INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.4.2527–2534.2001

Apr. 2001, p. 2527–2534 Vol. 69, No. 4

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Analysis of Chicken Mucosal Immune Response to Eimeria tenellaand Eimeria maxima Infection by Quantitative

Reverse Transcription-PCRFABRICE LAURENT,* ROSELYNE MANCASSOLA, SONIA LACROIX,

RITA MENEZES, AND MURIEL NACIRI

Unite de Pathologie Aviaire et de Parasitologie, Equipe des Maladies aProtozoaire, INRA, 37380 Nouzilly, France

Received 14 August 2000/Returned for modification 4 October 2000/Accepted 3 January 2001

The recent cloning of chicken genes coding for interleukins, chemokines, and other proteins involved inimmune regulation and inflammation allowed us to analyze their expression during infection with Eimeria. Theexpression levels of different genes in jejunal and cecal RNA extracts isolated from uninfected chickens andchickens infected with Eimeria maxima or E. tenella were measured using a precise quantitative reversetranscription-PCR technique. Seven days after E. tenella infection, expression of the proinflammatory cytokineinterleukin-1b (IL-1b) mRNA was increased 80-fold. Among the chemokines analyzed, the CC chemokinesK203 (200-fold) and macrophage inflammatory factor 1b (MIP-1b) (80-fold) were strongly upregulated in theinfected ceca, but the CXC chemokines IL-8 and K60 were not. However, the CXC chemokines were expressedat very high levels in uninfected cecal extracts. The levels of gamma interferon (IFN-g) (300-fold), induciblenitric oxide synthase (iNOS) (200-fold), and myelomonocytic growth factor (MGF) (50-fold) were also highlyupregulated during infection with E. tenella, whereas cyclooxygenase 2 showed a more modest (13-fold)increase. The genes upregulated during E. tenella infection were generally also upregulated during E. maximainfection but at a lower magnitude except for those encoding MIP-1b and MGF. For these two cytokines, nosignificant change in expression levels was observed after E. maxima infection. CD31 intraepithelial lympho-cytes may participate in the IFN-g upregulation observed after infection, since both recruitment and upregu-lation of the IFN-g mRNA level were observed in the infected jejunal mucosa. Moreover, in the chickenmacrophage cell line HD-11, CC chemokines, MGF, IL-1b, and iNOS were inducible by IFN-g, suggesting thatmacrophages may be one of the cell populations involved in the upregulation of these cytokines observed in vivoduring infection with Eimeria.

Chicken coccidiosis is caused by intracellular protozoan par-asites belonging to seven species of Eimeria. These parasitesinvade and reside in the lining of the intestine or ceca. Parasitedevelopment causes diarrhea, morbidity, and mortality, andthe impact of coccidiosis on the industry has serious economicconsequences. Thus far, chemoprophylaxis has controlled thedisease but has been complicated by the emergence of drugresistance. Infection by Eimeria promotes antibody and cell-mediated immune responses. However, cellular immunity me-diated by various cell populations, including T lymphocytes,NK cells, and macrophages, plays a major role in disease re-sistance (27). There is increasing evidence of CD41 and intra-epithelial lymphocyte (IEL) involvement during a primary in-fection, while T-cell receptor a- and b-chain-positive CD81

IEL play a key role in secondary infection (25). The develop-ment of a vaccine has been hampered by the lack of under-standing of the various components of the host immune systeminvolved in protective immunity.

The low level of homology between chicken genes and theirmammalian counterparts has made it difficult to discover im-munologically relevant chicken genes. However, there havebeen increasing numbers of chicken gene sequences appearing

in the databases due to the emergence of chicken genomeprojects. Among the cytokines cloned, one can find genes cod-ing for interleukins (interleukin-1b [IL-1b] [39], IL-2 [36], andIL-8 [20]) and interferons (alpha/beta interferon [IFN-a/b][34] and IFN-g [7]) and also for a macrophage growth factor(myelomonocytic growth factor [MGF]) (24) and three iso-forms of transforming growth factor b (TGF-b) (16–18). Inaddition, several members of the chemokine family have re-cently been cloned: C chemokine (unpublished data), CC che-mokines (macrophage inflammatory protein 1b [MIP-1b] [15]and K203 [35]), and CXC chemokines (K60 [35] and IL-8 [20]).A number of receptors have also been identified, including theIL-1 receptor (IL-1R) (12) and a putative chemokine receptor(Chem-R) (13). The development of chicken genome projectsin several countries and the use of DNA array technology willundoubtedly expedite the identification of components of thechicken immune response to a variety of pathogens. However,the analysis by reverse transcription-PCR (RT-PCR) of theexpression of an available panel of genes will provide initialclues about the development of the immune response to Eimeriainfection.

