JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2011, p. 1077–1082
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 3
Characteristics of Cryptosporidium Transmission in Preweaned Dairy
Cattle in Henan, China?
Rongjun Wang,1Helei Wang,1Yanru Sun,1Longxian Zhang,1* Fuchun Jian,1Meng Qi,1
Changshen Ning,1and Lihua Xiao2*
College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China,1and Division of
Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia 303332
Received 30 October 2010/Returned for modification 5 December 2010/Accepted 13 December 2010
To estimate the prevalence and public health significance of cryptosporidiosis in preweaned calves in China,
801 fecal samples from eight farms in seven areas in Henan Province were examined for Cryptosporidium
oocysts. The overall infection rate of Cryptosporidium was 21.5%, with the farm in Xinxiang having the highest
prevalence (40%). No significant difference in infection rates was observed between seasons. Cryptosporidium
spp. were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the small
subunit (SSU) rRNA gene and DNA sequencing of the 60-kDa glycoprotein (gp60) gene. The SSU rRNA-based
PCR identified four Cryptosporidium species, including Cryptosporidium parvum (54/172), C. bovis (65/172), C.
ryanae (19/172), and C. andersoni (12/172), and the occurrence of infections with mixed species (22/172). The
earliest detection of C. bovis was in calves of 1 week of age, showing that the prepatent period was shorter than
the previously stated 10 to 12 days. Infections with C. parvum peaked in summer, whereas C. bovis dominated
in autumn and winter. There was no apparent difference in the age of cattle infected with either C. parvum or
C. bovis. Sequencing analysis of the gp60 gene showed all 67 C. parvum samples belonged to subtype IIdA19G1.
These findings suggested that the transmission of Cryptosporidium spp. in preweaned calves in Henan, China,
appeared to be different from other areas both at genotype and subtype levels. Further molecular epidemiologic
studies (including samples from both calves and humans) are needed to elucidate the transmission dynamics
and public significance of C. parvum in cattle in China.
Cryptosporidium spp. are important gastrointestinal agents in
a wide spectrum of hosts, including humans, other mammals,
birds, reptiles, amphibians, and fish. There are extensive ge-
netic variations within the genus Cryptosporidium. In addition
to more than 20 recognized species of Cryptosporidium, more
than 60 Cryptosporidium genotypes with no designated species
names have been described (9).
Cattle is the common mammalian species in which Crypto-
sporidium infection was detected, and preweaned calves are
considered the most important reservoir for zoonotic infection.
Thus far, seven Cryptosporidium species and two genotypes
have been identified in cattle, including Cryptosporidium par-
vum, C. bovis, C. andersoni, C. ryanae, C. felis, C. hominis, C.
suis, a C. suis-like genotype, and the Cryptosporidium pig ge-
notype II (41). The former four species are mostly responsible
for bovine cryptosporidiosis. Studies conducted in numerous
industrialized nations suggest that there is an age-associated
distribution of the four common Cryptosporidium spp. Thus, C.
parvum is mostly found in preweaned calves and is a significant
cause of diarrhea (41), whereas C. bovis and C. ryanae usually
infect weaned calves and yearlings, with C. bovis being more
commonly seen than C. ryanae and both not associated with the
occurrence of diarrhea (31). In contrast, C. andersoni is com-
monly seen in adult cattle and has been associated with gas-
tritis, reduced milk yield, and poor weight gain (8).
