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Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr 5 6
Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China
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Rongjun Wang a,b,c,1, Guoquan Li b,c,1, Bin Cui b,c, Jianying Huang b,c, Zhaohui Cui b,c, Sumei Zhang b,c, Haiju Dong b,c, Daoyou Yue b,c, Longxian Zhang b,c,⇑, Changshen Ning b,c, Ming Wang a,⇑ a
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b c
College of Veterinary Medicine, China Agricultural University, Beijing 100193, China College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China International Joint Research Laboratory for Zoonotic Diseases of Henan, Zhengzhou 450002, China
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h i g h l i g h t s
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g r a p h i c a l a b s t r a c t
The overall prevalence of
Cryptosporidium in goats was 3.48% (44/1256). C. ubiquitum, C. andersoni and C. xiaoi were identified. The zoonotic XIIa subtype 2 of C. ubiquitum was found. C. ubiquitum and C. andersoni were the first identified species in goats. Age-associated distribution of Cryptosporidium was noticed in goats.
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Article history: Received 16 January 2014 Received in revised form 27 March 2014 Accepted 1 April 2014 Available online xxxx
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Keywords: Cryptosporidium Goats C. ubiquitum C. andersoni C. xiaoi SSU rRNA gp60
a b s t r a c t To estimate the prevalence and public health significance of cryptosporidiosis in goats in China, 1265 fecal samples from seven farms in Henan province and Chongqing city were examined for Cryptosporidium oocysts. The overall infection rate of Cryptosporidium spp. was 3.48% (44/1256). Significant difference was observed among age groups, with the post weaned kids having the highest infection rate (4.58%; q < 0.01). Cryptosporidium spp. were characterized by PCR–restriction fragment length polymorphism (RFLP) analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene. The SSU rRNA-based PCR identified three Cryptosporidium species, including Cryptosporidium ubiquitum (24/44) in Henan and Chongqing, and Cryptosporidium andersoni (16/44) and Cryptosporidium xiaoi (4/44) in Henan. Among which, the C. ubiquitum and C. andersoni were first identified in goats thus far and were found in all age groups except no C. andersoni being found in the postparturition nannies, whereas the C. xiaoi was detected in pre-weaned kids and pregnant nannies. Subtyping C. ubiquitum by DNA sequence analysis of the 60 kDa glycoprotein (gp60) gene suggested the isolates identified all belonged to zoonotic XIIa subtype 2. Thus, the dominant C. ubiquitum found in this study and the XIIa subtype 2 has been found in humans indicated goats are a potential source for zoonotic infections with the C. ubiquitum. More studies are needed for better understanding of differences in the transmission and public health significance of cryptosporidiosis in goats. Ó 2014 Published by Elsevier Inc.
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⇑ Corresponding authors. Address: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China. Fax: +86 371 63558180 (L. Zhang). Fax: +86 10 62733961 (M. Wang). E-mail addresses: [email protected], [email protected] (L. Zhang), [email protected] (M. Wang). 1 Rongjun Wang and Guoquan Li contributed equally to this work. http://dx.doi.org/10.1016/j.exppara.2014.04.001 0014-4894/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: Wang, R., et al. Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.001
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R. Wang et al. / Experimental Parasitology xxx (2014) xxx–xxx
1. Introduction Cryptosporidium spp. are important gastrointestinal protists in a wide spectrum of hosts, including humans, other mammals, birds, reptiles, amphibians, and fish. Infection is acquired following the ingestion of infective oocysts by the fecal-oral route, via either direct contact or ingestion of contaminated food or water (Xiao and Ryan, 2004). It is one of the common causes of diarrhea in humans and animals. Thus far, twenty-six Cryptosporidium species are considered valid, and more than 70 host-adapted genotypes with undetermined species status have been described (Fayer, 2009; Elwin et al., 2012b; Ren et al., 2012; Kvácˇ et al., 2013). The first Cryptosporidium infection case in goat was described in Australia in a 2-week-old kid with diarrhea (Mason et al., 1981). Since then, the infection has been reported in several continents, including Europe (Spain, Italy, Switzerland, Poland, Hungary, Netherland, Belgium, UK, France, and Czech Republic), Asia (India, Mongolia, Turkey, Korea, Iran, Cyprus, Sri Lanka, Iraq, Sultanate of Oman, and China), Africa (Egypt, Tunisia, Malawi, and Zambia), and America (USA, Brazil, and Trinidad & Tobago), with the infection rate ranging from 0% to 100% (average 17.92%, 980/5468) based on point prevalence data at different geographical areas (Noordeen et al., 2000; Geurden et al., 2008; Quílez et al., 2008; Robertson, 2009; Drumo et al., 2012; Giadinis et al., 2012; Marreros et al., 2012; Jafari et al., 2013; Maurya et al., 2013; Rieux et al., 2013). However, few studies have genotyped Cryptosporidium spp. from goats in the world. Previous studies indicated that Cryptosporidium parvum was the dominant Cryptosporidium species, as well as Cryptosporidium xiaoi, Cryptosporidium hominis, a goat genotype, and a new Cryptosporidium genotype have also been detected in goats (Ryan et al., 2005; Karanis et al., 2007b; Robertson, 2009). Goats play an important role in the agricultural economy of China. In 2011, the total goat population was 142.23 millions, ranking first in the world (http://kids.fao.org/glipha/). Unfortunately, little is known about the distribution and zoonotic transmission risk of Cryptosporidium spp. in goats, except for a few prevalence studies published in the Chinese language and two isolates identified as C. xiaoi (previously named as C. bovis-like genotype) and a new Q4 Cryptosporidium genotype (Karanis et al., 2007a,b). More recently, Li et al. (2014) developed a subtyping technique by whole-genome sequencing to characterize gp60 gene of Cryptosporidium ubiquitum from humans, various animals, and drinking source water, which produced six subtype families, XIIa–XIIf. Thus, the objective of this study was to identify the distribution and zoonotic potential of Cryptosporidium spp. in goats in Henan province and Chongqing city, China at the genotype and subtype levels.
2. Materials and methods 2.1. Sample and microscopy examination Fresh fecal sample for each animal was collected immediately after being defecated on the ground using a sterile disposal latex glove, and then placed in a disposable plastic bag individually. A total of 1265 fecal samples were obtained between July 2006 and July 2007 from four goat farms in two areas of Henan province and from three goat farms in three areas of Chongqing city in December 2011, including those from pre-weaned and postweaned kids, adult goats, pregnant nannies, and postparturition nannies (Table 1). Cryptosporidium oocysts were examined by microscopy after approximately 20 g fecal samples each animal were concentrated by the Sheather’s sugar flotation technique and were stained with the modified acid-fast staining. Cryptosporidium-positive samples were stored in 2.5% potassium dichromate at 4 °C prior to DNA extraction.
Table 1 Number of fecal samples examined for Cryptosporidium oocysts by microscopy on each farm and the distribution of Cryptosporidium spp. determined by PCR–RFLP and sequence analysis of the SSU rRNA gene. Collection site
Sample size
Cryptosporidium positive (%)
C. ubiquitum
C. andersoni
C. xiaoi
6 (24%) 2 (66.67%)
16 (64%)
3 (12%) 1 (33.32%)
16 (36.36%)
4 (9.09%)
HN-1 HN-2
790 138
25 (3.16%) 3 (2.17%)
HN-3 HN-4 CQ-1 CQ-2 CQ-3 Total
65 24 64 72 112 1265
0 0 1 (3.13%) 7 (9.72%) 8 (8.33%) 44 (3.48%)
1 (100%) 7 (100%) 8 (100%) 24 (54.55%)
2.2. DNA extraction
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For samples obtained between 2006 and 2007, Cryptosporidium oocysts were isolated from the positive fecal samples by the discontinuous density sucrose gradient centrifugation. Then, genomic DNA was extracted from the purified oocysts using the Mag Extractor Genome kit as described in previous study (Wang et al., 2008). As for samples collected in December 2011, genomic DNA was isolated from Cryptosporidium-positive samples using the E.Z.N.A.@ Stool DNA Kit (OMEGA Biotek Inc., USA) and the manufacturer recommended procedures (Wang et al., 2011b). The extracted DNA was kept at 20 °C before it was used in molecular analysis.
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2.3. Cryptosporidium genotyping and subtyping
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Cryptosporidium spp. were genotyped by amplifying the small subunit (SSU) rRNA gene by nested PCR and restriction fragment length polymorphism (RFLP) analysis using restriction enzymes SspI and VspI (Xiao et al., 2001). The PCR products were further sequenced directly with the secondary PCR primers on an ABI 3730 DNA Analyzer using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Sequence accuracy was confirmed by two-directional sequencing and by sequencing a new PCR product if necessary. C. ubiquitum was subtyped by using gp60 gene, with the sequences of primers being 50 -TTT ACC CAC ACA TCT GTA GCG TCG-30 (Ubi-18S-F1) and 50 -ACG GAC GGA ATG ATG TAT CTG A-30 (Ubi-18S-R1), and 50 -ATA GGT GAT AAT TAG TCA GTC TTT AAT-30 (Ubi-18S-F2) and 50 -TCC AAA AGC GGC TGA GTC AGC ATC-30 (Ubi-18SR2) for the primary and secondary PCR (Li et al., 2014). The PCR products of gp60 gene were sequenced using the secondary PCR primers as described above. Sequence analysis was performed by alignment of SSU rRNA sequences obtained in this study and reference sequences downloaded from GenBank using the program ClustalX 1.83 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/). Phylogenetic analyses were performed using the software Phylip 3.69 (http://evolution. genetics.washington.edu/phylip.html). Neighbour-joining trees were constructed based on the evolutionary distances calculated by Kimura-2-parameter model. The reliability of these trees was assessed using the bootstrap analysis with 1000 replicates. Sequences of the partial SSU rRNA and gp60 genes were deposited in GenBank under Accession Nos. EU926575–EU926602 and KJ622359.
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2.4. Statistical analysis
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The chi-square test was used to compare Cryptosporidium infection rates. Differences were considered significant when q < 0.05.
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Please cite this article in press as: Wang, R., et al. Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.001
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3. Results
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3.1. Prevalence of Cryptosporidium spp.
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Microscopic analysis of 1265 fecal samples showed the presence of Cryptosporidium oocysts in 44 samples (3.48%) on five out of seven goat farms (Table 1). The infection rate was 2.75% (28/ 1017) in Henan province and 6.45% (16/248) in Chongqing city (v2 = 8.12; q < 0.01), with the highest infection rate (9.72%) being observed on farm CQ-2 (v2 = 17.68; q < 0.01) (Table 1). The percentage of animals shedding oocysts was 2.11%, 4.58%, 1.90%, 4.44%, and 2.72% in pre-weaned kids, post-weaned kids, adult goats, pregnant nannies, and postparturition nannies, respectively (v2 = 14.23; q < 0.01) (Table 2).
