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\begin{document}

\title{Cryptosporidiosis outbreak caused by Cryptosporidium parvum subtype
IIdA20G1 in neonatal calves}



\author[1]{Zhenjie Zhang}%
\author[1]{Dongle Su}%
\author[1]{Xinan Meng}%
\author[1]{Ruobing Liang}%
\author[1]{Weijian Wang}%
\author[1]{Na Li}%
\author[1]{Yaqiong Guo}%
\author[2]{Aizhen Guo}%
\author[3]{Shujing Li}%
\author[3]{Zengyuan Zhao}%
\author[1]{Lihua Xiao}%
\author[1]{Yaoyu Feng}%
\affil[1]{Center for Emerging and Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University,}%
\affil[2]{State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University,}%
\affil[3]{Shijiazhuang Tianquan Elite Breeding Dairy Cow Co., LTD.,}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Cryptosporidium parvum is a major zoonotic pathogen responsible for
outbreaks of severe diarrhea in humans and calves. Almost all
investigations of cryptosporidiosis outbreaks caused by C. parvum have
focused on its IIa subtype family in industrialized nations. From
December 2018 to April 2019, approximately 200 neonatal calves on a
large cattle farm in Hebei Province, China presented watery diarrhea and
over 40 died. To investigate the cause of the outbreak, 179 and 223
fecal specimens were collected during and after the diarrhea outbreak
from the farm, including 40 and 56 from neonatal calves, respectively.
Among them, 18 fecal specimens from ill calves during the peak of the
outbreak were analyzed for four common enteric pathogens using enzymatic
immunoassay (EIA), 75 additional specimens from neonatal calves were
tested for rotavirus by EIA, and all specimens were analyzed for
Cryptosporidium spp. using PCR and sequencing techniques. Of the initial
18 specimens from sick calves, ten were positive for C. parvum, five for
rotavirus, and one for coronavirus. The overall prevalence of rotavirus
in neonatal calves was 20.0\% (15/75), with no significant differences
during (21.6\% or 8/37) and after (18.4\% or 7/38) the outbreak. In
contrast, the prevalence of C. parvum was significantly higher during
the outbreak (60.0\%, 24/40) than after the outbreak (30.4\%, 17/56). C.
parvum infection was associated with the occurrence of watery diarrhea
in neonatal calves (odds ratio = 11.19), while no association was
observed between C. bovis infection and diarrhea. All C. parvum isolates
were identified as subtype IIdA20G1. Older animals were infected with C.
bovis, C. ryanae, C. occultus, and C. andersoni. This is one of the few
reports of outbreaks of severe diarrhea caused by C. parvum IId subtypes
in calves. More attention should be directed toward preventing the
dissemination of C. parvum in China.%
\end{abstract}%



\sloppy


\textbf{Cryptosporidiosis outbreak caused by \emph{Cryptosporidium
parvum} subtype IIdA20G1inneonatal calves}

\textbf{Running title: Cryptosporidiosis outbreak in neonatal calves}

Zhenjie Zhang\textsuperscript{1,2}, Dongle Su\textsuperscript{1}, Xinan
Meng\textsuperscript{1}, Ruobing Liang\textsuperscript{1}, Weijian
Wang\textsuperscript{1}, Na Li\textsuperscript{1}, Yaqiong
Guo\textsuperscript{1}, Aizhen Guo\textsuperscript{3}, Shujing
Li\textsuperscript{4}, Zengyuan Zhao\textsuperscript{4}, Lihua
Xiao\textsuperscript{1, 2}*, Yaoyu Feng\textsuperscript{1,2}*

\textsuperscript{1} Center for Emerging and Zoonotic Diseases, College
of Veterinary Medicine, South China Agricultural University, Guangzhou
510642, China.

\textsuperscript{2} Guangdong Laboratory for Lingnan Modern Agriculture,
Guangzhou 510642, China.

\textsuperscript{3} State Key Laboratory of Agricultural Microbiology,
Huazhong Agricultural University, Wuhan 430070, China

\textsuperscript{4} Shijiazhuang Tianquan Elite Breeding Dairy Cow Co.,
LTD., Shijiazhuang 050227, China

* Corresponding authors: Yaoyu Feng (E-mail
addresses:\emph{yyfeng@scau.edu.cn} ), Lihua Xiao (E-mail
addresses:\emph{lxiao1961@gmail.com}).

\textbf{Summary}

\emph{Cryptosporidium parvum} is a major zoonotic pathogen responsible
for outbreaks of severe diarrhea in humans and calves. Almost all
investigations of cryptosporidiosis outbreaks caused by \emph{C. parvum}
have focused on its IIa subtype family in industrialized nations. From
December 2018 to April 2019, approximately 200 neonatal calves on a
large cattle farm in Hebei Province, China presented watery diarrhea and
over 40 died. To investigate the cause of the outbreak, 179 and 223
fecal specimens were collected during and after the diarrhea outbreak
from the farm, including 40 and 56 from neonatal calves, respectively.
Among them, 18 fecal specimens from ill calves during the peak of the
outbreak were analyzed for four common enteric pathogens using enzymatic
immunoassay (EIA), 75 additional specimens from neonatal calves were
tested for rotavirus by EIA, and all specimens were analyzed
for\emph{Cryptosporidium} spp. using PCR and sequencing techniques. Of
the initial 18 specimens from sick calves, ten were positive for
\emph{C. parvum} , five for rotavirus, and one for coronavirus. The
overall prevalence of rotavirus in neonatal calves was 20.0\% (15/75),
with no significant differences during (21.6\% or 8/37) and after
(18.4\% or 7/38) the outbreak. In contrast, the prevalence of \emph{C.
parvum} was significantly higher during the outbreak (60.0\%, 24/40)
than after the outbreak (30.4\%, 17/56). \emph{C. parvum} infection was
associated with the occurrence of watery diarrhea in neonatal calves
(odds ratio = 11.19), while no association was observed between \emph{C.
bovis}infection and diarrhea. All \emph{C. parvum} isolates were
identified as subtype IIdA20G1. Older animals were infected with
\emph{C. bovis} ,\emph{C. ryanae} , \emph{C. occultus} , and \emph{C.
andersoni} . This is one of the few reports of outbreaks of severe
diarrhea caused by\emph{C. parvum} IId subtypes in calves. More
attention should be directed toward preventing the dissemination of
\emph{C. parvum} in China.

