Introduction
Cryptosporidium is an important protozoan parasite, mainly causing gastrointestinal disease in humans and animals, including livestock, companion animals and wildlife (Fayer, Reference Fayer2010). Among domestic animals, cattle are recognized as the most common mammalian species to be infected by Cryptosporidium, and preweaned calves are considered an important reservoir for zoonotic Cryptosporidium infections (Xiao and Feng, Reference Xiao and Feng2008; Xiao, Reference Xiao2010; Imre et al., Reference Imre, Lobo, Matos, Popescu, Genchi and Darabus2011). Contamination of cattle manure has led to several food-borne and water-borne outbreaks of human cryptosporidiosis (Blackburn et al., Reference Blackburn, Mazurek, Hlavsa, Park and Tillapaw2006; Baldursson and Karanis, Reference Baldursson and Karanis2011). Cryptosporidium infections frequently result in morbidity, weight loss and delayed growth, and sometimes mortality of young animals.
To date, 37 valid Cryptosporidium species and over 70 genotypes have been described (Ryan et al., Reference Ryan, Fayer and Xiao2014; Kváč et al., Reference Kváč, Havrdová, Hlásková, Daňová, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová and McEvoy2016; Zahedi et al., Reference Zahedi, Durmic, Gofton, Kueh, Austen, Lawson, Callahan, Jardine and Ryan2017; Čondlová et al., Reference Čondlová, Horčičková, Sak, Květoňová, Hlásková, Konečný, Stanko, McEvoy and Kváč2018; Kváč et al., Reference Kváč, Vlnatá, Ježková, Horčičková, Konečný, Hlásková, McEvoy and Sak2018). Four of them, namely Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium andersoni and Cryptosporidium ryanae are the most common species that can infect cattle and cause bovine cryptosporidiosis. Occasionally, Cryptosporidium felis, Cryptosporidium hominis, Cryptosporidium suis, Cryptosporidium scrofarum and C. suis-like have also been detected in cattle (Trout and Santín, Reference Trout, Santín, Fayer and Xiao2008). Many studies conducted in industrialized nations demonstrate that the four common species have age-related distributions. Cryptosporidium parvum is mostly found in preweaned calves and is a significant cause of diarrhoea. Cryptosporidium bovis and C. ryanae usually infect postweaned calves and yearlings, although C. bovis is more prevalent than C. ryanae, and neither is associated with diarrhoea (Santín et al., Reference Santín, Trout and Fayer2008). In contrast, C. andersoni is commonly seen in adult cattle and has been associated with gastritis, reduced milk yield and poor weight gain (Esteban and Anderson, Reference Esteban and Anderson1995).
In China, Cryptosporidium infections have been reported in dairy cattle in Xinjiang, Ningxia, Gansu, Shaanxi, Heilongjiang, Henan, Shandong, Hubei and other provinces (Liu et al., Reference Liu, Wang, Li, Zhang, Shu, Zhang, Feng, Xiao and Ling2009; Wang et al., Reference Wang, Ma, Zhao, Lu, Wang, Zhang, Jian, Ning and Xiao2011a, Reference Wang, Wang, Sun, Zhang, Jian, Qi, Ning and Xiao2011b; Zhang et al., Reference Zhang, Wang, Yang, Zhang, Cao, Zhang, Hong, Liu and Shen2013, Reference Zhang, Tan, Zhou, Ni, Liu, Yang and Zhu2015; Zhao et al., Reference Zhao, Ren, Gao, Bian, Hu, Cong, Lin, Wang, Qi, Qi, Zhu and Zhang2013, Reference Zhao, Wang, Zhang, Liu, Cao, Shen, Yang and Zhang2014; Cui et al., Reference Cui, Wang, Huang, Wang, Zhao and Luo2014; Huang et al., Reference Huang, Yue, Qi, Wang, Zhao, Li, Shi, Wang and Zhang2014; Ma et al., Reference Ma, Li, Zhao, Xu, Wu, Wang, Guo, Wang, Feng and Xiao2015; Qi et al., Reference Qi, Wang, Jing, Wang, Wang and Zhang2015a, Reference Qi, Fang, Wang, Zhang, Wang, Du, Guo, Jia, Yao, Liu and Zhao2015b; Fan et al., Reference Fan, Wang, Koehler, Hu and Gasser2017), but no information is available on the prevalence or genotypes of Cryptosporidium infections in dairy cattle in Guangdong Province. In this study, we conducted the first molecular epidemiological survey of dairy cattle in Guangdong Province to identify the infection rates and species distribution of Cryptosporidium.
