Rodents as a reservoir of infection caused by multiple zoonotic species/genotypes of C. parvum, C. hominis, C. suis, C. scrofarum, and the first evidence of C. muskrat genotypes I and II of rodents in Europe


Danišová, Oľga.; Valenčáková, A.; Stanko, M.; Luptáková, L.; Hatalová, E.; Čanády, A.

Acta Tropica 172: 29-35

2017


Cryptosporidium spp. is an important causative agent of intestinal parasitoses-induced diarrhoea in humans and animals worldwide. Rodents (small mammals), the main reservoir of infections, are globally expanded and overpopulated, which increases the risk of transfer of human and zoonotic pathogens from the genus Cryptosporidium. In this study, Cryptosporidium was detected in wild immunocompetent asymptomatic small mammals. Altogether 262 fecal samples were collected from five areas in Eastern Slovakia from four different rodent species (Myodes glareolus, Apodemus agrarius, Apodemus flavicollis, Rattus norvegicus), eight samples originated from two insectivore species (Sorex araneus, Crocidura suaveolens), and two sample from a carnivore Mustela nivalis. The samples were examined using a method modified in our laboratory, based on the use of specific primers on a small subunit rRNA (18S rRNA) gene for species identification, and amplification of GP60 gene coding 60-kDa glycoprotein for genotype determination. The following species were identified: Cryptosporidium parvum (n=15), genotypes IIaA18G3R1 (n=11; KU311673), IIaA10G1R1 (n=1; KU311670), IIcA5G3a (n=1; KU311669), IIiA10 (n=2; KU311672); Cryptosporidium suis (n=4; KU311671); Cryptosporidium scrofarum (n=28); Cryptosporidium environment sp. (n=12; KU311677); Cryptosporidium muskrat genotype I (n=3; KU311675); Cryptosporidium muskrat genotype II (n=3; KU311676). From one of the rodent, the species Cryptosporidium hominis genotype IbA10G2 (KU311668) was identified for the first time. The results of this study indicate low host specificity of the detected Cryptosporidium species and imply the importance of free-living small mammals in urban and suburban habitats as a potential source of human cryptosporidiosis.

Acta
Tropica
172
(2017)
29-35
CTA
TR
A
OPICA
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lists
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at
ScienceDirect
Acta
Tropica
ELSEVT'
journal
homepage:
www.elsevier.com/locate/actatropica
Rodents
as
a
reservoir
of
infection
caused
by
multiple
zoonotic
species/
genotypes
of
C.
parvum,
C.
hominis,
C.
suis,
C.
scrofarum,
and
the
first
evidence
of
C.
muskrat
genotypes
I
and
II
of
rodents
in
Europe
Olga
DaniKova
,
Alexandra
Valeadkova
,
Michal
Stank°
,
Lenka
Luptakova
,
Elena
Hatalova
1,1
,
Alexander
danadyc'l
a
Department
of
Biology
and
Genetics,
University
of
Veterinary
Medicine
and
Pharmacy,
Komenskeho
73,
041
81
Kosice,
Slovak
Republic
b
Institute
of
Parasitology
and
Institute
of
Zoology,
Slovak
Academy
of
Science,
Hlinkovd
3,
040
01
Kosice,
Slovak
Republic
Faculty
of
Science,
Institute
of
Biology
and
Ecology,
Pavol
Josef
gafarik
University,
Moyzesovd
11,
040
02
Kosice,
Slovak
Republic
CrossMark
ARTICLE
INFO
ABSTRACT
Keywords:
Rodents
Zoonotic
Cryptosporidium
spp.
C.
muskrat
genotype
I
C.
muskrat
genotype
II
PCR
Cryptosporidium
spp.
is
an
important
causative
agent
of
intestinal
parasitoses-induced
diarrhoea
in
humans
and
animals
worldwide.
Rodents
(small
mammals),
the
main
reservoir
of
infections,
are
globally
expanded
and
overpopulated,
which
increases
the
risk
of
transfer
of
human
and
zoonotic
pathogens
from
the
genus
Cryptosporidium.
In
this
study,
Cryptosporidium
was
detected
in
wild
immunocompetent
asymptomatic
small
mammals.
Altogether
262
fecal
samples
were
collected
from
five
areas
in
Eastern
Slovakia
from
four
different
rodent
species
(Myodes
glareolus,
Apodemus
agrarius,
Apodemus
flavicollis,
Rattus
norvegicus),
eight
samples
originated
from
two
insectivore
species
(Sorex
araneus,
Crocidura
suaveolens),
and
two
sample
from
a
carnivore
Mustela
nivalis.
The
samples
were
examined
using
a
method
modified
in
our
laboratory,
based
on
the
use
of
specific
primers
on
a
small
subunit
rRNA
(18S
rRNA)
gene
for
species
identification,
and
amplification
of
GP60
gene
coding
60-kDa
glycoprotein
for
genotype
determination.
The
following
species
were
identified:
Cryptosporidium
pang=
(n
=
15),
genotypes
IIaA18G3R1
(n
=
11;
KU311673),
IMA10G1R1
(n
=
1;
KU311670),
IIcA5G3a
(n
=
1;
KU311669),
IliA10
(n
=
2;
KU311672);
Cryptosporidium
suis
(n
=
4;
KU311671);
Cryptosporidium
scrofarum
(n
=
28);
Cryptosporidium
environment
sp.
(n
=
12;
KU311677);
Cryptosporidium
muskrat
genotype
I
(n
=
3;
KU311675);
Cryptosporidium
muskrat
genotype
II
(n
=
3;
KU311676).
From
one
of
the
rodent,
the
species
Cryptosporidium
hominis
genotype
IbA10G2
(KU311668)
was
identified
for
the
first
time.
The
results
of
this
study
indicate
low
host
specificity
of
the
detected
Cryptosporidium
species
and
imply
the
importance
of
free-living
small
mammals
in
urban
and
suburban
habitats
as
a
potential
source
of
human
cryptosporidiosis.
