Genetic characterizations of Cryptosporidium spp. from children with or without diarrhea in Wenzhou, China: high probability of zoonotic transmission

Abstract

Background
Cryptosporidium is a highly pathogenic parasite responsible for diarrhea in children worldwide. Here, the epidemiological status and genetic characteristics of Cryptosporidium in children with or without diarrhea were investigated with tracking of potential sources in Wenzhou City, China.

Methods
A total of 1032 children were recruited, 684 of whom had diarrhea and 348 without, from Yuying Children’s Hospital in Wenzhou, China. Samples of stool were collected from each participant, followed by extraction of DNA, genotyping, and molecular identification of Cryptosporidium species and subtypes.

Results
Twenty-two of the 1032 (2.1%) children were infected with Cryptosporidium spp. with 2.5% (17/684) and 1.4% (5/348) in diarrhoeic and asymptomatic children, respectively. Four Cryptosporidium species were identified, including C. parvum (68.2%; 15/22), C. felis (13.6%; 3/22), C. viatorum (9.1%; 2/22), and C. baileyi (9.1%; 2/22). Two C. parvum subtypes named IIdA19G1 (n = 14) and IInA10 (n = 1), and one each of C. felis (XIXa) and C. viatorum (XVaA3g) subtype was found as well.

Conclusions
This is the first research that identified Cryptosporidium in children of Wenzhou, China, using PCR. Identification of zoonotic C. parvum, C. felis, C. viatorum, and their subtypes indicate potential cross-species transmission of Cryptosporidium between children and animals. Additionally, the presence of C. baileyi in children suggests that this species has a wider host range than previously believed and that it possesses the capacity to infect humans.

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Zhao et al. BMC Microbiology (2024) 24:113
https://doi.org/10.1186/s12866-024-03273-w BMC Microbiology
†Wei Zhao, Guangxu Ren and Weiyan Jiang contributed equally to
this work.
*Correspondence:
Yanyan Jiang
jiangyy@nipd.chinacdc.cn
Gang Lu
ganglu2018@163.com
Huicong Huang
hhc@wmu.edu.cn
Full list of author information is available at the end of the article
Abstract
Background Cryptosporidium is a highly pathogenic parasite responsible for diarrhea in children worldwide. Here,
the epidemiological status and genetic characteristics of Cryptosporidium in children with or without diarrhea were
investigated with tracking of potential sources in Wenzhou City, China.
Methods A total of 1032 children were recruited, 684 of whom had diarrhea and 348 without, from Yuying Children’s
Hospital in Wenzhou, China. Samples of stool were collected from each participant, followed by extraction of DNA,
genotyping, and molecular identication of Cryptosporidium species and subtypes.
Results Twenty-two of the 1032 (2.1%) children were infected with Cryptosporidium spp. with 2.5% (17/684) and 1.4%
(5/348) in diarrhoeic and asymptomatic children, respectively. Four Cryptosporidium species were identied, including
C. parvum (68.2%; 15/22), C. felis (13.6%; 3/22), C. viatorum (9.1%; 2/22), and C. baileyi (9.1%; 2/22). Two C. parvum
subtypes named IIdA19G1 (n = 14) and IInA10 (n = 1), and one each of C. felis (XIXa) and C. viatorum (XVaA3g) subtype
was found as well.
Conclusions This is the rst research that identied Cryptosporidium in children of Wenzhou, China, using PCR.
Identication of zoonotic C. parvum, C. felis, C. viatorum, and their subtypes indicate potential cross-species
transmission of Cryptosporidium between children and animals. Additionally, the presence of C. baileyi in children
suggests that this species has a wider host range than previously believed and that it possesses the capacity to infect
humans.