Subtype analysis based on the sequencing of the 60-kDa
glycoprotein (gp60) gene has indicated that IIa is the most
prevalent subtype family of C. parvum in calves worldwide. It
has been widely reported in cattle in the United States, Can-
ada, the United Kingdom, Ireland, Sweden, Germany, Bel-
gium, the Netherlands, Italy, Spain, Portugal, Hungary, Serbia,
and Montenegro, Slovenia, Japan, India, Australia, and New
Zealand (7, 17, 25, 33, 34, 44). Among IIa subtypes,
IIaA15G2R1 is the most common subtype in calves and has
also been commonly identified in human cases in these coun-
tries (44). In contrast, other subtype families such as IId and IIl
were uncommon and were only reported in small numbers of
cattle in Spain, Portugal, Belgium, the Netherlands, Sweden,
Germany, Hungary, Slovenia, and Serbia, and Montenegro
(33, 44). The only possible exception is Egypt, where a recent
small-scale study indicated that one IId subtype (IIdA20G1) of
C. parvum was prevalent on two dairy farms (3).
Dairy industry plays an important role in the agricultural
economy of China. In 2007, the total dairy cattle population
was 12.3 million (ranking fourth worldwide) and accounted for
5.0% of the total number of dairy cattle in the world (http:
//kids.fao.org/glipha/). However, only a few studies genetically
analyzed small numbers of Cryptosporidium isolates. In these
studies, C. andersoni (n ? 29) was identified in postweaned or
adult dairy cattle (21, 47) and C. bovis (n ? 4) and C. ryanae
* Corresponding author. Mailing address for L. Zhang: College of
Animal Science and Veterinary Medicine, Henan Agricultural Univer-
sity, Zhengzhou 450002, People’s Republic of China. Phone: 86-371-
63555387. Fax: 86-371-63558180. E-mail: firstname.lastname@example.org.
Mailing address for L. Xiao: Division of Foodborne, Waterborne, and
Environmental Diseases, National Center for Emerging and Zoonotic
Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, GA 30333. Phone: (404) 718-4161. Fax: (404) 718-4197.
?Published ahead of print on 22 December 2010.
(n ? 1) were identified in preweaned calves (11). Considering
calves are the most important source of zoonotic Cryptospo-
ridium infection, the objective of the present study was to
identify the species of Cryptosporidium present in preweaned
calves in Henan Province, which has the largest population of
dairy cattle in China.
MATERIALS AND METHODS
Sample collection and examination. A total of 801 fresh fecal samples were
collected between August 2008 and November 2009 from preweaned dairy cattle
on eight farms in seven areas in Henan Province, China (Table 1). One of the
farm (Zhengzhou A) was visited four times, and 369 samples taken in four
different seasons were used to assess the seasonal variation in the prevalence.
Cryptosporidium oocysts in fecal materials were concentrated by using both the
formalin-ethyl acetate sedimentation method and Sheather’s sugar flotation
technique. Cryptosporidium oocysts concentrated by the latter method were de-
tected by microscopy under ?400 magnification. Cryptosporidium-positive sam-
ples were stored in 2.5% potassium dichromate at 4°C prior to being used in
molecular biologic characterizations.
DNA extraction. Genomic DNA was extracted from Cryptosporidium-positive
feces samples by using an E.Z.N.A. Stool DNA kit (Omega Biotek, Inc.,
Norcross, GA) according to the manufacturer-recommended procedures.
Cryptosporidium genotyping and subtyping. Cryptosporidium species were de-
termined by nested PCR amplification of an ?830-bp fragment of the small
subunit (SSU) rRNA gene (the primary primers SSU-F2 [TTC TAG AGC TAA
TAC ATG CG] and SSU-R2 [CCC ATT TCC TTC GAA ACA GGA] and the
secondary primers SSU-F3 [GGA AGG GTT GTA TTT ATT AGA TAA AG]
and SSU-R4 [CTC ATA AGG TGC TGA AGG AGT A]) and restriction
fragment length polymorphism (RFLP) analysis using restriction enzymes SspI
and MboII (Fermentas, Shenzhen, China) (11). The presence of C. parvum, C.
bovis, C. ryanae, and C. andersoni was confirmed by DNA sequencing of PCR
product from one sample for each species.
All Cryptosporidium-positive samples were also analyzed by a nested PCR
targeting the gp60 gene (2). The previously established nomenclature system was
used in naming C. parvum subtype families and subtypes (44).
