parasites and chicks : the niah strain of e. tenella , 
 which is virulent and maintained at the laboratory of parasitic diseases , national institute 
 of animal health ( tsukuba , japan ) , was used throughout this study .
the parasites were 
 maintained by passage in 2- to 3-week - old chicks ( nisseiken , tokyo , japan ) .
the chicks were 
 housed in wire - floored cages in coccidian - free rooms and with free access to feed and water 
 that contained no anticoccidial drugs or antibiotics .
the animals were treated in accordance 
 with protocols approved by the animal care and use committee , niah ( approval nos .
they were orally inoculated with 2  10 oocysts , and feces were 
 collected after 68 days .
preparation of developmental zoites : the purified e. 
 tenella oocysts were incubated for various time periods ( 0
hr , 48 hr and 144 hr ) 
 at 28c in 2.5% potassium dichromate ( wako , osaka , japan ) , treated with sodium hypochlorite 
 ( nacalai tesque , kyoto , japan ) for 20 min at 4c and then washed with phosphate - buffered 
 saline ( pbs ) .
this chemical treatment was repeated twice , followed by washing with pbs 5 
 times , and processed for further analysis as described below .
for the purification of sporozoites , the sporulated oocysts were broken by vortexing with 
 glass beads for 12 min to release sporocysts .
the sporocysts were treated in excystation 
 medium , 0.25% ( w / v ) trypsin ( merck , darmstadt , germany ) and 1% ( w / v ) taurodeoxycholic acid 
 ( sigma , st .
louis , mo , u.s.a . ) in hanks balanced salt solution ( sigma ) , ph 7.4 , at 41c in 
 a 5% co2 incubator for 90100 min .
the sporozoites were obtained by purification 
 involving two steps , namely centrifugation at 450 g for 2 min according to 
 a modification of a previously described method 
 and a filtration protocol using 595 filter paper circles .
merozoites and schizonts of the second generation were purified by centrifugation using 
 percoll ( ge healthcare life sciences , piscataway , nj , u.s.a . ) as reported previously .
briefly , the chicks were orally inoculated with 2  
 10 oocysts , and the infected ceca were removed at 96 to 108 hr 
 post - inoculation after euthanasia of animals according to protocols approved by the animal 
 care and use committee , niah ( approval nos .
after removal of the 
 contents and washing with pbs , the ceca were cut into less than 5 mm pieces and filtered by 
 a wire mesh .
the filtered homogenate was mixed with 100% 
 percoll to make a 30% percoll - pbs homogenate and layered over 50% percoll - pbs , which were 
 finally layered over pbs .
a total of three layers was prepared and centrifuged at 1,300 
 g for 15 min .
after centrifugation , a high population of schizonts was 
 seen floating on the 30% percoll - pbs ; merozoites were concentrated on the 50% percoll - pbs , 
 while erythrocytes were collected as a red pellet at the bottom .
briefly , 2-week - old chicks in groups of three were inoculated with 2  
 10 oocysts , and the infected ceca were removed at 72 hr , 96 hr , 120 hr , 144 hr 
 and 168 hr post - inoculation and then fixed .
cloning and sequencing of eimeripain : the gene coding for eimeripain was 
 partially identified from the e. tenella gene index database ( sequence no . 

5 and 3 rapid amplification of cdna 
 ends ( race ) was performed using the smarter race cdna amplification kit ( takara , otsu , 
 japan ) according to the manufacturer s protocol using these primers : etcathep - f1 ; caa ctt 
 cga cca cgt gcc cat ttc tct t for the 5end and etcathep - f14 ; tac tgg cta gct gtg aac agc 
 tgg for the 3end .
the putative signal sequence of eimeripain was analyzed using the 
 prediction server signalp v4.1 . by sequence 
 analysis of race products , 1,536 bp of eimeripain
this 
 molecule also contained approximately 410 bp and 1,180 bp non - coding regions at the 5 and 
 3 ends , respectively , consisting of a 512 amino acid protein , with a signal peptide of 21 
 hydrophobic amino acids , a predicted molecular weight of 54.68 kda and a pi of 5.53 
 ( excluding signal peptide ) using peptidemass .
