- Email: [email protected]
Cryptosporidium species and Cryptosporidium parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey
Accepted Manuscript Cryptosporidium species and C. parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey Aysegul Taylan-Ozkan, Sibel Yasa-Duru, Selma Usluca, Colleen Lysen, Jianbin Ye, Dawn M. Roellig, Yaoyu Feng, Lihua Xiao PII:
S0014-4894(16)30136-9
DOI:
10.1016/j.exppara.2016.06.014
Reference:
YEXPR 7269
To appear in:
Experimental Parasitology
Received Date: 24 March 2016 Revised Date:
27 June 2016
Accepted Date: 29 June 2016
Please cite this article as: Taylan-Ozkan, A., Yasa-Duru, S., Usluca, S., Lysen, C., Ye, J., Roellig, D.M., Feng, Y., Xiao, L., Cryptosporidium species and C. parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey, Experimental Parasitology (2016), doi: 10.1016/ j.exppara.2016.06.014. 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.
RI PT
ACCEPTED MANUSCRIPT
Corum
Cattle: Farm 6 IIaA13G2R1 (3)
Cattle: Farm 8 IIaA13G2R1 (1)
Ankara
TE D
M AN U
SC
Balıkesir
AC C
EP
Cattle: Farms 1,2,9,10 IIaA13G2R1 (6), IIdA18G1 (2), IIdA20G1b (1)
Kırşehir
Goat: Farm 14 IIaA13G2R1 (1)
Kırıkkale Cattle: Farms 3,4,5,7 IIaA13G2R1 (10) Goat: Farms 11,12,13 IIaA13G2R1 (2), IIaA15G1R1 (2), IIdA22G1 (2), IIdA18G1 (1), mixed subtypes (1)
ACCEPTED MANUSCRIPT
Short Communication
2
Cryptosporidium species and C. parvum subtypes in dairy calves and
3
goat kids reared under traditional farming systems in Turkey
RI PT
1
4
Aysegul Taylan-Ozkan a, Sibel Yasa-Duru b, Selma Usluca c, Colleen Lysen d, Jianbin Ye d,e,
6
Dawn M. Roellig d, Yaoyu Feng e*, and Lihua Xiao d
7
a
8
Turkey
9
b
M AN U
SC
5
Department of Medical Microbiology, Faculty of Medicine, Hitit University, Corum 19200,
Department of Internal Medicine, Faculty of Veterinary Medicine, Kirikkale University,
Kirikkale, 71450, Turkey
11
c
12
Turkey
13
d
14
Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600
15
Clifton Rd, Atlanta, GA 30329, USA
16
e
17
on Chemical Process, School of Resources and Environmental Engineering, East China
18
University of Science and Technology, Shanghai 200237, China
TE D
10
National Parasitology Laboratory, Turkish National Public Health Agency, Ankara, 16100,
AC C
EP
Division of Foodborne, Waterborne and Environmental Diseases, National Center for
State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control
19 20
*Corresponding author. Tel: ++86 21 6425 0664; fax: +86 21 6425 0664.
21
E-mail address: [email protected] (Y. Feng).
1
ACCEPTED MANUSCRIPT
22
ABSTRACT
23 Molecular characterizations of Cryptosporidium spp. in ruminants reared under
25
traditional animal management systems are scarce and studies conducted thus far have revealed
26
largely an absence of the pathogenic and zoonotic species C. parvum in pre-weaned animals. In
27
this study, we examined Cryptosporidium species and subtype distribution in free-range pre-
28
weaned dairy calves and goat kids with diarrhea. Cryptosporidium-positive specimens from pre-
29
weaned calves on 10 farms and goat kids on 4 farms in Ankara, Balikesir, Corum, Kirikkale, and
30
Kirsehir Provinces, Turkey were genotyped by PCR-restriction length polymorphism analysis of
31
the small subunit rRNA gene, which identified C. parvum in 27 calves and 9 goat kids and C.
32
ryanae in 1 calf. Among the C. parvum isolates successfully subtyped by DNA sequence
33
analysis of the 60 kDa glycoprotein gene, three subtypes were detected in calves, including
34
IIaA13G2R1 (20/23), IIdA18G1 (2/23), and IIdA20G1b (1/23), and four subtypes were detected
35
in goat kids, including IIaA13G2R1 (3/8), IIaA15G1R1 (2/8), IIdA22G1 (2/8), and IIdA18G1
36
(1/8). Data of the study suggest that dairy calves reared in a traditional cow-calf system in
37
Turkey are mainly infected with a C. parvum subtype rarely seen elsewhere, whereas goat kids
38
are infected with diverse subtypes. As all five C. parvum subtypes found in this study are known
39
human pathogens, pre-weaned farm animals could play a potential role in the transmission of
40
human cryptosporidiosis.
42
SC
M AN U
TE D
EP
AC C
41
RI PT
24
Keywords: Cryptosporidium; cryptosporidiosis; genotype; subtype; cattle; goat; Turkey
43
2
ACCEPTED MANUSCRIPT
44
1. Introduction
45 Cryptosporidiosis is an important cause for diarrhea in both humans and farm animals
RI PT
46
(Cho, et al., 2013; Kotloff, et al., 2013). Several Cryptosporidium species are commonly found
48
in ruminants, including C. parvum, C. bovis, C. ryanae and C. andersoni in cattle, and C. parvum,
49
C. xiaoi and C. ubiquitum in sheep (Ryan, et al., 2014). Among them, C. parvum is the major
50
cause of zoonotic Cryptosporidium infections in humans (Chalmers and Giles, 2010; Xiao, 2010).
51
An age-associated distribution of Cryptosporidium species in dairy cattle has been well
52
documented in studies conducted in industrialized nations, with C. parvum dominating in pre-
53
weaned calves, C. bovis and C. ryanae in post-weaned calves and juveniles, and C. andersoni
54
mostly in adults (Fayer, et al., 2006, 2007; Santin, et al., 2004). Recent studies in Sweden, China,
55
and Egypt, however, indicate that the pattern of Cryptosporidium infections in dairy cattle could
56
be different in some areas, with an early occurrence of the non-pathogenic C. bovis and C.
57
ryanae in pre-weaned dairy calves reared under less intensive farming conditions (Amer, et al.,
58
2013; Silverlas, et al., 2013; Wang, et al., 2011).
M AN U
TE D
Few studies have been conducted to characterize Cryptosporidium spp. in goats, but data
EP
59
SC
47
obtained thus far suggest that the distribution of Cryptosporidium species in goats is similar to
61
that in sheep, with the occurrence of mainly C. parvum, C. xiaoi, and C. ubiquitum (Diaz, et al.,
62
2011, 2015; Koinari, et al., 2014; Mi, et al., 2014; Rieux, et al., 2013; Tzanidakis, et al., 2014;
63
Wang, et al., 2014). Geographic differences, however, exist in the distribution of
64
Cryptosporidium species in sheep, with C. parvum as the dominant species in some European
65
countries where animal management is more intensive, C. ubiquitum in Americas, C. xiaoi in
66
some developing countries, and all three species common in Australia (Ryan, et al., 2014). In
AC C
60
3
ACCEPTED MANUSCRIPT
67
addition, in a few European studies, C. parvum was more frequently found in ill lambs whereas
68
C. ubiquitum and C. xiaoi were more commonly seen in healthy lambs (Chalmers, et al., 2002;
69
Mueller-Doblies, et al., 2008; Quilez, et al., 2008a). To date, there are very few studies on Cryptosporidium species in farm animals in
RI PT
70
Turkey. In two studies conducted in Kars Province, northeastern Turkey, a small number of C.
72
parvum isolates from mostly pre-weaned calves were subtyped by gp60 sequence analysis (13
73
isolates) and multilocus microsatellite and minisatellite analysis (15 isolates) (Arslan and Ekinci,
74
2012; Tanriverdi, et al., 2006). The tools used in these earlier studies, however, are not expected
75
to detect other Cryptosporidium species in cattle. In the present small study, we genotyped and
76
subtyped Cryptosporidium spp. in microscopy-positive specimens from pre-weaned calves and
77
goats that were reared in traditional farm systems and had diarrhea at the time of sampling.
M AN U
SC
71
79
2. Materials and methods
80
83
EP
82
2.1. Specimen collection
Fecal specimens microscopically positive for Cryptosporidium spp. were used in this
AC C
81
TE D
78
84
study. They were collected in January 2012 and December 2013 from pre-weaned dairy calves
85
(one to four weeks in age) and pre-weaned kids (10-15 days in age) with diarrhea. Altogether, 14
86
and 19 bovine specimens from 2012 and 2013 and 6 and 3 caprine specimens from 2012 and
87
2013, respectively, were used. The dairy calves were mostly of the Holstein breed, with only
88
three of the Simental breed, whereas the kids were of the Hair Goat breed. Most calves were
89
reared under the traditional farming condition indoors together with their mothers, with breast
4
ACCEPTED MANUSCRIPT
milk as the diet. Calves on one farm, however, were recently separated from their dams and
91
bottle-fed with cow milk. Except for one animal, almost all goat kids in the study were kept with
92
their mothers on pastures and breast fed. These specimens came from 10 dairy farms and 4 goat
93
farms in Ankara, Balikesir, Corum, Kirikkale, and Kirsehir Provinces, Turkey, with 1 to 7
94
animals per farm (Table 1). They were diagnosed as positive for Cryptosporidium oocysts by
95
microscopy after staining with Kinyoun's carbol fuchsin solution (Heine, 1982). Positive
96
specimens were stored in 2.5% potassium dichromate at 4 °C before molecular analysis. This
97
study was approved by the Turkish National Public Health Agency.
