GENETICS Ii Gene expression profiles of two B-complex disparate, genetically inbred Fayoumi chicken lines that differ in susceptibility to Eimeria maxima
D. K. Kinl.* C. H. Kiin.t S. .T. Lanont4 C. L. Keeler .Jr.. and H. S. Lillehoj*I
AnzTnai Parasitic Disases Laboratory. Animal and i'vaturai Re,soaices 1i,.t,tatc, 115124, l3e1tsi ;,iie. AID 20705: tKorcan Biom formation Center. Korea Research Ins tit etc of Bioscience and Biotechnoloqy. Doe jeoi. 305-806. Republic of Korea: .f Department of Animal Science. iowa State Uiwersity, Arries 50011: ii o.dI)cparfmrrenf of Aiwaai and Food Sciences. (ni ic csitq of Delaware. Newark 19716
ABSTRACT This stud y Was conducted to compare the clown), and 92 (33 up. 59 down) mnRNA at the 3 time gene ixprussioli profiles, after Emmn crma maxima infection. points. Functional anal ysis using gene ontology catego- between 2 B-complex congcmc lines of Favonnui chick- rized the genes exhibiting the different expression pat- (-'115 that display differeiicis in disease resistance and terns between 2 chicken lines into several gene ontology innate immunity against avian coccidiosis using cD\A terms including imniunlity and defense. In summary, inicroarrav. When compared with uninfected controls transcriptional profiles showed that more gene expres- using a cutoff of >2.0-fold alteration (P < 0.05), l\I5.1 sion changes occurred with E. maxima infection in the demonstrated altered expression of I (downregulate(l). M15.2 than the A15.1 line. The roost gene expression 12 (6 up. 6 down). and 18 (5 up. 13 clown) infINA at 3. (liffmeices between the 2 chicken lines were exhibited 4. and 5 (II postiuifection. respectivel y. In the M15.2 line, at (1 4 and 5 after E. maxima infection. These results altered expression was observed iii 6 (3 up. 3 (lo\vn). demonstrate that differential gene expression patterns 29 (11 up. 18 down), and 32 (8 up. 24 clown) tran- associated wit,li the host, genetic clifkrence in coccicli- scripts at the 3 time points, compared with uninfected osis resistance provide insights into the host protective controls. Comparison of the expression levels between immune mechanisms and present a rational basis to [5.1 and N115.2 chickens after K. mo:rTrna infection r(, target specific genes and gene products to bolster host vealed alterations in 32 (10 up, 22 down). 98 (13 up. 55 defenses against avian coccidiosis. Key words: coccidiosis, Favounii. complementary DNA niicroarra y, intraepithelial lymphocyte 2009 Poult rv Science 88:1515-1579 (Ion 10.3382/1)s.2009-000 12
INTRODUCTION about the chicken immune sYstein at the molecular level is very limited. Avian coecidiosis is caused 1w infection uvif h pro- lii recent, years, t he chicken EST database has tozoa belonging to the genus Eimecw, amid presents a reached such a. level that anal ysis of the sequence data significant concern to the poultry industry due to its is feasible. There are now more than 580.000 chicken detrimental influence oil efficienc y affecting EST sequences front a wide variet y of tissues aiid dc- survivabilit y, nutrient absorption, growth rate, and egg velopniental stages publicly available (hmttp://www.tigi'. productioii of poultr y (Lill(,hoj and Li, 2004: Lillehoj ci org/tclh/t.(, i/). Therefore. it is now possible to ascribe al.. 2007). Until now, disease control strategies against provisional identities and functions I o genes on the ba- avian coccidiosis have mostl y depended oil prophylactic sis of sequence comparison. High-throughput gemlomn- chemotherapy and vaccination. but both methods have ic analyses have suggested a path toward increasing serious drawbacks (Alin et al. 2005). Genetic selection ideuitificat.ion of transcriptional regulations involved in for disease resistance and iiiiinune responses call time inimnune response (Degen et. al.. 2006). Large-scale to a reduction of drug use and risk of vaccination ill gene expression profiles of host responses to infection commercial poultry production. Hoivever, information could provide enonuuous information oil the iuuteractiomms bet veemm pathogens and the host imnmunie systeni. Time mnicroarray techmniqume is all tool for char- acterizing the biology of inminmumologic processes amid 20(]9 Poult,jv Scicncv A,sociat,ioii un. inunnie-relat ed diseases (van dee Pouw Kraa.n et al.. Received Ja in mv 9. 2(11)9. 111 Accepted April 1-1. 21109. 2004). chicken, several mnicroarm'ay studies have been (:orr('po1ahiu5 author: Hv till. LiIhlioj ii.iiIa.go\ performed for the infect ioums diseases such as March's
1565
1566 i'INi ET AL disease (Levy et al.. 2005: Sarsoii et al.. 2006: Heidari et RNA Extraction and Aminoallyl-Labeled al.. 2008: Sarson et al., 2008). West, Nile virus (Groves RNA Preparation et al., 2008). Salmonella (van Hemert et al., 2006. 2007). avian influenza virus (Degen et al., 2006: Groves et al.. After ent.iiamzation of time birds, jejunal intestines 2008: Zaffuto et al., 2008). avian infectious bronchitis were taken from 5 birds in each infected or noninfected virus (Dar et al., 2005). and Eimeria species (Niiii et group at O. 3. 4. and 5 (1 postinfection (dpi). Intestines al., 2003). were cut longitudinall y and washed 3 1 inies with ice- The Favoumi breed, which originated in Egypt, was cold Hanks' balanced salt solution coiit aimming 100 U/ imported to the United States in 1954 primarily be- mL of penicillin and 100 nmg/riL of streptomycin (5mg- cause of its reported resistance to the avian leukosis. nma. St. Louis. MO). The mucosal layer was carefully Subsequently, it was shown to develop a robust pro- scraped using a surgical scalpel and TEL were isolated tective response against Eimeria, tenella (Pinard-Van from the jejununi as described previously (Lee et al.. Der Laan et al., 1998). Derived from the original Fay- 2007). Total RNA was isolated from a constant number oumi breed, the M5.1 and M15.2 congenic pair lines are of TEL (5.0 x 10) using Trizol (Invitrogen. Carlsbad. highly inbred and geneticall y distant from broiler and CA) arid purified using the RNeasv Mini RNA Puri- Leghorn lines. They share all genetic back- fication Kit (Qiagen, Valencia, CA: \Vu et al.. 2004). ground but differ in the haplotype on chromosome 16 In preliminary experiments, we determined I hat equal carrying the MHC (Zhou and Lamont. 