funded by W. Stefański Institute of Parasitol-
ogy Polish Academy of Sciences.
Compliance with ethical standards
Conflict of interest WC and BM devised the study concept and con-
ducted part of the lab work; WC, BM, JB analyzed the results and

Acta Parasitologica
1 3
wrote the first draft. JB and KG, KB carried out the molecular study
and generated the sequences, designed the figures and edited the man-
uscript. KB was involved in the construction of the phylogenetic tree.
ŻSB and MB collected the samples. All authors have agreed with the
content of the manuscript. The authors declare no conflict of interests.
Ethical approval This article is not under consideration or published
elsewhere; no material in the paper has appeared or will appear on a
preprint server. All animals belong to the Witold Stefański Institute
of Parasitology, Polish Academy of Sciences. The Breeding Station is
located in Kosewo Górne. The experimental protocol was approved by
Scientific Board of the Witold Stefański Institute of Parasitology PAS.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.
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