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Parasites & Vectors

, 12:340 | Cite as

Comparative analysis of transcriptional profiles of Schistosoma japonicum adult worms derived from primary-infected and re-infected water buffaloes

  • Yudan Mao
  • Chuanchuan He
  • Hao Li
  • Ke Lu
  • Zhiqiang Fu
  • Yang Hong
  • Yamei Jin
  • Jiaojiao Lin
  • Xin Zhang
  • Jinming LiuEmail author
Open Access
Research

Abstract

Background

Schistosoma japonicum (S. japonicum) is an important zoonotic parasite that is prevalent in China and parts of Southeast Asia. Water buffaloes are an important reservoir and the main transmission sources of S. japonicum. However, self-curing and resistance to re-infection have been observed in water buffaloes.

Results

In this study, we compared the morphometry and differences in transcriptional expression of adult S. japonicum worms recovered from primary-infected and re-infected water buffaloes using Illumina RNA-sequencing (RNA-Seq) technology. Results of morphometry analysis revealed that adult S. japonicum worms recovered from re-infected water buffaloes were runtish with smaller organs. The ventral length of male worms was shorter in re-infected buffaloes (328 ± 13 vs 273 ± 8 µm, P < 0.05), and in female worms the oral sucker length (44 ± 3 vs 33 ± 5 µm, P < 0.05), ovary length (578 ± 23 vs 297 ± 27 µm, P < 0.05) and width (150 ± 8 vs 104 ± 9 µm, P < 0.05) were shorter, with fewer eggs in the uteri (41 ± 2 vs 12 ± 1, P < 0.05). Of 13,605 identified genes, 112 were differentially expressed, including 51 upregulated and 61 downregulated genes, in worms from re-infected compared with primary-infected water buffaloes. Gene ontology (GO) enrichment analysis revealed that GO terms such as “oxidation-reduction process”, “calcium-dependent phospholipid binding”, “lipid binding” and “calcium ion binding” were significantly enriched in downregulated genes, whereas GO terms related to metabolism and biosynthesis were significantly enriched in upregulated genes. The results revealed that the downregulation of some important genes might contribute to a reduction in worm numbers and maldevelopment of surviving worms in re-infected water buffaloes. Furthermore, upregulation of genes related to metabolic processes and biosynthesis might be a compensatory mechanism of worms in disadvantageous environments.

Conclusions

To our knowledge, our results present the first large-scale transcriptional expression study identifying the differences between adult S. japonicum worms from primary-infected and re-infected water buffaloes, and particularly emphasize differential expression that may affect the survival and growth of worms in re-infected water buffalo. This will provide new insight into screening for anti-schistosome targets and vaccine candidates.

Keywords

Comparative analysis Primary-infected Re-infected RNA-Seq Schistosoma japonicum Water buffalo 

Abbreviations

RNA-Seq

RNA-sequencing

qPCR

quantitative PCR

GO

Gene Ontology

PZQ

praziquantel

DEGs

differentially expressed genes

IL

interleukin

IFN

interferon

IgG

immunoglobulin G

RIN

RNA integrity number

SRA

Sequence Read Archive

Q20

Qpred ≤ 20

Q30

Qpred ≤ 30

FPKM

fragments per kilobase of transcript sequence per millions

FC

fold change

KEGG

Kyoto Encyclopedia of Genes and Genomes

ROS

reactive oxygen species

CcO

cytochrome c oxidase

ELOVL

elongation of the very long chain fatty acids

SEA

soluble egg antigens

SWA

soluble adult worm antigen

RNAi

RNA interference

AAALAC

Association for Assessment and Accreditation of Laboratory Animal Care International

Background

Schistosomiasis, caused by infections with Schistosoma species, is endemic in over 70 countries and territories located in tropical and subtropical regions and remains a major public health problem worldwide [1]. Schistosoma japonicum is a zoonotic parasite that is prevalent in China and parts of Southeast Asia [2]. It has been reported that over 40 species of wild and domestic animals can become infected with S. japonicum [3, 4]. Generally, praziquantel (PZQ) is the only effective chemotherapeutic drug against schistosomiasis. However, its utility is limited in areas with high re-infection and resistance rates. Additionally, low susceptibility of Schistosoma mansoni and S. japonicum to PZQ has been induced after mass drug administration programmes [5, 6]. Therefore, novel targets for drugs and vaccines to treat and eradicate schistosomiasis are urgently required.

Infection with S. japonicum causes serious economic losses to livestock farms. Many investigations have revealed that domestic animals, bovines in particular, are the major infectious source and play the most important role in the transmission of schistosomiasis in China [7, 8]. Thus, water buffaloes have been considered a target animal for schistosomiasis control in China for several decades [9]. A veterinary vaccine for domesticated bovines that blocks transmission would provide an effective approach to schistosomiasis control. Several research groups have focused on the development of efficient vaccines for buffaloes [10, 11]. However, the level of protection of these vaccines needs further improvement. Lack of knowledge about schistosome biology and host-parasite relationships in bovines remains an obstacle for vaccine development.

Recently, many public databases on the genomes, transcriptomes and proteomes of Schistosoma species, in particular S. mansoni and S. japonicum [12, 13], have been released, which has proved pivotal for the understanding of schistosome biology. In the past decade, a number of studies on gene expression and proteomic profiling of Schistosoma species have been performed using various analytical approaches. These studies mainly focus on expression patterns and features of sex-dependent, tissue-specific, host-associated and strain-specific genes [12, 14, 15, 16]. The findings have facilitated improved understanding of the molecular basis of schistosome developmental biology, host-parasite interactions and schistosomiasis pathogenesis. For example, studies from our research group have identified numerous differentially expressed genes (DEGs) that may influence parasite survival and development of schistosomula from susceptible BALB/c mice, less susceptible Wistar rats and resistant reed voles using comparative proteomic [17] and microarray analyses [18]. We have also examined gene expression profiles of S. japonicum worms derived from the natural hosts yellow cattle and water buffalo using comparative analysis of microarrays [19].

