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Molecular identification of Cryptosporidium spp. and Giardia duodenalis in grazing horses from Xinjiang, China
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
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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
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From August to September 2013, fresh fecal specimens were collected from
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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
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cr
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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
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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<0.01), as did the prevalence of G. duodenalis (χ2=10.29,
136 137 138
te
Ac ce p
135
d
131
0.01< p<0.05). All Cryptosporidium-positive specimens were from foals, whereas all G. duodenalis-positive specimens were from mares. Cryptosporidium prevalence was significantly associated with age (χ2=36.63, p<0.01). The prevalence of G. duodenalis was not significantly associated with sex (χ2=1.47, p>0.05) or age (χ2=0.80, p >0.05).
139
These findings are in agreement with other studies on horses, where the prevalence of
140
Cryptosporidium oocysts and G. duodenalis cysts varied from 1–31% and 0–35%,
141
respectively (Atwill et al., 2000; Burton et al., 2010; Santín et al., 2013). Similar to our
142
results, previous studies have shown that foals are more frequently infected with 7
Page 7 of 13
Cryptosporidium than older horses (de Souza et al., 2009; Perrucci et al., 2011). In the
144
present study, there was no statistically significant infection prevalence of G.
145
duodenalis based on age, a finding similar to those from studies on horses in the USA
146
(Santín et al., 2013). 18S
rRNA
gene
sequence
analysis
revealed
that
the
seven
cr
147
ip t
143
Cryptosporidium-positive samples belonged to the Cryptosporidium horse genotype.
149
They were further subtyped by gp60 sequence analysis as VIaA15G4, and their
150
sequences were identical to a foal isolated from Italy (KC559773). Similar to the
151
results of the present study, Cryptosporidium horse genotype was also found in foals
152
from New York State (Burton et al., 2010) and Italy (Caffara et al., 2013). In the few
153
genotyping studies conducted in horses to date, C. parvum was the most common
154
Cryptosporidium species identified, and is considered to be the most important
155
species involved in non-bacterial foal diarrhea (Grinberg et al., 2008; Perrucci et al.,
156
2011). However, in common with a study on horses from New York State, C. parvum
158 159 160
an
M
d
te
Ac ce p
157
us
148
was not found in horses in the present study. The Cryptosporidium horse genotype identified in a Przewalski’s wild horse foal was initially considered to be an equine-specific genotype (Burton et al., 2010); however, it has been found in patients with diarrhea (Caffara et al., 2013), suggesting that this genotype may pose a potential
161
zoonotic threat to human health. Based on phylogenetic analysis of gp60 sequences,
162
the following two subtype families (VIa and VIb) are now recognized as
163
Cryptosporidium horse genotype. In the present study, the gp60 gene sequences
164
showed that the genotype present in horses was subtype VIaA15G4, and this subtype 8
Page 8 of 13
165
has been reported in foals from Italy. Additionally, C. erinacei originally found in
166
hedgehogs, and it had also been found in horses (Kváč et al., 2014), therefore, an
167
example of host specificity being broader than previously thought. 16S rRNA gene sequence analysis of the four G. duodenalis PCR-positive
169
specimens identified as two assemblage A and two assemblage B. From the
170
nucleotide sequences of the tpi gene obtained from two horses, one was identified as
171
assemblage A and the other as assemblage B. These findings are similar to those
172
reported for horses from Colombia (Santín et al., 2013) and Western Australia
173
(Traub et al., 2005).The host specificity of G. duodenalis in horses is unclear
174
because the data for molecular characterization of G. duodenalis isolates from horses
175
are limited. An Italian study reported that all 20 G. duodenalis-positive isolates from
176
horses were assemblage E based on analysis of the 16S rRNA gene (Veronesi et al.,
177
2010). However, other surveys in Italy showed that of the 37 isolates identified using
178
16S rRNA and β-giardin(bg) genes, 16, 11, and 6 were identified as assemblages A,
180 181 182
cr
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an
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d
te
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179
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B, and E, respectively (Traversa et al., 2012). Consequently, horses might contribute to environmental contamination by shedding Giardia cysts, which is a potential zoonotic risk, especially around watershed areas (Traub et al., 2005). In the present study, isolates from two horses were genetically characterized at the tpi genes; one
183
isolate was identified as assemblage A and is identical to a wastewater isolate
184
(AY368157) from Milwaukee, USA, while the other, identified as assemblage B,
185
differs by two single nucleotide polymorphisms from a wastewater isolate
9
Page 9 of 13
186
(AY368167) from the same city. These findings support the standpoint that G.