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3.2. Distribution of Cryptosporidium species
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The SSU rRNA gene of Cryptosporidium spp. in all 44 microscopy-positive samples was successfully amplified by the nested PCR. RFLP and DNA sequence analyses of the SSU rRNA gene products revealed the presence of three Cryptosporidium species, including C. ubiquitum (24/44), Cryptosporidium andersoni (16/44), and C. xiaoi (4/44) (Table 1). Phylogenetic relationship analysis confirmed the identity of Cryptosporidium species (Fig. 1). Farms
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Table 2 Cryptosporidium species identified among different age groups of goats. Age group Pre-weaned kids Post weaned kids Adult goats Pregnant nannies Postparturition nannies Total
Sample size
Cryptosporidium positive (%)
C. C. C. ubiquitum andersoni xiaoi
229 393 368 174 101
17 (2.11%) 12 (4.58%) 7 (1.90%) 6 (4.44%) 2 (2.27%)
9 4 6 3 2
5 8 1 2 0
3 0 0 1 0
1265
44 (3.48%)
24
16
4
Fig. 1. Phylogenetic relationship of Cryptosporidium inferred by neighbour-joining analysis of the SSU rRNA based on Kimura 2-parameter model.
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in Henan province had infections of three Cryptosporidium species, with the C. andersoni appearing to be the predominant species. In contrast, only the C. ubiquitum was found in three Cryptosporidium-positive farms in Chongqing city (Table 1).
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3.3. Age patterns of Cryptosporidium species
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C. ubiquitum was the predominantly identified species, responsible for 45.55% of all Cryptosporidium infections. It was found in all age groups examined in this study, on all five Cryptosporidium-positive farms. C. andersoni was only detected in farm HN-1, found in four age groups (Table 2). In contrast, the proportion of C. xiaoi was lower, responsible for 9.09% of Cryptosporidium infections. The C. xiaoi was only found in pre-weaned kids and pregnant nannies (Table 2).
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3.4. Subtype of C. ubiquitum isolates
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A total of 15 C. ubiquitum isolates were amplified and sequenced at the gp60 locus, including the 8 isolates from HN-1 and HN-2, one from CQ-1, and each two from CQ-2 and CQ-3. All the 15 gp60 sequences were identical to each other and belonged to XIIa subtype 2, which shared 100% homology to Turkey sheep- and human-derived C. ubiquitum isolates (GenBank Accession Nos. KC204982 and JX412919), as well as the XIIa subtype 2 isolates from China sheep and yak, Nepal swamp deer, and South Africa impala and buffalo (Fig. 2 shown in Li et al., 2014). In contrast, the C. ubiquitum XIIa subtype 2 from goats found in this study had only two nucleotide differences in comparison with three China sheep XIIa subtype 1 isolates (GenBank Accession No. JX412921), and three nucleotide differences compared to Algeria goat XIIa subtype 3 isolate (GenBank Accession No. JX412917).
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4. Discussion
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A 3.48% overall infection rate of Cryptosporidium spp. was observed in this study, which was higher than 2.5% (6/237) of infection rate in Iran, or close to 3.54% (4/113) in Switzerland, and 3.5% (4/116) in India (Marreros et al., 2012; Jafari et al., 2013; Maurya et al., 2013). However, the present findings likely under represented the actual number of Cryptosporidium-positive goats because microscopy repeatedly has been shown to be less sensitive than molecular methods for detection of positive specimens. Summarizing the available data (n = 32) showed the overall infection rate of Cryptosporidium spp. was 17.92% (980/5468) in goats worldwide (Table 3). The difference of prevalence was observed in various countries (Table 3), which may be related to sample sizes, examination methods, different management practices, the timing of sample collection, and geographical ecological conditions. The highest levels of infection was seen in post weaned kids (4.58%) and pregnant nannies (4.44%), whereas the lowest infection rate was found in adult goats (1.90%) (q < 0.01). Thus, the difference associated with age groups might be partially attributed to the immunity or physical condition of animals at different ages. Five Cryptosporidium species/genotypes have been identified in goats thus far, including C. parvum, C. xiaoi, C. hominis, a goat genotype, and a new Cryptosporidium genotype (Ryan et al., 2005; Park et al., 2006; Karanis et al., 2007b; Quílez et al., 2008; Díaz et al., 2010). Among these species and genotypes identified in goats, C. parvum is the dominant Cryptosporidium species, which has been found in goats in Italy, Spain, Belgium, Czech Republic, the Netherlands, France, India, Sri Lanka, Zambia, and Egypt (Cacciò et al., 2000; Noordeen et al., 2002; Hajdušek et al., 2004; Ngouanesavanh et al., 2006; Goma et al., 2007; Geurden et al., 2008; Quílez et al., 2008; Giles et al., 2009; Drumo et al., 2012; Maurya et al., 2013;
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Table 3 Prevalence and Cryptosporidium species/genotypes in goats in reported studies. Area Italy Italy Spain Spain Spain Spain Spain Spain Hungary Poland Czech Republic Belgium United Kingdom England and Wales Switzerland France France France France Netherland, Italy, Czech Republic Romania India Mongolia Turkey China Korea Taiwan Iran Cyprus Sri Lanka Sri Lanka Iraq Tunisia Zambia Malawi Egypt Nigeria Trinidad and Tobago USA Brazil Brazil
253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279
No. positive/No. examined (%)
Cryptosporidium species/genotype (No.)
References
C. parvum (21)
Drumo et al. (2012) Rossanigo et al. (1987) Matos-Fernandez et al. (1993) Munoz-Fernandez et al. (1996) Castro-Hermida et al. (2007) Quílez et al. (2008) Sanz Ceballos et al. (2009) Díaz et al. (2010) Nagy et al. (1984) Majewska et al. (2000) Hajdušek et al. (2004) Geurden et al. (2008) Giles et al. (2009) Smith et al. (2010) Marreros et al. (2012) Delafosse et al. (2006) Castro-Hermida et al. (2005) Ngouanesavanh et al. (2006) Rieux et al. (2013) Cacciò et al. (2000) Bejan et al. (2009) Maurya et al. (2013) Burenbaatar et al. (2008) Ciçek et al. (2008) Karanis et al. (2007a,b) Park et al. (2006) Watanabe et al. (2005) Jafari et al. (2013) Giadinis et al. (2012) Noordeen et al. (2000) Noordeen et al. (2002) Mahdi and Ali (2002) Soltane et al. (2007) Goma et al. (2007) Banda et al. (2009) Shoukry et al. (2009) Pam et al. (2013) Kaminjolo et al. (1993) Caver et al. (1996) Vieira et al. (1997) Bomfim et al. (2005)
8/42 (19%) 40/367 (11%) 15/36 (42%) 9/116 (7.7%) C. parvum (17) 111/582 (19.1%) 2/5 (40%) 10/40 (25%) 0/46 14/148 (9.5%)
C. xiaoi (2)
C. parvum (1) C. parvum (11) C. hominis (1)
3/15 (20%) 4/113 (3.54%) 142/879 (16.2%) 13/100 (13%) C. parvum (7) C. xiaoi (18), C. parvum (1) C. parvum (15) 24% (99/412) 4/116 (3.5%) 0/16 6/56 (10.71%) 2/42 (4.76%) 3/7 (42.9%) 44/123 (35.8%) 6/237 (2.5%) 48/75 (64%) 291/1020 (28.5%)
C. parvum (4)
C. xiaoi (1), new genotype (1) C. hominis (3)
C. parvum (1) 6/45 (13.3%) 0/184 5/105 (4.8%) 23/225 (10.22%) 25.9% 36/150 (24%) 4/20 (20%) 5/19 (26%) 22/22 (100%) 5/105 (4.8%)
Rieux et al., 2013). In contrast, the other four species/genotypes were only detected in a few cases in Spain, the United Kingdom, Korea, Australia, France, and China (Park et al., 2006; Karanis et al., 2007b; Giles et al., 2009; Díaz et al., 2010; Rieux et al., 2013). In this study, both C. ubiquitum and C. andersoni represent the first identified Cryptosporidium species in goats, which have previously been found in sheep in Henan, China (Wang et al., 2000). C. ubiquitum has been found in a great many animals and humans in many geographic locations (Li et al., 2014) and has experimentally been transmitted from goats to cattle (Fayer et al., 2010). Although transmission of C. xiaoi from sheep to goats under experimental conditions was not successful (Fayer and Santin, 2009), C. xiaoi has been found in goats less than 21 days of age in Spain (Díaz et al., 2010). An age-associated distribution of Cryptosporidium was noticed in goats. In this study, all age groups of goats were infected by the C. ubiquitum, which was identical to our previous observations found in sheep (Wang et al., 2000). In contrast, C. xiaoi was only detected in kids and pregnant nannies (Table 2). A more recent study conducted in France showed that 18 out of 19 Cryptosporidium-positive isolates from pre-weaned kids were identified as C. xiaoi (Rieux et al., 2013). Thus, these findings might indicate a possible periparturient transmission for C. xiaoi infection. Indeed, a recent study conducted in sheep has suggested the periparturient transmission of C. xiaoi from ewes to lambs (Ye et al., 2013). Previously, no report compared the distribution of Cryptosporidium spp. in different age groups of goats although a few studies have conducted
C. parvum (1)
in sheep (Santin and Fayer, 2007; Mueller-Doblies et al., 2008; Wang et al., 2000). C. andersoni is the second common Cryptosporidium species, which was found in all age groups except for the postparturition nannies (Table 2). In fact, C. andersoni was one of the most common Cryptosporidium species identified in ruminants and other animals in China, such as cattle, sheep, bactrian camel, Golden hamster, Siberian hamster, and Campbell hamster (Lv et al., 2009; Wang et al., 2000, 2008, 2011a,b). Geographic differences in the distribution of Cryptosporidium spp. in goats are difficult to identify with accuracy because relatively few goats have been examined and many of those tested were from only a few farms in specific locations. C. parvum and C. xiaoi were found in Spain, and France (Quílez et al., 2008; Díaz et al., 2010; Rieux et al., 2013), C. hominis in the United Kingdom and Korea (Park et al., 2006; Giles et al., 2009), whereas C. parvum in the remaining countries (Table 3). In this study, only C. ubiquitum was identified in goats in Chongqing city, however C. ubiquitum, C. andersoni, and C. xiaoi were found in Henan province (Tables 1 and 2). In addition, no C. parvum infection was detected in goats in this study, which was identical to a previous study conducted in sheep in Henan, China (Wang et al., 2000). Therefore, the Cryptosporidium species distribution in goats in Henan, China is apparently different from the one seen in other countries. C. ubiquitum found in this study has potential public health importance. Indeed, the 15 C. ubiquitum isolates subtyped all belonged to zoonotic XIIa subtype 2, which has been detected in humans, as well as in some other animals (Li et al., 2014).
