Background

Cryptosporidium and Giardia are important gastrointestinal protists with a wide spectrum of hosts, including humans, livestock, companion animals, and wildlife. Infection is acquired via the fecal–oral route following the ingestion of infective oocysts or cysts, by either direct contact or the ingestion of contaminated food or water [12]. Thus, the distributions of Cryptosporidium spp. and the C. parvum subtype (all belonging to IIdA19G1) in dairy cattle differ considerably from those in other countries [13]. The objective of this study was to identify the species of Cryptosporidium and Giardia present in dairy cattle in the Ningxia Autonomous Region, northwest China.

Methods

Ethics statement

This study was conducted in accordance with the Chinese Laboratory Animal Administration Act of 1988. The research protocol was reviewed and approved by the Research Ethics Committee of the Henan Agricultural University. Permission was obtained from farm owners before collection of fecal samples.

Sample collection and examination

A fresh fecal sample was collected from each animal using a sterile disposal latex glove immediately after its defecation onto the ground, and was then placed individually into a disposable plastic bag. In total, 1366 fecal samples were collected between December 2011 and October 2012 from dairy cattle on 20 scale farms in the Ningxia Autonomous Region, northwestern China (Table 1). The Cryptosporidium oocysts in the fecal materials were concentrated with Sheather’s sugar flotation technique, with a further formalin–ethyl acetate sedimentation step included for the preweaned calf samples [9]. Giardia cysts were detected with Lugol’s iodine staining. Cryptosporidium- or Giardia-positive samples were stored in 2.5% potassium dichromate at 4°C before DNA extraction.

Table 1 Numbers of fecal samples examined with microscopy for Cryptosporidium oocysts and Giardia cysts on Chinese farms
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DNA extraction

The Cryptosporidium or Giardia-positive fecal specimens were washed three times with distilled water, and the genomic DNA was extracted from the fecal pellets with the E.Z.N.A.® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA), according to the manufacturer’s recommendations.

Cryptosporidium/Giardiagenoty** and subty**

The Cryptosporidium species were identified with PCR–restriction fragment length polymorphism (RFLP) analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene [17], and the genotype/subtype identities of the G. duodenalis samples were established by direct comparison of these sequences with reference sequences downloaded from the GenBank database.

DNA sequence analysis

PCR products were sequenced on an ABI Prism™ 3730 XL DNA Analyzer using the BigDye 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 aligned with the ClustalX 1.83 program. Representative nucleotide sequences have been deposited in GenBank under accession numbers KM067089–KM067096.

Statistical analysis

The χ2 test was used to compare the Cryptosporidium infection rates. Differences were considered significant at P <0.05.

Results

Prevalence of Cryptosporidium and Giardia

Microscopic analysis of 1366 fecal samples showed the presence of Cryptosporidium oocysts in 23 samples (1.68%) from nine farms, and the highest infection rate was 9.38% on farm 2 (Table 1). The infection rates of Cryptosporidium spp. were 10.22%, 0%, 0%, and 0.69% in preweaned, 3–11-month-old, 1–2-year-old, and > 2-year-old cattle, respectively (χ2 = 95.52, P <0.01).

Similarly, 29 samples from 12 farms were positive for Giardia, with an average infection rate of 2.12% (Table 1). The highest infection rate for Giardia was 7.94% on farm 5. The infection rates for Giardia were 10.22%, 2.53%, 0%, and 0% in preweaned, 3–11-month-old, 1–2-year-old, and > 2-year-old cattle, respectively (χ2 = 75.93, P <0.01).

Distribution of Cryptosporidium species/subtypes and G. duodenalisassemblages

The SSU rRNA genes of Cryptosporidium spp. in all 23 microscopy-positive samples were successfully amplified with nested PCR. RFLP and DNA sequence analyses of the SSU rRNA gene fragments revealed the presence of three Cryptosporidium species: C. parvum (n = 15) on six farms, C. bovis (n = 4) on three farms, and C. andersoni (n = 4) on one farm (Table 1). With the exception of farm 2, only one Cryptosporidium species was detected on all the Cryptosporidium-positive farms (Table 1). The gp60 sequencing analysis showed that the 15 C. parvum isolates all belonged to subtype IIdA15G1.