Genetic characterizations of Cryptosporidium
spp. from children with or without diarrhea
in Wenzhou, China: high probability
of zoonotic transmission
WeiZhao1†, GuangxuRen2,3,4†, WeiyanJiang5†, LongWang1, JiayangWang1, ZhongyingYuan6, LanzhuYan7,
YongtaiLi1, YanbinSun1, XinjieXue1, YanyanJiang6*, GangLu2,3,4* and HuicongHuang1*
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 2 of 9
Zhao et al. BMC Microbiology (2024) 24:113
Background
Cryptosporidium is a class of protozoan parasite with
the ability to infect epithelial cells of the gastrointestinal
tract of vertebrate species [1]. Cryptosporidiosis is often
a self-limiting infection in individuals with a fully func-
tioning immune system, but it can lead to severe diarrhea
and potentially cause death in individuals with Acquired
Immunodeciency Syndrome (AIDS), infants, and other
immunocompromised or decient individuals [2]. Cryp-
tosporidiosis is a signicant contributor to mortality
associated with moderate to severe diarrhea in children
residing in low- and middle-income countries and rep-
resents an important but often underestimated public
health threat in developed countries [3].
Cryptosporidium can infect a diverse range of animal
species and is distributed globally. e zoonotic trans-
mission of diseases from animals to humans is seen as a
signicant concern for public health [4]. e infectious
stage of Cryptosporidium, known as the oocyst, favors
direct transmission since it becomes infectious as soon as
it is excreted with the host’s feces [5]. Oocysts can endure
the surrounding environment and resist conventional
water treatment methods. ey may be transferred to
people either by interacting directly with animals or their
waste products, or by consuming water and food that
have been contaminated with oocysts [6]. e examina-
tion of sporadic instances and epidemics of cryptospo-
ridiosis has improved the present knowledge of the risk
factors and sources of infection.
In recent decades, there have been notable advance-
ments in molecular typing techniques, which have played
a crucial role in enhancing the current knowledge of epi-
demiology regarding cryptosporidiosis across various
seasonal, geographical, and socioeconomic contexts [7].
Currently, the most commonly used method for identi-
cation of the species is by the small subunit ribosomal
RNA (SSU rRNA) sequence with both highly variable and
conserved sequences [7]. At present, there have been a
total of 48 accepted species and more than 120 genotypes
of Cryptosporidium. Of these, 21 species and two geno-
types have been linked to human infection; C. hominis
and C. parvum represent around 95% of human infec-
tion cases [5]. Although humans are the main hosts of
C. hominis, this parasite is also reported in domestic and
wild animals, including sheep, goats, and cattle [5, 8]. C.
parvum mainly infects artiodactyls and humans, but it is
also frequently reported in various rodents and wild ani-
mals [5]. A number of subtyping instruments based on
the glycoprotein 60-kDa (gp60) gene were developed to
identify the human source of Cryptosporidium infection
precisely [9–11]. e proposed and widely adopted gp60
nomenclature system has greatly facilitated the analysis
of global epidemiology, as the variability in the sequence
of this marker has been instrumental in inferring trans-
mission routes [12]. However, there is still uncertainty
regarding the epidemiology of Cryptosporidium and
its spread in humans, particularly in underdeveloped
regions of the world, requiring further research.
A cumulative count of approximately 6,000 human
cases of cryptosporidiosis has been documented in 27
provinces of China [13], yet data involving molecular
studies remains limited. Out of the total of 68 studies
that were undertaken, only 10 focused on the molecu-
lar analysis of Cryptosporidium species [13–16]. Even
though fewer than 300 cases were genetically character-
ised, a total of nine distinct species of Cryptosporidium
were detected, including C. andersoni, C. felis, C. par-
vum, C. hominis, C. viatorum, C. meleagridis, C. canis, C.
occultus, and C. suis [13–16]. e isolates were subjected
to further classication into subtypes by the utilization
of sequence analysis of the gp60 gene, including fteen
C. hominis subtypes, three C. parvum subtypes, four C.
meleagridis subtypes, and one C. viatorum subtype [13–
16]. However, molecular data from many regions, such as
Wenzhou, remain unavailable. is study is the rst base-
line epidemiological study of human Cryptosporidium in
Wenzhou, focusing on exploring the characteristics of
Cryptosporidium in children with or without diarrhea.