expression of eimeripain : the open reading frame ( orf ) of eimeripain was 
 amplified by pcr from a cdna of sporulated oocysts using a set of primers [ forward : 
 reetcathep - f1 ( cgg ggt acc ccg atg ccc tcc gat gat ttg ggc ) , reverse : reetcathep - f2 ( ggg gta 
 ccc ctc ata ggt cct gcg ctg acg g ) ] containing kpn i restriction sites .
pcr was performed 
 for 5 min at 94c followed by 30 cycles of 30 sec at 94c , 30 sec at 63.4c and 2 min at 
 72c and a final elongation at 72c for 10 min .
the pcr product and the vector ptrchisb 
 ( invitrogen , carlsbad , ca , u.s.a . ) were digested by kpn i restriction enzymes .
the purified 
 pcr product was inserted into the kpn i sites of the vector ptrchisb using dna ligation kit 
 ( takara ) .
the 
 resultant plasmid was transformed into competent cells of escherichia coli 
 top10f strain ( invitrogen ) following the conventional method .
the expression of eimeripain 
 in e. coli with a polyhistidine - tag was performed according to the 
 procedure described by tsuji et al . .
briefly , the transfected cells were allowed to grow in sob medium ( bd , 
 franklin lakes , nj , u.s.a . ) containing 50 mg ampicillin / ml at 37c . to 
 induce recombinant protein expression ,
isopropyl - b - d - thiogalactopyranoside ( iptg ) was added 
 at 1 mm concentration , and the culture was grown for an additional 4 hr at 37c .
the culture 
 was then centrifuged at 10,000 g for 20 min and 4c , and then , the pellet 
 was resuspended in lysis buffer ( 20 mm sodium phosphate and 500 mm sodium chloride , ph 7.8 ) 
 with egg white lysozyme ( 100 g / ml ) .
the suspension was 
 sonicated on ice with an ultrasonic processor ( taitec , saitama , japan ) followed by freezing 
 and thawing . after three cycles of this treatment ,
the e. coli lysate was 
 centrifuged at 23,900 g for 30 min at 4c , and the supernatant was 
 collected . as a result
, eimeripain was successfully expressed in e. coli , 
 harvested as a soluble protein from bacterial cultures , separated on 12.5% sds 
page gel and 
 detected as an approximately 60 kda fusion protein by western blotting using an anti - his 
 monoclonal antibody ( nacalai tesque ) as described below .
the recombinant protein was 
 purified using probond resin ( invitrogen ) as described by the manufacturer .
page gel and detected as a single band by 
 staining with 0.2% coomassi brilliant blue r-250 ( wako ) ( fig .
protein concentration was calculated using the bca protein assay kit ( pierce , 
 rockford , il , u.s.a . ) , and protein was stored at 20c until further use .
sds - page of fusion - protein recombinant eimeripain after purification of probond 
 resin .
reverse transcription polymerase chain reaction ( rt - pcr ) : for rt - pcr , six 
 samples of unsporulated oocysts , sporulated oocysts incubated for 48 hr and 144 hr , and 
 purified sporozoites , merozoites and schizonts were used .
they were washed in pbs and 
 submerged in rlt lysis buffer ( qiagen , hilden , germany ) .
total rna from each sample was 
 extracted using the rneasy mini kit ( qiagen ) according to the manufacturer s protocol . 

single stranded cdna for mrna was prepared using the takara rna pcr kit ( amv ) ver.3.0 
 ( takara ) following the manufacturer s instructions .
four hundred nanograms of total rna was 
 used for rt before pcr . as a control , rna from chicken cells purified by the protocol for 
 schizonts and merozoites using non - infected ceca were used , because chicken cells might 
 contain purified schizonts or merozoites .
the synthesized cdnas were used for pcr using 
 eimeripain [ forward : etcathep - f5 ( att ctg cga gat gta gag aag gat att ) , reverse : etcathep - r5 
 ( ctg gat ctg aat aga aag aaa ggt aag t ) ] ( 499bp ) and e. tenella actin 
 ( 350bp )   specific oligonucleotides . as an 
 additional control ,
rna samples treated with the same protocol for synthesizing cdna without 
 transcriptase were used .
