Molecular epidemiology and risk factors of Anaplasma spp., Babesia spp. and Theileria spp. infection in cattle in Chongqing, China
Molecular epidemiology and risk factors of Anaplasma spp., Babesia spp. and Theileria spp. infection in cattle in Chongqing, China
Zuoyong ZhouID 0
Kai Li 0
Yingying Sun 0
Junge Shi 0
Hexian Li 0
Yiwang Chen 0
Haoyue Yang 0
Xiao Li 0
Bi Wu 0
Xiaoxia Li 0
Zhiying Wang 0
Fangjun Cheng 0
Shijun Hu 0
0 College of Animal Science, Rongchang Campus of Southwest University , Chongqing , China , 2 Veterinary Science Engineering Research Center of Chongqing , Chongqing , China
Tick-borne pathogens (TBPs) seriously affect cattle production and can be economically damaging. The epidemiology of these organisms in the Chongqing municipality of China is not well described. This study aimed to investigate the prevalence and risk factors of TBPs including Anaplasma spp., Babesia spp. and Theileria spp. in cattle in Chongqing municipality. The results showed that 43.48% (150/345) of cattle were infected with at least one TBP, of which single infections were detected in 104 (30.14%), double infections in 34 cattle (9.86%) and triple infections in 12 (3.48%) of the cattle. The overall prevalence of Anaplasma spp., Theileria spp. and B. bigemina were 22.32%, 23.19% and 7.24%, respectively. Among these, the prevalence of A. bovis, A. central, A. phagocytophilum, A. platys, A. marginale, T. sinensisi and T. orientalis were 8.41%, 7.83%, 4.93%, 4.35%, 2.61%, 22.32% and 2.60%, respectively. We could not detect B. bovis, T. annulata, T. luwenshuni or T. uilenbergi in cattle. Cattle 1-year-old were more likely to be infected with Theileria spp. [adjusted odd ratio (AOR) = 2.70, 95% CI = 1.12-6.56)] compared with younger cattle, while cattle 1-year-old had reduced susceptibility to B. bigemina (AOR = 0.14, 95% CI = 0.030.60). Cattle living at higher altitude ( 500 m) were more susceptible to B. bigemina (AOR = 6.97, 95% CI = 2.08-23.35) and Theileria spp. infection (AOR = 1.87, 95% CI = 1.06-3.32). The prevalence of Theileria spp. on farms with cats was significantly higher than that without cats (AOR = 2.56, 95% CI = 1.12-5.88). Infection with A. bovis and A. central were significantly associated with A. phagocytophilum infection. Furthermore, there were significant associations between A. bovis and A. central infection, T. sinensisi and A. marginale infection, and B. bigemina and T. orientalis infection. This study provides new data on the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in cattle in Chongqing, and for the first time we reveal a possible relationship between the afore-mentioned pathogens, which will help in formulating appropriate control strategies for these pathogens in this area.
Editor: Gordon Langsley, Institut national de la
sante? et de la recherche me?dicale - Institut Cochin,
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: National Key Research and Development
Program of China (2018YFD0501705), Chongqing
Social Undertakings and Livelihood Security
Technology Innovation Projects
(CSTC2015SHMSZX80020) provided funding for
this project. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Tick-borne pathogens (TBPs) have always attracted the attention of researchers, not only for
their damaging influence upon livestock production but also for their public health threat [
Among the tick-borne diseases, anaplasmosis, babesiosis and theileriosis are the most
important and are distributed widely. These organisms affect cattle worldwide [
]. Till now, five
Anaplasma pathogens (A. marginale, A. bovis, A. centrale, A. phagocytophilum, and A. platys)
have been reported to cause bovine anaplasmosis, of which A. phagocytophilum has been
shown to infect a variety of animals and humans [
]. Two mainly Babesia pathogens,
Babesia bovis and B. bigemina, were found responsible for bovine babesiosis [
], and three species
of Theileria including T. annulata, T. sinensis, and T. orientalis (also named T. sergenti) were
the main causative agents of bovine theileriosis [
], and recently, T. luwenshuni has also
been detected in blood samples from cattle and yaks .
Numerous studies have reported the infection and prevalence of Anaplasma spp., Babesia
spp. and Theileria spp. in cattle across many countries [
]. In China, there have also
been many studies [
]. However, these studies usually focus on single pathogen
infections, and records on pathogen co-infections, the risk factors, and the mutual influence of each
pathogen in cattle are absent. In addition, studies relating to the aforementioned pathogens in
cattle in China have mainly been restricted to the northwest region, while the information is
very limited for southwest China.