Please cite this article in press as: Wang, R., et al. Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.001
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Previously, C. ubiquitum has been identified in at least 40 sporadic cases, with 38 cases being reported in industrialized nations (Wang et al., 2008; Davies et al., 2009; Pollock et al., 2010; Molloy et al., 2010; Cieloszyk et al., 2012; Elwin et al., 2012a), and it is also one of the common Cryptosporidium spp. found in drinking source water in some countries, such as the United States and Canada (Jiang et al., 2005; Karanis et al., 2007a; Ruecker et al., 2007; Yang et al., 2008; Jellison et al., 2009; Van Dyke et al., 2012). However, the contribution of animal-derived C. ubiquitum to infection in humans is not entirely clear. Nevertheless, the more recent subtyping study indicated contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections (Li et al., 2014). In contrast, the public health importance of C. andersoni is low, only small number of human cases were reported in England and Australia (Leoni et al., 2006; Waldron et al., 2011). In conclusion, C. ubiquitum, C. andersoni, and C. xiaoi were detected in this study, with C. ubiquitum and C. andersoni being the first identified Cryptosporidium species in goats thus far. C. ubiquitum was the dominant Cryptosporidium species and belonged to the zoonotic XIIa subtype 2, which indicated this species has a potential for zoonotic transmission in the study area. More studies are needed for better understanding of differences in the transmission and public health significance of cryptosporidiosis in goats in the world.
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Acknowledgments
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This study was supported in part by the State Key Program of Q5 National Natural Science Foundation of China (31330079), the Q6 National Natural Science Foundation of China (U1204328, 313 02079), and the International Cooperation and Exchange Funds of the National Natural Science Foundation of China (3111 0103901).
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References
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Banda, Z., Nichols, R.A., Grimason, A.M., Smith, H.V., 2009. Cryptosporidium infection in non-human hosts in Malawi. Onderstepoort J. Vet. Res. 76, 363–375. Bejan, A., Mircean, V., Radu, C., Smaro, S., Cozma, V., 2009. Epidemiology of Cryptosporidium spp. infection in goat kids in the central and the northwest part of Romania. Rev. Scientia Parasitol. 10, 32–36. Bomfim, T.C., Huber, F., Gomes, R.S., Alves, L.L., 2005. Natural infection by Giardia sp. and Cryptosporidium sp. in dairy goats, associated with possible risk factors of the studied properties. Vet. Parasitol. 134, 9–13. Burenbaatar, B., Bakheit, M.A., Plutzer, J., Suzuki, N., Igarashi, I., Ongerth, J., Karanis, P., 2008. Prevalence and genotyping of Cryptosporidium species from farm animals in Mongolia. Parasitol. Res. 102, 901–905. Cacciò, S., Homan, W., Camilli, R., Traldi, G., Kortbeek, T., Pozio, E., 2000. A microsatellite marker reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum. Parasitology 120, 237–244. Castro-Hermida, J.A., Delafosse, A., Pors, I., Ares-Mazás, E., Chartier, C., 2005. Giardia duodenalis and Cryptosporidium parvum infections in adult goats and their implications for neonatal kids. Vet. Rec. 157, 623–627. Castro-Hermida, J.A., Almeida, A., González-Warleta, M., Correia da Costa, J.M., Rumbo-Lorenzo, C., Mezo, M., 2007. Occurrence of Cryptosporidium parvum and Giardia duodenalis in healthy adult domestic ruminants. Parasitol. Res. 101, 1443–1448. Caver, J.A., Hill, J.E., Thompson, S.J., 1996. Surveillance of Cryptosporidia in a veterinary diagnostic laboratory. J. Vet. Diagn. Invest. 8, 497–500. Ciçek, M., Körkoca, H., Gül, A., 2008. Investigation of Cryptosporidium sp. in workers of the Van municipality slaughterhouse and in slaughtered animals. Turkiye Parazitol. Derg. 32, 8–11. Cieloszyk, J., Goñi, P., García, A., Remacha, M.A., Sánchez, E., Clavel, A., 2012. Two cases of zoonotic cryptosporidiosis in Spain by the unusual species Cryptosporidium ubiquitum and Cryptosporidium felis. Enferm. Infecc. Microbiol. Clín. 30, 549–551. Davies, A.P., Campbell, B., Evans, M.R., Bone, A., Roche, A., Chalmers, R.M., 2009. Asymptomatic carriage of protozoan parasites in children in day care centers in the United Kingdom. Pediatr. Infect. Dis. J. 28, 838–840. Delafosse, A., Castro-Hermida, J.A., Baudry, C., Ares-Mazás, E., Chartier, C., 2006. Herd-level risk factors for Cryptosporidium infection in dairy-goat kids in western France. Prev. Vet. Med. 77, 109–121. Díaz, P., Quílez, J., Robinson, G., Chalmers, R.M., Díez-Baños, P., Morrondo, P., 2010. Identification of Cryptosporidium xiaoi in diarrhoeic goat kids (Capra hircus) in Spain. Vet. Parasitol. 172, 132–134.
5
Drumo, R., Widmer, G., Morrison, L.J., Tait, A., Grelloni, V., D’Avino, N., Pozio, E., Cacciò, S.M., 2012. Evidence of host-associated populations of Cryptosporidium parvum in Italy. Appl. Environ. Microbiol. 78, 3523–3529. Elwin, K., Hadfield, S.J., Robinson, G., Chalmers, R.M., 2012a. The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, 2000–2008. Epidemiol. Infect. 140, 673–683. Elwin, K., Hadfield, S.J., Robinson, G., Crouch, N.D., Chalmers, R.M., 2012b. Cryptosporidium viatorum n. sp. (Apicomplexa: Cryptosporidiidae) among travellers returning to Great Britain from the Indian subcontinent, 2007– 2011. Int. J. Parasitol. 42, 675–682. Fayer, R., 2009. Taxonomy and species delimitation in Cryptosporidium. Exp. Parasitol. 124, 90–97. Fayer, R., Santín, M., 2009. Cryptosporidium xiaoi n. sp. (Apicomplexa: Cryptosporidiidae) in sheep (Ovis aries). Vet. Parasitol. 164, 192–200. Fayer, R., Santín, M., Macarisin, D., 2010. Cryptosporidium ubiquitum n. sp. in animals and humans. Vet. Parasitol. 172, 23–32. Geurden, T., Thomas, P., Casaert, S., Vercruysse, J., Claerebout, E., 2008. Prevalence and molecular characterisation of Cryptosporidium and Giardia in lambs and goat kids in Belgium. Vet. Parasitol. 155, 142–145. Giadinis, N.D., Symeoudakis, S., Papadopoulos, E., Lafi, S.Q., Karatzias, H., 2012. Comparison of two techniques for diagnosis of cryptosporidiosis in diarrhoeic goat kids and lambs in Cyprus. Trop. Anim. Health Prod. 44, 1561–1565. Giles, M., Chalmers, R., Pritchard, G., Elwin, K., Mueller-Doblies, D., Clifton-Hadley, F., 2009. Cryptosporidium hominis in a goat and a sheep in the UK. Vet. Rec. 164, 24–25. Goma, F.Y., Geurden, T., Siwila, J., Phiri, I.G.K., Gabriel, S., Claerebout, E., Vercruysse, J., 2007. The prevalence and molecular characterisation of Cryptosporidium spp. in small ruminants in Zambia. Small Ruminant Res. 72, 77–80. Hajdušek, O., Ditrich, O., Slapeta, J., 2004. Molecular identification of Cryptosporidium spp. in animal and human hosts from the Czech Republic. Vet. Parasitol. 122, 183–192. Jafari, R., Maghsood, A.H., Fallah, M., 2013. Prevalence of Cryptosporidium infection among livestock and humans in contact with livestock in Hamadan district, Iran, 2012. J. Res. Health Sci. 13, 86–89. Jellison, K.L., Lynch, A.E., Ziemann, J.M., 2009. Source tracking identifies deer and geese as vectors of human-infectious Cryptosporidium genotypes in an urban/ suburban watershed. Environ. Sci. Technol. 43, 4267–4272. Jiang, J., Alderisio, K.A., Xiao, L., 2005. Distribution of Cryptosporidium genotypes in storm event water samples from three watersheds in New York. Appl. Environ. Microbiol. 71, 4446–4454. Kaminjolo, J.S., Adesiyun, A.A., Loregnard, R., Kitson-Piggott, W., 1993. Prevalence of Cryptosporidium oocysts in livestock in Trinidad and Tobago. Vet. Parasitol. 45, 209–213. Karanis, P., Kourenti, C., Smith, H., 2007a. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. J. Water Health 5, 1–38. Karanis, P., Plutzer, J., Halim, N.A., Igori, K., Nagasawa, H., Ongerth, J., Liqing, M., 2007b. Molecular characterization of Cryptosporidium from animal sources in Qinghai province of China. Parasitol. Res. 101, 1575–1580. Kvácˇ, M., Kestrˇánová, M., Pinková, M., Kveˇtonˇová, D., Kalinová, J., Wagnerová, P., Kotková, M., Vítovec, J., Ditrich, O., McEvoy, J., Stenger, B., Sak, B., 2013. Cryptosporidium scrofarum n. sp. (Apicomplexa: Cryptosporidiidae) in domestic pigs (Sus scrofa). Vet. Parasitol. 191, 218–227. Leoni, F., Amar, C., Nichols, G., Pedraza-Díaz, S., McLauchlin, J., 2006. Genetic analysis of Cryptosporidium from 2414 humans with diarrhoea in England between 1985 and 2000. J. Med. Microbiol. 55, 703–707. Li, N., Xiao, L., Alderisio, K., Elwin, K., Cebelinski, E., Chalmers, R., Santin, M., Fayer, R., Kvac, M., Ryan, U., Sak, B., Stanko, M., Guo, Y., Wang, L., Zhang, L., Cai, J., Roellig, D., Feng, Y., 2014. Subtyping Cryptosporidium ubiquitum, a zoonotic pathogen emerging in humans. Emerg. Infect. Dis. 20, 217–224. Lv, C., Zhang, L., Wang, R., Jian, F., Zhang, S., Ning, C., Wang, H., Feng, C., Wang, X., Ren, X., Qi, M., Xiao, L., 2009. Cryptosporidium spp. in wild, laboratory, and pet rodents in china: prevalence and molecular characterization. Appl. Environ. Microbiol. 75, 7692–7699. Mahdi, N.K., Ali, N.H., 2002. Cryptosporidiosis among animal handlers and their livestock in Basrah, Iraq. East Afr. Med. J. 79, 550–553. Majewska, A.C., Werner, A., Sulima, P., Luty, T., 2000. Prevalence of Cryptosporidium in sheep and goats bred on five farms in west-central region of Poland. Vet. Parasitol. 89, 269–275. Marreros, N., Frey, C.F., Willisch, C.S., Signer, C., Ryser-Degiorgis, M.P., 2012. Coprological analyses on apparently healthy Alpine ibex (Capra ibex ibex) from two Swiss colonies. Vet. Parasitol. 186, 382–389. Mason, R.W., Hartley, W.J., Tilt, L., 1981. Intestinal cryptosporidiosis in a kid goat. Aust. Vet. J. 57, 386–388. Matos-Fernandez, M.J., Pereira-Bueno, J., Ortega-Mora, L.M., Pilar-Izquierdo, M., Ferre, I., Rojo-Vazquez, F.A., 1993. Prevalencia de la infeccion por Cryptosporidium parvum en corderos, cabritos y terneros en la provincia de Leon. Acta Parasitol. Port. 1, 211. Maurya, P.S., Rakesh, R.L., Pradeep, B., Kumar, S., Kundu, K., Garg, R., Ram, H., Kumar, A., Banerjee, P.S., 2013. Prevalence and risk factors associated with Cryptosporidium spp. infection in young domestic livestock in India. Trop. Anim. Health Prod. 45, 941–946. Molloy, S.F., Smith, H.V., Kirwan, P., Nichols, R.A., Asaolu, S.O., Connelly, L., Holland, C.V., 2010. Identification of a high diversity of Cryptosporidium species genotypes and subtypes in a pediatric population in Nigeria. Am. J. Trop. Med. Hyg. 82, 608–613.