DNA sequence analysis. Sixty-seven PCR products of gp60 gene and one SSU
rRNA PCR product each of C. parvum, C. bovis, C. ryanae, and C. andersoni were
sequenced on an ABI Prism 3730 XL DNA analyzer (Applied Biosystems, Foster
City, CA), using a Big Dye Terminator v3.1 cycle sequencing kit (Applied
Biosystems). The sequence accuracy was confirmed by two-directional sequenc-
ing and by sequencing a new PCR product if necessary. The SSU rRNA and gp60
sequences obtained in the present study were aligned with reference sequences
downloaded from GenBank by using the program CLUSTAL X 1.83 (ftp://ftp
-igbmc.u-strasbg.fr/pub/ClustalX/). Representative nucleotide sequences have
been deposited in the GenBank under accession numbers HQ009805 to
Statistical analysis. A chi-square test was used to compare Cryptosporidium
infection rates. Differences were considered significant when P was ?0.05.
Prevalence of Cryptosporidium spp. Microscopic analysis of
801 fecal samples showed the presence of Cryptosporidium
oocysts in 172 samples (21.5%) on all eight farms (Table 1).
The highest infection rate (40%) was observed on farm Xinx-
iang, and the lowest infection rate (10.5%) was seen on farm
Luoyang (?2? 23.56; P ? 0.01) (Table 1). Cryptosporidium
oocysts were first observed in animals of 7 days. The Crypto-
sporidium infection rates in animals of 1, 2, 3, 4, 5, 6, 7, and 8
weeks were 8.0, 21.4, 21.5, 23.7, 28.4, 28.6, 7.1, and 22.2%,
respectively. The differences in infection rates among age
groups were not significant (?2? 14.68; P ? 0.05) (Fig. 1A).
Distribution of Cryptosporidium species. All 172 Cryptospo-
ridium-positive samples produced the expected PCR product
of the SSU rRNA gene. RFLP analysis of the PCR products
revealed the presence of four Cryptosporidium species, includ-
ing C. parvum (54/172) on four farms, C. bovis (65/172) on
TABLE 1. Infection rates of Cryptosporidium determined by microscopy on each farm and the distribution of Cryptosporidium species, as determined by PCR-RFLP analysis of the SSU
rRNA gene, and C. parvum subtypes, as identified by sequence analysis of the gp60 genea
No. of samples positive fora:
aMixed infections are indicated with a hyphen.
bTotal percentage values are given in parentheses.
1078WANG ET AL. J. CLIN. MICROBIOL.
seven farms, C. ryanae (19/172) on five farms, and C. andersoni
(12/172) on three farms; 22 samples from five farms had con-
current infection of mixed species (Table 1). With the excep-
tion of the farm Xinmi, all of the farms had more than one
Cryptosporidium species (Table 1). Cryptosporidium bovis was
the dominant species on Farm Zhengzhou A (?2? 71.95; P ?
0.01), whereas C. parvum was commonly seen on farms Zheng-
zhou B (?2? 39.62; P ? 0.01) and Shangqiu (?2? 10.89; P ?
0.01) (Table 1). DNA sequencing of the SSU rRNA gene PCR
products confirmed the identification of C. parvum, C. bovis, C.
ryanae, and C. andersoni.
Age patterns of Cryptosporidium species. Cryptosporidium
bovis was the most commonly identified Cryptosporidium, re-
sponsible for 37.8% of all Cryptosporidium infections. It was
found in all weekly age groups examined in the present study
(Fig. 1B). C. parvum, the second most common species, was
detected in seven age groups and accounted for 31.4% of all
Cryptosporidium infections (Fig. 1B). No significant difference
was observed in the infection rates of C. parvum and C. bovis
among the eight age groups (Fig. 1B). The initial detection of
C. bovis and C. ryanae was in calves aged 1 week and 2 weeks,
respectively (Fig. 1B). In contrast, C. andersoni was first de-
tected in animals of 5 weeks in age. The mixed infections were
mostly concentrated in calves 4 to 6 weeks and 8 weeks of age,
with C. parvum commonly seen in mixed infections (Fig. 1B).
Seasonal variation in the distribution of Cryptosporidium
spp. The highest infection rate (50%) was seen in summer and
the lowest (17.3%) in winter (?2? 7.17; P ? 0.05) (Fig. 2A).
Except for spring, the distribution of Cryptosporidium spp. dif-
fered among seasons, with C. parvum dominating in summer
and C. bovis in the autumn (?2? 10.89; P ? 0.01) and winter
(?2? 11.41; P ? 0.01) (Fig. 2B).
