Guo et al.

Parasites & Vectors (2022) 15:362

https://doi.org/10.1186/s13071-022-05468-4
Parasites & Vectors

RESEARCH Open Access

A novel promising diagnostic candidate

selected by screening the transcriptome
of Babesia gibsoni (Wuhan isolate) asexual
stages in infected beagles
Jiaying Guo1,2,4, Furong Yang1,2, Lingna Wang1,2, Xuenan Xuan3, Junlong Zhao1,2 and Lan He1,2*

Abstract
Background: Babesia gibsoni is one of the causative agents of canine babesiosis worldwide. Some dogs infected
with B. gibsoni show severe clinical signs with progressive anemia, hemoglobinuria and splenomegaly. However,
most infected dogs present a state of chronic infection and thereby may be a persistent pathogen carrier, increasing
the risk of pathogen spreading. To date, little is known about this pathogen, with genomic and transcriptomic data
in particular generally unavailable. This lack of knowledge extensively limits the development of effective diagnostic
strategies and vaccines.
Methods: High-throughput RNA sequencing of total RNA of B. gibsoni asexual stages collected from infected beagles
was performed. The unigenes were annotated in seven databases. The genes were sorted according to their frag-
ments per kilobase per million (FPKM) value, which was used as an indicator for expression level. The gene with the
highest FPKM value was cloned from the genome of B. gibsoni and further tested for immunogenicity, cellular localiza-
tion and efficacy as a potential diagnostic candidate for detecting B. gibsoni in sera collected from beagles.
Results: A total of 62,580,653 clean reads were screened from the 64,336,475 raw reads, and the corresponding
70,134 transcripts and 36,587 unigenes were obtained. The gene with the highest FPKM value was screened from
the unigenes; its full length was 1276 bp, and it was named BgP30. The BgP30 gene comprised three exons and
two introns, with a 786-bp open reading frame, and encoded 261 amino acids with a predicted molecular weight of
30 kDa. The cellular localization assay confirmed the existence of P30 protein in B. gibsoni parasites. Moreover, P30 was
detected in the serum of experimentally B. gibsoni-infected beagles, from 15 days up to 422 days post-infection, sug-
gesting its usefulness as a diagnostic candidate for both acute and chronic infections.
Conclusions: We sequenced the transcriptome of B. gibsoni asexual stages for the first time. The BgP30 gene was
highly expressed in the transcriptome screening experiments, with further studies demonstrating that it could induce
immune response in B. gibsoni-infected dogs. These results lead us to suggest that bgP30 may be a good diagnostic
candidate marker to detect both acute and chronic B. gibsoni infections.
Keywords: Babesia gibsoni, Canine babesiosis, Transcriptome, BgP30, Diagnostic candidate

*Correspondence: [email protected]
1
State Key Laboratory of Agricultural Microbiology, College of Veterinary
Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China
Full list of author information is available at the end of the article

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Guo et al. Parasites & Vectors (2022) 15:362 Page 2 of 12

