Accepted Manuscript Title: Molecular identification of Cryptosporidium spp. and Giardia duodenalis in grazing horses from Xinjiang, China Author: Meng Qi Huan Zhou Haiyan Wang Rongjun Wang Lihua Xiao Michael J. Arrowood Junqiang Li Longxian Zhang PII: DOI: Reference:
S0304-4017(15)00103-X http://dx.doi.org/doi:10.1016/j.vetpar.2015.02.030 VETPAR 7554
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
7-12-2014 26-2-2015 27-2-2015
Please cite this article as: Qi, M., Zhou, H., Wang, H., Wang, R., Xiao, L., Arrowood, M.J., Li, J., Zhang, L.,Molecular identification of Cryptosporidium spp. and Giardia duodenalis in grazing horses from Xinjiang, China, Veterinary Parasitology (2015), http://dx.doi.org/10.1016/j.vetpar.2015.02.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlight
1 2
4
1. This is the first report of Cryptosporidium and G. duodenalis in grazing horses from China.
ip t
3
2. Sequence analyses of 18S rRNA and gp60 genes revealed that seven horses were
6
positive for the presence of subtype VIaA15G4 of the Cryptosporidium horse
7
genotype.
9
us
3. G. duodenalis assemblages A and B were identified by using the 16S rRNA and
an
8
cr
5
tpi genes.
M
10
Ac ce p
te
d
11
1
Page 1 of 13
11
Molecular identification of Cryptosporidium spp. and Giardia duodenalis in
12
grazing horses from Xinjiang, China
13
Meng Qia,b, Huan Zhoua,b, Haiyan Wangc, Rongjun Wanga,b, Lihua Xiaod, Michael J.
15
Arrowoodd , Junqiang Lia,b, Longxian Zhanga.b
cr
ip t
14
us
16 17
19
a
20
Zhengzhou 450002, P. R. China
21
b
22
Zhengzhou 450002, P. R. China
23
c
24
451450, Henan, P. R. China
26 27 28
M
College of Animal Science and Veterinary Medicine, Henan Agricultural University,
te
d
International Joint Research Laboratory for Zoonotic Diseases of Henan,
Department of Animal Science, Henan Vocational College of Agriculture, Zhongmu
Ac ce p
25
an
18
d
Division of Foodborne, Waterborne, and Environmental Diseases, National Center
for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
29
*
30
Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002,
31
P. R. China. Tel.: +86-371-63555689; fax: +86-371-63558180. E-mail address:
32
[email protected]; [email protected]
Corresponding author. Mailing address for Longxian Zhang: College of Animal
2
Page 2 of 13
ABSTRACT
34
A total of 262 fecal specimens collected from grazing horses at five locations in
35
Xinjiang, China were examined by PCR for Cryptosporidium spp. and Giardia
36
duodenalis. The Cryptosporidium and G. duodenalis infection rates were 2.7% and
37
1.5%, respectively. Seven Cryptosporidium-positive specimens were found in foals
38
(16.3%), and four G. duodenalis-positive specimens were found in mares (2.5%).
39
Sequence analyses of 18S rRNA and gp60 genes revealed that seven animals were
40
positive for the subtype VIaA15G4 of Cryptosporidium horse genotype. Giardia
41
duodenalis assemblages A and B were identified by molecular characterization of the
42
16S rRNA and tpi genes. This is the first report of Cryptosporidium horse genotype
43
and G. duodenalis in grazing horses from China.
44
Key words: Assemblage, Genotype, Cryptosporidium, Giardia duodenalis, Horses,
45
PCR
47 48 49 50
cr
us
an
M
d
te
Ac ce p
46
ip t
33
51 52 53 54 3
Page 3 of 13
55
1. Introduction Cryptosporidium spp. and Giardia duodenalis (syn. G. lamblia, G. intestinalis)
57
are the causative agents of gastrointestinal illnesses in humans and other vertebrate
58
animals (Caffara et al., 2013; Wang et al., 2014). Horse cryptosporidiosis is
59
considered to be a potential cause of diarrhea in foals of a few weeks of age (Grinberg
60
et al., 2008; Perrucci et al., 2011). Giardia was first reported as a parasite of horses in
61
South Africa in 1921, but infected horses rarely show any clinical signs of giardiasis
62
(Santín et al., 2013).
an
us
cr
ip t
56
Most reports of cryptosporidiosis and giardiasis in horses are based on
64
microscopic analysis of fecal specimens (Atwill et al., 2000; de Souza et al., 2009).
65
Recently, the ability to identify and diagnose pathogens at the species or genotype
66
level using molecular tools has improved our understanding of their transmission.
