Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Ningxia, northwestern China.

BACKGROUND: Cryptosporidium spp. and Giardia duodenalis are important gastrointestinal protists in humans and animals worldwide. In China, bovine cryptosporidiosis and giardiasis are of increasing concern because cattle are important reservoirs of these parasites, which have become potential threats to public health and to large numbers of cattle in recent years.
RESULTS: A total of 1366 fecal samples from the Ningxia Autonomous Region were examined. The overall infection rates for Cryptosporidium spp. and G. duodenalis were 1.61% and 2.12%, respectively. Cryptosporidium was only detected in preweaned calves and adults older than 2 years, whereas G. duodenalis was only detected in calves aged less than 11 months. Cryptosporidium spp. were characterized with a PCR-restriction fragment length polymorphism analysis and DNA sequence analysis of the small subunit rRNA gene. Three Cryptosporidium species were identified: C. parvum (n = 15) and C. bovis (n = 4) in preweaned calves, and C. andersoni (n = 4) in adults aged over 2 years. A DNA sequence analysis of the gp60 gene suggested that the 15 C. parvum isolates all belonged to subtype IIdA15G1. Twenty-nine G. duodenalis isolates were analyzed by DNA sequencing of the triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Two G. duodenalis assemblages were identified, assemblages E (n = 15) and B (n = 4, one subtype B1 and three subtype B2) in preweaned calves, and assemblage E (n = 10) in 3-11-month-old calves.
CONCLUSIONS: The predominance of C. parvum detected in preweaned calves and the first identified subtype IIdA15G1 in dairy cattle, and the dominant G. duodenalis assemblage E in this study differed considerably from those found in Henan, Heilongjiang, and Shannxi Provinces. Our findings further confirm the dominance of C. parvum IId subtypes in China.

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 [1,2]. Diarrhea is the typical clinical symptom of human and animal cryptosporidiosis or giardiasis, as well as dehydration, fever, nausea, and anorexia.

Cattle are mammalian species commonly infected with Cryptosporidium, and preweaned calves are considered the most important reservoir for zoonotic infections. Large numbers of studies have suggested that C. parvum, C. bovis, C. andersoni, and C. ryanae are the most common species infecting cattle, although C. felis, C. hominis, C. suis, C. scrofarum (formerly pig genotype II), and C. suis-like genotype have also been detected [3]. The four common Cryptosporidium species have age-associated distributions. Cryptosporidium parvum is usually found in preweaned calves and is a significant cause of diarrhea. However, C. bovis and C. ryanae usually infect postweaned calves and yearlings, and C. bovis is detected more frequently than C. ryanae, although neither is associated with diarrhea [4]. In contrast, C. andersoni is commonly seen in adult cattle and is associated with gastritis, reduced milk yield, and poor weight gain [5].

Among the six known Giardia species, only G. duodenalis is recovered from humans and most other mammals [6]. Its molecular characterization has shown that G. duodenalis is a species complex comprising eight distinct ‘assemblages’ or genotypes: A to H [2]. Most of the assemblages (C to H) seem to be host specific for nonhuman species: assemblages C and D are specific for dogs, E for hoofed livestock, F for cats, G for rats, and H for seals. In cattle, the prevalence of giardiasis ranges from 2.2% to 50.7% worldwide, and assemblages A, B, and E have been detected, with assemblage E the predominant genotype in most countries [1,2].

In China, bovine cryptosporidiosis and giardiasis are of increasing concern because cattle are important reservoirs of these parasites, which have become potential threats to public health and to large numbers of cattle in recent years. Several studies of Cryptosporidium and Giardia infections in several areas have been published in the English language [7-11], and differences in Cryptosporidium distributions have been noted in different locations. For example, C. bovis rather than C. parvum is the predominant species in preweaned calves in Henan, whereas C. andersoni is the most common species of cattle in Heilongjiang and Shannxi Provinces [7,12]. 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 [9,10]. In contrast, there have been few molecular epidemiological studies of G. duodenalis in cattle [10,13]. 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 oocysts and cysts on Chinese farms

Farm	Sample size	Prevalence (%) Cryptosporidium / Giardia	Cryptosporidium species (no.)	Giardia duodenalis assemblage (no.)
1	45	2.22%/2.22%	C. bovis (1)*	B2 (1)*
2	32	9.38%/6.25%	C. parvum (1)**, C. bovis (2)**	B2 (2)*
3	57	3.51%/3.51%	C. parvum (2)***	E (1), B1 (1)***
4	46	4.35%/6.52%	C. parvum (2)	E (3)
5	63	3.17%/7.94%	C. parvum (2)	E (5)
6	106	0.94%/1.89%	C. parvum (1)	E (2)
7	110	6.36%/3.64%	C. parvum (7)	E (4)
8	166	0/1.20%		E (2)
9	94	4.26%/3.19%	C. andersoni (4)	E (3)
10	32	0/6.25%		E (2)
11	141	0/1.42%		E (2)
12	76	0/1.32%		E (1)
13	69	1.45%/0	C. bovis (1)
14-20	329	0/0
Total	1366	1.68%/2.12%	C. parvum (15), C. bovis (4), C. andersoni (4)

The distributions of Cryptosporidium spp. and G. duodenalis assemblages were determined with PCR–RFLP analysis of the SSU rRNA gene and sequence analysis of the tpi and gdh genes.

