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

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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
1 3
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).
Acta Parasitologica
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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
Acta Parasitologica
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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)
Acta Parasitologica
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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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... The Sarcocystis species were intensively studied in cervids [147], a taxonomic group in which this parasitic genus appears to be poorly host specific [148][149][150] and for which the definitive hosts and many epidemiological aspects are still unknown [151]. To date, at least 11 different Sarcocystis species, forming sarcocysts of five major morphological types, have been molecularly characterized in European red deers [135,148,152] (Table 2). ...
... Fewer studies focused on fallow deer [147,149,160]. Although Sarcocystis spp. ...
... Although Sarcocystis spp. have been identified in fallow deer in older studies, in Germany, Italy, Austria and Spain (detailed in de Las Cuevas [149] and Cabaj et al. [147]), the species were only morphologically described by and not identified by molecular methods. On the contrary, more recent studies applying DNA-based techniques found three identified species in this host: S. morae, S. entzerothi and S. gracilis (Table 2). ...
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Game meat is increasingly appreciated and consumed in Europe, also due to the growing population of wild ungulates. In addition to interesting nutritional properties and market opportunities, game meat is characterized by some specific public health issues. This review focuses on the etiology, epidemiology, public health aspects and risk management along the supply chain, including parasite detection at slaughtering and inactivation in meat, of three selected foodborne parasitic hazards (Alaria alata, Toxoplasma gondii and Sarcocystis spp.) in the main mammalian game meat species in the EU: wild boar (Sus scrofa), red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), Alpine chamois (Rupicapra rupicapra), moose (Alces alces), hare (Lepus europaeus) and wild rabbit (Oryctolagus cuniculus). The presented data point out the main issues, and knowledge gaps as well as the potential for improved control in order to contribute to the risk analysis process. To pursue an effective management of these parasitic zoonoses, awareness raising should involve all figures in the supply chain, including hunters, restaurateurs and consumers. Human behaviour and the lack of knowledge regarding meat borne parasitic zoonoses and the health risks they pose seem to be the most important factors responsible for human infections. However, detection methods, starting from the sampling procedure, should be further developed and standardized in order to improve the collection of accurate and up-to-date epidemiological data.
... The basic morphology (shape, size) of any cysts present were examined in wet mounts and collected under an inverted Olympus IX50 microscope fitted with a camera. The sarcocyst was individually preserved in sterile H 2 O in Eppendorf tubes for molecular investigation and stored at − 20 • C (Cabaj et al., 2020). ...
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European bison are susceptible to a range of pathogens which may influence their health, and hence, to ensure their protection, it is essential to provide effective monitoring of potential exposure. This study presents the first molecular confirmation of Sarcocystis cruzi infection in European bison based on PCR amplification of the cytochrome c oxidase subunit I (cox1) gene. A sample of heart tissue taken from one fifteen-year-old European bison cow was examined by light microscopy for the presence of heart sarcocysts. The genomic DNA isolated from any identified sarcocysts was subjected to PCR to amplify cox1 gene sequences, and the obtained amplicons were sequenced by Sanger dideoxy sequencing. Two partial cox1 sequences were obtained; they were identified as S. cruzi and deposited in the GenBank™ database under the accession numbers MW490605 and MW490606. BLAST analysis found them to demonstrate the closest similarity to S. levinei (MH255771-MH255779 and KU247874-KU247884), sharing an identity of 93.14–93.8 %. This is the first report to identify sarcocysts isolated from heart tissue of infected European bison living in the Białowieża forest to species level using cox1 analysis. Our findings confirm that the European bison is a natural intermediate host for S. cruzi. As such, coordinators of future conservation programmes should consider the impact of these diseases on reintroduced animals.
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We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
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Members of the genus Sarcocystis are frequently found infecting members of the family Cervidae. Although Sarcocystis species are generally host specific for their intermediate hosts, species in cervids appear to be less host specific. Here, we report fallow deer (Dama dama) as a new host for Sarcocystis morae, originally described from the red deer (Cervus elaphus). Tongues of 69 legally hunted animals in Spain were tested for sarcocysts, and the species were characterized by light microscopy, ultrastructurally and molecularly. Sarcocysts were identified in 66.7% of D. dama. Sarcocysts had thin (<2 μm thick) cyst wall with hair-like villar protrusions bifurcated at their tips resembling type 8a. Genetic sequences obtained for 18S rRNA and COI reached 99.6-100% and 97.9-98.7% similarity, respectively, to those of S. morae from the red deer. The present study provides new data concerning lower level of host specificity within Sarcocystis genus for cervids.