SC
RI PT
90
99
M AN U
98
2.2. DNA extraction and PCR amplification
100 101
Microscopy-positive fecal specimens were washed twice with distilled water by centrifugation to remove potassium dichromate prior to DNA isolation. DNA was extracted from
103
them using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA) and analyzed for
104
Cryptosporidium spp. by nested PCR targeting an approximately 830-bp fragment of the small
105
subunit (SSU) rRNA gene (Feng, et al., 2007). Cryptosporidium species were differentiated by
106
restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products with
107
SspI and MboII as previously described (Feng, et al., 2007). Each specimen was analyzed at least
108
twice using reagent water as the negative control and DNA of C. baileyi as the positive control.
109
The identity of Cryptosporidium species was confirmed by sequence analysis of the secondary
110
PCR products from representative specimens. Cryptosporidium-positive specimens at the SSU
111
rRNA locus were further analyzed by PCR amplification of the 60 kDa glycoprotein (gp60) gene
112
(Alves, et al., 2003).
AC C
EP
TE D
102
5
ACCEPTED MANUSCRIPT
113 114
2.3. DNA sequence analysis
115 All PCR products of the gp60 gene and selective PCR products of the SSU rRNA gene
RI PT
116
(from all specimens that produced positive SSU rRNA PCR results but negative gp60 PCR
118
results) were sequenced directly on an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster
119
City, CA). Bi-directional sequences obtained were assembled using the ChromasPro (version
120
1.5) software (http://technelysium.com.au/?page_id=27), and aligned with each other and
121
reference sequences of each gene downloaded from GenBank using ClustalX
122
(http://www.clustal.org/) to determine Cryptosporidium species (based on SSU rRNA sequences)
123
and C. parvum subtypes (based on gp60 sequences). The latter were named using the established
124
Cryptosporidium subtype nomenclature system (Sulaiman, et al., 2005). Representative
125
nucleotide sequences from this study were submitted to GenBank under accession numbers
126
KX443780-KX443784.
129 130 131 132
3. Results
AC C
128
EP
127
TE D
M AN U
SC
117
3.1. PCR detection of Cryptosporidium spp. in cattle and goats
Among the 33 specimens from pre-weaned calves examined in 2012 and 2013, 28 were
133
positive for Cryptosporidium spp. in SSU rRNA-based PCR analysis (Table 1). In contrast, all 9
134
specimens from pre-weaned goat kids were positive for Cryptosporidium spp. in SSU rRNA-
135
based PCR (Table 2). Cryptosporidium-positive specimens at the SSU rRNA locus were
6
ACCEPTED MANUSCRIPT
136
analyzed by gp60 PCR. Among them, 23 of 28 specimens from calves were positive in gp60
137
PCR, whereas all 9 from goat kids were positive.
139
3.2. Cryptosporidium species in cattle and goats
140
RFLP analyses of the SSU rRNA PCR products using restriction enzymes SspI and
SC
141
RI PT
138
MboII indicated the occurrence of two Cryptosporidium species in the 28 PCR-positive calves,
143
including C. parvum (27/28) and C. ryanae (1/28) (Table 1). In contrast, only C. parvum was
144
identified in the 9 PCR-positive specimens from goat kids (Table 2).
145
M AN U
142
Among the SSU rRNA PCR-positive but gp60 PCR-negative specimens, four were from C. parvum and one was from C. ryanae. The identification of C. parvum or C. ryanae in these
147
specimens was confirmed by DNA sequence analysis of the SSU rRNA PCR products. All four
148
C. parvum sequences obtained were identical to the GenBank reference sequence AF159111,
149
whereas the C. ryanae sequence obtained was identical to the reference sequence AY587166.
152 153
3.3. Cryptosporidium subtypes in cattle and goats
EP
151
AC C
150
TE D
146
Among those subtyped successfully, DNA sequence analysis of the gp60 PCR products
154
identified the presence of three subtypes in calves, including IIaA13G2R1 (20/23), IIdA18G1
155
(2/23), and IIdA20G1b (1/23) (Table 1). In goat kids, four gp60 subtypes were seen, including
156
IIaA13G2R1 (3/8), IIaA15G1R1 (2/8), IIdA22G1 (2/8), and IIdA18G1 (1/8). One specimen
157
from a goat kid had mixed infection with two undetermined subtypes (Table 2). The gp60
158
sequences obtained from IIaA13G2R1, IIaA15G1R1, IIdA18G1, and IIdA20G1b, and
7
ACCEPTED MANUSCRIPT
159
IIdA22G1subtypes were identical to GenBank sequences KF008184, AY738191, AY738194,
160
AY738185, and GU214368, respectively.
161
In calves, the three specimens of IId subtypes were collected from two farms in Ankara in 2013, as specimens collected in 2012 all belonged to the IIaA13G2R1 subtype (Table 1). In goat
163
kids, however, subtype IIaA13G2R1 was seen only in 2013 and IId subtypes were seen only in
164
2012 (Table 2).
RI PT
162
167
4. Discussion
Results of this small study indicate that despite the traditional, less intensive livestock
M AN U
166
SC
165
husbandry in the study areas, both pre-weaned calves and goat kids are mainly infected with C.
169
parvum. Previously, only two studies examined the occurrence of C. parvum in a small number
170
of dairy calves in Turkey (Arslan and Ekinci, 2012; Tanriverdi, et al., 2006). Thus, studies
171
conducted thus far all indicate a common occurrence of C. parvum in pre-weaned calves and
172
goat kids in Turkey. It is known that pre-weaned calves in less intensive management systems
173
have a common occurrence of C. bovis, C. ryanae, and C. andersoni in pre-weaned calves, in
174
addition to C. parvum (Meireles, et al., 2011; Muhid, et al., 2011; Qi, et al., 2015; Silverlas, et
175
al., 2013; Venu, et al., 2012; Wang, et al., 2011; Zhang, et al., 2013). We identified only C.
176
ryanae in this study in one pre-weaned calf. Similarly, we did not identify any C. xiaoi and C.
177
ubiquitum in pre-weaned goat kids, which often occur in these animals elsewhere, especially C.
178
xiaoi (Diaz, et al., 2011, 2015; Mi, et al., 2014; Parsons, et al., 2015; Rieux, et al., 2013;
179
Tzanidakis, et al., 2014; Wang, et al., 2014).
180 181
AC C
EP
TE D
168
In this study, most C. parvum isolates in calves were subtyped as IIaA13G2R1. Subtype IIaA13G2R1 is an uncommon subtype and has been only reported in a small number of calves in
8
ACCEPTED MANUSCRIPT
Canada, Belgium and the Netherlands and in Chincoteague ponies in the United States (Geurden,
183
et al., 2007; Trotz-Williams, et al., 2006; Wagnerova, et al., 2015; Wielinga, et al., 2008). It
184
nevertheless is probably common in dairy cattle in Turkey; among the 26 GenBank gp60
185
sequences of C. parvum from calves in Turkey, 23 are from the subtype IIaA13G2R1. These
186
sequences are from three unpublished studies conducted in calves in Burdur (JX826593-
187
JX826602), Konya (KF008176-KF008185), and Nevsehir (JQ034314-JQ034316) Provinces. The
188
only published subtyping study of C. parvum in cattle in Turkey showed a dominance (10/13) of
189
the subtype IIaA15G2R1 in Kars Province (Arslan and Ekinci, 2012). Elsewhere in
190
industrialized nations, subtype IIaA15G2R1 is the dominant subtype in pre-weaned dairy calves,
191
with the exception of the United Kingdom, Ireland, and Australia, where IIaA18G3R1 is the
192
most common subtype (Xiao, 2010).
193
M AN U
SC
RI PT
182
The other two subtypes identified in three calves in this study belong to the IId subtype family. They occurred on two farms from Ankara in 2013. C. parvum IId subtypes are common
195
in dairy calves in China, Egypt, Malaysia, and Romania (Amer, et al., 2013; Cui, et al., 2014;
196
Huang, et al., 2014; Muhid, et al., 2011; Qi, et al., 2015; Vieira, et al., 2015; Wang, et al., 2011;
197
Zhang, et al., 2013). They have been found at low frequencies in cattle in many European
198
countries (Bjorkman, et al., 2015; Broglia, et al., 2008; Geurden, et al., 2007; Misic and Abe,
199
2007; Plutzer and Karanis, 2007; Quilez, et al., 2008b; Silverlas, et al., 2013), but are largely
200
absent in cattle in other industrialized nations such as the United States and Canada (Xiao, 2010).
201
Previously, one IId subtype, IIdA15G1, was identified in a cow in Turkey (Arslan and Ekinci,
202
2012).
203 204
AC C
EP
TE D
194
In contrast, a high subtype diversity of C. parvum was seen in goat kids in this study. Four subtypes were identified among the nine C. parvum specimens, including two IIa subtypes
9
ACCEPTED MANUSCRIPT
(IIaA13G2R1 and IIa15G1R1) and two IId subtypes (IIdA18G1 and IIdA22G1). This is similar
206
to findings in Spain and China, where goat kids are known to have high genetic heterogeneity of
207
C. parvum (Diaz, et al., 2015; Mi, et al., 2014; Quilez, et al., 2008a, 2013). Like in the present
208
study, these studies also identified a high occurrence of IId subtypes in pre-weaned goat kids.
209
The difference in subtype distribution suggests that there is probably minimum cross-species
210
transmission of C. parvum between calves and goat kids in Turkey.
RI PT
205
Most of the C. parvum subtypes found in calves and goat kids in this study have been
212
found in humans. The dominant subtype IIaA13G2R1 has been reported in humans in the US
213
and Malaysia (Herges, et al., 2012; Iqbal, et al., 2012), whereas IIaA15G1R1 is the dominant
214
subtype in humans in Scotland (Deshpande, et al., 2015). Similarly, IId subtypes IIdA18G1,
215
IIdA20G1b, and IIdA22G1 have each been detected in some human cases (Chalmers, et al.,
216
2011; Sulaiman, et al., 2005; Vieira, et al., 2015). In Turkey, C. parvum has been identified in
217
humans, although subtype data are not yet available (Tamer, et al., 2007). More extensive
218
characterizations of Cryptosporidium spp. from farm animals and humans are needed to
219
understand the transmission of cryptosporidiosis in Turkey.
