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Veterinary Parasitology 193 (2013) 1– 7

Contents lists available at SciVerse ScienceDirect

Veterinary Parasitology

jo u rn al hom epa ge : www.elsev ier .com/ locate /vetpar

evelopment of transgenic lines of Eimeria tenella expressing2e-enhanced yellow fluorescent protein (M2e-EYFP)

ianyong Liu1, Jun Zou1, Guangwen Yin, Huali Su, Xiaoxi Huang, Jianan Li, Li Xie,ingqiong Cao, Yujuan Cui, Xun Suo ∗

ational Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District,eijing 100193, China

r t i c l e i n f o

rticle history:eceived 19 January 2012eceived in revised form6 November 2012ccepted 11 December 2012

eywords:ransgeneingle sporocyst isolationimeria tenellaaccine vehicle

a b s t r a c t

Eimeria parasites are obligate intracellular apicomplexan protists that can cause coccid-iosis, resulting in substantial economic losses in the poultry industry annually. As thecomponent of anticoccidial vaccines, seven Eimeria spp. of chickens are characterized withpotent immunogenicity. Whether genetically modified Eimeria spp. maintains this prop-erty or not needs to be verified. In this study, two identical transgenic lines of Eimeriatenella were developed by virtue of single sporocyst isolation from a stably transfectedpopulation expressing fused protein of M2 ectodomain of avian influenza virus (M2e) andenhanced yellow fluorescent protein (EYFP). The chromosomal integration and expressionof M2e-EYFP were confirmed by Southern blot, plasmid rescue and Western blot analysis.We found that the reproduction of transgenic parasites was higher than that of the parentalstrain. Chickens challenged with wild type E. tenella after immunization with 200 oocystsof transgenic parasites had similar performance compared to those in non-immunized andnon-challenged group. In another trial, the performance of transgenic parasite-immunized

birds was also comparable to that of the Decoquinate Premix-treated chickens. These resultssuggest that this transgenic line of E. tenella is capable of inducing potent protection againsthomologous challenge as a live anticoccidial vaccine. Taking together, our study indicatesthat transgenic eimerian parasites have the potential to be developed as a vaccine vehiclefor animal use in the future.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Eimeria species, parasitic protozoa of the Phylum Api-omplexa, are causative agents of coccidiosis that causesuge amount of losses to the poultry industry annually

Shirley et al., 2004). As an effective prevention strategyor the control of coccidiosis, vaccination with virulent orttenuated live oocyst vaccines has been administrated

∗ Corresponding author. Tel.: +86 10 62734325; fax: +86 10 62734325.E-mail addresses: suoxun@cau.edu.cn, suoxun3@yahoo.com.cn

X. Suo).1 These authors contributed equally.

304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.vetpar.2012.12.019

in field worldwide for more than 50 years (Shirley et al.,2005; Suo et al., 2006; Williams, 2002b). Anticoccidial vac-cines can be used in chickens as early as one-day-old andcan elicit full immune protection against field infection21–28 days after vaccination (Williams, 2002a). It has longbeen recognized that cell-mediated immunity provides themajority of protection when using live oocyst vaccines inchicken populations (Dalloul and Lillehoj, 2006).

Recently, genetic manipulation of Eimeria tenella provedto be feasible. Both transient and stable transfection of

E. tenella were confirmed to be successful (Kelleher andTomley, 1998; Hao et al., 2007; Yan et al., 2009); sev-eral molecular tools were also available for this parasite(Clark et al., 2008). For the reason that genetically identical

2 X. Liu et al. / Veterinary Parasitology 193 (2013) 1– 7

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opulation is the prerequisite for generally biological andmmunological study for eimerian parasites (Shirley andarvey, 1996), development of transgenic lines is neces-

ary when concerning transgenic manipulation of eimerianarasites.

Here we report for the first time the establishment ofransgenic E. tenella lines through single sporocyst isolationrom stably transfected parasites and the determination ofts immunogenicity as an anticoccidial vaccine.