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Mueller-Doblies, D., Giles, M., Elwin, K., Smith, R.P., Clifton-Hadley, F.A., Chalmers, R.M., 2008. Distribution of Cryptosporidium species in sheep in the UK. Vet. Parasitol. 154, 214–219. Munoz-Fernandez, M., Alvarez, M., Lanza, I., Carmenes, P., 1996. Role of enteric pathogens in the aethiology of neonatal diarrhoea in lambs and goat kids in Spain. Epidemiol. Infect. 117, 203–211. Nagy, B., Bozso, M., Palfi, V., Nagy, G., Sahibi, M.A., 1984. Studies on cryptosporidial infection of goat kids. In: Yvore, P., Perrini, G. (Eds.), Les maladies de la chevre. INRA Publishing, pp. 443–451. Ngouanesavanh, T., Guyot, K., Certad, G., Le Fichoux, Y., Chartier, C., Verdier, R.I., Cailliez, J.C., Camus, D., Dei-Cas, E., Bañuls, A.L., 2006. Cryptosporidium population genetics: evidence of clonality in isolates from France and Haiti. J. Eukaryot. Microbiol. 53, S33–S36. Noordeen, F., Rajapakse, R.P., Faizal, A.C., Horadagoda, N.U., Arulkanthan, A., 2000. Prevalence of Cryptosporidium infection in goats in selected locations in three agroclimatic zones of Sri Lanka. Vet. Parasitol. 93, 95–101. Noordeen, F., Horadagoda, N.U., Faizal, A.C., Rajapakse, R.P., Razak, M.A., Arulkanthan, A., 2002. Infectivity of Cryptosporidium parvum isolated from asymptomatic adult goats to mice and goat kids. Vet. Parasitol. 103, 217–225. Pam, V.A., Dakul, D.A., Karshima, N.S., Bata, S.I., Ogbu, K.I., Daniel, L.N., Udokaninyene, A.D., Kemza, S.Y., Igeh, C.P., Hassan, A.A., 2013. Survey of Cryptosporidium species among ruminants in Jos, Plateau state, north-central Nigeria. J. Vet. Adv. 3, 49–54. Park, J.H., Guk, S.M., Han, E.T., Shin, E.H., Kim, J.L., Chai, J.Y., 2006. Genotype analysis of Cryptosporidium spp. prevalent in a rural village in Hwasun-gun, Republic of Korea. Korean J. Parasitol. 44, 27–33. Pollock, K.G., Ternent, H.E., Mellor, D.J., Chalmers, R.M., Smith, H.V., Ramsay, C.N., Innocent, G.T., 2010. Spatial and temporal epidemiology of sporadic human cryptosporidiosis in Scotland. Zoonoses Public Health 57, 487–492. Quílez, J., Torres, E., Chalmers, R.M., Hadfield, S.J., Del Cacho, E., Sánchez-Acedo, C., 2008. Cryptosporidium genotypes and subtypes in lambs and goat kids in Spain. Appl. Environ. Microbiol. 74, 6026–6031. Ren, X., Zhao, J., Zhang, L., Ning, C., Jian, F., Wang, R., Lv, C., Wang, Q., Arrowood, M.J., Xiao, L., 2012. Cryptosporidium tyzzeri n. sp. (Apicomplexa: Cryptosporidiidae) in domestic mice (Mus musculus). Exp. Parasitol. 130, 274–281. Rieux, A., Paraud, C., Pors, I., Chartier, C., 2013. Molecular characterization of Cryptosporidium spp. in pre-weaned kids in a dairy goat farm in western France. Vet. Parasitol. 192, 268–272. Robertson, L.J., 2009. Giardia and Cryptosporidium infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination. Epidemiol. Infect. 137, 913–921. Rossanigo, E.G., Gialletti, L., Grelloni, V., Floroni, A., Rivero, V.B., 1987. Diagnosi di criptosporidiosi in alcuni allevamenti dell’ Italia Centrale. Riv. Zootecnia Vet. 15, 9–15. Ruecker, N.J., Braithwaite, S.L., Topp, E., Edge, T., Lapen, D.R., Wilkes, G., Robertson, W., Medeiros, D., Sensen, C.W., Neumann, N.F., 2007. Tracking host sources of Cryptosporidium spp. in raw water for improved health risk assessment. Appl. Environ. Microbiol. 73, 3945–3957. Ryan, U., Read, C., Hawkins, P., Warnecke, M., Swanson, P., Griffith, M., Deere, D., Cunningham, M., Cox, P., 2005. Genotypes of Cryptosporidium from Sydney water catchment areas. J. Appl. Microbiol. 98, 1221–1229. Santín, M., Fayer, R., 2007. Intragenotypic variations in the Cryptosporidium sp. cervine genotype from sheep with implications for public health. J. Parasitol. 93, 668–672.
Sanz Ceballos, L., Illescas Gómez, P., Sanz Sampelayo, M.R., Gil Extremera, F., Rodríguez Osorio, M., 2009. Prevalence of Cryptosporidium infection in goats maintained under semi-extensive feeding conditions in the southeast of Spain. Parasite 16, 315–318. Shoukry, N.M., Dawoud, H.A., Haridy, F.M., 2009. Studies on zoonotic cryptosporidiosis parvum in Ismailia Governorate, Egypt. J. Egypt. Soc. Parasitol. 39, 479–488. Smith, R.P., Chalmers, R.M., Mueller-Doblies, D., Clifton-Hadley, F.A., Elwin, K., Watkins, J., Paiba, G.A., Hadfield, S.J., Giles, M., 2010. Investigation of farms linked to human patients with cryptosporidiosis in England and Wales. Prev. Vet. Med. 94, 9–17. Soltane, R., Guyot, K., Dei-Cas, E., Ayadi, A., 2007. Prevalence of Cryptosporidium spp. (Eucoccidiorida: Cryptosporiidae) in seven species of farm animals in Tunisia. Parasite 14, 335–338. Van Dyke, M.I., Ong, C.S., Prystajecky, N.A., Isaac-Renton, J.L., Huck, P.M., 2012. Identifying host sources, human health risk and indicators of Cryptosporidium and Giardia in a Canadian watershed influenced by urban and rural activities. J. Water Health 10, 311–323. Vieira, L.S., Silva, M.B.O., Tolentino, A.C.V., Lima, J.D., Silva, A.C., 1997. Outbreak of cryptosporidiosis in dairy goats in Brazil. Vet. Rec. 140, 427–428. Waldron, L.S., Dimeski, B., Beggs, P.J., Ferrari, B.C., Power, M.L., 2011. Molecular epidemiology, spatiotemporal analysis, and ecology of sporadic human cryptosporidiosis in Australia. Appl. Environ. Microbiol. 77, 7757–7765. Wang, Y., Feng, Y., Cui, B., Jian, F., Ning, C., Wang, R., Zhang, L., Xiao, L., 2000. Cervine genotype is the major Cryptosporidium genotype in sheep in China. Parasitol. Res. 106, 341–347. Wang, R., Zhang, L., Ning, C., Feng, Y., Jian, F., Xiao, L., Lu, B., Ai, W., Dong, H., 2008. Multilocus phylogenetic analysis of Cryptosporidium andersoni (Apicomplexa) isolated from a bactrian camel (Camelus bactrianus) in China. Parasitol. Res. 102, 915–920. Wang, R., Ma, G., Zhao, J., Lu, Q., Wang, H., Zhang, L., Jian, F., Ning, C., Xiao, L., 2011a. Cryptosporidium andersoni is the predominant species in post-weaned and adult dairy cattle in China. Parasitol. Int. 60, 1–4. Wang, R., Wang, H., Sun, Y., Zhang, L., Jian, F., Qi, M., Ning, C., Xiao, L., 2011b. Characteristics of Cryptosporidium transmission in preweaned dairy cattle in Henan, China. J. Clin. Microbiol. 49, 1077–1082. Watanabe, Y., Yang, C.H., Ooi, H.K., 2005. Cryptosporidium infection in livestock and first identification of Cryptosporidium parvum genotype in cattle feces in Taiwan. Parasitol. Res. 97, 238–241. Xiao, L., Ryan, U.M., 2004. Cryptosporidiosis: an update in molecular epidemiology. Curr. Opin. Infect. Dis. 17, 483–490. Xiao, L., Bern, C., Limor, J., Sulaiman, I., Roberts, J., Checkley, W., Cabrera, L., Gilman, R.H., Lal, A.A., 2001. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J. Infect. Dis. 183, 492–497. Yang, W., Chen, P., Villegas, E.N., Landy, R.B., Kanetsky, C., Cama, V., Dearen, T., Schultz, C.L., Orndorff, K.G., Prelewicz, G.J., Brown, M.H., Young, K.R., Xiao, L., 2008. Cryptosporidium source tracking in the Potomac River watershed. Appl. Environ. Microbiol. 74, 6495–6504. Ye, J., Xiao, L., Wang, Y., Wang, L., Amer, S., Roellig, D.M., Guo, Y., Feng, Y., 2013. Periparturient transmission of Cryptosporidium xiaoi from ewes to lambs. Vet. Parasitol. 197, 627–633.