1999, 2003). In cell numbers and equal anmoimuts of total RNA were a previous study, we compared the typical disease pa- obtained per umut volume of gut mnmncosa. (L. K. Kim, rameters of avian coccidiosis. BW gain and fecal ooevst unpublished data). shedding, and transcriptional expression of cytokine Time airuinoahlyl-labeled RNA front IEL from genes between the Fa, voumi M5.1 and M15.2 lilies af- each group was prepared using the Amino Ahlvl Mes- ter oral infection with Eimeria maxima oocysts. The sage Amp 11 aRNA Amplification Kit (t Hoen et al., results demonstrated that line M5.1 is more resistant 2003) according to time protocol of the manufacturer against E. maxima than M15.2 and showed differential (Ainbion. Austin. TX). Briefl y, first strand eDNA was expression of immune-related cytokinies between the 2 prepared by reverse transcription from 2.0 gg of RNA genetically disparate chicken lines after infection. using a modified oligo-dT primer containing a 17 RNA The present study was conducted to compare gene polymerase binding site on the 5' end. followed b y see- expression profiles between these 2 chicken congenic ond strand eDNA synthesis. The double-stranded cDNA hues, which show a difference in disease resistance to gemmerated from first and second cDNA synthesis were coccidiosis, using our avian intestinal intraepithelial transcribed to arninoallyl-labeled RNA using T7 RNA lymphocyte eDNA nncroarray (AVIELA: Kim et al.. pohymerase with amiuioahl yl-iiridine triphosphate. Two 2008). The AVIELA is specific for mucosal transcrip- 20-gg aliquots of each aminoallvl-R NA sample were flu- tional investigations including imnnune response and oresceutly labeled with Alexa Fluor 555 or Alexa Fluor was constructed using an intraepithelial lymphocyte 647 (Invitrogeii) according to the instructions of time (TEL) eDNA library of Eimeria-infected chickens (Min manufacturer and labeled RNA were colunum-purified et al. 2005). Because TEL are the primarimmune cf- using the RNA Amplification Kit. (Ambion; 't. Hoemm lector cells in the gut and play a critical role in eliciting et al.. 2003). Concentration and labeling efficiencies of protective immunity to enteric pathogens, the results RNA were determined spectrophotonietricallv. contribute to comprehensive understanding of the in- nate immune responses in the chicken gut stimulated Microarray Hybridization by E. maxima infection. l3uthi of I lie RNA, labeled with 2 different fluores- MATERIALS AND METHODS cent d yes, from the mmoiiinfected control birds and the treated birds were hybridized to the AVIELA nnicroar- Birds and Experimental Eimeria Infection ray. Time AVIELA was constructed with 10.162 spot dc- meuits from the previously prepared TEL clones (Min et Chicks were bred and nmaint ainccl in the Poultry Ge- al.. 2005) and the imnuiune-related genes from hipopol- netics Program at Iowa State University (Ames). All saccharide (LPS )-activated macrophage (HD 11) eDNA matings were carried out by artificial insemination to library (Mimi et al.. 2003) as well as time direct PCR ensure pedigree accuracy and fl-complex genotypes clones of several cvtokimies and chmeniokiues (Min et al.. were confirmed by serological testing. Day-old chicks 2005). were air-shipped to the Animal and Natural Resources A total of 12 microarrays were used in this study. Ac- Institute. Chickens either remained uninfected or were cording to the reference design (McShane et al., 2003) orally inoculated at 4 wk of age with 1.0 x 104 sporu- with dye swap, 4 values were obtained for each time lated oocvsts/chicken of E. maxima. All experiments point (3 1 4. and 5 dpi) in a chicken line, 2 on I slide were approved by the Animal and Natural Resources and 2 on the dye-swapped slide because all elements Institute Institutional Animal Care and Use Commit- are duplicated oil AVIELA. Each treatment group tee. was normalized with the noninfected control of the
M1CH ARRAY ANALYSIS OF EIItIERIA INFECTION IN CHICKEN LINES 1567 same line. Two-color nhicroarrav It vbrklizat.ions were performed using Hyblt, hybridization buffer (Telechein Cl Cl International. Sunnyvale, CA) in Arravit reaction cas- settes (Telechem International) at 50°C overni ght. Af- ter Itybridization. the slides were rinsed in 0.5 x saline sodium citrate (SSC), 0.01% SDS at room tempera- ture, and washed once for 15 tiun in 0.2 x SSC. 0.2% SDS at 50°C. 3 times for 1 nun in 0.2 x SSC at room x - temperature, and 3 times for 1 nfin in distilled water at room temperature. Each sample had a- repeated It brid-
s izal ion using the alternate fluorescent (ic between the infected treatiiiemit amid immuiiilectcd control.
Microarray Scanning and Image Analysis — Images were acquired by laser confocal scanning using a ScanArrav Lit e microarray analysis s steni (Perkin- Elmer, Boston, MA) at a resolnt iou of 10 gnu. A 16-hit HH TIFF inlage was generated for each channel (Alexa Flit- Or 553 and Alexa Fluor 647). The scam imied iiiicroarrav iagesi) l for each cliaiinel were overlaid and quantified HH 10 5 0 U) w cc C, C) 0 o 10 z 15 2U 25 30 Figure 1. Nitoiber of transcripts oil avian intestinal intraepithelial lymphoc y te (DNA lmmicroarrav (AVIELA) showing the alteration of the expression levels in M5.1 and M15.2 lines with >2.0-fold changes after Euoer,a rna:elrna infect ion compared with uninfected controls. dpi = days post ill tect ion. Bioinformatic Analysis portion of these sequences have not defined their func- tion in chicken vet. The mapped gene ID were used for All sequence data files were ol:taiuccl from National analyses of pathway classification from thethe PANTHER Center for Biotechnology Information (NCBI). The database (http://www.pantherdb.org ). Having annota- TEL cDNA elements used to create the 1EL eDNA mi- tion derived from orthologous human genes means that croarray were mapped to the chicken genuine reference cross-species comparisons between chicken and human assembly (version 2.1) and reference RNA and protein array data niav be possible (Smith et al., 2006). sequences (formatted database for BLAST. May 2006) using NCBI BLAST (version 2.2.13). The criteria for Quantitative Reverse Transcription-PCR the acceptance of BLAST search results were alignment length >100 nucleotides and c-value 1e for DNA To confirm gene expression changes observed by mi- sequences and alignment length >30 amino acids and croarray analsis. quantitative real-time. (QRT- c-value Ic for protein sequences. Entrez gene data PCR) was performed as described (Hong et al.. 2006). and Homologene data (October 2007) from NCBI were Equivalent amounts of same RNA sauiples used for mi- used to gather gene information [identification num- croarray hybridizations were reverse-transcribed using ber (ID), symbol. anti name]. Gene Ontology (GO) the StrataScript first strand synthesis system (Strata.