Genetic Diversity of Cryptosporidium parvum in Neonatal Dairy Calves in Xinjiang, China

Cryptosporidium parvum has been identified as a major cause of diarrhea and diarrhea-associated deaths in young children and neonatal calves. Infections can remain asymptomatic but may lead to malnutrition and persistent growth retardation. To assess the relationship between C. parvum genetic diversity and pathogenicity in neonatal dairy calves and determine the cause of diarrhea among these calves, 232 fecal samples from neonatal dairy calves on 12 farms in Xinjiang, China, were characterized for Cryptosporidium presence based on the small subunit rRNA gene. The Cryptosporidium prevalence was 38.4% (89/232), and three species were detected with restriction fragment length polymorphism analysis, including C. parvum (the significantly dominant species), C. ryanae, and C. bovis. Cryptosporidium prevalence was significantly higher in neonatal dairy calves with diarrhea (52.6%, 51/97) than in calves without diarrhea (28.1%, 38/135). All C. parvum-positive samples were analyzed based on the 60 KDa glycoprotein gene, and IIdA15G1, IIdA20G1, IIdA14G1, and IIdA19G1 were successfully subtyped. These data indicate that C. parvum may be a major contributor to diarrheal disease in neonatal dairy calves, and C. parvum subtypes from neonatal dairy calves in Xinjiang exhibited high genetic diversity.