. Materials and methods

.1. Animals and parasites

One-day-old Arbor Acres broiler chickens were pur-hased from Arbor Acres Poultry Breed Company, Beijing,hina and raised under a coccidia-free condition untilse for passage or infection with eimerian oocysts. Dur-

ng the entire experimental process, chickens were givenood and water ad libitum. All experiments involving livehickens were performed in compliance with the currentnimal welfare legislations issued by China Agriculturalniversity. Oocysts of E. tenella BJ strain and transfectedopulation were passaged, collected, sporulated and storeds described elsewhere (Chapman and Shirley, 2003).

.2. Plasmid construction and sporozoite transfection

M2e, the coding sequence of ectodomain of M2 pro-ein of H5N1 subtype avian influenza virus (TJ strain, kindlyrovided by Prof. Jinhua Liu of China Agricultural Univer-ity), was fused in frame with enhanced yellow fluorescentrotein (EYFP) gene. The amplified chimeric M2e-EYFPene was inserted into an expression vector containing E.enella histone 4 promoter and Actin 3′ regulatory sequenceLiu et al., 2008). The final construct was named as pH4-

2e-EYFP-Actin (Fig. 1a). The maxi-prepared plasmid wasinearized with Nde I (New England Biolabs, Beijing, China).

Sporozoites of E. tenella BJ Strain were purified asescribed previously and suspended in complete cytomixuffer at concentration of 1 × 108 ml−1 (Shi et al., 2008).en million sporozoites, together with 100 �g linearizedH4-M2e-EYFP-Actin DNA and 100 U Nde I in a 4 mm gapuvette, were subjected to electroporation using a 2000 V,5 �, and 25 �F pulse of Gene Pulser X cellTM (Bio-Rad,

SA) according to previous studies (Black et al., 1995; Liut al., 2008). The electroporated parasites were inoculatednto the ileo-caecal opening of 2-week-old chickens at aose of 5 × 106.

onstruction and validation of transgenic lines of E. tenella Etm2e-2 and Etm2e-9ection of E. tenella. (b) Confocal images of sporulated Etm2e-2 oocysts. Images corom left to right panels. (c) Southern blot analysis of genomic DNA of transgennd EtM2e-9 was probed with Digaoxin-labeled YFP probe. “+” stands for the linelasmids from genomic DNA of EtM2e-2 and EtM2e-9. The genomic DNA was dompetent cells of E. coli. The rescued plasmids were analyzed by restriction anahows a band in size of 750 bp of EYFP gene. M, M1 and M2 stand for three differenn transgenic E. tenella by Western blot. The primary antibodies, i.e., anti-Mic2 ohe panels. HRP conjugated goat-anti-mouse, goat-anti-rabbit or goat-anti-chickerotein sample of parental strain.

ology 193 (2013) 1– 7 3

2.3. In vivo selection for transfectants

From seventh day to ninth day after infection withelectroporated sporozoites, oocysts shed in feces were col-lected and purified following standard procedures (Tomley,1997; Chapman and Shirley, 2003). The sporulated progenyoocysts were treated by sodium hypochloride solution(with 10% effective chlorine) for 5 min in an ice bath, andthen washed at least four times to remove the bleacher.

The purified oocysts were sieved through a 75-�m meshsieve following a 37.5-�m mesh sieve before subjectedto MoFlo high-speed cell sorter (Cytomation, Fort Collins,CO) equipped with a 488-nm argon laser. YFP emission isdetected using a 555SP dichroic mirror and a 530/40 nmbandpass filter. The oocysts expressing YFP were sortedand then passaged in chickens for the next round of sorting.After 5 successive passages, a population of oocysts stablyexpressing YFP was obtained.