Please cite this article in press as: Wang, R., et al. Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.001
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Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr 5 6
Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China
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Rongjun Wang a,b,c,1, Guoquan Li b,c,1, Bin Cui b,c, Jianying Huang b,c, Zhaohui Cui b,c, Sumei Zhang b,c, Haiju Dong b,c, Daoyou Yue b,c, Longxian Zhang b,c,⇑, Changshen Ning b,c, Ming Wang a,⇑ a
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b c
College of Veterinary Medicine, China Agricultural University, Beijing 100193, China College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China International Joint Research Laboratory for Zoonotic Diseases of Henan, Zhengzhou 450002, China
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h i g h l i g h t s
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g r a p h i c a l a b s t r a c t
The overall prevalence of
Cryptosporidium in goats was 3.48% (44/1256). C. ubiquitum, C. andersoni and C. xiaoi were identified. The zoonotic XIIa subtype 2 of C. ubiquitum was found. C. ubiquitum and C. andersoni were the first identified species in goats. Age-associated distribution of Cryptosporidium was noticed in goats.
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i n f o
Article history: Received 16 January 2014 Received in revised form 27 March 2014 Accepted 1 April 2014 Available online xxxx
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Keywords: Cryptosporidium Goats C. ubiquitum C. andersoni C. xiaoi SSU rRNA gp60
a b s t r a c t To estimate the prevalence and public health significance of cryptosporidiosis in goats in China, 1265 fecal samples from seven farms in Henan province and Chongqing city were examined for Cryptosporidium oocysts. The overall infection rate of Cryptosporidium spp. was 3.48% (44/1256). Significant difference was observed among age groups, with the post weaned kids having the highest infection rate (4.58%; q < 0.01). Cryptosporidium spp. were characterized by PCR–restriction fragment length polymorphism (RFLP) analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene. The SSU rRNA-based PCR identified three Cryptosporidium species, including Cryptosporidium ubiquitum (24/44) in Henan and Chongqing, and Cryptosporidium andersoni (16/44) and Cryptosporidium xiaoi (4/44) in Henan. Among which, the C. ubiquitum and C. andersoni were first identified in goats thus far and were found in all age groups except no C. andersoni being found in the postparturition nannies, whereas the C. xiaoi was detected in pre-weaned kids and pregnant nannies. Subtyping C. ubiquitum by DNA sequence analysis of the 60 kDa glycoprotein (gp60) gene suggested the isolates identified all belonged to zoonotic XIIa subtype 2. Thus, the dominant C. ubiquitum found in this study and the XIIa subtype 2 has been found in humans indicated goats are a potential source for zoonotic infections with the C. ubiquitum. More studies are needed for better understanding of differences in the transmission and public health significance of cryptosporidiosis in goats. Ó 2014 Published by Elsevier Inc.
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⇑ Corresponding authors. Address: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China. Fax: +86 371 63558180 (L. Zhang). Fax: +86 10 62733961 (M. Wang). E-mail addresses: [email protected], [email protected] (L. Zhang), [email protected] (M. Wang). 1 Rongjun Wang and Guoquan Li contributed equally to this work. http://dx.doi.org/10.1016/j.exppara.2014.04.001 0014-4894/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: Wang, R., et al. Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.001
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1. Introduction Cryptosporidium spp. are important gastrointestinal protists in a wide spectrum of hosts, including humans, other mammals, birds, reptiles, amphibians, and fish. Infection is acquired following the ingestion of infective oocysts by the fecal-oral route, via either direct contact or ingestion of contaminated food or water (Xiao and Ryan, 2004). It is one of the common causes of diarrhea in humans and animals. Thus far, twenty-six Cryptosporidium species are considered valid, and more than 70 host-adapted genotypes with undetermined species status have been described (Fayer, 2009; Elwin et al., 2012b; Ren et al., 2012; Kvácˇ et al., 2013). The first Cryptosporidium infection case in goat was described in Australia in a 2-week-old kid with diarrhea (Mason et al., 1981). Since then, the infection has been reported in several continents, including Europe (Spain, Italy, Switzerland, Poland, Hungary, Netherland, Belgium, UK, France, and Czech Republic), Asia (India, Mongolia, Turkey, Korea, Iran, Cyprus, Sri Lanka, Iraq, Sultanate of Oman, and China), Africa (Egypt, Tunisia, Malawi, and Zambia), and America (USA, Brazil, and Trinidad & Tobago), with the infection rate ranging from 0% to 100% (average 17.92%, 980/5468) based on point prevalence data at different geographical areas (Noordeen et al., 2000; Geurden et al., 2008; Quílez et al., 2008; Robertson, 2009; Drumo et al., 2012; Giadinis et al., 2012; Marreros et al., 2012; Jafari et al., 2013; Maurya et al., 2013; Rieux et al., 2013). However, few studies have genotyped Cryptosporidium spp. from goats in the world. Previous studies indicated that Cryptosporidium parvum was the dominant Cryptosporidium species, as well as Cryptosporidium xiaoi, Cryptosporidium hominis, a goat genotype, and a new Cryptosporidium genotype have also been detected in goats (Ryan et al., 2005; Karanis et al., 2007b; Robertson, 2009). Goats play an important role in the agricultural economy of China. In 2011, the total goat population was 142.23 millions, ranking first in the world (http://kids.fao.org/glipha/). Unfortunately, little is known about the distribution and zoonotic transmission risk of Cryptosporidium spp. in goats, except for a few prevalence studies published in the Chinese language and two isolates identified as C. xiaoi (previously named as C. bovis-like genotype) and a new Q4 Cryptosporidium genotype (Karanis et al., 2007a,b). More recently, Li et al. (2014) developed a subtyping technique by whole-genome sequencing to characterize gp60 gene of Cryptosporidium ubiquitum from humans, various animals, and drinking source water, which produced six subtype families, XIIa–XIIf. Thus, the objective of this study was to identify the distribution and zoonotic potential of Cryptosporidium spp. in goats in Henan province and Chongqing city, China at the genotype and subtype levels.
2. Materials and methods 2.1. Sample and microscopy examination Fresh fecal sample for each animal was collected immediately after being defecated on the ground using a sterile disposal latex glove, and then placed in a disposable plastic bag individually. A total of 1265 fecal samples were obtained between July 2006 and July 2007 from four goat farms in two areas of Henan province and from three goat farms in three areas of Chongqing city in December 2011, including those from pre-weaned and postweaned kids, adult goats, pregnant nannies, and postparturition nannies (Table 1). Cryptosporidium oocysts were examined by microscopy after approximately 20 g fecal samples each animal were concentrated by the Sheather’s sugar flotation technique and were stained with the modified acid-fast staining. Cryptosporidium-positive samples were stored in 2.5% potassium dichromate at 4 °C prior to DNA extraction.
Table 1 Number of fecal samples examined for Cryptosporidium oocysts by microscopy on each farm and the distribution of Cryptosporidium spp. determined by PCR–RFLP and sequence analysis of the SSU rRNA gene. Collection site
Sample size
Cryptosporidium positive (%)
C. ubiquitum
C. andersoni
C. xiaoi
6 (24%) 2 (66.67%)
16 (64%)
3 (12%) 1 (33.32%)
16 (36.36%)
4 (9.09%)
HN-1 HN-2
790 138
25 (3.16%) 3 (2.17%)
HN-3 HN-4 CQ-1 CQ-2 CQ-3 Total
65 24 64 72 112 1265
0 0 1 (3.13%) 7 (9.72%) 8 (8.33%) 44 (3.48%)
1 (100%) 7 (100%) 8 (100%) 24 (54.55%)
2.2. DNA extraction
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For samples obtained between 2006 and 2007, Cryptosporidium oocysts were isolated from the positive fecal samples by the discontinuous density sucrose gradient centrifugation. Then, genomic DNA was extracted from the purified oocysts using the Mag Extractor Genome kit as described in previous study (Wang et al., 2008). As for samples collected in December 2011, genomic DNA was isolated from Cryptosporidium-positive samples using the E.Z.N.A.@ Stool DNA Kit (OMEGA Biotek Inc., USA) and the manufacturer recommended procedures (Wang et al., 2011b). The extracted DNA was kept at 20 °C before it was used in molecular analysis.
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2.3. Cryptosporidium genotyping and subtyping
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Cryptosporidium spp. were genotyped by amplifying the small subunit (SSU) rRNA gene by nested PCR and restriction fragment length polymorphism (RFLP) analysis using restriction enzymes SspI and VspI (Xiao et al., 2001). The PCR products were further sequenced directly with the secondary PCR primers on an ABI 3730 DNA Analyzer using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Sequence accuracy was confirmed by two-directional sequencing and by sequencing a new PCR product if necessary. C. ubiquitum was subtyped by using gp60 gene, with the sequences of primers being 50 -TTT ACC CAC ACA TCT GTA GCG TCG-30 (Ubi-18S-F1) and 50 -ACG GAC GGA ATG ATG TAT CTG A-30 (Ubi-18S-R1), and 50 -ATA GGT GAT AAT TAG TCA GTC TTT AAT-30 (Ubi-18S-F2) and 50 -TCC AAA AGC GGC TGA GTC AGC ATC-30 (Ubi-18SR2) for the primary and secondary PCR (Li et al., 2014). The PCR products of gp60 gene were sequenced using the secondary PCR primers as described above. Sequence analysis was performed by alignment of SSU rRNA sequences obtained in this study and reference sequences downloaded from GenBank using the program ClustalX 1.83 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/). Phylogenetic analyses were performed using the software Phylip 3.69 (http://evolution. genetics.washington.edu/phylip.html). Neighbour-joining trees were constructed based on the evolutionary distances calculated by Kimura-2-parameter model. The reliability of these trees was assessed using the bootstrap analysis with 1000 replicates. Sequences of the partial SSU rRNA and gp60 genes were deposited in GenBank under Accession Nos. EU926575–EU926602 and KJ622359.
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2.4. Statistical analysis
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The chi-square test was used to compare Cryptosporidium infection rates. Differences were considered significant when q < 0.05.
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3. Results
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3.1. Prevalence of Cryptosporidium spp.
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Microscopic analysis of 1265 fecal samples showed the presence of Cryptosporidium oocysts in 44 samples (3.48%) on five out of seven goat farms (Table 1). The infection rate was 2.75% (28/ 1017) in Henan province and 6.45% (16/248) in Chongqing city (v2 = 8.12; q < 0.01), with the highest infection rate (9.72%) being observed on farm CQ-2 (v2 = 17.68; q < 0.01) (Table 1). The percentage of animals shedding oocysts was 2.11%, 4.58%, 1.90%, 4.44%, and 2.72% in pre-weaned kids, post-weaned kids, adult goats, pregnant nannies, and postparturition nannies, respectively (v2 = 14.23; q < 0.01) (Table 2).