In this study, we analyzed the local immune response ofleghorn chickens to two strains of Eimeria commonly found inpoultry farming, Eimeria tenella and E. maxima (33), by qual-itative RT-PCR followed by a precise quantitative RT-PCRmethod.

* Corresponding author. Mailing address: Unite de Pathologie Avi-aire et de Parasitologie, Equipe des Maladies a Protozoaire, INRA,37380 Nouzilly, France. Phone: (33) 02 47 42 77 45. Fax: (33) 02 47 4277 74. E-mail: laurent@tours.inra.fr.

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MATERIALS AND METHODS

Infection of chickens with Eimeria. Chickens used in this study are specific-pathogen-free White Leghorn (PA12) hatched in our animal facilities and keptin wire cages with water and food ad libitum. Three-week-old chickens wereorally infected with 2 3 104 oocysts of E. maxima (strain PAPm11) or E. tenella(strain PAPt38). Animals were killed by cervical dislocation 3, 7, or 13 days afterinfection.

RNA extraction. Three-centimeter-long intestinal fragments of duodenum(bottom of the duodenal loop), jejunum (3 cm above Meckel’s diverticulum),ileum (3 cm below Meckel’s diverticulum), or cecum (median part of the organ)were excised and cut longitudinally. To remove intestinal contents, fragmentswere washed in ice-cold phosphate-buffered saline (PBS) and immediately im-mersed in TRIzol solution (Life Technologies, Cergy Pontoise, France) for 3 minunder agitation. This technique allows the extraction of RNA from cells of theupper layer of the mucosa as assessed by microscopy. Total RNA extraction wasperformed according to the manufacturer’s recommendations.

RNA standards for quantitative RT-PCR. For the quantitation of mRNAlevels of the genes of interest, plasmids coding for truncated mRNA templates(standards) were constructed. In vitro transcription of these plasmids yieldsRNAs that carry primer sites identical to those that amplify target RNA. How-ever, the distances between specific 59 and 39 primer sites and, therefore, thesizes of the PCR amplification products differ from those of the standard andtarget RNAs. To generate a truncated template, we used a composite primermade of the upstream primer (59) followed by a sequence complementary to aregion located downstream in the RNA. The corresponding PCR product wasobtained with RNA extracted 7 days postinfection from the cecum of anE. tenella-infected chicken as a template, using the composite primer and thedownstream primer. The amplified fragment was cloned into plasmid pGEMeasy(Promega, Lyon, France). This procedure was performed for each of the 11plasmid constructs. Finally, to provide a poly(A) tail and a new unique HindIIIrestriction site at the 39 end of the coding sequence, the sequence encoded by twocomplementary oligonucleotides (59TCGACA20AAGCTTC and 59TCGAGAAGCTTT20G) was inserted at the SalI site of the plasmids. To generate standardRNA, plasmids were digested with HindIII and transcribed in vitro using T7RNA polymerase under conditions recommended by the supplier (Eurogentec,Angers, France).

Oligonucleotide primers for PCR amplification. Sequences of the oligonucle-otide primers used for PCR amplification and the sizes of the predicted PCRproducts from target and standard RNAs are given in Table 1. Primers weredesigned based on published sequences and obtained from Eurobio (Les Ulis,France). When genomic sequences were available in the databases, primers wereselected to either amplify fragments from cDNA that are distinguishable by sizefrom fragments amplified from genomic DNA or span exon-exon boundaries andtherefore do not amplify genomic DNA.

