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Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Beijing, China
Veterinary Parasitology 219 (2016) 61–65
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Research paper
Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Beijing, China Fuhuang Li a,1 , Haiyan Wang b,1 , Zhenjie Zhang b , Junqiang Li b , Chenrong Wang b , Jinfeng Zhao a,b , Suhui Hu b , Rongjun Wang b,∗ , Longxian Zhang b , Ming Wang a,∗∗ a b
College of Veterinary Medicine, China Agricultural University, Beijing 100193, China College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China
a r t i c l e
i n f o
Article history: Received 28 November 2015 Received in revised form 30 January 2016 Accepted 31 January 2016 Keywords: Cryptosporidium Giardia duodenalis Dairy cattle SSU rRNA gp60 tpi
a b s t r a c t 822 fecal samples from cattle in six areas of Beijing were examined with microscopy for Cryptosporidium oocysts and Giardia cysts. The overall infection rates for Cryptosporidium spp. and Giardia duodenalis were 2.55% and 1.09%, respectively. Cryptosporidium was only detected in calves and heifers, whereas G. duodenalis was found in all age groups. Cryptosporidium spp. were characterized with a PCR-restriction fragment length polymorphism analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene. Two Cryptosporidium species were identified: Cryptosporidium parvum (n = 12) and Cryptosporidium andersoni (n = 9). Six C. parvum isolates were successfully subtyped with the gp60 gene and three subtypes were detected: IIdA19G1 (n = 1), IIdA17G1 (n = 1), and IIdA15G1 (n = 4). Subtype IIdA17G1 is reported for the first time in cattle worldwide. Nine G. duodenalis isolates were analyzed by sequencing the triosephosphate isomerase (tpi) gene, and only G. duodenalis assemblage E was identified. Therefore, the predominance of C. parvum detected in calves was identical to that found in the Xinjiang Uyghur and Ningxia Hui Autonomous Regions, but differed considerably from that in Henan, Heilongjiang, and Shannxi Provinces. In contrast, the predominance of G. duodenalis assemblage E was more or less similar to its predominance in other areas of China or countries. Our findings confirm the unique character of the C. parvum IId subtypes in China. More systematic studies are required to better understand the transmission of Cryptosporidium and G. duodenalis in cattle in China. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cryptosporidium and Giardia are important gastrointestinal protists that can infect humans, livestock, companion animals, and wildlife. Infection occurs by the fecal–oral route after the ingestion of infective oocysts or cysts, by either direct contact or the ingestion of contaminated food or water (Feng and Xiao, 2011). Cattle are commonly infected with Cryptosporidium, and preweaned calves are considered the most important reservoir for zoonotic infections (Wang et al., 2011b). Many studies have suggested that Cryptosporidium parvum, Corynebacterium bovis, Cryptosporidium andersoni, and Cryptosporidium ryanae are the most common species infecting cattle, although Ctenocephalides
∗ Corresponding author. Fax: +86 371 63558180. ∗∗ Corresponding author. Fax: +86 10 62733961. E-mail addresses: [email protected] (R. Wang), [email protected] (M. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.vetpar.2016.01.023 0304-4017/© 2016 Elsevier B.V. All rights reserved.
felis, Cryptosporidium hominis, Chlamydia suis, Cryptosporidium scrofarum, and C. suis-like genotype have also been detected (Trout and Santín, 2008). The four common Cryptosporidium species have ageassociated distributions. C. parvum is usually found in preweaned calves and is a significant cause of diarrhea. However, C. bovis and C. ryanae usually infect post-weaned calves and yearlings, and C. bovis is detected more frequently than C. ryanae, although neither is associated with diarrhea (Santín et al., 2008). In contrast, C. andersoni is commonly seen in adult cattle and is associated with gastritis, reduced milk yield, and poor weight gain (Esteban and Anderson, 1995). Giardia duodenalis is a species complex comprising eight distinct ‘assemblages’ or genotypes, A–H (Feng and Xiao, 2011), which can infect humans and most other mammals (Adam, 2001). Most of the assemblages (C–H) seem to be host specific for nonhuman species: assemblages C and D are specific for dogs, E for hoofed livestock, F for cats, G for rats, and H for seals. In cattle, the prevalence of G. duodenalis ranges from 2.2% to 50.7% worldwide, and assemblages A, B, and E have been detected, with assemblage E the predominant
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genotype in most countries (Xiao and Fayer, 2008; Feng and Xiao, 2011). In China, Cryptosporidium and Giardia infections in cattle have been found in several areas, and differences in the Cryptosporidium distributions have been noted in different regions (Liu et al., 2009, 2012; Wang et al., 2011a,b; Zhang et al., 2013, 2015; Cui et al., 2014; Huang et al., 2014; Ma et al., 2015; Qi et al., 2015a,b). For example, C. bovis rather than C. parvum is the predominant species in preweaned calves in Henan (Wang et al., 2011b), Shaanxi (Qi et al., 2015b), Shandong (Ma et al., 2015), Gansu, and the Ningxia Hui Autonomous Region (Zhang et al., 2015), whereas C. andersoni is the most common species in cattle in Heilongjiang Province (Liu et al., 2009). Thus, the distributions of Cryptosporidium spp. and the C. parvum subtypes (IIdA19G1 and IIdA15G1) in dairy cattle differ more or less from those in other countries (Wang et al., 2011b; Liu et al., 2012; Huang et al., 2014; Zhang et al., 2015). In contrast, there have a limited number of molecular epidemiological studies of G. duodenalis in cattle (Liu et al., 2012, 2015a; Wang et al., 2014). The objective of this study was to identify the species of Cryptosporidium and Giardia present in dairy cattle in Beijing (the capital of the People’s Republic of China, and China’s political, economic, and cultural center), to assess the zoonotic potential of Cryptosporidium and G. duodenalis genotypes/subtypes for humans in this area.