Water buffaloes are numerous and widely distributed in schistosomiasis-endemic regions and are a major reservoir for Schistosoma species in China [8]. Compared with other natural hosts, such as yellow cattle, goats and rabbits, water buffaloes are less susceptible to infection [20]. In addition, previous researchers reported self-curing in water buffaloes, as they observed decreases in infection rates with age [21] and age-related resistance to re-infection after PZQ treatment [22]. Recently, we observed a reduction of over 97% in the S. japonicum worm burden, shorter female worms, and a reduction of over 87.7% in egg counts in re-infected buffaloes [23]. Strong type-2 immune responses at the site of cercarial penetration have also been reported in challenged water buffaloes [24], which is significantly different from observations in other animal models such as mice. We also found that re-infected water buffaloes had significantly higher levels of interleukin (IL)-4, IL-10, interferon (IFN)-γ and specific immunoglobulin G (IgG) antibodies before challenge infection [23]. Here, we performed comparative analyses of gene expression profiles of S. japonicum adult worms from primary-infected and re-infected water buffaloes using deep RNA-transcriptome sequencing (RNA-Seq) technology. Our results will be of significance for understanding the mechanism of resistance and host-parasite relationships in re-infected water buffaloes and could provide a valuable resource for the identification of genes functionally related to parasite development in water buffaloes as well as the screening of anti-schistosome drug targets and vaccine candidates.

Methods

Infection protocol and parasite collection

The parasites used in this study were collected from animals in the primary infection group (referred to in the previous study as the control group) and the re-infection group in the second trial of a study performed in 2017 to observe the degree of resistance of water buffaloes to re-infection with S. japonicum, as reported in our previous publication [23]. The infection protocol is summarized in Fig. 1 and briefly described as follows: six 15- to 18-month-old male water buffaloes (Bubalus bubalis) were purchased from non-schistosome-endemic areas in Nantong, Jiangsu Province, China and randomly divided into either the primary infection group or the re-infection group. Schistosoma japonicum cercariae used in all infections were shed from infected Oncomelania hupensis snails purchased from the Hunan Institute of Parasitic Diseases (Yueyang, China). Animals in the re-infection group were infected with 3000 S. japonicum cercariae on day 0 and on day 95 and treated with PZQ on day 54 and day 152. Water buffaloes in the primary infection group were not infected initially but were treated with PZQ at the same time points. In order to remove all S. japonicum worms in infected water buffaloes, PZQ was orally administered twice at 24 h intervals in each treatment at a dose of 25 mg/kg body weight up to a maximum of 10 g. On day 185, all animals were infected percutaneously with 3000 ± 100 cercariae. Animals were euthanized on day 255. Worms were obtained from three animals in the primary infection group and three animals in the re-infection group and designated C1, C2, C3, T1, T2 and T3, respectively. All parasites were manually washed twice with phosphate-buffered saline to remove residual host debris. Some worms were immediately preserved in RNAlater® (Invitrogen, Carlsbad, CA, USA) and stored in liquid nitrogen for subsequent RNA extraction. Five pairs of worms (five males and five females) from each animal were fixed with 10% neutral buffered formalin for morphological analysis.
Fig. 1

Schedule of animal infection, treatment and sacrifice. The primary infection and re-infection strategies for different groups were as shown. Briefly, re-infection group were infected with 3000 S. japonicum cercariae on day 0 and on day 95 and treated with PZQ on day 54 and day 152; primary infection group were not infected initially but were treated with PZQ at the same time points (on day 54 and day 152). On day 185, all animals were infected percutaneously with 3000 ± 100 cercariae. Animals were euthanized on day 255 (n = 3)

Morphological comparisons of worms collected from primary-infected and re-infected water buffaloes

A total of 30 pairs of worms, including 30 males and 30 females, were used for morphological comparisons. The paired worms were placed in ice water to separate the males and females. Slide specimens were prepared using the conventional method [25, 26] and observed under a microscope equipped with an automatic ACT-2U camera (Nikon, Tokyo, Japan). The total worm length, along with the dimensions of the oral sucker, ventral sucker and ovary were measured using NIS-Elements, a Nikon image analysis software. The number of eggs in the uterus of each female was counted. Data are expressed as the mean ± standard deviation. The results for each group were compared using Student’s t-tests in Excel 2013 (Microsoft Inc., Redmond, WA, USA) to examine intergroup differences. P-values < 0.05 were considered statistically significant.

Total RNA isolation, qualification and transcriptomic library construction

Total RNA was extracted from individual worms collected from each water buffalo using TRIzol Reagent (Invitrogen), and contaminating genomic DNA was removed from RNA samples by treatment with RNase-free DNase I (New England Biolabs, Ipswich, MA, USA). RNA degradation and contamination were monitored on 1% agarose gels, a NanoPhotometer® spectrophotometer (IMPLEN, Westlake Village, CA, USA) and a Qubit® RNA Assay Kit with a Qubit®2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). Before RNA library construction, RNA integrity was assessed using an RNA Nano 6000 Assay Kit and Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). RNA with an RNA integrity number (RIN) > 7.0 were considered of high enough quality for transcriptomic library construction and RNA sequencing according to the manufacturer’s instructions.

Transcriptomic library preparation

For library construction, mRNA was purified from 3 μg of total RNA of each sample using poly-T oligo-attached magnetic beads (New England Biolabs) [27]. Transcriptome sequencing libraries were generated using an Illumina NEBNext® UltraTM RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s instructions and index codes were added to attribute the sequences to the corresponding sample. Briefly, purified mRNA was cut into fragments and the cleaved mRNA fragments were reverse-transcribed into first-strand cDNA using random hexamers, followed by synthesis of double-strand cDNA. After blunting ends, the 250 to 300 bp fragments were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA). The purified cDNA fragments were then linked using an NEBNext Adaptor (New England Biolabs) with a hairpin loop structure and amplified by PCR. The AMPure XP system (Beckman Coulter) was used to purify the PCR products and the sample library quality was assessed using an Agilent Bioanalyzer 2100 system.