1.
Introduction
In
recent
years,
attention
has
focused
on
cryptosporidiosis
caused
by
zoonotic
species,
mainly
with
respect
to
host
specificity
and
therefore
the
possibility
of
mutual
transfer
of
infection
between
various
hosts
in
the
environment.
Protozoa
from
the
genus
Cryptosporidiwn
are
world-
wide
spread
parasites
causing
coccidiosis
with
manifestation
of
neona-
tal
enteritis,
destruction
of
intestinal
villi,
dehydration,
and
weight
loss.
They
are
characterized
by
dissemination
to
surrounding
organs
(liver,
gallbladder,
respiratory
system,
urinary
system)
and
immunosuppres-
sion
(Goodstein
et
al.,
1989).
The
main
reservoirs
of
Cryptosporidium
spp.
among
animals
are
farm
animals,
particularly
calves,
lambs,
kids,
and
weaner
pigs
(McAnulty
et
al.,
1994).
Largely,
they
also
act
as
vectors
for
the
zoonotic
species
of
this
pathogen.
Other
important
reservoirs
of
cryptosporidium
infections
are
rodents,
mainly
those
residing
in
the
vicinity
of
farms.
Reproduction
ability
of
rodents
is
enormous
and
they
can
serve
as
both
the
food
source
for
animals
and
source
of
infection.
This
is
why
their
proximity
to
farms,
sharing
sites
with
livestock
and
contaminating
drinking
water,
increases
the
possibility
of
transmitting
cryptosporidium
infection
(oocysts),
not
only
between
animals
but
also
to
humans
(Li
et
al.,
2014).
The
most
common
human
cryptosporidiosis
is
caused
by
species/
*
Corresponding
author
at:
Department
of
Biology
and
Genetics,
University
of
Veterinary
Medicine
and
Pharmacy,
Komenskeho
73,
041
81
Kosice,
Slovak
Republic.
E-mail
address
(0.
Dan&va).
1
Work
was
carried
out
on
Department
of
Biology
and
Genetics,
University
of
Veterinary
Medicine
and
Pharmacy,
Komenskeho
73,
041
81
Kosice,
Slovak
Republic
http://dx.doi.org/10.1016/j.actatropica.2017.04.013
Received
6
February
2017;
Accepted
14
April
2017
Available
online
20
April
2017
0001-706X/
@
2017
Published
by
Elsevier
B.V.
O.
Danilovd
et
al.
Acta
Tropica
172
(2017)
29-35
genotypes
C.
hominis,
C.
parvum,
C.
meleagridis,
C.
fells,
and
C.
cards
(Morgan-Ryan
et
al.,
2002).
Recently,
however,
cryptosporidium
infec-
tions
in
humans,
caused
by
species/genotypes
(Xiao
et
al.,
2004).
Infections
by
species
C.
parvum,
C.
hominis,
and
C.
ubiquitum
were
reported
in
several
places
in
Europe:
the
Czech
Republic
(Hajdukk
et
al.,
2004),
England
(Leoni
et
al.,
2006),
Denmark
(Enemark
et
al.,
2002),
France
(Guyot
et
al.,
2001),
the
Netherlands
(Homan
et
al.,
1999),
Northern
Ireland
(Lowery
et
al.,
2001),
Switzerland
(Fretz
et
al.,
2003),
Scotland
(Mallon
et
al.,
2003),
and
Poland
(Perec-Matysiak
et
al.,
2015).
Other
most
detected
species
were
C.
meleagridis,
C.
tyzzer
adaptable
and
the
host
genotype:
Cryptosporidium
mouse
genotype
II,
Cryptosporidium
rat
genotypes
I,
II,
III,
IV
(Foo
et
al.,
2007;
Kimura
et
al.,
2007;
Feng
et
al.,
2009;
Paparini
et
al.,
2012).
Species
C.
scrofarum
was
recently
identified
in
rodents
in
Slovalda
(Li
et
al.,
2014)
and
in
rats
on
the
Philippines
with
C.
suis-like
genotype,
C.
hamster
genotypes
(Ng-Hublin
et
al.,
2013).
C.
ubiquitum,
C.
muris
katawabi
genotype,
and
new
Naruko
genotype
were
detected
in
wild
rats
in
Japan
(Murakoshi
et
al.,
2013).
In
our
previous
studies
we
identified
C.
muris
(natural
host
-
rodents;
Miller
and
Schaefer,
2007)
in
children
from
Eastern
Slovakia
(Hasajova
et
al.,
2014),
and
also
species
C.
hominis,
C.
parvum
and
their
genotypes
(Petrincova
et
al.,
2015).
Infection
by
species
C.
muris
in
Europe
was
reported:
France
(Guyot
et
al.,
2001)
in
human
and
the
Czech
Republic
and
Poland
(Kva'd
et
al.,
2012;
Nemejc
et
al.,
2013;
Wagnerova
et
al.,
2015)
in
mice,
swine,
horses.
The
presented
data
indicate
that
Cryptosporidium
species
pathogenic
to
humans
circulate
in
the
environment
and
which
supports
their
zoonotic
potential
and
makes
them
a
public
health
threat.
The
aim
of
this
pilot
study
was
to
investigate
the
occurrence
of
Cryptosporidium
in
wild
small
mammals
in
the
Slovak
Republic.
2.
Materials
and
Methods
2.1.
Study
Population
-
Samples
Small
mammals
were
live-captured
in
Swedish
bridge
metal
traps
baited
with
sunflower
seeds,
in
five
different
sites
of
Eastern
Slovalda:
1.
t'ermer
valley
[208-600
masl
(meters
above
sea
level);
48°45'46.67
-
N;
21°8'8.1r
E]
mixed
forest
vegetation
with
a
predominance
of
beech
and
oak,
and
too
with
coniferous
forest
vegetation;
2.
FISTrov,
Hlbolca
valley
(500-750
masl;
48°44'22.80"
N;
21°4'18.90"
E);
mixed
forest
vegetation
with
a
predominance
of
beech,
hornbeam
and
spruce,
and
too
with
deciduous
forest
vegetation;
3.