Sequencing analyses of the tpi and gdh genes in G. duodenalis identified two assemblages: E (n = 25) on 10 farms and B (n = 4) on three farms (Table 1). Only farm 3 had both assemblages E and B.

Age distributions of Cryptosporidium and G. duodenalis

Cryptosporidium parvum was the most commonly identified Cryptosporidium species, responsible for 65.2% of all Cryptosporidium infections, and was only found in preweaned calves (Table 2). Cryptosporidium bovis and C. andersoni were found in preweaned calves and > 2-year-old cattle, respectively. In contrast, no Cryptosporidium-positive sample was identified in cattle aged between 3 months and 2 years.

Table 2 Cryptosporidium species identified in dairy cattle in different age groups
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Giardia duodenalis was only detected in preweaned and 3–11-month-old calves. Among these calves, G. duodenalis assemblage E was the dominant assemblage, responsible for 86.2% (25/29) of all Giardia-positive samples. In contrast, only four assemblage B infections (one subtype B1 and three subtype B2) were found in preweaned calves. Four mixed infections of Cryptosporidium and G. duodenalis were found in preweaned calves (Table 2).

Discussion

The overall infection rate for Cryptosporidium spp. was 1.68%, which is lower than those in Henan (13.0%, 276/2116) [12], Anhui (14.9%, 52/350), Jiangsu (20.7%, 251/1215), and Shanghai (12.5%, 55/440) [18]. The average infection rate for G. duodenalis was 2.12%, which is lower than those in Heilongjiang (5.2%, 42/814) [10] and Henan (7.2%, 128/1777) [13]. Oocysts shed at low intensity may be contributed to the low detecting rates of both parasites by microscopy. In general, it is difficult to explain the actual discrepancies in the prevalence of Cryptosporidium spp. and G. duodenalis among different studies because the infection rates are related to many factors, including the examination methods, age distributions of the animals, sample sizes, host health status at the time of sampling, the timing of specimen collection, and geo-ecological conditions. Nevertheless, the infection rates of both parasites were always higher in preweaned calves than in any other age group, which is consistent with previous observations [12]. The results of most previous studies, conducted in numerous countries, suggest that C. parvum is the predominant Cryptosporidium species in preweaned calves [12],[24],[13]. The results of most studies conducted in numerous countries, including Belgium, Denmark, Portugal, Spain, Sweden, Germany, UK, USA, Canada, Brazil, Australia, Rwanda, Egypt, Sri Lanka, and Malaysia, suggest that assemblage E is the predominant Giardia assemblage in cattle [13]. In general, assemblage B has been detected in only a small number of cattle in a few studies worldwide, including in Europe, Italy, Portugal, Uganda, New Zealand, and Canada [2],[30]. Assemblage B, one of the two major assemblages causing human giardiasis, has a broad host range, including cattle, sheep, pigs, horses, dogs, cats, and rabbits [2]. Although no strong evidence supporting the direct zoonotic transmission of G. duodenalis from animals to humans has been reported, case–control studies have shown that contact with farm animals increases the infection rate of human giardiasis [40],[41]. Considering how large the cattle industry is and the close contact that occurs between cattle and humans, assemblage B G. duodenalis identified in cattle may emerge as an important zoonotic pathogen in some areas of China.

Conclusion

In summary, C. parvum is the predominant species in preweaned calves in the study area, which clearly differs from the situation in Henan, Heilongjiang, and Shannxi Provinces. The presence of C. parvum subtype IIdA15G1, together with subtypes IIdA15G1 and IIdA19G1, previously found in rodents, ruminants, and humans, further confirms the dominance of the C. parvum IId subtypes in China. The dominant G. duodenalis assemblage, assemblage E, is similar to the dominant assemblages in other areas or countries.