Schistosoma japonicum EKLF/KLF1 is a potential immune target to tackle schistosomiasis

Background Interruption of parasite reproduction by targeting migrating schistosomula is a promising strategy for managing schistosomiasis. Hepatic schistosomula proteins previously identified based on second-generation schistosome DNA sequencing were found to hold excellent potential for schistosomiasis japonica diagnosis and as vaccine candidates. However, there are still many unknown schistosomula proteins that warrant further investigations. Herein, a novel schistosomula protein, the Schistosoma japonicum erythroid Krüppel-like factor (SjEKLF/KLF1), was explored. Methods Sequence alignment was carried out to detect the amino acid sequence characteristics of SjEKLF. The expression profile of SjEKLF was determined by western blot and immunofluorescence analysis. Enzyme-linked immunosorbent assay was used to determine the antigenicity of SjEKLF in hosts. Mice immunised with recombinant SjEKLF were challenged to test the potential value of the protein as an immunoprotective target. Results SjEKLF is defined as EKLF/KLF1 for its C-terminal DNA-binding domain. SjEKLF is mainly expressed in hepatic schistosomula and male adults and located within the intestinal intima of the parasites. Notably, high levels of SjEKLF-specific antibodies were detected in host sera and SjEKLF exhibited outstanding sensitivity and specificity for schistosomiasis japonica immunodiagnosis but failed to distinguish between ongoing infection and previous exposure. In addition, SjEKLF immunisation reduced the infection in vivo, resulting in decreased worm and egg counts, and alleviated body weight loss and hepatomegaly in infected mice. Conclusions Overall, these findings demonstrate that SjEKLF is critical for the infection of S. japonicum and may be a potential target to help control S. japonicum infection and transmission. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-023-05947-2.


Background
The prevalence of schistosomiasis has not diminished substantially over the past 2 to 3 decades [1].Results from an online survey of 365 experts on neglected tropical diseases suggested that current approaches focused primarily on mass drug administration may not be effective to eliminate schistosomiasis [2].Compared with schistosomiasis haematobium and schistosomiasis mansoni, the endemic region of schistosomiasis japonica is decreasing, primarily in Asian countries such as the Philippines and China [3,4].However, it is still necessary to recognize disease spreading patterns due to natural disasters and/or climate change.Thus, the development of improved diagnostic methods and efficacious vaccines is very important for preventing and controlling schistosomiasis.
Schistosomes have complex life cycles.In the host, schistosomula migrate from the infection site in the skin epidermis and travel along the blood vessels and then through the lungs.Schistosomula arrive at the hepatic portal system at approximately 14 days post infection (dpi), settle down and undergo sexual maturation.Eggs laid by female worms lead to the major pathologies associated with schistosomiasis [5,6].Migrating schistosomula are in direct contact with the host circulation and are directly exposed to the host humoral immune response; therefore, they are the main targets of schistosomiasis drugs and vaccines to prevent infection.
In a previous study, based on second-generation schistosome DNA chip data, we identified a set of genes whose expression was highest in the hepatic schistosomula, the early stage of Schistosoma japonicum, compared with that in cercariae, male adults, female adults and eggs [7].A group of hepatic schistosomula proteins encoded by these genes was successfully generated, and five exhibited excellent qualities as schistosomiasis japonica diagnostic and vaccine candidates.However, more than 60 proteins encoded by the identified genes have not been successfully prepared and investigated.In particular, the hepatic schistosomula transcriptional profile of a putative gene (GenBank ID: CNUS0000098854.1)was found to encode the S. japonicum KLF1 protein (SjEKLF, GenBank ID: TNN19877.1)by sequence alignment.
Krüppel-like factors (KLFs) belong to a family of transcription factors that comprise 18 proteins in humans [8][9][10], which are important constituents of the eukaryotic transcriptional machinery and regulate the expression of a wide variety of genes.The first mammalian KLF was identified in red blood cells and therefore was named erythroid Krüppel-like factor (EKLF) [11].EKLF, which is also known as Krüppel-like factor 1 (KLF1), consists of two domains: a N-terminal proline-rich transactivation domain and a C-terminal highly conserved DNAbinding domain comprising three C2H2 zinc fingers that recognise the consensus-binding site [12][13][14].EKLF plays a multifunctional and essential role at virtually all stages of erythrocyte development [15][16][17][18].Indeed, recent studies revealed that EKLF promotes the growth, migration and invasion of cancer cells [19][20][21].
To date, most studies on EKLF were performed in placental mammals and some in model organisms, such as zebrafish, but KLFs in pathogens have not been reported.SjEKLF was herein investigated further.The characteristics of this protein and its antigenicity during schistosome infection were explored in the present study.