Botanical
garden
in
KoNce
(208
masl;
48°44'6.84"
N;
21°14'16.14"
E)
with
a
predominance
of
hornbeam;
4.
KoNce,
Niine
Kapustniky
(200
masl;
48°42'48.19"
N;
21°1603.05"
E)
gardens
and
ruderal
habitat;
5.
Game-reserve
Rozhanovce
(215
masl;
48°4500"
N;
21°2100"
E),
an
ecotone
of
oak-
hornbeam
forest
and
a
field
around
animal
quarters.
Botanical
garden
and
Mine
Kapustniky
are
located
in
the
urban
area
of
the
town
of
Kosice,
while
t'ermer,
FISTrov
and
Rozhanovce
are
situated
in
suburban
and
rural
areas.
At
each
site,
50 traps
were
placed
5
m
apart
in
transects
(approxi-
mately
250
m)
for
two
consecutive
nights.
Captured
animals
were
transported
to
the
laboratory
where
species,
sex,
and
reproductive
status
were
recorded.
After
collecting
ectoparasites
(fleas,
ticks,
and
mites)
into
70%
ethanol,
the
rodents
were
euthanized
in
compliance
with
the
legislation
effective
in
the
Slovak
Republic
and
samples
of
their
feces
were
obtained.
Together
we
conducted
11
field
samplings
between
September
2012
and
September
2013.
During
this
period,
we
captured
and
examined
262
small
mammals
of
five
species
(Myodes
glareolus,
Apodemus
agrarius,
Apodemus
flavicol-
lis,
Rattus
norvegicus,
Microtus
subterraneus),
eight
specimens
of
two
insectivore
species
(Sorex
araneus,
Crocidura
suaveolens),
and
two
specimen
of
carnivore
-
weasel
(Mustela
nivalis,
Table
1).
The
evaluation
of
the
captured
animals
showed
predominance
of
three
types
of
rodents:
A.
agrarius
(39.3%),
M.
glareolus
(27.6%),
and
A.
flavicollis
(27.8%).
2.2.
Molecular
Analysis
2.2.1.
DNA
Isolation
Genomic
DNA
was
extracted
from
100
mg
fecal
samples
using
a
DNA-Sorb-B
nucleic
acid
extraction
kit
(AmpliSence,
Russia)
according
to
the
manufacturer's
instructions.
Before
extraction,
the
stool
and
disrupted
oocysts
were
homogenized
at
6500
rpm
for
90
s
with
addition
of
0.5
mm-glass
beads,
1.0
mm-zircon
beads,
and
300
µ.1
lysis
solution
in
a
homogenizer
Precellys
24
(Benin
technologies).
The
purified
DNA
was
stored
at
-
20°
C
until
required
for
nested
PCR.
2.2.2.
Nested
PCR,
Electrophoresis,
Sequencing
The
nested
PCR
was
conducted
using
a
protocol
modified
in
our
laboratory
to
amplify
the
SSU
region
of
DNA
of
Cryptosporidium
species.
In
the
first
and
second
reaction,
were
used
primers
Xiao
F1/
Xiao
R1
and
Xiao
F2/R2
from
the
protocol
by
Xiao
et
al.
(1999)
with
final
amplicon
826-864
bp
(depending
on
isolates).
We
made
the
third
reactions
to
higher
specificity
and
more
accurate
location
of
poly-
morphic
region
18S
rRNA
gene.
In
the
third
reaction,
we
used
VKSS
Fl
(AAT
TGG
AGG
GCA
AGT
CTG
63
as
a
forward primer;
external
primer
for
nested
PCR
from
the
protocol
by
Leetz
et
al.,
2007),
and
reverse
primer
VKSS
R2
(6TAA
ATA
CGA
AAT
GCC
CCo;
used
as
reverse
internal
primer
by
Leetz
et
al.,
2007).
The
combination
of
genus-specific
primers
VKSS
Fl/VKSS
R2
amplifies
the
gene
section
of
345-355
bp
in
length,
which
is
by
100
bp
larger
than
that
in
the
initial
reaction
by
Leetz
et
al.
(2007)
and
therefore
it
captures
a
larger
section
of
the
polymorphic
region
of
the
18S
rRNA
gene,
specific
for
the
identification
of
Cryptosporidium
spp.
Secondary
PCR
products
were
analyzed
by
electrophoresis
in
1.5%
agarose
gel
and
visualized
by
UV
light
of
wavelength
312
nm
(Xiao
et
al.,
2001).
For
confirmation
of
Cryptosporidium
spp.
after
the
nested
PCR
(VKSS
primers),
all
positive
samples
were
sent
for
sequencing.
The
sequences
were
compared
with
BLAST
to
NCBI
database
sequences.
The
samples
positive
for
Cryptosporidium
parvum
were
analyzed
again
by
nested
PCR
using
species-specific
primers
gp15
F1/
gp15
R1
(980-1000
bp),
and
gp15
F2/
gp15
F2/R2
(450
bp),
which
are
used
for
amplification
of
the
GP60
region
and
for
identification
of
genotype
and
subtype
of
C.
parvum
(Abe
et
al.,
2006)
species.
The
PCR
products
were
again
sent
for
sequencing
and
the
sequences
were
genotyped.
2.2.3.
PCR
Reaction
Mix
The
volume
of
the
PCR
reaction
mixtures
was,
in
both
cases,
50
µ1,
from
which
the
DNA
sample
was
5
µ1.
In
these
reactions,
we
used
primers
with
a
concentration
of
0.2
µM
and
5
U
Taq
DNA
polymerases
(FIREPol).
The
PCRs
were
run
in
a
thermo
cycler
(XP
Thermal
Cycler
Blocks)
with
an
initial
denaturation
at
95
°C
for
5
min,
followed
by
35
cycles
at
95
°C
for
1
min,
57/60/61/69
°C
(annealing)
for
1
min,
and
72
°C
for
2
min.