67
Polymerase chain reaction (PCR)-based molecular techniques have identified the
68
following Cryptosporidium species and genotypes in horses: C. parvum, C. erinacei
70 71 72
d
te
Ac ce p
69
M
63
and Cryptosporidium horse genotype (Grinberg et al., 2008; Burton et al., 2010; Kváč et al., 2014). Molecular characterization has identified eight assemblages (A–H) of G. duodenalis, and assemblages A, B, and E have been detected in horses (Traub et al., 2005; Veronesi et al., 2010; Santín et al., 2013).
73
Although molecular methods have recently been used to determine the
74
prevalence of Cryptosporidium spp. and G. duodenalis in horses, information
75
regarding their prevalence, species/genotypes, and zoonotic potential in horses
76
remains poorly understood. The purpose of this study was to investigate the 4
Page 4 of 13
distribution of Cryptosporidium species/genotypes and G. duodenalis assemblages in
78
horses from Xinjiang, China using molecular methods, and use the data obtained to
79
gain a better understanding of cryptosporidiosis and giardiasis in horses thereby
80
allowing a full assessment of the zoonotic potential of these parasites.
81
2. Materials and methods
82
2.1. Ethics statement
us
cr
ip t
77
This study was performed strictly according to the recommendations of the
84
Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China.
85
The research protocol was reviewed and approved by the Research Ethics Committee
86
of the Henan Agricultural University. Prior to fecal specimen collection, appropriate
87
permission was obtained from the animal owners whenever possible. The field studies
88
did not involve endangered or protected species.
89
2.2. Study area and sample collection
91 92 93 94
M
d
te
From August to September 2013, fresh fecal specimens were collected from
Ac ce p
90
an
83
horses in Wulumuqi, Kuerle, Tacheng, Aletai, and Yili cities in the Xinjiang Uyghur Autonomous Region (73°40’E–96°18’E, 34°25’N–48°10’N) of northwest China. The average altitude, annual temperature, and annual rainfall of the five cities vary from 548 m to 918 m, 4.2°C to 11.4°C, and 58.6 mm to 417 mm, respectively. Horses in the
95
areas were kept in pastures. A total of 262 fecal specimens were collected from the
96
horses. Fresh feces were collected from the ground if the animal was observed to
97
defecate, with care taken to avoid environmental contamination by sampling only
98
those portions of the fecal material that had not been in contact with the ground. No 5
Page 5 of 13
99
obvious clinical signs were observed in the sampled animals. Samples were
100
maintained at 4°C until DNA was extracted from them.
101
2.3. DNA extraction Ten grams feces of each sample were thoroughly mixed with 30 ml dH2O. The
103
suspension was passed through a 250 μm pore size wire mesh sieve and centrifuged
104
at 3000g for 5 min. The precipitates were used for DNA extraction. Genomic DNA
105
was extracted from 200mg precipitates of each sample using an E.Z.N.A.R® Stool
106
DNA Kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer’s
107
instructions. The extracted DNA was stored at −20°C.
108
2.4. PCR amplification
M
an
us
cr
ip t
102
PCR was used to differentiate Cryptosporidium species in the fecal specimens
110
by analysis of a ~830-bp fragment of the small subunit rRNA (18S rRNA) gene
111
(Alves et al., 2003). Cryptosporidium horse genotype was subtyped by sequence
112
analysis of the 60-kDa glycoprotein gene, gp 60 (Jiang et al., 2005).
114 115 116
te
Ac ce p
113
d
109
Giardia duodenalis genotypes were determined by nested PCR amplification of
the small subunit rRNA (16S rRNA) for each specimen (Wang et al., 2014). DNA from all 16S rRNA-positive specimens was subjected to PCR to amplify fragments of the triose phosphate isomerase (tpi) genes according to a previously published
117
method (Wang et al., 2014).
118
2.5. Sequence analysis
119
All secondary PCR amplicons were sequenced on an ABI PRISM™ 3730 XL
120
DNA Analyzer using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied 6
Page 6 of 13
Biosystems, Foster City, CA, USA). The sequencing accuracy was confirmed by
122
two-directional sequencing. Sequences were identified by alignment with reference
123
sequences downloaded from GenBank (http://www.ncbi.nlm.nih.gov) using MEGA 5
124
software (http://www.megasoftware.net/). The nucleotide sequences obtained in this
125
study
126
KM588592–KM588595.
127
2.6. Statistical analysis
in
GenBank
under
accession
numbers
cr
deposited
us
been
The χ2 test was used to compare the Cryptosporidium and G. duodenalis
an
128
have
ip t
121
infection rates and the differences were considered significant when p < 0.05.