187
duodenalis from horses poses a potential threat to human health. In conclusion, the presence of Cryptosporidium spp. and G. duodenalis in horses
189
provides new data on the possible role played by these animals as a source of
190
infection to human beings. Molecular tools are useful for understanding the
191
epidemiology of Cryptosporidium spp. and G. duodenalis parasites in horses. In
192
future studies, the data from such tools should contribute to a better understanding of
193
the population genetic substructure of Cryptosporidium spp. and G. duodenalis in
194
horses and their zoonotic potential.
M
195
an
us
cr
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188
Acknowledgments
197
This study was supported in part by the State Key Program of National Natural
198
Science Foundation of China (No. 31330079), Specialized Research Fund for the
199
Doctoral Program of Higher Education (No. 20124105120003), and the National
201 202 203
te
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200
d
196
Natural Science Foundation of China (Nos. U1204328, 31302079).
Conflict of interest
The authors declare no conflicts of interest.
204 205
References
206
Alves, M., Xiao, L., Sulaiman, I., Lal, A.A., Matos, O., Antunes, F., 2003.
207
Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo 10
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ruminants in Portugal. J. Clin. Microbiol. 41, 2744–2747. Atwill, E.R., McDougald, N.K., Perera, L., 2000. Cross-sectional study of faecal
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shedding of Giardia duodenalis and Cryptosporidium parvum among packstock
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in the Sierra Nevada Range. Equine. Vet. J. 32, 247–252.
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Burton, A.J., Nydam, D.V., Dearen, T.K., Mitchell, K., Bowman, D.D., Xiao, L.,
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2010. The prevalence of Cryptosporidium, and identification of the
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Cryptosporidium horse genotype in foals in New York State. Vet. Parasitol. 174,
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139–144.
an
us
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Caffara, M., Piva, S., Pallaver, F., Iacono, E., Galuppi, R., 2013. Molecular
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characterization of Cryptosporidium spp. from foals in Italy. Vet. J. 198,
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531–533.
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de Souza, P.N., Bomfim, T.C., Huber, F., Abboud, L.C., Gomes, R.S., 2009. Natural
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infection by Cryptosporidium sp., Giardia sp. and Eimeria leuckarti in three
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groups of equines with different handlings in Rio de Janeiro, Brazil. Vet.
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Parasitol. 160, 327–333.
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Kváč, M., Hofmannová, L., Hlásková, L., Květoňová, D., Vítovec, J., McEvoy, J.,
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Sak,
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Cryptosporidiidae) in hedgehogs. Vet. Parasitol. 201, 9–17.
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Cryptosporidium parvum infection in a mare and her foal with foal heat
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diarrhoea. Vet. Parasitol. 182, 333–336.
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Santín, M., Cortés Vecino, JA., Fayer, R., 2013. A large scale molecular study of Giardia duodenalis in horses from Colombia. Vet. Parasitol. 196, 31–36. Traub, R., Wade, S., Read, C., Thompson, A., Mohammed, H., 2005. Molecular
an
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characterization of potentially zoonotic isolates of Giardia duodenalis in horses.
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Traversa, D., Otranto, D., Milillo, P., Latrofa, M.S., Giangaspero, A., Di Cesare, A.,
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Paoletti, B., 2012. Giardia duodenalis sub-Assemblage of animal and human
242
origin in horses. Infect. Genet. Evol. 12, 1642–1646.
244 245 246 247
te
Veronesi, F., Passamonti, F., Cacciò, S.M., Diaferia, M., Piergili Fioretti, G., 2010.
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Epidemiological survey on equine Cryptosporidium and Giardia infections in Italy and molecular characterization of isolates. Zoonoses. Public. Health. 57, 510–517.
Wang, H., Zhao, G., Chen, G., Jian, F., Zhang, S., Feng, C., Wang, R., Zhu, J., Dong,
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H., Hua, J., Wang, M., Zhang, L., 2014. Multilocus genotyping of Giardia
249
duodenalis in dairy cattle in Henan, China. PLoS. One. 9, e100453.