The total number of cattle approximated 300 million at the end of 2015 in Chongqing, and
is one of the economic pillars of animal husbandry in this city. However, the prevalence of
Anaplasma spp., Babesia spp., and Theileria spp. in cattle in this area is unclear. The objectives
of this study were 1) to detect Anaplasma spp., Babesia spp., and Theileria spp. in cattle in
Chongqing, 2) to analyze the risk factors for infection of Anaplasma spp., Babesia spp., and
Theileria spp., and 3) to evaluate the associations of the aforementioned pathogens in cattle in
Materials and methods
Chongqing municipality is located in the southwest of China, between the northern latitudes
of 28.10??32.13?, and eastern longitudes of 105.11??110.11?. Its altitude ranges between 73.1
m at the Yangtze River in Wushan and 2796.8 m at Liangshan peak in Wuxi. The climate
tends to be subtropical, with a monsoon/humid climate and has an average annual
temperature of 16?18?C.
Blood sample collection and DNA extraction
Three hundred and forty five sodium citrate anticoagulated blood samples were collected from
10 ranches located in Tongnan, Rongchang, Jiangjing, Changshou, Liangping, Kaizhou,
Yunyang, Wushan, Fuling, and Qianjiang, from May 2016 to April 2017. The ranches were
selected based on the number of cattle ( 50) and convenience of sampling. The sampled
animals were randomly selected from apparently healthy cattle, and the information including
gender and age of cattle, as well as the altitude and the existence of cats in ranches were
recorded. The blood samples were sent back to the laboratory within an ice box. Whole blood
genome was extracted using a Wizard Genomic extraction kit (Promega, Madison, WI, USA)
according to the manufacturer?s instructions. This study was approved by the Ethics
Committee of Southwest. Consent was obtained from cattle owners before the collection of blood
samples from their cattle by an experienced, practicing veterinarian.
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PCR detection of Anaplasma spp., Babesia spp., and Theileria spp.
Anaplasma spp. (A. bovis, A. central, A. marginale, A. phagocytophilum and A. platys), Babesia
spp.(B. bovis and B. bigemina) and Theileria spp. (T. annulata, T. sinensis, T. orientalis, T.
luwenshuni and T. uilenbergi) infections were detected by PCR or nested PCR using the
primers reported in previous studies [
], the detail of primers can be found in S1 Table. The
primers were synthesized by Bioligo Biotechnology Co., Ltd (Shanghai, China). The PCRs
were performed according to the amplification programs in Table 1, with a volume of 12.5 ?L
in the reaction system including: 6.25 ?L Premix Taq (containing TaKaRa Taq, dNTP Mixture
and Taq Buffer) (Takara Dalian, China), 0.5 ?L of each forward and reverse primer (20 ?mol/
L), 1 ?L whole blood genome and 4.25 ?L ddH2O. The amplified PCR products were
photographed after electrophoresis in 1% agarose gels. The PCR amplification product were
randomly selected for sequencing to verify the reliability of test.
Risk factor analysis
Multivariable logistic regression was performed in SPSS for Windows (18.0 version, SPSS Inc.,
Chicago, IL, USA) to analyze factors associated with aforementioned infections. Adjusted odd
ratios (AOR) and 95% confidence intervals (CI) were calculated. A p-value of <0.05 was
considered statistical significant.
Prevalence of Anaplasma spp., Babesia spp., and Theileria spp. infection
A total of 345 cattle in Chongqing were included in this study. Detailed information pertaining
to infection is shown in Table 1 and Fig 1. The results showed that 43.48% (150/345) of cattle
were infected with at least one TBP, of which single infections were detected in 104 (30.14%),
double infections in 34 cattle (9.86%) and triple infections in 12 (3.48%) of the cattle. The
overall prevalence of Anaplasma spp., Theileria spp., and B. bigemina in cattle were 22.32% (77/
345), 23.19% (83/345), and 7.24% (25/345), respectively. Among the Anaplasma spp. detected,
A. bovis (29/345, 8.41%) was the most prevalent species recorded, followed by A. central (27/
345, 7.83%), A. phagocytophilum (17/345, 4.93%), and A. platys (15/345, 4.35%), while
infection with A. marginale (9/345, 2.61%) was the lowest. Among the Theileria spp., T. sinensisi
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and T. orientalis infections in cattle were 22.32% (77/345) and 2.60% (9/345), respectively. In
addition, we could not detect B. bovis, T. annulata, T. luwenshuni, or T. uilenbergi in this
Risk factor analysis based on blood sample data
The prevalence of Anaplasma spp. (25.13%) and Theileria spp. (27.27%) in male cattle was
significantly higher than that in females (Anaplasma spp.: AOR = 2.18, 95% CI = 1.05?4.52;
Theileria spp.: AOR = 3.27, 95% CI = 1.47?7.25). Cattle 1-year were more likely to be infected
with Theileria spp. than cattle aged below 1-year of age (25.00% vs. 20.99%), and the difference
was statistically significant (AOR = 2.70, 95% CI = 1.12?6.56). In contrast, cattle 1-year had a
lower risk of B. bigemina infection (AOR = 0.14, 95% CI = 0.03?0.60). Ranches at an altitude
500 m was found to be a risk factor for B. bigemina (AOR = 6.97, 95% CI = 2.08?23.35) and
Theileria spp. infection (AOR = 1.87, 95% CI = 1.06?3.32). With exception of Theileria spp.