Background Most dogs infected with B. gibsoni show mild signs of

Babesiosis as an emerging tick-borne zoonosis has disease, whereas some will show severe signs with pro-
received increasing attention due to its risks for severe gressive anemia, hemoglobinuria, thrombocytopenia,
clinical signs and even death. Canine babesiosis is an splenomegaly and hepatomegaly [25]. In most cases, B.
increasing babesiosis that occurs in dogs. Initially, the gibsoni infection is asymptomatic or at a stage of chronic
agents for canine babesiosis were reported according infection after an acute infection, with low parasitemia
size, with the larger being Babesia canis and the smaller, persisting in host tissue for several years. In this latter
Babesia gibsoni [1]. However, with increasing knowledge case, B. gibsoni is rarely cleared from the host and, con-
of these agents, some “B. canis” were found to be iden- sequently, the host becomes a persistent carrier with
tical in size but different in vectors, clinical manifesta- the risk of pathogen spreading. As such, subclinical dis-
tions, pathogenicity and geographic distribution [2]. ease and low parasitemia pose a challenge for diagnosis.
Improvements in molecular biology techniques has lead Currently, there are three known methods to diagnose
to the identification of subspecies of B. canis, including B. gibsoni infection: direct microscopy study, immuno-
B. canis, B. vogeli and B. rossi, based on the 18S rRNA logical detection methods and molecular methods. The
gene [3]. An additional two, yet unnamed “large” size microscopy examination is the most common and fastest
species were also identified with different geographic dis- and is always used as the first way to detect Babesia spp.
tributions in eastern and southeastern USA in immuno- However, this pathogen prefers to hide in the periph-
compromised dogs and the UK, respectively [4–6]. Two eral circulatory system to evade the immune system of
new members have also been added one by one in to the the host; thus, blood samples collected for microscopic
“smaller” group, B. conradae and B. microti-like piro- detection may lack the parasite, leading to false-negative
plasm (later named B. vulpes) [7–9]. Recently, a medium- results [26, 27]. Immunology-based diagnosis is based on
sized (between the “large” and “smaller” size) Babesia detecting antibodies that are produced in the infected
species, named B. negevi, was identified [10]. Due to this dogs, and a number of B. gibsoni antigens have been
great variety of Babesia spp., canine babesiosis is widely identified to be relatively effective as diagnostic mark-
distributed all over the world with global significance. ers, such as BgSA1, BgP50 and thrombospondin-related
Of the known species, B. gibsoni, which was first recog- adhesive protein (TRAP) [28–30]. However, antibodies
nized in India as a new piroplasm in 1910, is the causa- can only be detected in cases of chronic infection as time
tive agent for canine babesiosis and has been reported in is needed for the production of antibodies. Molecular
several regions in Europe, Africa, Middle East, Americas, methods consist mainly of the detection of Babesia DNA
Australia and Asia [11]. In China, the first cases of canine by PCR; as such these tests are more sensitive and faster
babesiosis caused by B. gibsoni was documented by in than the other methods [26]. However, once again, it is
1985 [12]. Since then, canine babesiosis caused by B. gib- hard to detect this parasite because it hides in the periph-
soni has been reported in succession in multiple cities eral circulatory system; consequently, molecular methods
and provinces in China, mainly in locations in the north- have the same limitation as the microscopy examination.
ern, eastern and central parts of China, including Liaon- It is therefore necessary to combine different methods
ing Province in 2011, Jiangsu Province in 2014 and 2015, to detect the parasite. So far, there are a number of anti-
Shandong, Anhui, Zhejiang and Fujian provinces in 2015, gens of B. gibsoni that have been identified as potential
Hubei Province in 2017, Jiangxi Province in 2015 and targets for diagnosis, such as P32, P38, P50, P57 and P47
2017, Henan Province in 2016 and 2019, Shaanxi Prov- that have mostly been tested by enzyme-linked immuno-
ince in 2020 and the municipalities of Shanghai in 2015 sorbent assay (ELISA) [31–34]. However, some of these
and Beijing in 2017 [13–20]. were ineffective or showed lower sensitivity, resulting in
Babesia gibsoni is a tick-borne pathogen and poten- almost no effective antigen that can be applied in clini-
tially transmitted by different tick species [21]. In addi- cal practice. Consequently, the diagnosis of B. gibsoni
tion to the traditional transmission route by ticks, remains challenging.
however, an increasing number of clinical investiga- The development of effective diagnostic candidates is
tions indicate that this pathogen can be opportunisti- closely related to a comprehensive understanding of B.
cally transmitted by other routes, including, for example, gibsoni biology. The current lack of information on the
blood transfusion, dog bites and transplacental routes genome and transcriptome of B. gibsoni extensively lim-
[22–24]. Those multiple transmission routes may repre- its the elucidation of the B. gibsoni mechanisms of its
sent the main factor underlying the high the prevalence life-cycle and thereby hinders the development of good
of B. gibsoni. diagnostic methods and vaccine candidates. In this arti-
The clinical manifestations for B. gibsoni infection are cle, we sequenced and analyzed the transcriptome of B.
varied depending on the host’s age and immune status. gibsoni (Wuhan isolate). We then selected an antigen that
Guo et al. Parasites & Vectors (2022) 15:362 Page 3 of 12