\textbf{Keywords:} \emph{Cryptosporidium parvum} ; Cryptosporidiosis;
IIdA20G1; Outbreak; Cattle; Diarrhea

\textbf{1. Introduction}

Neonatal calf diarrhea caused by intestinal pathogens including
bacteria, viruses and parasites is a leading cause of morbidity and
mortality in pre-weaned calves (Gomez \& Weese, 2017). Rotavirus,
coronavirus, \emph{Escherichia coli} , and\emph{Cryptosporidium} spp.
are considered the most important causes for calf diarrhea (Meganck,
Hoflack, \& Opsomer, 2014). Synergistic infections of these pathogens
were often identified during outbreaks of diarrhea in calves (Foster \&
Smith, 2009; Gomez \& Weese, 2017). Among them,\emph{Cryptosporidium}
spp. and rotavirus are the most frequently observed pathogens in fecal
specimens from neonatal calves worldwide (Mohamed, Mansour, El-Araby,
Mor, \& Goyal, 2017; Thomson et al., 2017). In addition,
moderate-to-severe diarrhea caused by \emph{Cryptosporidium}spp. is of
the leading causes for morbidity and mortality in young children in
developing countries (Kotloff et al., 2013).

\emph{Cryptosporidium} spp. are especially common in dairy calves. The
cumulative infection rate of \emph{Cryptosporidium} spp. in pre-weaned
dairy calves in the United States is 100.0\% (Santin, Trout, \& Fayer,
2008; Xiao \& Herd, 1994). In China, the average infection rate of
\emph{Cryptosporidium} spp. in dairy calves with diarrhea was 45.8\%,
much higher than that in cattle overall (14.5\%) (Wang, Zhao, Gong, \&
Zhang, 2017). Numerous cryptosporidiosis outbreaks have been reported in
neonatal calves in the United States, European countries, Australia,
India, and China, causing serious harms to the dairy industry (Brar et
al., 2017; Cui et al., 2014; Izzo et al., 2011; Li et al., 2019; Ouakli
et al., 2018; Randhawa, Randhawa, Zahid, Singla, \& Juyal, 2012).

Among the over 40 established \emph{Cryptosporidium} species (Feng,
Ryan, \& Xiao, 2018), \emph{C. parvum} , \emph{C. bovis} , \emph{C.
ryanae} and \emph{C. andersoni} are commonly found in cattle (Ryan,
Fayer, \& Xiao, 2014)\emph{.} Among the latter, \emph{C. parvum} is the
only major pathogenic and zoonotic species (Thomson et al.,
2017)\emph{.} The distribution of \emph{Cryptosporidium} spp. in cattle
in China, however, is very different from other countries, with \emph{C.
bovis} being the most common species in pre-weaned calves rather than
\emph{C. parvum}elsewhere (Feng \& Xiao, 2017). In addition, subtyping
of \emph{C. parvum} based on sequence analysis of the 60 kDa
glycoprotein (\emph{gp60} ) gene indicates that calves in China are
exclusively infected with IId subtypes (Feng \& Xiao, 2017). Elsewhere,
IIa subtypes are mostly identified in calves, while IId subtypes are
usually seen in lambs and goat kids (Xiao, 2010).

Thus far, almost all investigations of cryptosporidiosis outbreaks in
humans and calves caused by \emph{C. parvum} have focused on its IIa
subtype family in industrialized nations. The clinical importance of the
IId subtype family of \emph{C. parvum} is less clear. Two recent
outbreaks, however, have been reported in dairy calves in China,
responsible for substantial mortality (Cui et al., 2014; Li et al.,
2019). They were caused by IIdA15G1 and IIdA19G1 subtypes, two dominant
\emph{C. parvum} subtypes in calves and other animals in China (Feng \&
Xiao, 2017).

Here, we report the results of an investigation of a cryptosporidiosis
outbreak in neonatal calves on a large cattle farm in Hebei Province,
China caused by another IId subtype of \emph{C. parvum} . Approximately
20\% of neonatal calves on the farm died of watery diarrhea during the
outbreak period.

\textbf{2. Materials and Methods}

\textbf{2.1. Animals and Specimens}

The investigated farm is located in Hebei Province in northern China.
There were \textasciitilde{}2,000 cattle altogether, including about 350
calves on the farm. Among them, the majority of animals were of the
Holstein breed, together with fewer than 200 cattle of the Simmental
breed and 40 adult cattle of the Japanese Wagyu breed. There were no
vaccinations against rotaviruses, coronaviruses, and \emph{Escherichia
coli} prior to the occurrence of the outbreak of diarrhea.