Quantitation of mRNA levels. Quantitative RT-PCR was performed as de-scribed by Jung et al. (19). Briefly, serial dilutions of known quantities of stan-dard RNA molecules were mixed with 1 mg of total cellular RNA in a total

volume of 20 ml and reverse transcribed at 37°C (19). Two microliters of thereaction mixture was used in a 35-cycle PCR except for b-actin, which wasamplified for 28 cycles. Annealing temperature was 61°C for all primers exceptIFN-g primers (57°C). Sizes of the PCR amplification products differ by 25 to30% between standard and target RNAs; thus, the products can be easily sepa-rated on a 2% agarose gel and visualized by ethidium bromide staining. Bandintensities were quantitated by densitometry (GS-670 imaging densitometer;Bio-Rad, Ivry sur Seine, France). Ratios of the band intensities of the PCRproducts from the standard RNA and target RNA were plotted against thestarting number of standard RNA molecules on a double logarithmic scale.When the ratio of the band intensities equals 1, the number of target RNAmolecules is equivalent to the number of standard RNA molecules (19). Data areexpressed as the number of target mRNA molecules per microgram of totalsample RNA. On every RNA sample, a first set of serial 10-fold dilutions ofstandard was used in the reaction in order to determine the range in which thegene was expressed. Thereafter, six serial threefold dilutions of standard sur-rounding the estimated value were used. The quantitative RT-PCR was sensitiveto 103 mRNA molecules/mg of total RNA.

Immunohistochemistry of CD31 positive cells. Pieces of jejunum were fixed inPBS containing 4% paraformaldehyde and snap-frozen in liquid nitrogen. Seven-micrometer-thick frozen sections were incubated for 30 min with a mouse anti-CD3 antibody (clone CT-3; Southern Biotechnology, Birmingham, Ala.) diluted1/100 in PBS containing 0.05% Tween 20. After several washes, sections wereincubated with a goat anti-mouse-fluorescein isothiocyanate conjugate (Sigma,Saint Quentin Fallavier, France) for 30 min. Sections were slightly counter-stained with Evans blue (1/20,000) before microscopic examination.

Isolation of IEL. Chicken IEL were obtained as described by Bessay et al. (4).In brief, the small intestine between the duodenal loop and the region immedi-ately prior to Meckel’s diverticulum was excised, cut longitudinally, and washedin HBSS (Hank’s balanced salt solution; Gibco, Cergy Pontoise, France) mediumcontaining 4 g of glucose per liter and 2% fetal calf serum (FCS). Intestinalfragments of each chicken were treated separately, cut into 1- to 2-cm pieces, andincubated for 10 min in the same medium supplemented with 2 mM dithiothre-itol in order to eliminate the intestinal mucus. The supernatant was discarded,and the small pieces of intestine were incubated twice for 20 min at 41°C inmedium containing 2 mM dithiothreitol and 3 mM EDTA. Cells in the super-natant were washed and passed through nylon wool to remove most epithelialcells and cellular clusters. Cells were further purified on a Ficoll gradient (Sigma)(density of 1.077 g/ml, 30 min, 1,200 3 g) to remove red cells. Cell viability was.95% as determined by trypan blue exclusion.

Purification of CD31 IEL by magnetically activated cell sorting. IEL resus-pended in cold HBSS containing 2% FCS and 4 g of glucose per liter wereincubated 20 min with the mouse anti-chicken CD3 with a working dilution of 1mg/106 cells. After two washes, cells were then incubated for 20 min at 4°C witha rat anti-mouse immunoglobulin G1 (2 ml for 106 cells) conjugated with mag-netically activated cell sorting (MACS) superparamagnetic microbeads (Milte-nyi, Paris, France). Cells were washed twice in PBS containing 0.5% bovine

TABLE 1. Primers used for RT-PCR analysis of chicken mRNAs

TargetmRNA

Accessionno.