250 251 12
Page 12 of 13
Table 1. Prevalence of Cryptosporidium spp. and Giardia in horses from Xinjiang Giardia positive (%)
Mare
Stallion
Foal
Mare
Stallion
80
0/57(0)
0/17(0)
0/6(0)
0/57(0)
0/17(0)
Kuerle
7
Tacheng
22
Aletai Yili
0/16(0)
0/6(0)
2/16(12.5) 0/6(0)
35
0/18(0)
0/5(0)
7/12(58.3)
0/18(0)
0/5(0)
0/12(0)
118
0/70(0)
0/23(0)
0/25(0)
2/70(2.9)
0/23(0)
0/25(0)
0/161(0)
0/58(0)
4/161(2.5) 0/58(0)
0/43(0)
262
257 258 259 260
us
7/43(16.3)
te
d
4/262(1.5)
Ac ce p
256
cr
0/7(0)
253
255
0/6(0)
0/7(0)
7/262(2.7)
254
Foal
ip t
Wulumuqi
Total
Cryptosporidium positive (%) No. samples
an
Location
M
252
13
Page 13 of 13
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
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2.1. Ethics statement
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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.
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2.2. Study area and sample collection
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From August to September 2013, fresh fecal specimens were collected from
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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
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obvious clinical signs were observed in the sampled animals. Samples were
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maintained at 4°C until DNA was extracted from them.
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2.3. DNA extraction Ten grams feces of each sample were thoroughly mixed with 30 ml dH2O. The
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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
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DNA Kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer’s
107
instructions. The extracted DNA was stored at −20°C.
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2.4. PCR amplification
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PCR was used to differentiate Cryptosporidium species in the fecal specimens
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by analysis of a ~830-bp fragment of the small subunit rRNA (18S rRNA) gene
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(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).
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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).
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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
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two-directional sequencing. Sequences were identified by alignment with reference
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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
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study
126
KM588592–KM588595.
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2.6. Statistical analysis
in
GenBank
under
accession
numbers
cr
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The χ2 test was used to compare the Cryptosporidium and G. duodenalis
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infection rates and the differences were considered significant when p < 0.05.
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3. Results and discussion
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Of the 262 specimens analyzed, seven were Cryptosporidium positive (2.7%;
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95% CI: 2.7 ± 0.9%) and four were G. duodenalis positive (1.5%; 95% CI: 2.7 ±
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0.7%). Cryptosporidium prevalence among the collection sites showed a statistically
134
significant (χ2=46.65, p<0.01), as did the prevalence of G. duodenalis (χ2=10.29,
136 137 138
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0.01< p<0.05). All Cryptosporidium-positive specimens were from foals, whereas all G. duodenalis-positive specimens were from mares. Cryptosporidium prevalence was significantly associated with age (χ2=36.63, p<0.01). The prevalence of G. duodenalis was not significantly associated with sex (χ2=1.47, p>0.05) or age (χ2=0.80, p >0.05).
139
These findings are in agreement with other studies on horses, where the prevalence of
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Cryptosporidium oocysts and G. duodenalis cysts varied from 1–31% and 0–35%,
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respectively (Atwill et al., 2000; Burton et al., 2010; Santín et al., 2013). Similar to our
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results, previous studies have shown that foals are more frequently infected with 7
Page 7 of 13
Cryptosporidium than older horses (de Souza et al., 2009; Perrucci et al., 2011). In the
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present study, there was no statistically significant infection prevalence of G.
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duodenalis based on age, a finding similar to those from studies on horses in the USA
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(Santín et al., 2013). 18S
rRNA
gene
sequence
analysis
revealed
that
the
seven
cr
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Cryptosporidium-positive samples belonged to the Cryptosporidium horse genotype.