130
3. Results and discussion
M
129
Of the 262 specimens analyzed, seven were Cryptosporidium positive (2.7%;
132
95% CI: 2.7 ± 0.9%) and four were G. duodenalis positive (1.5%; 95% CI: 2.7 ±
133
0.7%). Cryptosporidium prevalence among the collection sites showed a statistically
134
significant (χ2=46.65, p
S0304-4017(15)00103-X http://dx.doi.org/doi:10.1016/j.vetpar.2015.02.030 VETPAR 7554
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
7-12-2014 26-2-2015 27-2-2015
Please cite this article as: Qi, M., Zhou, H., Wang, H., Wang, R., Xiao, L., Arrowood, M.J., Li, J., Zhang, L.,Molecular identification of Cryptosporidium spp. and Giardia duodenalis in grazing horses from Xinjiang, China, Veterinary Parasitology (2015), http://dx.doi.org/10.1016/j.vetpar.2015.02.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlight
1 2
4
1. This is the first report of Cryptosporidium and G. duodenalis in grazing horses from China.
ip t
3
2. Sequence analyses of 18S rRNA and gp60 genes revealed that seven horses were
6
positive for the presence of subtype VIaA15G4 of the Cryptosporidium horse
7
genotype.
9
us
3. G. duodenalis assemblages A and B were identified by using the 16S rRNA and
an
8
cr
5
tpi genes.
M
10
Ac ce p
te
d
11
1
Page 1 of 13
11
Molecular identification of Cryptosporidium spp. and Giardia duodenalis in
12
grazing horses from Xinjiang, China
13
Meng Qia,b, Huan Zhoua,b, Haiyan Wangc, Rongjun Wanga,b, Lihua Xiaod, Michael J.
15
Arrowoodd , Junqiang Lia,b, Longxian Zhanga.b
cr
ip t
14
us
16 17
19
a
20
Zhengzhou 450002, P. R. China
21
b
22
Zhengzhou 450002, P. R. China
23
c
24
451450, Henan, P. R. China
26 27 28
M
College of Animal Science and Veterinary Medicine, Henan Agricultural University,
te
d
International Joint Research Laboratory for Zoonotic Diseases of Henan,
Department of Animal Science, Henan Vocational College of Agriculture, Zhongmu
Ac ce p
25
an
18
d
Division of Foodborne, Waterborne, and Environmental Diseases, National Center
for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
29
*
30
Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002,
31
P. R. China. Tel.: +86-371-63555689; fax: +86-371-63558180. E-mail address:
32
[email protected]; [email protected]
Corresponding author. Mailing address for Longxian Zhang: College of Animal
2
Page 2 of 13
ABSTRACT
34
A total of 262 fecal specimens collected from grazing horses at five locations in
35
Xinjiang, China were examined by PCR for Cryptosporidium spp. and Giardia
36
duodenalis. The Cryptosporidium and G. duodenalis infection rates were 2.7% and
37
1.5%, respectively. Seven Cryptosporidium-positive specimens were found in foals
38
(16.3%), and four G. duodenalis-positive specimens were found in mares (2.5%).
39
Sequence analyses of 18S rRNA and gp60 genes revealed that seven animals were
40
positive for the subtype VIaA15G4 of Cryptosporidium horse genotype. Giardia
41
duodenalis assemblages A and B were identified by molecular characterization of the
42
16S rRNA and tpi genes. This is the first report of Cryptosporidium horse genotype
43
and G. duodenalis in grazing horses from China.
44
Key words: Assemblage, Genotype, Cryptosporidium, Giardia duodenalis, Horses,
45
PCR
47 48 49 50
cr
us
an
M
d
te
Ac ce p
46
ip t
33
51 52 53 54 3
Page 3 of 13
55
1. Introduction Cryptosporidium spp. and Giardia duodenalis (syn. G. lamblia, G. intestinalis)
57
are the causative agents of gastrointestinal illnesses in humans and other vertebrate
58
animals (Caffara et al., 2013; Wang et al., 2014). Horse cryptosporidiosis is
59
considered to be a potential cause of diarrhea in foals of a few weeks of age (Grinberg
60
et al., 2008; Perrucci et al., 2011). Giardia was first reported as a parasite of horses in
61
South Africa in 1921, but infected horses rarely show any clinical signs of giardiasis
62
(Santín et al., 2013).
an
us
cr
ip t
56
Most reports of cryptosporidiosis and giardiasis in horses are based on
64
microscopic analysis of fecal specimens (Atwill et al., 2000; de Souza et al., 2009).
65
Recently, the ability to identify and diagnose pathogens at the species or genotype
66
level using molecular tools has improved our understanding of their transmission.
67
Polymerase chain reaction (PCR)-based molecular techniques have identified the
68
following Cryptosporidium species and genotypes in horses: C. parvum, C. erinacei
70 71 72
d
te
Ac ce p
69
M
63
and Cryptosporidium horse genotype (Grinberg et al., 2008; Burton et al., 2010; Kváč et al., 2014). Molecular characterization has identified eight assemblages (A–H) of G. duodenalis, and assemblages A, B, and E have been detected in horses (Traub et al., 2005; Veronesi et al., 2010; Santín et al., 2013).