Primer Size of PCRproduct (bp)

59 39 Target Standard

b-Actin L08165 59-CATCACCATTGGCAATGAGAGG-39 59-GCAAGCAGGAGTACGATGAATC-39 353 271Chem L34552 59-GTCTTCTCCTTGGTCATGGTCA-39 59-CAAGGCAGAGCTGGCTCCATAA-39 403 303Chem-R AF029369 59-AGATGAGAGCAACCGCAGCATC-39 59-AAGCCAATGGCCTCTGTCACC-39 350COX-2 M64990 59-GGTGAGACTCTGGAGAGGCAAC-39 59-GTTGAACAGAAGCTCAGGGTCA-39 401 300IL-1b Y15006 59-GGCTCAACATTGCGCTGTAC-39 59-CCCACTTAGCTTGTAGGTGGC-39 350 270IL-1R M81846 59-TGATTCTCAAGAATTTACATCATACAT-39 59-CTTCTCCTGCTAAATCATTCCTC-39 353IL-8 AJ009800 59-ATGAACGGCAAGCTTGGAGCT-39 59-TCACAGTGGTGCATCAGAATTGA-39 312 238IFN-g Y07922 59-GCCGCACATCAAACACATATCT-39 59-CAGTAGGAGGTATAAATACTTTC-39 403 302iNOS U46504 59-AGGCCAAACATCCTGGAGGTC-39 59-TCATAGAGACGCTGCTGCCAG-39 371 285K60 Y14971 59-GGGCAAGGCTGTAGCTGCTG-39 59-TGGTGTCTGCCTTGTCCAGAAT-39 290 215K203 Y18692 59-ATGAAGCTCTCTGCAGTTGTTCT-39 59-TCAGTCCCGCTTGACGCTCTG-39 269 206Lymphotactin AF006742 59-ATGAAACTCCACGCCACAGTTCT-39 59-CTTCTTCTGGTAGTACGTCTTCTG-39 290MGF M85034 59-CTGCAGCTGTGCTGGCGCTG-39 59-CCAGCGAGTCGTGGTACGCG-39 320 189MIP-1b L34553 59-ATTGCCATCTGCTACCAGACCT-39 59-TCAGGTAGCTCTCCATGTCACA-39 322 230TGF-b2 X59080 59-TGCAGGATAATTGCTGCCTGCG-39 59-AGCTGCATTTGCAAGACTTTACAAT-39 305TGF-b3 M31154 59-CCCTCCTGCAGGAGAAAATCCT-39 59-TCAGATCATGAGTGAATGGCTCC-39 400TGF-b4 M31160 59-ACCTCGACACCGACTACTGCT-39 59-CTGCACTTGCAGGCACGGAC-39 340

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serum albumin and 2 mM EDTA and applied to the column. The cells werepurified as instructed by the manufacturer (Miltenyi). RNA was extracted fromCD31 cells as described previously. The mini-MACS separation allowed a purityof CD31 cells of 95% as controlled by flow cytometry.

Expression of recombinant IFN-g in COS7 cells. Chicken IFN-g coding se-quence was amplified by PCR from RNA extracted from E. tenella-infected cecaand cloned in the pcDNA3 vector (Invitrogen, Groningen, The Netherlands).The following primers were used: sense, 59-CTCGAATTCACCATGACTTGCCAGACTTACAACT-39; and anti-sense, 59-GTCCTCGAGTTAGCGGCCGCTGCAATTGCATCTCCTCTG-39.

COS7 cells were maintained in growth medium at 37°C (Dulbecco’s modifiedEagle medium (DMEM) containing 4.5 g of glucose per liter supplemented with10% FCS, 2 mM L-glutamine, 50 U of penicillin G per ml, and 50 mg ofstreptomycin per ml). Plasmids coding for IFN-g or b-galactosidase (b-Gal) as acontrol (pCMVb; Ozyme, Montigny-le-Bretonneux, France) were transfectedinto COS7 cells using LipofectAMINE (Gibco-BRL, Cergy Pontoise, France) asrecommended by the manufacturer. Briefly, serum-free DMEM containing lipid-DNA complexes were added to the COS7 cells for 5 h of incubation. FCS wasthen added to the incubation medium to reach a 10% concentration. Eighteenhours later, the medium was replaced with fresh growth medium. The superna-tants containing b-Gal or IFN-g activity were recovered 48 h later.

Activation of HD-11 cells with recombinant chicken IFN-g. HD-11 cells weremaintained at 41°C in growth medium (DMEM containing 1 g of glucose per litersupplemented with 8% FCS, 2% chicken serum, 2 mM L-glutamine, 50 U ofpenicillin G per ml, and 50 mg of streptomycin per ml).