2. Materials and methods 2.1. Sample collection and examination A fresh fecal sample was collected from each animal using a sterile disposal latex glove immediately after its defecation onto the ground, and placed individually into a disposable plastic bag, with the age of each animal being recorded. In total, 822 fecal samples were collected between January 2014 and December 2015 from 12 dairy cattle farms in Beijing, China (Table 1). The Cryptosporidium oocysts in the 25 g fecal materials were concentrated with Sheather’s sugar flotation technique, with a further formalin-ethyl acetate sedimentation step included for the samples from preweaned calves (Wang et al., 2011b). Giardia cysts were detected with Lugol’s iodine staining. The Cryptosporidiumor Giardia-positive fecal samples were stored in 2.5% potassium dichromate at 4 ◦ C until DNA extraction.
2.2. DNA extraction The 100 mg of Cryptosporidium- or Giardia-positive fecal samples were washed three times with distilled water, and the genomic DNA was extracted from the fecal pellets with the E.Z.N.A.® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA), according to the manufacturer’s recommendations.
2.3. Cryptosporidium/Giardia genotyping and subtyping The Cryptosporidium species were identified with a PCRrestriction fragment length polymorphism (RFLP) analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene (Feng et al., 2007). Cryptosporidium parvum was subtyped with nested PCR targeting the gp60 gene, and the previously established nomenclature was used to name the C. parvum subtype families and subtypes (Sulaiman et al., 2005; Xiao, 2010). G. duodenalis was identified by sequencing the triosephosphate isomerase (tpi) gene (Caccio et al., 2008), and the genotype/subtype identities of the G. duodenalis samples were established by direct comparison of the sequences with reference sequences downloaded from the GenBank database.
2.4. DNA sequence analysis The PCR products were sequenced on an ABI PrismTM 3730 XL DNA Analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster, CA, USA). Sequence accuracy was confirmed with two-directional sequencing and by sequencing a new PCR product if necessary. The sequences were aligned with the ClustalX 1.83 program. Representative nucleotide sequences have been deposited in GenBank under accession numbers KT964796–KT964801. 2.5. Statistical analysis The 2 test was used to compare the Cryptosporidium infection rates. Differences were considered significant at P < 0.05. 3. Results 3.1. Prevalence of Cryptosporidium and Giardia Cryptosporidium oocysts were found by microscopic analysis in 21 samples (2.55%) from six farms, and the highest infection rate was 12.5% on farm 5 (Table 1). The infection rates of Cryptosporidium spp. were 3.47%, 3.24%, and 0% in calves, heifers, and adult cattle, respectively (2 = 7.05, 0.01 < P < 0.05). Fourteen samples from four farms were positive for Giardia, with an average infection rate of 1.09% (Table 1). The highest infection rate for Giardia was 4.38% on farm 4. The infection rates for Giardia were 2.72%, 0.93%, and 0.50% in calves, heifers, and adult cattle, respectively (2 = 5.05, P > 0.05). 