Results
Prevalence of Cryptosporidium
According to the analysis of the SSU rRNA gene, Cryp-
tosporidium was identied in 2.1% (22/1032, 95% CI:
1.4–3.2%) of the samples, with 17/684 (2.5%, 95% CI: 1.6-
4.0%) in the patients with diarrhea and 5/348 (1.4%, 95%
CI: 0.6–3.3%) in the asymptomatic population (Table1).
e prevalence of Cryptosporidium in individuals experi-
encing diarrhea was found to be greater in comparison to
the prevalence seen in the asymptomatic group, but there
was no statistical dierence (χ2 = 1.216; p = 0.363). In the
group of children suering from diarrhea, the prevalence
was found to be 2.9% (11/376, 95% CI: 1.6–5.1%) among
boys and 1.9% (6/308, 95% CI: 0.9–4.2%) among girls.
Moreover, in the asymptomatic group, the prevalence
of Cryptosporidium was 2.1% (4/195, 95% CI: 0.8–5.2%)
and 0.7% (1/153, 95% CI: 0.1–3.6%) for boys and girls,
respectively. Statistical analysis revealed no signicant
dierence in the prevalence rate of Cryptosporidium was
observed through paired comparisons between boys and
girls (χ2 = 0.667; p = 0.414 for diarrhea group and p = 0.390
for asymptomatic group).
Keywords Cryptosporidium, Children, Genetic characteristics, China
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Zhao et al. BMC Microbiology (2024) 24:113
Genetic characterization based on the SSU rRNA gene
e present work involved the investigation of 22 Cryp-
tosporidium isolates, whereby the sequencing analysis
revealed the presence of four distinct Cryptosporidium
species, including C. viatorum, C. parvum, C. felis, and
C. baileyi (Table1). Among the examined specimens, it
was seen that C. parvum exhibited the highest preva-
lence rate, accounting for 21.3% (15/22). is was fol-
lowed by C. felis, which had a prevalence rate of 10.6%
(3/22). Additionally, C. viatorum and C. baileyi were
found to have equal prevalence rates of 2.1% (1/47) each.
C. parvum and C. viatorum were detected among both
the diarrhea patients and the asymptomatic population,
while C. felis was only found in diarrhea patients, and C.
baileyi was observed only in the asymptomatic individu-
als (Table1).
Fifteen isolates of C. parvum were represented by
seven sequences (OR815985 to OR815991). ese
sequences exhibited a high level of similarity, rang-
ing from 99.41 to 100% when compared to the pub-
lished C. parvum sequence (L16996). Among the seven
sequences obtained, a total of seven polymorphic sites
were also identied (Table2). Two out of the three C. felis
sequences (OR815984) acquired in this study exhibited
complete similarity to a sequence (MN394123) identied
in cat specimens from Turkey. e remaining sequence
(OR815983) showed a 99.08% similarity to MN394123,
diering by only three bases. e two C. viatorum iso-
lates obtained in this study shared a same sequence
(OR815982) which exhibited identical sequences to the
human-derived sequence MW014315 from China. Nei-
ther sequences of C. baileyi (OR815992 and OR815993)
obtained here had been described, but they exhib-
ited only minor dierences, with a single base dier-
ence at 240 and 359 sites compared with MH028033,
respectively.
Subtyping of C. parvum, C. viatorum, C. felis gp60 gene
e amplication of the gp60 gene was achieved in all
15 isolates of C. parvum, as well as in two out of the
three isolates of C. felis and two isolates of C. viatorum.