73
Although molecular methods have recently been used to determine the
74
prevalence of Cryptosporidium spp. and G. duodenalis in horses, information
75
regarding their prevalence, species/genotypes, and zoonotic potential in horses
76
remains poorly understood. The purpose of this study was to investigate the 4
Page 4 of 13
distribution of Cryptosporidium species/genotypes and G. duodenalis assemblages in
78
horses from Xinjiang, China using molecular methods, and use the data obtained to
79
gain a better understanding of cryptosporidiosis and giardiasis in horses thereby
80
allowing a full assessment of the zoonotic potential of these parasites.
81
2. Materials and methods
82
2.1. Ethics statement
us
cr
ip t
77
This study was performed strictly according to the recommendations of the
84
Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China.
85
The research protocol was reviewed and approved by the Research Ethics Committee
86
of the Henan Agricultural University. Prior to fecal specimen collection, appropriate
87
permission was obtained from the animal owners whenever possible. The field studies
88
did not involve endangered or protected species.
89
2.2. Study area and sample collection
91 92 93 94
M
d
te
From August to September 2013, fresh fecal specimens were collected from
Ac ce p
90
an
83
horses in Wulumuqi, Kuerle, Tacheng, Aletai, and Yili cities in the Xinjiang Uyghur Autonomous Region (73°40’E–96°18’E, 34°25’N–48°10’N) of northwest China. The average altitude, annual temperature, and annual rainfall of the five cities vary from 548 m to 918 m, 4.2°C to 11.4°C, and 58.6 mm to 417 mm, respectively. Horses in the
95
areas were kept in pastures. A total of 262 fecal specimens were collected from the
96
horses. Fresh feces were collected from the ground if the animal was observed to
97
defecate, with care taken to avoid environmental contamination by sampling only
98
those portions of the fecal material that had not been in contact with the ground. No 5
Page 5 of 13
99
obvious clinical signs were observed in the sampled animals. Samples were
100
maintained at 4°C until DNA was extracted from them.
101
2.3. DNA extraction Ten grams feces of each sample were thoroughly mixed with 30 ml dH2O. The
103
suspension was passed through a 250 μm pore size wire mesh sieve and centrifuged
104
at 3000g for 5 min. The precipitates were used for DNA extraction. Genomic DNA
105
was extracted from 200mg precipitates of each sample using an E.Z.N.A.R® Stool
106
DNA Kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer’s
107
instructions. The extracted DNA was stored at −20°C.
108
2.4. PCR amplification
M
an
us
cr
ip t
102
PCR was used to differentiate Cryptosporidium species in the fecal specimens
110
by analysis of a ~830-bp fragment of the small subunit rRNA (18S rRNA) gene
111
(Alves et al., 2003). Cryptosporidium horse genotype was subtyped by sequence
112
analysis of the 60-kDa glycoprotein gene, gp 60 (Jiang et al., 2005).
114 115 116
te
Ac ce p
113
d
109
Giardia duodenalis genotypes were determined by nested PCR amplification of
the small subunit rRNA (16S rRNA) for each specimen (Wang et al., 2014). DNA from all 16S rRNA-positive specimens was subjected to PCR to amplify fragments of the triose phosphate isomerase (tpi) genes according to a previously published
117
method (Wang et al., 2014).
118
2.5. Sequence analysis
119
All secondary PCR amplicons were sequenced on an ABI PRISM™ 3730 XL
120
DNA Analyzer using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied 6
Page 6 of 13
Biosystems, Foster City, CA, USA). The sequencing accuracy was confirmed by
122
two-directional sequencing. Sequences were identified by alignment with reference
123
sequences downloaded from GenBank (http://www.ncbi.nlm.nih.gov) using MEGA 5
124
software (http://www.megasoftware.net/). The nucleotide sequences obtained in this
125
study
126
KM588592–KM588595.
127
2.6. Statistical analysis
in
GenBank
under
accession
numbers
cr
deposited
us
been
The χ2 test was used to compare the Cryptosporidium and G. duodenalis
an
128
have
ip t
121
infection rates and the differences were considered significant when p < 0.05.
130
3. Results and discussion
M
129
Of the 262 specimens analyzed, seven were Cryptosporidium positive (2.7%;
132
95% CI: 2.7 ± 0.9%) and four were G. duodenalis positive (1.5%; 95% CI: 2.7 ±
133
0.7%). Cryptosporidium prevalence among the collection sites showed a statistically
134
significant (χ2=46.65, p