showed relative high expression to conduct subsequent Waltham, MA, USA), extracted by chloroform, precipi-
assays to explore its potential to be an effective diagnostic tated by isopropyl alcohol and ethanol and treated with
candidate. DNase I (Invitrogen, Thermo Fisher Scientific), thus
avoiding DNA contamination. The isolated RNA was
Methods processed with a preliminary quantitation, tested for
Experimental animals degradation and potential contamination and checked
Three 4-year-old healthy beagle dogs were purchased. for integrity and quantity. Total RNA was prepared by
They were subsequently tested and found to be free of messenger RNA (mRNA) enrichment using oligo beads.
Babesia infection using light microscopy, PCR (18S and The enriched mRNA was then fragmented randomly in
internal transcribed spacer primers), real-time PCR and fragmentation buffer, followed by complementary DNA
loop-mediated isothermal amplification (LAMP) meth- (cDNA) synthesis using random hexamers and reverse
ods [35, 36] (Additional file 1: Table S1). transcriptase. Construction of the cDNA library needed
Each beagle dog was injected intravenously with 1 ml for sequencing required a round of purification, terminal
of B. gibsoni (Wuhan strain)-infected blood that had been repair, tailing, ligation of sequencing adapters, size selec-
stored in liquid nitrogen (percentage parasitized erythro- tion and PCR enrichment, as well as accurate quantifica-
cytes [PPE]: 1%). At 1 week after injection, blood samples tion quantitative PCR. Sequencing of the cDNA library
from each beagle were tested by PCR and examined by was performed on an Illumina Hiseq X Ten sequencing
microscopy to monitor and calculate the PPE (Additional platform (Novogene Bioinformatics Technology Co., Bei-
file 2: Figure S2). When PPE reached 15%, blood from the jing, China).
three beagle dogs was collected into sterile vacuum tubes
containing anticoagulant with EDTA. After blood collec-
tion, each beagle dog was treated with diminazene acetu- Data analysis
rate and vitamins. The raw data obtained from the Illumina Hiseq X Ten
sequencing platform were transformed to sequenced
Blood preparation reads by base calling, followed by quality control, which
The procedure used for preparing blood in the present included the evaluation of error rate, of GC content dis-
study was modified from a previous study [34]. The tribution and of data filtering. Due to the absence of the
infected blood was centrifuged at 1500 rpm for 15 min at B. gibsoni genome as reference, clean reads were assem-
4 °C, and the supernatant and layer of white blood cells bled to obtain a reference sequence for subsequent analy-
removed completely. The pellet was resuspended in cold sis using Trinity software [37]. The obtained sequences
phosphate buffered saline (PBS) to the original volume were processed with hierarchical clustering using the
and gently mixed. These steps were repeated three more command-line program Corset. The RSEM software
times until the supernatant was clear and all white cells package was used to map reads back to the transcriptome
discarded [34]. and quantify the expression level, based on the FPKM
The infected red blood cells (RBCs) were lysed by Red (fragments per kilobase per million reads) value [38]. The
Blood Cell Lysis Buffer (Beyotime Biotechnology, Shang- annotation of the gene function was performed using
hai, China), following which the solution was left stand- several database: NR (NCBI non-redundant protein
ing for 1 h at 4 °C. The RBCs were then completely lysed sequences), PDB (Protein Data Bank), Swiss-Prot, PIR
by strenuous vibration. The lysate was then centrifuged at (Protein Information Resource), PRF (Protein Research
14,000 rpm for 10 min at 4 °C, and the supernatant was Foundation), COG (Cluster of Orthologous Groups of
discarded. The pellet was washed in PBS, and the above proteins), KOG (euKaryotic Orthologous Groups), GO
steps were repeated until the color of the pellet was white (Gene Ontology) and KEGG (Kyoto Encyclopedia of
rather than red or pink. The pellet was then resuspended Genes and Genome) [39–41]. CoDing Sequence (CDS)
in 1 ml PBS and filtered through a 2-μm nuclepore Track- prediction was carried out by Basic Local Alignment
Etch membrane filter (Whatman, Maidstone, UK). The Search Tool (BLAST; https:blast.ncbi.nlm.-nih.gov/Blast.
effluent liquid was collected and kept for further B. gib- cgi) unigenes based on the priority of the NR and Swiss-
soni RNA extraction. Prot databases. If there were no hits in BLAST, unigenes
were analyzed using the ESTScan (3.0.3) program to pre-
RNA isolation, complementary DNA library construction dict their coding regions and determine their sequence
and sequencing direction. A single nucleotide polymorphism and inser-
The RNA of B. gibsoni from the blood collected from tion-deletion were detected using the Genome Analy-
the three beagle dogs was individually isolated by TRI- sis Toolkit (GATK3) [42], and simple sequence repeat
zol reagent (Life Technologies, Thermo Fisher Scientific, detection of the unigenes was performed using the MISA
Guo et al. Parasites & Vectors (2022) 15:362 Page 4 of 12