pcr was performed for 5 min at 94c followed by 40 cycles of 30 sec 
 at 94c , 60 sec at 53c and 60 sec at 72c and finally elongation at 72c for 10 min .
polyclonal antisera against eimeripain : an antiserum against the 
 eimeripain was raised by immunizing balb / c mice subcutaneously with 100 g 
 of purified recombinant protein emulsified with complete freund s adjuvant ( difco 
 laboratories , detroit , mi , u.s.a . ) , followed by booster immunizations of 50 
 g at 2 weeks apart using the same route five times .
the immunized mice 
 were sacrificed 1 week after the last booster , and serum was collected and stored at 20c 
 for further use .
the peptide cggepkvpndknas , amino acids 257 to 270 of the mature domain of eimeripain as 
 previously reported , were synthesized and 
 coupled to keyhole limpet hemocyanin ( klh ) ( pierce ) .
this synthetic peptide was used to 
 raise polyclonal antibodies against the mature domain of eimeripain in a japanese white 
 rabbit by subcutaneous injection of 400 g of the antigen emulsified with 
 complete freund s adjuvant ( difco laboratories ) .
the protocol was the same as that described 
 above , except for the booster immunizations using 200 g of antigen .
western blotting : the sporulated oocysts ( 48 hr incubation ) and purified 
 sporozoites , merozoites and schizonts were resuspended in pbs . they were freeze - thawed five 
 times , sonicated in an ice bath with the ultrasonic processor ( taitec ) and centrifuged at 
 600 g for 5 min .
the concentration of crude antigens was determined by bca protein assay kit 
 ( pierce ) . these parasite lysates ( 1020 g ) and the purified recombinant 
 protein of eimeripain ( 2 g ) were separated through 12.5% sds - page gels 
 under reducing conditions , and the proteins were transferred onto nitrocellulose membranes 
 ( ge healthcare life sciences ) .
the membranes were blocked with 5% skim milk in tris - buffered 
 saline ( tbs ) for 30 min and then incubated with polyclonal antibodies against the 
 recombinant eimeripain or the peptide corresponding to the eimeripain mature domain 
 ( 1:1,000 ) for 1 hr at room temperature .
they were washed with tbs containing 0.05% tween 20 
 ( tbs - t ) and then incubated with alkaline phosphate - conjugated goat anti - mouse or rabbit igg 
 ( h + l ) ( zymed , south san francisco , ca , u.s.a . ) as a secondary antibody for 1 hr .
the 
 membrane was washed again with tbs - t , and the bound antibody was developed with nitroblue 
 tetrazolium/5-bromo-4-chloro-3-indolyl phosphate ( bcip / nbt , promega , madison , wi , 
 u.s.a . ) .
indirect immunofluorescence assay : the purified sporozoites and merozoites 
 were applied to slide glass , air - dried and fixed by methanol .
these prepared slides and 
 sections of infected ceca were blocked with 10% goat serum ( mp biomedicals , santa ana , ca , 
 u.s.a . ) for 30 min , washed three times with 0.05% tween 20 in pbs ( pbst ) and then incubated 
 with polyclonal antibodies ( diluted 1:1,000 ) against recombinant eimeripain and eimeripain 
 mature domain peptide for 1 hr .
the slides were washed three times with pbst and then 
 incubated with green fluorescence - labeled mouse and rabbit igg secondary antibodies [ alexa 
 fluor488 goat anti-(mouse igg ) ( h + l ) or anti-(rabbit igg ) ( h + l ) ; 
 invitrogen ] for 1 hr .
they were observed under a fluorescence microscope ( leica , wetzlar , 
 germany ) .
transcription of eimeripain : in the present study , the total rna that was 
 first extracted from several stages of e. tenella was subjected to rt - pcr 
 analysis for transcription profiling of eimeripain . by successful amplification of an 
 approximately 500 bp fragment
, we confirmed that eimeripain was expressed in all examined 
 stages , namely oocyst ( 0
hr , 48 hr and 144 hr after sporulation ) , sporozoite , schizont and 
 merozoite stages , but not in samples of chicken cells or in any of the controls ( fig .