From December 2018 to April 2019, an outbreak of diarrhea was noticed in
neonatal calves on the study farm (Fig. 1), with watery diarrhea
occurring in all \textasciitilde{}200 neonatal calves born during the
period. Forty-one calves died of diarrhea at 10 -- 30 days of age
despite antibiotic therapy. They experienced severe diarrhea before
death, with the excretion of watery feces. Most of them also had other
clinical signs such as poor appetite, weight loss and dehydration (Table
1). To identify the cause of the diarrhea, 402 fecal specimens were
collected from the farm during the outbreak in January 2019 (n = 173)
and after the outbreak in June 2019 (n = 229). Among the specimens
collected during the outbreak, 40 were from neonatal calves (1 - 4 weeks
in age) and 133 were from older cattle (\textgreater{} 4 weeks in age).
Similarly, 56 and 173 specimens collected after the outbreak were from
neonatal calves and older cattle, respectively. Altogether, 213
specimens were from calves under 24 weeks and 189 from older cattle. The
specimens from calves included formed feces from animals with no
diarrhea (n = 110), loose feces from animals with moderate diarrhea (n =
64), and liquid feces from animals with watery diarrhea (n = 39). The
older animals were mostly healthy.

Fecal specimens were collected directly from the rectum of each animal
using disposable gloves into 50-mL centrifuge tubes. Approximately 1.0 g
of each fecal specimen was transferred into a 1.5-mL tube and kept
frozen at -80 , while the remaining fecal material was stored in 2.5\%
potassium dichromate at 4 .

\textbf{2.2. Detection of Common Enteric Pathogens by Enzymatic
Immunoassay (EIA)}

To identify the possible etiological agent of calf diarrhea, 18
specimens from ill calves collected during the peak of the diarrhea
outbreak were examined for four common enteric pathogens. For this, 200
mg frozen fecal material was tested for rotavirus,
coronavirus,\emph{Escherichia coli} , and \emph{Cryptosporidium} spp.
using the Pathasure Enteritis EIA kit (Biovet, Saint-Hyacinthe, Canada).
In addition, rotavirus in another 75 fecal specimens collected during (n
= 37) and after (n = 38) the diarrhea outbreak from 1 to 4-week-old
calves were tested using the ProSpecT Rotavirus Kit (Thermo-Oxoid,
Basingstoke, UK).

\textbf{2.3. \emph{Cryptosporidium} Genotyping and Subtyping}

Fecal specimens preserved in potassium dichromate were washed three
times with distilled water by centrifugation at 2500\selectlanguage{ngerman}×g for 5 min.
Genomic DNA was extracted from 0.2 g washed fecal material using the
FastDNA SPIN Kit for Soil (MP Biomedicals, CA,
USA).\emph{Cryptosporidium} species were detected using polymerase chain
reaction (PCR) and sequence analysis of the SSU rRNA gene. To identify
the \emph{C. parvum} subtype involved, a \textasciitilde{}850 bp
fragment of the \emph{gp60} gene was amplified using nested PCR (Feng,
Li, Duan, \& Xiao, 2009). Reagent-grade water was used as the negative
control, whereas \emph{C.serpentis} DNA was used as the positive control
for the SSU rRNA PCR and \emph{C. hominis} DNA as the positive control
for the\emph{gp60} PCR. All positive PCR products of the SSU rRNA
and\emph{gp60} genes were sequenced bi-directionally on an ABI 3730
Genetic Analyzer (Applied Biosystems, CA, USA). Nucleotide sequences
generated were aligned together with reference sequences using ClustalX
2.1 (\emph{http://www.clustal.org/}) for the identification of
\emph{Cryptosporidium} species and \emph{C. parvum}subtypes.

\textbf{2.4. Statistical Analysis}

Data in this study were analyzed statistically using the software
packages R version 3.6.2 (\emph{https://www.r-project.org/}) and RStudio
version 1.1.463 (\emph{https://rstudio.com/}). The \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}
test was used to compare differences in\emph{Cryptosporidium} or
rotavirus infection rates by age group or diarrhea status. Logistic
regression was used to assess the association between putative risk
factors and the occurrence of diarrhea, using data from 213 pre-weaned
and post-weaned calves during and after the diarrhea outbreak.
Initially, the strength of association was assessed using univariate
analysis. Variables with \emph{P} [?] 0.2 were thereafter used to build a
multivariate model, with \emph{P} \textless{} 0.05 being considered
significant in the final model. Odds ratios (OR) with 95\% confidence
intervals (CI) were used to identify risk factors for diarrhea
occurrence.

\textbf{3. Results}

\textbf{3.1. Occurrence of Enteric Pathogens inIll Calves during the
Outbreak}

In the initial EIA analysis of major enteric pathogens in the 18
specimens from ill calves, 10 were positive for
\emph{Cryptosporidium}spp., five for rotavirus, one for coronavirus, and
none for enteropathogenic \emph{E. coli} . All five rotavirus-positive
specimens were also positive for \emph{Cryptosporidium} spp. (Table 2).
These 18 specimens were further analyzed for \emph{Cryptosporidium} spp.
using the SSU rRNA-based PCR, leading to the identification of \emph{C.
parvum} in 10 of them, including 9 of the 10 EIA-positive specimens. All
the \emph{C. parvum} -positive specimens were successfully subtyped as
IIdA20G1 by sequence analysis of the \emph{gp60} gene.