Transcriptome sequencing and data analysis

Equal amounts of the 12 transcriptomic libraries were pooled and sequenced using an Illumina HiSeq™ 2000 (Illumina, San Diego, CA, USA) sequencing platform [28]. Raw reads in fastq format were processed using in-house perl scripts. These sequencing data are available at the NCBI Sequence Read Archive (SRA) database (www.ncbi.nlm.nih.gov/sra) under the accession number SRP168979. After calculating the Qpred ≤ 20 (Q20), Qpred ≤ 30 (Q30) and GC content of each sequence, clean data were obtained by removing the adapter from each sequence as well as any reads containing poly-N. Low quality reads were removed and the high quality clean reads were independently mapped twice to the genomic data (WormBase ParaSite, http://parasite.wormbase.org/Schistosoma_japonicum_prjea34885/Info/Index/) using Hisat2 v.2.0.5 [29]. Mapped reads for each sample were assembled using StringTie v.1.3.3b with a reference-based approach [30]. The expression level of each gene was estimated using the fragments per kilobase of transcript sequence per millions (FPKM) method. The DESeq2 R package v.1.16.1 was used for differential expression analysis of three biological replicates per condition. The Benjamini and Hochberg’s approach was applied to calculate P-values and expression fold change (FC), and genes with P-values < 0.05 and FC > 2 were considered differentially expressed. DEGs were functionally annotated using Blast2GO at http://www.blast2go.de [31] and assessed for enrichment using the clusterProfiler R package. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for DEG pathway annotation and enrichment analysis (http://www.genome.jp/kegg/).

Verification of RNA sequencing data

The gene encoding S. japonicum NADH-ubiquinone reductase was employed as an endogenous reference gene [18]. The primer sequences used for qPCR were designed using Primer Express v.3.0 software (Applied Biosystems, Foster City, CA, USA) and are listed in Table 1. qPCR amplification was performed using a SYBR green kit (TaKaRa, Dalian, China) and an ABI 7500 Real-time System (Life Technologies). The qPCR cycling conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 2 min. Melt curve analysis from 72 to 95 °C was performed to ensure a specific product was amplified in each reaction. The relative expression level of the genes was calculated using the 2−ΔΔCT method [32].
Table 1

Primers used in quantitative PCR

Primer name

Sequence (5′–3′)

Product size (bp)

Upregulated genes

 Sjp_0125630F

CATGGATCTGTGCTCGCGTA

150

 Sjp_0125630R

GCAAACCCGTGATTGGCAAG

 Novel03105F

GAGCAGACCGCTTCGATGTA

75

 Novel03105R

CCGAATTTCGCTTGCCAGTC

 Novel00129F

ACTTCCCGCTCAAGATCACC

103

 Novel00129R

ACTCGTAAGAGTGGGGTCCA

 Novel01404F

TTCTCAACAGCGTACACCCC

77

 Novel01404R

GACCCAGTAGTGCAGACGTT

 Novel02684F

GGACGCCAGGTTAAGGAAGA

93

 Novel02684R

TCTACCAGCCGAAGGAGTGT

Downregulated genes

 Novel01481F

TTGGCCTCAATCGATCCTATCT

136

 Novel01481R

CTGTGATTGCTTACCGTTTTGC

 Novel00143F

TGTGGGATTAAGGCCTACCA

95

 Novel00143R

GCGAACGTAGAACAACTGCC

 Novel02333F

CGTTGTATGTGGATCATTTGTGC

70

 Novel02333R

TTCATCTTTGCATTCACTACTACAC

 Sjp_0116000F

TGCAATTGTTCACCGTGATG

86

 Sjp_0116000R

TGTCTCTGCAACATCTTGTGAT

 Novel00935F

CGCAATCAGATGTTCAGGGT

105

 Novel00935R

TCGGAAACCCAATGTCTGGAT

Results

Morphological differences between parasites from re-infected and primary-infected water buffaloes

Lengths of male and female worms were reported in our previous publication [23]. Here we analyzed other morphological characteristics and present the results from 30 pairs of worms (30 male and 30 female) in Table 2. Organs, including the oral sucker, ventral sucker and ovaries, of male and female worms from re-infected water buffaloes were smaller than those of worms from primary-infected animals. The differences were significant for the length of the ventral sucker in males (328 ± 13 vs 273 ± 8 µm; t(14) = 26.63, P = 0.02), the length of the oral sucker in females (44 ± 3 vs 33 ± 5 µm; t(14) = 8.52, P = 0.02) and the length (578 ± 23 vs 297 ± 27 µm; t(14) = 40.31, P = 0.005) and width (150 ± 8 vs 104 ± 9 µm; t(14) = 19.80, P = 0.02) of the ovary, but no other significant differences were found. We also observed that there were significantly fewer eggs in the uteri of female worms (41 ± 2 vs 12 ± 1; t(14) = 56.16, P < 0.0001) from re-infected water buffaloes.
Table 2

Morphometry of S. japonicum worms recovered from primary-infected and re-infected water buffaloes

Characters

Primary infection

Re-infection

Male worms

(n = 15)

(n = 15)

 Oral sucker length

261 ± 74

245 ± 23

 Oral sucker width

176 ± 34

170±23

 Ventral sucker length

328 ± 13

273 ± 8*

 Ventral sucker width

236 ± 46

193 ± 24

Female worms

(n = 15)

(n = 15)

 Oral sucker length

44 ± 3

33 ± 5*

 Oral sucker width

34 ± 1

24 ± 7

 Ventral sucker length

109 ± 8

94 ± 4

 Ventral sucker width

87 ± 8

73 ± 3

 Ovary length

578 ± 23

297 ± 27*

 Ovary width

150 ± 8

104 ± 9*

 No. of eggs in utero

41 ± 2

12 ± 1*

Note: Data are presented as the mean ± standard deviation; length and width in μm

P < 0.05

RNA extraction and RNA-Seq

The six RNA samples, including three from worms recovered from primary-infected water buffaloes and three from worms recovered from re-infected water buffaloes, had prominent 18S and 28S ribosomal peaks on agarose gels (data not shown). According to Bioanalyzer 2100 analysis, the RINs of all samples were > 7.0. These results showed that all RNA samples possessed high integrity and purity and could be used for further experiments.

cDNA was generated and sequenced using an Illumina sequencing platform. Q30 was > 90% for each sample. After removing adaptors and low-quality reads, we obtained approximately 305,888,154 high quality reads in all samples. The average mapping rate of clean reads (51,832,023 reads per sample) to S. japonicum genomic data in two independent analyses was 79.545% (Table 3). The correlation coefficient of the transcriptional profiles between three biological replicates of the six samples was 0.969–0.985 (Fig. 2).
Table 3