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3.2. Distribution of Cryptosporidium species
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The SSU rRNA gene of Cryptosporidium spp. in all 44 microscopy-positive samples was successfully amplified by the nested PCR. RFLP and DNA sequence analyses of the SSU rRNA gene products revealed the presence of three Cryptosporidium species, including C. ubiquitum (24/44), Cryptosporidium andersoni (16/44), and C. xiaoi (4/44) (Table 1). Phylogenetic relationship analysis confirmed the identity of Cryptosporidium species (Fig. 1). Farms
177 178 179 180 181 182 183 184
188 189 190 191 192 193
Table 2 Cryptosporidium species identified among different age groups of goats. Age group Pre-weaned kids Post weaned kids Adult goats Pregnant nannies Postparturition nannies Total
Sample size
Cryptosporidium positive (%)
C. C. C. ubiquitum andersoni xiaoi
229 393 368 174 101
17 (2.11%) 12 (4.58%) 7 (1.90%) 6 (4.44%) 2 (2.27%)
9 4 6 3 2
5 8 1 2 0
3 0 0 1 0
1265
44 (3.48%)
24
16
4
Fig. 1. Phylogenetic relationship of Cryptosporidium inferred by neighbour-joining analysis of the SSU rRNA based on Kimura 2-parameter model.
3
in Henan province had infections of three Cryptosporidium species, with the C. andersoni appearing to be the predominant species. In contrast, only the C. ubiquitum was found in three Cryptosporidium-positive farms in Chongqing city (Table 1).
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3.3. Age patterns of Cryptosporidium species
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C. ubiquitum was the predominantly identified species, responsible for 45.55% of all Cryptosporidium infections. It was found in all age groups examined in this study, on all five Cryptosporidium-positive farms. C. andersoni was only detected in farm HN-1, found in four age groups (Table 2). In contrast, the proportion of C. xiaoi was lower, responsible for 9.09% of Cryptosporidium infections. The C. xiaoi was only found in pre-weaned kids and pregnant nannies (Table 2).
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3.4. Subtype of C. ubiquitum isolates
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A total of 15 C. ubiquitum isolates were amplified and sequenced at the gp60 locus, including the 8 isolates from HN-1 and HN-2, one from CQ-1, and each two from CQ-2 and CQ-3. All the 15 gp60 sequences were identical to each other and belonged to XIIa subtype 2, which shared 100% homology to Turkey sheep- and human-derived C. ubiquitum isolates (GenBank Accession Nos. KC204982 and JX412919), as well as the XIIa subtype 2 isolates from China sheep and yak, Nepal swamp deer, and South Africa impala and buffalo (Fig. 2 shown in Li et al., 2014). In contrast, the C. ubiquitum XIIa subtype 2 from goats found in this study had only two nucleotide differences in comparison with three China sheep XIIa subtype 1 isolates (GenBank Accession No. JX412921), and three nucleotide differences compared to Algeria goat XIIa subtype 3 isolate (GenBank Accession No. JX412917).
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4. Discussion
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A 3.48% overall infection rate of Cryptosporidium spp. was observed in this study, which was higher than 2.5% (6/237) of infection rate in Iran, or close to 3.54% (4/113) in Switzerland, and 3.5% (4/116) in India (Marreros et al., 2012; Jafari et al., 2013; Maurya et al., 2013). However, the present findings likely under represented the actual number of Cryptosporidium-positive goats because microscopy repeatedly has been shown to be less sensitive than molecular methods for detection of positive specimens. Summarizing the available data (n = 32) showed the overall infection rate of Cryptosporidium spp. was 17.92% (980/5468) in goats worldwide (Table 3). The difference of prevalence was observed in various countries (Table 3), which may be related to sample sizes, examination methods, different management practices, the timing of sample collection, and geographical ecological conditions. The highest levels of infection was seen in post weaned kids (4.58%) and pregnant nannies (4.44%), whereas the lowest infection rate was found in adult goats (1.90%) (q < 0.01). Thus, the difference associated with age groups might be partially attributed to the immunity or physical condition of animals at different ages. Five Cryptosporidium species/genotypes have been identified in goats thus far, including C. parvum, C. xiaoi, C. hominis, a goat genotype, and a new Cryptosporidium genotype (Ryan et al., 2005; Park et al., 2006; Karanis et al., 2007b; Quílez et al., 2008; Díaz et al., 2010). Among these species and genotypes identified in goats, C. parvum is the dominant Cryptosporidium species, which has been found in goats in Italy, Spain, Belgium, Czech Republic, the Netherlands, France, India, Sri Lanka, Zambia, and Egypt (Cacciò et al., 2000; Noordeen et al., 2002; Hajdušek et al., 2004; Ngouanesavanh et al., 2006; Goma et al., 2007; Geurden et al., 2008; Quílez et al., 2008; Giles et al., 2009; Drumo et al., 2012; Maurya et al., 2013;
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Table 3 Prevalence and Cryptosporidium species/genotypes in goats in reported studies. Area Italy Italy Spain Spain Spain Spain Spain Spain Hungary Poland Czech Republic Belgium United Kingdom England and Wales Switzerland France France France France Netherland, Italy, Czech Republic Romania India Mongolia Turkey China Korea Taiwan Iran Cyprus Sri Lanka Sri Lanka Iraq Tunisia Zambia Malawi Egypt Nigeria Trinidad and Tobago USA Brazil Brazil
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No. positive/No. examined (%)
Cryptosporidium species/genotype (No.)
References
C. parvum (21)
Drumo et al. (2012) Rossanigo et al. (1987) Matos-Fernandez et al. (1993) Munoz-Fernandez et al. (1996) Castro-Hermida et al. (2007) Quílez et al. (2008) Sanz Ceballos et al. (2009) Díaz et al. (2010) Nagy et al. (1984) Majewska et al. (2000) Hajdušek et al. (2004) Geurden et al. (2008) Giles et al. (2009) Smith et al. (2010) Marreros et al. (2012) Delafosse et al. (2006) Castro-Hermida et al. (2005) Ngouanesavanh et al. (2006) Rieux et al. (2013) Cacciò et al. (2000) Bejan et al. (2009) Maurya et al. (2013) Burenbaatar et al. (2008) Ciçek et al. (2008) Karanis et al. (2007a,b) Park et al. (2006) Watanabe et al. (2005) Jafari et al. (2013) Giadinis et al. (2012) Noordeen et al. (2000) Noordeen et al. (2002) Mahdi and Ali (2002) Soltane et al. (2007) Goma et al. (2007) Banda et al. (2009) Shoukry et al. (2009) Pam et al. (2013) Kaminjolo et al. (1993) Caver et al. (1996) Vieira et al. (1997) Bomfim et al. (2005)
8/42 (19%) 40/367 (11%) 15/36 (42%) 9/116 (7.7%) C. parvum (17) 111/582 (19.1%) 2/5 (40%) 10/40 (25%) 0/46 14/148 (9.5%)
C. xiaoi (2)
C. parvum (1) C. parvum (11) C. hominis (1)
3/15 (20%) 4/113 (3.54%) 142/879 (16.2%) 13/100 (13%) C. parvum (7) C. xiaoi (18), C. parvum (1) C. parvum (15) 24% (99/412) 4/116 (3.5%) 0/16 6/56 (10.71%) 2/42 (4.76%) 3/7 (42.9%) 44/123 (35.8%) 6/237 (2.5%) 48/75 (64%) 291/1020 (28.5%)
C. parvum (4)
C. xiaoi (1), new genotype (1) C. hominis (3)
C. parvum (1) 6/45 (13.3%) 0/184 5/105 (4.8%) 23/225 (10.22%) 25.9% 36/150 (24%) 4/20 (20%) 5/19 (26%) 22/22 (100%) 5/105 (4.8%)
Rieux et al., 2013). In contrast, the other four species/genotypes were only detected in a few cases in Spain, the United Kingdom, Korea, Australia, France, and China (Park et al., 2006; Karanis et al., 2007b; Giles et al., 2009; Díaz et al., 2010; Rieux et al., 2013). In this study, both C. ubiquitum and C. andersoni represent the first identified Cryptosporidium species in goats, which have previously been found in sheep in Henan, China (Wang et al., 2000). C. ubiquitum has been found in a great many animals and humans in many geographic locations (Li et al., 2014) and has experimentally been transmitted from goats to cattle (Fayer et al., 2010). Although transmission of C. xiaoi from sheep to goats under experimental conditions was not successful (Fayer and Santin, 2009), C. xiaoi has been found in goats less than 21 days of age in Spain (Díaz et al., 2010). An age-associated distribution of Cryptosporidium was noticed in goats. In this study, all age groups of goats were infected by the C. ubiquitum, which was identical to our previous observations found in sheep (Wang et al., 2000). In contrast, C. xiaoi was only detected in kids and pregnant nannies (Table 2). A more recent study conducted in France showed that 18 out of 19 Cryptosporidium-positive isolates from pre-weaned kids were identified as C. xiaoi (Rieux et al., 2013). Thus, these findings might indicate a possible periparturient transmission for C. xiaoi infection. Indeed, a recent study conducted in sheep has suggested the periparturient transmission of C. xiaoi from ewes to lambs (Ye et al., 2013). Previously, no report compared the distribution of Cryptosporidium spp. in different age groups of goats although a few studies have conducted
C. parvum (1)
in sheep (Santin and Fayer, 2007; Mueller-Doblies et al., 2008; Wang et al., 2000). C. andersoni is the second common Cryptosporidium species, which was found in all age groups except for the postparturition nannies (Table 2). In fact, C. andersoni was one of the most common Cryptosporidium species identified in ruminants and other animals in China, such as cattle, sheep, bactrian camel, Golden hamster, Siberian hamster, and Campbell hamster (Lv et al., 2009; Wang et al., 2000, 2008, 2011a,b). Geographic differences in the distribution of Cryptosporidium spp. in goats are difficult to identify with accuracy because relatively few goats have been examined and many of those tested were from only a few farms in specific locations. C. parvum and C. xiaoi were found in Spain, and France (Quílez et al., 2008; Díaz et al., 2010; Rieux et al., 2013), C. hominis in the United Kingdom and Korea (Park et al., 2006; Giles et al., 2009), whereas C. parvum in the remaining countries (Table 3). In this study, only C. ubiquitum was identified in goats in Chongqing city, however C. ubiquitum, C. andersoni, and C. xiaoi were found in Henan province (Tables 1 and 2). In addition, no C. parvum infection was detected in goats in this study, which was identical to a previous study conducted in sheep in Henan, China (Wang et al., 2000). Therefore, the Cryptosporidium species distribution in goats in Henan, China is apparently different from the one seen in other countries. C. ubiquitum found in this study has potential public health importance. Indeed, the 15 C. ubiquitum isolates subtyped all belonged to zoonotic XIIa subtype 2, which has been detected in humans, as well as in some other animals (Li et al., 2014).