Introduction
Cryptosporidium spp. are common causative pathogens of gastroenteritis in humans and animals and is second only to the rotavirus as the causative pathogens of moderate-to-severe diarrhea in children aged younger than 2 years in the developing world [1,2]. Currently, at least 42 valid Cryptosporidium species have been recognized, and C. homisis and C. parvum cause most of the infections in humans [3,4]. Many studies have focused on cattle, with preweaned dairy calves being considered the most important reservoir for zoonotic infection [1,5]. Among common infections with C. parvum, C. bovis, C. andersoni, and C. ryanae in cattle, C. parvum has been associated with clinical disease in neonatal dairy calves, and C. parvum pathogenicity may be a consequence of host-microbe interactions from a long-term evolutionary perspective [6,7]. Moreover, C. xiaoi, C. meleagridis, C. hominis, and C. tyzzeri have been identified in dairy cattle in China. The overall infection rate of Cryptosporidium spp. in dairy cattle in China is 14.0%, with C. parvum and C. bovis being the predominant species [5].
Nearly 20 C. parvum subtype families have been identified via phylogenetic analysis of the 60-KDa glycoprotein (gp60, also known as gp40/15) gene [4,8]. Compared with other countries, accumulating Pathogens 2020, 9, 692 2 of 9 evidence shows a uniqueness of C. parvum subtype distributions in preweaned dairy calves in China. Early work has pointed to C. parvum IId subtypes being predominant in dairy calves in China, whereas C. parvum IIa subtypes were predominant in preweaned dairy calves in Europe, North America, and Australia [5,8]. One study demonstrated that C. parvum IId subtypes likely dispersed from Western Asia to other geographical regions through cattle introduction [9]. At least six C. parvum IId subtypes (IIdA14G1, IIdA15G1, IIdA17G1, IIdA19G1, IIdA20G1, and IIdA20G1) have been detected in dairy cattle in China [5]. Several studies have revealed that C. parvum subtype IIdA19G1 is the most commonly detected subtype in dairy cattle in eastern and central China. Additionally, cryptosporidiosis outbreaks were reported on two dairy farms in the Ningxia Hui Autonomous Region (hereinafter referred to as Ningxia) and Jiangsu Province, which were caused by C. parvum subtypes IIdA15G1 and IIdA19G1, respectively [7,10].
Located in northwest China (73 • 40 E-96 • 18 E, 34 • 25 N-48 • 10 N), Xinjiang Uygur Autonomous Region (hereinafter referred to as Xinjiang) is the largest Chinese administrative division, spanning over 1.6 million km 2 (0.64 million square miles). Historically, Xinjiang was a necessary part of the cattle trading route between Central Asia and China, and it presented a unique geographical advantage in the processes of cattle domestication and breeding. Holstein dairy cattle have been intensively farmed on a large scale in Xinjiang for at least 30 years. Unlike other provinces and municipalities in China, C. parvum subtypes IIdA14G1 and IIdA15G1 have been detected in dairy calves in Xinjiang [11]. To further address the knowledge gap regarding C. parvum genetic diversity and pathogenicity in this region, this study was conducted to examine the occurrence of Cryptosporidium spp. and determine the relationship between C. parvum subtypes and diarrhea in neonatal dairy calves and to assess the zoonotic transmission risk of C. parvum.
Four species of Cryptosporidium are commonly found in cattle: C. parvum, C. bovis, C. ryanae and C. andersoni, and more than 90% of the infection cases in preweaned dairy calves are attributed to C. parvum, which is reported to be a major cause of calf enteritis [4]. In China, severe diarrhea was observed in preweaned calves on a dairy farm in Ningxia in 2013, and C. parvum was the major cause of the outbreak [7]. Severe diarrhea was also reported in neonatal dairy calves on a large dairy farm in Jiangsu Province in 2016, and approximately 360 dairy calves died from watery diarrhea despite antibiotic therapy [10]. Additionally, in a longitudinal study in the USA, a group of dairy calves (n = 30), from birth to 24 months, exhibited the highest Cryptosporidium infection prevalence at 2 weeks of age, and C. parvum constituted 97% of the infections in the preweaned calves [25]. The Cryptosporidium (mainly C. parvum) infection rate was significantly higher in neonatal dairy calves with diarrhea than in calves without diarrhea in this study, further suggesting that C. parvum is associated with clinical disease in neonatal dairy calves. Generally, C. bovis and C. ryanae usually infect postweaned calves and yearlings, with no associated clinical disease [5,6]; however, the C. ryanae infection rate in preweaned dairy calves with diarrhea was significantly higher than in those without diarrhea on a farm that had a cryptosporidiosis outbreak in Jiangsu Province [10]. Therefore, more investigations are needed to clarify the C. ryanae pathogenicity for calves.
To date, seven C. parvum IId subtypes have been detected from cattle in China (Table 3), with subtypes IIdA14G1, IIdA15G1, IIdA19G1 and IIdA20G1 being zoonotic. IIdA15G1 was mostly found in Ningxia, Xinjiang, Heilongjiang Province, Sichuan Province, Beijing Municipality, and Gansu Province [7,11,13,[18][19][20]24]. Subtype IIdA19G1 was mostly found in Jiangsu Province, Henan Province, Shanghai Municipality, Xinjiang, Guangdong Province, Hebei Province, Tianjin Municipality, Beijing Pathogens 2020, 9, 692 5 of 9 Municipality, and Heilongjiang Province [10,12,15,16,[22][23][24]. Subtype IIdA20G1 was only found in Heilongjiang Province [13]. IIdA14G1, IIdA17G1, IIdA18G1, and IIdA21G1 were detected only in Xinjiang [11], Beijing Municipality [24], Tibet Autonomous Region [26], and Shandong Province (data unpublished), respectively.  (24), Sichuan (7), Gansu (1), Beijing (1) Shandong (1) 2 [7,11,13,[18][19][20]24] Neonatal dairy calf infections with different C. parvum subtypes can lead to diarrhea in calves in various countries and regions. Four subtypes (IIdA14G1, IIdA15G1, IIdA19G1, and IIdA20G1) found in our study can also cause diarrhea, which is consistent with previous data. Differences of virulence, varying with Cryptosporidium subtypes, is also no surprise as the evolution of the pathogen itself can be viewed as an emergent process [27]. In relatively separate geographic systems, such as Xinjiang, microbial pathogenesis over time can become increasingly unpredictable as a consequence of the host, microbes, and their interaction [28]. Data indicate that the C. parvum subtypes in dairy cattle in Xinjiang exhibit high genetic diversity and are more heterogeneous than in other research areas in China. We speculate that more subtypes may be detected in Xinjiang; therefore, more systematic epidemiological studies focusing on other animal species are needed to further clarify the genetic diversity and zoonotic transmission risk of Cryptosporidium spp. in this geographic region.

Ethics Approval
The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Henan Agricultural University (approval no. LVRIAEC 2017-019) and Tarim University (approval no. ECTU 2017-0013). All farm managers provided appropriate permission prior to fecal sample collection, and no specific permits were required for the described field studies. All field studies complied with local legislative guidelines for animal care and use, and no neonatal dairy calves were injured during the fecal sample collection.