222 223 224 225 226
M AN U
TE D
EP
221
Acknowledgements
AC C
220
SC
211
This research was supported by the National Natural Science Foundation of China
(Project No. 31229005 and 31110103901) and Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
10
ACCEPTED MANUSCRIPT
227 References
229
Alves, M., Xiao, L., Sulaiman, I., Lal, A. A., Matos, O., and Antunes, F., 2003. Subgenotype
230
analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal.
231
J Clin Microbiol 41, 2744-2747.
RI PT
228
Amer, S., Zidan, S., Adamu, H., Ye, J., Roellig, D., Xiao, L., and Feng, Y., 2013. Prevalence and
233
characterization of Cryptosporidium spp. in dairy cattle in Nile River delta provinces,
234
Egypt. Exp Parasitol 135, 518-523.
M AN U
SC
232
235
Arslan, M. O. and Ekinci, A. I., 2012. Determination of Cryptosporidium parvum subtypes in
236
cattle in Kars Province of Turkey. Kafkas Universitesi Veteriner Fakultesi Dergisi 18,
237
A221-A226.
239
Bjorkman, C., Lindstrom, L., Oweson, C., Ahola, H., Troell, K., and Axen, C., 2015. Cryptosporidium infections in suckler herd beef calves. Parasitology 142, 1108-1114.
TE D
238
Broglia, A., Reckinger, S., Caccio, S. M., and Nockler, K., 2008. Distribution of
241
Cryptosporidium parvum subtypes in calves in Germany. Vet Parasitol 154, 8-13.
242
Chalmers, R. M., Elwin, K., Reilly, W. J., Irvine, H., Thomas, A. L., and Hunter, P. R., 2002.
243
Cryptosporidium in farmed animals: the detection of a novel isolate in sheep. Int J
245 246
AC C
244
EP
240
Parasitol 32, 21-26.
Chalmers, R. M. and Giles, M., 2010. Zoonotic cryptosporidiosis in the UK - challenges for control. J Appl Microbiol 109, 1487-1497.
247
Chalmers, R. M., Smith, R. P., Hadfield, S. J., Elwin, K., and Giles, M., 2011. Zoonotic linkage
248
and variation in Cryptosporidium parvum from patients in the United Kingdom. Parasitol
249
Res 108, 1321-1325.
11
ACCEPTED MANUSCRIPT
250
Cho, Y. I., Han, J. I., Wang, C., Cooper, V., Schwartz, K., Engelken, T., and Yoon, K. J., 2013.
251
Case-control study of microbiological etiology associated with calf diarrhea. Vet
252
Microbiol 166, 375-385. Cui, Z., Wang, R., Huang, J., Wang, H., Zhao, J., Luo, N., Li, J., Zhang, Z., and Zhang, L., 2014.
254
Cryptosporidiosis caused by Cryptosporidium parvum subtype IIdA15G1 at a dairy farm
255
in Northwestern China. Parasit Vectors 7, 529.
RI PT
253
Deshpande, A. P., Jones, B. L., Connelly, L., Pollock, K. G., Brownlie, S., and Alexander, C. L.,
257
2015. Molecular characterization of Cryptosporidium parvum isolates from human
258
cryptosporidiosis cases in Scotland. Parasitology 142, 318-325.
M AN U
SC
256
259
Diaz, P., Quilez, J., Robinson, G., Chalmers, R. M., Diez-Banos, P., and Morrondo, P., 2011.
260
Identification of Cryptosporidium xiaoi in diarrhoeic goat kids (Capra hircus) in Spain.
261
Vet Parasitol 172, 132-134.
Diaz, P., Quilez, J., Prieto, A., Navarro, E., Perez-Creo, A., Fernandez, G., Panadero, R., Lopez,
263
C., Diez-Banos, P., and Morrondo, P., 2015. Cryptosporidium species and subtype
264
analysis in diarrhoeic pre-weaned lambs and goat kids from north-western Spain.
265
Parasitol Res 114, 4099-4105.
EP
TE D
262
Fayer, R., Santin, M., Trout, J. M., and Greiner, E., 2006. Prevalence of species and genotypes of
267
Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States. Vet
268
AC C
266
Parasitol 135, 105-112.
269
Fayer, R., Santin, M., and Trout, J. M., 2007. Prevalence of Cryptosporidium species and
270
genotypes in mature dairy cattle on farms in eastern United States compared with
271
younger cattle from the same locations. Vet Parasitol 145, 260-266.
12
ACCEPTED MANUSCRIPT
272
Feng, Y., Ortega, Y., He, G., Das, P., Xu, M., Zhang, X., Fayer, R., Gatei, W., Cama, V., and
273
Xiao, L., 2007. Wide geographic distribution of Cryptosporidium bovis and the deer-like
274
genotype in bovines. Vet Parasitol 144, 1-9. Geurden, T., Berkvens, D., Martens, C., Casaert, S., Vercruysse, J., and Claerebout, E., 2007.
276
Molecular epidemiology with subtype analysis of Cryptosporidium in calves in Belgium.
277
Parasitology 134, 1981-1987.
279
Heine, J., 1982. Eine einfache Nachweismethode für Kryptosporidien im Kot. Zbl. Vet. Med. B.
SC
278
RI PT
275
29, 324–327.
Herges, G. R., Widmer, G., Clark, M. E., Khan, E., Giddings, C. W., Brewer, M., and McEvoy,
281
J. M., 2012. Evidence that Cryptosporidium parvum populations are panmictic and
282
unstructured in the Upper Midwest of the United States. Appl Environ Microbiol 78,
283
8096-8101.
M AN U
280
Huang, J., Yue, D., Qi, M., Wang, R., Zhao, J., Li, J., Shi, K., Wang, M., and Zhang, L., 2014.
285
Prevalence and molecular characterization of Cryptosporidium spp. and Giardia
286
duodenalis in dairy cattle in Ningxia, northwestern China. BMC Vet Res 10, 292.
287
Iqbal, A., Lim, Y. A., Surin, J., and Sim, B. L., 2012. High diversity of Cryptosporidium
288
subgenotypes identified in Malaysian HIV/AIDS individuals targeting gp60 gene. PLoS
289
One 7, e31139.
291
EP
AC C
290
TE D
284
Koinari, M., Lymbery, A. J., and Ryan, U. M., 2014. Cryptosporidium species in sheep and goats from Papua New Guinea. Exp Parasitol 141, 134-137.
292
Kotloff, K. L., Nataro, J. P., Blackwelder, W. C., Nasrin, D., Farag, T. H., Panchalingam, S.,
293
Wu, Y., Sow, S. O., Sur, D., Breiman, R. F., Faruque, A. S., Zaidi, A. K., Saha, D.,
294
Alonso, P. L., Tamboura, B., Sanogo, D., Onwuchekwa, U., Manna, B., Ramamurthy, T.,
13
ACCEPTED MANUSCRIPT
Kanungo, S., Ochieng, J. B., Omore, R., Oundo, J. O., Hossain, A., Das, S. K., Ahmed,
296
S., Qureshi, S., Quadri, F., Adegbola, R. A., Antonio, M., Hossain, M. J., Akinsola, A.,
297
Mandomando, I., Nhampossa, T., Acacio, S., Biswas, K., O'Reilly, C. E., Mintz, E. D.,
298
Berkeley, L. Y., Muhsen, K., Sommerfelt, H., Robins-Browne, R. M., and Levine, M. M.,
299
2013. Burden and aetiology of diarrhoeal disease in infants and young children in
300
developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-
301
control study. Lancet 382, 209-222.
SC
RI PT
295
Meireles, M. V., de Oliveira, F. P., Teixeira, W. F., Coelho, W. M., and Mendes, L. C., 2011.
303
Molecular characterization of Cryptosporidium spp. in dairy calves from the state of Sao
304
Paulo, Brazil. Parasitol Res 109, 949-951.
M AN U
302
Mi, R., Wang, X., Huang, Y., Zhou, P., Liu, Y., Chen, Y., Chen, J., Zhu, W., and Chen, Z., 2014.
306
Prevalence and molecular characterization of Cryptosporidium in goats across four
307
provincial level areas in China. PLoS One 9, e111164.
TE D
305
Misic, Z. and Abe, N., 2007. Subtype analysis of Cryptosporidium parvum isolates from calves
309
on farms around Belgrade, Serbia and Montenegro, using the 60 kDa glycoprotein gene
310
sequences. Parasitology 134, 351-358.
EP
308
Mueller-Doblies, D., Giles, M., Elwin, K., Smith, R. P., Clifton-Hadley, F. A., and Chalmers, R.
312
M., 2008. Distribution of Cryptosporidium species in sheep in the UK. Vet Parasitol 154,
313
AC C
311
214-219.
314
Muhid, A., Robertson, I., Ng, J., and Ryan, U., 2011. Prevalence of and management factors
315
contributing to Cryptosporidium sp. infection in pre-weaned and post-weaned calves in
316
Johor, Malaysia. Exp Parasitol 127, 534-538.
14
ACCEPTED MANUSCRIPT
Parsons, M. B., Travis, D., Lonsdorf, E. V., Lipende, I., Roellig, D. M., Kamenya, S., Zhang, H.,
318
Xiao, L., and Gillespie, T. R., 2015. Epidemiology and molecular characterization of
319
Cryptosporidium spp. in humans, wild primates, and domesticated animals in the Greater
320
Gombe Ecosystem, Tanzania. PLoS Negl Trop Dis 9, e0003529.
321 322
RI PT
317
Plutzer, J. and Karanis, P., 2007. Genotype and subtype analyses of Cryptosporidium isolates from cattle in Hungary. Vet Parasitol 146, 357-362.