\textbf{3.2. Occurrence of Rotavirus inNeonatal (1-4-Week-Old) Calves}

During the outbreak, 8/37 (21.6\%) specimens from neonatal calves were
positive for rotavirus. In contrast, 7/38 (18.4\%) specimens from
neonatal calves were positive after the outbreak (Fig. 2). Altogether,
15 of the 75 fecal specimens from neonatal calves of 1 - 4 weeks were
positive for rotavirus by EIA. Among them, the infection rate was 21.1\%
(8/38) in calves with watery diarrhea, 0\% (0/6) in calves with moderate
diarrhea, and 22.6\% (7/31) in calves without diarrhea
(\selectlanguage{greek}\emph{χ\textsuperscript{2}}\selectlanguage{english} = 1.66, \emph{df} = 2, \emph{P} = 0.437).

\textbf{3.3. Occurrence of \emph{C. parvum} in Neonatal (1-4-Week-Old)
Calves}

During the outbreak, 27 of 40 (67.5\%) fecal specimens collected from
neonatal calves were positive for \emph{Cryptosporidium} spp. by PCR,
including \emph{C. parvum} (n = 24) and \emph{C. bovis} (n = 3). After
the outbreak, 35 of 56 (62.5\%) fecal specimens collected from neonatal
calves were positive for \emph{Cryptosporidium} spp., including \emph{C.
parvum} (n = 17), \emph{C. bovis} (n = 17) and \emph{C. ryanae} (n = 1)
(Fig. 2). The infection rate of \emph{C. parvum} during the outbreak
(60.0\%, 24/40) was significantly higher (\selectlanguage{greek}\emph{χ\textsuperscript{2}}\selectlanguage{english} =
8.38, \emph{df} = 1, \emph{P} = 0.004) than after the outbreak (30.4\%,
17/56) (Fig. 2). Animals older than 4 weeks had no \emph{C.
parvum}infection during and after the outbreak. Instead, they were
mostly infected with \emph{C. bovis} (n = 56) and \emph{C. ryanae} (n =
39), together with some occurrence of \emph{C. occultus} (n = 6) and
\emph{C. andersoni} (n = 5) (Table 3). Therefore, during the outbreak,
\emph{C. parvum} was the dominant species in neonatal calves, while
after the outbreak, \emph{C. bovis} and \emph{C. parvum} occurred at the
same frequency (Table 3).

Altogether, 39/41 specimens positive for \emph{C. parvum} were
successfully subtyped at the \emph{gp60} locus. The sequences obtained
were all identical to the reference sequence KU248815 of the IIdA20G1
subtype from GenBank.

\selectlanguage{greek}\textbf{3.4. Correlation between \emph{Cryptosporidium} Spp. and
Diarrhea Occurrence as Indicated byχ\textsuperscript{2}Analysis}\selectlanguage{english}

In neonatal (1-4-week-old) calves, \emph{Cryptosporidium} infection
rates were 66.7\% (26/39), 69.2\% (9/13) and 61.4\% (27/44) in animals
with watery diarrhea, moderate diarrhea and no diarrhea, respectively.
The differences among groups were not significant in
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} analysis (\selectlanguage{greek}\emph{χ\textsuperscript{2}}\selectlanguage{english} = 0.40,
\emph{df} = 2, \emph{P} = 0.820) (Tables 4). By\emph{Cryptosporidium}
species, \emph{C. parvum} infection rates were 61.5\% (24/39), 38.5\%
(5/13), and 27.3\% (12/44) in animals with watery diarrhea, moderate
diarrhea and no diarrhea, respectively. The difference in \emph{C.
parvum} infection rates between animals with watery diarrhea and those
with no diarrhea was significant (\selectlanguage{greek}\emph{χ\textsuperscript{2}}\selectlanguage{english} = 9.88,
\emph{df} = 1, \emph{P} = 0.002) (Tables 4). In contrast, the infection
rate of \emph{C. bovis} in animals with no diarrhea (31.8\%, 14/44) was
significantly higher than those with watery diarrhea (5.1\%, 2/39)
(\selectlanguage{greek}\emph{χ\textsuperscript{2}}\selectlanguage{english} = 9.46, \emph{df} = 1, \emph{P} = 0.002).
Infection rates of \emph{C. ryanae} were similar among the three groups
(\selectlanguage{greek}\emph{χ\textsuperscript{2}}\selectlanguage{english}= 1.19, \emph{df} = 2, \emph{P} = 0.550)
(Tables 4).

No \emph{C. parvum} infection nor watery diarrhea was observed in calves
older than 4 weeks. In this age group, the infection rates
of\emph{Cryptosporidium} spp. were 58.8\% (30/51) and 60.6\% (40/66) in
animals with moderate diarrhea and those without diarrhea, respectively
(\selectlanguage{greek}\emph{χ\textsuperscript{2}}\selectlanguage{english} = 0.04, \emph{df} = 1, \emph{P} = 0.845)
(Tables 4). By \emph{Cryptosporidium} species, the infection rates
of\emph{C. bovis} (\selectlanguage{greek}\emph{χ\textsuperscript{2}}\selectlanguage{english} = 0.078, \emph{df} =
1,\emph{P} = 0.780) or \emph{C. ryanae} (\selectlanguage{greek}\emph{χ\textsuperscript{2}}\selectlanguage{english} =
0.005, \emph{df} = 1, \emph{P} = 0.941) were not significantly different
between the two groups (Tables 4).