*Sample positive for a mixed infection of C. parvum and G. duodenalis subtype B2.

**One sample showed a mixed infection of C. parvum and G. duodenalis subtype B2, and another sample showed a mixed infection of C. bovis and G. duodenalis subtype B2.

***Sample positive for a mixed infection of C. parvum and G. duodenalis subtype B1.

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/Giardia genotyping and subtyping

The Cryptosporidium species were identified with PCR–restriction fragment length polymorphism (RFLP) analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene [14]. Cryptosporidium parvum was subtyped with nested PCR targeting the gp60 gene, and the previously established nomenclature system was used to name the C. parvum subtype families and subtypes [15,16]. The Giardia duodenalis genotypes/subtypes were identified by sequencing the triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes [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. duodenalis assemblages

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

species identified in dairy cattle in different age groups

Age group	Sample size	Cryptosporidium		G. duodenalis assemblage
		Prevalence	Species (no.)	Prevalence	Assemblage (no.)
Preweaned*	186	10.22%	C. parvum (15), C. bovis (4)	10.22%	E (15), B1 (1), B2 (3)
3–11 months	396	0	0	2.53%	E (10)
1–2 year	204	0	0	0	0
> 2 years	580	0.69%	C. andersoni (4)	0	0

*Among the positive samples from preweaned calves, one was a mixed infection of C. bovis and G. duodenalis subtype B2, one was a mixed infection of C. parvum and G. duodenalis subtype B1, and two were mixed infections of C. parvum and G. duodenalis subtype B2.

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) [8,9], Heilongjiang (15.0%, 99/658) [7,11], Shannxi (3.4%, 70/2071) [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 [8-10,19,20].

Among the preweaned calves, C. parvum was the predominant Cryptosporidium species, rather than C. bovis. This result clearly differs from the results reported in Henan and Heilongjiang, where C. bovis was the dominant species, and in Shannxi, where only C. andersoni was detected [9,11,12]. The results of most previous studies, conducted in numerous countries, suggest that C. parvum is the predominant Cryptosporidium species in preweaned calves [9]. In contrast, only C. andersoni was detected in adult cattle in the present study, which was basically similar to the reports of Cryptosporidium infections in cattle in Henan, Heilongjiang, Shannxi, and other countries [7,8,12,21].

The 15 C. parvum isolates were identified as subtype IIdA15G1 based on a sequence analysis of the gp60 gene, which differed from subtype IIdA19G1 found in dairy cattle in Henan and Heilongjiang provinces, China [9,11]. In fact, except for C. parvum IIa subtypes found in yaks [22], the C. parvum isolates characterized in China thus far have belonged to the IId subtypes, including IIdA15G1 in rodents [23], IIdA19G1 in cattle, humans, and urban wastewater [9,11,24,25]. Thus, the characteristics of C. parvum subtypes appear to be unique in China. Sequence analysis of the gp60 gene has been used extensively to characterize the molecular epidemiology of cryptosporidiosis in animals and humans, and at least 14 C. parvum subtype families have been identified: IIa, IIb, IIc, IId, IIe, IIf, IIg, IIh, IIi, IIk, IIl, IIm, IIn, and IIo [1,16]. Of these, IIa is the predominant subtype family in animals and humans worldwide, and IId is another major zoonotic subtype family reported in Europe (Hungary, Germany, Portugal, Sweden, Ireland, Spain, Belgium, Romania, United Kingdom, Netherlands, Slovenia, Serbia, and Montenegro), Asia (Kuwait, Iran, Jordan, India, Malaysia, and China), Egypt, and Australia [9,26-29]. In contrast, IIc and IIe are anthroponotic subtype families. Other subtype families of C. parvum are occasionally -seen in various animals or humans around the world [9,16].

Sequence analyses of the tpi and gdh genes showed that most of the dairy cattle were infected with livestock-specific G. duodenalis assemblage E (86.2%). This result is consistent with a previous study conducted in Heilongjiang and Henan, China [10,13]. 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 [2,30-39].

Giardia duodenalis assemblage B was only found in four preweaned calves in this study. In a previous study conducted in Heilongjiang Province, a higher rate of assemblage B was identified (18/43) in dairy cattle [10], whereas no assemblage B was found in Henan Province [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.