Study Area and Sample Collection
From April 2017 to April 2018, 232 fresh fecal samples (weighing approximately 20-40 g each) were randomly collected from 12 dairy cattle farms from the cities of Wushi, Alaer, Wensu, Shihezi, Hutubi, Tiemenguan, and Kuitun in Xinjiang, northwest China (Figure 1). The farms were of similar scale, consisting of 1400-1700 dairy cattle. The collected samples accounted for 50% of the number of neonatal dairy calves per farm. All calves were 7-28 days old and separately housed in calf hutches or raised in neonatal dairy calf cowsheds. All fecal samples were collected directly from the rectum of each calf with a sterile disposable latex glove. Diarrheal status was observed according to clinical symptoms, and neonatal dairy calves were divided into two groups: those with diarrhea and those without diarrhea. The samples were transported to the laboratory and stored at 4 °C for use in subsequent molecular analyses.

DNA Extraction and PCR Amplification
Genomic DNA was extracted from approximately 200 mg per sample using the E.Z.N.A ® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA). The DNA was stored at −20 °C prior to use in PCR amplification. All calves were 7-28 days old and separately housed in calf hutches or raised in neonatal dairy calf cowsheds. All fecal samples were collected directly from the rectum of each calf with a sterile disposable latex glove. Diarrheal status was observed according to clinical symptoms, and neonatal dairy calves were divided into two groups: those with diarrhea and those without diarrhea. The samples were transported to the laboratory and stored at 4 • C for use in subsequent molecular analyses.

DNA Extraction and PCR Amplification
Genomic DNA was extracted from approximately 200 mg per sample using the E.Z.N.A ® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA). The DNA was stored at −20 • C prior to use in PCR amplification.
The small subunit rRNA (SSU rRNA) gene was used to identify the Cryptosporidium species using previously described primers and conditions [29]. All SSU rRNA PCR products were further analyzed via RFLP method using the SspI and MboII restriction enzymes (TaKaRa Shuzo Co. Ltd., Otsu, Japan) [30]. C. parvum-positive samples, confirmed by DNA sequencing of the PCR products, were subtyped based on the sequence analysis of the 60-kDa glycoprotein gene (gp60) [31]. To improve the specificity and sensitivity of the PCR assays, two replicates were used per sample, and a positive sample in two replicates was considered infected. Positive (chicken-derived C. bailey DNA) and negative (containing no template DNA) controls were set during the amplification. The secondary PCR products were examined via electrophoresis using 1.5% agarose gel and staining with GelRed (Biotium Inc., Hayward, CA, USA).

Sequencing and Data Analysis
All positive PCR products based on SSU rRNA and gp60 genes were 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). Sequence accuracy was confirmed via two-directional sequencing, and by sequencing additional PCR products when necessary. The raw sequences obtained were edited with DNAstar Lasergene Editseq version 7.1.0 (http://www.dnastar.com/) and aligned Pathogens 2020, 9, 692 7 of 9 with reference sequences downloaded from GenBank (http://www.ncbi.nlm.nih.gov/) using Clustal X version 2.1 (http://www.clustal.org/).

Nucleotide Sequence Accession Numbers
Representative C. parvum subtype nucleotide sequences identified in the neonatal dairy calves were submitted to GenBank at the National Center for Biotechnology Information under accession numbers MT680896-MT680900.

Statistical Analysis
SPSS ver. 22.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Cryptosporidium infection rates were calculated and compared using the chi-square test. Differences were considered significant at p < 0.05.

Conclusions
Four C. parvum IId subtypes (IIdA14G1, IIdA15G1, IIdA19G1, and IIdA20G1) were detected, and the data indicate that C. parvum subtypes from neonatal dairy calves in Xinjiang have high genetic diversity. The statistically significant differences in Cryptosporidium prevalence between neonatal dairy calves with and without diarrhea indicate that C. parvum may be a major contributor to diarrheal disease in neonatal dairy calves. Our findings suggest that neonatal dairy calves may be a potential source of human Cryptosporidium infection and further evidence the uniqueness of C. parvum IId subtypes in dairy cattle in China.