Seasonal monitoring of Cryptosporidium species and genetic diversity in neonatal dairy calves on two large-scale farms in Xinjiang, China

Background: Neonatal dairy calves infected with Cryptosporidium can possess a signicant source of zoonotic infections and disease. To assess seasonal variations in the prevalence and genetic diversity of Cryptosporidium in neonatal dairy calves, 380 fecal samples from neonatal dairy calves on two large-scale farms in Xinjiang (Alaer and Wensu) were screened for the Cryptosporidium small subunit (SSU) rRNA gene. Results: The overall prevalence of Cryptosporidium was 48.7% (185/380): 48.6% (108/222) in Alaer and 48.7% (77/158) in Wensu. Cryptosporidium was most frequent in summer (56.8%, 54/95), followed by spring (50.0%, 44/88), winter (46.8%, 44/94), and autumn (41.7%, 43/103) (P > 0.05). Cryptosporidium was signicantly more prevalent in calves with diarrhea (72.4%, 113/156) than in those without (32.1%, 72/224) (P < 0.01). Based on a restriction fragment length polymorphism (RFLP) analysis, C. parvum (n = 173), C. bovis (n = 7), C. ryanae (n = 3), and co-infections of the three species (n = 2) were identied. Most (172/175) C. parvum samples were successfully sequenced at the 60-kDa glycoprotein gene (gp60), revealing two zoonotic subtypes: IIdA14G1 (n = 94) and IIdA15G1 (n = 7) in Alaer and IIdA15G1 (n = 71) in Wensu. Conclusions: These results showed that neonatal dairy calves were commonly infected with Cryptosporidium throughout the year, and there was a signicant association between the occurrence of diarrhea and Cryptosporidium infection. Presence of IIdA14G1 and IIdA15G1 indicated neonatal dairy calves may be a source of zoonotic C. parvum subtypes.

Autonomous Region (referred to as Ningxia hereafter) and Jiangsu Province, caused by C. parvum subtypes IIdA15G1 and IIdA19G1, respectively [10,11]. The IId subtypes have been detected in humans and various animal species, indicating that neonatal calves may be a reservoir for the transmission of human Cryptosporidium infections [12].
A previous study suggested that the C. parvum IId subtypes were probably dispersed from Western Asia to other geographic regions with the introduction of cattle [13]. As the largest Chinese administrative division in China, Xinjiang Uygur Autonomous region (referred to as Xinjiang hereafter) is the necessary route for cattle trading and domestication between Central Asia and China, and has an increasing number of large-scale dairy farms. However, the seasonal prevalence of C. parvum infections in neonatal dairy calves in China is poorly understood, with pre-weaned calves only analyzed in Henan and Guangdong [14,15]. To address the knowledge gap in the seasonal variations in C. parvum prevalence, the incidence of associated diarrhea, and the genetic subtypes present in neonatal dairy calves in Xinjiang, we conducted a series of experiments to characterize the seasonal features of Cryptosporidium infection in neonatal dairy calves. The data generated extend our understanding of the causes of diarrhea in calves and the possible role of cattle in the transmission of Cryptosporidium to humans.

Study area and sample collection
Between September 2017 and September 2018, a total of 380 fresh fecal samples were collected from female neonatal dairy calves (mainly 7-20 days old) on two large-scale dairy farms in Alaer and Wensu in the Aksu area (N 41°09', E 80°19') of Xinjiang. According to the number of calves born per month, 50%-80% of the neonatal dairy calves were randomly sampled once a month for a year, and four seasons were divided according to the northern hemisphere. In Alaer, the calves were raised separately in a calf hutch with straw bedding that was not changed often, whereas in Wensu, the calves were raised separately in a calf hutch for a week, and then mixed breeding in an open outdoor stall. Fecal samples (approximately 30-50 g each) were collected directly from the rectum of each calf with disposable gloves. The fresh feces were placed into clean plastic bags marked with the date, calf age, and farm. All the samples were immediately transported to the laboratory and stored at 4 °C before genomic DNA extraction.