Animals and parasites
Snails infected with S. japonicum (Oncomelania hupensis) were provided by the Jiangxi Provincial Institute of Parasitic Diseases, Jiangxi, China.Freshly released cercariae stimulated by light were immediately harvested.Six-week-old pathogen-free male BALB/c mice and New Zealand white rabbits (both from Vital River Laboratory Animal Technology Co., Beijing, China) were percutaneously infected with cercariae (40 ± 2 per mouse and 1000 ± 100 per rabbit).Serum samples were collected from the infected animals at 0, 7, 14, 21, 28, 35, 42 and 56 dpi.Hepatic schistosomula (14 dpi) and adult worms (42 dpi) were manually isolated by portal perfusion under a light microscope via the vascular system of the infected mice.Eggs were purified from the liver tissues of infected mice at 42 dpi by enzyme digestion, as previously described [22].

Human samples
Patients with schistosomiasis japonica (n = 20), echinococcosis (n = 15) and healthy volunteers (n = 20) were recruited in Hunan Province, Xinjiang Uyghur Autonomous Region and Heilongjiang (a province non-endemic for schistosomiasis japonica), China, from March 2016 to February 2017.Sera were collected from all participants at enrolment and again 3 months after praziquantel treatment in the case of schistosomiasis patients.All schistosomiasis cases were confirmed by examining EPG (eggs per gram of faeces) using the Kato-Katz method.Only three patients were moderately infected (EPG > 120), and the rest were mildly infected.After 3 months of praziquantel treatment, EPG was 0 in all patients except one case with 2. Cystic echinococcosis was confirmed by clinical diagnosis, parasitological detection and medical history records.Patient information is presented in Table 1.

Quantitative real-time polymerase chain reaction (qRT-PCR)
QRT-PCR was performed as previously described [24].Briefly, total RNA from parasites were extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany) and reversetranscribed into cDNA using the Invitrogen SuperScript III reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.The 26S proteasome non-ATPase regulatory subunit 4 (PSMD4; GenBank ID: FN320595) was used as reference gene [25].Reactions detecting the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank ID: FN324551) in standard cDNA (equally mixed cDNA from parasites at each developmental stage) were used as standard controls.The primers for the genes were designed using Primer BLAST (https:// www.ncbi.nlm.nih.gov/ tools/ primer-blast/) and were the following: SjEKLF: forward primer, 5′-GAG TCA CAC ATC CGA ACT GA-3′ and reverse primer, 5′-ACA TGT GCA ACC TGA CAA CTG-3′; PSMD4: 5′-CCT CAC CAA CAA TTT CCA CATCT-3′, 5′-GAT CAC TTA TAG CCT TGC GAA CAT -3′; GAPDH:5′-ATG GAA CAA GGA TGG TGC TGAG-3′; 5′-CAA CAA ACA TGG GTG CGT CT-3′.QRT-PCR was performed in technical triplicates using Brilliant II SYBR Green QPCR Master Mix Kit (Agilent Technologies, Santa Clara, CA, USA) and an Applied Biosystems 7500 Real-time PCR System (Thermo Fisher Scientific), according to the manufacturers' instructions.The data were analysed using Applied Biosystems 7500 system software version 1.3.1, and relative copy numbers were computed according to the 2 −ΔΔCt method using a statistical confidence interval of 95%.Gene expression values within parasites of specified developmental stages were normalised to the standard control (relative expression value = (relative copy number of SjEKLF in parasites of specific stage/relative copy number of GAPDH in the standard parasite cDNA) × 100%).

