Occurrence and molecular characterization of Cryptosporidium spp. in yaks (Bos grunniens) in China

Veterinary Parasitology 202 (2014) 113–118

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Occurrence and molecular characterization of Cryptosporidium spp. in yaks (Bos grunniens) in China Jingbo Ma a , Jinzhong Cai b , Jiawen Ma a , Yaoyu Feng a,∗∗ , Lihua Xiao c,∗ a State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China b Qinghai Academy of Veterinary Medicine and Animal Science, Xining 810016, China c Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA

a r t i c l e

i n f o

Article history: Received 17 December 2013 Received in revised form 24 March 2014 Accepted 26 March 2014

Keywords: Cryptosporidium Qinghai Yaks 18S rRNA gene Nested PCR Genotype

a b s t r a c t Compared with dairy and beef cattle, few data are available on the occurrence and distribution of Cryptosporidium species in yaks, which live in a very different habitat. In this study, 327 fecal specimens were collected from yaks in 4 counties in Qinghai Province of China and screened for Cryptosporidium by nested PCR analysis of the 18S rRNA gene. A total of 98 (30.0%) specimens were positive for Cryptosporidium. The occurrence of Cryptosporidium varied significantly among age groups; infection rates were 49.3% in weaned calves, 31.7% in yearlings, and 17.4% in adults. PCR products of all Cryptosporidium-positive specimens were successfully sequenced, with 56 specimens (57.1%) having C. bovis, 33 (33.7%) having C. ryanae, 2 (2.0%) having C. andersoni, 1 (1.0%) having C. ubiquitum, 1 (1.0%) having C. xiaoi, 2 (2.0%) having a novel genotype, and 3 (3.1%) having mixed infections of C. bovis and C. ryanae. There were some age-related differences in the distribution of Cryptosporidium species in post-weaned yaks examined. To our knowledge, this is the first report of C. andersoni, C. ubiquitum, C. xiaoi and a novel Cryptosporidium genotype in yaks. Published by Elsevier B.V.

1. Introduction Cryptosporidium spp. are common parasites of humans, farm animals, and other vertebrates, causing the disease cryptosporidiosis (Fayer, 2010; Plutzer and Karanis, 2009; Xiao, 2010). Cryptosporidium oocysts are released in feces by infected hosts and transmitted via direct contact with infected persons or animals or through contaminated food or water (Baldursson and Karanis, 2011; Chalmers and Davies, 2010). Cryptosporidiosis is self-limiting in

∗ Corresponding author. Tel.: +1 404 718 4161; fax: +1 404 718 4197. ∗∗ Corresponding author. Tel.: +86 21 6425 0664; fax: +86 21 6425 0664. E-mail addresses: [email protected] (Y. Feng), [email protected], [email protected] (L. Xiao). http://dx.doi.org/10.1016/j.vetpar.2014.03.030 0304-4017/Published by Elsevier B.V.

immunocompetent patients but can be life-threatening in immunocompromised and young individuals (Chalmers and Davies, 2010). Cryptosporidiosis is difficult to control as effective treatment is not widely available and Cryptosporidium oocysts are resistant to many environmental conditions and disinfection treatments (Cacciò and Pozio, 2006). In cattle, cryptosporidiosis is associated with the occurrence of diarrhea, weight loss and delayed growth, and sometimes mortality of calves, leading to economic losses (Anderson, 1998; Singh et al., 2006). Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium andersoni are the four major Cryptosporidium species in dairy cattle. There is an age-related distribution of the four species in cattle in most areas: C. parvum predominates in pre-weaned calves, C. bovis and