detection of eimeripain ( 499 bp ) ( a ) and e. 
 tenella actin ( 350 bp ) ( b ) specific rna .
l ; dna marker , 1 ; rna from 
 unsporulated oocysts , 2 ; from oocysts sporulated at 48 hr , 3 ; from oocysts sporulated 
 at 144 hr , 4 ; from purified sporozoites , 5 ; from purified schizonts , 6 ; from purified 
 merozoites , 7 and 8 ; from chicken cells separated on a 30% , and 50% percoll layer 
 using uninfected chicken ceca , respectively , 9 ; only pcr buffer , 10 ; genomic dna from 
 sporulated oocysts . ) , although the expression between mrna and protein did not always correlate . 

e. tenella actin gene showed the same results with those of eimeripain 
 ( fig .
2b ) , except for e. 
 tenella genomic dna . expression of eimeripain genes during the developmental stages of e. 
 tenella .
detection of eimeripain ( 499 bp ) ( a ) and e. 
 tenella actin ( 350 bp ) ( b ) specific rna .
l ; dna marker , 1 ; rna from 
 unsporulated oocysts , 2 ; from oocysts sporulated at 48 hr , 3 ; from oocysts sporulated 
 at 144 hr , 4 ; from purified sporozoites , 5 ; from purified schizonts , 6 ; from purified 
 merozoites , 7 and 8 ; from chicken cells separated on a 30% , and 50% percoll layer 
 using uninfected chicken ceca , respectively , 9 ; only pcr buffer , 10 ; genomic dna from 
 sporulated oocysts .
endogenous form of eimeripain : in order to identify the endogenous form of 
 eimeripain , anti - eimeripain antibodies were prepared using recombinant eimeripain and a 
 peptide corresponding to the mature domain of eimeripain . by western blotting analysis , the 
 two polyclonal antibodies generated against eimeripain recognized the recombinant eimeripain 
 fusion protein as a 60 kda band ( fig .
3.western blot analysis using antisera with anti - eimeripain ( a ) and anti - peptide 
 against eimeripain mature domain ( b ) .
l : molecular weight marker , 1 ; sporulated 
 oocysts , 2 , purified sporozoites , 3 and 4 ; purified schizonts and merozoites of the 
 second generation , 5 ; purified recombinant eimeripain .
arrows , arrowhead and arrows 
 with broken line show 33 kda mature eimeripain , 54 kda promature eimeripain and 60 kda 
 recombinant eimeripain protein , respectively . , lane 5 ) , and thus , these epitopes were confirmed to be present within the amino acid 
 sequence of eimeripain .
consequently , a 54 kda band of promature eimeripain , previously 
 reported , was detected in all stages of sporulated 
 oocysts , sporozoites , merozoites and schizonts examined with the antiserum against 
 recombinant eimeripain ( fig .
a 33 
 kda band , which has been reported as the active form of eimeripain , was recognized only in sporulated oocysts and schizonts ( fig .
unexpectedly , the 54 kda band 
 of promature eimeripain was also detected in schizonts ( fig .
these results show that antisera against recombinant eimeripain 
 and eimeripain mature domain recognize promature and mature eimeripain , respectively , and 
 that mature eimeripain is undetectable or does not exist in invasive zoites like sporozoites 
 and merozoites , but only in sporulated oocysts and developmental schizonts .
western blot analysis using antisera with anti - eimeripain ( a ) and anti - peptide 
 against eimeripain mature domain ( b ) .
l : molecular weight marker , 1 ; sporulated 
 oocysts , 2 , purified sporozoites , 3 and 4 ; purified schizonts and merozoites of the 
 second generation , 5 ; purified recombinant eimeripain .
arrows , arrowhead and arrows 
 with broken line show 33 kda mature eimeripain , 54 kda promature eimeripain and 60 kda 
 recombinant eimeripain protein , respectively .
endogenous localization of eimeripain in invasive zoites : promature 
 eimeripain was detected in the cytoplasm of sporozoites and merozoites by incubating with 
 the antiserum against recombinant protein ( fig . 