(v1.0) tool. Quantitative real-time PCR was conducted to The pE-sumo-P30 was then induced by isopropyl-β-d-
verify the results obtained by sequencing. thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis,
MO, USA) at 37 °C for 4 h and analyzed by sodium dode-
Gene amplification and recombinant protein expression cyl sulphat-polyacrylamide gel electrophoresis (SDS-
Due to incomplete information on the genome of B. PAGE). The pE-sumo-P30 was His-tagged and purified
gibsoni, the sequence of the reference genome was also by ProteinPure Ni–NTA Resin (TransGen Biotech, Bei-
based on other Babesia species whose genome infor- jing, China) and stored at − 80 °C.
mation had been submitted to the NCBI database, such
as Babesia bovis, Babesia ovata, Babesia bigemina, B. Polyclonal antibody production
microti, among others. The transcriptome level of the Two Japanese white rabbits, aged 2–3 months, were pre-
different genes was indicated by the FPKM value [43]. pared to generate antibodies against the recombinant
The genes were sorted by FPKM values; the one that was BgP30 (rBgP30). For the first immunization procedure,
highly expressed at the transcriptome level was anno- 400 μg rBgP30 that had been completely mixed with an
tated to the hypothetical protein of B. bovis (NCBI refer- equal volume of Freund’s complete adjuvant (Sigma-
ence sequence: XP_001611467) using the NR database. In Aldrich) was subcutaneously injected into the back of
order to obtain the gene sequence in B. gibsoni, the amino the rabbits. The rabbits were subsequently given three
acid sequence of the hypothetical protein of B. bovis was boosters (each 150 μg in Freund’s incomplete adjuvant)
regarded as the query sequence and BLAST was used at 2-week intervals. The serum was collected before and
to screen the high similarity sequence in the incomplete 2 weeks after the final booster and the titers of antibody
assembly genome of B. gibsoni. The gene thus obtained tested using ELISA.
was analyzed using bioinformatic tools. Based on the
obtained sequence in the genome, primers were designed Enzyme‑linked immunosorbent assay
to clone the gene in B. gibsoni. Due to the presence of two Blood was collected from each beagle dog infected with
introns, the gene was truncated and expressed from 749 B. gibsoni from day 1 up to day 422 post infection. The
to 1276 bp. The truncated gene was amplified from the serum collected on the different days was collected
cDNA of B. gibsoni and then ligated with the pE-sumo separately and stored at − 20 °C. The concentration of
vector to construct the recombinant plasmid by the rBgP30 was evaluated using the BCA Protein Assay Kit
homologous recombination method. The forward primer (Beyotime Biotechnology). rBgP30 was diluted by coating
for cloning the P30 gene was 5′-CAC CGC GAA CAG buffer (25 mM carbonate buffer solution, pH 9.6) to 1 μg/
ATT GGA GGT CGG GAT CCA TAC GGC TAT GAG- ml, and each well of the 96-well (flat-bottom) plate was
3′ and the reverse primer was 5′-TCG AAT TCG GAT coated with purified rBgP30 at 4 °C and allowed to stand
CCT CTA GTT CAG CGT GGC ACA CGA CGT TC-3′. overnight. The plate was then blocked by 1% bovine
The forward primer for cloning pE-sumo was 5′-ACC serum albumin (BSA) at 37 °C for 30 min, followed by
TCC AAT CTG TTC GCG GTG-3′and the reverse incubation with the serum collected on different days
primer was 5′-ACT AGA GGA TCC GAA TTC GA-3′. post infection, diluted 1:1000 in 0.1% BSA/PBS, with
The PCR cycling parameters for the P30 gene were: an each well containing 200 μl, at 37 °C for 1 h. The second-
initial denaturation for 2 min at 98 °C; followed by 35 ary antibody, horseradish peroxidase (HRP)-labeled goat
cycles of denaturation at 98 °C for 10 s, annealing at 55 °C anti-canine (Beyotime Biotechnology), diluted 1:5000 in
for 10 s and extension at 72 °C for 10 s; with a final exten- 0.1% BSA/PBS, was added to each well at 37 °C for 1 h.
sion for 10 min at 72 °C. The PCR cycling parameters for Then, TMB (Sigma-Aldrich), a HRP substrate, and ­H2O2
the pE-sumo vector were: 95 °C for 3 min; followed by 35 were added to each well, and the reaction was stopped by
cycles of 95 °C for 15 s, 56 °C for 15 s and 72 °C for 6 min; the addition of 0.25% hydrofluoric acid. The absorbance
with a final extension for 1 min at 72 °C. The amplicon values were measured at OD630.
product for the P30 gene was ligated to the pE-sumo vec-
tor by the ClonExpress II One Step Cloning Kit (Vazyme Immunodetection of rBgP30
Biotech, Nanjing, China). The recombinant plasmid con- The rBgP30 protein was separated by SDS-PAGE and
struct was confirmed by enzyme digestion and PCR for transferred to a PVDF membrane (Thermo Fisher Scien-
integration. The validated pE-sumo-P30 product was tific), then blocked with 5% skim milk at 4 °C overnight.
transfected into Escherichia coli BL21 (DE3) for recom- The membrane was subsequently incubated with the
binant protein expression. Based on the ratio of 1:100, serum of B. gibsoni-infected beagle dogs diluted 1:500
the pE-sumo-P30 bacterial solution was transferred to in PBS at 37 °C for 3 h. The secondary antibody, HRP-
LB medium containing ­Amp+ (1:1000) for incubation at labeled goat anti-canine (Beyotime Biotechnology), was
37 °C for about 3 h to reach an absorbance of 0.5 at ­A600. incubated with the membranes. The membranes were
Guo et al. Parasites & Vectors (2022) 15:362 Page 5 of 12