2.4. Single-sporocyst isolation

Totally 106 freshly sporulated oocysts were sorted froma pH4-M2e-EYFP-Actin stably transfected E. tenella popu-lation (6th generation). These oocysts were broken usinga tissue grinder. The released sporocysts were dilutedwith PBS (pH 7.4) to about 1–3 sporocysts per microliter.Glass slide attached with pieces of transparent thin plas-tic scraps (∼0.5 cm × 0.5 cm) was set on the microstat ofinverted fluorescence microscope (IX71, Olympus, Tokyo).The microstat was adjusted to let the fluorescent beamtransmit through the center of one scrap. Then 0.25 �l ofdiluted sporocyst suspension was dropped onto the cen-ter of the scrap. Under the 10× objective lens, the wholedrop was thoroughly checked to confirm if there was onlyone fluorescent sporocyst within it. When a drop con-taining only one fluorescent sporocyst was confirmed bytwo researchers independently, it was covered immedi-ately with 3 �l 50% glycerol (in water, v/v). The scrap wasthen removed from the slide with a muscle forceps andgiven orally to a 1-day-old chicken. Totally ten birds wereinfected with single sporocyst following the same proce-dure.

Seven and a half days post the inoculation, the intesti-nal contents and the mucosa scrapings of each pair of caecawere collected together from each killed bird. The mixtureof each bird was incubated with 5 ml PBS (pH 7.4) sup-

plemented with trypsin (final con. 1.5%, m/v) at 37 ◦C for1 h, and then filtered through bilayer gauze into a 35 mmpetri dish. Half a milliliter of K2Cr2O7 solution (5%, v/v)was added to the petri dish. After sporulation for 48 h at

. (a) Schematic map of plasmid pH4-M2e-EYFP-Actin used for the trans-rresponding to YFP fluorescent filter, bright field and merged are showedic lines. EcoR I digested genomic DNA of parental strain (WT), EtM2e-2arized pH4-M2e-EYFP-Actin plasmid. (d) Restriction digestion of rescuedigested by EcoR I and then ligated by T4 DNA ligase, transformed intolysis with Nde I (lines 1 and 3) or Sac II-Not I (lines 2 and 4). The arrowt DNA markers. (e) Detection of the expression of M2e-YFP fused protein

r anti-M2e were incubated with each strap of membrane as indicated inn IgG was employed as secondary antibody, respectively. WT stands for

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4 X. Liu et al. / Veterinary

28 ◦C, the petri dish was checked under inverted fluores-cence microscope. Fluorescent progeny oocysts obtainedfrom the single sporocyst were then passaged 2 generationsin chickens to obtain a large number of oocysts.

2.5. Southern blot analysis

For either transgenic or parental parasites, one hundredmillion oocysts were ground with liquid nitrogen, and thenlysed by CTAB buffer (10% hexadecyl trimethyl ammoniumbromide in 0.7 M NaCl) at 60 ◦C overnight with occa-sional agitation. After cooling to room temperature, thelysis was extracted twice by phenol/chloroform/isoamylalcohol (25:24:1, v/v) and precipitated with alcohol.Following RNase H treatment, the genomic DNA wasdigested by EcoR I at 37 ◦C overnight and then reclaimedfrom phenol/chloroform extraction and alcohol precip-itation. Then the digested genomic DNA of transgenicor wild-type E. tenella was subjected to Southernblot analysis using DIG High Prime DNA Labeling andDetection Starter Kit II following the manufacturer’sinstruction manual (Roche, Basel, Switzerland). Briefly,the gel fractionated genomic DNA was transferred ontoHybondTM-N+ membrane (Amersham Biosciences, NJ,USA). The UV-crosslinked membrane was hybridized withDigoxigenin-labeled YFP-specific probe, and incubatedwith Anti-Digoxigenin-AP conjugated antibody. X-rayexposure was carried out after CSPD was added to themembrane.

2.6. Restriction analysis for the rescued plasmids

If the transfected plasmid DNA inserted into the chro-mosome of the transgenic parasites, this plasmid couldbe rescued by virtue of its pUC element and karamycinresistant gene both responsible for the replication withinE. coli in culture medium with kanamycin (Yan et al., 2009).The genomic DNA of two transgenic lines EtM2e-2 andEtM2e-9 was digested by EcoRI, which is absent within thelinear plasmid pH4-M2e-EYFP-Actin, at 37 ◦C overnight,and then extracted by phenol/chloroform. After ligationby T4 DNA ligase, the digested genomic DNA was trans-formed into competent cells of E. coli. The positive coloniesfrom kanamycin-added agar plates were used for plasmidextract. The rescued plasmid DNA was single-digested withNde I or double-digested with Sac II-Not I and analyzed bygel electrophoresis.