3.2. Distribution of Cryptosporidium species/subtypes and G. duodenalis assemblage The SSU rRNA genes of the Cryptosporidium spp. in all 21 microscopy-positive samples were successfully amplified with nested PCR. RFLP and DNA sequence analyses of the SSU rRNA gene fragments revealed the presence of two Cryptosporidium species: C. parvum (n = 12) on four farms and C. andersoni (n = 9) on two farms (Table 1). Only one Cryptosporidium species was detected on each of the Cryptosporidium-positive farms (Table 1). The six C. parvum isolates successfully subtyped with the gp60 sequencing analysis belonged to subtypes IIdA19G1 (n = 1), IIdA17G1 (n = 1), and IIdA15G1 (n = 4). The sequencing analyses of the tpi gene of G. duodenalis identified assemblage E (n = 14) on four farms (Table 1). 3.3. Age distributions of Cryptosporidium and G. duodenalis C. parvum was the most commonly identified Cryptosporidium species in calves, whereas C. andersoni was the dominant Cryptosporidium species in heifers (Table 2). In contrast, no Cryptosporidium-positive sample was identified in adult cattle. G. duodenalis was detected in all age groups, but calves had the highest infection rate. Only G. duodenalis assemblage E was found in the different age groups (Table 2). 4. Discussion The overall infection rate for Cryptosporidium spp. was 2.55%, which is lower than the rate of 9.68% (18/186) reported previously in Beijing (Jiang et al., 1989), and the rates reported in Henan (13.0%, 276/2116) (Wang et al., 2011a,b), Heilongjiang (15.0%, 99/658) (Liu et al., 2009; Zhang et al., 2013), Shannxi (3.4%, 70/2071) (Zhao et al., 2013), Anhui (14.9%, 52/350), Jiangsu (20.7%, 251/1215), Shanghai (12.5%, 55/440) (Chen and Huang, 2012), Gansu and Ningxia (5.09%,
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Table 1 Numbers of fecal samples examined microscopically for Cryptosporidium oocysts and Giardia cysts on Chinese farms. The distributions of Cryptosporidium spp. and G. duodenalis assemblages were determined with a PCR–RFLP analysis of the SSU rRNA gene and sequence analysis of the tpi genes. Farm 1 2 3 4 5 6 7 8 9 10 11 12 Total
Sample size
Prevalence (%)Cryptosporidium/Giardia
Cryptosporidium species (n)
Giardia duodenalis (n)
51 79 92 23 66 61 79 33 103 111 66 58 822
1.96%/0 10.1%/2.53% 1.09%/3.26% 8.69%/4.38% 12.1%/12.1% 0/0 0/0 1.03%/0 0/0 0/0 0/0 0/0 2.55%/1.09%
Cryptosporidium andersoni (1) Cryptosporidium andersoni (8) Cryptosporidium parvum (1) Cryptosporidium parvum (2) Cryptosporidium parvum (8)
Assemblage E (2) Assemblage E (3) Assemblage E (1) Assemblage E (8)
Cryptosporidium parvum (1)
Cryptosporidium andersoni (9), Cryptosporidium parvum (12)
Assemblage E (14)
Table 2 Cryptosporidium species identified in dairy cattle in different age groups. Age groupa
calf heifer adult a
Sample size
404 216 202
Cryptosporidium
G. duodenalis assemblage
Prevalence
Species (n)
Prevalence
Assemblage (n)
3.47% 3.24% 0
C. andersoni (4), C. parvum (10) C. andersoni (5), C. parvum (2)
2.72% 0.93% 0.50%
E (11) E (2) E (1)
Calf (<1 year old), heifer (1–2 years old), and adult (>2 years old).