A
final
elongation
step
at
72
°C
for
7
min
was
included
to
ensure
complete
extension
of
the
amplified
products.
The
PCR
products
were
directly
sequenced
in
both
directions.
The
sequences
were
aligned
and
completed
using
Chromas
Pro
Programme
and
compared
to
known
sequences
in
the
National
Centre
for
Biotechnology
Information
GenBank
database.
The
annealing
temperatures
used
with
primers
Xiao
Fl/R1
and
Xiao
F2/Xiao
R2
were
60
and
57
°C
with
VKSS
Fl/VKSS
R2,
61
°C
with
gp15
Fl/gp
15
R1,
and
69
°C
with
gp15
F2/
gp
15
R2.
2.2.4.
Phylogenetic
Analysis
The
sequenced
data
were
processed
to
form
a
sequence
alignment
for
identifying
similarities
using
MEGA6
software
in
subdirectory
Align
with
CLUSTALW
option.
Subsequently
the
phylogenetic
tree
was
constructed
also
with
MEGA6
software
using
a
Phylogeny
menu
and
30
O.
Danilovd
et
al.
Acta
Tropica
172
(2017)
29-35
Table
1
Oyptosporidiwn
species
and
genotypes
identified
in
wild
immature/mature
rodents
(Apodemus
agrariu.s,
Apodemus
flavicolis,
Myodes
glareolus)
and
insectivore
(Sorex
araneus)
in
this
study.
Isolate
code
Location
Hosts
species
Sex
Age
Identity
at
the
gene
locus
18
S
rRNA
GP
60
1-m
Botanical
garden
Apodemus
agrarius
M
Immature
Oyptosporidiwn
sorofarwn
2-m
Botanical
garden
Apodemus
agrarius
M
Immature
Oyptosporidiwn
sorofarwn
9-m
Botanical
garden
Apodemus
agrarius
M
Immature
Oyptosporidiwn
hominis
IbAl
0G2
10-m
Botanical
garden
Apodemus
agrarius
M
Mature
Oyptosporidiwn
sorofarwn
11-m
Botanical
garden
Apodemus
agrarius
M
Immature
Oyptosporidiwn
sorofarwn
12-m
Botanical
garden
Apodemus
agrarius
M
Immature
Oyptosporidiwn
sorofarwn
14-m
Botanical
garden
Apodemus
agrarius
F
Mature
Oyptosporidiwn
sorofarwn
15-m
Botanical
garden
Apodemus
flavicollis
F
Immature
Oyptosporidiwn
sorofarwn
16-m
Botanical
garden
Apodemus
agrarius
F
Mature
Oyptosporidiwn
sorofarwn
19-m
Botanical
garden
Apodemus
agrarius
M
Immature
Oyptosporidiwn
parvwn
IIcA5G3a
43-m
Rozhanovce
Myodes
glareolus
F
Immature
Oyptosporidiwn
parvwn
BaA18G3R1
44-m
Rozhanovce
Apodemus
agrarius
M
Immature
Oyptosporidiwn
parvwn
BaA18G3R1
51-m
Rozhanovce
Apodemus
agrarius
M
Immature
Oyptosporidiwn
parvwn
BaA18G3R1
52-m
Rozhanovce
Myodes
glareolus
F
Immature
Oyptosporidiwn
parvwn
BaA18G3R1
53-m
Rozhanovce
Apodemus
agrarius
F
Immature
Oyptosporidiwn
parvwn
BaA18G3R1
60-m
Rozhanovce
Apodemus
agrarius
M
Immature
Oyptosporidiwn
parvwn
BaA18G3R1
62-m
Rozhanovce
Apodemus
agrarius
F
Immature
Oyptosporidiwn
parvwn
BaA18G3R1
65-m
Rozhanovce
Apodemus
flavicollis
F
Mature
Oyptosporidiwn
parvwn
BaA18G3R1
70-m
Rozhanovce
Apodemus
agrarius
F
Immature
Oyptosporidiwn
parvwn
BaA18G3R1
81-m
Rozhanovce
Apodemus
flavicollis
F
Mature
Oyptosporidiwn
parvwn
BaA18G3R1
83-m
Rozhanovce
Apodemus
agrarius
F
Immature
Oyptosporidiwn
parvwn
BaA18G3R1
95-m
Rozhanovce
Apodemus
agrarius
M
Immature
Oyptosporidiwn
sorofarwn
96-m
Rozhanovce
Apodemus
agrarius
F
Mature
Oyptosporidiwn
sorofarwn
97-m
Rozhanovce
Sorex
araneus
M
Immature
Oyptosporidium
sorofarwn
100-m
Nikre
Kapustniky
Apodemus
agrarius
M
Immature
Oyptosporidiwn
sorofarwn
106-m
Nikre
Kapustniky
Apodemus
agrarius
M
Immature
Oyptosporidiwn
sorofarwn
107-m
Nikre
Kapustniky
Apodemus
agrarius
F
Immature
Oyptosporidiwn
sorofarwn
108-m
Nikre
Kapustniky
Apodemus
agrarius
M
Immature
Oyptosporidiwn
sorofarwn
110-m
Nikre
Kapustniky
Apodemus
agrarius
F
Immature
Oyptosporidiwn
sorofarwn
111-m
Nikre
Kapustniky
Apodemus
agrarius
F
Mature
Oyptosporidiwn
sorofarwn
112-m
Nikre
Kapustniky
Apodemus
agrarius
F
Immature
Oyptosporidiwn
sorofarwn
122-m
Nikre
Kapustniky
Apodemus
agrarius
M
Mature
Oyptosporidiwn
sorofarwn
123-m
termer
valley
Myodes
glareolus
F
mature
Oyptosporidiwn
sorofarwn
138-m
termer
valley
Myodes
glareolus
M
Immature
Oyptosporidiwn
sorofarwn
142-m
termer
valley
Myodes
glareolus
M
Immature
Oyptosporidiwn
environment
isolate
147-m
termer
valley
Apodemus
flavicollis
M
Immature
Oyptosporidiwn
environment
isolate
148-m
termer
valley
Myodes
glareolus
F
Immature
Oyptosporidiwn
environment
isolate
149-m
termer
valley
Myodes
glareolus
F
Immature
Oyptosporidiwn