visualized using diaminobenzidine (ZSGB-BIO, Beijing, reads and reads containing N, which could not be used
China). for further analysis. We therefore filtered the raw reads to
remove these types of reads from the dataset and obtain
Immunofluorescence antibody assay clean reads. In this study, 62,580,653 clean reads were fil-
Polyclonal antibody was prepared for detection of BgP30 tered from the raw reads, accounting for 97.28% of data,
in the B. gibsoni by immunofluorescence antibody assay with an average data volume of 9.39 G. The average Q20
(IFAT). The RBCs from B. gibsoni-infected beagle dogs and Q30 values (percentage of bases with a quality score
was smeared onto the slide. The smears were fixed with > 20 and > 30, respectively) and GC content were 96.79,
95% methanol and 5% acetone (v:v) at − 20 °C for 30 min, 91.97 and 52.7%, respectively. The clean reads were then
permeabilized for 10 min with Triton X-100 in PBS and spliced to obtain the transcripts as references for subse-
then washed three times in PBS. The rabbit polyclonal quent analyses. However, as information of the B. gib-
antibodies against rP30 diluted 1:500 in 1% BSA/PBS soni genome was not available at the time of the study,
were added to the smears at 37 °C for 2 h. The control we had no reference genome for splicing and assembly.
groups were incubated with the serum of naïve rabbits. In this study, we used the “Trinity” tool for splicing the
After the smears were washed three times in PBS, they clean reads [37], and obtained 70,134 transcripts and
were simultaneously incubated with the Alexa Fluor 36,587 unigenes. The lengths of the transcripts and uni-
488-conjugated secondary antibody (Life Technologies, genes were analyzed, and most of the transcripts were
Thermo Fisher Scientific) diluted 1:2000 in 1% BSA/ found to be between 200 and 500 bp (57%); for the uni-
PBS as the secondary antibody and Hoechst (Invitrogen, genes, we focused on those 500–1000 bp in length (29%).
Thermo Fisher Scientific) for nuclear staining at 37 °C for All of the unigene studies were performed with the gene
1 h in the dark. The slides were analyzed by fluorescence functional annotation according to seven databases (NR,
microscopy or stored at 4 °C for further analysis. Nt [NCBI nucleotide sequences], Pfam [protein family],
KOG, Swiss-Prot, KEGG and GO [Gene Ontology]). For
Results GO classification, the unigenes were annotated in three
RNA sequencing and de novo assembly major categories (Fig. 1): (i) biological processes; (ii) cel-
The initial data obtained from the RNA sequencing were lular components; and (iii) molecular functions. For the
raw reads, with 64,336,475 raw reads obtained from the biological process category, most of the unigenes were
RNA sequencing transcripts of B. gibsoni. However, the matched to cellular process and metabolic process. For
raw data contained low-quality reads, adapter-related the cellular component category, most of the unigenes

Fig. 1 Gene function classification of transcriptome data on Babesia gibsoni during the asexual stages in infected beagle dog. Red indicates the
biological process category, green indicates the cellular component category and blue indicates the molecular function category
Guo et al. Parasites & Vectors (2022) 15:362 Page 6 of 12

were assigned to cell and cell part. For the molecu- in the uncomplete genome of B. gibsoni. For the query
lar function category, binding and catalytic activity sequences of B. bigemina proteins (XP_012766105 and
accounted for the largest proportion. For KOG classifica- XP_012766347), there were no complete open reading
tion, the functions of “general function prediction only,” frame (ORF) sequences when screening in the unassem-
“posttranslational modification, protein turnover, chap- bled genome of B. gibsoni. After comprehensive analysis
erones” and “signal transduction mechanisms” were the of the three sequences, we determined only to study the
dominant functions (Fig. 2). Most of the unigenes were hypothetical protein of B. bovis (XP_001611467) in sub-
predicted to be involved in signal transduction and trans- sequent analyses.
lation processes in the KEGG classification. Through NR
annotation, the unigenes could be matched to sequences P30 of B. gibsoni
that had a high similarity and had been deposited in the The amino acid sequence of this hypothetical pro-
database to conduct gene functional annotation. Of the tein of B. bovis was screened in the genome of B. gib-
unigenes, 13.4% could be matched to B. bigemina genes soni. Through comprehensive analysis of the results of
and 2.8% could be matched to B. bovis genes. The clean tBLASTn in the genome, the full-length of the 467-like
reads were then mapped to the reference sequences that gene in B. gibsoni was 1276 bp, comprising three exons,
were the splicing results of Trinity by RESM software. with lengths 120, 138 and 528 bp, respectively. Two
The number of “readcount” of every gene obtained in introns, with lengths 31 and 459 bp, respectively, were
this process was switched to FPKM. The FPKM value interspersed among the three exons. The length of the
is currently the most common indicator for the expres- ORF was 786 bp, encoding 261 amino acids with the
sion level of unigenes [43–45]. The genes matched to the predicted molecular weight of 30 kDa. The theoretical
Babesia species were sorted based on the FPKM value isoelectric point was 9.67. The 467-like gene in B. gib-
(Additional file 3: Table S3). Of these, the gene with the soni was thus named P30. Forward and reverse primers
highest FPKM value was the G-beta repeat domain-con- were designed to clone the P30 gene from the genomic
taining protein of B. bigemina (XP_012766105), followed DNA and cDNA of B. gibsoni, respectively. A specific
by a hypothetical protein of B. bigemina (XP_012766347) band of approximately 1276 bp was amplified from the
and a hypothetical protein of B. bovis (XP_001611467). gDNA (Fig. 3). To further study this gene, the 528-bp
The amino acid sequences of these three proteins were exon was cloned from the cDNA and ligated to the
all screened as query sequences and BLAST-searched pE-sumo vector. The pE-sumo-BgP30 was His-tagged