150/2945) (Zhang et al., 2015), and Shandong (24.3%, 36/148) (Ma et al., 2015). The average infection rate for G. duodenalis was 1.09%, which is lower than those reported in Heilongjiang (5.2%, 42/814) (Liu et al., 2012), Henan (7.2%, 128/1777) (Wang et al., 2014), and northeast China (7.9%, 52/655) (Liu et al., 2015a). Low-intensity oocyst shedding may have contributed to the low detection rates for both parasites with microscopy. Many factors have contributed to the discrepancies in the prevalence of Cryptosporidium and G. duodenalis among different studies, including the examination method used, the age distribution of the animals examined, the sample size, the host health status at the time of sampling, the timing of specimen collection, and geo-ecological conditions. However, in this study, the infection rates of both parasites were higher in calves than in any other age group, which is consistent with previous findings (Wang et al., 2011a,b, 2014; Liu et al., 2012; Geurden et al., 2012; Minetti et al., 2013; Huang et al., 2014). C. parvum was the predominant Cryptosporidium species among the calves, which is similar to the findings of recent studies conducted in the Xinjiang Uyghur Autonomous Region (Qi et al., 2015a) and the Ningxia Hui Autonomous Region (Huang et al., 2014), whereas it clearly differs from the results reported in Henan (Wang et al., 2011b), Heilongjiang (Zhang et al., 2013), Shaanxi (Qi et al., 2015b), Shandong (Ma et al., 2015), Gansu, and the Ningxia Hui Autonomous Region (Zhang et al., 2015), where C. bovis was the dominant species, and from a study in Shannxi, where only C. andersoni was detected (Zhao et al., 2013). The results of most previous studies, conducted in several countries, suggest that C. parvum is the predominant Cryptosporidium species in calves, especially in preweaned calves (Wang et al., 2011b). In contrast, C. andersoni was the dominant species in heifers in this study, which is basically similar to reports of Cryptosporidium infections in cattle in Henan, Heilongjiang, Shannxi, Gansu, and the Ningxia Hui Autonomous Region (Xiao and Fayer, 2008; Liu et al., 2009; Wang et al., 2011a; Zhao et al., 2013; Huang et al., 2014; Zhang et al., 2015). The six C. parvum isolates successfully subtyped were identified as subtypes IIdA19G1, IIdA17G1, and IIdA15G1 based on a sequence analysis of the gp60 gene. In China, subtype IIdA19G1 has previously been found in cattle, humans, and urban wastewater (Wang et al., 2011b, 2013; Li et al., 2012; Zhang et al., 2013), and sub-
type IIdA15G1 in cattle and rodents (Lv et al., 2009; Huang et al., 2014). Therefore, the present study confirms the unique character of the C. parvum subtypes in China. This is also the first report of C. parvum subtype IIdA17G1 in dairy cattle worldwide. The IIdA17G1 is a zoonotic subtype, and has been found in humans in the United Kingdom (Chalmers et al., 2011), in goats in Spain (Díaz et al., 2015), and in treated water samples (Lobo et al., 2009). Sequence analyses of the gp60 gene have been used extensively to characterize the molecular epidemiology of cryptosporidiosis in animals and humans, and at least 14C. parvum subtype families have been identified: IIa, IIb, IIc, IId, IIe, IIf, IIg, IIh, IIi, IIk, IIl, IIm, IIn, and IIo (Xiao and Ryan, 2008; Xiao, 2010; Liu et al., 2015b). Of these, IIa is the predominant subtype family in animals and humans worldwide, and IId is another major zoonotic subtype family reported in Europe (Hungary, Germany, Portugal, Sweden, Ireland, Spain, Belgium, Romania, United Kingdom, Netherlands, Slovenia, Serbia, and Montenegro), Asia (Kuwait, Iran, Jordan, India, Malaysia, and China), Egypt, and Australia (Insulander et al., 2013; Wang et al., 2011b; Amer et al., 2013; Iqbal et al., 2012; Imre et al., 2013; Vieira et al., 2015). In contrast, IIc and IIe are anthroponotic subtype families. The other subtype families of C. parvum are occasionally seen in various animals or humans around the world (Xiao, 2010; Wang et al., 2011b). A sequence analysis of the tpi gene showed that all the dairy cattle were infected with livestock-specific G. duodenalis assemblage E. This result is consistent with previous studies conducted in Heilongjiang, Henan, and northeast China (Jilin, Changchun, Shenyang, Jiutai, Daqing, and Ningxia) (Liu et al., 2012, 2015a; Wang et al., 2014; Huang et al., 2014). The results of most studies conducted in numerous countries (including Belgium, Denmark, Portugal, Spain, Sweden, Germany, United Kingdom, United States of America, Canada, Brazil, Australia, Rwanda, Egypt, Sri Lanka, Malaysia, and Bangladesh) suggest that assemblage E is the predominant Giardia assemblage in cattle (Feng and Xiao, 2011; Budu-Amoako et al., 2012; Mark-Carew et al., 2012; Muhid et al., 2012; Paz e Silva et al., 2012; Santin et al., 2012; Amer, 2013; Fava et al., 2013; Gillhuber et al., 2013; Abeywardena et al., 2014; Hogan et al., 2014; Ehsan et al., 2015).