Materials and methods
Study area and specimen collection
Guangdong Province is located at the southern end of mainland China. The annual average temperature is above 20 °C and rainfall is abundant in this region. In this study, 1440 fecal specimens (approximately 20 g each) were collected from eight large-scale dairy cattle farms and two small dairy cattle farms in the cities of Huizhou, Guangzhou, Shenzhen and Qingyuan in Guangdong Province in April 2016 (Table 1). The collected specimens represented 10–15% of the total cattle on each farm. A fresh fecal specimen was collected from each animal using a sterile disposable latex glove immediately after its defecation onto the ground, and the sample was then placed individually into a disposable plastic bag. The cattle were divided into age groups: preweaned calves (<2 months old), postweaned calves (2–6 months old), heifers (7 months to 2 years old) and adult cattle (>2 years old). All the specimens were stored in 2.5% potassium dichromate at 4 °C before DNA extraction.
Table 1. Infection rates and distribution of Cryptosporidium species in dairy cattle of different collection sites and age groups in Guangdong Province
DNA extraction and PCR amplification
All fecal specimens collected were washed three times with distilled water by centrifugation at 1500 × g for 10 min at room temperature. The genomic DNA was extracted from 200 mg of each specimen using the E.Z.N.A.® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA), according to the manufacturer's instructions. The eluted DNA was stored at −20 °C until PCR analysis.
All DNA samples were screened for Cryptosporidium using nested PCR amplification of an approximate 830-bp fragment of the small subunit rRNA (SSU rRNA) gene, as previously described (Alves et al., Reference Alves, Xiao, Sulaiman, Lal, Matos and Antunes2003). Positive and negative controls were included in each PCR analysis. Then, the samples positive for C. andersoni at the SSU rRNA locus were subtyped by amplifying the four minisatellite/microsatellite targets including MS1 (coding for hypothetical protein), MS2 (coding for 90 kDa heat shock protein), MS3 (coding for hypothetical protein) and MS16 (coding for leucine-rich repeat family protein) according to the previously described nested PCR (polymerase chain reaction) protocols (Feng et al., Reference Feng, Yang, Ryan, Zhang, Kvác, Koudela, Modrý, Li, Fayer and Xiao2011; Zhao et al., Reference Zhao, Ren, Gao, Bian, Hu, Cong, Lin, Wang, Qi, Qi, Zhu and Zhang2013). KOD-Plus amplification enzyme (Toyobo Co., Ltd., Osaka, Japan) was used for PCR amplification. The secondary PCR products were examined by agarose gel electrophoresis and visualized after GelRed™ staining (Biotium Inc., Hayward, CA, USA).
DNA sequence analysis
Positive secondary PCR products were sequenced on an ABI Prism™ 3730 XL DNA Analyzer using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster, CA, USA). Sequence accuracy was confirmed with bi-directional sequencing and by sequencing a new PCR product if necessary. The sequences were identified by alignment with reference sequences downloaded from GenBank (http://www.ncbi.nlm.nih.gov) using the ClustalX2.13. The C. andersoni subtypes were named according to the repeat characteristics of minisatellite repeats in four genetic loci by Feng et al. (Reference Feng, Yang, Ryan, Zhang, Kvác, Koudela, Modrý, Li, Fayer and Xiao2011).
Statistical analysis
The Cryptosporidium infection rates were evaluated using Regression Analysis in Statistic Package for Social Science (SPSS) for Windows with 95% confidence intervals (CI). Probability level (P) of <0.05 were considered as statistically significant. The linkage disequilibrium (LD) across all four loci was assessed with the standardized index of association (I SA) proposed by Haubold and Hudson (Reference Haubold and Hudson2000). LD was tested with LIAN version 3.7 (http://guanine.evolbio.mpg.de/cgi-bin/lian/lian.cgi.pl) using a parametric method for the four microsatellite/minisatellite loci.