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Members of the genus Sarcocystis are frequently found infecting members of the family Cervidae. Although Sarcocystis species are generally host specific for their intermediate hosts, species in cervids appear to be less host specific. Here, we report fallow deer (Dama dama) as a new host for Sarcocystis morae, originally described from the red deer (Cervus elaphus). Tongues of 69 legally hunted animals in Spain were tested for sarcocysts, and the species were characterized by light microscopy, ultrastructurally and molecularly. Sarcocysts were identified in 66.7% of D. dama. Sarcocysts had thin (<2 μm thick) cyst wall with hair-like villar protrusions bifurcated at their tips resembling type 8a. Genetic sequences obtained for 18S rRNA and COI reached 99.6-100% and 97.9-98.7% similarity, respectively, to those of S. morae from the red deer. The present study provides new data concerning lower level of host specificity within Sarcocystis genus for cervids.
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Along with the increase in consumption of raw animal meat, the prevalence of food poisoning is increasing. A 67-year-old Japanese man had eaten raw venison 4 hours prior to the beginning of vomiting. Many white cysts were discovered in the venison, with numerous bradyzoites being detected after the cysts were punctured. The presence of the Sarcocystis spp. 18S rRNA gene was detected by polymerase chain reaction, and Sarcocystis truncata was isolated from the venison. Sarcocystis truncata has not previously been identified in sika deer (Cervus nippon) in Japan. This is the first report of possible Sarcocystis truncata-induced food poisoning following consumption of venison.
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Worldwinde, cervids are considered an important source of infection and dissemination of a wide variety of pathogens, both for farm animals and humans. Among this diseases is sarcosporidiosis, which is a parasitic disease caused by Sarcocystis spp. (Protozoa: Apicomplexa). Most frequent clinical signs are hemolytic anemia, weakness, weight loss and decrease of growth and some species of Sarcocystis might cause abortions. The clinical disease in ruminants is fairly rare but the infection is very frequent. Infections are accumulative and the parasite does not generate immunity in any of the hosts. Ovine sarcosporidiosis is a serious issue in the some regions of Chile due to the macrocysts located in the muscle which means condemnation of the whole carcass. Sarcocystis spp. has been widely reported in red deer and other cervid species but in Chile the situation remains unknown. Nowadays there is little to no evidence of Sarcocystis in foreign deer in Chile and there is only one report of the parasite on pudu. The main goal of this study is to demonstrate the presence of Sarcocystis spp. in myocardium of red deer and fallow deer in Chile, and confirm the presence of Sarcocystis spp. in pudu. All cervid cases from 1994 to 2013 of the Institute of Animal Pathology of the Universidad Austral de Chile were reviewed. The animals selected were those in which a myocardium sample was taken. From the histopathological samples observed, it was found that 5 of the 9 red deer, 1 of the 4 fallow deer and in 11 of the 23 pudu there were Sarcocystis cysts in the myocardium. This study represents the first record for Chile of Sarcocystis spp. in myocardium of red deer and fallow deer. Stablishing the red deer, fallow deer and pudu as hosts of Sarcocystis aids to have a better understanding of the parasite epidemiology in Chile and the role of wild and captive cervids in the maintenance and spread of these parasites.
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Samples of muscle tissue from the diaphragm, oesophagus and/or heart of eight adult red deer (Cervus elaphus hispanicus) from the Quintos de Mora Park in Toledo, Central Spain, were screened for sarcocysts by means of the compression method. From positive samples, individual sarcocysts were excised and examined in wet mounts under a light microscope (LM) in order to study their basic morphology before being preserved for molecular studies. In all red deer examined, only microscopic sarcocysts were found. Those in the diaphragm and oesophagus were spindle-shaped and about 1 × 0.1 mm in size, while those in cardiac muscle were sac-like and 500–800 × 80–180 μm. By LM, the sarcocysts either had densely packed, about 8-μm-long, hair-like protrusions (type 1), sparsely distributed indistinct projections (fuzzy outline; type 2) or no visible protrusions (smooth surface; type 3). In cardiac muscle, only sarcocysts without visible protrusions were found. One of the latter sarcocysts was examined by scanning electron microscopy (SEM) and found to possess thin ribbon-like protrusions. Forty-eight sarcocysts isolated from the diaphragm, oesophagus and heart of one red deer, as well as 55 sarcocysts from the heart of three other red deer, 103 sarcocysts in total, were characterized molecularly through PCR amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of the mitochondrial genome, revealing the presence of six major cox1 sequence types. Each type comprised either a single sequence (three types) or a collection of several identical or nearly identical sequences. From selected isolates possessing each of these cox1 sequence types, the complete 18S ribosomal RNA (rRNA) gene was amplified and sequenced directly and/or after cloning of the 5′ end half. Supported by the sequence data from the latter gene, as well as the morphology of the sarcocysts from which the sequences originated, the six cox1 sequence types were considered to represent six separate Sarcocystis spp. Two cox1 sequence types were identified as belonging to the previously characterized species Sarcocystis hjorti (one sequence/sarcocyst) and Sarcocystis linearis (38 sequences/sarcocysts), respectively, whereas four sequence types were new. One of the latter types was assigned to the previously named species Sarcocystis cervicanis from red deer, since this sequence type was obtained from 52 sarcocysts