parvwn
BaA
1
0G1R1
150-m
termer
valley
Myodes
glareolus
F
Immature
Oyptosporidiwn
environment
isolate
151-m
termer
valley
Apodemus
agrarius
F
Immature
Oyptosporidiwn
environment
isolate
153-m
termer
valley
Myodes
glareolus
M
Immature
Oyptosporidiwn
environment
isolate
156-m
termer
valley
Myodes
glareolus
M
Immature
Oyptosporidiwn
sorofarwn
162-m
Hyrov-Hlboka
valley
Myodes
glareolus
F
Immature
Oyptosporidiwn
sorofarwn
165-m
Hyrov-Hlboka
valley
Myodes
glareolus
M
Immature
Oyptosporidiwn
muscrat
genotype
I
166-m
Hyrov-Hlboka
valley
Apodemus
agrarius
M
Immature
Oyptosporidiwn
sorofarwn
167-m
Hyrov-Hlboka
valley
Myodes
glareolus
M
Mature
Oyptosporidiwn
muscrat
genotype
I
169-m
Hyrov-Hlboka
valley
Myodes
glareolus
F
Immature
Oyptosporidiwn
muscrat
genotype
I
170-m
Hyrov-Hlboka
valley
Apodemus
flavicollis
M
mature
Oyptosporidiwn
parvwn
IIiA10
176-m
Hyrov-Hlboka
valley
Myodes
glareolus
M
Immature
Oyptosporidiwn
environment
isolate
177-m
Hyrov-Hlboka
valley
Apodemus
agrarius
M
Mature
Oyptosporidiwn
muscrat
genotype
II
178-m
Hyrov-Hlboka
valley
Apodemus
agrarius
F
Immature
Oyptosporidiwn
muscrat
genotype
II
179-m
Hyrov-Hlboka
valley
Apodemus
agrarius
M
Immature
Oyptosporidiwn
muscrat
genotype
II
180-m
Hyrov-Hlboka
valley
Apodemus
agrarius
F
Immature
Oyptosporidiwn
parvwn
IIiA
1
0
193-m
Hyrov-Hlboka
valley
Apodemus
flavicollis
M
Mature
Oyptosporidiwn
environment
isolate
198-m
termer
valley
Myodes
glareolus
F
Immature
Oyptosporidiwn
environment
isolate
205-m
Rozhanovce
Apodemus
agrarius
M
Mature
Oyptosporidiwn
environment
isolate
261-m
termer
valley
Apodemus
flavicollis
F
Immature
Oyptosporidiwn
suis
263-m
termer
valley
Apodemus
flavicollis
F
Mature
Oyptosporidiwn
suis
264-m
termer
valley
Apodemus
flavicollis
M
Mature
Oyptosporidiwn
suis
265-m
termer
valley
Apodemus
flavicollis
F
Mature
Oyptosporidiwn
sorofarwn
266-m
termer
valley
Apodemus
flavicollis
F
Mature
Oyptosporidiwn
suis
270-m
termer
valley
Apodemus
agrarius
F
Mature
Oyptosporidiwn
environment
isolate
271-m
termer
valley
Apodemus
flavicollis
F
Mature
Oyptosporidiwn
environment
isolate
276-m
termer
valley
Apodemus
flavicollis
M
Mature
Oyptosporidiwn
sorofarwn
277-m
termer
valley
Apodemus
flavicollis
F
Immature
Oyptosporidiwn
sorofarwn
279-m
termer
valley
Apodemus
flavicollis
F
Mature
Oyptosporidiwn
sorofarwn
maximum
likelihood
method.
and
KU311677.
The
sequence
generated
in
this
study
has
been
deposited
in
the
GenBank
database
under
accession
numbers
KU311668,
KU311669,
KU311670, KU311671, KU311672,
KU311673, KU311675,
KU311676,
31
O.
Danifovd
et
al.
Acta
Tropica
172
(2017)
29-35
2.3.
Statistical
Analysis
All
statistical
analyses
were
performed
using
MS
Excel
2003
for
Windows
XP
and
the
statistical
analysis
system
GraphPad
Prism,
version
5.01
(GraphPad
Software,
Inc.,
San
Diego,
California,
USA).
Statistical
comparison
of
categorical
variables
was
carried
out
with
the
chi-square
(x
2
)
test
or
Fisher's
exact
test
and
p
values
of
less
than
0.05
were
considered
significant.
We
tested
differences
in
prevalence
mainly
in
three
dominant
species
of
rodents
(Apodemus
agrarius,
A.
flavicollis,
and
Myodes
glareolus).
Similarly,
prevalence
was
tested
between
rodent
sexes
and
between
urban
and
suburban
areas.
3.
Results
By
using
specific
primers
in
our
modified
method,
the
samples
were
analyzed
by
nested
PCR
for
the
amplification
of
18S
rRNA
gene
for
the
species
determination,
and
amplification
of
GP60
gene
for
genotyping.
After
DNA
sequencing
and
comparison
of
the
sequences
with
the
GenBank
sequences,
we
identified
six
different
Cryptosporiclium
spp.
in
65
rodents:
Apodemus
flavicolis
(15);
Apodemus
agrarius
(34);
Myodes
glareolus
(16);
and
in
one
insectivore
(Sorex
araneus)
(Table
1).
Mean
prevalence
of
small
mammals
in
examined
material
was
24.3%,
with
the
highest
values
in
black-striped
field
mouse
(A.
agrarius).
In
next
two
species,
A.
flavicollis
and
M.
glareolus,
value
of
prevalence
were
lower
(Table
1),
but
tested
differences
among
three
dominant
rodents
were
statistical
non-significant
(X
2
=
3.847,
df
=
2,
p
=
0.146).