Results
Prevalence of Cryptosporidium
Eight of the 10 farms tested were positive for Cryptosporidium, with infection rates of 1.34–27.5% (P < 0.01; Table 1). Among the Cryptosporidium-positive farms, farm HZ-3 had the highest prevalence and farms GZ-4 and QY-1 had no Cryptosporidium infections. The infection rates of the different age groups were 6.4% (95% CI 3.60–9.20) in preweaned calves, 6.19% (95% CI 4.41–8.24) in postweaned calves, 1.48% (95% CI 0.03–2.92) in heifers and 2.06% (95% CI 0.54–3.59) in adult cattle (P < 0.01, Table 1).
Distribution of Cryptosporidium species and subtypes
Sequence analysis of the 18S rRNA gene fragment revealed the presence of three Cryptosporidium species: C. andersoni (n = 33) on seven farms, C. bovis (n = 22) on five farms and C. ryanae (n = 8) on four farms (Table 1). Except for farm SZ-1, C. andersoni was detected on all the Cryptosporidium-positive farms and was the only species found on three farms.
Cryptosporidium andersoni was found in all age groups of dairy cattle, responsible for 52.4% (33/63) of all Cryptosporidium infections in this study. The prevalence of C. andersoni in preweaned calves, postweaned calves, heifers and adult cattle was 1.01% (95% CI 0–2.15), 3.56% (95% CI 1.99–5.14), 1.48% (95% CI 0.03–2.92) and 2.06% (95% CI 0.54–3.59), respectively. In contrast, C. bovis and C. ryanae were only found in dairy calves, with infection rates of 2.65% (95% CI 1.56–3.75) and 0.96% (95% CI 0.30–1.63), respectively (Table 1).
At the four microsatellite/minisatellite loci (MS1, MS2, MS3 and MS16), 24, 25, 24 and 25 DNA preparations were sequenced successfully, respectively, and three, two, two and one haplotypes were identified, respectively. In total, 24 of 33 C. andersoni isolates were successfully subtyped at all four loci, and three C. andersoni MLST subtypes were identified. Of these, MLST subtype A4,A4,A4,A1 was most prevalent (87.5%, 21/24) in the dairy cattle, appearing on all C. andersoni-positive farms, demonstrating an extensive distribution in the investigated area. However, the other two MLST subtypes, A2,A5,A2,A1 (n = 1) and A1,A4,A4,A1 (n = 2), were only detected on farms GZ-2 and GZ-3, respectively. MLST subtype A4,A4,A4,A1 was predominant in all age groups of dairy cattle and was the only subtype detected in dairy calves. MLST subtypes A2,A5,A2,A1 and A1,A4,A4,A1 were only found in heifers and adult cattle, respectively (Table 2).
Table 2. The distribution of MLST subtypes of Cryptosporidium andersoni in dairy cattle of different collection sites and age groups in Guangdong Province
a Subtyped no. indicates the number of C. andersoni isolates subtyped successfully at all the four loci (MS1, MS2, MS3 and MS16) with PCR; amplified no. indicates the number of C. andersoni isolates analysed with PCR at all the four loci (MS1, MS2, MS3 and MS16).
b Haplotypes are arranged in the order of the gene loci amplified: MS1, MS2, MS3 and MS16.
LD analysis of C. andersoni
The 24 C. andersoni isolates successfully subtyped at all the four loci were included in the LD analysis. The standardized index of association (I SA = 0.4109) was >0 and the pairwise variance (V D = 0.7431) was greater than the 95% confidence limit (L = 0.4527), indicating the presence of LD and the clonal population structure of C. andersoni in Guangdong Province.