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In conclusion, C. parvum is the predominant Cryptosporidium species in preweaned calves in the study area, which is similar to the situation in the Xinjiang Uyghur Autonomous Region. The presence of subtypes IIdA19G1, IIdA17G1, and IIdA15G1 confirms the unique character of the C. parvum subtypes in China, with subtype IIdA17G1 reported here in dairy cattle for the first time worldwide. The predominance of G. duodenalis E is similar to its distribution in other areas and countries. More molecular epidemiological studies are required to better understand the transmission and public health significance of Cryptosporidium spp. and G. duodenalis in China. Acknowledgements This study was supported, in part, by the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-37), the State Key Program of National Natural Science Foundation of China (31330079), and the Specialized Research Fund for the Doctoral Program of Higher Education (20124105120003). 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Research paper
Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Beijing, China Fuhuang Li a,1 , Haiyan Wang b,1 , Zhenjie Zhang b , Junqiang Li b , Chenrong Wang b , Jinfeng Zhao a,b , Suhui Hu b , Rongjun Wang b,∗ , Longxian Zhang b , Ming Wang a,∗∗ a b
College of Veterinary Medicine, China Agricultural University, Beijing 100193, China College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China
a r t i c l e
i n f o
Article history: Received 28 November 2015 Received in revised form 30 January 2016 Accepted 31 January 2016 Keywords: Cryptosporidium Giardia duodenalis Dairy cattle SSU rRNA gp60 tpi
a b s t r a c t 822 fecal samples from cattle in six areas of Beijing were examined with microscopy for Cryptosporidium oocysts and Giardia cysts. The overall infection rates for Cryptosporidium spp. and Giardia duodenalis were 2.55% and 1.09%, respectively. Cryptosporidium was only detected in calves and heifers, whereas G. duodenalis was found in all age groups. Cryptosporidium spp. were characterized with a PCR-restriction fragment length polymorphism analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene. Two Cryptosporidium species were identified: Cryptosporidium parvum (n = 12) and Cryptosporidium andersoni (n = 9). Six C. parvum isolates were successfully subtyped with the gp60 gene and three subtypes were detected: IIdA19G1 (n = 1), IIdA17G1 (n = 1), and IIdA15G1 (n = 4). Subtype IIdA17G1 is reported for the first time in cattle worldwide. Nine G. duodenalis isolates were analyzed by sequencing the triosephosphate isomerase (tpi) gene, and only G. duodenalis assemblage E was identified. Therefore, the predominance of C. parvum detected in calves was identical to that found in the Xinjiang Uyghur and Ningxia Hui Autonomous Regions, but differed considerably from that in Henan, Heilongjiang, and Shannxi Provinces. In contrast, the predominance of G. duodenalis assemblage E was more or less similar to its predominance in other areas of China or countries. Our findings confirm the unique character of the C. parvum IId subtypes in China. More systematic studies are required to better understand the transmission of Cryptosporidium and G. duodenalis in cattle in China. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cryptosporidium and Giardia are important gastrointestinal protists that can infect humans, livestock, companion animals, and wildlife. Infection occurs by the fecal–oral route after the ingestion of infective oocysts or cysts, by either direct contact or the ingestion of contaminated food or water (Feng and Xiao, 2011). Cattle are commonly infected with Cryptosporidium, and preweaned calves are considered the most important reservoir for zoonotic infections (Wang et al., 2011b). Many studies have suggested that Cryptosporidium parvum, Corynebacterium bovis, Cryptosporidium andersoni, and Cryptosporidium ryanae are the most common species infecting cattle, although Ctenocephalides
∗ Corresponding author. Fax: +86 371 63558180. ∗∗ Corresponding author. Fax: +86 10 62733961. E-mail addresses: [email protected] (R. Wang), [email protected] (M. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.vetpar.2016.01.023 0304-4017/© 2016 Elsevier B.V. All rights reserved.
felis, Cryptosporidium hominis, Chlamydia suis, Cryptosporidium scrofarum, and C. suis-like genotype have also been detected (Trout and Santín, 2008). The four common Cryptosporidium species have ageassociated distributions. C. parvum is usually found in preweaned calves and is a significant cause of diarrhea. However, C. bovis and C. ryanae usually infect post-weaned calves and yearlings, and C. bovis is detected more frequently than C. ryanae, although neither is associated with diarrhea (Santín et al., 2008). In contrast, C. andersoni is commonly seen in adult cattle and is associated with gastritis, reduced milk yield, and poor weight gain (Esteban and Anderson, 1995). Giardia duodenalis is a species complex comprising eight distinct ‘assemblages’ or genotypes, A–H (Feng and Xiao, 2011), which can infect humans and most other mammals (Adam, 2001). Most of the assemblages (C–H) seem to be host specific for nonhuman species: assemblages C and D are specific for dogs, E for hoofed livestock, F for cats, G for rats, and H for seals. In cattle, the prevalence of G. duodenalis ranges from 2.2% to 50.7% worldwide, and assemblages A, B, and E have been detected, with assemblage E the predominant
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genotype in most countries (Xiao and Fayer, 2008; Feng and Xiao, 2011). In China, Cryptosporidium and Giardia infections in cattle have been found in several areas, and differences in the Cryptosporidium distributions have been noted in different regions (Liu et al., 2009, 2012; Wang et al., 2011a,b; Zhang et al., 2013, 2015; Cui et al., 2014; Huang et al., 2014; Ma et al., 2015; Qi et al., 2015a,b). For example, C. bovis rather than C. parvum is the predominant species in preweaned calves in Henan (Wang et al., 2011b), Shaanxi (Qi et al., 2015b), Shandong (Ma et al., 2015), Gansu, and the Ningxia Hui Autonomous Region (Zhang et al., 2015), whereas C. andersoni is the most common species in cattle in Heilongjiang Province (Liu et al., 2009). Thus, the distributions of Cryptosporidium spp. and the C. parvum subtypes (IIdA19G1 and IIdA15G1) in dairy cattle differ more or less from those in other countries (Wang et al., 2011b; Liu et al., 2012; Huang et al., 2014; Zhang et al., 2015). In contrast, there have a limited number of molecular epidemiological studies of G. duodenalis in cattle (Liu et al., 2012, 2015a; Wang et al., 2014). The objective of this study was to identify the species of Cryptosporidium and Giardia present in dairy cattle in Beijing (the capital of the People’s Republic of China, and China’s political, economic, and cultural center), to assess the zoonotic potential of Cryptosporidium and G. duodenalis genotypes/subtypes for humans in this area.