Recombinant protein production and polyclonal antibody generation
The SjEKLF-encoding gene was cloned using Gateway Technology with Clonase II (Invitrogen, Thermo Fisher Scientific, Waltham, MA; USA) according to the manufacturer's instructions, as previously described [26].Primers were designed using Primer BLAST (forward primer: 5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GTC ACA CAT TTG GAA TGC TAG A-3′; reverse primer: 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA GGT ATG AAT TCG ATA ATG AGC C-3′).His-tagged fusion proteins were purified using Ni-NTA Agarose (Qiagen, Hilden, Germany), according to the manufacturer's instructions.The molecular weights of the recombinant and natural proteins were predicted using ProtParam (https:// web.expasy.org/ protp aram/).The selected gene fragments were amplified from schistosome cDNA using high-fidelity Phusion DNA polymerase (Finnzymes Oy, Finland).The amplified product was purified using DNA Gel Extraction Kit (Axygen, CA, USA) and then cloned into the entry plasmid pDONR TM 221 by the BP recombination reaction.Entry plasmids were then used to perform the LR recombination reaction to transfer the gene fragments into expression plasmid pDEST TM 17. Positive clones in Escherichia coli Transetta (DE3) (TransGen Biotech, Beijing, China) were selected for sequencing to obtain the expression plasmids with the correct reading frame.The fusion proteins with His-tags were purified with Ni-NTA Agarose (QIAGEN) according to the manufacturer's instruction.Proteins were analysed by western blotting using monoclonal antibodies against His-tag (Cell Signalling Technology, Danvers, MA, USA).Recombinant S. japonicum secreted protein 13 (SjSP-13) was generated as previously described [27].Rabbit polyclonal antibodies were prepared by Beijing Protein Innovation (Beijing, China) by immunising New Zealand white rabbits with the recombinant proteins.

In vitro cultivation of parasites
Mice were infected with male cercariae to generate hepatic schistosomulum (200 ± 20 per mouse) and male adults (40 ± 2 per mouse).Hepatic schistosomulum and male adults were obtained at 14 dpi and 42 dpi respectively.Then, schistosomulum (100 per well) and adults (20 per well) were cultured in 24-well plates in RPMI 1640 for 8 h.The supernatants were collected and concentrated using Amicon Ultra-15 Centrifugal Filters (Ultracel-10K, Merck Millipore, Carrigtwohill, IRL).These concentrations were then used for western blot.

Western blot
Parasites were homogenised and incubated with lysis buffer (8 M urea, 4% CHAPS, 1% dithiothreitol, 1% EDTA, 10 mM Tris and 35 μg/ml phenylmethylsulfonyl fluoride).Protein concentration was quantified using a BCA kit (Pierce Biotechnology, Waltham, MA, USA) in accordance with the manufacturer's instructions.Recombinant or extracted proteins were separated on 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA).Sera from S. japonicum-infected humans, mice and rabbits (diluted 1:500) with sera from uninfected individuals and animals as negative controls, rabbit polyclonal antibodies against recombinant SjEKLF (rSjEKLF; diluted 1:1000) and anti-actin antibody produced in rabbit affinity isolated antibody (Sigma-Aldrich, St. Louis, MO, USA) with rabbit IgG as a control were used as primary antibodies.IRDye 800 CW conjugated goat anti-human IgG (H + L) antibodies, goat anti-mouse IgG (H + L) antibodies and goat anti-rabbit IgG (H + L) antibodies (all from Li-COR Biosciences, Lincoln, NE, USA) were used for the final incubation.Detection was performed using an Odyssey system (Li-COR Biosciences).

Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed as previously described [7].Briefly, plates (96 well) were coated with 1 μg/ml rSjEKLF overnight at 4 °C.Human IgG, mouse IgG and rabbit IgG were used as positive controls.Serum samples (diluted 1:100) were added to the wells.Goat anti-mouse, anti-rabbit and anti-human polyvalent immunoglobulin (α-, γ-, and μ-chain specific) conjugated to alkaline phosphatase (Sigma-Aldrich) were used as secondary antibodies (1:10,000).Optical density (OD) values were measured at 405 nm.The OD values on different plates were weighted by the OD value of the control IgG at 0.1 μg/ml.The cutoff value of the positive test was set at 2.1 times the mean OD value of serum samples from healthy individuals [28].Sensitivity was defined as true positives/(true positives + false negatives) and specificity as true negatives/ (false positives + true negatives).

Immunofluorescence
Immunofluorescence was performed to detect the localisation of SjEKLF.Serial cryosections (5-7 μm) were obtained and fixed in 4% formaldehyde.Total IgG from the sera of rabbits immunised with rSjEKLF was purified using a Protein A Sepharose Fast Flow Kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer's instructions.The sections were blocked with 5% bovine serum albumin buffer (in phosphate-buffered saline [PBS]) for 2 h at 25 °C and then incubated with rabbit polyclonal antibodies against rSjEKLF (2 mg/ml, 1:500 dilution) and control IgG in blocking solution overnight at 4 °C.Cells were detected using Alexa Fluor 555 donkey anti-rabbit IgG (H + L) and 4′,6-diamidino-2-phenylindole (DAPI, all from Invitrogen) and observed using a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Immunization and challenge experiments
SjEKLF-immunised and control groups (10 mice per group) were first immunised with 60 µg rSjEKLF or an equal volume of PBS emulsified with complete Freund's adjuvant by subcutaneous injection and then with 30 µg protein or PBS emulsified with incomplete Freund's adjuvant, respectively, every 2 weeks for a total of three immunisations.After immunisation, the mice were infected with cercariae (40 ± 2 parasites per mouse) released by infected O. hupensis.Adult worms and eggs were isolated and counted at 42 dpi.The experiment was repeated twice.