Before RNA extraction, 106 HD-11 cells were activated for 6 h with 2 ml ofdiluted (1/30) supernatant from IFN-g- or b-Gal- transfected COS7 cells. At thatdilution, b-Gal-transfected cell supernatant had no effect on nitrate (NO2) andnitrite (NO3) release, whereas IFN-g-transfected cell supernatant induced themaximal level (NOx 5 NO2 1 NO3 5 90 mM) as determined by the Greissreaction according to the previously described protocol (10).

RESULTS

Inflammatory gene expression in different intestinal regionsat homeostasis and during infection with E. tenella or E. max-ima. In pathogen-free animals, genes are differentially ex-pressed in organs reflecting the normal physiologic conditions.Figure 1A shows that while b-actin, MIP-1b, IFN-g, induciblenitric oxide synthase (iNOS), and MGF were expressed atsimilar levels in the cecum and the jejunum, IL-1b, cyclooxy-genase 2 (COX-2), K60, K203, and especially IL-8 were morehighly expressed in the cecum. Only the putative chemokineChem was expressed at higher levels in the jejunum than in thececum (Fig. 1A). IL-8 and K60, which belong to the CXCchemokine family, were found to be highly expressed (about108 copies/mg of total RNA) in uninfected ceca (Fig. 3, day 0).Expression was apparently restricted to the part of the intes-tine colonized by the parasite, i.e., the cecum for E. tenella (Fig.1B) and the small intestine for E. maxima. Although E. max-ima infects more specifically the midintestinal area, the para-site can spread to the duodenal loop and to the lower ileum ifthe infection is severe (Fig. 1B). RNA extracted from thechicken jejunum was selected for the gene expression analysisduring E. maxima infection. The level of cytokine response wasdependent on the dose of inoculation. For example, whenchickens were infected with 2,000 or 20,000 E. tenella oocysts,IFN-g expression in the cecum 7 days after infection increased50- or 300-fold, respectively, compared to the control value.Values were determined by quantitative RT-PCR on a pool ofRNA extracted from five animals. Similar data were obtainedwith E. maxima infection 7 days after infection with 2,000 or20,000 oocysts, IFN-g expression in the jejunum increased 85-or 200-fold, respectively, compared to the control value. Thehigher dose of inoculation (20,000 oocysts) was used for fur-ther experiments with both Eimeria strains.

Expression of iNOS, COX-2, and inflammatory cytokines inE. tenella-infected ceca and E. maxima-infected jejunum. Adaily time course of gene expression during the infectionwas performed after inoculation of 3-week-old chickens with20,000 E. tenella or E. maxima oocysts, which leads to severeinfection. Four time points were further selected (days 0, 3, 7,and 13) for the following reasons. On day 3 after E. tenellainfection, blood was not detected in the cecum. Maximal up-regulation of expression of almost all genes investigated in thisstudy occurred by 7 days postinfection for both Eimeria strains.Finally, by day 13, oocyst excretion had ceased. For each timepoint, six to eight chickens were used in order to allow forindividual variations and to prepare a pool for the quantitativeRT-PCR.

Expression of the genes studied during E. tenella infectionvaried little between chickens in the same treatment group(Fig. 2). During E. maxima infection, there was greater varia-tion in mRNA expression of IL-1b and IFN-g between theanimals at days 3 and 13 postinfection, probably due to a slightshift in the kinetic of the response (Fig. 2). Expression of theproinflammatory cytokine IL-1b was increased 80- and 27-fold7 days after infection with E. tenella and E. maxima, respec-tively (Fig. 3). Little or no increase was detected for the IL-1R

FIG. 1. Cytokine expression along the chicken intestine. (A) Ratiobetween gene expression in the cecum and the jejunum at homeostasis.Pools of RNA extracted from the jejunum or cecum of eight PA12chickens were prepared. Quantitative RT-PCRs were performed forthe 11 genes in both RNA pools, and the number of mRNA copiesexpressed per microgram of total RNA was determined. For eachgene, the ratio between gene expression in the cecum and the jejunumwas calculated. Data were obtained from a representative experimentperformed twice. (B) IFN-g mRNA expression in the different parts ofchicken intestine infected with 20,000 oocysts of E. maxima (E.m) orE. tenella (E.t). Data presented are from representative chickens; sim-ilar data were obtained with four other animals for both Eimeriastrains. D, duodenal loop; J, jejunum; I, ileum, C, cecum.