- annotations were extracted from gene to GO data in gene. La Jolla. CA). Amplification and detection were NCBI and 7.878 elements were annotated iii a total of carried out with the M0000P system and Brilliant 10,162 spots on AVIELA. To anal ysis pathway informa- SYBR Green QBT-PCH master mix (Stratagene). tion. chicken Entrez gene ID >2.0-fold differentially ex- Standard curves were generated using base-10 loga- pressed between M5.1 and M15.2 were mapped to Ho- rithin-diluted standard RNA and levels of individual mologene ID (locus link ID) for human because a large transcripts were normalized to those of glyceraldehyde \ITCROARRAY ANALYSIS OF EIMERL4 INFECTION IN CHICKEN LINES 1569 3-phosphate (lehivdrogenase b y the Q-gene prograili and identified with comninoli gene names mapped using (Muller et al.. 2002). human gene ID are listed in Table 2 and 3. The nuni- For the calculation of fold changes between control bers of the genes annotated aniong >2.0401d altered and treated group. the. normalized c ycle threshold val- ones are 1. 8, and 17 in \I3.1 and 3, 24. and 22 in M13.2 ues of target gene to glvceraldehvde 3-pliospliat e (IeliV- at each times point. respectively. drogeiiase were calibrated to time relevant, control line Comparing birds of line M5.1 with N11.5.2 after E. values. Oligonucleot.ide primers for QJIT-PCI{ are listed ira-name infection revealed differential expression in in Table 1. Each uuualvsis was perfoimned in triplicate. 32 (10 upregulated. 22 dowuregulated ) . 98 (43 up. 55 down), and 92 (33 up. 59 down) inRNA. using a cut off of >2.0401d at time 3 time points (Figure 2). The >2.0- RESULTS fold altered genes between the 2 lines at 3 time points Gene Expression Profile Induced exannned are shown in Table 4. 5. and 6. respectively. by E. maxima Infection Gene Ontology Annotation In this stud ' v, differential expression levels of tram!- and Bioinformatics Analysis scripts causedby E. rruuima infection in intestinal lET. of M5.1 and N115.2 line chickens were analyzed using The elements that corresponded to In unan gene II) AVTELA nucroarrav. and that were differentiall y modulated >2.040ld be- When compared with tuilinle('t ud controls and using tween lilies N15.1 and 1\115.2 at any of the time points a cutoff of >2.0-fohl differential expression between after E. ma.iiina infection were distributed iii the infected and iioiuiifected. M5.1 (leummolistratecl altered branch of biological process according to the GO index expression of I (downregulated), 12 (6 up. 6 down). using the PANTHER databases (Mi et al.. 2003). In the and 18 (5 up. 13 down) mRNA at 3. 4. and 5 dpi. branches of biological process, two of the largest parts respect ivelv. In the case of the M15.2 line, altered ex- were involved in the categories of protein metabolism pression was observed in 6 (3 up. 3 down). 29 (11 up. and modification (14 up. 1 I down), and nucleoside. nu- 18 down). and 32 (8 up. 2-1 down) transcripts at. the 3 cleotide. and nucleic acid metabolism (9 lij), a down) in tiiiie points compared with uninfected controls (Figure both up- and downregulated genes iii i\ 15.1 compared 1). The genes with differential expression after infection with M15.2 (Figure 3). Generally, a larger number of Table 2. The di Hire Iii dlv cx )ri 'eui 1-cla's iil nt ifie I u i III(' correal Xliii lei it i( lent liv iianie of liii iiiafl gC'iiea ii ft cr Eu 711 (. PU) maxima(1 iitectioii in Pavoiiiii.i N15.1 hun' compared \\'il ii iiuiinfected controls I '2.O-foIcl ('liaiiges) (1eiiBank no. N01-11iaIi7ec I ratio I'-valoe (a'nc syitihol' 3 d posi infect ion CFO 749 19 0.3 0.03 11A52 Ilvaliiroiiati ant lasi' 2 1 d post infection C0738831 9.7 (liii PUANII P In isplioglvcerate nail oar 1 (brain) UD720890 9.6 0.115 Nit AS .\e, irohlast.oiiia HAS viral (v-rita I uncogelle lioniolog (1075017 3.11 0.0:t 11.1311 A I I nterleukiu i 3 receptor. is 1 C'D732030 2.1 0.0 1) L0C'423827 Similar to TUBA (117075109 0.1 (1.1)1 LOC4 19615 Splicing factor. argunne/seriuie-ricli 3 ('1)7:19019 0.3 0.1)3 QFRTD 1 Qucu0 ic I H NA-ril,osvltransferaac (lonlain containing I ('0728022 0.3 0.03 LOC'423895 Similar to glycerol-3-pliosphatc acvllriitisfeu'ase, iiiitocliotiilrial CD736338 0.3 002 A COAl Apohipoprotciii A-1\' 5 0 postiuifu'et it)ii ('0736627 11.0 0.01 RARIIESI iieti,oic acid receptor responder (tazarotene induced) ('F07501 7 4.3 0.01 ELi 311A I Interlenkin 13 receptor. (1 1 CD737602 3.2 0.03 PPP3CB Protci ii pliosphat ase 3 ( forii ierlv 213) catal ytic subunit 3 isoforni CD73761 3 2.8 0.01 LO( '7711135 Similar to smallest subunit of tuliiqninol-cvt.ocliroioe c ri'iliuctase (1075075 2.4 0.03 E1F.111 Fiukarvot ii' translation initiation fact or Ill C'F0751 09 0.5 0.0:1 LOCI 198 1 Splicing factor. arginine/serine-rich :1 CD73 1060 11.5 0.02 1.0(769871 Similar to transcriptional adaptor I (1 EEl 1 hoinolog. yeast )-like CF'07 1926 0.5 11.02 L0('777308 Similar to eukarvotic translation lInt iat.iolu factor 3 1) 12 subunit CF075105 0-1 0.11.1 [.0(719210 Similar to ribosomal prot ciii 59 CD740249 0.4 0.04 L0('4 m 9391) Similar to cnfianui'r of split-related prot vin-7 CF075134 0.1 11.03 FYN I'YN oiicogeu(i' related to SIIC'. FUR. YES C'D737563 0.3 0.05 L0('42345l) 11vpotlietiil L()C.123l511 CD728772 0.3 (1.1)1 ('hro. 40465 (93 1766 1(1 \ I ,-clocken intestinal lvniplioeyte Callus qalfiis cDNA clone IGAL_60E0 7 5'. niRNA sequence ('D729710 0.2 0.03 TSR1 TSR 1, 208 nt N A flCii ill ii il,ut ion. lionuolog ( Sacc/oi.romyces (('It., '(51(1(.) CD729636 0.2 0.01 (.-fps Similar to c-fps proto 01 (ogeni' CD729635 0.2 0.0.1 KCN.132 Pot assitnii voltage-gated channel. sI,,,lo'r-n'lat ''l su)family. 13 imui'irilx'r 2 ('D7399. 