\textbf{3.5. Association of \emph{C. parvum} Infection with Diarrhea
Occurrence as Indicated by Univariate and Multivariate Analyses}

In the univariate analysis of data, \emph{C. parvum} -positive calves
had a significantly higher occurrence of diarrhea (odds ratio =
3.20,\emph{P} = 0.002, Table 5), especially watery diarrhea (odds ratio
= 14.78, \emph{P} = 0.000). There was no correlation
between\emph{Cryptosporidium} infection and occurrence of moderate
diarrhea. In addition, neonatal calves were more likely to have watery
diarrhea (odds ratio = 34.12, \emph{P} = 0.001, Table 5) and moderate
diarrhea (odds ratio = 3.09, \emph{P} = 0.000, Table 5) than older
animals. Further analysis using the multivariate model showed that
\emph{C. parvum}infection was the only species associated with the
occurrence of diarrhea (odds ratio = 2.98, \emph{P} = 0.016, Table 5),
especially watery diarrhea (odds ratio = 11.19, \emph{P} = 0.000, Table
5). In addition, age was an important risk factor associated with the
occurrence of watery diarrhea (odds ratio = 40.00, \emph{P} = 0.001,
Table 5) and moderate diarrhea (odds ratio = 2.91, \emph{P} = 0.001,
Table 5) in these animals.

\textbf{4. Discussion}

Cryptosporidiosis was identified as the cause for outbreak of severe
diarrhea in neonatal calves in this study. The initial EIA analysis of
18 fecal specimens from ill calves at the peak of the outbreak showed
that \emph{C. parvum} and rotavirus were possible etiological agents
rather than \emph{E. coli} and coronavirus. The role of rotavirus was
excluded in comparative detection of the pathogen in specimens from
neonatal calves during and after the outbreak. There was no significant
difference in infection rates of rotavirus during and after the
outbreak, and rotavirus infection was not significantly associated with
the occurrence of moderate or watery diarrhea in these animals. In
contrast, the infection rate of \emph{C. parvum} in neonatal calves was
higher during the outbreak than after it, and the infection was
significantly associated with the occurrence of watery diarrhea in these
animals. In addition, all \emph{C. parvum} infections were observed in
calves of 1-4 weeks in concurrence with watery diarrhea, reinforcing the
role of \emph{C. parvum} in the occurrence of the outbreak.

The present study constitutes one of several lines of evidence for the
pathogenicity of the IId subtype family of \emph{C. parvum} . The
pathogenicity of IIa subtypes of \emph{C. parvum} has been well
established, as cryptosporidiosis outbreaks have been reported in
neonatal calves in many industrialized countries where they are
prevalent (Holzhausen, Lendner, Gohring, Steinhofel, \& Daugschies,
2019; Izzo et al., 2011; Niine, Dorbek-Kolin, Lassen, \& Orro, 2018).
The pathogenicity of IId subtypes of \emph{C. parvum} , however, has
only been indicated in molecular epidemiological investigations of
\emph{Cryptosporidium} spp. in China, which have shown a dominance of
the IId subtype family and association of it with the occurrence of
diarrhea in pre-weaned calves (Cai et al., 2017; Wu et al., 2020).
Recently, the IId subtype family was implicated as the cause for two
outbreaks of cryptosporidiosis in calves in China, including one by the
IIdA15G1 subtype in Ningxia, leading to the death of 356 calves (Cui et
al., 2014), and one by the subtype IIdA19G1 in Jiangsu, leading to the
death of 360 calves (Li et al., 2019). In the present study, the
IIdA20G1 subtype was identified as the cause of cryptosporidiosis
outbreak in calves in China.

IIdA20G1 appears to be an emerging \emph{C. parvum} subtype. In China,
it has been recently reported in cattle in Heilongjiang, Hebei and
Xinjiang and deer in Hubei (Tao et al., 2018; Wu et al., 2020; Xie et
al., 2019; Zhang et al., 2020). Elsewhere, it has been reported in
livestock and humans in Egypt, Kuwait, Qatar, Jordan, Turkey, Iran,
Italy and Romania (Amer et al., 2010; Amer, Zidan, Adamu, et al., 2013;
Amer, Zidan, Feng, et al., 2013; Boughattas et al., 2017; Diaz et al.,
2018; Helmy, Krucken, Nockler, von Samson-Himmelstjerna, \& Zessin,
2013; Hijjawi, Mukbel, Yang, \& Ryan, 2016; Ibrahim, Abdel-Ghany,
Abdel-Latef, Abdel-Aziz, \& Aboelhadid, 2016; Imre et al., 2013; Kiani
et al., 2017; Mahfouz, Mira, \& Amer, 2014; Majeed et al., 2018; Naguib
et al., 2018; Taylan-Ozkan et al., 2016). Most of these countries are
located in the Middle East. Nevertheless, this is the first report of
cryptosporidiosis outbreak caused by \emph{C. parvum}IIdA20G1 subtype.

As with the \emph{C. parvum} IIa subtype family, young age is associated
with diarrhea caused by the IIdA20G1 subtype. In present study, \emph{C.
parvum} all appeared in neonatal calves under four weeks of age. This is
likely due to the immature immune system in these animals, which also
makes them prone to the occurrence of clinical signs after \emph{C.
parvum} infection (Santin et al., 2008). In the present study, neonatal
calves had high occurrence of \emph{C. bovis} after the outbreak. This,
however, was not associated with the occurrence of diarrhea. Previously,
in a few reports, \emph{C. bovis}and \emph{C. ryanae} had been
associated with the occurrence of moderate diarrhea (Lee et al., 2016;
Li et al., 2019; Silverlas \& Blanco-Penedo, 2013; Silverlas,
Bosaeus-Reineck, Naslund, \& Bjorkman, 2013).