Statistical analysis
Data were analysed using Prism 5.0 (GraphPad, San Diego, CA, USA) and Microsoft Excel 2010 (Microsoft, Redmond, WA, USA).The statistical significance of the experimental data was evaluated between two groups using two-tailed paired Student's t-test or two-tailed Mann-Whitney test, and among more groups using oneway analysis of variance.Statistical significance was set at P < 0.05.

Protein of SjEKLF is mainly expressed in hepatic schistosomula and male adults
Analysis of the transcriptional expression of SjEKLF revealed that the protein was highly transcriptionally expressed in the hepatic schistosomula compared with the other four stages of the parasite (cercariae, male adults, female adults and eggs, Fig. 2A).Notably, the C-terminal of SjEKLF was highly conserved, in particular the amino acid positions 194-289 that encoded three Krüppel-like C2H2 zinc fingers (Fig. 1).To ensure the specificity of the detection of polyclonal antibodies, gene fragments of SjEKLF encoding the protein fragment comprising the 6-214 amino acids were amplified to construct the clones.A recombinant protein (approximately 30 kDa) of SjEKLF was obtained and further verified by western blotting (Additional file 1: Fig. S1A).Western blotting showed that the natural SjEKLF was approximately 40 kDa (Fig. 2B), which is smaller than the mammal EKLF, but similar to that predicted by the ProtParam tool.SjEKLF was found to be mainly expressed in the hepatic schistosomula and male adults of S. japonicum with very low expression in cercariae, female adults and eggs (Fig. 2B).SjEKLF present in the culture supernatant of both hepatic schistosomulum and male adult in vitro (Additional file 1: Fig. S1B).Moreover, immunofluorescence analysis showed that SjEKLF localized within the intestinal intima of the hepatic schistosomula and male adults (Fig. 3).

SjEKLF exhibits promising qualities for schistosomiasis japonica immunodiagnosis
Sera from S. japonicum-infected patients, BALB/c mice and New Zealand rabbits at 42 dpi specifically recognised rSjEKLF (Additional file 1: Fig. S1A).Assessment of the dynamics of SjEKLF-specific IgG in mouse and rabbit sera was carried out by ELISA.The results revealed that the antibody titres in the serum peaked at 42 and 28 dpi in S. japonicum-infected mice (Fig. 4A) and rabbits (Fig. 4B), respectively, and then declined.However, the SjEKLF antibody levels at 56 dpi were still significantly higher than those at 0 dpi.Antibodies against SjEKLF in human sera were also detected using ELISA.Serum samples collected from 20 S. japonicum-infected patients before and 3 months after praziquantel treatment, 15 echinococcosis patients and 20 healthy individuals were evaluated.Sera from healthy volunteers were used as negative controls, whereas sera from patients with echinococcosis were used to assess test specificity and crossreactivity.rSjEKLF exhibited outstanding sensitivity (95% [95% confidence intervals, CI: 73.1-99.7%],twotailed Student's t-test) and specificity (100% [95% CI: 80.0-100%]) (Fig. 4C).SjSP-13 was recently identified as a novel protein with excellent sensitivity and specificity for the diagnosis of schistosomiasis japonica [28] and was herein used as a control.The results of SjSP-13 (sensitivity: 90% [95% CI: 66.9-98.2%]and specificity: 100% [95% CI: 80.0-100%], two-tailed Student's t-test) (Fig. 4D) in this study were close to the results (90.4% sensitivity and 98.9% specificity) reported by Xu et al. [28].Comparison of matching serum samples before and after drug treatment showed that antibody titres against rSjEKLF were not significantly affected.