Two representative gp60 gene sequences were observed
among the 15 C. parvum isolates, comprising OR963532
(n = 14) and OR963531 (n = 1). e observed sequences
(OR963532 and OR963531) exhibited complete simi-
larity, reaching 100%, with the subtypes IIdA19G1
(KM199738) and IInA10 (KU852717), respectively.
e presence of subtype IInA10 was identied in a boy
with symptoms of diarrhea. In the present investiga-
tion, it was shown that the sequence (OR963530) of the
two C. felis isolates exhibited a similarity of 99.81% with
the sequences MW351828 (XIXa-89) and MT458682
(XIXa-E1). e sequence (OR963529) of two isolates of
C. viatorum examined in this investigation exhibited a
Table 1 Prevalence and species distribution of Cryptosporidium among children from Wenzhou of China by counties clinical
symptoms and gender
Clinical symptoms No.Positive/No. Examined (%,
95%CI)
Cryptosporidium species (n)/gp60 subtypes (n)
Diarrhea
Boy 11/376 (2.9, 1.6–5.1) C. parvum (8)/IIdA19G1 (7), IInA10 (1); C. viatorum (1)XVaA3g (1); C. baileyi (2)/-
Girl 6/308 (1.9, 0.9–4.2) C. parvum (6)/IIdA19G1 (6)
Subtotal 17/684 (2.5, 1.6-4.0) C. parvum (14)/IIdA19G1 (13), IInA10 (1); C. viatorum (1)/XVaA3g (1); C. baileyi
(2)/-
Non-Diarrhea
Boy 4/195 (2.1, 0.8–5.2) C. parvum (1)/ IIdA19G1 (1); C. felis (2)/XIXa (2), C. viatorum (1)/XVaA3g (1)
Girl 1/153 (0.7, 0.1–3.6) C. felis (1)/XIXa (1)
Subtotal 5/348 (1.4, 0.6–3.3) C. parvum (1)/ IIdA19G1 (1); C. felis (3)/XIXa (3); C. viatorum (1)/XVaA3g (1)
Total 22/1032 (2.1, 1.4–3.2) C. parvum (15)/IIdA19G1 (14), IInA10 (1); C. viatorum (2)/XVaA3g (2); C. felis (3)/
XIXa (3); C. baileyi (2)/-
Table 2 Variation at seven polymorphic sites within SSU rRNA gene sequences of human-derived Cryptosporidium parvum isolates
obtained in the present study
GenBank accession no. Nucleotide at position (relative to the start of that sequence)
82 203 232 283 309 311 331
OR815985 G G T A T A T
OR815986 G G T A T C T
OR815987 G A T A T T T
OR815988 A G T A T T T
OR815989 A G T A T T C
OR815990 A G T C T T T
OR815991 G G A A C C T
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Zhao et al. BMC Microbiology (2024) 24:113
complete similarity of 100% to the sequence of XVaA3g
(MK796004).
Discussion
is work represents the rst molecular investigation of
Cryptosporidium among children residing in Wenzhou,
China. e ndings indicate that the average rate of
infection was 2.1%, a rate that is comparatively lower than
the combined prevalence of Cryptosporidium infection
observed in children across China (2.9%) [14]. Variations
are reported in the prevalence of Cryptosporidium infec-
tion among children throughout dierent provinces in
China. As an example, the prevalence of Cryptosporidium
infection in Henan province was recorded at 0.9% [14].
However, in Inner Mongolia, it exhibited a much higher
rate of 8.9% [14]. Overall, the prevalence of Cryptosporid-
ium infection among Chinese children is notably lower
compared to rates observed in children from certain
developing countries. For instance, Bangladesh reported
a rate of 77% [18], India’s under-2-year-olds exhibited a
rate of 92.4% [19], rural western Kenya’s 6 to 24-month-
olds had a rate of 88.7% [20], and children under 2 years
old from eight countries across Africa, Asia, and South
America demonstrated an approximate rate of 65% [21].
Furthermore, when comparing China’s prevalence to cer-
tain developed countries, the rates are also lower, such
as Australia’s Aboriginal children had a rate of 8.2% [22],
and children with diarrhea in Switzerland had a rate of
5.5% [23]. However, reports from asymptomatic children
in developed countries reveal relatively low prevalence
rates, such as 0.9% in Madrid, Spain [24], 1.3% among
children under 5 years old in Canada [25], and 1.3%
among asymptomatic preschool children in the UK [26].