(AOR = 2.56, 95% CI = 1.12?5.88), there were no significant associations between presence of
cats and infection with Anaplasma spp., or B. bigemina, (Table 2).
Risk factor analysis based on pathogen co-infection
In order to evaluate the effect of specific pathogen infections and how they influence other
pathogen infections within the same host, we considered each tested pathogen species as a
potential risk factor in the analysis. The results of correlation analyses between each species of
pathogen in infected cattle in Chongqing, are shown in Table 3. Infection with A. bovis and A.
central were significantly associated with A. phagocytophilum infection, and A.
Fig 1. An UpSetR plot of Anaplasma spp., Babesia spp. and Theileria spp. infection in cattle from Chongqing of southwest China
(n = 345). The blue horizontal coordinate columns represent the number of positive samples infected by pathogens. The purple vertical
coordinate columns represent the number of positive samples infected by a single pathogen or multiple pathogens. The black dots represent
the corresponding pathogens, and the dots connected by lines indicate co-infection of pathogens.
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phagocytophilum was more likely to increase the risk of A. central infection (AOR = 3.80, 95%
CI = 1.10?13.18, p = 0.035). However, A. central was less likely to impact upon infection with
A. phagocytophilum (AOR = 3.50, 95% CI = 0.97?12.59, p = 0.055). Furthermore, there was a
significant association between A. bovis and A. central infection, T. sinensisi and A. marginale
PLOS ONE | https://doi.org/10.1371/journal.pone.0215585
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Note: AOR: adjusted odds ratio; APH: A. phagocytophilum; AC: A. central; AB: A. bovis; AM: A. marginale; APL: A. platys; TS: T. sinensisi; TO: T. orientalis; BB: B.
infection, and B. bigemina and T. orientalis infection (p<0.05). There were no significant
associations between other pathogens that we aimed to identify.
For the first time, this systematic study investigated the epidemiology of Anaplasma spp.,
Babesia spp. and Theileria spp. infection in cattle in Chongqing, China. The infection rate of
Anaplasma spp. in our study was lower than that reported in Algeria [
] and in Tunisia [
but higher than that reported in northwest China [
]. The prevalence of A. bovis (8.41%) in
cattle in Chongqing was higher than that of cattle reported in other locations, where the
prevalence varied from 3.9% to 6.2% [
]. In contrast, the prevalence of A. centrale (7.83%)
was lower than that of cattle in previous studies (range between 12.1%-39.4%) [
Compared to the high prevalence of A. marginale in cattle in Madagascar (89.7%),
north-eastern Uganda (82.9%) , South Africa (57%) [
], Thailand (39.1%) [
] and in China
], we demonstrated a relatively low infection rate of A. marginale (2.61%) in
Chongqing. In addition, 4.93% of cattle tested positive for A. phagocytophilum in this study, which
was similar to the positivity rate (5.3%) of this pathogen in white yaks [
]. A. platys infection
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in cattle was first reported in Algeria [
], while Ben et al. reported a prevalence of A. platys-like
species (3.5%, 13/367) in cattle in Tunisia [
]. In this study, we noted a prevalence of A. platys
(4.35%, 15/345) in cattle for the first time in Chongqing.
The prevalence of B. bigemina in this study was similar to previous research by Liu et al
], and is lower than that reported in other provinces of China [
], South Africa ,
and in Tanzania [
]. However, the prevalence in our study was higher than that recorded in
the Philippines [
]. In this survey, only T. sinensisi and T. orientalis were detected, with the
prevalence being lower than T. sinensisi and T. orientalis infection rates recorded elsewhere
]. Similar to the previous report [
], we did not detected B. bovis infection in cattle.