4.immunofluorescent staining of sporozoites ( a ) and merozoites ( b ) with antibody 
 against promature eimeripain ( arrows ) . left and right figures show fluorescent and 
 bright field photos .
they were observed as several dots , but not all over the entire region of 
 sporozoite and merozoite bodies , although these reactivities were not strong .
the antibody 
 against the eimeripain mature domain did not show any reactivity with these zoites ( data not 
 shown ) .
immunofluorescent staining of sporozoites ( a ) and merozoites ( b ) with antibody 
 against promature eimeripain ( arrows ) . left and right figures show fluorescent and 
 bright field photos . scale bar is 10 m .
endogenous localization during asexual stages : during the second 
 generation , in sections at 72 hr , 96 hr and 120 hr post - infection , promature eimeripain was 
 detected only in early schizonts at around 10 m in size , as constellations 
 of dots in the cytoplasm ( fig . 5afig .
5.immunofluorescent staining of asexual stages , the second generation schizonts ( a ) and 
 the third generation schizonts ( b ) with antisera against promature ( upper photos ) and 
 mature ( bottom photos ) eimeripain .
figures a and d are small immature schizonts of the 
 second generation , b and e are large immature schizonts , and c and f are mature 
 schizonts .
arrowheads and arrows 
 show smaller immature schizonts and larger immature ones of the third generation in 
 fig . h. scale bar is 10 m . ) ; it was not detected in schizonts over 15 m ( fig . 5b and 5c ) .
on the other hand , mature eimeripain was observed 
 over the entire cytoplasm surrounding the nuclei in small immature schizonts and in 
 developmental second - generation schizonts of middle size ( around 20 m ) 
 with the strongest reactivity ( fig . 5d and 5e ) . in 
 mature schizonts ,
this reactivity became weak and seemed to be present only between 
 merozoites in the mature schizonts ( fig .
reactivity of promature eimeripain was similar to 
 that of the second generation , and this enzyme was observed as dots in the cytoplasm .
mature 
 eimeripain was present as dots in the cytoplasm of small immature third schizonts and 
 observed over the entire cytoplasm of large immature ones ( fig .
5 g and 5h ) , but disappeared in mature third schizonts ( data not 
 shown ) .
immunofluorescent staining of asexual stages , the second generation schizonts ( a ) and 
 the third generation schizonts ( b ) with antisera against promature ( upper photos ) and 
 mature ( bottom photos ) eimeripain .
figures a and d are small immature schizonts of the 
 second generation , b and e are large immature schizonts , and c and f are mature 
 schizonts .
arrowheads and arrows 
 show smaller immature schizonts and larger immature ones of the third generation in 
 fig .
endogenous localization during the sexual stage : at the sexual stage , 
 reactivities were found to be similar between promature and mature eimeripain ( fig .
6.immunofluorescent staining of sexual stage with antibodies against promature ( a ) and 
 mature ( b ) eimeripain .
arrowheads , arrows and asterisks show macro - gametocytes with a 
 prominent wall forming body , micro - gametocytes and zygotes , respectively .
scale bar is 10 m . ) and different from those of the asexual stages described above .
both types of 
 eimeripain were observed in the wall - forming bodies and cytoplasm of immature 
 macro - gametocytes , only in the wall - forming bodies of mature macro - gametocytes , and across 
 the cytoplasm of micro - gametocytes . in zygotes signals were present over the entire 
 cytoplasm , except in the nuclei . at the oocyst formation stage , the final step of sexual 
 development , which is seen as a distorted shape , eimeripain was weakly observed in the 
 cytoplasm around the inner oocyst wall as spots ( fig .
immunofluorescent staining of sexual stage with antibodies against promature ( a ) and 
 mature ( b ) eimeripain .
arrowheads , arrows and asterisks show macro - gametocytes with a 
 prominent wall forming body , micro - gametocytes and zygotes , respectively .
eimeripain has been proposed as a candidate drug target to be exploited against chicken 
 coccidiosis in the future [ 8 , 10 ] . in those studies ,
a panel of inhibitors was tested against the 
 enzyme and three new inhibitors were identified . 