Germany, has a prevalence level similar to our study, at
2.5% [27]. ese disparities could be attributed to various
factors, including the environment, sanitary conditions,
and living habits of dierent countries. We hypothesize
that the relatively lower occurrence of Cryptosporidium
infection in China can be attributed to the widespread
access to safe drinking water, with approximately 90%
of drinking water being boiled in the country [28]. is
practice signicantly reduces the risk of ingesting infec-
tious Cryptosporidium oocysts, thereby mitigating the
potential harm. However, it is imperative not to over-
look this issue, as incidences of infection persist among
Chinese children. It is noteworthy that the detection of
Cryptosporidium in asymptomatic children also has a
signicant public health implications. erefore, regular
monitoring of Cryptosporidium infection in the popula-
tion should be continued.
Cryptosporidium infection is well recognized as a
prominent etiological factor contributing to the occur-
rence of diarrhea in pediatric populations [29]. In this
study, the proportion of children with diarrhea was 2.5%,
which was higher than non-diarrheal children. Other
studies have drawn similar conclusions. For example, in
Saudi Arabia, the proportion of asymptomatic children
infected with Cryptosporidium was 4.7%, while the chil-
dren with diarrhea exhibited a proportion of 32.0% [30].
In Uganda, 25.0% of diarrheal children were infected with
C. parvum, while only 8.5% of non-diarrheal children
were infected with the same pathogen [31]. is obser-
vation is present not only among pediatric populations
but also among adults. e prevalence of Cryptosporid-
ium infection in individuals suering from diarrhea was
notably higher compared to those without diarrhea [29].
Consequently, it is imperative to enhance the detection
of Cryptosporidium in individuals with diarrhea, as this
would facilitate a more comprehensive assessment of its
impact and enable proactive clinical management of diar-
rhea-related complications.
is study found no statistically signicant disparity
in the prevalence of Cryptosporidium infection between
boys and girls. However, it is worth noting that infec-
tion rates were somewhat higher among males in both
groups. Several other studies have also shown that sex is
not the main cause of the dierences in Cryptosporidium
infection rates [14, 16].
e ndings of this investigation revealed that C. par-
vum was the most prevalent Cryptosporidium species
identied in the studied population with15/22 (68.2%)
of the positive ndings. C. parvum infects an extensive
range of hosts, including ungulates and wildlife, and is
recognized as the primary zoonotic species aecting
humans [4]. is is especially signicant in rural regions
where individuals frequently come into touch with live-
stock [5]. In this study, two subtypes were identied,
including IIdA19G1 and IInA10. e IInA10 subtype
was rst found in China here and is presently limited to
the Tanzania/Sweden population [32]. However, there is
a lack of data on infected animals, making it dicult to
determine the exact source of infection. ere exist two
distinct subtypes within family IIn, namely IInA8 and
IInA10, both of which lack any documented instances of
infected animals [32, 33]. It is not possible to determine
whether this genotype can spread between humans and
animals, so further investigation of the data is needed to
clarify its host range and to complete the traceability of
the source of infection. Despite the scarcity of data on
C. parvum gp60 subtypes in the population of China,
the existing evidence has substantiated the ability of
the IIdA19G1 subtype to infect human beings [13, 34].
Moreover, this particular subtype has been identied in
children and individuals aicted with AIDS in China
[13–15, 35]. Simultaneously, it is observed that the preva-
lence of C. parvum infection in animals originating from
China is extensive, with a notable abundance of gp60 sub-
types. Particularly, the IIdA19G1 subtype shows a high
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Zhao et al. BMC Microbiology (2024) 24:113
frequency among animals in China and demonstrates a
broad geographic dispersion [34]. e subtype IIdA19G1
has been identied as a signicant contributor to the ris-
ing fatality rates observed in several cryptosporidiosis
outbreaks in dairy calves in China [34, 36, 37]. Hence,
it is imperative to enhance the routine surveillance of
individuals and animals, taking into account the aspects
of animal and human health. is should be particularly
emphasized for vulnerable populations such as children
and those aected by AIDS.