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C. ryanae in post-weaned calves, and C. andersoni in adult cattle (Santin, 2013). In addition, contact with pre-weaned calves is a major risk factor for C. parvum infections in humans (Chalmers and Giles, 2010). Yaks (Bos grunniens) belong to the genus Bos, thus are related to cattle. Yaks living on the Qinghai-Tibetan Plateau of China (about 3000 m above sea level) make up 90% of the global yak population (13 million). Domesticated yaks are usually kept in close contact with local residents, primarily for milk and meat, and as draft animals, raising public health concerns (Liu et al., 2008). The free-range nature and cold habitat of yak farming suggest that the distribution of Cryptosporidium species in these animals could in theory be different from the widely studied dairy cattle. Thus far, few studies have been done to genotype Cryptosporidium spp. in yaks. Most cryptosporidiosis studies in yaks have been done by microscopy or enzyme immunoassays in China (Bai et al., 2001; Dou, 2007; Ma et al., 2011; Wang and Liu, 2007; Zhang et al., 2006; Zhou et al., 2009). Only three studies used molecular tools to characterize Cryptosporidium spp. in yaks and identified three species: C. parvum, C. bovis, and C. ryanae (Feng et al., 2007; Karanis et al., 2007; Mi et al., 2013). In this study, we used an 18S rRNA-based genotyping tool to characterize Cryptosporidium spp. in post-weaned yaks in Qinghai Province of China, and identified the occurrence of C. andersoni, a novel Cryptosporidium genotype, and two species common in sheep: Cryptosporidium ubiquitum and Cryptosporidium xiaoi. 2. Materials and methods 2.1. Study area and sample collection Fresh fecal specimens were collected from May to July 2013 from yaks in Dari, Yushu, Qilian and Chendi Counties in Qinghai Province (31–39◦ N, 88–103◦ E), Northwestern China. The average altitude, annual temperature, and annual rainfall of the 4 counties range from 3100 to 4600 m, −1.0 to 8.5 ◦ C, and 360 to 550 mm, respectively. Yaks in the areas were mostly kept outdoor year around, and shared pastures with sheep and wild animals. A total of 327 fecal specimens were collected from yaks of <1 year (weaned calves), 1–2 years (yearlings), and >2 years (adults) of age in this study. No clinical signs were observed in the sampled animals. Specimens were stored in 2.5% potassium dichromate at 4 ◦ C before molecular analysis.

rRNA gene as previously described (Ryan et al., 2003). Some PCR-positive specimens were re-analyzed by a nested PCR targeting an ∼830 bp fragment of the 18S rRNA (Feng et al., 2007). For both assays, the PCR mixture consisted of 1 ␮l DNA (primary PCR) or 2 ␮l primary PCR product (secondary PCR), 3 mM MgCl2 , 2.5 U Taq DNA polymerase (Thermo Scientific, Pittsburgh, PA), 1× PCR buffer, 0.2 mM of each dNTP, 200 nM primers and 400 ng/␮l of bovine serum albumin (Sigma–Aldrich, St. Louis, MO) in a final volume of 50 ␮l. Each specimen was analyzed at least twice using DNA of C. baileyi as the positive control and reagent water as the negative control. PCR products were examined by electrophoresis on 1.5% agarose gel stained with ethidium bromide. 2.4. Sequence and phylogenetic analysis All positive secondary PCR products of the 18S rRNA gene were sequenced in both directions with the secondary primers using an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA) and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequences obtained were aligned with each other and published 18S rRNA gene sequences of Cryptosporidium spp. using the software ClustalX (http://www.clustal.org/) to determine Cryptosporidium species. Phylogenetic relationship of the new Cryptosporidium genotype to established species and common genotypes was assessed using the Neighbor-Joining (NJ) analysis implemented in MEGA 5.0 (http://www.megasoftware.net/) based on genetic distances calculated by the Kimura 2-parameter model. The sequence generated from PCR analysis of the ∼830 bp fragment of the 18S rRNA gene was used in the phylogenetic analysis. Representative nucleotide sequences (from the ∼830 bp PCR product for C. bovis, C. ryanae, and the novel genotype, and ∼587 bp PCR products for other species) generated in the study were deposited in the GenBank database under accession numbers KF971356 and KJ531688-KJ531692. 2.5. Statistical analysis Data were analyzed using SPSS 19.0 for Windows (SPSS Inc., Chicago, USA). The 2 test was used to analyze differences in infection rates among different counties and age groups. Differences were considered significant when P < 0.05.

2.2. DNA extraction

3. Results

DNA was extracted from 500 ␮l of fecal material after the specimens were washed off potassium dichromate with distilled water by centrifugation at 2000 × g for 10 min. The FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA) was used for extraction of genomic DNA as previously described (Jiang et al., 2005).