Information on sequencing and mapping of reads to genomic data

Sample name

C1

C2

C3

T1

T2

T3

Total readsa

62,194,716

51,418,256

39,854,922

42,581,418

57,908,184

51,930,658

Total mapped (%)b

49,530,227 (79.64)

40,967,457 (79.67)

31,302,966 (78.54)

33,901,809 (79.62)

46,380,422 (80.09)

41,394,770 (79.71)

Multiple mapped (%)c

1,634,122 (2.63)

1,263,823 (2.46)

1,009,725 (2.53)

1,110,332 (2.61)

1,478,461 (2.55)

1,404,997 (2.71)

Uniquely mapped (%)d

47,896,105 (77.01)

39,703,634 (77.22)

30,293,241 (76.01)

32,791,477 (77.01)

44,901,961 (77.54)

39,989,773 (77.01)

Read-1 (%)

24,189,990 (38.89)

20,074,308 (39.04)

15,311,530 (38.42)

16,478,614 (38.7)

22,663,081 (39.14)

20,193,218 (38.88)

Read-2 (%)

23,706,115 (38.12)

19,629,326 (38.18)

14,981,711 (37.59)

16,312,863 (38.31)

22,238,880 (38.4)

19,796,555 (38.12)

Reads map to ‛+ʼ (%)e

23,840,204 (38.33)

19,786,847 (38.48)

15,100,441 (37.89)

16,352,368 (38.4)

22,390,777 (38.67)

19,940,268 (38.4)

Reads map to ‛-ʼ (%)f

24,055,901 (38.68)

19,916,787 (38.73)

15,192,800 (38.12)

16,439,109 (38.61)

22,511,184 (38.87)

20,049,505 (38.61)

aSampling sequence filtered by sequencing data (clean data)

bStatistics on the number of sequencing sequences that can be mapped to the genome and the number in parentheses indicates the number of sequences (total mapped reads) as a percentage (%) of clean reads

cQuantitative statistics of sequencing sequences with multiple alignment positions on the reference sequence

dQuantitative statistics of sequencing sequences with unique alignment positions on the reference sequence

eThe statistics of the sequencing sequence alignment to the positive strands on the genome

fThe statistics of the sequencing sequences aligned to the negative strands on the genome

Fig. 2

Heat map of correlation coefficients for all samples. C1, C2 and C3 represent samples from primary-infected water buffaloes. T1, T2 and T3 represent samples from re-infected water buffaloes. R2 represents the Pearsonʼs correlation coefficient

We identified a total of 16,605 genes, including 3867 novel genes. Of these, 13,310 genes were expressed in adult worms recovered from both primary-infected and re-infected water buffaloes; 256 were only expressed in adult worms recovered from re-infected water buffaloes and 219 were only expressed in adult worms recovered from primary-infected water buffaloes (Fig. 3a). Using a P-value cutoff of < 0.05 and an FC cutoff of > 2, 112 genes were identified as DEGs (Fig. 3b). Compared with worms recovered from primary-infected water buffaloes, 51 genes were upregulated and 61 genes were downregulated in worms recovered from re-infected water buffaloes (Fig. 3b). DEGs that were successfully mapped to homologous proteins in the Gene DB database (http://www.genedb.org/Homepage) or annotated using the Swiss-Prot database (http://www.gpmaw.com/html/swiss-prot.html) are listed in Tables 4 and 5. Only genes that were successfully annotated using the Swiss-Prot database or found to have homologous proteins in the Gene DB database are listed. Cluster analysis for these DEGs in all six samples is shown in Fig. 3c.
Fig. 3

Transcription profile analysis of significantly differentially expressed genes. a Venn diagram showing the total number of expressed genes in each group in the large circles, and the number of genes expressed in both groups are shown in overlapping portion of the circles. b Volcano map showing significantly differentially expressed genes represented by red dots (upregulated) and green dots (downregulated). Genes that were not significantly differentially expressed are represented by blue dots. Abscissas indicate fold changes in gene expression between samples. Ordinates represent the statistical significance of the difference in gene expression. c Hierarchical clustering map of DEGs showing the overall FPKM. log10 (FPKM+1) values are scaled and clustered, with red representing upregulated genes and blue representing downregulated genes. Colors from red to blue indicate log10 (FPKM+1) values from largest to smallest. Abbreviations: C, worms from primary-infected water buffaloes; T, re-infected water buffaloes

Table 4

Upregulated genes in schistosomes recovered from re-infected water buffaloes normalized to primary-infected water buffaloes