In the present study, C. felis was found in three stool
samples. C. felis is typically harbored by feline hosts and
has been documented in HIV-infected individuals in
Ethiopia [38], both HIV-infected and non-infected indi-
viduals in Nigeria [39], and children under the age of 5
in Kenya [40]. Research ndings indicate that there is a
signicant likelihood of zoonotic transmission of C. felis.
e parasite is found in other animal species, such as
non-human primates, foxes, horses, and calves, in addi-
tion to cats and people, indicating the potential for zoo-
notic transmission [10, 41]. Furthermore, it was observed
that there are ve distinct subtype families of C. felis,
namely XIXa, XIXb, XIXc, XIXd, and XIXe. Notably,
two of these subtypes (XIXa and XIXb) were shown to
be present in both humans and cats, providing evidence
for the possibility of zoonotic transmission [10, 41]. Con-
versely, the other three subtypes (XXc, XIXd and XIXe)
may be specic for human-to-human transmission [41].
e two C. felis identied in the current research belong
to the XIXa subtype, indicating a risk of cat-to-human
transmission. Pet owners should, therefore, be educated
regarding the risk of cryptosporidiosis from their pet
cats.
In this study, two children were infected with C. via-
torum, a species originally reported in 2012 in travel-
ers returning from India [42]. To date, C. viatorum has
been documented in the human population throughout
a multitude of nations, surpassing a total of 13 coun-
tries, including China [43–45]. e species was previ-
ously believed to only aect humans due to its infrequent
occurrence in animals. However, recent research con-
ducted in Australia and China has shown the presence
of this species in wild rats [46–48]. ese ndings imply
that C. viatorum has the potential to transmit between
rodents and humans. Subtype partitioning supports the
potential for animal-borne transmission of C. viato-
rum between rodents and humans due to the presence
of its three subtypes (XVaA3g, XVaA3h, and XVcA2G1)
in both humans and rodents [43]. e present nding
showed that the XVaA3g was present in two children,
further supporting the hypothesis that C. viatorum can
complete reciprocal transmission between humans and
rodents. To gain a more comprehensive understanding
of the distribution of rodents as hosts for C. viatorum, it
is essential to conduct screenings throughout wider geo-
graphic regions.
Although C. baileyi has already been found in humans,
its identication of two children here is nevertheless
intriguing [49]. e nding suggests the possibility of
human infection from avian to Cryptosporidium oocysts.
C. baileyi, which is recognized as a parasite restricted
to avian species, can infect over 20 dierent bird hosts,
including poultry, domesticated animals, and wild birds
[50]. A recent study showed that the absolute infection
rate of C. baileyi in broilers was as high as 96.1% [51].
us, humans coming in contact with poultry chicken
or with eggs contaminated with chicken feces may have
played a role in the transmission of Cryptosporidium
infection. Additional studies are required to determine
the potential of C. baileyi to infect humans and to under-
stand its transmission patterns. It is anticipated that fur-
ther research in this eld will bring more enlightenment
to public health.
Conclusions
is work was the rst molecular examination conducted
on Cryptosporidium infection among children residing
in Wenzhou, China. e mean prevalence of Cryptospo-
ridium in children was found to be 2.1%. Nonetheless, it
was observed that children presenting with diarrhea were
more likely to have Cryptosporidium compared with
asymptomatic children. A total of four distinct species of
Cryptosporidium were found, namely C. parvum, C. felis,
C. viatorum and C. baileyi. Among them, C. parvum and
its subtype, IIdA19G1, have well-known zoonotic prop-
erties. Furthermore, the identication of subtypes XIXa
of C. felis and XVaA3g of C. viatorum further revealed
the possibility of transmission of cryptosporidiosis from
animals to humans. is work was the rst to document
the prevalence of C. baileyi infection in Chinese children.
is study presents compelling data on the potential
origin of Cryptosporidium infection among children in
Wenzhou, particularly emphasizing the potential trans-
mission via animals, including cats, rats, and chickens. It
is imperative to adopt a heightened level of vigilance in
implementing ecacious preventive measures aimed at
mitigating the likelihood of infection in children.