1 11.2 0.03 LOCI 19 127 Similar to HlltFN eDNA 9.130))I5GI)) Gene names for liii o ian mapped liv chicken Li it rez gene ident i (hat ion ninnbers. I 7() KIAI El AL. Table 3. The differentiall y expressed genes identified using the correspondent identity name of lllinlaii genes after Eiiuera maxitna infect ion in FaYoumi M15.2 line compared with iii liii ft'ei ed controls ( >2.0-fold changes) GcnB,m,k ',orimmalized rat me I '-you me Gene svimml,ol Deaiiit ioll 1 (I iiilc,t i, I CF07470! 2.8 (1.03 TNI4COI_3 '1 rinimeli' (ide repeat umitmuilig, (ill CF074848 2.3 0.03 RC.'.JMI9I l ._lkll( Similar( .0 1(asGAP-,,ctivm,m,p-Iike prote imm 1 CD733250 2.0 0.1)3 ANKRDIO Ankvrin repeat domain 10 ('0740095 0.5 0.02 LOC 1837 18 Similar to Finkel-Biskis-f(ejll y mnurine sarcoma virus ubiquitously expressed CD729 173 0.5 0.02 IP1F Peptidvlprolvl isomnerase 1" (cvclnplmilimi F) 4 d liostinfectioii CF074761 ((.0! TNR COB Trinucleot itO' repeat containing GB ('0733216 0.00 L0C426427 Similar to N1AA0556 protein CD733250 ((((2 ANKTI D10 At k yrt n repeat tlonmain 10 ('F071943 0.03 LOCT7((561 ArcImaimi I CD728893 ((.01 R .A SSF2 ((as Ossocma( ioii (I) alCDS/AF-G) domain famoil v 2 (0738017 ().05 L0C42242 I Similar to F1..J38482 protein CD738198 0.04 1.00770216 Adaptor-related protein complex 2, o I subunit. CF074848 0114 JIC.1MR04_I k19 Similar to RasC1AP-activating-like protein 1 CF074844 0.04 H P85 Rihosomal pi'o( 'mn S5 CD729 173 0.01 PPIF Peptidvlprolvl isomimerase F (cvclopliilin F) ('0 735567 ((.03 PIGX Pliosphatidvli ni sit ol glycan anchor I.aosvnthesjs. class X CD 736593 ((.03 LOC 776552 Similar to MADM I CD 7126866 ((.00 1.0(115975 Similar to hrelel,l,mm-resistammt Arf-G F F' 2h CD733075 0.11 Z\\ 10 ZW 0, kinemochome associated. Immammolog (Drosophila) CD736:356 (1.03 LO( 122525 Similar to 1IINEN cDXA 2:1100085110 CD731 124 0.0.1 BDNF Brain-derived nemmrotrophic factor ('1)734663 0.03 CDKSRAP2 '0 KS regtmlatorv , mbummit associated protein 2 CD 72 890 1 0.115 3F1 Bru Ion agaimmmaglohi mlii ci mini t rod mm' kinase C 07-Il 1.18.1 0.112 ADIIOI)R2 Adip mo:ctimi reccp( or 2 CD 738626 0.02 ('1)30 CD36 molecule ft hm'omimhospommdimm receptor) CD738977 0.01 L0C4266 14 Similar to N1AAI8I5 protein CD7350 (3 0.03 1.0(115791 Similar to carhommic amilmulrase \'II CD 732 103 0.01 RCJ\1004_30g1 7 Similar (o ,l-1,cxosarmminidase a chain precursor (N-imcet0_:3_ glucosaimmimiidase ) ( I-N-acetvll)cxosammmimmidase) (hexos,miiimm i,l ,ism' ('1)73.1891 0.3 0.02 Ilad23a B AD23,i lmnmmmolng ( Sacc/totrimti.ijccs ccrem.'itdo,c) S d post infect ion CD738017 2.6 0.02 L0( '.02121 Similar to F1...138482 protein CF074761 2.5 0.03 TNHCOR 'I'rimnmcleotide repeat containing GB ('F071848 2.5 0.02 l(C.JSIB0I_11c19 Similar to RasCAP-activating-like protein 1 CD737969 2.5 0.03 TCEH(41 Transcription elongation regulator I ('0740789 2.3 0.01 LOC•120075 Similar to KIAA0096 CD734282 2.2 0.05 L0C42.1735 Siimmilam to S'I'AG 1 varimmnt protein CD 1-281 1 .1 2.1 ((.02 L0C42309 1 Sin, miar to transient receptor potential m'ation cliammnel. subfannlv M. mmmemmiher 5 CFO7 1926 0.01 L( (C 777308 Similar to eukarvotmc translation initiation factor 3 p42 subunit, CD726837 0.05 IA)C4 18899 Similar to regulator factor X-associated protein CD736696 ((.03 TXNL5 [hioredoxnm -likc 5 ("D731350 ((.00 HCP9 C'alcitonmmm gene-related peptide-receptor component protein ("D73.5013 (1.03 LCC4! 5791 Similar to carhum,ic a.mmhvdrase VI! CD733203 ((.0.1 LOC 116956 Simrmilar (a KIAA0G. 15 pro) elmi CD 72) 173 ((.01 PPIF PeptmdvllmmolvI isommmerase F (cvclophilimm F) CD73.1801 ((.11.1 11am 123a l'fA D23a homt clog (S. cr...ct'isiuc) CD733404 (1.02 LOC 11)8(147 Tlivmmiosiii 3 4 CD728880 (((1.5 I-'TCD3 Pemmtatricopept dc repeat dommm,m i mm 3 C 1.) 736593 0.01 L00776552 Similar to .\IADSIL CD728901 0.01 lIEN Bruton aga.nmmmma tyrosine,glohtmhmieimmia kina.se CD735567 0.00 PICuX Plmospliatidvlimiosit.ol glvcaa anchor bios ynthesis, class X CD738626 ((1)1) ('l.)30 CD36 moleci Ic (tImmombospondin receptor) CD 732 103 0.01 l(C'.JNll(o l....3((gli Similar to I-hexosamninirlaoe a chiumm precursor (N-acetyl-3_ 11tmm'osamamtmiilisc ) ( 1-N-acetv!hexosam'nini(iase) (lmexosaimmimmidasm' A) 'Gene names for human ,,m,pped (a chicken Emitres gene i,Ient,itium,tioti m,utulmcrs. genes upregulated in M5.1 (M5.1 > M15.2) were classi- (M5. 1 > A115.2) and downregulatccl (1\15. 1 < M15.2) fied in each GO index than downregulated genes (1\15.1 genes in M5.1. < 1\115.2) except apoptosis and electron transport. In In the GO classification for the >2.0-fold altered the 3 terms of GO, cell structure and motility (8 up, genes between the M5.1 and M15.2 lines. 7 transcripts 1 down), intracellular protein traffic (5 ill). 1 down), are grouping into the category of immunity and de- and signal transduction (12 up, 3 down), the consider- fense. Among those genes, 4 upregulat ed genes includ- able differences were shown between the number of up- ing growth factor receptor-hound protein 2 (GRB2), MTCROARRAY ANALYSIS OF EJMERIA INFECTION IN CHICKEN LINES 1,571 60 40 20 U) w C w a) 'I- 0 d 20 z 40 60 80 3dpi 4dpi 5dpi Figure 2. Nninlar of transcripts oil tlti: avni intestinal iitraep,thelial Ivntphocvtu ,DNA tt,iiroarrav r\\'IEJ..\ I sliioving tin alter,ttiot, of the expression levels between MS. 1 and Ml 5.2 lines ivit I >2.0-fold changes alter Liot ru' flier, 100 infection. dpi = days posthifeptioii. Table 4. The annotated genes altered with >2.0 changes between Favoutni M5.1 and M15.2 lines al 3 d Post Eiirtr Jut (tlU.i'lJit(t itift'e- tioti (P < 0.01) (;enB,,nk no. 5,(I,,,,li,,'(l ratio (etje SVttii)Ol Desci'ipt 0], 115.1 1115.2 CD726!17 J.00770701 Reich- like 25 Dtosu1,/, do i ('Fo7sfr I (IAPDH Glvceralilelivdi'-3-pI ii ispi ate ileliilriigenase ('1)7:36911 A1)IPOR I Adiponecti u receptor 1 CD 72Sa-l7 TTL Itibulin tvros, e I igaae ('1)7:355!! LO('421 -1-17 Similar to Ephx I pi'oteiIi CD 7126890 NI) AS Neitroblastonta RAS viral (v-ma) oncogene liotnolog P1)729173 PPIF I 'ept idviprolvl isonlera,se F ( cvclophulin F) ('D736353 L()C.119543 Similar to dehvdroclolicl vi dipliosphate synt base CFO7 19:33 PP l'2 F! SC Protein phospliat asi' 2. regulatory subunit B'. isoforin (D7:16260 CRB2 Growth factor receptor-hoi nid protein 2 (D737331 1)l245stt Similar to liv pot bet ical protein FL.i 10597 CF075002 RPLS Ribosomal Protein L5 CF075075 ElF) H Eukaryotic translation initiation factor 411 CD729060 MPST llercaptopvruvate stil furt no isferase CD71D 122 P1.EK1lll I F'leckstr,n homology doniain containing. famil y M (wit ii RUN don,aiii ) member 1 CD 7:30633 ZWTL('H Zvilch. kinet ocliorc associa ted. honiolog (Drosophila) ('1)73109:3 I ,O(426882 Similar to SMARCD I protein 115.1 < M15.2 CD733216 —2.1 J,OCl26427 Similar to KIAA0556 protein ('D735777 —2.2 LO('42-I065 FIvpoI hetical LOC•421065 CF075058 —2.1 LOP] 16900 Similar to \ISTI1028 CD729397 —2.2 L0C415957 Similar to ItIKEN eDNA 1110007C09 C'D733805 —2.3 EBEG Epiregulim, CD7281lG —2.6 PFL_4320 GUDEI" domain protein CF07.19211 —3.0 RPL21 Bihosnmtnd protein L21 CD729636 —3.8 c-fps Similar to c-fps proto ommi'iiguiie 'Gene miames for lotmmiami mapped liv chicken Em,tre'i gene identification numbers. .5 721 NL\l El' AL. Table 5. The annotated genes altered with >2.0 changes between Fa outni M5.1 and M15.2 lines at 4 d post Eimerui rnaxsnw infec- tion (P < 0.01) Normalized Genl3auk no. ratio Gene svnibol Description II> 1 > M15.2 (1)734677 lISPS-I !