Qi, M., Wang, H., Jing, B., Wang, D., Wang, R., and Zhang, L., 2015. Occurrence and molecular
324
identification of Cryptosporidium spp. in dairy calves in Xinjiang, Northwestern China.
325
Vet Parasitol 212, 404-407.
M AN U
SC
323
326
Quilez, J., Torres, E., Chalmers, R. M., Hadfield, S. J., Del Cacho, E., and Sanchez-Acedo, C.,
327
2008a. Cryptosporidium genotypes and subtypes in lambs and goat kids in Spain. Appl
328
Environ Microbiol 74, 6026-6031.
Quilez, J., Torres, E., Chalmers, R. M., Robinson, G., Del Cacho, E., and Sanchez-Acedo, C.,
330
2008b. Cryptosporidium species and subtype analysis from dairy calves in Spain.
331
Parasitology 135, 1613-1620.
TE D
329
Quilez, J., Vergara-Castiblanco, C., Monteagudo, L., del Cacho, E., and Sanchez-Acedo, C.,
333
2013. Host association of Cryptosporidium parvum populations infecting domestic
334
ruminants in Spain. Appl Environ Microbiol 79, 5363-5371.
AC C
EP
332
335
Rieux, A., Paraud, C., Pors, I., and Chartier, C., 2013. Molecular characterization of
336
Cryptosporidium spp. in pre-weaned kids in a dairy goat farm in western France. Vet
337 338 339
Parasitol 192, 268-272.
Ryan, U., Fayer, R., and Xiao, L., 2014. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 141, 1667-1685.
15
ACCEPTED MANUSCRIPT
340
Santin, M., Trout, J. M., Xiao, L., Zhou, L., Greiner, E., and Fayer, R., 2004. Prevalence and
341
age-related variation of Cryptosporidium species and genotypes in dairy calves. Vet
342
Parasitol 122, 103-117.
344
Silverlas, C., Bosaeus-Reineck, H., Naslund, K., and Bjorkman, C., 2013. Is there a need for
RI PT
343
improved Cryptosporidium diagnostics in Swedish calves? Int J Parasitol 43, 155-161. Sulaiman, I. M., Hira, P. R., Zhou, L., Al-Ali, F. M., Al-Shelahi, F. A., Shweiki, H. M., Iqbal, J.,
346
Khalid, N., and Xiao, L., 2005. Unique endemicity of cryptosporidiosis in children in
347
Kuwait. J Clin Microbiol 43, 2805-2809.
SC
345
Tamer, G. S., Turk, M., Dagci, H., Pektas, B., Guy, E. C., Guruz, A. Y., and Uner, A., 2007. The
349
prevalence of cryptosporidiosis in Turkish children, and genotyping of isolates by nested
350
polymerase chain reaction-restriction fragment length polymorphism. Saudi Med J 28,
351
1243-1246.
M AN U
348
Tanriverdi, S., Markovics, A., Arslan, M. O., Itik, A., Shkap, V., and Widmer, G., 2006.
353
Emergence of distinct genotypes of Cryptosporidium parvum in structured host
354
populations. Appl Environ Microbiol 72, 2507-2513.
TE D
352
Trotz-Williams, L. A., Martin, D. S., Gatei, W., Cama, V., Peregrine, A. S., Martin, S. W.,
356
Nydam, D. V., Jamieson, F., and Xiao, L., 2006. Genotype and subtype analyses of
357
Cryptosporidium isolates from dairy calves and humans in Ontario. Parasitol Res 99,
AC C
358
EP
355
346-352.
359
Tzanidakis, N., Sotiraki, S., Claerebout, E., Ehsan, A., Voutzourakis, N., Kostopoulou, D., Stijn,
360
C., Vercruysse, J., and Geurden, T., 2014. Occurrence and molecular characterization of
361
Giardia duodenalis and Cryptosporidium spp. in sheep and goats reared under dairy
362
husbandry systems in Greece. Parasite 21, 45.
16
ACCEPTED MANUSCRIPT
363
Venu, R., Latha, B. R., Basith, S. A., Raj, G. D., Sreekumar, C., and Raman, M., 2012.
364
Molecular prevalence of Cryptosporidium spp. in dairy calves in Southern states of India.
365
Vet Parasitol 188, 19-24. Vieira, P. M., Mederle, N., Lobo, M. L., Imre, K., Mederle, O., Xiao, L., Darabus, G., and
367
Matos, O., 2015. Molecular characterisation of Cryptosporidium (Apicomplexa) in
368
children and cattle in Romania. Folia Parasitol (Praha) 62.
RI PT
366
Wagnerova, P., Sak, B., McEvoy, J., Rost, M., Sherwood, D., Holcomb, K., and Kvac, M., 2015.
370
Cryptosporidium parvum and Enterocytozoon bieneusi in American Mustangs and
371
Chincoteague ponies. Exp Parasitol 162, 24-27.
M AN U
SC
369
372
Wang, R., Wang, H., Sun, Y., Zhang, L., Jian, F., Qi, M., Ning, C., and Xiao, L., 2011.
373
Characteristics of Cryptosporidium transmission in preweaned dairy cattle in Henan,
374
China. J Clin Microbiol 49, 1077-1082.
Wang, R., Li, G., Cui, B., Huang, J., Cui, Z., Zhang, S., Dong, H., Yue, D., Zhang, L., Ning, C.,
376
and Wang, M., 2014. Prevalence, molecular characterization and zoonotic potential of
377
Cryptosporidium spp. in goats in Henan and Chongqing, China. Exp Parasitol 142, 11-
378
16.Wielinga, P. R., de Vries, A., van der Goot, T. H., Mank, T., Mars, M. H., Kortbeek,
379
L. M., and van der Giessen, J. W., 2008. Molecular epidemiology of Cryptosporidium in
380
humans and cattle in The Netherlands. Int J Parasitol 38, 809-817.
382
EP
AC C
381
TE D
375
Xiao, L., 2010. Molecular epidemiology of cryptosporidiosis: An update. Exp Parasitol 124, 8089.
383
Zhang, W., Wang, R., Yang, F., Zhang, L., Cao, J., Zhang, X., Ling, H., Liu, A., and Shen, Y.,
384
2013. Distribution and genetic characterizations of Cryptosporidium spp. in pre-weaned
385
dairy calves in Northeastern China's Heilongjiang Province. PLoS One 8, e54857.
386 17
Table 1
388
Cryptosporidium species and C. parvum subtypes in dairy cattle in Turkey
Breed
Nutrition
1
Kalecik/Ankara
Holstein
Breast milk
2
Kalecik/Ankara
Simental, Holstein
Breast milk
Holstein
Breast milk
4
5
Keskin/Kırıkkale
Kaman/Kırıkkale
Hasandede/Kırıkkale
Holstein
Holstein
Breast milk Breast milk
In barn with mother In barn with mother In barn with mother In barn with mother In barn with mother Initially in barn with mother In barn with mother In barn
EP
3
Care
Manyas/Balıkesir
Holstein
7
Merkez/Kırıkkale
Holstein
Breast milk
8
Alaca/Çorum
Simental
Breast
AC C
6
Breast milk, bottle milk
Sampling time
2012
M AN U
Location
2012
2012
TE D
Farm ID
No. No. microscopyspecimen positive PCRspecimen positive examined
SC
387
RI PT
ACCEPTED MANUSCRIPT
6
4
2
C. parvum
C. parvum subtype*
C. ryanae
4
IIaA13G2R1 (4)
0
2
IIaA13G2R1 (2)
0
2
IIaA13G2R1 (2)
0
3
IIaA13G2R1 (3)
0
2 3 2
2012
3 3
2013
7
6
6
IIaA13G2R1 (4), unknown (2)
0
2013
5
5
4
IIaA13G2R1 (3), unknown (1)
1
2013
1
1
IIaA13G2R1 (1)
0
2013
1
1
IIaA13G2R1
0
18
1 1
ACCEPTED MANUSCRIPT
Kalecik/Ankara
Simental, Holstein
Breast milk
Holstein
Breast milk
In barn with mother
2013
AC C
EP
TE D
Total 389 *Unknown subtype: gp60 subtyping was unsuccessful. 390
2013
RI PT
10
Kalecik/Ankara
(1)
M AN U
9
with mother In barn with mother
19
4
3
1
1
SC
milk
33
3
1
28
27
IIdA18G1 (1), IIdA20G1b (1), (unknown (1)
0
IIdA18G1 (1)
0
IIaA13G2R1 (20), IIdA18G1 (2), IIdA20G1b (1), unknown (4)
1
392
Cryptosporidium parvum subtypes in goats in Turkey
Location
Breed
Nutrition
Merkez/Kırıkkale
Hair Goat
Breast milk
11 Sulakyurt/Kırıkkale
Hair Goat
Keskin/Kırıkkale
Hair Goat
Akpınar/ Kırşehir
Hair Goat
12 13
Breast milk 1/2 diluted cow's milk
Pasturing with mother Pasturing with mother Pasturing with mother Pasturing with mother
C. parvum
C. parvum subtype
4
4
4
IIaA15G1R1 (2), IIdA18G1 (1), mixed subtypes (1)
2012
2
2
2
IIdA22G1 (2)
2013
2
2
2
IIaA13G2R1 (2)
1
1
1
IIaA13G2R1 (1)
9
IIaA13G2R1 (3), IIaA15G1R1 (2), IIdA22G1 (2), IIdA18G1 (1), mixed subtypes (1)
Sampling time
2012
2013
Total 393
AC C
EP
14
Breast milk
Care
No. specimen PCRpositive
TE D
Farm ID
No. microscopy -positive specimen examined
SC
Table 2
M AN U
391
RI PT
ACCEPTED MANUSCRIPT
9
20
9
ACCEPTED MANUSCRIPT Highlights Preweaned calves and goat kids reared in traditional farming systems were examined
•
Cryptosporidium parvum was identified in 27 calves and 9 goat kids
•
IIaA13G2R1 (20/23) was the dominant subtype in calves
•
A high C. parvum subtype diversity was found in goat kids
•
All five C. parvum subtypes found are known human pathogens
AC C
EP
TE D
M AN U
SC
RI PT
•
S0014-4894(16)30136-9
DOI:
10.1016/j.exppara.2016.06.014
Reference:
YEXPR 7269
To appear in:
Experimental Parasitology
Received Date: 24 March 2016 Revised Date:
27 June 2016
Accepted Date: 29 June 2016
Please cite this article as: Taylan-Ozkan, A., Yasa-Duru, S., Usluca, S., Lysen, C., Ye, J., Roellig, D.M., Feng, Y., Xiao, L., Cryptosporidium species and C. parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey, Experimental Parasitology (2016), doi: 10.1016/ j.exppara.2016.06.014. 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.