Methods
Sample Collection
From March 2021 to January 2022, a total of 1032 chil-
dren were recruited at Yuying Children’s Hospital in
Wenzhou, China (Fig. 1). Among these children, 348
were found to be asymptomatic, while 684 had symp-
toms of diarrhea (Table1). e parents/guardians were
guided for the collection of the fecal material in a plastic
fecal collector with appropriate labeling, having the date
of collection and relevant details of the patient, such as
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Zhao et al. BMC Microbiology (2024) 24:113
age and sex. Following the collecting process, the samples
were then kept at 4°C.
DNA extraction and PCR amplication
e genomic DNA was obtained from fecal material
weighing between 180 and 200mg using a QIAamp DNA
Stool Mini Kit (QIAgen, Hilden, Germany). following
the instructions provided by the manufacturer. e pres-
ence of Cryptosporidium was identied in the collected
DNA by the utilization of nested PCR amplication tar-
geting a specic region of the Cryptosporidium genome,
approximately ∼ 587 bp of the partial SSU rRNA gene
fragment. e primers used were described previously in
a study by Ryan et al. (2003) [17]. e PCR amplications
in this study were performed using TaKaRa Taq DNA
Polymerase (TaKaRa Bio Inc., Tokyo, Japan). A negative
control consisting of dH2O was included, while a posi-
tive control was prepared using DNA extracted from C.
xiaoi obtained from goats. e isolates of C. parvum, C.
felis, and C. viatorum were subjected to subtyping using
nested PCR of the gp60 gene [9–11]. e primers and
PCR program settings utilized in the current investiga-
tion are shown in Table3. e PCR products underwent
electrophoresis on a 1.5% agarose gel and were observed
using a Gel Doc EZ UV-gel imaging equipment manu-
factured by Bio-Rad Inc. (USA). To facilitate visibility,
GelRed (Biotium Inc., Hayward, CA) was used to dye the
gel.
Fig. 1 A map of the sampling hospital location in Wenzhou, China. The gure was originally designed by the authors under the software ArcGIS 10.4. The
original vector diagram imported in ArcGIS was adapted from National Geomatics Center of China (http://www.ngcc.cn). The map has been modied and
assembled according topermission and attribution guidelines using the softwares of Microsoft PowerPoint 2003 and Adobe Photoshop CS6.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 7 of 9
Zhao et al. BMC Microbiology (2024) 24:113
Sequence and statistical analyses
All PCR products of the expected size were successfully
puried using the DNA Gel Purication Kit (Sangon,
Shanghai, China) and sent for a bi-directional Sanger
sequencing analysis (performed by Sangon, Guangzhou,
China). Sequencing was conducted using the BigDy-
eTerminator v3.1 Cycle Sequencing Kit (from Applied
Bio systems, Carlsbad, CA, USA) on an ABI Prism 3730
XL DNA Analyzer. DNASTAR Lasergene EditSeq v7.1.0
(http://www.dnastar.com/) was employed for editing the
generated sequences, while Clustal X v2.1 (http://www.
clustal.org/) was utilized for aligning them with reference
sequences obtained from GenBank.
e data analysis was conducted using SPSS version
22.0 (SPSS Inc., IL, USA). e chi-square test and 95%
condence intervals (CIs) were employed to compare the
prevalence of Cryptosporidium between diarrheic and
asymptomatic individuals, as well as between boys and
girls. A P-value less than 0.05 was considered to be statis-
tically signicant.
Nucleotide sequence accession numbers
e representative nucleotide sequences obtained in the
present study were deposited in GenBank database under
the following accession nos.: OR815982 to OR815993
(SSU), OR963529 to OR963532 (gp60).
Abbreviations
SSU rRNA Small subunit ribosomal RNA
gp60 Glycoprotein 60-kDa
Acknowledgements
Not applicable.
Author contributions
Conceived and designed the experiments: HH, GL, YJ. Performed the
experiments: WZ, GR, WJ, LW, JW. Analyzed the data: WZ, GR, YL, YS, XX, LY.