Gene_ID

Protein homology

Annotation

Fold Change

Novel02058

Reverse transcriptase SjR1

na

na

Sjp_0123960

Immunogenic miracidial antigen 8I

na

15.20106

Sjp_0125630

Uncharacterized protein

Virion assembly

9.447286

Novel00553

Magnesium transporter nipa

Magnesium ion transmembrane transporter activity

6.277108

Novel01404

Protein TraX

Integral component of membrane

5.105315

Novel03501

Channel-forming transporter/TpsB family cytolysins activator

Protein transport

4.772681

Novel02684

Uncharacterized protein LOC106012791

Zinc ion binding

4.759467

Novel02475

Gag-Pol polyprotein

Threonine kinase

4.240105

Sjp_0110350

Asparagine-rich protein

na

3.560797

Sjp_0124980

SJCHGC04245 protein

Heterocyclic compound binding

3.293907

Sjp_0078930

Centrosomal protein of 162 kDa

Bacteriocin immunity, toxic substance binding

3.189433

Sjp_0132490

Retinol dehydrogenase 12

Catalytic activity

3.096417

Novel00396

Reverse transcriptase

Intrinsic component of membrane

2.91319

Novel02372

Polyprotein

DNA binding

2.854029

Sjp_0067470

Trehalose-6-phosphate hydrolase

Cation binding

2.620605

Novel03138

Ribonuclease III, putative

na

2.601241

Novel01504

SJCHGC02001 protein

Integral component of membrane

2.403939

Sjp_0002070

Malignant fibrous histiocytoma-amplified sequence 1 homolog

Purine nucleotide binding

2.379569

Sjp_0126430

ATP synthase subunit a

Purine nucleoside monophosphate biosynthetic process

2.360512

Sjp_0128790

Cytochrome c oxidase subunit 1

Oxidoreductase activity

2.287703

Sjp_0128350

Periodic tryptophan protein 2 homolog

na

2.270484

Novel02961

TPA: endonuclease-reverse transcriptase

Transferase activity

2.251209

Sjp_0127400

Cytochrome c oxidase subunits 1+2

Generation of precursor metabolites and energy, cytochrome-c oxidase activity

2.228696

Novel00551

Centrosomal protein of 162 kDa

Serine-type carboxypeptidase activity

2.226072

Sjp_0036210

Glycoprotein 3-alpha-l-fucosyltransferase A

Acting on L-amino acid peptides

2.111693

Sjp_0128800

ATP synthase subunit a

Purine nucleoside triphosphate biosynthetic

2.076854

Novel00613

Hypothetical protein MS3_03474

Porphobilinogen synthase activity

2.068952

Novel03809

Reverse transcriptase

RNA-directed DNA polymerase activity, nucleic acid binding

2.067948

Novel03549

TPA: endonuclease-reverse transcriptase

na

2.033408

Sjp_0126760

Glutamic acid-rich protein

Nucleobase-containing compound biosynthetic process

2.021043

Notes: Only genes that were successfully annotated using the Swiss-Prot database or found to have homologous proteins in the Gene DB database are listed

Abbreviation: na, not available

Table 5

Downregulated genes in schistosomes recovered from re-infected water buffaloes normalized to primary-infected water buffaloes

Gene_ID

Protein homology

Annotation

Fold change

Sjp_0008840

na

Integral component of membrane

na

Sjp_0126930

SJCHGC00271 protein

Transmembrane

0.0264

Novel00143

Uncharacterized protein

Mitochondrion

0.090296

Novel02333

Zinc finger and BTB domain-containing protein 16 (Fundulus heteroclitus (killifish; mummichog))

Integral component of membrane

0.09335

Sjp_0071350

Egg protein P3811

Transmembrane

0.09468

Novel00935

Egg protein CP11 (Schistosoma japonicum)

na

0.109659

Novel03624

Putative membrane protein

Transmembrane

0.131051

Novel00443

Transcriptional regulator

Regulation of transcription, DNA-templated

0.161175

Sjp_0048330

Beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-N-acetylglucosaminyltransferase 3

Transferase activity

0.224891

Novel01156

Uncharacterized protein, SJCHGC08959 protein

Establishment of localization

0.262939

Sjp_0123650

CAP-Gly domain-containing linker protein 2, gene name:MS3_05295

Response to abiotic stimulus

0.266222

Sjp_0128890

Cytochrome c oxidase subunit 2

Aerobic respiration

0.295064

Novel02380

Gap-Pol polyprotein

na

0.297261

Novel02465

Reverse transcriptase

na

0.3384

Novel00176

SJCHGC04823 protein

na

0.339669

Sjp_0072400

Uncharacterized protein

Transmembrane

0.34707

Sjp_0031650

Probable delta-1-pyrroline-5-carboxylate synthase

Oxidoreductase activity

0.363745

Novel00063

Transposon Ty3-G Gag-Pol polyprotein

na

0.368695

Sjp_0113480

Annexin A3

Secretion by cell

0.371285

Sjp_0107330

Uncharacterized protein

Transmembrane

0.373661

Novel01784

Uncharacterized protein, HYPBUDRAFT_236782

Cytoskeleton

0.380482

Sjp_0095040

Amide-activated amiloride-sensitive sodium channel

Sodium ion transmembrane transporter activity

0.380508

Sjp_0084800

Beta-catenin-like protein 1

na

0.391179

Novel01664

Gag-Pol polyprotein

na

0.39513

Sjp_0122740

Annexin

na

0.414344

Novel03120

Hypothetical protein N336_01965, partial (Phalacrocorax carbo)

na

0.418181

Novel00971

Hypothetical protein Smp_144270 (Schistosoma mansoni)

na

0.431371

Novel01286

Uncharacterized protein

Transmembrane

0.434793

Sjp_0077960

Annexin A10

na

0.439215

Sjp_0122640

NADH-ubiquinone oxidoreductase chain 1

Inorganic cation transmembrane transporter activity

0.457011

Novel02095

Mitogen-activated protein kinase 15

na

0.467131

Sjp_0077970

Annexin A3

Phospholipase activity

0.468623

Novel01808

Hypothetical protein Smp_153120

na

0.472374

Novel02008

Reverse transcriptase

na

0.481698

Novel03665

Putative glycosyltransferase, partial

na

0.487137

Sjp_0071740

Uncharacterized protein

Transmembrane

0.487779

Novel03376

Hypothetical protein M959_01698, partial

Photosystem I reaction center

0.488354

Sjp_0076310

Tight junction protein ZO-1

Transcription factor TFIID complex

0.49107

Novel00873

Hypothetical protein MS3_01562

NADH dehydrogenase (ubiquinone) activity

0.496237

Notes: Only genes that were successfully annotated using the Swiss-Prot database or found to have homologous proteins in the Gene DB database are listed

Abbreviation: na, not available

GO and KEGG enrichment analysis of differentially expressed genes

To obtain a better understanding of the enriched functions of DEGs, GO term enrichment analysis was performed. Of all identified genes, a total of 9012 (65.37%) were annotated (Additional file 1: Table S1). A total of 127 GO terms were enriched with P < 0.05 (Additional file 2: Table S2). Annotations of DEGs from the Swiss-Prot database are listed in Tables 4 and 5.