from cardiac muscle, which matched the original morphological description (smooth surface) and habitat of this species. The remaining three sequence types were assigned to the three new species Sarcocystis iberica (one sequence/sarcocyst) Sarcocystis venatoria (10 sequences/sarcocysts) and Sarcocystis morae (one sequence/sarcocyst), respectively. The two species S. iberica and S. venatoria shared the same sarcocyst morphology (type 1) and habitat (diaphragm) and had virtually identical 18S rRNA gene sequences, but differed by 4% at cox1, which was considered sufficient to regard them as separate species. The single sarcocyst of S. morae (from the oesophagus) examined by LM had a smooth wall and this species was therefore believed to have the same type of ribbon-like protrusions (ultrastructurally) as sarcocysts of S. cervicanis and S. linearis, which were also the species most closely related to S. morae at cox1. Thus, there seems to be three species with similar ribbon-like cyst wall protrusions in red deer (S. cervicanis, S. linearis, S. morae), as well as three species with similar hair-like protrusions (S. hjorti, S. iberica, S. venatoria). Sarcocysts of S. cervicanis were only identified in cardiac muscle, whereas sarcocysts of S. linearis were found mainly in the diaphragm and oesophagus and rarely in the heart. The relative number of cox1 haplotypes was greater among sequences/isolates of S. linearis (17/38) than among isolates of S. cervicanis (7/52). Four of the species examined (S. cervicanis, S. linearis, S. iberica, S. venatoria) possessed considerable intra-isolate (intra-genomic) sequence variation (insertions/deletions, substitutions) in the 18S rRNA gene.
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Fresh (frozen/thawed) muscle samples from four 2–12-year-old roe deer (Capreolus capreolus) from the Sondrio province in north-eastern Italy were examined under a dissecting microscope, and about 180 sarcocysts were isolated and identified to morphological type in wet mounts by light microscopy (LM). Seventy-seven of these sarcocysts were subsequently examined by molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of all isolates, as well as PCR amplification, cloning and sequencing of the complete18S ribosomal RNA (rRNA) gene of two isolates of each species found. By LM, three major sarcocyst types were recognised: spindle-shaped sarcocysts, 0.5–3 mm long, either with no clearly recognisable protrusions (thin-walled) or with finger-like protrusions (thick-walled); and slender, thread-like sarcocysts, 2–3 mm long, with hair-like protrusions. Sequencing of cox1 revealed that the sarcocysts belonged to four different species. Those with no visible protrusions either belonged to Sarcocystis gracilis (n = 24) or to a Sarcocystis taeniata-like species (n = 19), whereas those with finger- and hair-like protrusions belonged to Sarcocystis silva (n = 27) and Sarcocystis capreolicanis (n = 7), respectively. The 19 cox1 sequences of the S. taeniata-like species, comprising five haplotypes, differed from each other at 0–16 of 1038 nucleotide positions (98.5–100% identity). They differed from 25 previous cox1 sequences of S. taeniata from moose and sika deer (with 98.0–100% intraspecific identity), at 33–43 nucleotide positions (95.9–96.8% interspecific identity), and there were 20 fixed nucleotide differences between the two populations. In the phylogenetic analysis based on cox1 sequences, the two populations formed two separate monophyletic clusters. The S. taeniata-like species in roe deer was therefore considered to represent a separate species, which was named Sarcocystis linearis n. sp. At the 18S rRNA gene, however, the two species could not be clearly separated from each other. Thus, there was considerable intraspecific sequence variation in the 18S rRNA gene of S. linearis (98.1–99.9% identity between 24 sequences), which was similar both in magnitude and nature to the variation previously found in this gene of S. taeniata. The new 18S rRNA gene sequences of S. linearis shared an identity of 97.9–99.6% with those of S. taeniata (overlap between intra- and interspecific identity), and in the phylogenetic tree, sequences of the two species were interspersed. By scanning electron microscopy (SEM), the sarcocysts of S. linearis were found to possess regularly spaced, thin and narrow ribbon-like cyst wall protrusions (about 2.8–3.2 μm long, 0.3–0.4 μm wide and about 0.02–0.03 μm thick), terminating in a plate-like structure of the same thickness but with an elliptic outline (about 0.3–0.4 μm wide and 0.7–0.9 μm long). The terminal plates were connected in the middle with the band-like portion of the protrusions like the board of a seesaw (tilting board). The terminal plates of adjacent protrusions were neatly arranged in a hexagonal pattern resembling tiles on a roof. Together, they formed an outer roof-like layer facing the surrounding cytoplasm of the host cell and completely covering the band-like proximal portion of the protrusions, which overlapped and were stacked in three to four layers close to the cyst surface. The sarcocyst morphology of S. linearis was consistent with that of an unnamed Sarcocystis sp. in roe deer previously found by transmission electron microscopy in several countries, including Italy. A few sarcocysts of S. gracilis and S. silva were also examined by SEM, confirming the presence of regularly distributed, short knob-like protrusions in S. gracilis (as seen in previous SEM studies) and revealing tightly packed, erect 6–7-μm-long villus-like protrusions having regularly distributed round depressions on their surface in S. silva. The sequencing of cox1 of 7, 24 and 27 new isolates of S. capreolicanis, S. gracilis and S. silva, respectively, recovered 7, 11 and 10 new haplotypes from each of the three species and expanded our knowledge on the intraspecific sequence variation at this marker. Similarly, the study revealed a more extensive intragenomic sequence variation at the 18S rRNA gene of S. capreolicanis and S. silva than known from previous studies and confirmed a near absence of such variation in the 18S rRNA gene of S. gracilis.