Discussion
The overall Cryptosporidium prevalence in this study was 4.38%, which is lower than most rates reported in dairy cattle in Heilongjiang (17.33%, 257/1483), Anhui (14.9%, 52/350), Shanghai (12.5%, 55/440), Jiangsu (20.7%, 251/1215), Henan (13.0%, 276/2116), Xinjiang (16.0%, 82/514) and Shaanxi (5.24%, 122/2329) (Liu et al., Reference Liu, Wang, Li, Zhang, Shu, Zhang, Feng, Xiao and Ling2009; Wang et al., Reference Wang, Ma, Zhao, Lu, Wang, Zhang, Jian, Ning and Xiao2011a, Reference Wang, Wang, Sun, Zhang, Jian, Qi, Ning and Xiao2011b; Chen and Huang, Reference Chen and Huang2012; Zhang et al., Reference Zhang, Wang, Yang, Zhang, Cao, Zhang, Hong, Liu and Shen2013; Zhao et al., Reference Zhao, Wang, Zhang, Liu, Cao, Shen, Yang and Zhang2014; Qi et al., Reference Qi, Wang, Jing, Wang, Wang and Zhang2015a, Reference Qi, Fang, Wang, Zhang, Wang, Du, Guo, Jia, Yao, Liu and Zhao2015b; Zhang et al., Reference Zhang, Tan, Zhou, Ni, Liu, Yang and Zhu2015), but higher than that in Ningxia (3.76%, 115/3054) (Zhao et al., Reference Zhao, Ren, Gao, Bian, Hu, Cong, Lin, Wang, Qi, Qi, Zhu and Zhang2013; Huang et al., Reference Huang, Yue, Qi, Wang, Zhao, Li, Shi, Wang and Zhang2014; Cui et al., Reference Cui, Wang, Huang, Wang, Zhao and Luo2014; Qi et al., Reference Qi, Fang, Wang, Zhang, Wang, Du, Guo, Jia, Yao, Liu and Zhao2015b). The infection rate of 6.4% in preweaned calves was lower than all previously reported studies, in which the prevalence was 10.22–47.68% (Wang et al., Reference Wang, Wang, Sun, Zhang, Jian, Qi, Ning and Xiao2011b; Zhang et al., Reference Zhang, Wang, Yang, Zhang, Cao, Zhang, Hong, Liu and Shen2013; Huang et al., Reference Huang, Yue, Qi, Wang, Zhao, Li, Shi, Wang and Zhang2014; Qi et al., Reference Qi, Wang, Jing, Wang, Wang and Zhang2015a, Reference Qi, Fang, Wang, Zhang, Wang, Du, Guo, Jia, Yao, Liu and Zhao2015b; Fan et al., Reference Fan, Wang, Koehler, Hu and Gasser2017). However, the infection rate in postweaned calves (6.19%) was higher than that in Heilongjiang (5.5%) (Liu et al., Reference Liu, Wang, Li, Zhang, Shu, Zhang, Feng, Xiao and Ling2009), but lower than that in Henan (11.3%) and Xinjiang (16.2%) (Wang et al., Reference Wang, Ma, Zhao, Lu, Wang, Zhang, Jian, Ning and Xiao2011a; Qi et al., Reference Qi, Wang, Jing, Wang, Wang and Zhang2015a). Many factors, including specimen size, diagnostic technique, management system, season and geographic area, may be responsible for the differences in the prevalence of Cryptosporidium observed in different areas of China. These results were also consistent with previous studies, which showed that the prevalence of Cryptosporidium was higher in preweaned calves than in any other age group (Wang et al., Reference Wang, Ma, Zhao, Lu, Wang, Zhang, Jian, Ning and Xiao2011a, Reference Wang, Wang, Sun, Zhang, Jian, Qi, Ning and Xiao2011b; Huang et al., Reference Huang, Yue, Qi, Wang, Zhao, Li, Shi, Wang and Zhang2014; Zhang et al., Reference Zhang, Tan, Zhou, Ni, Liu, Yang and Zhu2015) (Table 1).