The role of rotavirus in causing the diarrhea outbreak appears to be
minor. Although rotavirus was commonly found in neonatal calves in the
present study, there was no significant difference in its infection
rates during and after the outbreak. In statistical analyses, its
occurrence was not correlated with the occurrence of diarrhea.
Nevertheless, rotavirus is a well-known cause of diarrhea in
1-2-week-old calves (Cho, Han, Wang, Cooper, \& Yoon, 2013). It has been
frequently identified in co-infections with \emph{C. parvum} ,
contributing to the occurrence of diarrhea in infected calves (Brar et
al., 2017; Izzo et al., 2011). Thus, the concurrence of rotavirus in
neonatal calves could have worsen the deleterious impact of \emph{C.
parvum} infection in the investigated outbreak of diarrhea.

\textbf{5. Conclusion}

This represents the first report of an outbreak of bovine
cryptosporidiosis caused by the \emph{C. parvum} subtype IIdA20G1, which
appears to be an emerging pathogen in China and the Middle East.
Significant morbidity and mortality have been associated with this
outbreak, possibly as a result of concurrence of rotavirus on the farm.
Control measures should be implemented to effectively reduce the
transmission of \emph{C. parvum} , which still occurs on only a portion
of the dairy farms in China. Further studies are required to examine the
clinical significance of rotavirus and \emph{C. parvum} coinfections in
neonatal calves.

\textbf{Acknowledgements}

This study was supported by the National Natural Science Foundation of
China (32030109, U1901208 and 31820103014), 111 Project (D20008), and
Innovation Team Project of Guangdong University (2019KCXTD001).

\textbf{Conflict of Interest Statement}

The authors declare that they have no competing interests.

\textbf{Ethics Statement}

Fecal specimens from cattle in this study were collected with the
permission of the farm managers as part of the technical assistance in
the investigation of an outbreak of diarrhea. The animals were handled
in accordance with the Animal Ethics Procedures and Guidelines of the
People's Republic of China. The research protocol was reviewed and
approved by the Ethics Committee of the South China Agricultural
University.

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\textbf{Legends to Figures}

\textbf{Fig. 1} Calves with diarrhea on a cattle farm in Hebei, China

\textbf{Fig. 2} Infection rates of rotavirus and
\emph{Cryptosporidium}spp. in 1-4-week-old calves during and after the
outbreak of watery diarrhea in Hebei, China

\textbf{Table 1.} Number of births and deaths of calves during a
diarrhea outbreak provided by the farm owner\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Time}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Number of} \textbf{newborn calves}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{No. of dead calves}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{No. of deaths due to diarrhea}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Sept. 2018\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
23\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Oct. 2018\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
19\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Nov. 2018\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
43\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
11\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dec. 2018*\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
27\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
12\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
9\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Jan. 2019*\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
71\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Feb. 2019*\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
45\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Mar. 2019*\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
41\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
13\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
13\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Apr. 2019*\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
16\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
11\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
May 2019\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Jun. 2019\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Jul. 2019\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
45\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Total\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
386\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
79\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
54\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

*The time period during which the outbreak of diarrhea was noticed.

\textsuperscript{a}Sampling time during the diarrhea outbreak.

\textsuperscript{b}Sampling time after the diarrhea outbreak.

\textbf{Table 2.} Detection of common enteric pathogens in fecal
specimens from 18 seriously ill calves during a diarrhea outbreak on a
cattle farm in Hebei, China using an enzymatic immunoassay (EIA) test
and PCR\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\textbf{Specimen} & \textbf{Breed} & \textbf{Age (weeks)} & \textbf{EIA
test} & \textbf{EIA test} & \textbf{EIA test} & \textbf{EIA test} &
\emph{Cryptosporidium} \textbf{PCR} & \emph{Cryptosporidium}
\textbf{PCR} & \emph{Cryptosporidium} \textbf{PCR}\tabularnewline
\midrule
\endhead
& & & \textbf{Rotavirus} & \textbf{Coronavirus} & Escherichia coli &
Cryptosporidium & \textbf{Positivity} & \textbf{Species} &
\textbf{Subtype}\tabularnewline
12359 & Holstein & 1 & - & - & - & + & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12406 & Holstein & 1 & - & - & - & - & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12394 & Holstein & 2 & + & - & - & + & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12407 & Holstein & 2 & - & - & - & + & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12401 & Holstein & 2 & - & - & - & - & - & &\tabularnewline
12403 & Holstein & 2 & - & - & - & - & - & &\tabularnewline
12404 & Holstein & 2 & - & - & - & - & - & &\tabularnewline
12385 & Holstein & 1 & - & + & - & - & - & &\tabularnewline
12386 & Holstein & 1 & - & - & - & - & - & &\tabularnewline
12532 & Simmental & 1 & - & - & - & - & - & &\tabularnewline
12533 & Holstein & 1 & - & - & - & + & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12537 & Holstein & 1 & - & - & - & + & - & &\tabularnewline
12539 & Simmental & 1 & - & - & - & - & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12531 & Simmental & 1 & + & - & - & + & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12534 & Simmental & 1 & + & - & - & + & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12536 & Simmental & 1 & + & - & - & + & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12540 & Simmental & 1 & + & - & - & + & + & \emph{C. parvum} &
IIdA20G1\tabularnewline
12541 & Holstein & 1 & - & - & - & - & - & &\tabularnewline
\bottomrule
\end{longtable}

+, positive; -, negative.