Of the upregulated genes in worms recovered from re-infected water buffaloes, 110 GO terms were significantly enriched with P < 0.05 (Fig. 4, Additional file 3: Table S3). Specifically, 83 GO terms, such as “metabolic process”, “cellular metabolic process”, “nitrogen compound metabolic process” and “cellular biosynthetic process”, were overrepresented in the “biological process” category; 24 GO terms, including “hydrogen ion transmembrane transporter activity”, “monovalent inorganic cation transmembrane transporter activity”, “inorganic cation transmembrane transporter activity” and “cation transmembrane transporter activity”, were overrepresented in the “molecular function” category; and three GO terms, including “viral procapsid”, “Cdc73/Paf1 complex” and “transcription elongation factor complex”, were overrepresented in the “cellular component” category.
Fig. 4

GO enrichment analysis of differentially expressed genes. ac Upregulated genes. df Downregulated genes. GO terms were categorized as biological processes (a, d), cellular components (b, e) or molecular functions (c, f) using the Blast2Go program. Genes with no assigned GO terms were excluded from the graph. The ordinate shows the enriched GO term, and the abscissa shows the number of differentially expressed genes associated with the term

Of the downregulated genes in worms recovered from re-infected water buffaloes, a total of 35 GO terms were significantly enriched with P < 0.05 (Fig. 4, Additional file 4: Table S4). In the “biological process” category, 11 GO terms, including “oxidation-reduction process”, “monovalent inorganic cation transport” and “response to ionizing radiation”, were significantly enriched; 19 GO terms, such as “transmembrane transporter activity”, “calcium-dependent phospholipid binding” and “phospholipid binding”, were significantly enriched in the “molecular function” category; and five GO terms, such as “photosystem I reaction center”, “transcription factor TFIID complex” and “exocyst”, were significantly enriched in the “cellular component” category.

Functional enrichment analyses of GO terms of DEGs were compared between the upregulated and downregulated genes in worms recovered from re-infected water buffaloes (Fig. 4, Table 6). Only DEGs that were successfully annotated using the Swiss-Prot database are listed. The analysis indicated that a large number of GO terms associated with metabolic processes and biosynthesis were enriched only in the upregulated genes. By contrast, the GO term oxidation-reduction was enriched only in downregulated genes. GO terms related to transportation and binding were significantly enriched in both the upregulated and downregulated genes. However, we found that upregulated and downregulated genes were associated with different binding functions. Upregulated genes were related to iron ion binding, heme binding and tetrapyrrole binding, whereas the downregulated genes were related to calcium-dependent phospholipid binding, lipid binding and calcium ion binding.
Table 6

Summary of GO enrichment analysis of differentially expressed genes

Function

Upregulated genes

Downregulated genes

Enrichment terms

Major genes

Enrichment terms

Major genes

Metabolism

GO:0008152, GO:0009404, GO:0071941, GO:0006807

Sjp_0126430, Novel00613, Sjp_0127400, Sjp_0067470, Sjp_0078930, Novel02961, Sjp_0036210, Novel02108, Novel02684, Sjp_0132490, Novel02475, Novel00129, Sjp_0128790, Sjp_0126760, Novel00551, Sjp_0128800, Novel0296

None

None

Transportation

GO:0015078, GO:0015077, GO:0022890, GO:0008324, GO:0015075 GO:0022891

Sjp_0127400, Sjp_0128800, Sjp_0128790, Sjp_0126430

GO:0022857, GO:0005215, GO:0015077, GO:0022890, GO:0008324, GO:0015078

Sjp_0095040, Sjp_0128890, Novel01156, Sjp_0122640

Biosynthesis

GO:0044249, GO:1901576, GO:0009058, GO:0019438, GO:0018130, GO:1901362, GO:0006754

Novel02684, Novel00551, Sjp_0128800, Sjp_0036210, Sjp_0126760, Novel02475, Sjp_0126430, Novel00613

None

None

Binding

GO:0005506, GO:0020037, GO:0046906, GO:0015643

Sjp_0128790, Sjp_0127400, Sjp_0078930

GO:0005544, GO:0005543, GO:0008289, GO:0005509

Sjp_0113480, Sjp_0077960, Sjp_0077970

Oxidation-reduction

None

None

GO:0055114

Sjp_0128890, Sjp_0031650, Sjp_0122640

Note: Only differentially expressed genes that were successfully annotated using the Swiss-Prot database are listed

KEGG pathway enrichment analysis of DEGs indicated that both upregulated and downregulated genes were involved in oxidative phosphorylation and metabolic pathways. In addition, upregulated genes were also involved in extracellular matrix-receptor interactions, N-glycan biosynthesis and ribosome biogenesis in eukaryotes, and downregulated genes were associated with arginine and proline metabolism, biosynthesis of amino acids and spliceosome (Fig. 5, Additional file 5: Table S5).
Fig. 5

KEGG enrichment pathway analysis of differentially expressed genes. a Number of upregulated genes. b Number of downregulated genes

qPCR validation of RNA sequencing data

To validate RNA-Seq data, qPCR was performed for 10 genes with different biological functions and expression patterns, including five upregulated genes and five downregulated genes, using NADPH as the housekeeping gene for internal normalization. The qPCR results corresponded with the RNA-Seq data (Fig. 6a), with a significant correlation coefficient of 0.9405 (P < 0.0001, n = 10) (Fig. 6b).
Fig. 6

The expression of 10 selected genes with different expression patterns was quantified by qPCR analysis. a Ten selected genes with different expression patterns. b Correlation analysis of qPCR results and RNA-Seq data. Error bars represent the standard deviation for three technical replicates

Discussion

Intriguingly, water buffaloes are less susceptible to S. japonicum infection than yellow cattle, goats and rabbits [33] and self-curing has been observed in water buffaloes after infection [21]. In our previous publication, we reported a reduction in worm burdens of over 97.4% (P < 0.05) in re-infected compared to primary-infected water buffaloes [23]. Additionally, surviving adult female worms in water buffaloes previously exposed to S. japonicum were shorter in length. Here we further observed that organs including the oral sucker, ventral sucker and ovaries of both male and female worms were smaller in re-infected buffaloes than in primary-infected buffaloes, with a significant difference (P < 0.05) observed in the length of the ventral sucker of males, the length of the oral sucker of females and the length and width of the ovary. We also observed that there were significantly fewer eggs in the uteri of female worms of re-infected water buffaloes (P < 0.05). These results revealed that worm development was inhibited in water buffaloes previously exposed to S. japonicum. The susceptibility varies among diverse mammalian hosts to S. japonicum infection and the development of the worms in these hosts is different; for example, in susceptible hosts the development of the worm is favorable and in less susceptible hosts it is repressed [3, 20, 34, 35]. The growth environment of the worm in re-infected buffalo may be similar to that in an unsuitable host. Therefore, the worm development may be inhibited in the host environment.