Cryptosporidium andersoni is the predominant species in postweaned calves and adult cattle, which had been confirmed in China, Mongolia, Egypt and some European countries (Burenbaatar et al., Reference Burenbaatar, Bakheit, Plutzer, Suzuki, Igarashi, Ongerth and Karanis2008; Ondráčková et al., Reference Ondráčková, Kváč, Sak, Květoňová and Rost2009; Wang et al., Reference Wang, Ma, Zhao, Lu, Wang, Zhang, Jian, Ning and Xiao2011a; Amer et al., Reference Amer, Zidan, Adamu, Ye, Roellig, Xiao and Feng2013; Zhao et al., Reference Zhao, Ren, Gao, Bian, Hu, Cong, Lin, Wang, Qi, Qi, Zhu and Zhang2013; Ma et al., Reference Ma, Li, Zhao, Xu, Wu, Wang, Guo, Wang, Feng and Xiao2015; Qi et al., Reference Qi, Wang, Jing, Wang, Wang and Zhang2015a, Reference Qi, Fang, Wang, Zhang, Wang, Du, Guo, Jia, Yao, Liu and Zhao2015b). In contrast, in other countries, C. bovis was considered the predominant species in postweaned calves (Enemark et al., Reference Enemark, Ahrens, Lowery, Thamsborg and Enemark2002; Feng et al., Reference Feng, Ortega, He, Das, Xu, Zhang, Fayer, Gatei, Cama and Xiao2007). Cryptosporidium andersoni was the only species detected in heifers and adult cattle in this study, which was identical to previous studies conducted in Heilongjiang, Shaanxi and Henan Provinces (Liu et al., Reference Liu, Wang, Li, Zhang, Shu, Zhang, Feng, Xiao and Ling2009; Wang et al., Reference Wang, Ma, Zhao, Lu, Wang, Zhang, Jian, Ning and Xiao2011a; Zhao et al., Reference Zhao, Ren, Gao, Bian, Hu, Cong, Lin, Wang, Qi, Qi, Zhu and Zhang2013). The potential zoonotic transmission of C. andersoni is unknown, but the species has been isolated from humans with diarrhoea (Leoni et al., Reference Leoni, Amar, Nichols, Pedraza-Diaz and McLauchlin2006; Jiang et al., Reference Jiang, Ren, Yuan, Liu, Zhao, Liu, Chu, Pan, Cao, Lin and Shen2014).
Several studies have reported that zoonotic C. parvum is responsible for the majority of Cryptosporidium infections in preweaned calves (Santín et al., Reference Santín, Trout, Xiao, Zhou, Greiner and Fayer2004; Fayer et al., Reference Fayer, Santín, Trout and Greiner2006; Santín et al., Reference Santín, Trout and Fayer2008; Trout and Santín, Reference Trout, Santín, Fayer and Xiao2008; Amer et al., Reference Amer, Zidan, Adamu, Ye, Roellig, Xiao and Feng2013). However, other studies have shown that C. bovis is the species most commonly found in preweaned calves (Feng et al., Reference Feng, Ortega, He, Das, Xu, Zhang, Fayer, Gatei, Cama and Xiao2007; Silverlas et al., Reference Silverlas, Naslund, Bjorkman and Mattsson2010; Rieux et al., Reference Rieux, Chartier, Pors and Paraud2013). In the present study, C. bovis, rather than C. parvum, was the most abundant species in preweaned calves, which was in concordance with the results from Shaanxi, Heilongjiang, Henan and Hubei Provinces, as well as Shanghai (Wang et al., Reference Wang, Wang, Sun, Zhang, Jian, Qi, Ning and Xiao2011b; Zhang et al., Reference Zhang, Wang, Yang, Zhang, Cao, Zhang, Hong, Liu and Shen2013; Qi et al., Reference Qi, Fang, Wang, Zhang, Wang, Du, Guo, Jia, Yao, Liu and Zhao2015b; Fan et al., Reference Fan, Wang, Koehler, Hu and Gasser2017), but differed from Xinjiang and Ningxia (Cui et al., Reference Cui, Wang, Huang, Wang, Zhao and Luo2014; Huang et al., Reference Huang, Yue, Qi, Wang, Zhao, Li, Shi, Wang and Zhang2014; Qi et al., Reference Qi, Wang, Jing, Wang, Wang and Zhang2015a, Reference Qi, Fang, Wang, Zhang, Wang, Du, Guo, Jia, Yao, Liu and Zhao2015b).
However, C. parvum was not detected in the present study, which was also reported in early studies from China and abroad (Maikai et al., Reference Maikai, Umoh, Kwaga, Lawal, Maikai, Cama and Xiao2011; Feng et al., Reference Feng, Karna, Dearen, Singh, Adhikari, Shrestha and Xiao2012; Murakoshi et al., Reference Murakoshi, Xiao, Matsubara, Sato, Kato, Sasaki, Fukuda, Tada and Nakai2012; Nguyen et al., Reference Nguyen, Fukuda, Tada, Sato, Duong, Nguyen and Nakai2012; Abeywardena et al., Reference Abeywardena, Jex, Koehler, Rajapakse, Udayawarna, Haydon, Stevens and Gasser2014; Wegayehu et al., Reference Wegayehu, Karim, Anberber, Adamu, Erko, Zhang and Tilahun2016; Fan et al., Reference Fan, Wang, Koehler, Hu and Gasser2017). The reason for the absence of C. parvum remains unclear. The failure to detect C. parvum in preweaned calves in Guangdong Province suggests that the dairy cattle in this province have low zoonotic potential for the transmission of Cryptosporidium to humans. However, larger sample of dairy cattle from this province should be analysed by PCR to confirm the findings of the present study.