Subtypes of the C. parvum. Sequences of the gp60 gene were
successfully obtained from 54 C. parvum-positive samples and
13 samples of mixed infections (Table 1). All of them belonged
to the subtype IIdA19G1 (Table 1).
The prevalence of cryptosporidiosis in dairy cattle varies
among countries in the world. However, a general trend was
observed in many studies: the prevalence of Cryptosporidium
declined with increases in age (17, 18, 31, 32). In the present
study, a 21.5% infection rate was seen in preweaned dairy
calves, which was much higher than the 5.6% (27/485) in post-
weaned and adult dairy cattle reported in a recent Chinese
RFLP and sequence analyses of the SSU rRNA identified
four Cryptosporidium species in the 172 positive samples,
namely, C. parvum, C. bovis, C. ryanae, and C. andersoni.
Among the species detected, C. parvum and C. bovis were
the two most common species, with C. bovis having a higher
infection rate (37.8% versus 31.4%) (Fig. 1B). Previously,
the results of most studies conducted in numerous countries
suggested that C. parvum was the predominant Cryptospo-
ridium species in preweaned calves (Table 2). The only
exception was a recent study conducted in Sweden, in which
54 of 73 Cryptosporidium-positive samples from preweaned
calves had C. bovis (33). In the present study, the youngest
calf infected with C. bovis was 1 week old, indicating that the
prepatent period of C. bovis is shorter than the previously
stated 10 to 12 days (10). Thus, the distribution of Crypto-
FIG. 1. Infection rates and distribution of Cryptosporidium spp. in
preweaned dairy calves in Henan, China. (A) Infection rates of Cryp-
tosporidium spp. in calves at 1 to 8 weeks of age. (B) Distribution of C.
parvum, C. bovis, C. ryanae, C. andersoni, and mixed infections by age.
N, number of samples examined.
FIG. 2. Seasonal variation in infection rates of Cryptosporidium
spp. in preweaned dairy calves in Henan, China. (A) Infection rates of
Cryptosporidium spp. in calves in different seasons. (B) Distribution of
C. parvum and C. bovis in calves by season.
VOL. 49, 2011CRYPTOSPORIDIUM SPP. IN DAIRY CATTLE1079
sporidium species in preweaned dairy calves in Henan,
China appears to be different from that seen in most other
countries. The reason for the high occurrence of C. bovis in
this and the Swedish studies is not clear. Feng et al. sug-
gested that in areas where C. parvum is endemic the high
infection rate and shedding intensity of C. parvum in
preweaned calves probably had masked the concurrent in-
fection of these animals by C. bovis or C. ryanae (11).
Sequence analysis of the gp60 gene has been used exten-
sively in characterizing the molecular epidemiology of crypto-
sporidiosis in calves and humans (45). In recent years, data
generated from numerous studies suggested that IIa was the
TABLE 2. Distribution of Cryptosporidium species/genotypes and subtypes in pre-weaned dairy cattle in different countries
Cryptosporidium species (no. of
C. parvum subtype(s) (no. of isolates)Reference
United States 161C. parvum (138), C. bovis (14), C. ryanae
(8), C. andersoni (1)
C. parvum (6), C. bovis (9), C. ryanae
(5), C. bovis-C. ryanae (3)
C. parvum (175)
United States23 IIaA15G2R1 (6) 11
United States175IIaA15G2R1 (135), IIaA15G2R2 (11), IIaA11G2R1 (11), IIaA17G2R1
(10), IIaA18G2R1 (7), IIaA19G2R1 (4)
C. parvum (107), C. bovis (3)
C. parvum (44), C. bovis (25),
C. ryanae (5)
C. parvum (44)
Canada44IIaA15G2R1 (10), IIaA16G2R1 (9), IIaA16G3R1 (8), IIaA16G1R1
(4), IIaA13G2R1 (2), IIaA17G2R1 (2), IIaA18G3R1 (1)
Belgrade, Serbia, and
5C. parvum (5)
C. parvum (62)
2362IIaA16G1R1 (6), IIlA16 (4), IIaA18G1R1 (2), IIaA20G1R1 (2),
IIdA18G1b (2), IIlA17 (2)
IIaA15G2R1 (52), IIaA16G2R1 (2), IIaA14G2R1 (1), IIaA13G2R1 (1)