Fig. 2 KOG function classification. A total of 26 groups of KOG functions were identified in the transcriptome data of B. gibsoni. The percentage of
genes that annotated to the groups are shown on the Y-axis. KOG, euKaryotic Orthologous Groups database
Guo et al. Parasites & Vectors (2022) 15:362 Page 7 of 12

Immunoreactivity of BgP30
In the western blotting assay, rBgP30 was blotted onto
the nitrocellulose membranes and then incubated with
serum collected from healthy and B. gibsoni-infected
beagle dogs, respectively. A corresponding band of about
32 kDa (consistent with the predicted molecular weight
of rBgP30) was specifically recognized by the serum of
the B. gibsoni-infected beagle dog, but not by the serum
of the healthy beagle dog (Fig. 5). These results showed
that P30 had good immunoreactivity that could effec-
tively induce the production of antibodies in the beagle
dog.

Localization of P30 in B. gibsoni

The localization of P30 in B. gibsoni was determined
by IFAT using the rabbit polyclonal antibodies against
Fig. 3 Amplification of the B. gibsoni P30 gene (BgP30). a Lanes: M, rP30. Both for extracellular and intracellular merozo-
marker; 1, cloned BgP30 gene from the complementary DNA (cDNA) ites, the green fluorescence (P30 of B. gibsoni) simulta-
of B. gibsoni; 2, cloned BgP30 gene from the genomic DNA (gDNA) neously localized with the blue fluorescence (nucleus of
of B. gibsoni. b Lanes: M, marker; 1, the truncated BgP30 gene cloned
B. gibsoni) (Fig. 6), but there was no green fluorescence
from gDNA; 2, the truncated BgP30 gene cloned from cDNA
detected by when rabbit polyclonal antibodies against
rP30 were incubated with the serum of healthy rabbits.

with the predicted molecular weight of 32 kDa and was Determination of the antibody response of BgP30
mainly expressed as the inclusion body at 37 °C (Fig. 4). The serum of the experimental beagle dog that was
The secondary structure of BgP30 was predicted and infected with B. gibsoni was collected from day 1 to day
analyzed by online software. No potential signal pep- 422 post infection to determine the antibody response
tides, transmembrane regions or GPI anchors were of rBgP30 by ELISA. As shown in Fig. 7a, all three dogs
observed. were determined to have a detectable level of antibody
response to rBgP30 on day 5 post infection and have a
significant antibody response to rBgP30 on day 7 post

Fig. 4 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

analysis of protein expression of the recombinant BgP30 protein Fig. 5 Determination of the immunogenicity of BgP30. Lanes:
(rBgP30) in Echerischia coli. Lanes: M, molecular weight marker; 1, M, molecular weight marker; 1, rBgP30 incubated with serum of
supernatant form of rBgP30; 2, inclusion body form of rBgP30; 3, B. gibsoni-infected dog; 2, rBgP30 incubated with the serum of B.
purified form of rBgP30 gibsoni-naïve dog
Guo et al. Parasites & Vectors (2022) 15:362 Page 8 of 12

Fig. 6 Cellular localization of the P30 protein in B. gibsoni. a During the extracellular stage, BgP30 with green fluorescence co-located with the
nucleus of merozoites with blue fluorescence. b When the merozoite resided in the red blood cell, the poly-antibody against rBgP30 detected
P30 in the intraerythrocytic merozoite of B. gibsoni. c At the post-invasion stage of paired parasites, P30 was also detected with green fluorescence
adjacent to the merozoite nucleus. d Negative control: infected erythrocytes were incubated with the serum of B. gibsoni-naïve rabbits. Scale bar:
1 μm