We
try
to
compare
differences
in
cryptosporidium
infection
between
sexes
of
dominant
rodent
species
in
examined
material
A.
agrarius
(63
males,
44
females),
A.
flavicollis
(41
males,
32
females),
and
M.
glareolus
(47
males,
31
females).
Nevertheless,
no
significant
differences
between
males
and
females
by
using
Fisher's
exact
test
were
observed
in
this
study
(A.
agrarius:
p
=
0.843;
A.
flavicollis:
p
=
0.571,
and
for
M
glareolus:
p
=
0.272).
Individual
cryptosporidium
genotypes
were
con-
firmed
so
between
mature
rodents
as
well
in
the
samples
of
immature
rodent
specimens
(Table
2).
3.1.
Kafice
Urban
Area
Together
42
samples
of
feces
in
urban
area
coming
from
two
rodent
species
were
examined
(Table
2)
with
high
mean
prevalence
on
cryptosporidium
prevalence
(42.8%).
In
the
samples
of
feces
collected
from
19
rodents
captured
in
KoNce
Botanical
garden
we
identified
C.
parvum
in
one
fecal
sample,
C.
hominis
in
one
fecal
sample
from
and
C.
scrofarum
in
eight
rodents.
C.
scrofarum
was
also
detected
in
feces
from
eight
rodents
captured
in
Niine
Kapustniky.
3.2.
Kafice
Suburban
and
Rural
Areas
A
total
of
230
feces
samples
of
eight
mammals
were
examined
from
three
localities
(Table
1).
Of
the
134
samples
collected
in
Rozhanovce,
infection
with
C.
parvum
(11).
C.
scrofarum
was
confirmed
in
two
samples
of
rodents
and
one
specimens
of
Sorex
araneus.
Sampling
performed
in
Rozhanovce
detected
also
C.
environment
sp.
in
two
rodents.
In
the
samples
collected
from
Hyrov
(Hlbolca
valley),
the
species
C.
parvum
(2),
C.
scrofarum
(2),
and
C.
environment
sp.
(2)
were
also
identified.
In
t'
ermer
valley
we
collected
fecal
samples
from
58
rodents
in
which
C.
parvum
(1),
C.
scrofarwn
(7),
and
C.
environment
sp.
(9;
KU311677)
were
identified.
In
addition
to
this
C.
suis
(KU311671)
was
identified
in
four
rodents.
The
comparison
of
prevalence
between
localities
from
urban
and
suburban
areas
was
confirmed
high
significant
difference,
i.e.
less
than
0.01
(p
=
0.005
by
Fisher's
exact
test).
In
contrast,
no
significant
differences
between
males
and
females
were
observed
in
this
study
(A.
agrarius:
p
=
0.679;
A.
flavicollis:
p
=
0.398,
and
for
M.
glareolus:
p
=
0.252).
When
identifying
the
sequence
obtained
from
Apodemus
agrarius
captured
in
Botanical
garden
(KoNce)
by
the
BLAST
program
used
for
comparison
with
GenBank
sequences
we
observed
a
percentage
agree-
ment
with
the
reference
samples
C.
hominis
(KM
215744.1),
interpreted
by
BLAST
as
the
highest
percentage
agreement.
After
the
detection
of
C.
hominis,
we
analyzed
the
sample
once
more
by
nested
PCR
with
primers
amplifying
the
GP60
gene
region.
After
sequencing,
based
on
the
order
of
repetitions,
we
identified
genotype
IbAl
0G2
(KU311668),
which
has
never
been
recorded
in
Apodemus
agrarius.
In
all
samples
positive
for
species
C.
parvum,
after
the
analysis
of
the
18S
rRNA
gene,
we
analyzed
the
GP60
gene
and
determined
genotypes
IIaA18G3R1
(n
=
11;
KU311673),
IIaA10G1R1
(n
=
1;
KU311670),
IIcA5G3a
(n
=
1;
KU311669),
and
IIiA10
(n
=
2;
KU311672).
3.3.
Phylogenetic
Analysis
3.3.1.
Phylogenic
Tree
for
Partial
Fragment
of
the
GP60
Gene
For
creating
a
phylogenetic
tree
were
used
16
sequences
identified
by
BLAST
as
Cryptosporidium
spp.,
11
reference
samples
from
NCBI,
as
the
best
matching
species
and
four
sequences
identified
in
Slovakia:
C.
parvum
IIaAl
3G1T1R1
(KT355488),
C.
hominis
IaAl
1G2R8
(KT355489),
C.
hominis
IbAl0G2T1
(KT355490),
and
C.
hominis
IbA11G2
(KT355491),
by
Petrincova
et
al.(2015;
Fig.
1).
4.
Discussion
Our
study
showed
that
in
addition
to
farm
animals
rodents
are
also
important
reservoirs
of
cryptosporidium
infections.
We
identified
six
different
Cryptosporiclium
species
in
66
fecal
samples
collected
from
rodents.
The
highest
prevalence
was
observed
for
A.
agrarius
(31.8%),
and
the
prevalence
found
in
two
other
rodents
for
A.
flavicollis
(20.5%)
and
M.
glareolus
(21.3%)
was
approximately
the
same.
In
one
case
we
confirmed
the
presence
of
Cryptosporichum
in
a
shrew
S.
araneus.
Table
2
Number
of
small
mammals
positive
for
Czyptosporidium
spp.
by
PCR
and
number
of
trapped
rodents
used
for
molecular
analysis
at
model
sites
in
years
2012-2013;
prevalence
of
infection
(%).