_ hiquain specific peptidase 54 1)726897 1,0(770701 Kelch-like 25 (Drosophila) '1)737602 1' P1 '3CB Protein pliosphatase 3 (formerl y 2B) catal ytic submit. 3 isoform ('1)7:17041 LCPI Lyio j ilioc.t'tc' cytosohc pr()tm-'in 1 (L-plastin) ('1)7:18026 (:D36 (.1)30 molecule ( tliromm ibospondin receptor) 1)739823 L0C42i4ll2 Similar to adenosine t riphospliate-hinding cassette subfamily (1 nmcmmmher 8 D737778 1.0(4237311 ('at echol- 0-met liy!transfera.se (1010010 containing 1 1)736563 PPP2tI'A Protein phosphatase 2 (formerly 2A) . ca)a!vtic subunit. cm isoforni ('I )735620 RCS2 Regulator of C-protein signaling 2. 24kDa 1)736352 DNSI 1 L Dynammin Mike ( 1737576 HPRI I Hvpoxaiithine phosphoribosylt.ransferase 1 1)735718 LOt '4278:18 liv pot hetical LOC427838 1)735496 A N K!) D13 Ankvrin repeat domain 43 D735615 [1 L5111 Bleoiiiycimi hvdrolase 1)710791 LOC4S1 656 Similar to potential phosplmolipmd-transporung ATPase identification number (ATP;ise class I type 813 member 2) (iimi,s ge/his) 2.,1, (fl'!:) Gene trap locus :3 (mouse 1)735377 2.7 CO ()it.'a'iu kinase 1. 1 (1)737186 2.7 FItSIDIB F 111151 domain containing 41.3 ('0729722 2.1) L0C115075 Similar to brefeldin-resistant ArI-CEF 21) CD 726866 T(P2A lopoisoiiierase (DNA) II rs 170k Da CD729219 2.1) 2.5 C130052I12RTK1CLPTMI!. CLP'I'5l 1-like ('1)737494 1.CC4 16833 Similar to MC('5.0999 protein CD727217 23 Rad23a I) .5D2:ba lo)Inolog ( Sucr/>ai'o'rr>yces cereuisiae) CD 7:3489 1 2.3 R.'\B 10 Il_SB It). member RAS oncogene family CD739023 2.3 2.3 I.()('121F39 Similar to V-ATPase C2 subunit CD728308 ('RYI. I 'rvst dliii. X I CD738543 2.2 CF075038 2.2 DDXG 1) EAT) (Asp-GIn-Ala-Asp) box polvpeptide 6 2. J.0C423737 Similar to Ca2 '> /cahmuxlulin-depcndeta protein kinase (EC 2.7.1.123) 11 -E - bunion CD737673 2.! 1-()C.11615.4 Similar to intestinal 15 kDa protein: I-lIP CD7293.12 CD7373 I-i 2.1 LOC.124093 Similar to M0(1,83858 protein 2.1 SCYL2 SCY 1-like 2 (S. cer(viswe) CID 729537. 2.1 LOC4 1877.1 Similar to propionyl-eoenzynie A carhoxvlsam, n polypept.ide CD736595 CD731530 2.0 L()('121055 Similar to hand 4. I-like protein 3 (4.113) (differentiall y expressed iii adenocarcumouia of the lung protein 1) (DAL-1) 515.1 < 5115.2 CD732036 -2.0 SAF.2 Sf15101 activating enzyme subunit 2 ('D7:31 958 -2.0 LOt' 119119 Similar to RP5-977131 .6 CD732691 -2.0 CCDCI 28 Coiled-coil domain containing 128 CD7386'12 -2.1 ICL\' Ig light chain variable region CD 733218 NIJBP2 Nucleotide binding protein 2 (MinD lioniolog, Eschem'ichia cob) ('1)734942 L0('.1194:19 SS'eaklv similar to NI'796:3..lO.2 protein-coupled receptor 157 (Alas mns_sci>lus) CD7.1079S -2.1 L0077755 Similar to JINIIPC protein CD737016 -2.1 L0C3953 10 Pr000nix>-like protein CD738651 -2.2 I"OS v-los FTI.J mnhirine osteosarcomoa viral oncogenc honmolog CD734506 -2.2 P20K Quiescence-specific protein CD 726889 -2.2 LOC116715 Similar to brain-selective and closel y mapped on the counter allele of CSIAI' in cyst itmn cluster CD726888 -2.4 L0C420129 Similar to interferon. ,-indimcible protein Nt CF075071 -2.4 P0 Acidic ribosomal phosplioproteimm CD 73325 2 -2.1 CMAS C'ytidine monophosphate. N-acet ylneuraminic acid syntbeta,se CD739766 -2.4 L()C423773 Similar to 3-ioicrosi'nminoprotein precursor (prostate secreted seminal plasma protein) (prostate secretor y protein PSP91) (PSP-94 ) (seminal plasma :3-immliibiim) I imnunoglobmilin-hin(Iiug factor) (l'N44 CD736633 -2.4 L0C125$00 Euksm'votic translation initiation factor 213. subunit 4 A, 67 kDa CD7281 09 -2.4 SPCS 1 Signal peptidase complex subunit 1 liomnolog (S. (e','ecis>a e) CD736370 -2.4 LOC426$ 13 CRAM domain containing 3 CD 727877 -2.5 EPASI Endothelial PAS domain protein I CF075 1.56 -2.7 TI) BA 1 C Itiholni, o 0' CD740371 -2.7 RBM16 RNA binding motif protein 16 CD 736859 -2.7 LOC' 16104 ('erebloim CF074794 -2.7 \VWOX W\V doumain containing oxidoreducta"e CD72881 8 -2.8 LOC-I 17.158 Similar to cvtidiue dc'am'ninsse ('D".;33216 -2.9 1,0('12642-, Similar to K1AA0556 protein CD728075 -2.9 LOC771339 Fituslied cDNA, clone C'liEST567a2:3 CD7:34909 -2.9 COPG C>istomoer protein complex. smmlnmmiit CD735777 -3.0 L0C424065 Hypothetical L0C424065 CF074761 -3.1 TNRC6B I rinueleot ide repeat containing 6)1 CD 739 117 -3.8 1.0(124142 Similar to rnitocbondrial adenusine tripllospll;ite s'titliime sul.,iimiit 9 CD 7326. 18 -.1.0 HNIIPAB Heterogeneous nuclear rihonucleoproteimi A/B CD730 156 -5.0 EIFIAX Eukam'votic translation initiation factor 1A. X-linked CD727(87 -6.1 LDC4 1726$ Similar to solute carrier fammmilv 31. member 1 I Gene names for human mapped b y chicken Entrez gene identification numbers. liCflOARRAY ANALYSIS OF EIMERIA INFECTION IN CHICKEN LINES 1573 Table 6. The annotated genes altered with >2.0 changes between Favounii MS.) and NI 15.2 lines at 5 rI post Euinci')a ma.wna infec- tion (P < 0.01) Nt ti'i iial iZt.'d (lenBank no. raIl, Gene symbol Duscriptioti M5.1 3115.2 C0736627 sO RAIIRES! Retinuic acid receptor responder (tazarot<'imi' in(Iuced) ('1)733718 -1.5 L0C427838 llvpotliet cal L0C427838 CI)736698 1.6 AZIN I Atit iZ\'iii(' inhibitor 1 (07-10791 4.3 [0(131651! Similar to potential phospliolipid-t ralisporting ATPa,se i(,'ntilication number ) ATPase class 1 type SR member 2) (Callus yailis) ('1)726890 NIIAS Neuroblastom miii 13 AS viral (v-i'as) oncogcne boniolog ('1)736325 LOCI 26 107 Similar to [-cl'!) receptor A <'ham ('0731 120 1.0(417235 Similar to t,ubulin, 3 8 isoform 2 ( )Iuca,'i, in u/aID) ('1)7358.15 1,) )( '121:109 Similar to enhancer of poI'comb liomnolog 2 ('1)73! 