infection. The antibody titer maintained a relatively sta- reside in a parasitophorous vacuole to separate them-
ble value until day 422 post infection, even when the dog selves from the content of the host cell, research indi-
had reached the chronic infection stage with a low level cates that Babesia do not form a vacuole in the RBCs
of parasitemia (Fig. 7B). [46]. These differences suggest that Babesia have a simple
life-cycle and a simple invasion mechanism that allows
Discussion it to reside in the RBC. However, current knowledge on
Babesia spp. are included in phylum Apicomplexa, and Babesia is relatively limited, and only the genomes of
compared to other members of this phylum, such as Plas- only a few Babesia species have been sequenced, includ-
modium, which can invade both hepatocytes and RBCs, ing those of B. bovis, B. divergens, B. bigemina, B. ovata,
and Toxoplasma gondii, which can invade all nucleated B. microti and Babesia sp. Xinjiang [48–53]. For tran-
cells, Babesia spp. can only invade RBCs, which have no scriptome studies, only the transcriptome of B. bovis, B.
nucleus, thereby providing space for hemoglobin, which divergens, B. ovata, B. microti and Babesia sp. Xinjiang
accounts for up to 96% of the total protein in RBCs [46, are available in databases [48, 49, 54, 55]. Comparison
47]. Also, unlike Plasmodium and T. gondii, which will of these genomes suggests that the transcriptome will
Guo et al. Parasites & Vectors (2022) 15:362 Page 9 of 12

Fig. 7 Detection of antibody response to rBgP30 in the serum of beagle dogs experimentally infected with B. gibsoni, by enzyme-linked
immunosorbent assay. a Details of antibody response from day 1 to day 28 post infection. b Results for antibody response from day 1 to day 422
post infection. P/N, Mean OD of the test specimen reacted on antigen (P)/mean OD of the control serum reacted on antigen (N)

change to adapt with changes in the environment to a obtained were then annotated by NR, Nt, KOG, Swiss-
certain degree, in a manner that will better reflect gene Prot, KEGG and GO analysis [61, 62]. The FPKM value,
expression. Babesia expresses different genes in response which is the common indicator for expression level, was
to its presence either in a vertebrate host or in ticks [56, used to screen some genes with relative high expres-
57]. Therefore, transcriptome data will greatly contribute sion level in the transcriptome of B. gibsoni [43, 45]. The
to our understanding of the mechanism of how Babesia selected genes with high FPKM value as query sequences
spp. can reside in RBCs. For B. gibsoni, one of the main were successively BLAST-searched in the unassem-
agents of babesiosis in dogs, complete information on the bled genome of B. gibsoni. A gene with a full length of
genome and transcriptome is currently unavailable. Most 1276 bp was screened in the genome of B. gibsoni with
of the current research on B. gibsoni has focused on anti- the query sequence of high FPKM value of B. bovis
gen identification, drug treatment and the establishment (XP_001611467). The ORF of this gene was 786 bp and
of a related ELISA, PCR and/or other molecular level comprised three exons encoding 261 amnio acids. The
methods for diagnosis purposes. Some antigens have predicted molecular weight of the encoded protein was
been identified as potential diagnostic candidates, such 30 kDa, and the gen was named BgP30. The amino acid
as P12 for serodiagnosis, and other B. gibsoni antigens, sequence of the BgP30 protein was BLAST-searched in
as P32, P38 and P50, which have been evaluated using the sequences database in NCBI, and 13 sequences were
ELISAs [31, 32, 36, 58, 59]. However, the mechanism identified with significant alignments, including proteins
for its asexual life in RBCs and sexual life in the vector in B. bovis (XP_001611467), B. ovata (XP_028865573),
tick remain unknown [60]. The unavailability of genome B. sp. Xinjiang (XP_028872314), Theileria equi
and transcriptome information has extensively increased (XP_004832370), Theileria orientalis (UKK01334,
research on diagnostic markers. XP_009691561, PVC49716 and UKJ88966), Theileria
In this study, the transcriptome of B. gibsoni (Wuhan parva (XP_763348), Theileria annulata (XP_955014), B.
isolate) during the asexual stages in beagle dogs was bigemina (XP_012768809), B. microti (XP_012647546)
sequenced and analyzed. A total of 62,580,653 clean and Cryptosporidium andersoni (OII78288), with B. gib-
reads and 36,587 unigenes were obtained. The unigenes soni being the exception. All of the aligned sequences
Guo et al. Parasites & Vectors (2022) 15:362 Page 10 of 12