3.1. Occurrence of Cryptosporidium spp.

2.3. PCR detection of Cryptosporidium For the detection of Cryptosporidium, a nested PCR protocol was used to amplify an ∼587-bp fragment of the 18S

Of the 327 fecal specimens examined, 98 (30.0%) were positive for Cryptosporidium. Cryptosporidium infection rates in the 4 study locations ranged from 8.0% to 44.7% (Table 1). Higher infection rates were recorded in Dari (44.7%) and Qilian counties (28.0%) than Chengdi (17.3%) and Yushu counties (8.0%). Results of the ␹2 test showed that the difference in infection rates was statistically significant (P < 0.05). Cryptosporidium infection rates also varied significantly among age groups (Table 2). Weaned

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Table 1 Occurrence of Cryptosporidium infection in yaks in Qinghai Province, China by county. Location

Sample size

Cryptosporidium positive (%)

C. bovis

C. ryanae

C. andersoni

C. bovis + C. ryanae

C. ubiquitum

C. xiaoi

New genotype

Dari Yushu Qilian Chengdi Total (%)

132 50 93 52 327

59 (44.7) 4 (8.0) 26 (28.0) 9 (17.3) 98 (30.0)

27 2 22 5 56/98 (57.1)

26 2 3 2 33/98 (33.7)

1 0 0 1 2/98 (2.0)

2 0 0 1 3/98 (3.1)

1 0 0 0 1/98 (1.0)

0 0 1 0 1/98 (1.0)

2 0 0 0 2/98 (2.0)

calves had a significantly higher infection rate (49.3%) than yearlings (31.7%; P = 0.014), whereas the latter had a significantly higher infection rate than adults (17.4%; P = 0.008).

3.2. Distribution of Cryptosporidium species Of the 98 Cryptosporidium-positive specimens, all were successfully sequenced, leading to the identification of C. bovis, C. ryanae, C. andersoni, C. ubiquitum, C. xiaoi and a novel genotype. The partial 18S rRNA gene sequences of C. bovis, C. ryanae, C. andersoni, C. ubiquitum, C. xiaoi were identical to GenBank sequences AY741305, EU410344, DQ448631, HM209375 and EF613338, respectively. No sequence intra-species sequence variations were seen in the 18S rRNA gene in C. bovis (56 specimens) and C. ryanae (33 specimens) in this study. The most common species were C. bovis (57.1%) and C. ryanae (33.7%), which were found in all 4 study locations. Other Cryptosporidium species and the novel genotype were each found in only 1–2 animals (Table 1). C. bovis and C. ryanae were found in all 3 age groups. C. bovis was more commonly seen in calves and yearlings than C. ryanae. In contrast, C. ryanae (47.8%) was more frequently seen in adults than C. bovis (34.8%). The remaining Cryptosporidium species and the novel genotype were seen in either yearlings or adults (Table 2).

3.3. Phylogenetic analysis of the novel genotype The novel Cryptosporidium genotype had 30–73 nucleotide differences from other known Cryptosporidium species and genotypes in the partial ∼830 bp fragment of the 18S rRNA gene. In the NJ analysis, the new Cryptosporidium genotype formed the basal branch of the cluster containing most intestinal Cryptosporidium species (Fig. 1).

4. Discussion Data from the present study suggest that Cryptosporidium infection is common in yaks in Qinghai Province. The overall infection rate of 30.0% is similar to rates reported by Ma et al. (2011), Bai et al. (2001) and Dou (2007), but lower than rates reported by Zhang et al. (2006) and higher than rates reported by Karanis et al. (2007), Zhou et al. (2009), Wang and Liu (2007) and Mi et al. (2013) (Table 3). Like in cattle (Khan et al., 2010; Santin et al., 2008, 2004), we show that weaned calves and yearlings have a higher Cryptosporidium infection rate (49.3 and 31.7%, respectively) than adults (17.4%). Thus far, in addition to the four common Cryptosporidium species (C. parvum, C. andersoni, C. bovis, and C. ryanae), C. hominis, C. felis, C. suis, and C. scrofarum have also been identified in a small number of cattle (Wang et al., 2011a). In the present study, five Cryptosporidium species (C. bovis, C. ryanae, C. andersoni, C. ubiquitum, C. xiaoi) and a novel genotype were identified in yaks. The most abundant species were C. bovis (57.1%) and C. ryanae (33.7%). C. bovis was also identified as the most common species in yaks examined in the same area in a recent study, although the latter identified a significantly lower occurrence of C. ryanae (Mi et al., 2013). In many recent studies, a dominance of C. bovis or C. ryanae in cattle reared under a less intensive traditional management system such as beef and native cattle have mostly identified a low occurrence of C. parvum in calves (Ayinmode et al., 2010; Fayer et al., 2010a; Feltus et al., 2008; Feng et al., 2012; Geurden et al., 2006; Maikai et al., 2011; Murakoshi et al., 2012; Nguyen et al., 2012; Rieux et al., 2013). The lack of C. parvum in yaks examined in the current study may also be attributed to the inclusion of animals over 3 months of age in the weaned calve group (Table 2). However, Mi et al. (2013) did find C. parvum in some post-weaned yaks. Unlike the recent study of Cryptosporidium spp. in yaks (Mi et al., 2013), we have also identified the occurrence of C. andersoni, C. ubiquitum, C. xiaoi and a novel genotype in yaks in the present study. To our knowledge, this is the first