DNA extraction and PCR ampli cation
The samples were concentrated by centrifugation at 1,500 × g for 10 min, and the genomic DNA was extracted from approximately 200 mg of each sample with the E.Z.N.A. ® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA), according to the manufacturer's instructions. The extracted DNA samples were then frozen at −20 °C before their molecular analysis for Cryptosporidium.
To characterize Cryptosporidium, a nested PCR assay was performed based on the small subunit rRNA (SSU rRNA) gene with previously described primers and reaction conditions [16]. The Cryptosporidium isolates were further identi ed with the restriction fragment length polymorphism (RFLP) method using the SspI and MboII restriction enzymes (TaKaRa Shuzo Co. Ltd., Otsu, Japan) [17]. To determine the C. parvum subtypes, all C. parvum-positive samples were characterized to the subtype level based on a sequence analysis of an approximately 850-bp fragment of the 60-kD glycoprotein gene (gp60) [18]. To ensure the accuracy of ampli cation, positive controls (chicken-derived C. bailey DNA) and negative controls (containing no template DNA) were included in the PCR analysis of each sample.
All secondary PCR products were stained with GelRed (Biotium Inc., Hayward, CA, USA), separated with 1% agarose gel electrophoresis and visualized on a UV transilluminator. All secondary PCR ampli cation products of the expected size (∼840 bp) were then bidirectionally sequenced on an ABI PRISM™ 3730 XL DNA Analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

Molecular analysis
To identify the species of Cryptosporidium and the subtypes of C. parvum, the raw DNA sequences were edited with DNAStar Lasergene EditSeq version 7.1.0 (http://www.dnastar.com/) and aligned with reference sequences available from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) using ClustalX version 2.1 (http://www.clustal.org/). Statistical analysis SPSS ver. 22.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. The infection rates and their 95% con dence intervals (CIs) were calculated with the Wald method. Differences in infection rates among seasons or symptoms were evaluated with the Chi-square test, and differences were considered signi cant at P < 0.05.