395 ZA20l)2 ZF\Nl)5 Zinc finger. AN 1-type domain 5 1 [3 73 73 76 H P itT 1 Hvpoxanthimic plmosplioribosyltra,misferase I M -128880 P1(3)3 F'entatricopeptide repcim). domain 3 ('1)73211)3 IICJIIB04_30g17 Similarto 3-liexosaniinidase ci chant precursor (N-acetvt-(3-ghicosaniimmi(la,se) ((3-N- acctvl!ii'xosaniinidiise) ( Iiexosanimnidase A) (1)736593 1,0(776552 Similar to MAD NIL NM. 2050•l6 LI )(':Ii'SO II Lvinphotacrin C17071-1772 13 I'Ll> 11 ihosomital protein L15 ('1)728315 L0069:1257 liI'-I\'siii C 1)738700 LOCIGST12 I DCI domain tamily, member 16 (1)733357 011(313 as tiomnolog gt'iit' family, mcmxiher B ('1)738626 ( D36 CD:16 molecule ) r.hrornhospond iii receptor) (3)7:17(0! LC['I! l,vmiipliocvtc' t'vtosolic pro! miii I ( m.-plast iii) ('1)731023 [0(4201:13 Sinular to m yosin 1XB (1)730542 086811 Ileparami sulfate 6-0-simtfotransfera,se I (:D7272l4 (;A1)PA GA binding protemim transcription factor. ci sitluimat 60kDt< ('1075081 Al) 1'(' lB Act iii related protein 2/3 complex, subunitt. If), 4! kDa (1)7:16565 L0C4201 19 Similar to K IA A 1585 protein CD7291 I) L( )( '12(1)7 Hypothetical I .( 3(420097 ('D730482 ('Itt!! ('ore-binding factor, ii subunit (.1)7:15615 13L\l11 lileomnvcin It 0 rolase (3)728986 LOCI 19:338 Breast <'arc onma amplihm'il sequence 1 (3)728235 BC.) II B0'i_ 2<' 12 Similar to K IA A0776 protein CD736321 SIR 38L Si'riime/) lir,'onin,' kinase :38-like Xlii 418246.2 1.0(120129 Similar to intcrleromm. -inducibk' protein 30 ) L.00-l20129) (3)73561)2 I C135 ititegrin. 0 5 CD73.116t3 L0C425(l 7 Similar to mall a_se-gluco,unvlase CD737055 AQI) Aiiiiarii is honmolog (mouse N5 I ..)010))1:115 T,0('108047 'flivitmosin 13 .1 (L0C4080.17) CD73r1530 LCC 121055 Similar to hand 4.1-like protein 3 ) lID) )diftem'entitsllv expressed in 'adcnocai','iuoma of the lung protein 1)(DAL-1) CD730117 BAN I 13(1,2-antagonist "killer I (0732989 N(.:P214 N ucl<'iil)oi'i i 214 Ma NSLOI) 1001315 LO C10,047 Th\ iiiosiii 3 -1 (LOC.lOS)) 17) CD732701 L.1lL!11I)! 1.11111)1 doillaill contaillilig I 315.1 3115.2 ('01:14004 -2.!) 1.( )( '12680:3 iivpot lietn'al gene supported by (11388998 CD7295-13 -2.1 ('('DC 134 Coiled-coil doiiiaiii containing 134 ('0720.49)) --2.2 (1 PR 17-I O pi-Otciii-coupled receptor 171 CD73 1526 -2.2 LOC423Slti Sinnlar to 1(1 A A09 10 protein ('073751)1 -2.2 DNA.) Al Dna) ( HspiO) hoinolog subfamily A member I ('1)736407 -2.2 13 ihoiiimm- lease 111 CD 738866 -2.3 NDUI-'A! 0 N A Dii d,'liv,li'o genase (obiquinone) I a subcomplex, 10. -12 kDa ('1)737774 2.3 [[NP 1:3 H ing finger limit ciii 1:1 ('1)7:14283 -2.3 LOP 119:356 Finished eDNA. clone CIES'l'4951)1 I CD739480 -2.3 MON1A 11(3 Ni liowolog A (yeast) CD739l66 -2.-! [0CM 11)39 Similar to c'entrosonmal protein 76 ('1)7:11792 -2.4 LOP 125561 Similar to death receptor :3 ('1)73978 2 -2.4 RCJ 110(14_i 0<11 Peptidase D ('1)710029 -2.5 ACOX1 .Acvl-coenzviae A oxidase I, painmitoyl (.'D736633 -2.5 1.0('12-5800 Ei.mkarvotii' translation initiation factor 20. Subunit 4 A. 67 kfia CD738 12)) -2.7 \l1"AP3 liuicm'ofihrmllar-a.ssncial,e,l protein 3 (lD735820 -2.7 L0('126 168 Similar to \lGC69002 protein CD736528 'ii) S'I'Fd 713 Serine/'tliri'oimiiie kinase 171, ('1)7:10156 ElF lAX Eukarrot it' translation initiation factor IA. X-linkc' 1574 KIM ET AL. 0 M5.1 Amino acid metabolism Apoptosis Carbohydrate metabolism Cell adhesion Cell cycle Cell proliferation and differentiation Cell structure and motility Coenzyme and prosthetic group metabolism Developmental processes Electron transport Homeostasis Immunity and defense Intracellular protein traffic Lipid, fatty acid, and steroid metabolism Muscle contraction Neuronal activities Nucleoside, nucleotide, and nucleic acid metabolism Oncogenesis Protein metabolism and modification Sensory perception Signal transduction Transport Biological process unclassified D 2 4 6 8 10 12 14 16 18 20 No. of genes Figure 3. Gene ontology (GO) mal ' v.'is of genes niapped for human exiohi tiiig >2.0-fold altered expIesiofl levels between It 15.1 and N115.2 lines ihirmg Ehneviu inocima infection using the GO l ernis of biological process. integrin 3 5, protein pliosphatase 3 catalytic SUI)Uuit 4 Expression of APOA4 was more repressed in N15.1 isoform, peptidyiprolyl isornerase F and 1 downregulat- than M15.2 at 4 dpi of E. maxima, as measured by both ed gene. DnaJ (Hsp40) liomolog subfanilv A member AVIELA and QflT-PCR (Figure 4A). Pathway analysis 1, are annotated with comnion gene names for human indicated that APOA4 is related with the statin path- (Table 7). way. Statins are highly effective in lowering serum cho- lesterol concentrations and preventing ischemic heart QRT-PCR disease (Pignone et. al., 2000). The expression of FYN transcripts was significantly downregimla.ted in AVIELA We selected 4 genes and followed the kinetics of data and RT-PCR. analysis (P < 0.05) at 3 dpi in the their corresponding transcript, levels after E. maxima 1\15.1 line. In contrast, it was upregulated in the M15.2 infection. All of the genes selected showed alteration line (Figure 413). The gene FYN is supposed to he in- of expression with >2.0-fold change in the normalized volved in several interleukin signaling pathways, integ- data of AVTELA at more than one time point exam- rin signaling pathways, and immune cell receptor path- ined (P < 0.05). Three of those genes, apolipoprotein ways. In our study. NRAS was nma.rkedly upregulated in A-IV (APOA4); FYN oncogenc related to SRC, FGR. M5.1 chickens at 4 dpi, measured by using microarray; YES (FYN) and neurohlast.orria RAS vital oncogene however, its expression was only slightly increased as homolog (NRAS), are involved in more than one spe- quantified by RT-PCR (Figure 4C). In the analysis us- cific pathway by the analysis using GeneSpring GX 7.3 ing GeneSpring CX 7.3, the epidernial growth factor software. I signaling pathway and niit.ogen-activated protein ki- \1TCROARRAY ANALYSIS OF EIMERIA INFECTION TN CHICKEN LINES 1575 hi Table 7. The genes classified into the gene ontology (GO) term of ilnini ii dl v mid defense with >2.0-fobd changes between Fa oti nO - N15.1 am! M15.2 lines using the PANThER database Ceiic 113 1 (baie (liuniaii ) Gene name symbol Score2 t3io1oiiiiil process Pat Iiaiv Lpregiiliited lice eptor protein tvrosine kliil)Si' hifliiiiiinitinii n,eii)ited 1) N, clieniokine sigiialiiig pathuivav and cvt i >k iiic 5)l in) ing pat liway— Growth I. factor receptor- hound protein S1uiIIuK.H.tait.lIrI!!U P13 kinase pathwav—'Grb2 F(IF signaling pathway—'Crh2 lnterleukin signal ii ig pat hvav—giowt h factor receptor hi oiling protein 2 T-cell acrivat.ion—Grh2 Dnpamnmne receptor-mediated signaling lil1LIiay—gnc>'t Ii tact or receptor-bound protein 2 2885 Growth factor (11)132 1.