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Previously, C. ubiquitum has been identified in at least 40 sporadic cases, with 38 cases being reported in industrialized nations (Wang et al., 2008; Davies et al., 2009; Pollock et al., 2010; Molloy et al., 2010; Cieloszyk et al., 2012; Elwin et al., 2012a), and it is also one of the common Cryptosporidium spp. found in drinking source water in some countries, such as the United States and Canada (Jiang et al., 2005; Karanis et al., 2007a; Ruecker et al., 2007; Yang et al., 2008; Jellison et al., 2009; Van Dyke et al., 2012). However, the contribution of animal-derived C. ubiquitum to infection in humans is not entirely clear. Nevertheless, the more recent subtyping study indicated contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections (Li et al., 2014). In contrast, the public health importance of C. andersoni is low, only small number of human cases were reported in England and Australia (Leoni et al., 2006; Waldron et al., 2011). In conclusion, C. ubiquitum, C. andersoni, and C. xiaoi were detected in this study, with C. ubiquitum and C. andersoni being the first identified Cryptosporidium species in goats thus far. C. ubiquitum was the dominant Cryptosporidium species and belonged to the zoonotic XIIa subtype 2, which indicated this species has a potential for zoonotic transmission in the study area. More studies are needed for better understanding of differences in the transmission and public health significance of cryptosporidiosis in goats in the world.
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Acknowledgments
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This study was supported in part by the State Key Program of Q5 National Natural Science Foundation of China (31330079), the Q6 National Natural Science Foundation of China (U1204328, 313 02079), and the International Cooperation and Exchange Funds of the National Natural Science Foundation of China (3111 0103901).
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References
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Banda, Z., Nichols, R.A., Grimason, A.M., Smith, H.V., 2009. Cryptosporidium infection in non-human hosts in Malawi. Onderstepoort J. Vet. Res. 76, 363–375. Bejan, A., Mircean, V., Radu, C., Smaro, S., Cozma, V., 2009. Epidemiology of Cryptosporidium spp. infection in goat kids in the central and the northwest part of Romania. Rev. Scientia Parasitol. 10, 32–36. Bomfim, T.C., Huber, F., Gomes, R.S., Alves, L.L., 2005. Natural infection by Giardia sp. and Cryptosporidium sp. in dairy goats, associated with possible risk factors of the studied properties. Vet. Parasitol. 134, 9–13. Burenbaatar, B., Bakheit, M.A., Plutzer, J., Suzuki, N., Igarashi, I., Ongerth, J., Karanis, P., 2008. Prevalence and genotyping of Cryptosporidium species from farm animals in Mongolia. Parasitol. Res. 102, 901–905. Cacciò, S., Homan, W., Camilli, R., Traldi, G., Kortbeek, T., Pozio, E., 2000. A microsatellite marker reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum. Parasitology 120, 237–244. Castro-Hermida, J.A., Delafosse, A., Pors, I., Ares-Mazás, E., Chartier, C., 2005. Giardia duodenalis and Cryptosporidium parvum infections in adult goats and their implications for neonatal kids. Vet. Rec. 157, 623–627. Castro-Hermida, J.A., Almeida, A., González-Warleta, M., Correia da Costa, J.M., Rumbo-Lorenzo, C., Mezo, M., 2007. Occurrence of Cryptosporidium parvum and Giardia duodenalis in healthy adult domestic ruminants. Parasitol. Res. 101, 1443–1448. Caver, J.A., Hill, J.E., Thompson, S.J., 1996. Surveillance of Cryptosporidia in a veterinary diagnostic laboratory. J. Vet. Diagn. Invest. 8, 497–500. Ciçek, M., Körkoca, H., Gül, A., 2008. Investigation of Cryptosporidium sp. in workers of the Van municipality slaughterhouse and in slaughtered animals. Turkiye Parazitol. Derg. 32, 8–11. Cieloszyk, J., Goñi, P., García, A., Remacha, M.A., Sánchez, E., Clavel, A., 2012. Two cases of zoonotic cryptosporidiosis in Spain by the unusual species Cryptosporidium ubiquitum and Cryptosporidium felis. Enferm. Infecc. Microbiol. Clín. 30, 549–551. Davies, A.P., Campbell, B., Evans, M.R., Bone, A., Roche, A., Chalmers, R.M., 2009. Asymptomatic carriage of protozoan parasites in children in day care centers in the United Kingdom. Pediatr. Infect. Dis. J. 28, 838–840. Delafosse, A., Castro-Hermida, J.A., Baudry, C., Ares-Mazás, E., Chartier, C., 2006. Herd-level risk factors for Cryptosporidium infection in dairy-goat kids in western France. Prev. Vet. Med. 77, 109–121. Díaz, P., Quílez, J., Robinson, G., Chalmers, R.M., Díez-Baños, P., Morrondo, P., 2010. Identification of Cryptosporidium xiaoi in diarrhoeic goat kids (Capra hircus) in Spain. Vet. Parasitol. 172, 132–134.
5
Drumo, R., Widmer, G., Morrison, L.J., Tait, A., Grelloni, V., D’Avino, N., Pozio, E., Cacciò, S.M., 2012. Evidence of host-associated populations of Cryptosporidium parvum in Italy. Appl. Environ. Microbiol. 78, 3523–3529. Elwin, K., Hadfield, S.J., Robinson, G., Chalmers, R.M., 2012a. The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, 2000–2008. Epidemiol. Infect. 140, 673–683. Elwin, K., Hadfield, S.J., Robinson, G., Crouch, N.D., Chalmers, R.M., 2012b. Cryptosporidium viatorum n. sp. (Apicomplexa: Cryptosporidiidae) among travellers returning to Great Britain from the Indian subcontinent, 2007– 2011. Int. J. Parasitol. 42, 675–682. Fayer, R., 2009. Taxonomy and species delimitation in Cryptosporidium. Exp. Parasitol. 124, 90–97. Fayer, R., Santín, M., 2009. Cryptosporidium xiaoi n. sp. (Apicomplexa: Cryptosporidiidae) in sheep (Ovis aries). Vet. Parasitol. 164, 192–200. Fayer, R., Santín, M., Macarisin, D., 2010. Cryptosporidium ubiquitum n. sp. in animals and humans. Vet. Parasitol. 172, 23–32. Geurden, T., Thomas, P., Casaert, S., Vercruysse, J., Claerebout, E., 2008. Prevalence and molecular characterisation of Cryptosporidium and Giardia in lambs and goat kids in Belgium. Vet. Parasitol. 155, 142–145. Giadinis, N.D., Symeoudakis, S., Papadopoulos, E., Lafi, S.Q., Karatzias, H., 2012. Comparison of two techniques for diagnosis of cryptosporidiosis in diarrhoeic goat kids and lambs in Cyprus. Trop. Anim. Health Prod. 44, 1561–1565. Giles, M., Chalmers, R., Pritchard, G., Elwin, K., Mueller-Doblies, D., Clifton-Hadley, F., 2009. Cryptosporidium hominis in a goat and a sheep in the UK. Vet. Rec. 164, 24–25. Goma, F.Y., Geurden, T., Siwila, J., Phiri, I.G.K., Gabriel, S., Claerebout, E., Vercruysse, J., 2007. The prevalence and molecular characterisation of Cryptosporidium spp. in small ruminants in Zambia. Small Ruminant Res. 72, 77–80. Hajdušek, O., Ditrich, O., Slapeta, J., 2004. Molecular identification of Cryptosporidium spp. in animal and human hosts from the Czech Republic. Vet. Parasitol. 122, 183–192. Jafari, R., Maghsood, A.H., Fallah, M., 2013. Prevalence of Cryptosporidium infection among livestock and humans in contact with livestock in Hamadan district, Iran, 2012. J. Res. Health Sci. 13, 86–89. Jellison, K.L., Lynch, A.E., Ziemann, J.M., 2009. Source tracking identifies deer and geese as vectors of human-infectious Cryptosporidium genotypes in an urban/ suburban watershed. Environ. Sci. Technol. 43, 4267–4272. Jiang, J., Alderisio, K.A., Xiao, L., 2005. Distribution of Cryptosporidium genotypes in storm event water samples from three watersheds in New York. Appl. Environ. Microbiol. 71, 4446–4454. Kaminjolo, J.S., Adesiyun, A.A., Loregnard, R., Kitson-Piggott, W., 1993. Prevalence of Cryptosporidium oocysts in livestock in Trinidad and Tobago. Vet. Parasitol. 45, 209–213. Karanis, P., Kourenti, C., Smith, H., 2007a. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. J. Water Health 5, 1–38. Karanis, P., Plutzer, J., Halim, N.A., Igori, K., Nagasawa, H., Ongerth, J., Liqing, M., 2007b. Molecular characterization of Cryptosporidium from animal sources in Qinghai province of China. Parasitol. Res. 101, 1575–1580. Kvácˇ, M., Kestrˇánová, M., Pinková, M., Kveˇtonˇová, D., Kalinová, J., Wagnerová, P., Kotková, M., Vítovec, J., Ditrich, O., McEvoy, J., Stenger, B., Sak, B., 2013. Cryptosporidium scrofarum n. sp. (Apicomplexa: Cryptosporidiidae) in domestic pigs (Sus scrofa). Vet. Parasitol. 191, 218–227. Leoni, F., Amar, C., Nichols, G., Pedraza-Díaz, S., McLauchlin, J., 2006. Genetic analysis of Cryptosporidium from 2414 humans with diarrhoea in England between 1985 and 2000. J. Med. Microbiol. 55, 703–707. Li, N., Xiao, L., Alderisio, K., Elwin, K., Cebelinski, E., Chalmers, R., Santin, M., Fayer, R., Kvac, M., Ryan, U., Sak, B., Stanko, M., Guo, Y., Wang, L., Zhang, L., Cai, J., Roellig, D., Feng, Y., 2014. Subtyping Cryptosporidium ubiquitum, a zoonotic pathogen emerging in humans. Emerg. Infect. Dis. 20, 217–224. Lv, C., Zhang, L., Wang, R., Jian, F., Zhang, S., Ning, C., Wang, H., Feng, C., Wang, X., Ren, X., Qi, M., Xiao, L., 2009. Cryptosporidium spp. in wild, laboratory, and pet rodents in china: prevalence and molecular characterization. Appl. Environ. Microbiol. 75, 7692–7699. Mahdi, N.K., Ali, N.H., 2002. Cryptosporidiosis among animal handlers and their livestock in Basrah, Iraq. East Afr. Med. J. 79, 550–553. Majewska, A.C., Werner, A., Sulima, P., Luty, T., 2000. Prevalence of Cryptosporidium in sheep and goats bred on five farms in west-central region of Poland. Vet. Parasitol. 89, 269–275. Marreros, N., Frey, C.F., Willisch, C.S., Signer, C., Ryser-Degiorgis, M.P., 2012. Coprological analyses on apparently healthy Alpine ibex (Capra ibex ibex) from two Swiss colonies. Vet. Parasitol. 186, 382–389. Mason, R.W., Hartley, W.J., Tilt, L., 1981. Intestinal cryptosporidiosis in a kid goat. Aust. Vet. J. 57, 386–388. Matos-Fernandez, M.J., Pereira-Bueno, J., Ortega-Mora, L.M., Pilar-Izquierdo, M., Ferre, I., Rojo-Vazquez, F.A., 1993. Prevalencia de la infeccion por Cryptosporidium parvum en corderos, cabritos y terneros en la provincia de Leon. Acta Parasitol. Port. 1, 211. Maurya, P.S., Rakesh, R.L., Pradeep, B., Kumar, S., Kundu, K., Garg, R., Ram, H., Kumar, A., Banerjee, P.S., 2013. Prevalence and risk factors associated with Cryptosporidium spp. infection in young domestic livestock in India. Trop. Anim. Health Prod. 45, 941–946. Molloy, S.F., Smith, H.V., Kirwan, P., Nichols, R.A., Asaolu, S.O., Connelly, L., Holland, C.V., 2010. Identification of a high diversity of Cryptosporidium species genotypes and subtypes in a pediatric population in Nigeria. Am. J. Trop. Med. Hyg. 82, 608–613.