RI PT
ACCEPTED MANUSCRIPT
Corum
Cattle: Farm 6 IIaA13G2R1 (3)
Cattle: Farm 8 IIaA13G2R1 (1)
Ankara
TE D
M AN U
SC
Balıkesir
AC C
EP
Cattle: Farms 1,2,9,10 IIaA13G2R1 (6), IIdA18G1 (2), IIdA20G1b (1)
Kırşehir
Goat: Farm 14 IIaA13G2R1 (1)
Kırıkkale Cattle: Farms 3,4,5,7 IIaA13G2R1 (10) Goat: Farms 11,12,13 IIaA13G2R1 (2), IIaA15G1R1 (2), IIdA22G1 (2), IIdA18G1 (1), mixed subtypes (1)
ACCEPTED MANUSCRIPT
Short Communication
2
Cryptosporidium species and C. parvum subtypes in dairy calves and
3
goat kids reared under traditional farming systems in Turkey
RI PT
1
4
Aysegul Taylan-Ozkan a, Sibel Yasa-Duru b, Selma Usluca c, Colleen Lysen d, Jianbin Ye d,e,
6
Dawn M. Roellig d, Yaoyu Feng e*, and Lihua Xiao d
7
a
8
Turkey
9
b
M AN U
SC
5
Department of Medical Microbiology, Faculty of Medicine, Hitit University, Corum 19200,
Department of Internal Medicine, Faculty of Veterinary Medicine, Kirikkale University,
Kirikkale, 71450, Turkey
11
c
12
Turkey
13
d
14
Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600
15
Clifton Rd, Atlanta, GA 30329, USA
16
e
17
on Chemical Process, School of Resources and Environmental Engineering, East China
18
University of Science and Technology, Shanghai 200237, China
TE D
10
National Parasitology Laboratory, Turkish National Public Health Agency, Ankara, 16100,
AC C
EP
Division of Foodborne, Waterborne and Environmental Diseases, National Center for
State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control
19 20
*Corresponding author. Tel: ++86 21 6425 0664; fax: +86 21 6425 0664.
21
E-mail address: [email protected] (Y. Feng).
1
ACCEPTED MANUSCRIPT
22
ABSTRACT
23 Molecular characterizations of Cryptosporidium spp. in ruminants reared under
25
traditional animal management systems are scarce and studies conducted thus far have revealed
26
largely an absence of the pathogenic and zoonotic species C. parvum in pre-weaned animals. In
27
this study, we examined Cryptosporidium species and subtype distribution in free-range pre-
28
weaned dairy calves and goat kids with diarrhea. Cryptosporidium-positive specimens from pre-
29
weaned calves on 10 farms and goat kids on 4 farms in Ankara, Balikesir, Corum, Kirikkale, and
30
Kirsehir Provinces, Turkey were genotyped by PCR-restriction length polymorphism analysis of
31
the small subunit rRNA gene, which identified C. parvum in 27 calves and 9 goat kids and C.
32
ryanae in 1 calf. Among the C. parvum isolates successfully subtyped by DNA sequence
33
analysis of the 60 kDa glycoprotein gene, three subtypes were detected in calves, including
34
IIaA13G2R1 (20/23), IIdA18G1 (2/23), and IIdA20G1b (1/23), and four subtypes were detected
35
in goat kids, including IIaA13G2R1 (3/8), IIaA15G1R1 (2/8), IIdA22G1 (2/8), and IIdA18G1
36
(1/8). Data of the study suggest that dairy calves reared in a traditional cow-calf system in
37
Turkey are mainly infected with a C. parvum subtype rarely seen elsewhere, whereas goat kids
38
are infected with diverse subtypes. As all five C. parvum subtypes found in this study are known
39
human pathogens, pre-weaned farm animals could play a potential role in the transmission of
40
human cryptosporidiosis.
42
SC
M AN U
TE D
EP
AC C
41
RI PT
24
Keywords: Cryptosporidium; cryptosporidiosis; genotype; subtype; cattle; goat; Turkey
43
2
ACCEPTED MANUSCRIPT
44
1. Introduction
45 Cryptosporidiosis is an important cause for diarrhea in both humans and farm animals
RI PT
46
(Cho, et al., 2013; Kotloff, et al., 2013). Several Cryptosporidium species are commonly found
48
in ruminants, including C. parvum, C. bovis, C. ryanae and C. andersoni in cattle, and C. parvum,
49
C. xiaoi and C. ubiquitum in sheep (Ryan, et al., 2014). Among them, C. parvum is the major
50
cause of zoonotic Cryptosporidium infections in humans (Chalmers and Giles, 2010; Xiao, 2010).
51
An age-associated distribution of Cryptosporidium species in dairy cattle has been well
52
documented in studies conducted in industrialized nations, with C. parvum dominating in pre-
53
weaned calves, C. bovis and C. ryanae in post-weaned calves and juveniles, and C. andersoni
54
mostly in adults (Fayer, et al., 2006, 2007; Santin, et al., 2004). Recent studies in Sweden, China,
55
and Egypt, however, indicate that the pattern of Cryptosporidium infections in dairy cattle could
56
be different in some areas, with an early occurrence of the non-pathogenic C. bovis and C.
57
ryanae in pre-weaned dairy calves reared under less intensive farming conditions (Amer, et al.,
58
2013; Silverlas, et al., 2013; Wang, et al., 2011).
M AN U
TE D
Few studies have been conducted to characterize Cryptosporidium spp. in goats, but data
EP
59
SC
47
obtained thus far suggest that the distribution of Cryptosporidium species in goats is similar to
61
that in sheep, with the occurrence of mainly C. parvum, C. xiaoi, and C. ubiquitum (Diaz, et al.,
62
2011, 2015; Koinari, et al., 2014; Mi, et al., 2014; Rieux, et al., 2013; Tzanidakis, et al., 2014;
63
Wang, et al., 2014). Geographic differences, however, exist in the distribution of
64
Cryptosporidium species in sheep, with C. parvum as the dominant species in some European
65
countries where animal management is more intensive, C. ubiquitum in Americas, C. xiaoi in
66
some developing countries, and all three species common in Australia (Ryan, et al., 2014). In
AC C
60
3
ACCEPTED MANUSCRIPT
67
addition, in a few European studies, C. parvum was more frequently found in ill lambs whereas
68
C. ubiquitum and C. xiaoi were more commonly seen in healthy lambs (Chalmers, et al., 2002;
69
Mueller-Doblies, et al., 2008; Quilez, et al., 2008a). To date, there are very few studies on Cryptosporidium species in farm animals in
RI PT
70
Turkey. In two studies conducted in Kars Province, northeastern Turkey, a small number of C.
72
parvum isolates from mostly pre-weaned calves were subtyped by gp60 sequence analysis (13
73
isolates) and multilocus microsatellite and minisatellite analysis (15 isolates) (Arslan and Ekinci,
74
2012; Tanriverdi, et al., 2006). The tools used in these earlier studies, however, are not expected
75
to detect other Cryptosporidium species in cattle. In the present small study, we genotyped and
76
subtyped Cryptosporidium spp. in microscopy-positive specimens from pre-weaned calves and
77
goats that were reared in traditional farm systems and had diarrhea at the time of sampling.
M AN U
SC
71
79
2. Materials and methods
80
83
EP
82
2.1. Specimen collection
Fecal specimens microscopically positive for Cryptosporidium spp. were used in this
AC C
81
TE D
78
84
study. They were collected in January 2012 and December 2013 from pre-weaned dairy calves
85
(one to four weeks in age) and pre-weaned kids (10-15 days in age) with diarrhea. Altogether, 14
86
and 19 bovine specimens from 2012 and 2013 and 6 and 3 caprine specimens from 2012 and
87
2013, respectively, were used. The dairy calves were mostly of the Holstein breed, with only
88
three of the Simental breed, whereas the kids were of the Hair Goat breed. Most calves were
89
reared under the traditional farming condition indoors together with their mothers, with breast
4
ACCEPTED MANUSCRIPT
milk as the diet. Calves on one farm, however, were recently separated from their dams and
91
bottle-fed with cow milk. Except for one animal, almost all goat kids in the study were kept with
92
their mothers on pastures and breast fed. These specimens came from 10 dairy farms and 4 goat
93
farms in Ankara, Balikesir, Corum, Kirikkale, and Kirsehir Provinces, Turkey, with 1 to 7
94
animals per farm (Table 1). They were diagnosed as positive for Cryptosporidium oocysts by
95
microscopy after staining with Kinyoun's carbol fuchsin solution (Heine, 1982). Positive
96
specimens were stored in 2.5% potassium dichromate at 4 °C before molecular analysis. This
97
study was approved by the Turkish National Public Health Agency.
SC
RI PT
90
99
M AN U
98
2.2. DNA extraction and PCR amplification
100 101
Microscopy-positive fecal specimens were washed twice with distilled water by centrifugation to remove potassium dichromate prior to DNA isolation. DNA was extracted from
103
them using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA) and analyzed for
104
Cryptosporidium spp. by nested PCR targeting an approximately 830-bp fragment of the small
105
subunit (SSU) rRNA gene (Feng, et al., 2007). Cryptosporidium species were differentiated by
106
restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products with
107
SspI and MboII as previously described (Feng, et al., 2007). Each specimen was analyzed at least
108
twice using reagent water as the negative control and DNA of C. baileyi as the positive control.
