The protozoon genus Sarcocystis
is part of the apicomplexan family. To
complete their life cycle, Sarcocystis
species need two hosts, typically in a predator-prey relationship. The
predator gets infected by eating prey, causing intestinal disease and shedding
sporocysts in stool. Prey is infected by ingesting contaminated vegetables or
water and develops tissue cysts in muscle tissue (CDC, 2017). More than 189 Sarcocystis
spp. have been described and new species are continuously being identified (Gjerde, 2016; Gjerde et al., 2017;
Odening, 1998). Cattle
serve as intermediate hosts of S. cruzi,
S. sinensis, S. hirsuta and S. hominis with
canids, domestic cats, felids and humans as final hosts, respectively (Gjerde & Hilali, 2016; Moré et
al., 2014). In
cattle, sarcocystosis is a chronic, often asymptomatic infection. Therefore,
mixed infections are often observed (Chiesa et al., 2013; Hornok et al.,
2015; Moré et al., 2014). The
consumption of S. hominis infected
beef can result in intestinal sarcocystosis. Human infections are often
asymptomatic, but nausea, vomiting, acute and chronic enteritis can be observed.

Little is known about the duration and quantity of excretion of oocysts, though
reports of 21 and 12 months were cited (Fayer et al., 2015).

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            In the European Union, recent data on the
prevalence of Sarcocystis spp. in
cattle is far from complete and reported numbers on prevalence vary greatly.

The European Food Safety Authority (EFSA) only obtained data from Belgium
reporting a prevalence of 0,012%: 107 out of 874,948 cattle samples tested in
slaughterhouses contained Sarcocystis (EFSA, 2016). In contrast, studies performed in Italy, Hungary and Germany report
a prevalence as high as 66 to 91%, although these numbers are based on
relatively small panels: 44, 151 and 257 samples of cattle meat tested,
respectively (Chiesa et al., 2013; Hornok et al.,
2015; Moré et al., 2014). In the
Netherlands, the presence of Sarcocystis
is currently not being monitored in cattle meat. The most recent research dates
from 1993, reporting a prevalence of 100% (RIVM, 1993). Therefore, in 2015 a pilot study was performed on 17 cattle meat
samples obtained from a Dutch slaughterhouse, with 12 out of 17 samples testing
positive. (Mineur, 2016).

The presence of S.

hominis cysts in cattle is evident for a complete transmission cycle. To
interrupt transmission, cattle meat for human consumption must be subjected to
meat inspection and condemned when infected with S. hominis. Therefore, identification
of S. hominis must be both sensitive
and specific. Using traditional microscopy, the observation of thin-walled
cysts are indicative for S. cruzi (Moré et al, 2008). Thick-walled sarcocysts are typically observed in S. sinensis, S. hirsuta and S. hominis infections
and can be further differentiated using molecular techniques (Moré et al., 2014). Most techniques described for molecular identification of Sarcocystis spp. in cattle target the small-subunit
(SSU or 18S) rRNA gene (Ho et al., 1996; Moré et al., 2013;
Yang et al., 2001). Many
nucleotide sequences are available in public databases for reliable
identification and establishing phylogenetic relationships. Targeting the 18S
rRNA gene, interspecific variations have been described between strains
isolated from different hosts (Yang et al., 2001). Intraspecific variations of closely related Sarcocystis spp. isolated from the same host have also been
observed. Gjerde et al. (2013)
have successfully elucidated these variations targeting the mitochondrial cytochrome
c oxidase subunit I (cox1) gene. In
this study, the cox1 gene of 22 Sarcocystis spp. has been sequenced. The
cysts microscopically identified as S.

hominis appeared to be S. hirsuta
and S. sinensis based on 18S rDNA
sequence analysis. Therefore, the cox1 gene of S. hominis was not determined (Gjerde, 2013). To date, the sequence of
the S. hominis cox1 gene is not available
in public databases, hence the relevance of this target for the identification Sarcocystis spp. in cattle remains to be

Tissue cysts are not evenly distributed in
muscle tissue and for this reason testing larger amounts of meat will increase
the probability of detection. For PCR, DNA is extracted from 25 – 100 mg of
meat using commercially available DNA extraction kits (Chiesa et al., 2013; Hornok et al., 2015). Moré et al (2014)
enlarge the amount of meat to 5 grams using pepsin digestion prior to DNA
extraction, thereby increasing the probability of detection (Moré et al., 2014). Opsteegh et al.

combine tissue homogenization of 100 g of meat sample with sequence-specific
magnetic capture for the detection and genotyping of Toxoplasma gondii in meat samples. This method is based on
hybridization of DNA using target-specific biotin-labled oligonucleotides. With
streptavidin-labeled magnetic beads the specific DNA can be extracted from the
sample (Opsteegh et al., 2010). Application of this
technique could further increase the probability of detecting Sarcocystis spp.

in meat samples. The aim of this study is to develop a
method for sensitive and specific molecular detection and species
identification of Sarcocystis spp. in
meat in order to provide data on the prevalence of Sarcocystis spp. in cattle meat for human consumption in The