()()E- 110 Insulin/insiiliii-like growth factor receptor-hOiliicl pal }iiviv-i lit ogcmi-aclivated 1u)t)in protein 2 kinasi' kinase/niitogen-acrivatedprotein kinasc crscade—'growth factor receptor bound protein 2 Ras pa thwav—growt Ii fact or receptor- bound protein-2 1'-cell active t ion — Ga l B-cell ui-I ivat ion—Grl i2 .-\ iigmogemiesis—'Crowl h fact or Receptor- bound prdit ciii 2 let egrin sigmialmg patliway-growl Ii lii-! or receptor hound protein 2 PDGF signilnig pal hwav—growth factor R ecept or himici tug protein 2 Ft : F receptor signaling pathway—'Urb2 Angiogenesis—growth factor receptor- hound protein 2 3693 I nngrii . .1 5 11(1135 l)OOE+fJO Cell adhesion-mediated signaling Integnin signaling pathcvav-ointegnin 3 Cell ad I iesioii Blood coagulation—.glycoprotein lIla Blood clotting ('ci) niiitjhty 5532 Protein pliospi hit use I t' 'SC II tt.t)()E-t- Ott Regulation of carbohydrate 13-cell activation—'calcineurin 3 (lornicrlv 213 iii eta) iolisin cat alvl ic subuiii t .3 Other polysaccharide inclabolism '1-cell activation--cialcincnrin isoformn (cnlcilleurill tilvcogcn nictabolisiii \\mit signaling parhwav—calciieurm A 3) odIN A transcription Protein phosphor , vl it) Oil iriiislationiil rcgimllit ion Other intracellular signaliig citsdiiiic St less respolise \leiosis ('ill cycle control \litiisis liomi heist ask 10115 l'ept Ic kIproir I PP IF 0 POE +01) Protein folding- isolnerase I' Nil( 1c;11 Ira iis)iciiI ciclopliiliii 1-') liii liii nit y iuid delensc Docvi iregulated 3003 Granzvine K GZMK POPE-i-DO Prot i'olvsis (granzvnie 3; tryptase T-cell-nieilii iii innnnni lv ii) Natural killer cell-mediated imii iii liii v (Irniiiliiivt e-iiiecliated iinmnunitv Apo Di isis 10437 Interferon, '-inihmciblc IF(S)) POPE-gPO Prolvolvi, protein 30 51 l-l('Il-irieihated inimnunitv 3301 Dna.J (11sp40) DNAJA 1 0.00E+00 Pn >11)0 folding lioiiio)og siiiiiamiiii' A St re's rc'spoi li-s member I Homiiologene identification numbers for human mapped by chicken Entrez gene iilcis lieu The low scores closing wit Ii II indicate the high level of confidence in the relation between gene and (10 term. 15 76 KL\1 LI AL. tiase signaling pathway were searched as NRAS associ- of Eimer)a-infected chickens and LPS-stiniulated mac- ated pathway. Both pathways play vital roles as liripor- rophages (\[in et al.. 2003. 2005). Intraepithelial lviii- I ;iiit regulators in response to a diverse range of stimuli. phiocyt es pla y an important role iii protect iye iuiimmmnitv itch as cytokines, growth factors, neurotransmitters, to avian coccidiosis (Lillehoj and Lillehoj. 2000). Li- cellular stress, and cell adherence during development, popolysaccharides. a component of bacteria membrane. cellular proliferation, survival, and migration (Orion provoke all shock and induce a strong response et al.. 2005: Marks et al., 2007). Finally, expression of in animal immune svst ems. It causes the secretion of CD36 was (lownregulated in the i\115.2 line at 4 dpi us- proinflanunatorv cvtokines b y a receptor complex coin- ag 1 iL AVI L1.\ and real-time RT-PCR (Figure 4D). posed of LPS-binding protein CDI4 and Toll-like re- ceptor 1 in mnacrophiages (Beutler. 2000). Furthermore, DISCUSSION there are the selected cytokines and chemokine genes from the direct PCR clones oil inicroarrav. >iuviui lv reported that 2 genet icillv disparate Therefoi'e. AV1LlA is a suitable tool for the host gene Idvourui chicken lines. MS. 1 and M15.2. show differ- expression profiling of the interact ion between host and ences in susceptibility against avian coccicliosis (Kim et parasite in the chicken gut. iii.. 2008). The MHC molecules play important roles in In the current study. the number of genes with >2- the regulation of the immune response by conununicat- fold changes in TEL was increased with time after Eimc- ing among different cellular componentsiponent.s of the immune na infection (Figure 1). 'l'lie altered gene Iluinber was system: T cells, B cells, and antigen-presenting cells larger in line Ml 5.2 tItan in line MS. 1 birds at any (Lamont, 1998). Because M5.1 and M15.2 are highly dpi and the latter line is more resi stant compared with identical congelliC lines except the chromosome. includ- M15.2. The genes changed in tramiscriptiommal regulation ag MHC, the differentially expressed genes after the are increased after the conditions of coccidiosis and F. maxima infection might he related with avian irn- the higher number of differentially expressed genes ap- ii inity. Accordingly, the current study was conducted peared in the more susceptible line. These data suggest (a analyze the gene expression profiles induced by time that the greater pathology induces, Ili( , higher gene ex- infection of E. maxima oncvst.s iii these 2 13-coin plex pression changes. However, more Functional approaches congemc chicken hues by eDNA microarray technique. needed to clarify thus assumption. Avian intestinal TEL are fabricated with cDNA clones Comparing the expression patterns of transcripts be- ,-,elected from a (, DNA library of'chicken intestim al JET. tween I lie 2 ('luckeD lines. t lie nittitliet' of alt cued gelies A C APOA4 NRAS 1.5 12 * 10 C) C) O 1 " C C 8 * 6 C) C) 0.5 AVIELA 4 or a_ • AVIELA QRT-PCR . 0 Eaii1II I! ' 0 •- IL U- I- I F . 3 dpi 4 dpi 5 dpi 3 dpi 4 dpi 5 dpi 3 dpi 4 dpi 5 dpi 3 dpi 4 dpi 5 dpi M5.1 M15.2 M5.1 M15.2 B I] FYN CD36 3 1.5 2.5 II, C) 0 ') C 2 C 1 E1.5 C) 1 * •AVIELA 0.5 AVIELA 0.5 •0 • nQRT-pCR V 3 0 I I I- IL U- i i I 3 dpi 4 dpi 5 dpi 3 dpi 4 dpi 5 dpi 3 dpi 4 dpi 5 dpi 3 dpi 4 dpi 5 dpi M5.1 M15.2 M5.1 M15.2 Figure 4. Quantitative aiialvsis of niRNA levels of selected genes hv real-time reverse traliscripi 011 PCB after E0ri rw maxima infection at 3. 4. and 5 d postinfection (dpi).p (A) A olipoprotein A-I\' (APOA4). (B) FYN oncogene related to SRC, FGR. y (FYN). (C) Neuroblastorna hAS viral olicogene lioino]og NRAS). (D) ('1336. *1) < 005 in the 1]licrnarray analysis. AVIELA - avian intestinal intraepithelial lymphocyte (DNA microarrav: QR'l-P( Ii = quantitative real-time P('II MICROARRAY ANALYSIS OF EIML'RL4 INFECTION IN CHICKEN LINES 1577 was highest at 4 dpi. rather than 5 dpi (Figure 2). This (Samnelson. 