The reason may be that 1) B. bovis infection in tick is usually lower than B. bigemina, which
result a lower transmission rates of B. bovis, and 2) B. bovis-infected red blood cells usually
accumulate in the capillary bed and leading to low parasitemia in circulating blood [
the reasons that T. annualata, transmitted by Hyalomma anatolicum anatolicum, is mainly
distributed in Northern China [
], T. luwenshuni and T. uilenbergi, both transmitted by
Haemaphysalis qinghaiensis and H. longicornis, usually infected sheep and goats in China [
there is no evidence of above ticks existence in Chongqing. It was not strange that we did not
detected T. annulata, T. luwenshuni, or T. uilenbergi infection in cattle from Chongqing.
There were 117 described species in the Chinese tick, 38 of which carry multiple pathogens
], and most of the ticks including H. anatolicum, H. qinghaiensis, H. longicornis, H.
bispinosa, Rhipicephalus (Boophilus) microplus, R. sanguineus, Dermacentor abaensis, D. silvarum
and D. nuttalli were founded in northwest, northeast or central of China [
], and these
ticks are responsible for transmission of a large amount of TBPs. However, the only reported
tick specie in Chongqing was R. microplus [
], which was recorded to be the vector of A.
phagocytophilumin, A. marginale, B. bigemina and B. bovis in China [
]. The differences in
the prevalence of some parasites in this study compared to that reported previously in other
studies in China or other countries, might be associated with geographical difference and
variation in tick species.
Risk factor analysis revealed a significant correlation of altitude and age with the prevalence
of B. bigemina and Theileria spp., which supported a previous report that there was a trend in
increased seropositivity for B. bigemina infection with age [
]. In addition, gender is a risk
factor associated with prevalence of Anaplasma spp. and Theileria spp., in cattle, which showed
that male cattle had higher risk for these two type of pathogens infection, and the presence of
cats in farm had positive effect on Theileria spp. infection in cattle from Chongqing, and the
reasons for these phenomenon are not clear.
This study first took a single infection as a risk factor in evaluating the impact on infection
with other pathogens. We found that cattle infected with A. bovis or A. central were more likely
to be infected with A. phagocytophilum, and there was also a strong association between A.
bovis and A. central infection. In addition, a very close relationship was observed for
co-infection with T. sinensisi and A. marginale, and B. bigemina and T. orientalis. Anaplasma spp.,
Babesia spp., and Theileria spp. are all tick borne pathogens (TBPs), and some ticks can harbor
mixed TBPs [
]. For the reasons that one species of TBP can be spread by different
types of ticks, and equally that the same type of tick may also be the transmission vector for
many species of TBPs, the significant correlation of the aforementioned pathogens might be
attributed to the fact that infected cattle were bitten by ticks carrying different pathogens.
From current data, it is not possible to estimate the chronological order of the aforementioned
pathogen infections but there does appear to be significant relationships among some of these
pathogens during infection of cattle. Parasite-parasite interaction may modify the impact of
the pathogenic species and affect the performance and survival of host [
]. It is a pity that
this study failed to evaluated the effect of above TBPs on health of cattle, since all the sampled
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animals in this study were apparently healthy, and we did not track the outcome of these cattle
and the causes of their death. Further research should be conducted to elucidate the type of
ticks present in Chongqing and the proportion of ticks that carry TBPs. Furthermore, attempts
should be made to confirm whether a pathogen significantly increases the incident infection of
other pathogens and the effects on production performance of cattle.
The results of the present survey indicated that infection of cattle with Anaplasma spp., Babesia
spp., and Theileria spp. is widespread in Chongqing. We provide a possible relationship
between afore-mentioned pathogenic infections, which will help in formulating appropriate
control strategies for these pathogens in this area.
S1 Table. Primers used for Anaplasma spp., Babesia spp. and Theileria spp. detection in
We really appreciate Prof. Kui Nie and Dr. Cailiang Fan for their practical support, Mrs.
Chunxia Dong and Mr. Hongquan Lin for their assistant in sample collection. We also thank
Prof. Jianjun Wen for English-language editing of this manuscript.
Investigation: Kai Li, Yingying Sun, Haoyue Yang, Xiao Li, Bi Wu, Xiaoxia Li.
Supervision: Zuoyong Zhou, Fangjun Cheng, Shijun Hu.
Writing ? original draft: Zuoyong Zhou, Kai Li, Yingying Sun, Junge Shi, Hexian Li, Yiwang
Writing ? review & editing: Zuoyong Zhou, Zhiying Wang, Fangjun Cheng, Shijun Hu.
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