2. Materials and methods 2.1. Sample collection and examination A fresh fecal sample was collected from each animal using a sterile disposal latex glove immediately after its defecation onto the ground, and placed individually into a disposable plastic bag, with the age of each animal being recorded. In total, 822 fecal samples were collected between January 2014 and December 2015 from 12 dairy cattle farms in Beijing, China (Table 1). The Cryptosporidium oocysts in the 25 g fecal materials were concentrated with Sheather’s sugar flotation technique, with a further formalin-ethyl acetate sedimentation step included for the samples from preweaned calves (Wang et al., 2011b). Giardia cysts were detected with Lugol’s iodine staining. The Cryptosporidiumor Giardia-positive fecal samples were stored in 2.5% potassium dichromate at 4 ◦ C until DNA extraction.
2.2. DNA extraction The 100 mg of Cryptosporidium- or Giardia-positive fecal samples were washed three times with distilled water, and the genomic DNA was extracted from the fecal pellets with the E.Z.N.A.® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA), according to the manufacturer’s recommendations.
2.3. Cryptosporidium/Giardia genotyping and subtyping The Cryptosporidium species were identified with a PCRrestriction fragment length polymorphism (RFLP) analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene (Feng et al., 2007). Cryptosporidium parvum was subtyped with nested PCR targeting the gp60 gene, and the previously established nomenclature was used to name the C. parvum subtype families and subtypes (Sulaiman et al., 2005; Xiao, 2010). G. duodenalis was identified by sequencing the triosephosphate isomerase (tpi) gene (Caccio et al., 2008), and the genotype/subtype identities of the G. duodenalis samples were established by direct comparison of the sequences with reference sequences downloaded from the GenBank database.
2.4. DNA sequence analysis The PCR products were sequenced on an ABI PrismTM 3730 XL DNA Analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster, CA, USA). Sequence accuracy was confirmed with two-directional sequencing and by sequencing a new PCR product if necessary. The sequences were aligned with the ClustalX 1.83 program. Representative nucleotide sequences have been deposited in GenBank under accession numbers KT964796–KT964801. 2.5. Statistical analysis The 2 test was used to compare the Cryptosporidium infection rates. Differences were considered significant at P < 0.05. 3. Results 3.1. Prevalence of Cryptosporidium and Giardia Cryptosporidium oocysts were found by microscopic analysis in 21 samples (2.55%) from six farms, and the highest infection rate was 12.5% on farm 5 (Table 1). The infection rates of Cryptosporidium spp. were 3.47%, 3.24%, and 0% in calves, heifers, and adult cattle, respectively (2 = 7.05, 0.01 < P < 0.05). Fourteen samples from four farms were positive for Giardia, with an average infection rate of 1.09% (Table 1). The highest infection rate for Giardia was 4.38% on farm 4. The infection rates for Giardia were 2.72%, 0.93%, and 0.50% in calves, heifers, and adult cattle, respectively (2 = 5.05, P > 0.05). 3.2. Distribution of Cryptosporidium species/subtypes and G. duodenalis assemblage The SSU rRNA genes of the Cryptosporidium spp. in all 21 microscopy-positive samples were successfully amplified with nested PCR. RFLP and DNA sequence analyses of the SSU rRNA gene fragments revealed the presence of two Cryptosporidium species: C. parvum (n = 12) on four farms and C. andersoni (n = 9) on two farms (Table 1). Only one Cryptosporidium species was detected on each of the Cryptosporidium-positive farms (Table 1). The six C. parvum isolates successfully subtyped with the gp60 sequencing analysis belonged to subtypes IIdA19G1 (n = 1), IIdA17G1 (n = 1), and IIdA15G1 (n = 4). The sequencing analyses of the tpi gene of G. duodenalis identified assemblage E (n = 14) on four farms (Table 1). 