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They were further subtyped by gp60 sequence analysis as VIaA15G4, and their
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sequences were identical to a foal isolated from Italy (KC559773). Similar to the
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results of the present study, Cryptosporidium horse genotype was also found in foals
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from New York State (Burton et al., 2010) and Italy (Caffara et al., 2013). In the few
153
genotyping studies conducted in horses to date, C. parvum was the most common
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Cryptosporidium species identified, and is considered to be the most important
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species involved in non-bacterial foal diarrhea (Grinberg et al., 2008; Perrucci et al.,
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2011). However, in common with a study on horses from New York State, C. parvum
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was not found in horses in the present study. The Cryptosporidium horse genotype identified in a Przewalski’s wild horse foal was initially considered to be an equine-specific genotype (Burton et al., 2010); however, it has been found in patients with diarrhea (Caffara et al., 2013), suggesting that this genotype may pose a potential
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zoonotic threat to human health. Based on phylogenetic analysis of gp60 sequences,
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the following two subtype families (VIa and VIb) are now recognized as
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Cryptosporidium horse genotype. In the present study, the gp60 gene sequences
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showed that the genotype present in horses was subtype VIaA15G4, and this subtype 8
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has been reported in foals from Italy. Additionally, C. erinacei originally found in
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hedgehogs, and it had also been found in horses (Kváč et al., 2014), therefore, an
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example of host specificity being broader than previously thought. 16S rRNA gene sequence analysis of the four G. duodenalis PCR-positive
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specimens identified as two assemblage A and two assemblage B. From the
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nucleotide sequences of the tpi gene obtained from two horses, one was identified as
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assemblage A and the other as assemblage B. These findings are similar to those
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reported for horses from Colombia (Santín et al., 2013) and Western Australia
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(Traub et al., 2005).The host specificity of G. duodenalis in horses is unclear
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because the data for molecular characterization of G. duodenalis isolates from horses
175
are limited. An Italian study reported that all 20 G. duodenalis-positive isolates from
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horses were assemblage E based on analysis of the 16S rRNA gene (Veronesi et al.,
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2010). However, other surveys in Italy showed that of the 37 isolates identified using
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16S rRNA and β-giardin(bg) genes, 16, 11, and 6 were identified as assemblages A,
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B, and E, respectively (Traversa et al., 2012). Consequently, horses might contribute to environmental contamination by shedding Giardia cysts, which is a potential zoonotic risk, especially around watershed areas (Traub et al., 2005). In the present study, isolates from two horses were genetically characterized at the tpi genes; one
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isolate was identified as assemblage A and is identical to a wastewater isolate
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(AY368157) from Milwaukee, USA, while the other, identified as assemblage B,
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differs by two single nucleotide polymorphisms from a wastewater isolate
9
Page 9 of 13
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(AY368167) from the same city. These findings support the standpoint that G.
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duodenalis from horses poses a potential threat to human health. In conclusion, the presence of Cryptosporidium spp. and G. duodenalis in horses
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provides new data on the possible role played by these animals as a source of
190
infection to human beings. Molecular tools are useful for understanding the
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epidemiology of Cryptosporidium spp. and G. duodenalis parasites in horses. In
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future studies, the data from such tools should contribute to a better understanding of
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the population genetic substructure of Cryptosporidium spp. and G. duodenalis in
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horses and their zoonotic potential.
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Acknowledgments
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This study was supported in part by the State Key Program of National Natural
198
Science Foundation of China (No. 31330079), Specialized Research Fund for the
199
Doctoral Program of Higher Education (No. 20124105120003), and the National
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Natural Science Foundation of China (Nos. U1204328, 31302079).
Conflict of interest
The authors declare no conflicts of interest.
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Table 1. Prevalence of Cryptosporidium spp. and Giardia in horses from Xinjiang Giardia positive (%)
Mare
Stallion
Foal
Mare
Stallion
80
0/57(0)
0/17(0)
0/6(0)
0/57(0)
0/17(0)
Kuerle
7
Tacheng
22
Aletai Yili
0/16(0)
0/6(0)
2/16(12.5) 0/6(0)
35
0/18(0)
0/5(0)
7/12(58.3)
0/18(0)
0/5(0)
0/12(0)
118
0/70(0)
0/23(0)
0/25(0)
2/70(2.9)
0/23(0)
0/25(0)
0/161(0)
0/58(0)
4/161(2.5) 0/58(0)
0/43(0)
262
257 258 259 260
us
7/43(16.3)
te
d
4/262(1.5)
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256
cr
0/7(0)
253
255
0/6(0)
0/7(0)
7/262(2.7)
254
Foal
ip t
Wulumuqi
Total
Cryptosporidium positive (%) No. samples
an
Location
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