SjEKLF immunization can relieve schistosomiasis japonica in mice
BALB/c mice were immunised with His-tagged rSjEKLF and were then challenged with S. japonicum (40 cercariae per mouse).Overall, the treated animals showed significantly reduced body weight loss (Fig. 5A) and hepatomegaly (Fig. 5B and Additional file 2: Fig. S2) compared with animals non-immunised with rSjEKLF, but this treatment approach had no effect on splenomegaly (Fig. 5C and Additional file 2: Fig. S2).The cercariae used in the challenge experiment were highly infective as the average ratio of adult worms to cercariae in the PBSimmunised group reached 72.5% (Fig. 4D).Immunisation with rSjEKLF significantly reduced the number of worms with the worm reduction rate reaching 34.9% (Fig. 5D, E).In contrast with rSjEKLF immunization, SjSP-13 showed no protective effects (Fig. 5D, E).

Discussion
The major drug and vaccine strategies for schistosomiasis aim to prevent schistosome infection and/or reduce egg production by interrupting parasite reproduction by targeting migrating schistosomula and adult females [29].Proteins critical for schistosomula development are promising candidates for schistosomiasis control.
Our previous work revealed the transcriptional profile of hepatic schistosomula [7], which includes a putative gene encoding the S. japonicum EKLF protein.EKLF is essential for definitive erythropoiesis [15,16] and is a critical transcription factor for the expression of the β-globin coding gene in mice and humans [30,31].Several studies have elucidated more potential EKLF target genes, including dematin and other components of the red cell cytoskeleton, transcription factors, blood group antigens, heme synthesis enzymes and genes involved in cell cycle regulation [32][33][34].Moreover, EKLF is upregulated in cervical and gastric cancer cells, thereby promoting cancer cell proliferation, migration and invasion [20,35].EKLF was also reported to promote the proliferation, migration and invasion of human lens epithelial cells [21].A protein with a high homology degree to EKLF is highly expressed in the epidermal layer of the skin and epithelial cells of the tongue, palate, oesophagus, stomach and colon in mice [36].SjEKLF was found to locate within the intestinal intima of the parasites, indicating SjEKLF may be involved in the regulation of intestinal endothelial cells.
Herein, we describe the EKLF protein of a pathogen (S. japonicum) for the first time to our knowledge.The challenge assay revealed that SjEKLF immunization can relieve schistosomiasis japonica in mice, indicating the important roles of SjEKLF in the parasite infection and development.Specifically, SjEKLF was detected in the culture supernatant of both schistosomula and male adult in vitro and was found to locate in the intestinal lining of the hepatic schistosomula and male adults of S. japonicum.ELISA assays showed high levels of SjEKLFspecific antibodies in host sera.These results suggest that SjEKLF may be transported into the schistosome intestinal tract and discharged into the host circulation, thereby triggering a host immune response.Therefore, we hypothesize that SjEKLF may regulate the biological function of intestinal endothelial cells and participate in the digestion and/or absorption of host components and that specific antibodies binding to SjEKLF may block its pathway of action, thus affecting the development of worms.The specific function of SjEKLF in schistosome development still needs further investigation.
Several proteins of S. japonicum have been found to have encouraging performance in the immunodiagnosis of schistosomiasis japonica, including SjSP-13 [28], SjSAPLP4, SjSAPLP5 [27], SjScP80, SjScP84 and SjScP88 [7].The herein described rSjEKLF, a non-conserved fragment of naïve SjEKLF, was also found to exhibit excellent sensitivity (95%) and specificity (100%) for the diagnosis of S. japonicum.Furthermore, ELISA assay based on rSjEKLF can effectively identify mildly infected patients, as only three patients included in this study were moderately infected and the rest were mildly infected.In addition, animal experiments showed that the serum titres of SjEKLF antibodies decreased significantly after reaching a peak at 42dpi or 28dpi, while our previous work found SjSP13 antibodies remained high at 56dpi [7].These results suggest that SjEKLF antibody levels are higher during acute infection at 28-42 dpi but declined when the chronic infection progresses.However, schistosomiasis japonica patients with acute infection were hard to recruit; patients often do not seek medical attention in a timely manner.All patients involved in this study have developed liver lesions and are in a state of chronic infection.Unfortunately, there was no significant difference in serum SjEKLF antibody titres in schistosome patients before and after drug treatment; thus, SjEKLF could not be used to distinguish between ongoing infection and previous exposure.
The highly conserved C-terminal domain of this protein makes it difficult to design drugs that can specifically act on schistosome EKLF without affecting mammalian EKLF activity.In addition to drugs, vaccines represent the most cost-effective method for the long-term control of schistosomiasis.Many candidates from S. mansoni and Schistosoma haematobium have been subjected to clinical trials, such as S. haematobium 28-kD glutathione S-transferase (rSh28GST) [37], S. mansoni 14-kDa fatty acid-binding protein (Sm14) [38], S. mansoni tetraspanin (SmTSP-2) [39] and the large subunit of S. mansoni calpain (Smp80) [40].To date, no candidate for schistosomiasis japonica has been under clinical trials.The important regulatory function of EKLF in cell biology and the high expression of SjEKLF in migrating larvae and hepatic schistosomula make this protein a valuable target for schistosomiasis vaccine design.The sequence outside the N-and C-terminal domains is highly nonconserved, making it possible to design schistosomiasis vaccines targeting SjEKLF.Overall, in vivo immunization with rSjEKLF reduced the number of worms and eggs in S. japonicum-infected mice.Schistosomula that are severely affected by SjEKLF antibodies may be eliminated by the host immune system, resulting in fewer worms and eggs in the challenge experiments, while the ones mildly affected may successfully resist the host immune system and eventually mature.Many S. japonicum antigens are currently under research, such as triosephosphate isomerase (SjTPI) [41], cytosolic fatty acid-binding protein (SjFABPc) [42], 23-kDa integral membrane protein (Sj23) [43] and schistosomulum protein 25 (SjScP25) [7].However, the protective effects of these S. japonicum antigens in animal studies remain limited, with the highest worm and egg reduction rates reaching only 50% and 65%, respectively, in S. japonicum-infected mice.Therefore, research on vaccines for schistosomiasis japonica still requires comprehensive investigations.
In summary, our study revealed that SjEKLF is critical for the infection of S. japonicum larvae and exhibits potential value for schistosomiasis japonica immunodiagnosis and immunoprotection.Hence, SjEKLF may pave the way for the development of new strategies to manage S. japonicum infection and transmission.However, the specific function of SjEKLF in the development of schistosome still needs further investigation.
(n = 10 per group).After immunisation, the mice were challenged with cercariae (40 ± 2 per mouse).The PBS group was immunised with PBS and did not receive cercariae.The mice were killed 42 days post infection.The morphology of the livers and spleen from each group of mice is shown.Scale bar indicates 5 mm.