3.3. Age distributions of Cryptosporidium and G. duodenalis C. parvum was the most commonly identified Cryptosporidium species in calves, whereas C. andersoni was the dominant Cryptosporidium species in heifers (Table 2). In contrast, no Cryptosporidium-positive sample was identified in adult cattle. G. duodenalis was detected in all age groups, but calves had the highest infection rate. Only G. duodenalis assemblage E was found in the different age groups (Table 2). 4. Discussion The overall infection rate for Cryptosporidium spp. was 2.55%, which is lower than the rate of 9.68% (18/186) reported previously in Beijing (Jiang et al., 1989), and the rates reported in Henan (13.0%, 276/2116) (Wang et al., 2011a,b), Heilongjiang (15.0%, 99/658) (Liu et al., 2009; Zhang et al., 2013), Shannxi (3.4%, 70/2071) (Zhao et al., 2013), Anhui (14.9%, 52/350), Jiangsu (20.7%, 251/1215), Shanghai (12.5%, 55/440) (Chen and Huang, 2012), Gansu and Ningxia (5.09%,
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Table 1 Numbers of fecal samples examined microscopically for Cryptosporidium oocysts and Giardia cysts on Chinese farms. The distributions of Cryptosporidium spp. and G. duodenalis assemblages were determined with a PCR–RFLP analysis of the SSU rRNA gene and sequence analysis of the tpi genes. Farm 1 2 3 4 5 6 7 8 9 10 11 12 Total
Sample size
Prevalence (%)Cryptosporidium/Giardia
Cryptosporidium species (n)
Giardia duodenalis (n)
51 79 92 23 66 61 79 33 103 111 66 58 822
1.96%/0 10.1%/2.53% 1.09%/3.26% 8.69%/4.38% 12.1%/12.1% 0/0 0/0 1.03%/0 0/0 0/0 0/0 0/0 2.55%/1.09%
Cryptosporidium andersoni (1) Cryptosporidium andersoni (8) Cryptosporidium parvum (1) Cryptosporidium parvum (2) Cryptosporidium parvum (8)
Assemblage E (2) Assemblage E (3) Assemblage E (1) Assemblage E (8)
Cryptosporidium parvum (1)
Cryptosporidium andersoni (9), Cryptosporidium parvum (12)
Assemblage E (14)
Table 2 Cryptosporidium species identified in dairy cattle in different age groups. Age groupa
calf heifer adult a
Sample size
404 216 202
Cryptosporidium
G. duodenalis assemblage
Prevalence
Species (n)
Prevalence
Assemblage (n)
3.47% 3.24% 0
C. andersoni (4), C. parvum (10) C. andersoni (5), C. parvum (2)
2.72% 0.93% 0.50%
E (11) E (2) E (1)
Calf (<1 year old), heifer (1–2 years old), and adult (>2 years old).
150/2945) (Zhang et al., 2015), and Shandong (24.3%, 36/148) (Ma et al., 2015). The average infection rate for G. duodenalis was 1.09%, which is lower than those reported in Heilongjiang (5.2%, 42/814) (Liu et al., 2012), Henan (7.2%, 128/1777) (Wang et al., 2014), and northeast China (7.9%, 52/655) (Liu et al., 2015a). Low-intensity oocyst shedding may have contributed to the low detection rates for both parasites with microscopy. Many factors have contributed to the discrepancies in the prevalence of Cryptosporidium and G. duodenalis among different studies, including the examination method used, the age distribution of the animals examined, the sample size, the host health status at the time of sampling, the timing of specimen collection, and geo-ecological conditions. However, in this study, the infection rates of both parasites were higher in calves than in any other age group, which is consistent with previous findings (Wang et al., 2011a,b, 2014; Liu et al., 2012; Geurden et al., 2012; Minetti et al., 2013; Huang et al., 2014). C. parvum was the predominant Cryptosporidium species among the calves, which is similar to the findings of recent studies conducted in the Xinjiang Uyghur Autonomous Region (Qi et al., 2015a) and the Ningxia Hui Autonomous Region (Huang et al., 2014), whereas it clearly differs from the results reported in Henan (Wang et al., 2011b), Heilongjiang (Zhang et al., 2013), Shaanxi (Qi et al., 2015b), Shandong (Ma et al., 2015), Gansu, and the Ningxia Hui Autonomous Region (Zhang et al., 2015), where C. bovis was the dominant species, and from a study in Shannxi, where only C. andersoni was detected (Zhao et al., 2013). The results of most previous studies, conducted in several countries, suggest that C. parvum is the predominant Cryptosporidium species in calves, especially in preweaned calves (Wang et al., 2011b). In contrast, C. andersoni was the dominant species in