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Mueller-Doblies, D., Giles, M., Elwin, K., Smith, R.P., Clifton-Hadley, F.A., Chalmers, R.M., 2008. Distribution of Cryptosporidium species in sheep in the UK. Vet. Parasitol. 154, 214–219. Munoz-Fernandez, M., Alvarez, M., Lanza, I., Carmenes, P., 1996. Role of enteric pathogens in the aethiology of neonatal diarrhoea in lambs and goat kids in Spain. Epidemiol. Infect. 117, 203–211. Nagy, B., Bozso, M., Palfi, V., Nagy, G., Sahibi, M.A., 1984. Studies on cryptosporidial infection of goat kids. In: Yvore, P., Perrini, G. (Eds.), Les maladies de la chevre. INRA Publishing, pp. 443–451. Ngouanesavanh, T., Guyot, K., Certad, G., Le Fichoux, Y., Chartier, C., Verdier, R.I., Cailliez, J.C., Camus, D., Dei-Cas, E., Bañuls, A.L., 2006. Cryptosporidium population genetics: evidence of clonality in isolates from France and Haiti. J. Eukaryot. Microbiol. 53, S33–S36. Noordeen, F., Rajapakse, R.P., Faizal, A.C., Horadagoda, N.U., Arulkanthan, A., 2000. Prevalence of Cryptosporidium infection in goats in selected locations in three agroclimatic zones of Sri Lanka. Vet. Parasitol. 93, 95–101. Noordeen, F., Horadagoda, N.U., Faizal, A.C., Rajapakse, R.P., Razak, M.A., Arulkanthan, A., 2002. Infectivity of Cryptosporidium parvum isolated from asymptomatic adult goats to mice and goat kids. Vet. Parasitol. 103, 217–225. Pam, V.A., Dakul, D.A., Karshima, N.S., Bata, S.I., Ogbu, K.I., Daniel, L.N., Udokaninyene, A.D., Kemza, S.Y., Igeh, C.P., Hassan, A.A., 2013. Survey of Cryptosporidium species among ruminants in Jos, Plateau state, north-central Nigeria. J. Vet. Adv. 3, 49–54. Park, J.H., Guk, S.M., Han, E.T., Shin, E.H., Kim, J.L., Chai, J.Y., 2006. Genotype analysis of Cryptosporidium spp. prevalent in a rural village in Hwasun-gun, Republic of Korea. Korean J. Parasitol. 44, 27–33. Pollock, K.G., Ternent, H.E., Mellor, D.J., Chalmers, R.M., Smith, H.V., Ramsay, C.N., Innocent, G.T., 2010. Spatial and temporal epidemiology of sporadic human cryptosporidiosis in Scotland. Zoonoses Public Health 57, 487–492. Quílez, J., Torres, E., Chalmers, R.M., Hadfield, S.J., Del Cacho, E., Sánchez-Acedo, C., 2008. Cryptosporidium genotypes and subtypes in lambs and goat kids in Spain. Appl. Environ. Microbiol. 74, 6026–6031. Ren, X., Zhao, J., Zhang, L., Ning, C., Jian, F., Wang, R., Lv, C., Wang, Q., Arrowood, M.J., Xiao, L., 2012. Cryptosporidium tyzzeri n. sp. (Apicomplexa: Cryptosporidiidae) in domestic mice (Mus musculus). Exp. Parasitol. 130, 274–281. Rieux, A., Paraud, C., Pors, I., Chartier, C., 2013. Molecular characterization of Cryptosporidium spp. in pre-weaned kids in a dairy goat farm in western France. Vet. Parasitol. 192, 268–272. Robertson, L.J., 2009. Giardia and Cryptosporidium infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination. Epidemiol. Infect. 137, 913–921. Rossanigo, E.G., Gialletti, L., Grelloni, V., Floroni, A., Rivero, V.B., 1987. Diagnosi di criptosporidiosi in alcuni allevamenti dell’ Italia Centrale. Riv. Zootecnia Vet. 15, 9–15. Ruecker, N.J., Braithwaite, S.L., Topp, E., Edge, T., Lapen, D.R., Wilkes, G., Robertson, W., Medeiros, D., Sensen, C.W., Neumann, N.F., 2007. Tracking host sources of Cryptosporidium spp. in raw water for improved health risk assessment. Appl. Environ. Microbiol. 73, 3945–3957. Ryan, U., Read, C., Hawkins, P., Warnecke, M., Swanson, P., Griffith, M., Deere, D., Cunningham, M., Cox, P., 2005. Genotypes of Cryptosporidium from Sydney water catchment areas. J. Appl. Microbiol. 98, 1221–1229. Santín, M., Fayer, R., 2007. Intragenotypic variations in the Cryptosporidium sp. cervine genotype from sheep with implications for public health. J. Parasitol. 93, 668–672.
Sanz Ceballos, L., Illescas Gómez, P., Sanz Sampelayo, M.R., Gil Extremera, F., Rodríguez Osorio, M., 2009. Prevalence of Cryptosporidium infection in goats maintained under semi-extensive feeding conditions in the southeast of Spain. Parasite 16, 315–318. Shoukry, N.M., Dawoud, H.A., Haridy, F.M., 2009. Studies on zoonotic cryptosporidiosis parvum in Ismailia Governorate, Egypt. J. Egypt. Soc. Parasitol. 39, 479–488. Smith, R.P., Chalmers, R.M., Mueller-Doblies, D., Clifton-Hadley, F.A., Elwin, K., Watkins, J., Paiba, G.A., Hadfield, S.J., Giles, M., 2010. Investigation of farms linked to human patients with cryptosporidiosis in England and Wales. Prev. Vet. Med. 94, 9–17. Soltane, R., Guyot, K., Dei-Cas, E., Ayadi, A., 2007. Prevalence of Cryptosporidium spp. (Eucoccidiorida: Cryptosporiidae) in seven species of farm animals in Tunisia. Parasite 14, 335–338. Van Dyke, M.I., Ong, C.S., Prystajecky, N.A., Isaac-Renton, J.L., Huck, P.M., 2012. Identifying host sources, human health risk and indicators of Cryptosporidium and Giardia in a Canadian watershed influenced by urban and rural activities. J. Water Health 10, 311–323. Vieira, L.S., Silva, M.B.O., Tolentino, A.C.V., Lima, J.D., Silva, A.C., 1997. Outbreak of cryptosporidiosis in dairy goats in Brazil. Vet. Rec. 140, 427–428. Waldron, L.S., Dimeski, B., Beggs, P.J., Ferrari, B.C., Power, M.L., 2011. Molecular epidemiology, spatiotemporal analysis, and ecology of sporadic human cryptosporidiosis in Australia. Appl. Environ. Microbiol. 77, 7757–7765. Wang, Y., Feng, Y., Cui, B., Jian, F., Ning, C., Wang, R., Zhang, L., Xiao, L., 2000. Cervine genotype is the major Cryptosporidium genotype in sheep in China. Parasitol. Res. 106, 341–347. Wang, R., Zhang, L., Ning, C., Feng, Y., Jian, F., Xiao, L., Lu, B., Ai, W., Dong, H., 2008. Multilocus phylogenetic analysis of Cryptosporidium andersoni (Apicomplexa) isolated from a bactrian camel (Camelus bactrianus) in China. Parasitol. Res. 102, 915–920. Wang, R., Ma, G., Zhao, J., Lu, Q., Wang, H., Zhang, L., Jian, F., Ning, C., Xiao, L., 2011a. Cryptosporidium andersoni is the predominant species in post-weaned and adult dairy cattle in China. Parasitol. Int. 60, 1–4. Wang, R., Wang, H., Sun, Y., Zhang, L., Jian, F., Qi, M., Ning, C., Xiao, L., 2011b. Characteristics of Cryptosporidium transmission in preweaned dairy cattle in Henan, China. J. Clin. Microbiol. 49, 1077–1082. Watanabe, Y., Yang, C.H., Ooi, H.K., 2005. Cryptosporidium infection in livestock and first identification of Cryptosporidium parvum genotype in cattle feces in Taiwan. Parasitol. Res. 97, 238–241. Xiao, L., Ryan, U.M., 2004. Cryptosporidiosis: an update in molecular epidemiology. Curr. Opin. Infect. Dis. 17, 483–490. Xiao, L., Bern, C., Limor, J., Sulaiman, I., Roberts, J., Checkley, W., Cabrera, L., Gilman, R.H., Lal, A.A., 2001. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J. Infect. Dis. 183, 492–497. Yang, W., Chen, P., Villegas, E.N., Landy, R.B., Kanetsky, C., Cama, V., Dearen, T., Schultz, C.L., Orndorff, K.G., Prelewicz, G.J., Brown, M.H., Young, K.R., Xiao, L., 2008. Cryptosporidium source tracking in the Potomac River watershed. Appl. Environ. Microbiol. 74, 6495–6504. Ye, J., Xiao, L., Wang, Y., Wang, L., Amer, S., Roellig, D.M., Guo, Y., Feng, Y., 2013. Periparturient transmission of Cryptosporidium xiaoi from ewes to lambs. Vet. Parasitol. 197, 627–633.
Please cite this article in press as: Wang, R., et al. Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.001
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