Fig. 1
Fig. 1 Sequence analysis of Schistosoma japonicum erythroid Krüppel-like factor (SjEKLF). a Schematic representation of the SjEKLF (TNN19877.1)amino acid sequence.The dark blue block indicates the domain of SFP1 super family and the light blue blocks indicate C2H2 zinc fingers.b The sequences of EKLFs from Schisotosoma japonicum, S. mansoni, H. sapiens, M. musculus, R. norvegicus and B. taurus were obtained and aligned.Pink indicates homology 100%, grey indicates homology > 75% and blue indicates homology > 50%

Fig. 2
Fig. 2 Expression of Schistosoma japonicum erythroid Krüppel-like factor (SjEKLF) in different developmental stages of the parasite.a Transcriptional expression of the SjEKLF-encoding gene in the five developmental stages of S. japonicum detected by quantitative real-time polymerase chain reaction.Data are shown as mean + SD. *, #, Δ and Φ indicate comparison with the E, C, M and F groups, respectively.*, #, Δ and Φ indicate P < 0.01 by Mann-Whitney test.b Expression of SjEKLF and actin in the five developmental stages of S. japonicum were detected by western blot with 20 µg protein lysate uploaded per lane for SjEKLF detection and 5 µg for actin detection.Red arrows indicate the target protein bands.Abbreviations: E, egg; C, cercariae; S, hepatic schistosomula; M, adult male; F, adult female

Fig. 3 Fig. 4 Fig. 5
Fig. 3 Location of Schistosoma japonicum erythroid Krüppel-like factor (SjEKLF) detected by immunofluorescence.Cryosections of male adult (a) and hepatic schistosomulum (b) samples obtained from S. japonicum-infected BALB/c mice were incubated with rabbit anti-SjEKLF polyclonal antibodies; cryosections of male adults were incubated with rabbit IgG as control (c) and then with Alex Flour 555 donkey anti-rabbit IgG (green fluorescence).DAPI (blue) was used to stain the nucleus.Red arrows indicate positive staining.These results are representative of three independent experiments

Table 1
Clinical characteristics of the enrolled subjects whose sera were used in ELISA for diagnosis Eggs per gram of stool (assessed by Kato-katz technique) * Median ± SD #