2.7. Reproduction test of transgenic parasites

To assess the reproduction of the transgenic parasites,two groups of 5 birds were each infected with 1000 sporu-lated oocysts of EtM2e-2 or wild type, respectively. Fecalsamples were collected every 24 h between 132 h and312 h. Oocyst per gram (OPG) was counted using MacMas-ter chamber and oocysts shed per bird was calculated.

2.8. Western blot analysis

To validate the expression of M2e peptide in trans-genic E. tenella, Western blot analysis was performed. Ten

ology 193 (2013) 1– 7

million E. tenella oocysts of EtM2e-2 or the wild type wereground with liquid nitrogen and suspended in 200 �l PBS.The suspension was sonicated using SONICS VC 250 (Sonics& Materials, Newtown, USA) for 5 pulses (40% power with2 s on/3 s off pulse). The lysis was centrifuged at 10,000 × gfor 10 min, and the supernatant was subjected to SDS-PAGEand immunoblot analysis following standard protocols. Pri-mary antibodies employed in this assay included mousemonoclonal antibody specific for microneme 2 (Mic2) ofE. tenella (AbMax Biotechnology, Beijing, China), rabbitpolyclonal antibody for GFP (which can also recognizeYFP; from Protech, purified poly Ab, diluted by 1:1500)and chicken anti-sera against M2e-KLH (Keyhole limpethemocyanin conjugated M2e peptide; antisera from KLHconjugated M2e peptide immunize SPF White LeghornChicken, diluted by 1:2000)); while HRP-conjugated goatanti-mouse IgG (Proteintech, USA), goat anti-rabbit IgG(Proteintech, USA) and goat anti-chicken IgG (KPL, USA)were used as secondary antibodies for the immunoblotdetection.

2.9. Animal trials and data collection

To test the immunogenicity of the transgenic E. tenella,two trials were carried out using transgenic line EtM2e-2.In both trials, 120 chickens were randomly allocated intotwo groups, each of which contains six replicates of 10chickens.

In Trial 1, each bird in the experimental group wasorally inoculated with 200 sporulated oocysts of trans-genic E. tenella at the age of 4 days; while the controlgroup received distilled water as placebo. At the age of 18days, immunized chickens were challenged with 50,000sporulated oocysts of E. tenella BJ strain; while the con-trol group was still inoculated with distilled water. Thebody weight, feed consumption and survival number ofeach replicate were recorded at day 4, 18, 25 and 42,respectively.

In Trial 2, every chicken in the experimental group wasimmunized with 200 transgenic parasites at the age of 4days, while birds in the control group were given antic-occidial drug Decoquinate Premix (product of Qilu AnimalHealth Products Co., Ltd., China) in feed at concentration of30 mg/kg from the age of 4–37 days. The body weight, feedconsumption and survival number of each replicate wererecorded at day 4 and 42, respectively. At the age of 42 days,two chickens from each replicate of both groups were chal-lenged with 1 × 105 oocysts of E. tenella BJ strain. At the ageof 49 days, all challenged birds were killed. Caecal lesionwas recorded following a previous procedure (Johnson andReid, 1970).

2.10. Statistic analysis

Data were statistically analyzed using SPSS 11.5 forWindows. After one-way analysis of variance (ANOVA),Tukey’s multiple range test was used to test significancebetween different treatments at p < 0.05.

X. Liu et al. / Veterinary Parasit

Fig. 2. Oocyst output in transgenic line EtM2e-2 infected chickens. Onegroup of five chickens were infected with 1000 transgenic parasites(d

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genic line retains its immunogenicity as an anticoccidial

EtM2e-2), another group received 1000 wild type parasites. Oocyst shed-ing was measured every 24 h between 120 h and 336 h post infection.