heifers in this study, which is basically similar to reports of Cryptosporidium infections in cattle in Henan, Heilongjiang, Shannxi, Gansu, and the Ningxia Hui Autonomous Region (Xiao and Fayer, 2008; Liu et al., 2009; Wang et al., 2011a; Zhao et al., 2013; Huang et al., 2014; Zhang et al., 2015). The six C. parvum isolates successfully subtyped were identified as subtypes IIdA19G1, IIdA17G1, and IIdA15G1 based on a sequence analysis of the gp60 gene. In China, subtype IIdA19G1 has previously been found in cattle, humans, and urban wastewater (Wang et al., 2011b, 2013; Li et al., 2012; Zhang et al., 2013), and sub-
type IIdA15G1 in cattle and rodents (Lv et al., 2009; Huang et al., 2014). Therefore, the present study confirms the unique character of the C. parvum subtypes in China. This is also the first report of C. parvum subtype IIdA17G1 in dairy cattle worldwide. The IIdA17G1 is a zoonotic subtype, and has been found in humans in the United Kingdom (Chalmers et al., 2011), in goats in Spain (Díaz et al., 2015), and in treated water samples (Lobo et al., 2009). Sequence analyses of the gp60 gene have been used extensively to characterize the molecular epidemiology of cryptosporidiosis in animals and humans, and at least 14C. parvum subtype families have been identified: IIa, IIb, IIc, IId, IIe, IIf, IIg, IIh, IIi, IIk, IIl, IIm, IIn, and IIo (Xiao and Ryan, 2008; Xiao, 2010; Liu et al., 2015b). Of these, IIa is the predominant subtype family in animals and humans worldwide, and IId is another major zoonotic subtype family reported in Europe (Hungary, Germany, Portugal, Sweden, Ireland, Spain, Belgium, Romania, United Kingdom, Netherlands, Slovenia, Serbia, and Montenegro), Asia (Kuwait, Iran, Jordan, India, Malaysia, and China), Egypt, and Australia (Insulander et al., 2013; Wang et al., 2011b; Amer et al., 2013; Iqbal et al., 2012; Imre et al., 2013; Vieira et al., 2015). In contrast, IIc and IIe are anthroponotic subtype families. The other subtype families of C. parvum are occasionally seen in various animals or humans around the world (Xiao, 2010; Wang et al., 2011b). A sequence analysis of the tpi gene showed that all the dairy cattle were infected with livestock-specific G. duodenalis assemblage E. This result is consistent with previous studies conducted in Heilongjiang, Henan, and northeast China (Jilin, Changchun, Shenyang, Jiutai, Daqing, and Ningxia) (Liu et al., 2012, 2015a; Wang et al., 2014; Huang et al., 2014). The results of most studies conducted in numerous countries (including Belgium, Denmark, Portugal, Spain, Sweden, Germany, United Kingdom, United States of America, Canada, Brazil, Australia, Rwanda, Egypt, Sri Lanka, Malaysia, and Bangladesh) suggest that assemblage E is the predominant Giardia assemblage in cattle (Feng and Xiao, 2011; Budu-Amoako et al., 2012; Mark-Carew et al., 2012; Muhid et al., 2012; Paz e Silva et al., 2012; Santin et al., 2012; Amer, 2013; Fava et al., 2013; Gillhuber et al., 2013; Abeywardena et al., 2014; Hogan et al., 2014; Ehsan et al., 2015).
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In conclusion, C. parvum is the predominant Cryptosporidium species in preweaned calves in the study area, which is similar to the situation in the Xinjiang Uyghur Autonomous Region. The presence of subtypes IIdA19G1, IIdA17G1, and IIdA15G1 confirms the unique character of the C. parvum subtypes in China, with subtype IIdA17G1 reported here in dairy cattle for the first time worldwide. The predominance of G. duodenalis E is similar to its distribution in other areas and countries. More molecular epidemiological studies are required to better understand the transmission and public health significance of Cryptosporidium spp. and G. duodenalis in China. Acknowledgements This study was supported, in part, by the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-37), the State Key Program of National Natural Science Foundation of China (31330079), and the Specialized Research Fund for the Doctoral Program of Higher Education (20124105120003). 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