Occurrence and genotyping of Giardia duodenalis and Cryptosporidium in pre-weaned dairy calves in central Sichuan province, China

Giardia duodenalis and Cryptosporidium spp. are common human and animal pathogens. They have increasingly been reported in dairy calves in recent years; however, multilocus genotyping information for G. duodenalis and Cryptosporidium infecting pre-weaned dairy calves in southwestern China is limited. In the present study, the prevalence of G. duodenalis and Cryptosporidium spp. in pre-weaned dairy calves in central Sichuan province was determined and the pathogens were analyzed molecularly. Of 278 fecal samples from pre-weaned dairy calves, 26 (9.4%) were positive for G. duodenalis and 40 (14.4%) were positive for Cryptosporidium spp. Cryptosporidium bovis (n = 28), Cryptosporidium ryanae (n = 5) and Cryptosporidium parvum (n = 7) were detected. All seven C. parvum isolates were successfully subtyped based on the gp60 gene sequence, and only IIdA15G1 was detected. Multilocus sequence typing of G. duodenalis based on beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes revealed 19 different assemblage E multilocus genotypes (two known and 17 unpublished genotypes). Based on eBURST analysis, a high degree of genetic diversity within assemblage E was observed in pre-weaned dairy calves in Sichuan province. To the best of our knowledge, this is the first study using multilocus sequence typing and eBURST analysis to characterize G. duodenalis in pre-weaned dairy calves in southwestern China.


Introduction
Protists of the genera Giardia and Cryptosporidium infect a wide range of animals as well as humans [3,12,19]. Typically, the infection is acquired following the ingestion of highly resilient, infective stages (oocysts or cysts) via the fecal-oral route [3,4]. Disease is commonly associated with clinical signs including diarrhea, dehydration, fever, inappetence and anorexia. Infections are often self-limiting in immune-competent individuals [2,31], but can be chronic and severe in infants, elderly people, and immune-compromised individuals [9,16].
Ruminants are recognized as a significant reservoir of Giardia and Cryptosporidium taxa that infect animals and humans [19,21]. Current data indicate that of the eight assemblages within Giardia duodenalis, assemblages A and E and the Cryptosporidium species C. parvum, C. andersoni, C. ryanae, and C. bovis predominate in cattle worldwide [4,20].
Unlike in other countries (e.g. Australia, Sudan, Japan and India) [6,14,15,24], where C. parvum is known to be the predominant species in pre-weaned calves, this does not appear to be the case everywhere in China. Some studies have shown that C. parvum is a major species in pre-weaned calves in some regions, whereas C. bovis is a major species in other regions [5,25,28].
According to the National Bureau of Statistics of the People's Republic of China, in 2016, the total population of dairy cattle in Sichuan Province was 176 thousand heads. However, no information about G. duodenalis and Cryptosporidium infection of pre-weaned dairy calves was previously available in Sichuan Province. We undertook a molecular epidemiological study to obtain a preliminary snap-shot of the prevalence of G. duodenalis assemblages and Cryptosporidium genotypes in pre-weaned calves in Sichuan province, China.

Sample collection
We collected 278 rectal fecal samples from pre-weaned dairy calves (<1 month of age) from 10 farms with a history of bovine diarrhea in 10 regions in Sichuan province, southwestern China, between June 2016 and March 2017. Collection sites included: and Deyang (104°39 0 E, 31°13 0 N). The 10 farms are distributed in central Sichuan Province (Fig. 1). The city-level map was provided by the National Geomatics Centre of China (National Geomatics Centre of China, Beijing, China, http://ngcc.sbsm. gov.cn/).
Of the 10 farms, six (Chengdu, Hongya, Aba, Mianyang, Ziyang, and Qionglai) are intensive feeding farms, while the other four are free-ranging. For intensive farms, there were approximately 1000-2500 cattle per farm, with more than 100 pre-weaned dairy calves, and the fecal samples were randomly collected from about 20% in each farm. For freeranging farms, there were approximately 100-120 cattle per farm and the herd sizes of pre-weaned dairy calves were less than 50; in this case we collected fecal samples from all of the pre-weaned dairy calves at each farm (Table S1). In intensive farms, calves were bred in different calf stalls, with one hour outdoor time after eating and excretion in the morning and afternoon, respectively. Calves shared one yard during the outdoor time in intensive farms. In free-ranging farms, calves were kept in a field with a half cover and were raised together. The farms we selected had solely cattle, and no other animals.
Fecal samples were collected from the rectum using disposable gloves, transferred into disposable plastic bags, and stored in 2.5% potassium dichromate at 4°C.

DNA extraction
Before DNA extraction, feces were washed with distilled water to remove potassium dichromate. Genomic DNA was extracted from 250 mg (approximately) of individual samples using the Power Soil DNA isolation kit (MOBIO, USA), according to the manufacturer's instructions, and frozen at À20°C until use.