. Results

.1. Transgenic E. tenella lines expressing M2e-YFP wereeveloped via single sporocyst isolation

After single-sporocyst isolation, caecal content samplesf two chickens from ten of single sporocyst inoculatedirds were found to be YFP positive under fluorescenticroscope, and the number of oocysts was about 600 for

ach sample. After two generations of passage in chickenseparately, 1.4 × 106 and 2.9 × 106 oocysts were recovered,espectively. These two transgenic lines were named astm2e-2 and Etm2e-9 (Fig. 1b). As shown in Fig. 1c, DIG-abeled yfp probe detected the yfp-containing plasmid DNAragment, which was a single band in both Etm2e-2 andtm2e-9 group. The size of both bands is more than 10 kbnd in the parallel position on the membrane. PCR andestriction digestion of the rescued plasmids demonstratedame bands for the fused M2e-EYFP gene in both linesFig. 1d). These results strongly suggest that the plasmidNA have integrated into the same locus of chromosomalNA in both transgenic lines, i.e., Etm2e-2 and Etm2e-9 arerobably identical in the sense of integration and expres-ion of the fused M2e-EYFP gene.

Immunoblotting assay also confirmed the expression ofFP and M2e tag in transgenic lines Etm2e-2 and Etm2e-

(Fig. 1e). Both anti-M2e and anti-YFP antibodies reactedith three protein bands at size between 25 and 36 kDa,

uggesting the existence of truncated M2e-YFP protein.

.2. Transgenic line Etm2e-2 produces more progenyocysts than the wild type

Transgenic line Etm2e-2 showed a similar pattern ofocyst output, with a peak of output at 144 h after infection.hen the area under the curve of oocyst output was calcu-

ated, average number of oocysts shed by each bird infected

ith transgenic parasites were about 7.7 × 107, muchigher than those of wild type parasite group (4.8 × 107)see Fig. 2).

ology 193 (2013) 1– 7 5

3.3. Transgenic E. tenella line expressing M2e-YFP ishighly immunogenic against homologous infection

In the trial 1, transgenic parasite immunized groupshowed similar weight gain and feed conversion ratio(FCR) when compared to that of the control group dur-ing the entire process, with exception at the age of 18–25days, during which chicken growth was interfered byheavy auto-reinfection of oocysts excreted in the feces ofimmunized chicks (see Table 1). Similarly, performance oftransgenic parasite immunized chickens showed no obvi-ously difference between 4 and 42 days against that of theDecoquinate medicated ones (see Table 2). As expected, inthe Decoquinate treated group, the average lesion scorewas 3.82 ± 0.45, while no lesion was observed in the immu-nized group when both group of birds were challenged with1 × 105 oocysts of E. tenella BJ strain (see Table 2).

4. Discussion

Vaccination provides the most potent measure againstinfectious diseases (Liniger et al., 2007). Live vaccine vehi-cles, based almost exclusively on recombinant virusesand bacteria expressing defined pathogen-derived anti-gens, represent powerful vaccine candidates for futurevaccine developing strategies (Liniger et al., 2007; Medinaand Guzman, 2001). When the transfection manipulationcame true to Eimeria species (Kelleher and Tomley, 1998;Hao et al., 2007), it provides the possibility to developEimeria-based eukaryotic vaccines (Liu et al., 2008; Huanget al., 2011; Clark et al., 2012). To achieve this great aim,recombinant eimerian parasites with ascertained geneticbackground, i.e. transgenic lines of Eimeria spp., are needed,and its potent immunogenicity is also a prerequisite. Theestablishment of transgenic E. tenella lines in this study is abig step to the stable transfection of E. tenella (Clark et al.,2008; Yan et al., 2009).