Contributed reagents/materials/analysis tools: ZY, YJ, GL. Wrote the paper: WZ.
All authors reviewed the manuscript.
Funding
This work was supported by the Department of Education Scientic Research
Project of Zhejiang (Y202249687), the Basic scientic research project
of Wenzhou (Y2023070), National Natural Science Foundation of China
(82273693). The funding sponsors had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Data availability
The representative nucleotide sequences obtained in the present study were
deposited in GenBank database under the following accession nos.: OR815982
to OR815993 (SSU), OR963529 to OR963532 (gp60).
Declarations
Ethics approval and consent to participate
The necessary ethical approvals for these investigations were received from
the Ethics Committees of Wenzhou Medical University under the reference
number SCILLSC-2021-01. Each participant, including the 17-year-old
participants, provided written informed consent. For the below the age of 17
years, consent was gained from their parents or guardian. Prior to obtaining
consent, participants or their parents/guardians received information about
the study’s objectives and methods.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1Department of Parasitology, School of Basic Medical Sciences, Wenzhou
Medical University, Wenzhou, Zhejiang 325035, China
2Department of Pathogenic Biology, Hainan Medical University, Haikou,
Hainan, China
3Hainan Medical University-The University of Hong Kong Joint Laboratory
of Tropical Infectious Diseases, Hainan Medical University, Haikou, Hainan,
China
4Key Laboratory of Tropical Translational Medicine of Ministry of
Education, Hainan Medical University, Haikou 571199, China
5The Second School of Medical, Aliated Hospital and Yuying Children’s
Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
6National Institute of Parasitic Diseases, Chinese Center for Disease
Control and Prevention (Chinese Center for Tropical Diseases Research),
NHC Key Laboratory of Parasite and Vector Biology, WHO Collaborating
Centre for Tropical Diseases, National Center for International Research on
Tropical Diseases, Shanghai 200025, China
7Department of Laboratory and Pathology, Hebei Provincial Corps
Hospital of Chinese People’s Armed Police Force, Shijiazhuang,
Hebei 050081, China
Table 3 The primers employed for identifying Cryptosporidium spp., and subtypes of C. parvum, C. viatorum, C. felis in the present study
Usage (s) Primer sequence (5′ to 3′) Fragment length (bp) Annealing temperature (°C) Gene Ref
Cryptosporidium genus
specic
18SiCF2: G A C A T A T C A T T C A A G T T T C T G A C
18SiCR2: C T G A A G G A G T A A G G A A C A A C C
18SiCF1: C C T A T C A G C T T T A G A C G G T A G G
18SiCR1: T C T A A G A A T T T C A C C T C T G A C T G
∼ 587 58
58
SSU rRNA [17]
Subtyping of C. parvum AGP-F1: A T A G T C T C C G C T G T A T T C
AGP-R1: G G A A G G A A C G A T G T A T C T
AGP-F2: T C C G C T G T A T T C T C A G C C
AGP-R2: G C A G A G G A A C C A G C A T C
∼ 850 55
58
gp60 [9]
Subtyping of C. viatorum CviatF2: T T C A T T C T G A C C C C T T C A T A G
CviatR5: G T C T C C T G A A T C T C T G C T T A C T C
CviatF3: G A G A T T G T C A C T C A T C A T C G T A C
CviatR8: C T A C A C G T A A A A T A A T T C G C G A C
∼ 950 55
52
gp60 [11]
Subtyping of C. felis GP60CF_F1: T T T C C G T T A T T G T T G C A G T T G C A
GP60CF_R1: A T C G G A A T C C C A C C A T C G AAC
GP60CF_F2: G G G C G T T C T G A A G G A T G T AA
GP60CF_R2: C G G T G G T C T C C T C A G T C T T C
∼ 900 55
55
gp60 [10]
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 8 of 9
Zhao et al. BMC Microbiology (2024) 24:113
Received: 29 December 2023 / Accepted: 22 March 2024
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