Results
Prevalence of Cryptosporidium spp.
In Alaer, the proportions of Cryptosporidium species identi ed in the four seasons were C. parvum 84.6% (22/26)
In general, Cryptosporidium was signi cantly associated with calves younger than 2 weeks with diarrhea [29]. A previous study of a diarrhea outbreak among neonatal dairy calves in Jiangsu suggested that the prevalence of Cryptosporidium was signi cantly higher during the outbreak (80.3%) than after the outbreak (22.7%), and that the prevalence of Cryptosporidium in dairy calves less than 3 weeks old was much higher than in any other age group [11]. In a similar cryptosporidiosis outbreak in Ningxia, the highest prevalence of Cryptosporidium (83.3%, 40/48) was seen in 2-3-week-old calves with severe diarrhea [10]. In the present study, the neonatal dairy calves ranged in age from 1 week to 3 weeks, and this may have contributed to the higher prevalence of Cryptosporidium than has been seen in other studies in China. Another factor that may have in uenced the prevalence of Cryptosporidium in this study is that the neonatal dairy calves were sampled continuously for a year, whereas dairy calves were sampled only once in most of the other studies.
Data on the seasonal variations in the infection rates of Cryptosporidium in pre-weaned dairy calves in Henan [14], Guangdong (without autumn) [15], and Western Australia [30] suggested that there is no signi cant seasonal difference in Cryptosporidium infections in pre-weaned dairy calves. The data in the present study were consistent with those studies. A previous study showed that the highest prevalence of Cryptosporidium in dairy calves was observed in summer in Henan, China (50.0%) [14], which was similar to our result, which showed a peak in occurrence in summer (56.8%, 54/95). In contrast, in New York State, dairy cattle were signi cantly more likely to be infected by Cryptosporidium in winter than in summer [31]. Surprisingly, the farm in Alaer displayed its highest prevalence in December (71.4%) and lowest in late January (23.8%). Further research is required to clarify the cause. Whether C. parvum or C. bovis is the dominant Cryptosporidium species in pre-weaned dairy calves is controversial among publications that document Cryptosporidium prevalence among cattle in China. Cryptosporidium bovis was more common in central China (Henan and Hubei) [14,25], eastern China (Shanghai) [27], southwestern China (Sichuan) [26], and southern China (Guangdong) [15,28], whereas C. parvum is more common in northeastern China (Heilongjiang) [32], northern China (Beijing, Hebei, and Tianjin) [20,33], and most of northwestern China (Ningxia and Xinjiang) [10,22,24]. Dairy calves are most commonly infected with C. parvum within 1 month of birth, whereas C. bovis is mainly associated with dairy calves aged 2-3 months [6]. The differences in the distributions of Cryptosporidium species in preweaned dairy calves in China can be attributed to the ages of the calves sampled. In most studies, samples were collected from cattle < 3 months old, but the numbers of pre-weaned dairy calves sampled that were < 1 month are unclear.
Data on the seasonal distributions of Cryptosporidium species in pre-weaned dairy calves in Henan suggested that they varied as C. parvum was predominant in summer and C. bovis was predominant in autumn and winter [14]. However, the seasonal shift in pre-weaned dairy calves in New York showed that C. bovis was most common in summer and C. parvum was dominant in spring and winter [34]. However, in Guangdong, C. parvum was dominant in pre-weaned dairy calves in three seasons (not autumn) [15]. Our ndings indicate that C. parvum was clearly predominant in every season, whereas C. bovis and C. ryanae were found only occasionally. This is similar to two studies of cryptosporidiosis outbreaks in Ningxia and Jiangsu, in which the occurrence of C. parvum was signi cantly higher than that of C. bovis or C. ryanae [10,11]. Subtyping C. parvum revealed two zoonotic subtypes, IIdA14G1 and IIdA15G1. Among the six IId family subtypes detected in dairy cattle in China (IIdA14G1, IIdA15G1, IIdA17G1, IIdA20G1, and IIdA21G1), IIdA14G1 has only been seen in Xinjiang [24]. IIdA15G1 was rst detected in Ningxia and then in Xinjiang [24], Heilongjiang [32], Sichuan [26], and Beijing [33]. There have been several reports of human infections with IIdA14G1 and IIdA15G1 [35,36]. The presence of IIdA14G1 and IIdA15G1 indicates the genetic diversity of C. parvum in neonatal dairy calves in Xinjiang, and the data also suggest that the IId family of C. parvum subtypes derived from cattle is distributed uniquely in China. Therefore, neonatal dairy cattle infected with C. parvum may be a signi cant source of zoonotic infections and disease.

Conclusion
In this study, neonatal dairy calves on two farms in Xinjiang were commonly infected with Cryptosporidium throughout the year. The prevalence of Cryptosporidium was signi cantly higher in calves with diarrhea than in those without diarrhea. C. parvum was clearly predominant in every season. The detection of zoonotic subtypes IIdA14G1 and IIdA15G1 suggests that dairy cattle are a crucial reservoir in the transmission human Cryptosporidium infections. These results extend our understanding of the epidemiology of cryptosporidiosis and the causes of diarrhea in dairy calves.

Abbreviations
SSU rRNA, small subunit rRNA; gp60, the 60 kDa glycoprotein gene; RFLP, restriction fragment length polymorphism; CI, con dence intervals Declarations Ethics approval and consent to participate The research protocol was reviewed and approved by the Research Ethics Committee of Henan Agricultural University (Approval No: LVRIAEC 2017-019). The eld studies did not involve endangered or protected species. Consent to participate was obtained from all the participating farm owners before sample collection and no speci c permits were required for the eld studies described. No neonatal dairy calves were injured and all efforts were made to minimize pain and suffering during the collection of fecal samples.

Consent for publication
Not applicable.

Availability of data and materials
All data used or analyzed during this study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Tables Table 1. Seasonal characterization of Cryptosporidium in neonatal dairy calves on two farms in Xinjiang, China