Due to variations in the susceptibility of disparate mammalian hosts to S. japonicum infection, several recent comparative studies using proteomic analysis [36] and microarray analysis [19, 37] of S. japonicum from different hosts have been conducted, and numerous DEGs that may influence parasite survival and development have been identified. Yang et al. [19] compared the transcriptional profiles of adult schistosome worms recovered from yellow cattle and water buffaloes and found that several genes involved in transcription, transport, lipid metabolism, energy metabolism, nucleotides and energy and signaling pathways were differentially expressed in worms from these two hosts. In the present study, we compared the gene expression profiles of adult worms recovered from re-infected and primary-infected water buffaloes using RNA-Seq. We identified 112 DEGs in worms recovered from re-infected water buffaloes, including 61 upregulated genes and 51 downregulated genes. Genes related to transport, binding and oxidation-reduction were downregulated in adult worms from re-infected water buffaloes. For example, the GO terms “calcium-dependent phospholipid binding” (Sjp_0113480, Sjp_0077960, Sjp_0077970), “lipid binding” (Sjp_0113480, Sjp_0077960, Sjp_0077970) and “calcium ion binding” (Sjp_0077970, Sjp_0113480, Sjp_0077960) were enriched only in downregulated genes. Schistosoma japonicum parasites are unable to synthesize some key nutrient molecules such as fatty acids, sterols, purines and nine essential amino acids, including arginine and tyrosine [38]. Previous reports suggested that schistosomes cannot produce long-chain fatty acids and only obtain complex lipids for the synthesis of sterols and fatty acids from the host [39]. We hypothesize that the downregulation of these genes contributed to the reduction in worm numbers and the maldevelopment of surviving worms in re-infected water buffaloes by mechanisms other than acquired immunity [23]. Our functional enrichment analyses indicated that a large number of GO terms enriched in the upregulated genes were associated with metabolic processes and biosynthesis. In addition, we observed the overexpression of genes with a variety of binding-related activities, such as “zinc ion binding” (uncharacterized protein LOC106012791), “heterocyclic compound binding” (SJCHGC04245 protein), “DNA binding” (polyprotein), “cation binding” (trehalose-6-phosphate hydrolase), “purine nucleotide binding” (malignant fibrous histiocytoma-amplified sequence 1 homolog) and “nucleic acid binding” (reverse transcriptase). We speculated that the overexpression of genes related to metabolic processes and biosynthesis in surviving worms recovered from re-infected water buffaloes might be a compensatory mechanism by these worms to adapt to disadvantageous environments. Compensation mechanisms have been extensively studied in many diseases of humans and animals, such as Parkinson’s disease [40], facet joints arthritis [41] and unilateral kidney pathology[42]. In addition, synaptic plasticity deficit studies of Alzheimer’s disease have shown that the recruitment of nitric oxide (NO) serves a compensatory role to boost synaptic transmission and plasticity during early AD stages[43]. Presumably, in order to cope with the host’s unfavorable host environment, re-infected buffalo-derived worms employ a compensatory mechanism to integrate some molecules involved in metabolism and synthesis. Our results are consistent with the biology of S. japonicum and will help to clarify the basic molecular mechanisms underlying resistance to re-infection in water buffaloes, which might provide new insight into the genes functionally related to development as well as aid in screening of anti-schistosome drug targets and vaccine candidates.

Among the 112 DEGs, we observed that the transcript Sjp_0008840 was not expressed and that the transcript Novel02058 was expressed only in worms recovered from re-infected buffaloes. According to GO annotation analysis, transcript Sjp_0008840 is an integral membrane component. The transcript Novel02058 has 73.3% similarity at the gene level with reverse transcriptase SjR1 in S. japonicum [44] and, according to the UniProt database, functions in anaerobic aromatic compound degradation. We also found that the gene (Sjp_0078930) encoding toxic substance binding protein (centrosomal protein of 162 kDa) was upregulated in worms recovered from re-infected buffaloes. According to GO functional enrichment analysis, this centrosomal protein of 162 kDa is a critical molecule in centriole duplication during cell proliferation and development. Gudi et al. [45] showed that accurate centriole duplication is important for many cellular and physiological activities such as cell division and ciliogenesis. According to gene functional annotation analysis using the Gene DB database (http://www.genedb.org) [46], the gene is the DNA double-strand break repair protein RAD50 ATPase. DNA double-strand breaks are induced by environmental agents such as ionizing radiation and genotoxic chemicals and occurring spontaneously during DNA replication, threatening genomic stability [47]. DNA damage repair is fundamental to cell survival and cancer prevention [47, 48]. Thus, the effect of differential expression of these three genes on schistosome development is worthy of further investigation.

Reactive oxygen species (ROS) are naturally produced in cell compartments such as the peroxisome and mitochondria as well as at the plasma membrane and play key roles in signaling cell fate, growth and survival [49]. The observed increase in hydrogen peroxide (H2O2) detoxification capacity and resistance to multiple sources of ROS during S. mansoni development in the vertebrate host suggested that adult parasites are exposed to greater redox challenges than immature parasites [50, 51]. Similarly, we speculate that redox challenges in re-infected water buffaloes are greater than in primary-infected animals. Cytochrome c oxidase (CcO), which is a highly regulated enzyme, is believed to regulate mitochondrial oxidative metabolism and ATP synthesis [52, 53]. A previous study suggested that CcO dysfunction is associated with increased mitochondrial ROS production and cellular toxicity. Under normal physiological conditions, CcO is rate-limiting in the respiratory chain and its activity is an indicator of the oxidative capacity of cells [53]. Previous work indicated that the survival mechanisms of schistosomes in their definitive hosts included the production of protective antioxidant proteins, which neutralized oxidative damage caused by the host immune response as well as by worm-generated oxygen radicals [54, 55]. Oliveira et al. [49] found that S. mansoni employed a unique antioxidant network that is key to parasite survival and may be a valuable chemotherapeutic target. In the present study, three genes (Sjp_0128890, Sjp_0031650 and Sjp_0122640) related to oxidation-reduction processes were downregulated in worms recovered from re-infected water buffaloes, whereas the opposite expression pattern was observed for genes (Sjp_0127400 and Sjp_0128790) related to the redox pathway, including genes related to cytochrome c oxidase subunit 1 and cytochrome c oxidase subunit 1+2. These results indicate that redox reactions and ROS produced by circulating immune cells might play an essential role in parasite killing. Therefore, surviving worms have an increased ability to detoxify ROS and avoid redox imbalances and parasite cell death triggered by the host immune system.