The reproduction and pattern of oocyst output are twoimportant characters for eimerian parasites when compar-ing a precocious line or a newly isolated population with aparental strain or a reference strain (Long, 1972; Yan et al.,2009). The transgenic E. tenella line Etm2e-2 developed inthis study showed an increased fecundity, which is contraryto another population of stably transfected parasites (Yanet al., 2009). This change of fecundity is probably due tothe random selection of the transgenic line from the sta-bly transfected population by single-sporocyst isolation,which led to the selection of a population with relativelyhigher fecundity. However, uncovering the mechanismbehind this phenotype needs the mining of the genomicinformation, including the integration locus of the trans-gene.

When concerning the immunogenicity of the transgenicE. tenella, chickens immunized with Etm2e-2 showed sim-ilar performance to that of the wild type parasites, whichis in accord with live oocyst vaccines applied in the field(Williams et al., 1999, 2000), indicating that the trans-

vaccine. Meanwhile, the normal performance of chickensimmunized with transgenic parasites may suggest thattransgenic E. tenella is harmless to hosts like its parental

6 X. Liu et al. / Veterinary Parasitology 193 (2013) 1– 7

Table 1Chicken performance in Trial 1.

Age of days Transgenic oocysts immunizeda Non-immunized

Weight gain (g/per bird) Feed conversion ratio Survival rate Weight gain (g/per bird) Feed conversion ratio Survival rate

4–18 393.97 ± 11.83 1.51 ± 0.28 97.5 419.07 ± 14.09 1.43 ± 0.06 99.218–25 302.67 ± 33.57 2.23 ± 0.15 99.1 357.79 ± 38.35 1.88 ± 0.19 98.325–42 1002.75 ± 70.52 2.96 ± 0.22 96.0 1025.61 ± 55.85 3.15 ± 0.52 94.8

4–42 1700.38 ± 58.93 2.32 ± 0.16 93.3 1802.47 ± 85.39 2.20 ± 0.15 93.3

a Birds were immunized with oocysts of line EtM2e-2 at day 4 and then challenged with oocysts of wild type E. tenella.All values between the two groups are p > 0.05 except those for age of 18–25 days shown in bold text.

Table 2Chicken performance and lesion score in Trial 2.

Treatment Age of days Performance Lesion score

Weight gain (kg/per bird) Feed conversion ratio Survival rate

re.

Decoquinate medicated 4–42 2.27 ± 0.12

Transgenic oocysts immunized 4–42 2.19 ± 0.06

All values between the two groups are p > 0.05 except those for lesion sco

strain, though much more work needs to be done for testingits biological safety.

Recently, many studies confirmed that M2e ectodomainexpressed and deliveried in vivo could induce protectiveimmune responses against both homologous or heterol-ogous avian influenza viruses challenge (Huleatt et al.,2008; Denis et al., 2008). These reports encouraged us toco-express M2e in transgenic eimerian parasites and testits immunogenicity. Unfortunately, the M2e monomer co-expressed with YFP failed to stimulate either antibody orcellular responses in chickens (data not shown). If strate-gies such as increasing the copy number of M2e andconstructing a M2e-secreting parasites become successful,immune responses would be induced by M2e-expressingtransgenic parasites.

In summary, we successfully developed transgenic E.tenella lines expressing M2e-YFP via single-sporocyst iso-lation and the transgenic line retained its immunogenicityas a live oocyst vaccine. Our findings pave the way for eval-uating transgenic Eimeria lines in terms of immunogenicityand safety, and for further study of its immunogenicityagainst avian influenza virus.

Conflict of interest

The authors declare that they have no conflict of interestwith the contents of this paper in any respect.

Acknowledgements

We thank Jingxia Suo in our laboratory for her gener-ous assistance during the study. This study was supportedby the National Natural Science Foundation of China(30471298, 30671579 and 30871862), the National High

Technology Research and Development Program of China(2011AA10A209), Chinese Universities Scientific Fund(2009-2-02) and Yangtze River Scholar and InnovationResearch Team Development Program (IRT0945).

1.69 ± 0.05 98.3 3.82 ± 0.451.73 ± 0.08 98.3 0

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