C. parvum (67), C. bovis (6)
C. parvum (74), 12 C. bovis (12), C.
ryanae (3), atypical isolate (1)
C. parvum (21), 1 C. ryanae (1)
Hungary 22IIaA16G1R1 (15), IIaA17G1R1 (3), IIaA18G1R1 (1), IIdA19G1 (1),
IIaA15G2R1 (43), IIaA14G2R1 (2), IIaA17G2R1 (2), IIaA18G2R1
(2), IIaA21R1 (1), IIaA22G1R1 (1), IIaA16G1R1 (1),
IIaA15G2R1 (27), IIaA16G1R1 (6), IIaA13R1 (5), IIaA16R1 (3),
IIIA16R2 (2), IIlA18R2 (2)
C. parvum (134)
Slovenia 51C. parvum (45), C. bovis (3),
C. ryanae (3)
C. parvum (1)
C. parvum (160)
IIaA15G2R1 (89), IIaA17G1R1 (14), IIaA16G3R1 (6), IIaA13G2R1
(2), IIaA14G2R1 (2), IIaA17G2R1 (2), IIaA18G4R1 (2), IIaA18R1
(2), IIaA19G2R1 (2), IIaA11G2R1 (1), IIaA12G2R1 (1),
IIaA16G1R1 (1), IIaA16G2R1 (1), IIaA18G3R1 (1), IIaA19G1R1
(1), IIaA21G3R1 (1), IIlA24 (1)
IIaA15G2R1 (106), IIaA16G3R1 (14), IIaA18G3R1 (8), IIaA16G2R1
(4), IIaA19G3R1 (2), IIdA23G1 (2)
IIaA15G2R1 (26), IIaA13G1R1 (1)
IIaA18G1R1 (2), IIaA21G1R1 (2), IIdA20G1e (2), IIdA23G1 (2),
IIdA16G1c (1), IIaA16G1R1 (1)
IIaA18G3R1 (120), IIaA15G2R1 (28), IIaA17G2R1 (19), IIaA19G4R1
(15), IIaA20G3R1 (6), IIaA19G3R1 (5), IIaA17G3R1 (5),
IIaA20G5R1 (3), IIaA18G2R1 (2), IIaA20G2R1 (2), IIaA16G3R1
(1), IIaA17G1R1 (1), IIaA18R1 (1), IIaA19G2R (1), IIaA20G4R1
(1), IIaA21G2R1 (1), IIa-unknown (5)
IIaA15G2R1 (35), IIaA17G1R1 (7), IIaA16G3R1 (4), IIaA19G1R1
(2), IIaA18G1R1 (2), IIaA14G2R1 (1)
IIaA17G1R1 (10), IIaA15G2R1 (3)
Spain149 C. parvum (147), C. bovis (2) 30
C. parvum (27)
C. parvum (15), C. bovis (54),
C. ryanae (4)
C. parvum (213), C. bovis (8),
C. ryanae (3)
England 54C. parvum (50), C. bovis (3),
C. ryanane (1)
C. parvum (29), C. bovis (2)
C. parvum (22), C. bovis (10)
C. parvum (7)
C. parvum (24), C. andersoni (2)
C. parvum (33)
C. parvum (7)
C. parvum (124)
England and Wales
IIdA20G1 (23), IIaA15G2R1 (1)
IIaA15G2R1 (2), IIaA18G3R1 (5)
IIaA19G3R1a (80), IIaA20G2R1 (9), IIaA20G4R1 (9), IIaA23G3R1
(9), IIaA16G3R1 (7), IIaA18G2R1a (2), IIaA18G2R1b (1),
IIaA18G4R1 (1), IIaA19G3R1b (1), IIaA20G3R1 (1), IIaA21G3R1
(1), IIaA17G2R1 (1)
3 C. parvum (3)
C. parvum (32), C. bovis (1)
C. bovis (4), 1 C. ryanae (1)
C. bovis (5), C. parvum (1)
C. parvum (138)
C. parvum (6), C. bovis (1), C. ryanae (1)
C. parvum (34), C. bovis (4),
C. andersoni (1), C. parvum
aMixed infections are indicated with a hyphen.
bCryptosporidium-positive samples were collected from animals younger than 10 weeks of age.
cThe 134 samples were positive for oocysts by microscopic analysis.
dThis isolate was from a ?3-month-old calf.
eThe mean age of cattle was 13 days (range, 2 to 125 days), with only 7% of cattle older than 3 weeks.