Species
of
small
mammals
No.
of positive
small
mammals/no.
of
total
trapped
small
mammals/prevalence
(%)
Locality
1
termer
valley
Locality
2
Hyrov
valley
Locality
3
Botanical
garden
Locality
4
Niir
Kapustnilcy
Locality
5
Rozhanovce
Total/%
Apodemus
agrarius
2/8
5/6
9/16
8/22
10/55
34/107
31.8
Apodemus
flavicollis
10/18
2/11
1/3
0/1
2/40
15/73
20.5
Rattus
norvegicus
0/0
0/0
0/0 0/0
0/4 0/4
0
Myodes
glareolus
9/28
5/17
0/0 0/0
2/30
16/75
21.3
Microtus
subtermneus
0/1
0/1
0/0 0/0
0/1
0/3
0
Sorex
araneus
0/2
0/3
0/0 0/0
1/2
1/7
14.3
Crocidura
suaveolens
0/0
0/0
0/0 0/0
0/1 0/1
0
Mustela
nivalis
0/1
0/0
0/0 0/0
0/1
0/2
0
21/58
12/38
10/19
8/23
15/134
66/272
24.3
32
O.
Danifovd
et
al.
Acta
Darden
172
(2017)
29-35
isollte
9
in,
C
hoininisibAl0G2,11CU3115581
59
C.
homing
IbAlOG2T1
IKT3554901,
isolate
front
Homo
sapiens,
Slovakia
39
P4
C
hominis
IbAl0G2
1E1727780h
isolate
from
cattle
C
hominis
IbAlOG2
IJF7277881,
isolate
from
Homo
sapiens
C.
/mms
IbAl0G4TF7277521,
isolate
from
Homo
sapiens
a
pansy?),
HcA5G2R2
IGU2143651,
isolate
from
Homo
sapiens
87
isolate
83-in,
C
pansy»,
HaA18G3R1
59
isollte
6
1
111,C.
Fumy,
HaAl8G3R1
isolate
70411,C
pansy»,
liaAISG3R1
87
isolate
65-tn,C
partsan
HaAISG3R1
isolate
81-tit,
C
pan.=
HaAISG3R1
87
Isolate
51-m,
C
pansim
HaAl8G3R1
IKU311673I
isolate
53-tn,C
pansy,
HaAl8G3R1
12
isolate
43-m,
C.
pansentIlaAl8G3R1
101
ta
60-rn,
C.
parrinn
HaAl8G3R1
isolate
52-m,
C
layman
HaAl8G3R1
88
isolite
44-m,
C
pan=
1.1aA18G3R1
72
C.
pansign
HcA5G53
IFJ839876],
isolate
from
Homo
sapiens
isohte
19-m,
C
pansy»,
11cA5G3a
1E:U3116681
C.
sp
He
clonel1315390401
100
Isolate
149-m,
C
pansan
HaALOGIR1
IKU3116701
C.
puma
HaA10G1R11109971421,
isolate
from
cattle
100
57
C.
pan=
HaAISG3R1IN36249-11,
isolate
from
cattle
59
C.
pansan
HaA1SG3R1IGQ986657l,
isolate
from
Homo
sapiens
C
parrum
IlaA18G3R1
IEF576960I,
isolate
from
Bos
taurus
C.
hominis
IbAlOG2T1
IKT3554901,
isolate
from
Homo
sapiens,
Slovakia
78
C
pampa
HaA13G1T1R1
VT3554881,
isolate
from
Homo
sapiens,
Slovakia
59
C.
hominis
IaA11G2128
103554891,
isolate
front
Homo
sapiens,
Slovakia
52
isohte
170-m,
C.
prim»
HiA10
C.
pansun
HiA10
'H8737821,
isolate
from
Homo
sapiens
IKU311673l
isohte
180-m,
C
pinyon
Hi
a10
tF
Fig.
1.
Evolutionary
relationships
among
reference
genotypes
of
C.
pcamen,
C.
hominis,
genotypes
of
C.
parvam,
C.
hominis
detected
on
Slovalda
(Petrincova
et
aL,
2015)
and
our
genotypes
of
Oyptosporidiwn
spp.
obtained
from
a
partial
fragment
of
the
GP60
gene.
The
species
most
commonly
identified
in
our
study
was
C.
scrofar-
um.
It
was
detected
in
27
samples
from
rodents
and
in
one
sample
collected
from
a
brown
shrew
(C.
scrofarwn,
accession
No.
KF
597530.1,
KJ790201.1).
Identification
of
C.
scrofarwn
at
five
sampling
places
(Botanical
garden,
Rozhanovce,
Niine
ICapustniky,
t'ermer
valley,
Holbolca
valley)
focused
out
attention
to
spreading
of
species
normally
pathogenic
to
pigs.
Wild
boars
(Sus
scrofa)
living
in
the
above
mentioned
sampling
areas
and
in
their
vicinity
may
constitute
a
focus
of
cryptosporidium
infection
caused
by
C.
suis
and
C.
scrofarum,
but
mice
may
also
act
as
a
reservoir
of
infection,
causing
cryptosporidiosis
by
this
zoonotic
species,
as
was
confirmed
in
studies
by
Li
et
al.
(2014).
Recently,
C.
scrofarum
was
not
identified
only
by
us
in
many
species
of
small
rodents,
but
also
in
weaner
pigs
and
sows
(Quilez
et
al.,
1996;
Kva'd
et
al.,
2009;
Danikova
et
al.,
2016),
as
well
as
in
rats
in
Philippines
(Ng-Hublin
et
al.,
2013),
and
was
reported
also
by
other
authors.
Furthermore,
it
should
be
stressed
that
this
species
was
not
identified
only
in
immunosuppressed
humans,
but
also
in
an
immunocompetent
one
(Kva'd
et
al.,
2009b), which
confirms
the
zoonotic
potential
of
C.
scrofarwn.
Twelve
sequences
from
samples
collected
near
water
sources
in
ermer
valley
and
Rozhanovce
were
identified
as
C.
environment
sp.,
identical
with
reference
samples
accession
No.
EF061292.1.
Unexpected
was
the
finding
of
C.
muskrat
genotype
I
and
C.
muskrat
genotype
II
in
samples
from
Hyrov
(Hlbolca
valley).
Their
sequences
were
identical
with
isolates
from
the
environment
and
from
animals
living
in
water
or
in
its
proximity
(Chalmers
et
al.,
2010;
Ruecker
et
al.,
2012;
Wilkes
et
al.,
2013).