Compared with cattle, fewer parasites are able to survive and mature in water buffaloes. The upregulation of elongase of very long-chain fatty acids (ELOVL) in adult schistosomes from water buffaloes might be a compensatory mechanism by parasites in less susceptible hosts to enable worm growth and development [56]. Here we found that genes related to metabolism and biosynthesis were upregulated in surviving worms in re-infected water buffaloes, which might be another compensatory mechanism by parasites in re-infected or immunized animals. Regarding metabolism, GO terms enriched in upregulated genes included metabolic processes, nitrogen compound metabolic processes, heterocycle metabolic processes, organic cyclic compound metabolic processes and carbohydrate derivative metabolic processes. Importantly, we found that three genes (Sjp_0067470, Sjp_0036210 and Sjp_0128800) were associated with most of these metabolic processes. For biosynthesis, GO terms enriched in upregulated genes included cellular biosynthetic processes, chemical component (i.e. organic substances, aromatic compounds, heterocycle and organic cyclic compounds) biosynthetic processes, energy substance (i.e. carbohydrate derivatives, ATP, purine nucleoside monophosphate, purine ribonucleoside monophosphate, nucleoside triphosphate and purine nucleoside triphosphate) biosynthetic processes and glycosyl compound biosynthetic processes. In particular, glycosyl compound biosynthetic processes are important for schistosome development, and schistosome glycoconjugates play an important role in the evasion mechanisms that worms utilize to evade host immunological responses [57, 58]. Regarding resistance to re-infection and self-curing in water buffaloes, it is generally accepted that these phenomenon are mainly due to specific immune responses produced by the animals in response to schistosome infection [59]. Studies have reported that some individuals develop partial resistance to re-infection after schistosomiasis infection and treatment and that high levels of isotype antibody IgE and low levels of IgG4 are closely related to soluble egg antigens (SEA) and adult worm antigen (SWA) in the resistant population [33, 60, 61]. We also found that a subset of transcripts involved in glycosylation, including beta-1, 3-galactosyl-O-glycosyl-glycoprotein beta-1, 6-N-acetylglucosaminyltransferase 3 (Sjp_0048330) and putative glycosyltransferase (Novel03665), were downregulated in worms recovered from re-infected water buffaloes. Glycosylation is a complicated biological process [62] and the global level of glycosylation and the functions of glycoconjugates in worms recovered from re-infected water buffaloes or other hosts that are resistant to re-infection require further study

Annexins belong to a multigene family of calcium-dependent phospholipid-binding proteins, many members of which have been identified in important eukaryotic phyla [63]. In humans, annexins interact closely with various cell membrane components to form networks on the cell surface that participate in the regulation of membrane organization, cell differentiation, migration and intracellular signaling by enzyme modulation and ion fluxes [64, 65]. Although annexins lack related signal peptides for secretion, many extracellular family members have been found to act as receptors for serum proteases on the endothelium and as inhibitors of neutrophil migration and blood coagulation [63]. In addition, some human annexin isoforms are involved in immunoregulatory functions such as the resolution of inflammation [66]. We identified four annexin domain-containing protein-encoding genes (Sjp_0113480, Sjp_0122740, Sjp_0077960 and Sjp_0077970) that were downregulated in worms recovered from re-infected hosts. This result indicates that the differential expression of these genes might affect the survival and development of schistosomes in their definitive host.

Information on the DEGs identified in the present study is essential to understanding the development of schistosomes in re-infected and primary-infected water buffaloes. In future studies, we will further investigate the S. japonicum genes identified in this study using in vitro culture or in vivo infection experiments using RNA interference (RNAi). We expect this to further contribute to our understanding of host-parasite relationships in re-infected and primary-infected water buffaloes and to provide helpful information for screening bovine schistosome vaccine candidates.

Conclusions

In summary, we employed an Illumina RNA-Seq platform to identify DEGs in adult S. japonicum worms recovered from re-infected and primary-infected water buffaloes. We found that the downregulation of some important genes may have contributed to reductions in worm counts and the maldevelopment of surviving worms in re-infected water buffaloes. Furthermore, worms may have developed compensatory mechanisms to survive in the disadvantageous host environment. The identification of these DEGs provides new insights into mechanisms of resistance of water buffaloes to re-infection as well as useful information for comprehending the interactions between S. japonicum and its host.

Notes

Acknowledgements

We would like to thank the members of our laboratory for their kind assistance with animal infections, worm perfusion and collection. We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.

Authors’ contributions

YDM and JML conceived and designed the experiments. YDM, CCH, HL, KL, ZQF, JJL and YMJ performed the experiments and analyzed the data. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Number 31572218) and the Scientific and Technological Innovation Programme of the Chinese Academy of Agricultural Sciences.

Ethics approval and consent to participate

All procedures performed on animals in this study were conducted according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The study protocol was approved by the Institutional Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, People’s Republic of China.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Supplementary material

13071_2019_3600_MOESM1_ESM.xlsx (2.1 mb)
Additional file 1: Table S1. Blast2GO annotation of all identified genes. Shown are genes from Blast2GO analysis.
13071_2019_3600_MOESM2_ESM.xlsx (239 kb)
Additional file 2: Table S2. Significantly enriched GO terms (P < 0.05).
13071_2019_3600_MOESM3_ESM.xlsx (236 kb)
Additional file 3: Table S3. Significantly enriched 110 GO terms (P < 0.05).
13071_2019_3600_MOESM4_ESM.xlsx (231 kb)
Additional file 4: Table S4. Significantly enriched 35 GO terms (P < 0.05).
13071_2019_3600_MOESM5_ESM.xlsx (231 kb)
Additional file 5: Table S5. KEGG pathway enrichment analysis of differently expressed genes.

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Yudan Mao
    • 1
  • Chuanchuan He
    • 1
  • Hao Li
    • 1
  • Ke Lu
    • 1
  • Zhiqiang Fu
    • 1
  • Yang Hong
    • 1
  • Yamei Jin
    • 1
  • Jiaojiao Lin
    • 1
  • Xin Zhang
    • 1
  • Jinming Liu
    • 1
    Email author
  1. 1.Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal ParasitologyMinistry of AgricultureShanghaiPeople’s Republic of China

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