were hypothetical or uncharacterized proteins that have immunoreactivity. The results suggest that BgP30 can be
not been further studied and annotated with specific and regarded as a potential candidate for diagnostic purposes.
detailed functions. The alignment results suggested that These results may provide new insight into B. gibsoni
BgP30 had no homology with any sequences that had research and promote the development of an effective
been annotated and studied in B. gibsoni, and that it was diagnosis and control of B. gibsoni infection.
a new gene that could be further studied. It should be
noted that the sequences with high similarities to BgP30
Abbreviations
in other species had also not been deeply studied with cDNA: Complementary DNA; BLAST: Basic Local Alignment Search Tool; BSA:
regards to specific functions. Bovine serum albumin; ELISA: Enzyme-linked immunosorbent assay; FPKM:
BgP30 was subjected to detailed assays in an attempt to Fragments per kilobase per million; HRP: Horseradish peroxidase; IFAT: Immu-
nofluorescent antibody test; IPTG: Isopropyl-β-d-thiogalactopyranoside; LAMP:
elucidate related functions. In a first step, this protein was Loop-mediated isothermal amplification; PBS: Phosphate buffered saline; PPE:
expressed as a His-tagged fusion protein in E. coli. The Percentage parasitized erythrocytes; rRNA: Ribosomal RNA.
His-tagged protein was then used for antibody detection
in subsequent assays. IFAT was conducted using antibod- Supplementary Information
ies against BgP30. The specific green fluorescence (indi- The online version contains supplementary material available at https://​doi.​
cator of BgP30) was detected in the B. gibsoni by IFAT, org/​10.​1186/​s13071-​022-​05468-4.
thereby confirming that P30 was present in B. gibsoni.
Additional file1: Table S1. PCR primers for Babesia detection.
These results confirm the western blotting assay can be
Additional file2: Figure S2. Parasitemia for each beagle dog.
used to specifically detect rBgP30 in the serum of beagle
Additional file3: Table S3. The list of genes that matched to the Babesia
dogs infected with B. gibsoni, revealing that BgP30 could
species.
induce the production of antibodies in the host. Moreo-
ver, the serum B. gibsoni-infected Beagle dogs on day 5
Acknowledgements
post infection could even detect rBgP30, and the serum Our sincere thanks to Dr. Heba Alzan, Parasitology and Animal Diseases
on day 7 post infection could significantly recognize the Department, National Research Center, Dokki, Giza, Egypt for editing the
rBgP30 by ELISA. These results indicate that BgP30 has English.
the potential to be an early diagnosis antigen for B. gib- Author contributions
soni; they are also consistent with the results of the tran- Samples collection: JG and LW. Experiments: JG, FY and LW. Data analysis:
scriptome study showing a high expression level of BgP30 JG, FY and LW. Manuscript editing: JG, XX, JZ and LH. All authors read and
approved the final manuscript.
during the asexual stages in the RBCs of infected bea-
gle dogs. Moreover, when the B. gibsoni-infected Beagle Funding
dogs were in the chronic stage of infection, BgP30 could This study was supported by the National Natural Science Foundation of
China (Grant No. 31930108), and the Fundamental Research Funds for the
be also recognized by the serum on day 422 post infec- Central Universities in China (Project 2662020DKPY016 and 2662022DKYJ001).
tion, suggesting P30 might be continuously expressed in
B. gibsoni and may be considered as a good diagnostic Availability of data and materials
All data obtained in this study had been deposited to the National Center for
candidate. Biotechnology Information (NCBI) with the accession number SRR12433308,
The growing significance of pets to humans and the SRR12433307 and SRR12433306. The GenBank accession number of P30 of B.
ever increasing frequency at which dogs are a traveling gibsoni was OM925517.
companion to humans extensively increase the risk of
B. gibsoni spreading. However, there is still no effective Declarations
commercial diagnostic kit available for B. gibsoni. This Ethics approval and consent to participate
study aimed to screen a candidate marker to be used for The experimental animals were housed and treated in accordance with the
B. gibsoni diagnosis from the transcriptome of B. gibsoni. stipulated rules for the regulation of the administration of affair concerning
experimental animals of P. R. China. All experiments were performed under
BgP30, with a high expression level, was confirmed by the approval of Laboratory Animals Research Centre of Hubei Province and
assays to be a potential candidate not only for early diag- the Ethics Committee of Huazhong Agricultural University (Permit number:
nosis but also during the chronic stage. The results of this SCXK (Hubei) 2020–0019).
study may be useful and provide the theoretical founda- Consent for publication
tion for the development of diagnostic kits. Not applicable.

Competing interests
Conclusions The authors declare that they have no competing interests.
To our knowledge, we report here the first results on the
Author details
transcriptome of B. gibsoni endemic to Wuhan, China. 1
State Key Laboratory of Agricultural Microbiology, College of Veterinary
Based on transcriptome results, BgP30 was screened Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
2
for further study regarding its cellular localization and Key Laboratory of Preventive Veterinary Medicine in Hubei Province,
Guo et al. Parasites & Vectors (2022) 15:362 Page 11 of 12

Wuhan 430070, Hubei, China. 3 National Research Center for Protozoan 22. Stegeman JR, Birkenheuer AJ, Kruger JM, Breitschwerdt EB. Transfusion-
Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, associated Babesia gibsoni infection in a dog. J Am Vet Med Assoc.
Hokkaido 080‑8555, Japan. 4 Present Address: Northeast Agricultural University, 2003;222:959–63.
Harbin 150000, Heilongjiang, China. 23. Fukumoto S, Suzuki H, Igarashi I, Xuan X. Fatal experimental transplacen-
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Received: 22 June 2022 Accepted: 1 September 2022 24. Birkenheuer AJ, Correa MT, Levy MG, Breitschwerdt EB. Geographic
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