2002) and time GR132 SF13 dommiaimis bring suggests that the transcriptome conversion (luring the various higamids to the sites of active signaling (Koi'e- early stage of avian coccidiosis before 4 dpi is crucial tzky. 1097). The eDNA sequence of chicken GRB2 was for the resistance due to host innate immune system ill determined in 1993199:3 (\Vasenins et al.. 1993): however. the chicken gut. Similar results were observed b y Mm functional studies have not vet beemi performed. Tnt - et al. (2003). The monitored transcriptional changes grimi. 13 5 plays a key role in time regulation ofof apop- between I and 4 (1 post primary infection with Eirnera tosis of endothelial cells by binding with annexin V. oocvsts in chicken TEL using a eDNA microarrav con- which activates protein kimia,se C cr 2 to stimulate taining 400 chicken genes and showed that the largest apopt ot.ic events (Cardo-Vila et al. 2003) and medi- changes of transcript number occurred at 4 dpi of pri- ate phagocytosis of apoptotic cells (Singhi et al. 2007). mary infection. In protein phosphatase 3 catal ytic subunit 3 isofbrni Quantitative real-time BT-PCR for the selected genes knockout mice, total peripheral T-cell numbers were was performed to confirm the differential expression significantly reduced and interleukiim-2 production in patterns that we found iii AVIELA analysis between response to phorbol 12-in , yristate 13- acetate/ ionomycin the 2 Favounu chicken lines. It has ])cell that and T-cell receptor cross-hnikiug were defective (Buemio APOA4 is am I endogenous ant imiflainniatorv protein et al.. 2002). Lin and Lechleit er (2002) reported that (Vovinkel et. al.. 2004) and it shows impaired expression peptidylprolyl isonmerase F has a protective role against in inflainniatory bowel disease niucosa, even in the non- apoptosis that is mnediated by one or more targets other inflamed regions (Orso et al. 2007). lii mast cells, the than the adenine nucleotide I ranslocator. The cvtosohc absence of FYN and phiosphaticlylinositol 3-kinase. or chaperone pair of hsp70 and Dima j (Hsp40) homnolog the inhibition of p35 mitogen-activated protein kinase sumbfainily A member 1 prevents NO-mediated alx)pto- activity, (lei iiomistrated that they are essential for H()- sis I lpstreani of cvtochrome c release from niitochiondria. driven interleukin-4 production (Frossi et al, 2007). (C;otohm et al.. 2004). Gm'anzvme K triggers rapid cell Mit.ogen-activated protein kinase activity is subject to deatli by rapid extem'mmahizat-iomm of phosplmatidvlsem'imme. regulation even iii \-raf murine sarcoma viral oncogene nuclear morphological changes, amid simigle-stranded honmolog 111 (131IAF)/NRAS niutant melanoma cells DNA nicks (Zhao et al.. 2007). The enz yme encoded 1) (Edlundh-Rose et al.. 2006). It has been found that interferon, 1-iimduicihhe protein 30. is a lysosomal I luol CD36 facilitates file uptake of specific lipid molecules reductase that has air role imi l\ IHC class II- and adheres to macromolecules with subsequent I rans- restricted antigen processing (Sum et al.. 2005). cluction of intracellular signals with releviniN to infli-un- In i'oumcliisiomi, transcriptional profiling revealed dif- inat,ion, phagocytosis, and endocytosis (Huseinann et ferential expression of genes possessing various func- al.. 2002). The APOA4. FYN. NRAS. and CD36 tran- tional roles including imnmunitv and defense between scripts showed similar l)1ttermls when compared with 2 13-complex disparate. geneticall y inbred, Fayoummii niicm'oarray data iii the transcriptional changes between chicken himies after Li ruci'ia infection. 2 chicken lines, although the normalized values from Because of time similarit y of genetic background of mincroarray analysis amid QRT-PCII are.not perfctly the lines, and their difference in coccicliosis susceptibil- correspondent. It might he caused from the differences it y. some of the transcripts altered in this study may of the normalization methods or the fluorescent dyes to be associated with genes controlling (occidiosis disease detect gene expression between mnicroarray and QR.T- resistance. Therefore. future studies based oil re- PCR, or both. The normalization method using the sults will contribute to comprehensive understanding expression levelslevels of the housekeeping gene is geIlel'?dlv of time imimiate immune s stem in the chicken gilt and applied in QRT-PCR,. However, it has been argued to facilitate the development of novel strategies targeting not. always he accurate as a negative comparison coil- major protective immune pathways important in avilu I trol (Lee et al.. 2002). cc )c( i (1 i( )sjs Iii a prelinunary analysis, 110 differential genie ex- pression between the noninfect ccl controls of N15.1 and ACKNOWLEDGMENTS N115.2 hivbi'idized to AVEELA appeared (D. K. Kim. unpublished data.). The differential expressed elements We I hiamik -Mar jorie Nichols (Amiinial Parasitic Discus- ill after infection between 2 lines could be es Laboratory. Animal and Natural Resources Institute, highly related with disease resistance, even though the ABS. USDA) for technical assistance. This work was number of genes that changed was narrow. supported ha' Korea Research Foundation Grant fund- In the results. 7 differentiall y altered genes between ccl by the Korean govermimmiemit . Mimust r' of Education chicken lines were classified by GO terimis of ininunntv and Human Resource Development (Seoul. Republic of and (lelinse (Table 7). Growth factor receptor- boumid Korea: number KR F-2006-2 1 4-F000 17). protein 2 is all adaptor protein involved in the fornia- tion of multiprotein complexes at receptors and adaptor proteins. lmi T cells, the S112 dontain of CR112 activates REFERENCES T-cell receptor by binding with phiosphorylated t y lt(Itl(r. B. 2000. liii: (eiii la] coil IJ)oIsllt of ill(' ,olc l1011LlnlaliamL -rosine on the adaptor protein linker of activated T cells LPS s(iisOr. Cur. Opimi . imnoimiol. I 2:2t) 26. 15 7? KIM Li AL. Bneno, 0. F., E. B. Brandt. Al. E. Rothenberg. and J. D. Niolkentin. for avian anemic (ia's. 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