PCR amplification and sequencing
G. duodenalis was detected by nested PCR amplification of the bggene. The bg-positive samples were further characterized by amplifications of gdh and tpi. Genotyping of Cryptosporidium was based on amplification of the small subunit (SSU) rRNA gene by nested PCR and subsequent sequence analysis. All the C. parvum isolates were further characterized by amplification of the gp60 gene. The primers and amplification conditions in this study were described previously [1,11,23]. Positive and negative controls were included in each test. The secondary PCR products were visualized under UV light after electrophoresis on a 1% agarose gel mixed with Golden View.
All positive secondary PCR products were sent to Invitrogen (Shanghai, China) and sequenced in both directions. Sequences were aligned with reference sequences from GenBank using BLAST (http://blast.ncbi.nlm.nih.gov) and ClustalX. A previous nomenclature system was used to name subtypes at each genetic locus [29,30]. Specimens that were successfully subtyped at all three loci were included in multilocus genotyping of G. duodenalis. The genetic pedigree of the assemblage E multilocus genotypes (MLGs) was assessed by using eBURST 3.0 (http://eBURST.mlst.net).

Statistical analysis
The v 2 test was used to compare the infection rates of G. duodenalis and Cryptosporidium in different feeding patterns. Differences were considered significant at p < 0.05.

Results and discussion
G. duodenalis was detected in 9.4% of 278 pre-weaned dairy calves on 6 of 10 farms, with prevalences ranging from 7.7% to 46.4% (Table 1). Its prevalence shows substantial   [7,13,18,26,29]. In this study, the overall infection rate in southwestern China was close to the prevalence in northwestern (9.7% [18]), northeastern (13.3% [13]) and north China (7.1% [7]), but much lower than the infection rates in central (17.6% [26]) and southeastern (60.1% [29]) China. Prior to the present study, these results were interpreted as related to differences in geographic distribution, environmental management and cultivation scale [7,13,18,26,29]. Cattle were kept in groups or in free stalls, which might promote the transmission of G. duodenalis infection among animals and lead to the high infection rates [26,29]. Furthermore, we analyzed the infection rates between intensive feeding and free-ranging farms; there was no significant difference between the two breeding patterns (X 2 = 0.629, df = 1, p = 0.428). Cryptosporidium was detected in 14.4% of 278 fecal samples, on 9 out of 10 farms, with prevalences ranging from 4.2% to 35.0% (Table 1). The overall infection rate for Cryptosporidium is lower than the average prevalence of 19.5% reported previously in pre-weaned cattle in China [5], but similar to the infection rate reported in Xinjiang (15.6%) [17], and much higher than the rate in Hebei and Tianjin, China (1.0%) [7]. Prevalence of Cryptosporidium was significantly different (X 2 = 4.924, df = 1, p = 0.026) between intensive feeding and free-ranging farms in this study, which suggests that cultivation scale may lead to differences in infection rates with Cryptosporidium. Other studies also showed that geographic distribution and host health status may lead to the difference [5,10,17]. Three species of Cryptosporidium (28 C. bovis, 7 C. parvum [subtype IIdA15G1] and 5 C. ryanae) were identified in this study. Previous studies have shown that C. parvum is a major species in pre-weaned calves in Beijing [10], Xinjiang [17] and Ningxia [8], whereas C. bovis predominated in pre-weaned calves in this study, similar to reports from Henan [27] and Heilongjiang [32].
For G. duodenalis, multi-locus genotyping analysis suggested a high genetic diversity of assemblage E in pre-weaned  Table 2). A high degree of nucleotide variation in assemblage E has been also detected in previous studies [18,22,26,29]. Of the 19 MLGs, 17 were unpublished MLGs. The majority of MLGs were MLG-E3 and MLG-E13, which have also been detected in dairy calves in Shanghai [29]. To further analyze the evolutionary descent of the 19 assemblage E MLGs, we used eBURST analysis of the 19 assemblage E MLGs and 58 reference MLGs, which revealed two clonal complexes and seven singletons (Fig. 2). MLG-E3 is the primary founder of clonal complex 1, which is consistent with findings in a previous study in Shanghai [29]. The majority of MLGs (14/19) originated from MLG-E3. Furthermore, MLG-E60 is a variant of clonal complex 2, and MLG-E59 and MLG-E70 were singletons. The latter three MLG subtypes showed distant evolution from other assemblage E MLGs, which may indicate substantial differences in their evolutionary divergence [29].

Conclusion
This is the first study to genotype G. duodenalis and Cryptosporidium in pre-weaned dairy calves in Sichuan province. C. bovis and G. duodenalis assemblage E are the dominant species in pre-weaned dairy calves in Sichuan, and high genetic diversity of assemblage E MLGs was observed.

Statements Ethics approval and consent to participate
This study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University under permit number DYY-S20174604.

Consent for publication
Not applicable.

Availability of data and material
The datasets supporting the conclusions of this article are included within the article. Cryptosporidium and G. duodenalis sequences were deposited in the GenBank database under accession numbers MF671870-MF671908.

Competing interests
The authors declare that they have no conflict of interest. Acknowledgements. The authors thank Guangwen Yan for giving advice on sample collection. We sincerely thank Robin B Gasser  Table S1 is available at https://www.parasite-journal.org/ 10.1051/parasite/2018023/olm