109
The identity of Cryptosporidium species was confirmed by sequence analysis of the secondary
110
PCR products from representative specimens. Cryptosporidium-positive specimens at the SSU
111
rRNA locus were further analyzed by PCR amplification of the 60 kDa glycoprotein (gp60) gene
112
(Alves, et al., 2003).
AC C
EP
TE D
102
5
ACCEPTED MANUSCRIPT
113 114
2.3. DNA sequence analysis
115 All PCR products of the gp60 gene and selective PCR products of the SSU rRNA gene
RI PT
116
(from all specimens that produced positive SSU rRNA PCR results but negative gp60 PCR
118
results) were sequenced directly on an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster
119
City, CA). Bi-directional sequences obtained were assembled using the ChromasPro (version
120
1.5) software (http://technelysium.com.au/?page_id=27), and aligned with each other and
121
reference sequences of each gene downloaded from GenBank using ClustalX
122
(http://www.clustal.org/) to determine Cryptosporidium species (based on SSU rRNA sequences)
123
and C. parvum subtypes (based on gp60 sequences). The latter were named using the established
124
Cryptosporidium subtype nomenclature system (Sulaiman, et al., 2005). Representative
125
nucleotide sequences from this study were submitted to GenBank under accession numbers
126
KX443780-KX443784.
129 130 131 132
3. Results
AC C
128
EP
127
TE D
M AN U
SC
117
3.1. PCR detection of Cryptosporidium spp. in cattle and goats
Among the 33 specimens from pre-weaned calves examined in 2012 and 2013, 28 were
133
positive for Cryptosporidium spp. in SSU rRNA-based PCR analysis (Table 1). In contrast, all 9
134
specimens from pre-weaned goat kids were positive for Cryptosporidium spp. in SSU rRNA-
135
based PCR (Table 2). Cryptosporidium-positive specimens at the SSU rRNA locus were
6
ACCEPTED MANUSCRIPT
136
analyzed by gp60 PCR. Among them, 23 of 28 specimens from calves were positive in gp60
137
PCR, whereas all 9 from goat kids were positive.
139
3.2. Cryptosporidium species in cattle and goats
140
RFLP analyses of the SSU rRNA PCR products using restriction enzymes SspI and
SC
141
RI PT
138
MboII indicated the occurrence of two Cryptosporidium species in the 28 PCR-positive calves,
143
including C. parvum (27/28) and C. ryanae (1/28) (Table 1). In contrast, only C. parvum was
144
identified in the 9 PCR-positive specimens from goat kids (Table 2).
145
M AN U
142
Among the SSU rRNA PCR-positive but gp60 PCR-negative specimens, four were from C. parvum and one was from C. ryanae. The identification of C. parvum or C. ryanae in these
147
specimens was confirmed by DNA sequence analysis of the SSU rRNA PCR products. All four
148
C. parvum sequences obtained were identical to the GenBank reference sequence AF159111,
149
whereas the C. ryanae sequence obtained was identical to the reference sequence AY587166.
152 153
3.3. Cryptosporidium subtypes in cattle and goats
EP
151
AC C
150
TE D
146
Among those subtyped successfully, DNA sequence analysis of the gp60 PCR products
154
identified the presence of three subtypes in calves, including IIaA13G2R1 (20/23), IIdA18G1
155
(2/23), and IIdA20G1b (1/23) (Table 1). In goat kids, four gp60 subtypes were seen, including
156
IIaA13G2R1 (3/8), IIaA15G1R1 (2/8), IIdA22G1 (2/8), and IIdA18G1 (1/8). One specimen
157
from a goat kid had mixed infection with two undetermined subtypes (Table 2). The gp60
158
sequences obtained from IIaA13G2R1, IIaA15G1R1, IIdA18G1, and IIdA20G1b, and
7
ACCEPTED MANUSCRIPT
159
IIdA22G1subtypes were identical to GenBank sequences KF008184, AY738191, AY738194,
160
AY738185, and GU214368, respectively.
161
In calves, the three specimens of IId subtypes were collected from two farms in Ankara in 2013, as specimens collected in 2012 all belonged to the IIaA13G2R1 subtype (Table 1). In goat
163
kids, however, subtype IIaA13G2R1 was seen only in 2013 and IId subtypes were seen only in
164
2012 (Table 2).
RI PT
162
167
4. Discussion
Results of this small study indicate that despite the traditional, less intensive livestock
M AN U
166
SC
165
husbandry in the study areas, both pre-weaned calves and goat kids are mainly infected with C.
169
parvum. Previously, only two studies examined the occurrence of C. parvum in a small number
170
of dairy calves in Turkey (Arslan and Ekinci, 2012; Tanriverdi, et al., 2006). Thus, studies
171
conducted thus far all indicate a common occurrence of C. parvum in pre-weaned calves and
172
goat kids in Turkey. It is known that pre-weaned calves in less intensive management systems
173
have a common occurrence of C. bovis, C. ryanae, and C. andersoni in pre-weaned calves, in
174
addition to C. parvum (Meireles, et al., 2011; Muhid, et al., 2011; Qi, et al., 2015; Silverlas, et
175
al., 2013; Venu, et al., 2012; Wang, et al., 2011; Zhang, et al., 2013). We identified only C.
176
ryanae in this study in one pre-weaned calf. Similarly, we did not identify any C. xiaoi and C.
177
ubiquitum in pre-weaned goat kids, which often occur in these animals elsewhere, especially C.
178
xiaoi (Diaz, et al., 2011, 2015; Mi, et al., 2014; Parsons, et al., 2015; Rieux, et al., 2013;
179
Tzanidakis, et al., 2014; Wang, et al., 2014).
180 181
AC C
EP
TE D
168
In this study, most C. parvum isolates in calves were subtyped as IIaA13G2R1. Subtype IIaA13G2R1 is an uncommon subtype and has been only reported in a small number of calves in
8
ACCEPTED MANUSCRIPT
Canada, Belgium and the Netherlands and in Chincoteague ponies in the United States (Geurden,
183
et al., 2007; Trotz-Williams, et al., 2006; Wagnerova, et al., 2015; Wielinga, et al., 2008). It
184
nevertheless is probably common in dairy cattle in Turkey; among the 26 GenBank gp60
185
sequences of C. parvum from calves in Turkey, 23 are from the subtype IIaA13G2R1. These
186
sequences are from three unpublished studies conducted in calves in Burdur (JX826593-
187
JX826602), Konya (KF008176-KF008185), and Nevsehir (JQ034314-JQ034316) Provinces. The
188
only published subtyping study of C. parvum in cattle in Turkey showed a dominance (10/13) of
189
the subtype IIaA15G2R1 in Kars Province (Arslan and Ekinci, 2012). Elsewhere in
190
industrialized nations, subtype IIaA15G2R1 is the dominant subtype in pre-weaned dairy calves,
191
with the exception of the United Kingdom, Ireland, and Australia, where IIaA18G3R1 is the
192
most common subtype (Xiao, 2010).
193
M AN U
SC
RI PT
182
The other two subtypes identified in three calves in this study belong to the IId subtype family. They occurred on two farms from Ankara in 2013. C. parvum IId subtypes are common
195
in dairy calves in China, Egypt, Malaysia, and Romania (Amer, et al., 2013; Cui, et al., 2014;
196
Huang, et al., 2014; Muhid, et al., 2011; Qi, et al., 2015; Vieira, et al., 2015; Wang, et al., 2011;
197
Zhang, et al., 2013). They have been found at low frequencies in cattle in many European
198
countries (Bjorkman, et al., 2015; Broglia, et al., 2008; Geurden, et al., 2007; Misic and Abe,
199
2007; Plutzer and Karanis, 2007; Quilez, et al., 2008b; Silverlas, et al., 2013), but are largely
200
absent in cattle in other industrialized nations such as the United States and Canada (Xiao, 2010).
201
Previously, one IId subtype, IIdA15G1, was identified in a cow in Turkey (Arslan and Ekinci,
202
2012).
203 204
AC C
EP
TE D
194
In contrast, a high subtype diversity of C. parvum was seen in goat kids in this study. Four subtypes were identified among the nine C. parvum specimens, including two IIa subtypes
9
ACCEPTED MANUSCRIPT
(IIaA13G2R1 and IIa15G1R1) and two IId subtypes (IIdA18G1 and IIdA22G1). This is similar
206
to findings in Spain and China, where goat kids are known to have high genetic heterogeneity of
207
C. parvum (Diaz, et al., 2015; Mi, et al., 2014; Quilez, et al., 2008a, 2013). Like in the present
208
study, these studies also identified a high occurrence of IId subtypes in pre-weaned goat kids.
209
The difference in subtype distribution suggests that there is probably minimum cross-species
210
transmission of C. parvum between calves and goat kids in Turkey.
RI PT
205
Most of the C. parvum subtypes found in calves and goat kids in this study have been
212
found in humans. The dominant subtype IIaA13G2R1 has been reported in humans in the US
213
and Malaysia (Herges, et al., 2012; Iqbal, et al., 2012), whereas IIaA15G1R1 is the dominant
214
subtype in humans in Scotland (Deshpande, et al., 2015). Similarly, IId subtypes IIdA18G1,
215
IIdA20G1b, and IIdA22G1 have each been detected in some human cases (Chalmers, et al.,
216
2011; Sulaiman, et al., 2005; Vieira, et al., 2015). In Turkey, C. parvum has been identified in
217
humans, although subtype data are not yet available (Tamer, et al., 2007). More extensive
218
characterizations of Cryptosporidium spp. from farm animals and humans are needed to
219
understand the transmission of cryptosporidiosis in Turkey.