\textbf{Table 3.} Prevalence of \emph{Cryptosporidium} infection in
cattle during and after a diarrhea outbreak on a farm in Hebei, China\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{Time} & \textbf{Age group} & \textbf{No. positive/No. sampled
(\%)} & \emph{Cryptosporidium} \textbf{spp.} & \emph{Cryptosporidium}
\textbf{spp.} & \emph{Cryptosporidium} \textbf{spp.} &
\emph{Cryptosporidium} \textbf{spp.} & \emph{Cryptosporidium}
\textbf{spp.}\tabularnewline
\midrule
\endhead
& & & C. parvum & C. bovis & C. ryanae & C. occultus & C.
andersoni\tabularnewline
\textbf{During the outbreak} & Neonatal calves (1 -- 4 weeks) & 27/40
(67.5) & 24 & 3 & 0 & 0 & 0\tabularnewline
& Older cattle* (\textgreater{} 4 weeks) & 78/133 (58.6) & 0 & 39 & 32 &
6 & 1\tabularnewline
\textbf{After the outbreak} & Neonatal calves (1 -- 4 weeks) & 35/56
(62.5) & 17 & 17 & 1 & 0 & 0\tabularnewline
& Older cattle* (\textgreater{} 4 weeks) & 28/173 (16.2) & 0 & 17 & 7 &
0 & 4\tabularnewline
\textbf{Total} & \textbf{-} & \textbf{168/402 (41.8)} & \textbf{41} &
\textbf{76} & \textbf{40} & \textbf{6} & \textbf{5}\tabularnewline
\bottomrule
\end{longtable}

*Including calves of 5 -- 24 weeks, heifers (7 -- 12 months) and adults
(\textgreater{} 12 months)