Book
Sarcocystis is one of the most prevalent parasites of livestock and also infects many wild mammals, birds, and humans. Written by the authors who pioneered studies of Sarcocystosis of domestic animals, Sarcocystosis of Animals and Humans, Second Edition provides a current and comprehensive review of Sarcocystis and the infections it causes in animals and humans. The book reviews the history, structure, life cycle, pathogenesis, lesions, clinical signs, diagnosis, immunity, epidemiology, treatment, prevention, and control of Sarcocystosis. See What’s New in the Second Edition: •New section on molecular diagnosis and DNA characterization of Sarcocystis species •New section on clinical sarcocystosis outbreaks in humans is added with a summary of all reports, symptoms, diagnosis, and treatment •New section on acute fatal outbreaks of sarcocystosis in birds •Complete description of the life cycles of all Sarcocystis species •List of all species whose life cycles are known •Comprehensive information on diagnosis, including molecular diagnosis •Additional information on zoonoses •In-depth coverage of treatment, control, and prevention Maintaining the format that made the first edition so popular, this new edition covers recent developments and excludes information that has become redundant. The authors include all literature and provide a comprehensive review of biology, clinical disease, economic losses, public health concerns, diagnosis, treatment, and prevention. They have tabulated information on all Sarcocystis species by host and listed species that should be considered species inquirende/invalid.
Article
Fresh muscle samples from water buffalo (Bubalus bubalis) aged 2–15, from Giza Province, Egypt; were examined for Sarcocystis infection. Macroscopic ovoid sarcocysts embedded in the muscle tissues of the examined buffaloes were detected; they measured 152–230 (210 ± 7) µm in length and 37–119 (95 ± 3) µm in width. The esophagus was the most infected organ followed by the diaphragm, and tongue, while the heart muscles were the least infected. The cyst cavity was compartmentalized by septa derived from the ground substance located under the primary cyst wall. Using transmission electron microscopy, the primary cyst wall bordered sarcocysts were determined to be 0.08–0.22 µm in thickness, raised from the parasitophorous vacuolar membrane, and surrounded by a secondary cyst wall of host origin. The primary cyst wall had irregular wall folds with numerous cauliflower-like projections of variable sizes and shapes accompanied by knob-like electron-dense elevations. 18S rRNA gene expression studies confirmed that the present parasite isolates belonged to the genus Sarcocystis. The sequence data showed significant identities (>90%) with archived gene sequences from many Eimeriidae organisms, and a dendogram showing the phylogenetic relationship was constructed. The most closely related species was Sarcocystis fusiformis KR186117, with an identity percentage of 98%. The recovered sequences were deposited in the GenBank under the accession number MG572125. The present study, to our knowledge, is the first collective ultrastructural and molecular study that confirmed the taxonomy of sarcocysts isolated from water buffaloes in Egypt as Sarcocystis fusiformis.
Article
Recent investigations have shown that the intensity of Sarcocystis infection in game animals used for food is high and in some countries even exceeds 90%. Sarcosporidia found in the muscle tissue of domestic and wild animals pose a potential risk to humans. Therefore, their investigations are rather important. Infection with Sarcosporidia (Sarcocystis spp.) cysts was evaluated in the main species of wild animals used for food: elks (n = 27), red deer (n = 54), sika deer (n = 45), roe (n = 36), and wild boars (n = 127). Sarcocystis cysts were found in all examined groups of muscles: esophagus, diaphragm, heart, neck, mandible, dorsum and leg. Depending on the animal species and examined groups of muscles, the prevalence of infection, determined microscopically in 1,279 muscle samples by compression slide method using methylene blue as an indicator dye, ranged from 46.43% to 100.0%.