222 223 224 225 226
M AN U
TE D
EP
221
Acknowledgements
AC C
220
SC
211
This research was supported by the National Natural Science Foundation of China
(Project No. 31229005 and 31110103901) and Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
10
ACCEPTED MANUSCRIPT
227 References
229
Alves, M., Xiao, L., Sulaiman, I., Lal, A. A., Matos, O., and Antunes, F., 2003. Subgenotype
230
analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal.
231
J Clin Microbiol 41, 2744-2747.
RI PT
228
Amer, S., Zidan, S., Adamu, H., Ye, J., Roellig, D., Xiao, L., and Feng, Y., 2013. Prevalence and
233
characterization of Cryptosporidium spp. in dairy cattle in Nile River delta provinces,
234
Egypt. Exp Parasitol 135, 518-523.
M AN U
SC
232
235
Arslan, M. O. and Ekinci, A. I., 2012. Determination of Cryptosporidium parvum subtypes in
236
cattle in Kars Province of Turkey. Kafkas Universitesi Veteriner Fakultesi Dergisi 18,
237
A221-A226.
239
Bjorkman, C., Lindstrom, L., Oweson, C., Ahola, H., Troell, K., and Axen, C., 2015. Cryptosporidium infections in suckler herd beef calves. Parasitology 142, 1108-1114.
TE D
238
Broglia, A., Reckinger, S., Caccio, S. M., and Nockler, K., 2008. Distribution of
241
Cryptosporidium parvum subtypes in calves in Germany. Vet Parasitol 154, 8-13.
242
Chalmers, R. M., Elwin, K., Reilly, W. J., Irvine, H., Thomas, A. L., and Hunter, P. R., 2002.
243
Cryptosporidium in farmed animals: the detection of a novel isolate in sheep. Int J
245 246
AC C
244
EP
240
Parasitol 32, 21-26.
Chalmers, R. M. and Giles, M., 2010. Zoonotic cryptosporidiosis in the UK - challenges for control. J Appl Microbiol 109, 1487-1497.
247
Chalmers, R. M., Smith, R. P., Hadfield, S. J., Elwin, K., and Giles, M., 2011. Zoonotic linkage
248
and variation in Cryptosporidium parvum from patients in the United Kingdom. Parasitol
249
Res 108, 1321-1325.
11
ACCEPTED MANUSCRIPT
250
Cho, Y. I., Han, J. I., Wang, C., Cooper, V., Schwartz, K., Engelken, T., and Yoon, K. J., 2013.
251
Case-control study of microbiological etiology associated with calf diarrhea. Vet
252
Microbiol 166, 375-385. Cui, Z., Wang, R., Huang, J., Wang, H., Zhao, J., Luo, N., Li, J., Zhang, Z., and Zhang, L., 2014.
254
Cryptosporidiosis caused by Cryptosporidium parvum subtype IIdA15G1 at a dairy farm
255
in Northwestern China. Parasit Vectors 7, 529.
RI PT
253
Deshpande, A. P., Jones, B. L., Connelly, L., Pollock, K. G., Brownlie, S., and Alexander, C. L.,
257
2015. Molecular characterization of Cryptosporidium parvum isolates from human
258
cryptosporidiosis cases in Scotland. Parasitology 142, 318-325.
M AN U
SC
256
259
Diaz, P., Quilez, J., Robinson, G., Chalmers, R. M., Diez-Banos, P., and Morrondo, P., 2011.
260
Identification of Cryptosporidium xiaoi in diarrhoeic goat kids (Capra hircus) in Spain.
261
Vet Parasitol 172, 132-134.
Diaz, P., Quilez, J., Prieto, A., Navarro, E., Perez-Creo, A., Fernandez, G., Panadero, R., Lopez,
263
C., Diez-Banos, P., and Morrondo, P., 2015. Cryptosporidium species and subtype
264
analysis in diarrhoeic pre-weaned lambs and goat kids from north-western Spain.
265
Parasitol Res 114, 4099-4105.
EP
TE D
262
Fayer, R., Santin, M., Trout, J. M., and Greiner, E., 2006. Prevalence of species and genotypes of
267
Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States. Vet
268
AC C
266
Parasitol 135, 105-112.
269
Fayer, R., Santin, M., and Trout, J. M., 2007. Prevalence of Cryptosporidium species and
270
genotypes in mature dairy cattle on farms in eastern United States compared with
271
younger cattle from the same locations. Vet Parasitol 145, 260-266.
12
ACCEPTED MANUSCRIPT
272
Feng, Y., Ortega, Y., He, G., Das, P., Xu, M., Zhang, X., Fayer, R., Gatei, W., Cama, V., and
273
Xiao, L., 2007. Wide geographic distribution of Cryptosporidium bovis and the deer-like
274
genotype in bovines. Vet Parasitol 144, 1-9. Geurden, T., Berkvens, D., Martens, C., Casaert, S., Vercruysse, J., and Claerebout, E., 2007.
276
Molecular epidemiology with subtype analysis of Cryptosporidium in calves in Belgium.
277
Parasitology 134, 1981-1987.
279
Heine, J., 1982. Eine einfache Nachweismethode für Kryptosporidien im Kot. Zbl. Vet. Med. B.
SC
278
RI PT
275
29, 324–327.
Herges, G. R., Widmer, G., Clark, M. E., Khan, E., Giddings, C. W., Brewer, M., and McEvoy,
281
J. M., 2012. Evidence that Cryptosporidium parvum populations are panmictic and
282
unstructured in the Upper Midwest of the United States. Appl Environ Microbiol 78,
283
8096-8101.
M AN U
280
Huang, J., Yue, D., Qi, M., Wang, R., Zhao, J., Li, J., Shi, K., Wang, M., and Zhang, L., 2014.
285
Prevalence and molecular characterization of Cryptosporidium spp. and Giardia
286
duodenalis in dairy cattle in Ningxia, northwestern China. BMC Vet Res 10, 292.
287
Iqbal, A., Lim, Y. A., Surin, J., and Sim, B. L., 2012. High diversity of Cryptosporidium
288
subgenotypes identified in Malaysian HIV/AIDS individuals targeting gp60 gene. PLoS
289
One 7, e31139.
291
EP
AC C
290
TE D
284
Koinari, M., Lymbery, A. J., and Ryan, U. M., 2014. Cryptosporidium species in sheep and goats from Papua New Guinea. Exp Parasitol 141, 134-137.
292
Kotloff, K. L., Nataro, J. P., Blackwelder, W. C., Nasrin, D., Farag, T. H., Panchalingam, S.,
293
Wu, Y., Sow, S. O., Sur, D., Breiman, R. F., Faruque, A. S., Zaidi, A. K., Saha, D.,
294
Alonso, P. L., Tamboura, B., Sanogo, D., Onwuchekwa, U., Manna, B., Ramamurthy, T.,
13
ACCEPTED MANUSCRIPT
Kanungo, S., Ochieng, J. B., Omore, R., Oundo, J. O., Hossain, A., Das, S. K., Ahmed,
296
S., Qureshi, S., Quadri, F., Adegbola, R. A., Antonio, M., Hossain, M. J., Akinsola, A.,
297
Mandomando, I., Nhampossa, T., Acacio, S., Biswas, K., O'Reilly, C. E., Mintz, E. D.,
298
Berkeley, L. Y., Muhsen, K., Sommerfelt, H., Robins-Browne, R. M., and Levine, M. M.,
299
2013. Burden and aetiology of diarrhoeal disease in infants and young children in
300
developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-
301
control study. Lancet 382, 209-222.
SC
RI PT
295
Meireles, M. V., de Oliveira, F. P., Teixeira, W. F., Coelho, W. M., and Mendes, L. C., 2011.
303
Molecular characterization of Cryptosporidium spp. in dairy calves from the state of Sao
304
Paulo, Brazil. Parasitol Res 109, 949-951.
M AN U
302
Mi, R., Wang, X., Huang, Y., Zhou, P., Liu, Y., Chen, Y., Chen, J., Zhu, W., and Chen, Z., 2014.
306
Prevalence and molecular characterization of Cryptosporidium in goats across four
307
provincial level areas in China. PLoS One 9, e111164.
TE D
305
Misic, Z. and Abe, N., 2007. Subtype analysis of Cryptosporidium parvum isolates from calves
309
on farms around Belgrade, Serbia and Montenegro, using the 60 kDa glycoprotein gene
310
sequences. Parasitology 134, 351-358.
EP
308
Mueller-Doblies, D., Giles, M., Elwin, K., Smith, R. P., Clifton-Hadley, F. A., and Chalmers, R.
312
M., 2008. Distribution of Cryptosporidium species in sheep in the UK. Vet Parasitol 154,
313
AC C
311
214-219.
314
Muhid, A., Robertson, I., Ng, J., and Ryan, U., 2011. Prevalence of and management factors
315
contributing to Cryptosporidium sp. infection in pre-weaned and post-weaned calves in
316
Johor, Malaysia. Exp Parasitol 127, 534-538.
14
ACCEPTED MANUSCRIPT
Parsons, M. B., Travis, D., Lonsdorf, E. V., Lipende, I., Roellig, D. M., Kamenya, S., Zhang, H.,
318
Xiao, L., and Gillespie, T. R., 2015. Epidemiology and molecular characterization of
319
Cryptosporidium spp. in humans, wild primates, and domesticated animals in the Greater
320
Gombe Ecosystem, Tanzania. PLoS Negl Trop Dis 9, e0003529.
321 322
RI PT
317
Plutzer, J. and Karanis, P., 2007. Genotype and subtype analyses of Cryptosporidium isolates from cattle in Hungary. Vet Parasitol 146, 357-362.
Qi, M., Wang, H., Jing, B., Wang, D., Wang, R., and Zhang, L., 2015. Occurrence and molecular
324
identification of Cryptosporidium spp. in dairy calves in Xinjiang, Northwestern China.
325
Vet Parasitol 212, 404-407.