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in infected chickens. Among the chemokines analyzed, lym-photactin, the putative chemokine Chem, and the CXC chemo-kines K60 and IL-8 exhibited unchanged or modest increasesin mRNA expression during infection with either strain ofEimeria. In contrast, the CC chemokines K203 (200-fold) andMIP-1b (80-fold) were strongly upregulated during E. tenellainfection, suggesting a role for these molecules in the mucosalimmune response. After E. maxima infection, K203 mRNA

expression was also clearly upregulated (100-fold) in the jeju-num when MIP-1b mRNA expression showed only low up-regulation. The putative chemokine receptor (Chem-R) was weak-ly expressed in the intestinal mucosa compared to the spleen(data not shown). However, a moderate increase in mucosalexpression was observed following infection with E. tenella.

A clear dichotomy between the MGF response to E. tenellaand E. maxima infection exists, since a 50-fold upregulation

FIG. 2. Qualitative RT-PCR amplification of mRNAs extracted from E. tenella-infected cecum (A) or E. maxima-infected jejunum (B). Thedata shown are the results of individual RT-PCRs on total RNA extracted from six to eight PA12 chickens at 0, 3, 7, and 13 days postinfection(p.i.). Thirty-five amplification cycles were performed, except for b-actin (28 cycles).

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was observed in infected ceca whereas no significant changeswere detected in infected jejunum. The strongest upregulationwas measured for IFN-g (300-fold) and iNOS (200-fold) ex-pression 7 days after E. tenella infection. Although IFN-g wasalso strongly (200-fold) upregulated during E. maxima infec-tion 7 days after infection, at the same time, iNOS expressionwas increased only slightly (10-fold) in the jejunum. COX-2mRNA expression increased 13-fold in the infected ceca,whereas little or no increase was measured in the E. maximainfected jejunum. The mRNA expression of the three TGF-bisoforms did not seem to be regulated during infection withEimeria, although minor increases can only be seen by quan-titative RT-PCR.

Upregulated expression of IFN-g in CD31 IEL from E. maxi-ma-infected jejunum. The number of CD31 cells increased in

the infected jejunal mucosa, as shown by immunohistochemis-try in Fig. 4. Among these cells, an increasing number of CD31

IEL was seen in the infected epithelium (Fig. 4). CD31 IELfrom a 7-day E. maxima-infected chicken overexpressed IFN-gmessenger 27-fold compared to CD31 IEL from an uninfectedchicken. Quantitative RT-PCR values were 2.0 3 106 6 1.0 3106 (n 5 4) and 5.4 3 107 6 2.9 3 107 (n 5 4) copies/mg oftotal RNA from CD31 IEL isolated from control and in-fected chickens, respectively. b-Actin expression measured inthe same samples was stable: 2.1 3 108 6 1.1 3 108 for allsamples analyzed.

IFN-g upregulates cytokine expression by macrophages.The presence of a large quantity of IFN-g in the mucosa iscapable of stimulating the synthesis of proinflammatory cyto-kines and chemokines. We analyzed whether the upregulated

FIG. 3. Quantitative RT-PCR determination of the time course of gene expression during Eimeria infection. Pools of total RNA extracted fromthe cecum and jejunum of six to eight PA12 chickens infected with E. tenella (A) and E. maxima (B), respectively, were prepared. QuantitativeRT-PCRs were performed for the 11 genes on both RNA pools for the four time points, and the number of mRNA copies expressed per microgramof total RNA was determined as described in Materials and Methods. Ratios between gene expression at days 7 and 0 are indicated in parentheses.Data were obtained from a representative experiment performed twice.