moreover , it was confirmed that eimeripain was expressed in the extracellular stage during 
 sporulation ; however , no other life cycle stage 
 was examined for its expression so far . in the present study , we generated two antisera 
 against eimeripain ; one recognized promature eimeripain , while the other recognized 
 eimeripain by western blotting . as a result
, the eimeripain was detected as protein of 
 promature at examined in all examined stages of sporulated oocysts , and purified 
 sporozoites , merozoites and schizonts . while , mature protein was identified as 33 kda 
 molecule only at sporulated oocysts and schizonts , but not sporozoites and merozoites . 

probably , the epitope recognized by anti - peptide sera against eimeripain mature domain might 
 be masked by the modification of carbohydrate chain or protein folding in promature 
 eimeripain before maturation .
interestingly , this polyclonal antibody also identified a 54 
 kda band in schizonts , but did not react with sporozoites or merozoites .
there is a 
 possibility that purified schizonts represent many developmental stages from immature to 
 mature second - generation schizonts , and thus , mature eimeripain of 54 kda might be contained 
 before complete maturation .
these results suggest that mature eimeripain do not exist in 
 invasive zoites , but only in sporulated oocysts and developmental schizonts . by indirect immunofluorescence assay ,
the reactivities of promature eimeripain in 
 sporozoites and merozoites were similar to that seen in transfected sporozoites , as 
 previously reported , but mature eimeripain was 
 not .
in intracellular developmental stages , the promature enzyme was detected in the 
 cytoplasm of early schizonts of the second and third generations , but disappeared during 
 their respective developments . meanwhile , mature protein was present in the entire 
 cytoplasm , but later was not detected .
thus , the promature protein is present as dots in the 
 early asexual schizonts and probably sporozoites and merozoites as well , and active 
 eimeripain might spread throughout the cytoplasm during development or differentiation of 
 schizonts .
these findings tempt speculation that eimeripain might play a key role in the 
 differentiation of intracellular zoites in the ceca in addition to the extracellular 
 stage .
cathepsins , which are cysteine proteases related to papain - like enzymes ( clan ca , family 
 c1 ) , are major virulence factors expressed by apicomplexan parasites [ 1 , 3 , 11 ] .
for instance , in toxoplasma gondii , antisense or 
 inhibitors of cathepsin b can block the invasion of tachyzoites into host cells and cause 
 abnormal rhoptry morphology . in higher eukaryotic 
 cells ,
however , the lysosomal system of e. tenella , like that of 
 toxoplasma gondii , has not been characterized . in our study ,
eimeripain 
 was expressed in all examined intracellular stages at both mrna and protein levels .
although 
 more detailed functional characteristics remain to be analyzed , this enzyme might correlate 
 with asexual and sexual development with a fundamental biological function .
therefore , the 
 results reported here offer evidence that compounds of inhibitors could be effective 
 candidates for novel drug discovery against chicken coccidiosis .
 abstracthemorrhagic diarrhea in 
 poultry is caused by eimeria tenella , the most pathogenic avian coccidian 
 parasite , and new approaches to treat the disease are continually being sought . 
 although 
 eimeripain , a cathepsin b - like cysteine protease from e. tenella , has 
 recently been identified as a novel anticoccidial drug target , its localization during the 
 intracellular development of parasites remains unclear . 
 here , we demonstrate the 
 expression of eimeripain during asexual and sexual development of e. tenella in 
 vivo . 
 promature eimeripain was detected only in the early immature second 
 generation of schizonts . 
 in contrast , the mature eimeripain was most strongly detected in 
 the middle - sized immature second generation of schizonts . 
 both promature and mature 
 eimeripain disappeared depending on the maturation level of second generation of 
 schizonts , but were strongly expressed again in the third generation of schizonts . in the 
 sexual stage , 
 both promature and mature eimeripain were detected in the cytoplasm of 
 micro- and macro - gametocytes and zygotes , but expression became weak in zoites forming 
 oocysts . 
 collectively , our findings suggest that eimeripain might play a key role in the 
 differentiation of intracellular zoites in the ceca and could be an interesting candidate 
 to develop a novel , effective anti - coccidian drug . 