M AN U
SC
323
326
Quilez, J., Torres, E., Chalmers, R. M., Hadfield, S. J., Del Cacho, E., and Sanchez-Acedo, C.,
327
2008a. Cryptosporidium genotypes and subtypes in lambs and goat kids in Spain. Appl
328
Environ Microbiol 74, 6026-6031.
Quilez, J., Torres, E., Chalmers, R. M., Robinson, G., Del Cacho, E., and Sanchez-Acedo, C.,
330
2008b. Cryptosporidium species and subtype analysis from dairy calves in Spain.
331
Parasitology 135, 1613-1620.
TE D
329
Quilez, J., Vergara-Castiblanco, C., Monteagudo, L., del Cacho, E., and Sanchez-Acedo, C.,
333
2013. Host association of Cryptosporidium parvum populations infecting domestic
334
ruminants in Spain. Appl Environ Microbiol 79, 5363-5371.
AC C
EP
332
335
Rieux, A., Paraud, C., Pors, I., and Chartier, C., 2013. Molecular characterization of
336
Cryptosporidium spp. in pre-weaned kids in a dairy goat farm in western France. Vet
337 338 339
Parasitol 192, 268-272.
Ryan, U., Fayer, R., and Xiao, L., 2014. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 141, 1667-1685.
15
ACCEPTED MANUSCRIPT
340
Santin, M., Trout, J. M., Xiao, L., Zhou, L., Greiner, E., and Fayer, R., 2004. Prevalence and
341
age-related variation of Cryptosporidium species and genotypes in dairy calves. Vet
342
Parasitol 122, 103-117.
344
Silverlas, C., Bosaeus-Reineck, H., Naslund, K., and Bjorkman, C., 2013. Is there a need for
RI PT
343
improved Cryptosporidium diagnostics in Swedish calves? Int J Parasitol 43, 155-161. Sulaiman, I. M., Hira, P. R., Zhou, L., Al-Ali, F. M., Al-Shelahi, F. A., Shweiki, H. M., Iqbal, J.,
346
Khalid, N., and Xiao, L., 2005. Unique endemicity of cryptosporidiosis in children in
347
Kuwait. J Clin Microbiol 43, 2805-2809.
SC
345
Tamer, G. S., Turk, M., Dagci, H., Pektas, B., Guy, E. C., Guruz, A. Y., and Uner, A., 2007. The
349
prevalence of cryptosporidiosis in Turkish children, and genotyping of isolates by nested
350
polymerase chain reaction-restriction fragment length polymorphism. Saudi Med J 28,
351
1243-1246.
M AN U
348
Tanriverdi, S., Markovics, A., Arslan, M. O., Itik, A., Shkap, V., and Widmer, G., 2006.
353
Emergence of distinct genotypes of Cryptosporidium parvum in structured host
354
populations. Appl Environ Microbiol 72, 2507-2513.
TE D
352
Trotz-Williams, L. A., Martin, D. S., Gatei, W., Cama, V., Peregrine, A. S., Martin, S. W.,
356
Nydam, D. V., Jamieson, F., and Xiao, L., 2006. Genotype and subtype analyses of
357
Cryptosporidium isolates from dairy calves and humans in Ontario. Parasitol Res 99,
AC C
358
EP
355
346-352.
359
Tzanidakis, N., Sotiraki, S., Claerebout, E., Ehsan, A., Voutzourakis, N., Kostopoulou, D., Stijn,
360
C., Vercruysse, J., and Geurden, T., 2014. Occurrence and molecular characterization of
361
Giardia duodenalis and Cryptosporidium spp. in sheep and goats reared under dairy
362
husbandry systems in Greece. Parasite 21, 45.
16
ACCEPTED MANUSCRIPT
363
Venu, R., Latha, B. R., Basith, S. A., Raj, G. D., Sreekumar, C., and Raman, M., 2012.
364
Molecular prevalence of Cryptosporidium spp. in dairy calves in Southern states of India.
365
Vet Parasitol 188, 19-24. Vieira, P. M., Mederle, N., Lobo, M. L., Imre, K., Mederle, O., Xiao, L., Darabus, G., and
367
Matos, O., 2015. Molecular characterisation of Cryptosporidium (Apicomplexa) in
368
children and cattle in Romania. Folia Parasitol (Praha) 62.
RI PT
366
Wagnerova, P., Sak, B., McEvoy, J., Rost, M., Sherwood, D., Holcomb, K., and Kvac, M., 2015.
370
Cryptosporidium parvum and Enterocytozoon bieneusi in American Mustangs and
371
Chincoteague ponies. Exp Parasitol 162, 24-27.
M AN U
SC
369
372
Wang, R., Wang, H., Sun, Y., Zhang, L., Jian, F., Qi, M., Ning, C., and Xiao, L., 2011.
373
Characteristics of Cryptosporidium transmission in preweaned dairy cattle in Henan,
374
China. J Clin Microbiol 49, 1077-1082.
Wang, R., Li, G., Cui, B., Huang, J., Cui, Z., Zhang, S., Dong, H., Yue, D., Zhang, L., Ning, C.,
376
and Wang, M., 2014. Prevalence, molecular characterization and zoonotic potential of
377
Cryptosporidium spp. in goats in Henan and Chongqing, China. Exp Parasitol 142, 11-
378
16.Wielinga, P. R., de Vries, A., van der Goot, T. H., Mank, T., Mars, M. H., Kortbeek,
379
L. M., and van der Giessen, J. W., 2008. Molecular epidemiology of Cryptosporidium in
380
humans and cattle in The Netherlands. Int J Parasitol 38, 809-817.
382
EP
AC C
381
TE D
375
Xiao, L., 2010. Molecular epidemiology of cryptosporidiosis: An update. Exp Parasitol 124, 8089.
383
Zhang, W., Wang, R., Yang, F., Zhang, L., Cao, J., Zhang, X., Ling, H., Liu, A., and Shen, Y.,
384
2013. Distribution and genetic characterizations of Cryptosporidium spp. in pre-weaned
385
dairy calves in Northeastern China's Heilongjiang Province. PLoS One 8, e54857.
386 17
Table 1
388
Cryptosporidium species and C. parvum subtypes in dairy cattle in Turkey
Breed
Nutrition
1
Kalecik/Ankara
Holstein
Breast milk
2
Kalecik/Ankara
Simental, Holstein
Breast milk
Holstein
Breast milk
4
5
Keskin/Kırıkkale
Kaman/Kırıkkale
Hasandede/Kırıkkale
Holstein
Holstein
Breast milk Breast milk
In barn with mother In barn with mother In barn with mother In barn with mother In barn with mother Initially in barn with mother In barn with mother In barn
EP
3
Care
Manyas/Balıkesir
Holstein
7
Merkez/Kırıkkale
Holstein
Breast milk
8
Alaca/Çorum
Simental
Breast
AC C
6
Breast milk, bottle milk
Sampling time
2012
M AN U
Location
2012
2012
TE D
Farm ID
No. No. microscopyspecimen positive PCRspecimen positive examined
SC
387
RI PT
ACCEPTED MANUSCRIPT
6
4
2
C. parvum
C. parvum subtype*
C. ryanae
4
IIaA13G2R1 (4)
0
2
IIaA13G2R1 (2)
0
2
IIaA13G2R1 (2)
0
3
IIaA13G2R1 (3)
0
2 3 2
2012
3 3
2013
7
6
6
IIaA13G2R1 (4), unknown (2)
0
2013
5
5
4
IIaA13G2R1 (3), unknown (1)
1
2013
1
1
IIaA13G2R1 (1)
0
2013
1
1
IIaA13G2R1
0
18
1 1
ACCEPTED MANUSCRIPT
Kalecik/Ankara
Simental, Holstein
Breast milk
Holstein
Breast milk
In barn with mother
2013
AC C
EP
TE D
Total 389 *Unknown subtype: gp60 subtyping was unsuccessful. 390
2013
RI PT
10
Kalecik/Ankara
(1)
M AN U
9
with mother In barn with mother
19
4
3
1
1
SC
milk
33
3
1
28
27
IIdA18G1 (1), IIdA20G1b (1), (unknown (1)
0
IIdA18G1 (1)
0
IIaA13G2R1 (20), IIdA18G1 (2), IIdA20G1b (1), unknown (4)
1
392
Cryptosporidium parvum subtypes in goats in Turkey
Location
Breed
Nutrition
Merkez/Kırıkkale
Hair Goat
Breast milk
11 Sulakyurt/Kırıkkale
Hair Goat
Keskin/Kırıkkale
Hair Goat
Akpınar/ Kırşehir
Hair Goat
12 13
Breast milk 1/2 diluted cow's milk
Pasturing with mother Pasturing with mother Pasturing with mother Pasturing with mother
C. parvum
C. parvum subtype
4
4
4
IIaA15G1R1 (2), IIdA18G1 (1), mixed subtypes (1)
2012
2
2
2
IIdA22G1 (2)
2013
2
2
2
IIaA13G2R1 (2)
1
1
1
IIaA13G2R1 (1)
9
IIaA13G2R1 (3), IIaA15G1R1 (2), IIdA22G1 (2), IIdA18G1 (1), mixed subtypes (1)
Sampling time
2012
2013
Total 393
AC C
EP
14
Breast milk
Care
No. specimen PCRpositive
TE D
Farm ID
No. microscopy -positive specimen examined
SC
Table 2
M AN U
391
RI PT
ACCEPTED MANUSCRIPT
9
20
9
ACCEPTED MANUSCRIPT Highlights Preweaned calves and goat kids reared in traditional farming systems were examined
•
Cryptosporidium parvum was identified in 27 calves and 9 goat kids
•
IIaA13G2R1 (20/23) was the dominant subtype in calves
•
A high C. parvum subtype diversity was found in goat kids
•
All five C. parvum subtypes found are known human pathogens
AC C
EP
TE D
M AN U
SC
RI PT
•