\textbf{Table 4.} Associations between \emph{Cryptosporidium} or
rotavirus infection and occurrence of moderate or watery diarrhea in
neonatal and older calves during and after a diarrhea outbreak on a farm
in Hebei, China, as indicated by results of \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}analysis\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllll@{}}
\toprule
\textbf{Factors} & \textbf{Factors} & \textbf{No diarrhea} & \textbf{No
diarrhea} & & \textbf{Moderate diarrhea} & \textbf{Moderate diarrhea} &
\textbf{Moderate diarrhea} & \textbf{Moderate diarrhea} & &
\textbf{Watery diarrhea} & \textbf{Watery diarrhea} & \textbf{Watery
diarrhea} & \textbf{Watery diarrhea}\tabularnewline
\midrule
\endhead
& & \textbf{n} & \selectlanguage{greek}\textbf{χ\textsuperscript{2}}\selectlanguage{english} & & \textbf{n} &
\selectlanguage{greek}\textbf{χ\textsuperscript{2}}\selectlanguage{english} & \textbf{OR (95\% CI)} & P & & \textbf{n}
& \selectlanguage{greek}\textbf{χ\textsuperscript{2}}\selectlanguage{english} & \textbf{OR (95\% CI)} &
P\tabularnewline
\textbf{Neonatal calves (1 -- 4 weeks)} & \textbf{Neonatal calves (1 --
4 weeks)} & \textbf{Neonatal calves (1 -- 4 weeks)} & \textbf{Neonatal
calves (1 -- 4 weeks)} & \textbf{Neonatal calves (1 -- 4 weeks)} &
\textbf{Neonatal calves (1 -- 4 weeks)} & \textbf{Neonatal calves (1 --
4 weeks)} & \textbf{Neonatal calves (1 -- 4 weeks)} & \textbf{Neonatal
calves (1 -- 4 weeks)} & \textbf{Neonatal calves (1 -- 4 weeks)} &
\textbf{Neonatal calves (1 -- 4 weeks)} & \textbf{Neonatal calves (1 --
4 weeks)} & \textbf{Neonatal calves (1 -- 4 weeks)} & \textbf{Neonatal
calves (1 -- 4 weeks)}\tabularnewline
& \textbf{Sample size} & 44 & - & & 13 & - & - & - & & 39 & - & - &
-\tabularnewline
& \emph{Cryptosporidium} spp. & 27 & Reference & & 9 & 0.27 & 0.71
(0.19, 2.66) & 0.605 & & 26 & 0.25 & 0.79 (0.32, 1.96) &
0.616\tabularnewline
& \emph{C. parvum} & 12 & Reference & & 5 & 0.60 & 0.60 (0.16, 2.20) &
0.438 & & 24 & 9.88 & 1.89 (1.22, 2.93) & 0.002\tabularnewline
& \emph{C. bovis} & 14 & Reference & & 4 & 0.01 & 1.05 (0.28, 4.00) &
0.943 & & 2 & 9.46 & 0.72 (0.58, 0.89) & 0.002\tabularnewline
& \emph{C. ryanae} & 1 & Reference & & 0 & 0.30 & 0.98 (0.93, 1.02) &
0.583 & & 0 & 0.90 & 0.98 (0.93, 1.02) & 0.344\tabularnewline
& Rotavirus & 7 & Reference & & 0 & 3.06 & 1.08 (1.00, 1.18) & 0.081 & &
8 & 1.44 & 2.03 (0.74, 5.89) & 0.23\tabularnewline
\textbf{Older cattle (5 -- 24 weeks)} & \textbf{Older cattle (5 -- 24
weeks)} & \textbf{Older cattle (5 -- 24 weeks)} & \textbf{Older cattle
(5 -- 24 weeks)} & \textbf{Older cattle (5 -- 24 weeks)} & \textbf{Older
cattle (5 -- 24 weeks)} & \textbf{Older cattle (5 -- 24 weeks)} &
\textbf{Older cattle (5 -- 24 weeks)} & \textbf{Older cattle (5 -- 24
weeks)} & \textbf{Older cattle (5 -- 24 weeks)} & \textbf{Older cattle
(5 -- 24 weeks)} & \textbf{Older cattle (5 -- 24 weeks)} & \textbf{Older
cattle (5 -- 24 weeks)} & \textbf{Older cattle (5 -- 24
weeks)}\tabularnewline
\textbf{Sample size} & \textbf{Sample size} & 66 & - & & 51 & - & - & -
& & 0 & - & - & -\tabularnewline
& \emph{Cryptosporidium} spp. & 40 & Reference & & 30 & 0.04 & 1.08
(0.51, 2.27) & 0.845 & & - & - & - & -\tabularnewline
& \emph{C. bovis} & 21 & Reference & & 15 & 0.08 & 1.12 (0.51, 2.48) &
0.780 & & - & - & - & -\tabularnewline
& \emph{C. ryanae} & 19 & Reference & & 15 & 0.01 & 0.97 (0.43, 2.17) &
0.941 & & - & - & - & -\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 5.} Association between \emph{Cryptosporidium} infection
and occurrence of diarrhea in neonatal and older calves during and after
a diarrhea outbreak on a farm in Hebei, China, as indicated by results
of univariate and multivariate analyses\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{Diarrhea status} & \textbf{Factor} & \textbf{Univariate model} &
\textbf{Univariate model} & \textbf{Univariate model} &
\textbf{Multivariate model} & \textbf{Multivariate model} &
\textbf{Multivariate model}\tabularnewline
\midrule
\endhead
& & \textbf{OR} & \textbf{95\% CI} & P & \textbf{OR} & \textbf{95\% CI}
& P\tabularnewline
\textbf{Watery diarrhea} & Cryptosporidium & 1.28 & 0.63 - 2.74 & 0.505
& NA & NA & NA\tabularnewline
& \emph{C. parvum} & 14.78 & 6.66 - 34.33 & 0.000 & 11.19 & 4.90 - 26.91
& 0.000\tabularnewline
& \emph{C. bovis} & 0.12 & 0.02 - 0.4 & 0.004 & 0.29 & 0.04 - 1.11 &
0.115\tabularnewline
& No \emph{Cryptosporidium} & 1 & Reference & & 1 & Reference
&\tabularnewline
& \textbf{Age} & & & & & &\tabularnewline
& Neonatal (1 - 4 weeks) & 34.12 & 4.32 -- 269.51 & 0.001 & 40.00 & 3.92
-- 245.43 & 0.001\tabularnewline
& Other ages (5 - 24 weeks) & 1 & Reference & & 1 & Reference
&\tabularnewline
\textbf{Moderate diarrhea} & Cryptosporidium & 0.94 & 0.52 - 1.73 &
0.839 & NA & NA & NA\tabularnewline
& \emph{C. parvum} & 0.27 & 0.09 - 0.66 & 0.009 & 0.82 & 0.23 -- 2.66 &
0.74\tabularnewline
& \emph{C. bovis} & 1.28 & 0.66 - 2.44 & 0.461 & NA & NA &
NA\tabularnewline
& \emph{C. ryanae} & 1.97 & 0.93 -- 4.15 & 0.074 & 1.01 & 0.45 - 2.22 &
0.989\tabularnewline
& No \emph{Cryptosporidium} & 1 & Reference & & 1 & Reference
&\tabularnewline
& \textbf{Age} & & & & & &\tabularnewline
& Neonatal (1 - 4 weeks) & 3.09 & 1.93 -- 5.16 & 0.000 & 2.91 & 1.63 --
5.59 & 0.001\tabularnewline
& Other ages (5 - 24 weeks) & 1 & Reference & & 1 & Reference
&\tabularnewline
\textbf{Any diarrhea} & Cryptosporidium & 1.1 & 0.63 - 1.91 & 0.741 & NA
& NA & NA\tabularnewline
& \emph{C. parvum} & 3.2 & 1.56 - 6.91 & 0.002 & 2.98 & 1.24 -- 7.45 &
0.016\tabularnewline
& \emph{C. bovis} & 0.55 & 0.29 - 1.02 & 0.060 & 0.72 & 0.37 - 1.37 &
0.315\tabularnewline
& \emph{C. ryanae} & 0.77 & 0.36 - 1.59 & 0.477 & NA & NA &
NA\tabularnewline
& No \emph{Cryptosporidium} & 1 & Reference & & 1 & Reference
&\tabularnewline
& \textbf{Age} & & & & & &\tabularnewline
& Neonatal (1 - 4 weeks) & 0.74 & 0.50 - 1.09 & 0.125 & 1.04 & 0.66 --
1.65 & 0.87\tabularnewline
& Other ages (5 - 24 weeks) & 1 & Reference & & 1 & Reference
&\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Cry-Outbreak-Cattle-Hebei-Fig1/Cry-Outbreak-Cattle-Hebei-Fig1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Cry-Outbreak-Cattle-Hebei-Fig2/Cry-Outbreak-Cattle-Hebei-Fig2}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