To date, 21 MLST subtypes have been identified in C. andersoni isolates from animals, 17 of which occur in cattle (Feng et al., Reference Feng, Yang, Ryan, Zhang, Kvác, Koudela, Modrý, Li, Fayer and Xiao2011; Wang et al., Reference Wang, Jian, Zhang, Ning, Liu, Zhao, Feng, Qi, Wang, Lv, Zhao and Xiao2012; Zhao et al., Reference Zhao, Ren, Gao, Bian, Hu, Cong, Lin, Wang, Qi, Qi, Zhu and Zhang2013; Qi et al., Reference Qi, Wang, Jing, Jian, Ning and Zhang2016). In the present study, the MLST subtype A4,A4,A4,A1 was the most prevalent subtype in dairy cattle in Guangdong, which was in agreement with the finding in Heilongjiang and other areas of China (Wang et al., Reference Wang, Jian, Zhang, Ning, Liu, Zhao, Feng, Qi, Wang, Lv, Zhao and Xiao2012; Zhao et al., Reference Zhao, Wang, Zhang, Liu, Cao, Shen, Yang and Zhang2014), whereas subtype A2,A4,A2,A1 was predominant in dairy cattle in Xinjiang and A1,A4,A4,A1 in Shaanxi (Zhao et al., Reference Zhao, Ren, Gao, Bian, Hu, Cong, Lin, Wang, Qi, Qi, Zhu and Zhang2013; Qi et al., Reference Qi, Wang, Jing, Jian, Ning and Zhang2016). These differences may be related to the number of samples examined and geographic segregation. MLST subtype A2,A5,A2,A1 was found for the first time in cattle in this study, which was also identified in sheep, and further suggested that C. andersoni might circulate between cattle and sheep (Wang et al., Reference Wang, Jian, Zhang, Ning, Liu, Zhao, Feng, Qi, Wang, Lv, Zhao and Xiao2012).
In this study, the samples successfully amplified at all four loci were included in the LD analysis, which showed that C. andersoni isolated from dairy cattle in Guangdong Province had a clonal genetic population structure. The result differed from previous findings that C. andersoni population in cattle from Xinjiang and other geographical regions of China had an epidemic genetic structure (Wang et al., Reference Wang, Jian, Zhang, Ning, Liu, Zhao, Feng, Qi, Wang, Lv, Zhao and Xiao2012; Qi et al., Reference Qi, Wang, Jing, Jian, Ning and Zhang2016). However, it was consistent with C. andersoni isolates in cattle from Shaanxi and Heilongjing Provinces (Zhao et al., Reference Zhao, Ren, Gao, Bian, Hu, Cong, Lin, Wang, Qi, Qi, Zhu and Zhang2013; Zhao et al., Reference Zhao, Wang, Zhang, Liu, Cao, Shen, Yang and Zhang2014). A clonal genetic population structure indicated that the prevalence of C. andersoni in cattle in Guangdong Province was not attributable to the introduction of cattle.
In conclusion, Cryptosporidium is common in dairy cattle in Guangdong province. Sequence analysis revealed the presence of C. andersoni, C. bovis and C. ryanae infection, with C. andersoni as the most prevalent species. Three MLST subtypes of C. andersoni were identified, and subtype A2,A5,A2,A1 was found for the first time in cattle. Cryptosporidium andersoni in dairy cattle in Guangdong presented a clonal genetic structure. The findings in this study provided valuable basic data for developing strategies and measures to control Cryptosporidium infection in dairy cattle and evaluate the risk of Cryptosporidium infection to humans.
Financial support
This study was partly supported by the National Natural Science Foundation of China (31330079, 31672548), the National Key Research and Development Program of China (2017|YFD0501305, 2016YFD0500707) and the Natural Science Foundation of Henan Province (162300410129).
Conflict of interest
None.
Ethical standards
This study was performed in accordance with the Chinese Laboratory Animal Administration Act of 1988. Before the experiments, the protocol of the study was reviewed and approved by the Research Ethics Committee of Henan Agricultural University. All the fecal samples were collected from animals with the permission of the farm owners.