In
animals
these
genotypes
were
identified
for
the
first
time
in
muskrats
(Ondatra
zibethicus;
Ruecker
et
al.,
2012),
after
which
they
were
named.
Shortly
afterwards,
Jellison
et
al.
(2009)
identified
both
genotypes
in
humans.
Four
different
genotypes
of
C.
parvwn
(15)
were
identified
from
four
sampling
areas
(
'able
1).
In
samples
from
11
rodents
of
different
species
from
Rozhanovce
we
identified
C.
parvum
genotype
HaA18G3R1,
which
was
detected
in
a
human
by
Jex
et
al.
(2011),
Koehler
et
al.
(2013)
in
human
and
Wielinga
et
al.
(2008)
in
cattle.
In
another
sampling
area
(t'ermer
valley)
we
detected
C.
parvum
genotype
HaAl0G1R1
in
one
species
of
small
rodents
(Myodes
glareolus).
This
genotype
was
first
identified
in
a
calf
in
Poland
(Kaupke
and
Rzeiutica,
2015).
The
study
conducted
in
children
by
Akiyoshi
et
al.
(2006)
showed
presence
of
C.
parvum
genotype
IIiA10,
which
was
found
in
our
33
O.
Danifovd
et
al.
Acta
Tropica
172
(2017)
29-35
study
in
two
samples
of
feces
collected
from
Apodemus
agrarius
(area:
Hyrov,
Hlboka
valley).
Another
surprising
finding
was
the
detection
of
species
isolated
from
small
rodents
feces
captured
in
Botanical
garden
in
Ko'Sice.
Of
the
19
samples
collected,
nine
were
positive
for
three
different
species
and
their
genotypes
(C.
parvum,
C.
hominis,
C.
scrofarum).
The
C.
parvum
genotype
IIcA5G3a,
identified
in
our
study,
was
recorded
previously
in
a
human
(Abe
et
al.,
2006;
Pangasa
et
al.,
2010)
and
in
European
hedgehog
(Erinaceus
europaneus;
Dyachenko
et
al.,
2010),
which
suggests
potential
zoonotic
transmission
between
insectivores
and
people.
The
genotype
IdAl0G2
of
C.
hominis
has
not
been
previously
described
in
rodents,
but
was
identified
in
a
European
hedgehog
(Erinaceus
europaneus)
in
Netherlands
(Krawczyk
et
al.,
2015)
and
in
humans
(Koehler
et
al.,
2013;
Pangasa
et
al.,
2010;
Waldron
et
al.,
2011).
This
information
is
alarming
due
to
almost
50%
prevalence
of
zoonotic
species
of
Cryptosporidium
in
small
rodents
in
the
areas
where
the
visiting
rates
and
movement
of
people
(especially
children)
are
high
as
this
can
lead
to
additional
spreading
of
the
infection.
All
genotypes
of
the
species
C.
parvum
and
C.
hominis
identified
in
small
rodents
(Fig.
1)
were
detected
in
people,
rodents
and
insectivores
in
many
different
places
(the
above
citations)
which
raises
concern
about
zoonotic
transmission
in
which
rodents
and
insectivores
serve
as
a
vector
of
cryptosporidiosis
as
it
was
demonstrated
in
this
study.
Worldwide,
the
prevalence
of
infection
is
1-62%
in
mice
and
2.3-32.8%
in
rats
(Yamura
et
al.,
1990;
Sinski
et
al.,
1993;
Chalmers
et
al.,
1997;
Chilvers
et
al.,
1998;
Kimura
et
al.,
2007;
Lv
et
al.,
2009).
The
most
commonly
described
were
BALD
and
SCID
mice
experimen-
tally
infected
with
C.
muris
and
C.parvum
(Miller
et
al.,
2007;
Codices
et
al.,
2013).
Compared
with
other
studies,
the
positivity
in
our
study
reached
24.3%,
in
urban
habitats
reached
infectivity
values
up
42%
in
our
material,
which
indicates
the
importance
of
this
study,
although
it
was
conducted
in
a
relatively
small
area
of
Slovakia,
and
the
significance
of
wild
rodents
acting
as
a
source
of
cryptosporidium
infection.
5.
Conclusion
The
detection
and
identification
of
such
diversity
of
individual
species
of
Cryptosporidium
in
a
group
composed
of
272
samples
of
feces
from
rodents
and
insectivores
is
alarming.
Mainly
the
identification
of
zoonotic
species
C.
parvum,
C.
hominis,
C.
scrofarum,
and
C.
suis
focuses
the
attention
on
small
mammals
as
an
important
source
of
cryptospor-
idiosis.
Rodents
are
commonly
situated
near
farm
animals,
drinking
water
sources,
and
living
spaces.
Although
the
epidemiologic
studies
identi-
fied
rodents
and
some
insectivores
as
reservoirs
of
zoonotic
Cryptosporidium
species,
monitoring
of
their
role
in
spreading
of
this
infection
is
insufficient
which
may
be
related
to
difficulties
with
their
capture.
The
results
of
our
study
showed
that
rodents
(regardless
of
the
rodent
species;
Table
1)
can
be
important
sources
of
Cryptosporidiosis-
causing
pathogens
and
thus
have
serious
impact
on
public
health.
Competing
Interests
The
authors
declare
that
they
have
no
competing
interests.
Acknowledgments
The
study
was
supported
by
grants
VEGA
No.1/0063/13,
APVV-15-
0134
and
APVV-14-0274.
The
authors
thank
to
L.
Mo'SanskST,
J.
Kraljik
and
M.
Onderova
for
their
assistance
during
field
work.
The
small
mammals
were
handled
in
compliance
with
the
legislative
provisions
effective
in
the
Slovak
Republic,
under
the
licenses
of
the
Ministry
of
Environment
of
the
Slovak
Republic
No.
6743/2008-2.1
and
No.
4874/
2011-2.2.
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