Table 2 Occurrence of Cryptosporidium infection in yaks in Qinghai Province, China by age group. Age (year)

Weaned calves (<1) Yearlings (1–2) Adults (>2)

Sample size

75 120 132

Cryptosporidium positive (%)

37 (49.3) 38 (31.7) 23 (17.4)

No. positive/No. genotyped (%)

C. bovis

C. ryanae

C. andersoni

C. bovis + C. ryanae

C. ubiquitum

C. xiaoi

New genotype

23/37 (62.2) 25/38 (65.8) 8/23 (34.8)

13/37 (35.1) 9/38 (23.7) 11/23 (47.8)

0 1/38 (2.6) 1/23 (4.3)

1/37 (2.7) 2/38 (5.3) 0

0 0 1/23 (4.3)

0 1/38 (2.6) 0

0 0 2/23 (8.7)

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Fig. 1. Phylogenetic relationship between the novel Cryptosporidium genotype in yaks and other common Cryptosporidium species or genotypes, inferred by neighbor-joining (NJ) analysis of the partial (∼830 bp) 18S rRNA gene. There were 762 positions in the final dataset. A sequence from Selenidium terebellae (AF196709) was used as the outgroup. Bootstrap values greater than 50% from 1000 replicates are shown. The novel Cryptosporidium genotype in this study is indicated by “䊉”.

report of C. ubiquitum, C. xiaoi and the novel genotype in bovine animals. Because C. ubiquitum and C. xiaoi are common Cryptosporidium species in sheep, which share pasture with yaks in the study area, these two species could be of sheep origin. We have temporarily named the novel Cryptosporidium genotype in yaks as the Cryptosporidium yak

genotype. However, wild ruminants could be the source of infections with the novel Cryptosporidium genotype. Only two cases of C. andersoni were detected in this study, a yearling yak and an adult yak. In contrast, a dominance of C. andersoni has been observed in dairy cattle in several locations in China (Liu et al., 2009; Wang

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Table 3 Infection rates of Cryptosporidium infection in yaks in China in published studies. Location

Method

Qinghai Qinghai Qinghai Qinghai Qinghai, Shanghai Zoo Qinghai Qinghai Qinghai Qinghai

Microscopy Microscopy Microscopy Microscopy Microscopy ELISA PCR Microscopy, PCR Microscopy, PCR

Total no. of specimens

No. of positive specimens

No. of specimen genotyped

80 151 85 190 402

26 (32.5) 60 (39.7) 26 (30.6) 32 (16.8) 42 (10.4)

0 0 0 0 0

1094 1 17 586

368 (33.6) 1 (100) 3 (17.6) 142 (24.2)

0 1 1 55

et al., 2011b; Zhao et al., 2013), indicating the distribution of Cryptosporidium species could be different between dairy cattle and yaks in China. In Egypt, a similar difference in the distribution of Cryptosporidium species has also been observed between dairy cattle and water buffalos reared under different management systems (Amer et al., 2013a,b). Although the major zoonotic species C. parvum was not detected in yaks in this study, Mi et al. (2013) found C. parvum in some yaks in the same study area. In addition, a few cases of C. andersoni infections were reported in humans (Leoni et al., 2006; Morse et al., 2007). A more recent study showed a dairy farmer was infected with C. bovis (Khan et al., 2010). C. ubiquitum has been found in many human cases in the United Kingdom, Slovenia, United States, Canada, and New Zealand (Fayer et al., 2010b). Therefore, Cryptosporidium spp. from yaks may pose a potential threat to human health. In conclusion, there is a frequent occurrence of Cryptosporidium in yaks in Qinghai Province of China. Although the common Cryptosporidium species in these animals are also common in cattle, the dominant species for cryptosporidiosis could be different between in yaks in Qinghai and dairy cattle in other areas of China. More thorough studies are needed before we can have a better picture on the distribution and clinical significance of Cryptosporidium species in bovine animals in China.

Acknowledgments This work was supported by National Natural Science Foundation of China (31110103901 and 31229005), National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204), and Fundamental Research Funds for the Central Universities, China (No. WB1214073). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

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