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gene expression that occurred in vivo following infection withEimeria could be reproduced by stimulating macrophages withIFN-g. Lipopolysaccharide (LPS) is a well-known strong in-ducer of macrophages and was used as positive control. Sixhours after stimulation, IFN-g and LPS activated HD-11 cellsupregulated mRNA expression for K203, MIP-1b, IL-1b,MGF, and iNOS (Fig. 5). However, unlike K203, MIP-1b wasalready well expressed in nonstimulated HD-11 cells (Fig. 5).Although the b-Gal COS7 supernatant dilution used did notinduce NOx release by HD-11 cells as detected by the Greissreaction, a small nonspecific stimulation of several cytokinesand of iNOS gene expression was observed after the incuba-tion. This discrepancy was probably due to the difference insensitivity of the two methods.

DISCUSSION

The intestinal mucosa provides both a physiologic and im-munologic barrier to pathogens. Coccidia of the genus Eimeriacomplete their life cycles within the epithelial cells of thechicken intestine. Although E. tenella sporozoites are some-times found in macrophages or IEL, this is regarded as a routeby which the parasite can be translocated within these cells intothe lamina propria and gain access to the crypt epithelial cells(37). The first line of defense against Eimeria is provided by theinfected epithelial cells and the cells in close contact with themsuch as IEL and fibroblasts. The RNA extraction method thatwe used allowed us to detect mainly the immune response inthe more apical part of the mucosa, although we cannot ex-clude some contamination with cells located deeper in thelamina propria.

The inflammation observed in Eimeria-infected intestine isassociated with an infiltration of macrophages and T cells (38),accompanied by edema and a thickening of the mucosa (27).IL-1b is a powerful proinflammatory cytokine secreted bymany different cell types, with stimulated macrophages beingthe main producer. IL-1b stimulates the secretion of chemo-kines by fibroblasts (39), macrophages (32), and epithelial cells(9), which can then attract inflammatory cells including mac-rophages, neutrophils, and lymphocytes, thus amplifying theimmune response. The upregulation of IL-1b mRNA was mea-sured during both E. tenella and E. maxima infection and mightcontribute to the chemokine upregulation observed. We andothers have previously shown that human intestinal epithelialcells upregulate IL-8 mRNA expression after infection withCryptosporidium parvum and Toxoplasma gondii (6, 22). In thepresent study, the mRNA levels for the CXC chemokines IL-8and K60 were unchanged or increased slightly compared tothe CC chemokines K203 and MIP-1b. CC chemokines aremore specifically involved in the recruitment of macrophages,whereas CXC chemokines participate in the recruitment ofneutrophils at inflammatory sites. Our data complement invivo observations that macrophages are the main inflammatorycells in the Eimeria-infected chicken mucosa (38). Moreover,we have shown that IFN-g-activated HD-11 cells display up-regulated mRNA expression for IL-1b and the CC chemo-kines. Although macrophages are most probably activated invivo after Eimeria infection, their relative participation in ourRNA extract is not known.

Another set of molecules involved in the mucosal immuneresponse in addition to chemokines are prostaglandins. Pros-taglandins are important inflammation mediators and regula-tors of gastrointestinal fluid secretion (8). Their synthesis fromarachidonic acid is dependent on the activities of an enzymethat exists in two isoforms, the constitutive COX-1 and theinducible COX-2. High-level expression of COX-2 can be in-duced in macrophages and in intestinal epithelial cells by stim-ulators like IL-1 and TNF (11, 14). In addition, human intes-tinal epithelial cells produce prostaglandins E2 and F2a via theinduction of COX-2 following infection with intestinal patho-gens such as C. parvum (23). Our present data show that theinducible cyclooxygenase mRNA was moderately upregulatedduring infection, suggesting that prostaglandin productioncould occur in response to both strains of Eimeria. However, to

FIG. 5. Upregulation of K203, MIP-1b, MGF, IL-1b, and iNOSexpression in chicken macrophage HD-11 cells stimulated with IFN-g.HD-11 cells were incubated for 6 h with COS7 supernatant containingb-Gal (2) or IFN-g (1) activity or incubated for 18 h with or withoutLPS (5 mg/ml). LPS was used as a positive control of HD-11 cellactivation. After RNA extraction and reverse transcription reaction, 35cycles of PCR were performed and products were resolved on 2%agarose gels.

