Genetic diversity of Cryptosporidium parvum in neonatal calves in Xinjiang, China CURRENT STATUS:

Background The Cryptosporidium causes infection in wide spectrum of vertebrate hosts and is well known for leading to diarrhea and other gastrointestinal illness. To assess Cryptosporidium genetic diversity in neonatal calves and probe the cause for diarrhea of them, a total of 232 fecal samples from neonatal calves in 12 farms in Xinjiang were characterized for the presence of Cryptosporidium . The prevalence of Cryptosporidium was 38.4% (89/232), and three species were detected with SSU rRNA gene, including C. parvum (n = 88), C. ryanae (n = 9), and C. bovis (n = 1). Prevalence of C . parvum neonatal calves with diarrhea (52.6%, 51/97) was significantly higher than calves without diarrhea (28.1%, 38/135). All the C . parvum -positive samples were analyzed based on gp60 gene, IIdA15G1 (n = 35), IIdA20G1 (n = 21), IIdA14G1 (n = 17), and IIdA19G1 (n = 13) were successfully subtyped in this study. These data indicated that C . parvum was a major contributor in diarrheal disease in neonatal calves, and C . parvum subtypes from neonatal calves in Xinjiang were high genetic diversity. Additionally, our findings implicating neonatal calves could be a potential source of human Cryptosporidium infection and provide further evidence for the uniqueness of C . parvum IId subtypes in cattle in China.


Abstract Background
The Cryptosporidium causes infection in wide spectrum of vertebrate hosts and is well known for leading to diarrhea and other gastrointestinal illness. To assess Cryptosporidium genetic diversity in neonatal calves and probe the cause for diarrhea of them, a total of 232 fecal samples from neonatal calves in 12 farms in Xinjiang were characterized for the presence of Cryptosporidium .

Conclusions
These data indicated that C . parvum was a major contributor in diarrheal disease in neonatal calves, and C . parvum subtypes from neonatal calves in Xinjiang were high genetic diversity. Additionally, our findings implicating neonatal calves could be a potential source of human Cryptosporidium infection and provide further evidence for the uniqueness of C . parvum IId subtypes in cattle in China.

Background
Cryptosporidium is common causative pathogen caused gastroenteritis in humans and animals, especially responsible for significant morbidity and mortality in children, immuno-compromised individuals, and HIV patients [1]. Cryptosporidium is second only to Rotavirus as the cause pathogens of moderate-to-severe diarrhea in children younger than 2 years in the developing world [2].
Currently, 38 valid Cryptosporidium species and over 40 Cryptosporidium genotypes have been discovered, the most infections in human were Cryptosporidium hominis and C. parvum [3,4]. Many studies have been drawn attention to cattle, with pre-weaned calves were considered the most important reservoir for zoonotic infection [5]. Among of C. parvum, C. bovis, C. andersoni, and C. ryanae were common infection in cattle, only C. parvum being associated with clinical disease in neonatal calves [6].
To date, based on the 60 KDa glykoprotein (gp60, also known as gp40/15) gene, nearly twenty C. parvum subtype families have been identified by phylogenetic analysis [4,7]. Compared with other countries, evidence is accumulating for the uniqueness of C. parvum subtypes distribution in China, especially in pre-weaned dairy calves. Early work pointed to IId subtypes is dominant in dairy calves in China, while pre-weaned dairy calves are most commonly infected with IIa subtypes in Europe, North America, and Australia [5,7]. It was demonstrated by the study that C. parvum IId subtypes likely dispersed from Western Asia to other geographical regions by cattle introduction [8].
Previous study showed that C. parvum subtypes IIdA14G1 and IIdA15G1 in dairy calves in the Xinjiang Uyghur Autonomous Region (referred to as Xinjiang hereafter) were zoonotic subtypes [9]. In history, Xinjiang plays as a necessary part of trading route for cattle between Central Asia and China. To address the knowledge gap for C. parvum genetic diversity and pathogenicity in the region, this study was conducted to examine the occurrence of Cryptosporidium and determine C. parvum subtypes in neonatal calves, and to assess the zoonotic transmission risk of C. parvum.
Nine samples from three farms were detected with mixed infection, one isolate co-infection of C.
parvum and C. ryanae with diarrhea, while seven isolates co-infection of C. parvum and C. ryanae, one isolate co-infection of C. bovis and C. ryanae without diarrhea ( Table 1, Table 2).
parvum is the dominant species in pre-weaned dairy calves from Ningxia [12,18], Xinjiang [9], Beijing [23], Hebei and Tianjin [21], and Heilongjiang [11]. In this study, C. parvum was also the dominant species while C. bovis was only detected in one sample here. In addition, C. ryanae and C. andersoni can be occasionally isolated from pre-weaned dairy calves in China [5]. C. parvum mainly infections dairy calves within one month, while C. bovis and C. ryanae were more commonly detected in dairy calves 2-3 months old [6,24]. So the differences of infection species between different locations may attribute to the age of dairy calves sampled.
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 pre-weaned dairy calves are attributed to C.
parvum, which has been reported as a major cause of calf enteritis [4]. In China, severe diarrhea was observed in pre-weaned calves on a dairy farm in the Ningxia in 2013, and C. parvum was the major cause for the outbreak [12]. In another report, severe diarrhea was observed in neonatal dairy calves on a large dairy farm in Jiangsu Province (East China), and approximately 360 calves died due to watery diarrhea despite antibiotic therapy [25]. Additionally, in a longitudinal study of the USA, a group of calves (n = 30) from birth to 24 months showed that the highest prevalence of Cryptosporidium infection was at 2 weeks of age and C. parvum constituted 97% of infections in preweaned calves [26]. The C. parvum infection rate of neonatal calves with diarrhea was significantly higher than calves without diarrhea in this study, which further suggests that C. parvum was associated with clinical disease in neonatal calves.
Nearly twenty C. parvum subtype families have been identified based on gp60 sequencing analysis [7]. In a series of subtyping studies published, there is a high prevalence of IIa subtype family in both humans and cattle in Europe, North America and Australia [4]. However, IId subtypes were major subtype identified from C. parvum isolates in dairy cattle of China, and the distribution of C. parvum subtype families in dairy cattle seems to be distinct in different areas [5].

Statistical analysis
The infection rates of Cryptosporidium were calculated and compared by the Chi-square test.
Differences were considered significant at P < 0.05.

Ethics approval and consent to participate
The experiment protocol was performed strictly according to the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. In accordance with the field study guidelines of local legislation, written consent to participate was obtained from all participating farm owners in our study. In addition, the research protocol was reviewed and approved by the Research Ethics Committee of Henan Agricultural University (approval number IRC-HENAU-20160225).
No neonatal calves were injured during the fecal samples collection.

Funding
This work was supported in part by the National Natural Science Foundation of China (31702227); the Program for Young and Middle-aged Leading Science, Technology, and Innovation of Xinjiang Production & Construction Group (2018CB034); and the President Foundation of Tarim University (TDZKCX201701). The sponsors had no role in study design, in the collection, analysis, or interpretation of data, in the writing of the report, or in the decision to submit the article for publication.
All authors read and approved the final version of the manuscript.