1080 WANG ET AL.J. CLIN. MICROBIOL.
predominant subtype family of C. parvum in calves. Within the
IIa family, the subtype IIaA15G2R1 was shown to be the most
prevalent C. parvum subtype in preweaned dairy calves in the
United States, Canada, Belgium, Netherlands, Spain, Portugal,
Slovenia, Germany, and Japan (Table 2). Several other C.
parvum IIa subtypes were predominant in other countries (Ta-
ble 2). In the present study, all 67 gp60 PCR positive samples
belonged to one single subtype (IIdA19G1), which was de-
tected previously in one case in Hungary (28). There was no
nucleotide difference between the IIdA19G1 isolates of two
sources. The source of C. parvum in calves in Henan is unclear.
Generally, IId is not as common as the major zoonotic subtype
family IIa (44). Thus, only for Hungary (IIdA19G1 and
IIdA22G1), Belgium (IIdA22G1), Portugal (IIdA17G1),
Spain (IIdA23G1), Sweden (IIdA20G1e, IIdA23G1, and
IIdA16G1c), Germany (IIdA22G1), Egypt (IIdA20G1), and
Serbia and Montenegro (IIdA18G1b), members of this sub-
type family were recorded in small numbers of dairy calves (3,
4, 23, 30, 33, 44). Previously, the IId subtype family of C.
parvum was known mostly as a parasite of sheep and goats in
southern Europe (29). The only exception is a recent study in
Egypt, in which it was shown that 23 of 24 of C. parvum-
infected preweaned dairy calves had excreted IIdA20G1 (3). In
China, 10 C. parvum isolates from pet Siberian chipmunks and
hamsters in Henan were identified as IIdA15G1 (22). Despite
its rare occurrence in dairy cattle, the IId subtype family is
common in humans in the Middle East (14, 37), and has also
been reported in a few human cases in Portugal, Ireland, the
United Kingdom, Belgium, the Netherlands, and Australia
(44). Thus, parasites of the subtype IId may be responsible for
zoonotic transmission of cryptosporidiosis in some areas.
The results of the present study suggested that there was no
significant seasonal difference in Cryptosporidium infection in
preweaned calves. However, there appeared to be a seasonal
shift in the dominant Cryptosporidium species in preweaned
calves, with C. parvum peaking in summer and C. bovis peaking
in autumn and winter. This finding is somewhat different from
the recent observation of C. bovis dominance in summer and C.
parvum dominance in spring and winter in dairy cattle in New
York (38). More large studies in different areas are needed to
determine whether these differences are attributable to differ-
ence in animal management.
In conclusion, our findings suggest that the transmission of
Cryptosporidium spp. in preweaned calves in China is probably
different from that in other countries at both the species and
subtype levels. Although C. parvum is common in preweaned
dairy calves, the public health significance of C. parvum iden-
tified here is still unclear, since no C. parvum infection has
been seen in humans in China (27, 42). In other countries the
IIa subtype family has been a more important zoonotic patho-
gen than the IId family prevalent in the present study. There-
fore, additional molecular epidemiologic studies in cattle and
humans are needed to understand the transmission dynamics
of Cryptosporidium spp. in China and the public health signif-
icance of C. parvum in cattle.
This study was supported in part by the Ph.D. Program Funds of the
Ministry of Education of China (no. 20094105110003), Key National
Science and Technology Specific Projects (no. 2008ZX10004-011), the
National Natural Science Foundation of China (no. 30771881,
30871863, and 30928019), and the Henan Province Special Fund of
Public Welfare (no. 81100912300).
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