FIG. 4. Localization of CD31 cells in the jejunal mucosa. Frozensections of control (A) and E. maxima-infected (B) chickens werelabeled with a mouse anti-chicken CD3 and then stained with a fluo-rescein isothiocyanate-conjugated goat anti-mouse antibody. Arrowsindicate IEL; arrowheads indicate parasites in the infected mucosa.Slides were slightly counterstained with Evans blue. Magnification,3400.

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confirm that hypothesis, prostaglandins must be measured andtheir relative contributions to inflammation and diarrhea dur-ing coccidiosis must to be investigated.

The TGF-b isoforms are important regulators of inflamma-tion, being proinflammatory at low concentrations and anti-inflammatory at high concentrations (30). These molecules areinvolved in differentiation and proliferation of T and B cells(21) and have been shown to delay and decrease the barrierdisruption caused by C. parvum (31). In a recent study, a slightincrease in TGF-b4 mRNA expression was observed in IELisolated from E. acevulina-infected SC chickens; however, thisupregulation was dependent on the chicken strain used (5).Although only qualitative RT-PCR measurements have beenperformed for the different TGF isoforms, in our hands, noclear upregulation of gene expression seems to occur during E.tenella and E. maxima infection in PA12 chickens.

IFN-g is a major factor in the development of resistance toEimeria, as it inhibits E. tenella development in vitro (26) andreduces oocyst production and body weight loss following E.acervulina infection (26, 28). In a recent study, IFN-g transcriptlevels were shown to be upregulated in the cecal tonsils, spleen,and intestinal IEL during the course of E. tenella infection(43). In the present study, 7 days after infection, we observeda recruitment of CD31 cells in the lamina propria and theepithelium of the E. maxima-infected jejunum. CD31 IELisolated from the infected jejunum produced 27-fold moreIFN-g mRNA than CD31 IEL isolated from uninfected jeju-num. The strong (200-fold) upregulation of IFN-g expressionin the jejunum of E. maxima-infected chicken was thereforeprobably due in large part to both the recruitment and stimu-lation of these cells. This high production of IFN-g may con-tribute to clearance of the infection and the development ofimmunity to reinfection. IFN-g induces iNOS expression inseveral cells types, including epithelial cells (40) and macro-phages (41). During E. maxima infection, levels of nitrite andnitrate reach peak values at about 6 days postinoculation (3),which concurs with our findings on the levels of iNOS mRNAmeasured in infected tissues. Although free radical species areproduced in response to Eimeria infections, their efficacyagainst the parasite in vivo is more debatable (1–3). We ob-served that iNOS mRNA expression was much more importantduring E. tenella than E. maxima infection. This may contributeto the hemorrhage frequently observed after E. tenella infec-tion, by causing vasodilatation in the cecum (2). The strongupregulation of MGF in infected cecum but not in infectedjejunum may contribute to the differences observed in iNOSupregulation in both regions of the intestine. Chickens admin-istered a live recombinant fowlpox virus that expresses MGFexhibited a marked and sustained increase in the number ofcirculating blood monocytes as well as enhanced phagocyticactivity and elevated production of nitric oxide (42). In thisstudy, we showed that recombinant chicken IFN-g was able toinduce both iNOS and MGF mRNA expression in HD-11 cells.These results suggest that IFN-g and MGF may both contrib-ute to iNOS induction in vivo.

The data presented here give an overview of the immuno-logically relevant gene-specific response to two strains of Ei-meria commonly found in poultry livestock and also providenew insights into a possible use for cytokines as therapeuticagents against this pathogen. The potential uses of cytokine

therapy in poultry via delivery with live vectors (viral andbacterial), naked DNA injection, or injection of the recombi-nant protein is currently being investigated by several groups(26, 29). The populations of cells upregulating cytokine geneexpression will have to be identified in order to further char-acterize the mechanisms by which the natural protective im-mune response against Eimeria occurs in vivo.

ACKNOWLEDGMENTS

We thank Genevieve Fort for expert technical help with the animals,Yves Le Vern for flow cytometric analysis, and Michele Peloille forperforming the sequencing during construction of the RT-PCR plas-mids. We are also very grateful to Declan McCole (UCSD) for criticalreview of the manuscript.

Rita Menezes was supported by a fellowship from the CAPES,Brasilia, Brazil.

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